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PurposeVascular endothelial growth factor (VEGF) causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization (NV). Basic fibroblast growth factor (bFGF) also has been implicated in the production of ocular NV. This study was performed to investigate the ability of simultaneous sustained intravitreal release of both VEGF and bFGF to induce robust retinal NV in the rabbit.MethodsIntravitreal implantation of sustained-release Hydron polymeric pellets containing both 20 microg of VEGF and 20 microg of bFGF was performed on adult male Dutch belted rabbits. In other animals either 20 microg or 50 microg bFGF-containing pellets was implanted intravitreally; also, either 20 microg VEGF or 50 microg VEGF-containing pellets was implanted. Control rabbits received either blank polymeric pellets or a pellet containing 30 microg bovine serum albumin. Eyes were examined by indirect ophthalmoscopy after surgery at 24 hrs, 48 hrs, 4 days, 7 days, 14 days, 21 days, and 28 days. Findings were documented by color fundus photography and fluorescein angiography (FA). Eyes were enucleated and prepared for histologic analysis at 28 days following intravitreal implantation of the VEGF/bFGF-containing pellets.ResultsIn all eyes implanted with VEGF/bFGF pellets, dilation and tortuosity of existing blood vessels were observed within 48 hrs after pellet implantation. The progression of retinal vascular changes was rapid and occurred over the entire optic disk and medullary rays between 4 and 7 days. Hemorrhage occurred as early as 14 days after VEGF/bFGF pellet implantation. In eyes with massive hemorrhage, total traction retinal detachment developed after the second week. The presence of abnormal tissues at the vitreo-retinal interface within 28 days was demonstrated by light microscopy while FA showed profuse leakage of dye from anomalous vessels within the first week. Neither bFGF-exposed eyes nor control eyes showed any vascular changes. Eyes that received only VEGF-containing pellets exhibited tortuosity of existing vessels, but neither hemorrhaging nor retinal detachment occurred.ConclusionsThese results demonstrate that retinal vascular changes leading to hemorrhaging is produced rapidly in the rabbit by simultaneous intravitreal release of both VEGF and bFGF. Understanding how these growth factors induce retinal NV may suggest novel therapeutic treatment strategies.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered M r 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a - Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.

Background: Pathogenic variants in TTN cause a spectrum of autosomal dominant and recessive cardiovascular, skeletal muscle and cardioskeletal disease with symptom onset across the lifespan. The aim of this study was to characterise the genotypes and phenotypes in a cohort of TTN +paediatric patients., Methods: Retrospective chart review was performed at four academic medical centres. Patients with pathogenic or truncating variant(s) in TTN and paediatric-onset cardiovascular and/or neuromuscular disease were eligible., Results: 31 patients from 29 families were included. Seventeen patients had skeletal muscle disease, often with proximal weakness and joint contractures, with average symptom onset of 2.2 years. Creatine kinase levels were normal or mildly elevated; electrodiagnostic studies (9/11) and muscle biopsies (11/11) were myopathic. Variants were most commonly identified in the A-band (14/32) or I-band (13/32). Most variants were predicted to be frameshift truncating, nonsense or splice-site (25/32). Seventeen patients had cardiovascular disease (14 isolated cardiovascular, three cardioskeletal) with average symptom onset of 12.9 years. Twelve had dilated cardiomyopathy (four undergoing heart transplant), two presented with ventricular fibrillation arrest, one had restrictive cardiomyopathy and two had other types of arrhythmias. Variants commonly localised to the A-band (8/15) or I-band (6/15) and were predominately frameshift truncating, nonsense or splice-site (14/15)., Conclusion: Our cohort demonstrates the genotype-phenotype spectrum of paediatric-onset titinopathies identified in clinical practice and highlights the risk of life-threatening cardiovascular complications. We show the difficulties of obtaining a molecular diagnosis, particularly in neuromuscular patients, and bring awareness to the complexities of genetic counselling in this population., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Single-nucleotide variants (SNVs) in the gene encoding Kinesin Family Member 5A (KIF5A), a neuronal motor protein involved in anterograde transport along microtubules, have been associated with amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive and fatal neurodegenerative disease that primarily affects the motor neurons. Numerous ALS-associated KIF5A SNVs are clustered near the splice-site junctions of the penultimate exon 27 and are predicted to alter the carboxy-terminal (C-term) cargo-binding domain of KIF5A. Mis-splicing of exon 27, resulting in exon exclusion, is proposed to be the mechanism by which these SNVs cause ALS. Whether all SNVs proximal to exon 27 result in exon exclusion is unclear. To address this question, we designed an in vitro minigene splicing assay in human embryonic kidney 293 cells, which revealed heterogeneous site-specific effects on splicing: only 5' splice-site (5'ss) SNVs resulted in exon skipping. We also quantified splicing in select clustered, regularly interspaced, short palindromic repeats-edited human stem cells, differentiated to motor neurons, and in neuronal tissues from a 5'ss SNV knock-in mouse, which showed the same result. Moreover, the survival of representative 3' splice site, 5'ss, and truncated C-term variant KIF5A (v-KIF5A) motor neurons was severely reduced compared with wild-type motor neurons, and overt morphological changes were apparent. While the total KIF5A mRNA levels were comparable across the cell lines, the total KIF5A protein levels were decreased for v-KIF5A lines, suggesting an impairment of protein synthesis or stability. Thus, despite the heterogeneous effect on ribonucleic acid splicing, KIF5A SNVs similarly reduce the availability of the KIF5A protein, leading to axonal transport defects and motor neuron pathology., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Kinesin family member 5A (KIF5A) is an essential, neuron-specific microtubule-associated motor protein responsible for the anterograde axonal transport of various cellular cargos. Loss of function variants in the N-terminal, microtubule-binding domain are associated with hereditary spastic paraplegia and hereditary motor neuropathy. These variants result in a loss of the ability of the mutant protein to process along microtubules. Contrastingly, gain of function splice-site variants in the C-terminal, cargo-binding domain of KIF5A are associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease involving death of upper and lower motor neurons, ultimately leading to degradation of the motor unit (MU; an alpha motor neuron and all the myofibers it innervates) and death. These ALS-associated variants result in loss of autoinhibition, increased procession of the mutant protein along microtubules, and altered cargo binding. To study the molecular and cellular consequences of ALS-associated variants in vivo, we introduced the murine homolog of an ALS-associated KIF5A variant into C57BL/6 mice using CRISPR-Cas9 gene editing which produced mutant Kif5a mRNA and protein in neuronal tissues of heterozygous (Kif5a +/c.3005+1G>A ; HET) and homozygous (Kif5a c.3005+1G>A/c.3005+1G>A ; HOM) mice. HET and HOM mice appeared normal in behavioral and electrophysiological (compound muscle action potential [CMAP] and MU number estimation [MUNE]) outcome measures at one year of age. When subjected to sciatic nerve injury, HET and HOM mice have delayed and incomplete recovery of the MUNE compared to wildtype (WT) mice suggesting an impairment in MU repair. Moreover, aged mutant Kif5a mice (aged two years) had reduced MUNE independent of injury, and exacerbation of the delayed and incomplete recovery after injury compared to aged WT mice. These data suggest that ALS-associated variants may result in an impairment of the MU to respond to biological challenges such as injury and aging, leading to a failure of MU repair and maintenance. In this report, we present the behavioral, electrophysiological and pathological characterization of mice harboring an ALS-associated Kif5a variant to understand the functional consequences of KIF5A C-terminal variants in vivo., (Copyright © 2023. Published by Elsevier Inc.)

Numerous pediatric neurogenetic diseases may be optimally treated by in utero gene therapy (IUGT); but advancing such treatments requires animal models that recapitulate developmental physiology relevant to humans. One disease that could benefit from IUGT is the autosomal recessive motor neuron disease spinal muscular atrophy (SMA). Current SMA gene-targeting therapeutics are more efficacious when delivered shortly after birth, however postnatal treatment is rarely curative in severely affected patients. IUGT may provide benefit for SMA patients. In previous studies, we developed a large animal porcine model of SMA using AAV9 to deliver a short hairpin RNA (shRNA) directed at porcine survival motor neuron gene (Smn) mRNA on postnatal day 5. Here, we aimed to model developmental features of SMA in fetal piglets and to demonstrate the feasibility of prenatal gene therapy by delivering AAV9-shSmn in utero. Saline (sham), AAV9-GFP, or AAV9-shSmn was injected under direct ultrasound guidance between gestational ages 77-110 days. We developed an ultrasound-guided technique to deliver virus under direct visualization to mimic the clinic setting. Saline injection was tolerated and resulted in viable, healthy piglets. Litter rejection occurred within seven days of AAV9 injection for all other rounds. Our real-world experience of in utero viral delivery followed by AAV9-related fetal rejection suggests that the domestic sow may not be a viable model system for preclinical in utero AAV9 gene therapy studies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Using a spatial analysis methodology, we analyzed sport participation through membership data of a Provincial Sport Organization (PSO) in Ontario, Canada. Specifically, our analysis brings attention to the participation of women and girls in Row Ontario and the urban and rural nature of the PSO's membership. This research was conducted in partnership with the PSO to provide insights into how contextual factors influence sport participation and how these findings can inform future sport development efforts. Our results demonstrate that women and girls represented the majority of participants within the PSO and highlight an opportunity to use participant centered approaches for sport development to grow women and girls' participation. This is a promising outcome as women and girls are generally underrepresented in sport and may face greater participation barriers in rural contexts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rich, Moore, Boggs and Pegoraro.)

Background: Longitudinal access to cerebrospinal fluid (CSF) is useful for biomarker discovery in neurological disorders or diseases affecting CSF composition. Here, we aim to test a new method for insertion of a permanent intrathecal catheter, facilitating longitudinal collection of CSF., New Method: We surgically placed a permanent intrathecal catheter into the cisterna magna of anesthetized neonatal piglets. The thecal sac was accessed at the L5-S1 spinal level and a radiopaque catheter was inserted under fluoroscopic x-ray guidance to position the tip at the cisterna magna. A titanium access port was connected to the catheter and anchored subcutaneously. Immediately after surgery, we confirmed CSF flow through the catheter and port via needle aspiration. Catheter patency over a two-month study period was determined through periodic CSF collection from the port., Results: Frequent (up to 3 times weekly), longitudinal sampling of CSF was achievable in neonatal piglets up to 60 days after implantation. CSF was readily accessible through the port without major adverse events. Catheterized piglets demonstrated slower, but normal, weight gain compared to control piglets. Post-operative complications were managed with standard access precautions and medications. There were no complications involving the implanted hardware., Comparison With Existing Method(s): This method fills a critical gap in the existing methods for longitudinal CSF sampling through an implanted intrathecal catheter system in neonatal piglets., Conclusions: This novel method is both safe and effective for longitudinal CSF access in the domestic piglet. Catheter patency and access to CSF is maintained over multiple months without major adverse events., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: To retrospectively evaluate the utility of serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NfL) and phosphorylated neurofilament heavy chain (pNfH) as biomarkers for spinal muscular atrophy (SMA) progression and response to nusinersen treatment., Methods: NfL and pNfH levels were quantified using single molecular array (SIMOA) in CSF of 33 adult SMA patients (SMN copy number 3-5) before and in response to nusinersen treatment. In 11 of the patients, blood serum samples were also collected. CSF NfL and pNfH from patients were compared to CSF Nfs from age-matched controls without neurological disease (n = 6). For patients, pearson correlation coefficients (r) were calculated to investigate associations between Nf levels and other functional outcome measures., Results: Nf levels were similar between SMA and control adults and showed no change in response to nusinersen treatment in CSF or serum. Cross-sectional analyses showed an increase in CSF NfL and pNfH with age in patients (NfL p = 0.0013; pNfH p = 0.0035) and an increase in CSF NfL in controls (p = 0.002). In non-ambulatory patients, baseline serum pNfH showed a negative correlation with multiple strength and functional assessment metrics including Revised Upper Limb Module (r = -0.822, p = 0.04), upper extremity strength (r = -0.828, p = 0.042), lower extremity strength (r = -0.860, p = 0.028), and total strength (r = -0.870, p = 0.024)., Conclusions: Nf levels did not change in response to nusinersen in adults with SMA and were not different from controls. In patients and controls, we detected an age-related increase in baseline CSF NfL and pNfH levels. Though some associations were identified, our results suggest Nf levels are not preditive or prognostic biomarkers in this population.

Community is a context for much research in sport, sport management, and sport policy, yet relatively few authors explicitly articulate the theoretical frameworks with which they interrogate the concept. In this paper, we draw from communitarian theory and politics in order to contribute to a robust discussion and conceptualization of community in and for sport management research and practice. We provide a synthesis of current sport management and related research in order to highlight contemporary theoretical and methodological approaches to studying community. We distinguish between community as a context, as an outcome, as a site for struggle or resistance, as well as a form of regulation or social control. We then advance a critical communitarian agenda and consider the practical implications and considerations for research and practice. This paper synthesizes current research and establishes a foundation upon which sport management scholars and practitioners might critically reflect on community and deliberatively articulate its implications in both future research and practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rich, Spaaij and Misener.)

The availability of disease modifying therapies for spinal muscular atrophy (SMA) has created an urgent need to identify clinically meaningful biomarkers. Biomarkers present a means to measure and evaluate neurological disease across time. Changes in biomarkers provide insight into disease progression and may reveal biologic, physiologic, or pharmacologic phenomena occurring prior to clinical detection. Efforts to identify biomarkers for SMA, a genetic motor neuron disease characterized by motor neuron degeneration and weakness, have culminated in a number of putative molecular and physiologic markers that evaluate biological media (eg, blood and cerebrospinal fluid [CSF]) or nervous system function. Such biomarkers include SMN2 copy number, SMN mRNA and protein levels, neurofilament proteins (NFs), plasma protein analytes, creatine kinase (CK) and creatinine (Crn), and various electrophysiology and imaging measures. SMN2 copy number inversely correlates with disease severity and is the best predictor of clinical outcome in untreated individuals. SMN mRNA and protein are commonly measured in the blood or CSF of patients receiving SMA therapies, particularly those aimed at increasing SMN protein expression, and provide insight into current disease state. NFs have proven to be robust prognostic, disease progression, and pharmacodynamic markers for SMA infants undergoing treatment, but less so for adolescents and adults. Select plasma proteins are altered in SMA individuals and may track response to therapy. CK and Crn from blood correlate with motor function and disease severity status and are useful for predicting which individuals will respond to therapy. Electrophysiology measures comprise the most reliable means for monitoring motor function throughout disease course and are sensitive enough to detect neuromuscular changes before overt clinical manifestation, making them robust predictive and pharmacodynamic biomarkers. Finally, magnetic resonance imaging and muscle ultrasonography are non-invasive techniques for studying muscle structure and physiology and are useful diagnostic tools, but cannot reliably track disease progression. Importantly, biomarkers can provide information about the underlying mechanisms of disease as well as reveal subclinical disease progression, allowing for more appropriate timing and dosing of therapy for individuals with SMA. Recent therapeutic advancements in SMA have shown promising results, though there is still a great need to identify and understand the impact of biomarkers in modulating disease onset and progression., Competing Interests: Declaration of Conflicting Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: SJK has received compensation for consulting from Biogen, Roche, and Novartis., (© The Author(s) 2021.)

Objective: To report the frequency of amyotrophic lateral sclerosis (ALS) genetic variants in a nationwide cohort of clinic-based patients with ALS with a family history of ALS (fALS), dementia (dALS), or both ALS and dementia (fALS/dALS)., Methods: A multicenter, prospective cohort of 573 patients with fALS, dALS, or fALS/dALS, underwent genetic testing in the ALS Genetic Access Program (ALS GAP), a clinical program for clinics of the Northeast ALS Consortium. Patients with dALS underwent C9orf72 hexanucleotide repeat expansion (HRE) testing; those with fALS or fALS/dALS underwent C9orf72 HRE testing, followed by sequencing of SOD1 , FUS , TARDBP , TBK1 , and VCP ., Results: A pathogenic (P) or likely pathogenic (LP) variant was identified in 171/573 (30%) of program participants. About half of patients with fALS or fALS/dALS (138/301, 45.8%) had either a C9orf72 HRE or a P or LP variant identified in SOD1 , FUS , TARDBP , TBK1 , or VCP . The use of a targeted, 5-gene sequencing panel resulted in far fewer uncertain test outcomes in familial cases compared with larger panels used in other in clinic-based cohorts. Among dALS cases 11.8% (32/270) were found to have the C9orf72 HRE. Patients of non-Caucasian geoancestry were less likely to test positive for the C9orf72 HRE, but were more likely to test positive on panel testing, compared with those of Caucasian ancestry., Conclusions: The ALS GAP program provided a genetic diagnosis to ∼1 in 3 participants and may serve as a model for clinical genetic testing in ALS., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Background: Physicians report discomfort when interacting with patients with disabilities, which can negatively impact the quality of healthcare they provide., Objective/hypothesis: An intervention structured around a formative clinical encounter was assessed for its effectiveness in changing comfort towards treating patients with disabilities. It was predicted that this encounter would have a positive short- and long-term impact on medical students., Method: During the 2017-2018 academic year, 169 third-year medical students conducted a patient encounter with a person who had a disability. Students met individually with the "patient" and completed a brief social and medical history as if they were meeting a new patient to establish care. A measure of perceived comfort caring for patients with disabilities was administered to students before and after the encounter. One year after the patient encounter, 59 students were surveyed about their satisfaction and the impact of the patient encounter., Results: The impact of encountering people with disabilities in a clinical setting was positive, with statistically significant improvements across all items on the measure of perceived comfort. Students were highly satisfied with the experience and anticipated feeling more confident, more comfortable, less awkward, and more skilled and efficacious when encountering a person with a disability in their future practice. A thematic analysis of the one year follow-up data suggest that students valued the encounter and desired more content on disability throughout their education., Conclusions: Medical education should include dedicated exposure to persons with disabilities and a simulated patient experience allowing for a safe environment to gain skills and confidence., Competing Interests: Declaration of competing interest The authors report no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Genome-wide association studies (GWAS) and rare variant association studies (RVAS) are applied across many areas of complex disease to analyze variation in whole genomes of thousands of unrelated patients. These approaches are able to identify variants and/or biological pathways which are associated with disease status and, in contrast to traditional linkage studies or candidate gene approaches, do so without requiring multigenerational affected families, prior hypotheses, or known genes of interest. However, the novel associations identified by these methods typically have lower effect sizes than those found in classical family studies. In the motor neuron disease amyotrophic lateral sclerosis (ALS), GWAS, and RVAS have been used to identify multiple disease-associated genes but have not yet resulted in novel therapeutic interventions. There is significant urgency within the ALS community to identify additional genetic markers of disease to uncover novel biological mechanisms, stratify genetic subgroups of disease, and drive drug development. Given the widespread and increasing application of genetic association studies of complex disease, it is important to recognize the strengths and limitations of these approaches. Here, we review ALS gene discovery via GWAS and RVAS., Competing Interests: SK has received compensation for consulting from Biogen Idec, and AveXis. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rich, Roggenbuck and Kolb.)

Background: Variants in TTN are frequently identified in the genetic evaluation of skeletal myopathy or cardiomyopathy. However, due to the high frequency of TTN variants in the general population, incomplete penetrance, and limited understanding of the spectrum of disease, interpretation of TTN variants is often difficult for laboratories and clinicians. Currently, cardiomyopathy is associated with heterozygous A-band TTN variants, whereas skeletal myopathy is largely associated with homozygous or compound heterozygous TTN variants. Recent reports show pathogenic variants in TTN may result in a broader phenotypic spectrum than previously recognized., Methods: Here we report the results of a multisite study that characterized the phenotypes of probands with variants in TTN. We investigated TTN genotype-phenotype correlations in probands with skeletal myopathy and/or cardiomyopathy. Probands with TTN truncating variants (TTNtv) or pathogenic missense variants were ascertained from two academic medical centers. Variants were identified via clinical genetic testing and reviewed according to the American College of Medical Genetics criteria. Clinical and family history data were documented via retrospective chart review. Family studies were performed for probands with atypical phenotypes., Results: Forty-nine probands were identified with TTNtv or pathogenic missense variants. Probands were classified by clinical presentation: cardiac (n = 30), skeletal muscle (n = 12), or both (cardioskeletal, n = 7). Within the cardioskeletal group, 5/7 probands had heterozygous TTNtv predicted to affect the distal (3') end of the A-band. All cardioskeletal probands had onset of proximal-predominant muscle weakness before diagnosis of cardiovascular disease, five pedigrees support dominant transmission., Conclusion: Although heterozygous TTNtv in the A-band is known to cause dilated cardiomyopathy, we present evidence that these variants may in some cases cause a novel, dominant skeletal myopathy with a limb-girdle pattern of weakness. These findings emphasize the importance of multidisciplinary care for patients with A-band TTNtv who may be at risk for multisystem disease., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Understanding the historical framework in which species interactions have diversified across landscapes may help to partition the effects of vicariance and geographically variable selection in shaping the geographical mosaic of coevolving species. We used phylogeographical analyses of the pollinating seed parasite Greya politella (Lepidoptera: Prodoxidae) to define the historical processes that may have structured interactions of this species with its host plants across major biogeographical breaks in western North America. Using 648 bp of cytochrome oxidase I and amplified fragment length polymorphisims, we identified deep genetic breaks among some populations consistent with some definitions of cryptic species. A combination of phylogenetic and population genetic approaches indicates that different historical processes may have structured G. politella genetic diversity in four regions: northern Pacific Northwest, southern Oregon, southern Sierra Nevada, and the remainder of California. The northern Pacific Northwest had high genetic diversity likely due to glacial refugia and subsequent spatial expansion, concordant with some other taxa. Populations in southern Oregon possessed unique, closely related haplotypes with restricted gene flow, possibly indicating a long-standing set of populations in this endemic-rich region. Analyses of California populations showed evidence of restricted gene flow and spatial expansion with many closely related haplotypes that occupy a broad geographical range. Southern Sierra Nevada populations were genetically distinct and highly diverse, possibly due to a localized glacial refugium. Together, these results suggest that vicariance and population expansion, possibly in combination with geographically variable selection, have shaped the diversification of G. politella and its interactions with its host plants.

Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components. The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes. In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy. If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles. The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria. These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER. Internalization of metabolically arrested cytoplasmic L. monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin. Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway. Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation.

Purpose: We investigated the effects of 35 weeks of a cholesterol diet in apolipoprotein E (apoE)-deficient mice on their ERG response., Methods: C57BL/6J and apoE-deficient mice were fed regular mouse chow (C57-R and ApoE-R, respectively) or a cholesterol-containing diet (C57-C and ApoE-C, respectively). Retinal function was assessed by dark-adapted electroretinography (ERG). Retina tissue was also analyzed by immunohistochemical staining and nucleic acid array expression analysis performed by gene array technology., Results: ApoE-C mice had diminished a- and b-wave amplitudes (60.7% +/- 8.4% (p < 0.005) and 44.8% +/- 10% (p < 0.005) of control values, respectively). Gene expression profiling revealed upregulation of several pro-apoptotic genes. Furthermore, immunohistochemistry showed increased Bax immunoreactivity., Conclusions: In the hypercholesterolemic mice, we demonstrated a loss of ERG response and induction of apoptotic activity at the gene and protein levels. Our current and previous findings suggest that cholesterol metabolism plays an important role in retinal function.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used as analgesics. They inhibit cyclooxygenases (COX), preventing the formation of prostaglandins, including prostacyclin and thromboxane. A serious side effect of COX-1 and COX-2 is renal damage. We report here that both a nonselective NSAID (aspirin, acetylsalicylic acid) and COX-2 selective NSAIDs (celecoxib and NS-398) diminished renal prostacyclin and thromboxane concentration in the renal medulla. NSAIDs failed to change COX-2 and iNOS (the inducible form of NO synthase) expression. A NO donor, B-NOD, preserved renal prostacyclin and thromboxane after administration of aspirin. PGI2 and COX-2 protein were mainly expressed in the renal medulla, whereas iNOS expression was greater in the cortex. B-NOD preserved renal prostacyclin levels after administration of NSAIDs.

Appendicitis is a frequent pediatric surgical condition for which there is great variability among practitioners regarding diagnosis and postoperative management. With this in mind, the authors designed and implemented an evidence-based appendicitis clinical pathway at their institution. Establishment of the pathway resulted in decreased hospital cost, reduced hospital stay, and fewer unnecessary laboratory tests. The purpose of the current study was to determine the sustainability of the pathway beyond its initial implementation phase. The authors showed that several, but not all, favorable outcomes of the pathway were sustained. These data suggest that a clinical pathway for appendicitis at the authors' institution results in sustained beneficial effects in some but not all outcome parameters. Ongoing monitoring of pathway compliance, continued education of practitioners and nursing personnel, and identification of key pathway team member(s) responsible for the pathway system might result in a greater long-term impact of these guidelines., (Copyright 2002 by W.B. Saunders Company)

Purpose: Several studies have shown an overexpression of cyclooxygenase-2 (COX-2) and elevated levels of prostacyclin (PGI(2)) and thromboxane (TXA(2)) in colon cancer. In this report, we determined the distribution of inducible form of nitric oxide synthase (iNOS), PGI(2), and TXA(2) in cancerous and adjoining areas of specimens from human colon and breast cancer obtained during surgery. Additionally, we investigated differences in expression and histological localization of COX-2 in colon and breast cancer., Experimental Design: Specimens were obtained during surgery, one centrally located, the second from an adjacent, cancer-free area. Activity of iNOS was determined, using the conversion of L-[(14)C]arginine to L-[(14)C]citrulline. PGI(2) and TXA(2) were measured as their stable metabolites, using enzyme immunoassay. A standard immunoperoxidase method was used for immunohistochemical expression of COX-2., Results: Significant differences in iNOS, PGI(2), and TXA(2) expressions between colon and breast cancer were noted, with an enhanced expression of COX-2 in colon cancer, including the cancerous, adjoining, and stromatous fields., Conclusions: Increased expression of iNOS and production of prostanoids in colon cancer parallels the increase in COX-2, confirming the importance of this enzyme in colon cancer. The overexpression of COX-2, prostanoids, and nitric oxide in areas adjoining the tumor indicates increased metastatic potential for neoplastic cells in this area. Inflammatory changes in the tissue adjoining the cancer may play a role. COX-2 may result in the formation of new blood vessels and the spread of cancer.

Purpose: To examine the pathologic changes in the retina of apolipoprotein E (apoE)-deficient mice fed a high-cholesterol diet., Methods: ApoE-deficient mice (ApoE) were maintained on either regular mouse chow (ApoE-R) or a high-cholesterol diet (ApoE-C) for 25 weeks. Age-matched control C57BL/6J mice (C57) were also maintained on either regular mouse chow (C57-R) or a cholesterol-containing diet (C57-C). Retinal function was assessed by dark-adapted electroretinography (ERG). The eyes were embedded, sectioned, and analyzed by histologic and immunohistochemical methods, as well as by light and transmission electron microscopy., Results: After the 25-week feeding period, ERG tracings of ApoE-C mice revealed significant increases of a- and b-wave implicit times when compared with the C57-R group of mice. In addition, there were reductions in oscillatory potential (OP) amplitudes in the ApoE-C group. However, a- and b-wave amplitudes appeared to be unchanged among the four groups of mice. Light microscopic examination of the retinas showed that compared with control C57-R mice, ApoE-C mice had significantly lower cell numbers in the inner and outer nuclear layers (85.1% +/- 4.6%, P < 0.05 and 81.4% +/- 3.7%, P < 0.01 of C57-R controls, respectively). Transmission electron microscopy of apoE-deficient mice revealed cells of the inner nuclear layer with condensation of nuclear chromatin and perinuclear vacuolization in focal areas. Bruch's membrane was also found to be thicker, and its elastic lamina appeared disorganized and discontinuous. Immunohistochemistry demonstrated diminished or no immunoreactivity for carbonic anhydrase II and calretinin in the retinal layers of apoE-deficient mice., Conclusions: Overall, there were increasing abnormalities of retinal function and cellular morphology among the four groups of mice in the order of C57-R < C57-C < ApoE-R < ApoE-C. These findings suggest that apoE and/or cholesterol play an important role in retinal function.

Purpose: The aim of this study is to isolate and characterize cDNA clones and the genes that encode mouse RPE retinal G protein-coupled receptor (RGR) and to analyze expression of the RGR gene in the developing mouse retina. The conserved amino acid sequences of RGR from various mammals can be compared to the amino acid sequence motif of G protein-coupled receptors., Methods: Mouse RGR cDNA and gene clones were isolated from a retina cDNA library and 129SV genomic DNA library, respectively. The expression of RGR in the developing C57BL/6J mouse retina was analyzed by immunohistochemical staining with a polyclonal antipeptide antibody., Results: The deduced amino acid sequence of mouse RGR is 78% and 81% identical to that of bovine and human RGR, respectively. The mouse RGR gene is split into seven exons and extends about 11 kb. Two predominant mRNA transcripts, 1.9 and 1.7 kb in length, and a third, relatively faint, 5.5-kb transcript were detected in mouse eye by hybridization to a RGR cDNA probe. Frozen sections of C57BL/6J mouse retina at various stages of development were incubated with a mouse RGR antipeptide antibody. RGR immunoreactivity was first seen at postnatal day 2 (P2) in centrally located RPE cells. From day P6 to P12, there was an increase in the number and intensity of immunoreactive RPE cells in the central and mid-peripheral regions of the retina, while the most peripheral RPE cells were still negative. By day P16, the length of the RPE monolayer was immunoreactive, and staining of the central RPE cells was markedly more intense than at younger ages., Conclusions: Mouse and human RGR are highly conserved. A gradient of RGR expression in RPE extends from the central to the peripheral retina during development. In reference to the appearance of melanin-positive differentiated RPE cells, the induction of RGR expression is a relatively late event in the maturation of the retina.

Mouse retinal photoreceptor cell generation and morphogenesis take place in a well-characterized temporal sequence. Both rod and cone photoreceptor differentiation and synaptogenesis occur postnatally, but the relative timing of these events has been difficult to document due to the paucity of cell-specific markers. We have found that antibodies to neuron-specific enolase (NSE) preferentially label a subpopulation of photoreceptors in the outer nuclear layer (ONL) of the mouse retina in addition to labeling ganglion, amacrine, bipolar, and horizontal cells within the inner layers of the retina. The appearance of NSE immunoreactivity in the different classes of retinal neurons during development showed a close temporal relationship to the onset of expression of the synaptic vesicle-associated protein SV2 and clearly preceded the sequential development of synaptic connections in both inner and outer synaptic layers. The NSE-immunoreactive photoreceptors were identified as cones by dual labeling of their inner segments with the lectin peanut agglutinin or by colabeling with antisera to cone photopigments. Axonal extensions of NSE-labeled cone cells were shown to interact with those of differentiating horizontal cells as early as postnatal day 3 (P3). Colocalization of NSE with SV2 indicated that cone cells began to make synaptic contacts with horizontal cell processes several days prior to the development of rod synaptic terminals. Between P4 and P11, cone photoreceptor cell nuclei were observed to be scattered at various levels throughout the ONL and thus appeared to have become displaced from their previous position directly beneath the outer limiting membrane (OLM). By P12, the cone nuclei had migrated sclerad once again and were now observed to be neatly aligned adjacent to the OLM. In the rd mouse mutant, this migratory process was delayed, so that, at P12, positioning of the cone cell nuclei within the ONL was still quite irregular. Thus, we have identified a late migratory phase for cone photoreceptors during the second week after birth that correlates with the timing of maturation of the rod synaptic terminals just prior to eye opening. The types of cues used by maturing cone cells for their eventual sclerad location remain to be elucidated.

Purpose: A retroviral marker was used to label daughter cells arising from individual neuroblasts in the rd mouse retina, in order to investigate the hypothesis that a clonal relationship exists among degenerating photoreceptor cells., Methods: On the day of birth, a single injection of retrovirus with a lac Z (beta-galactosidase) reporter construct was injected into the retina in the vicinity of the subretinal space. Descendants of single neuroblasts were identified histochemically by examining the retinas at P14 (postnatal day 14). Light and electron microscopic studies were used to identify the retrovirally-induced marker beta-galactosidase using Bluo-gal dye. Double-labeling of degenerating cone cells was accomplished by taking 100 microns vibratome sections of retrovirally-injected eyes and using either FITC-PNA or HRP-PNA to visualize clusters of degenerating cones as well as Bluo-gal labeled clones of photoreceptor cells on the same tissue section., Results: A relatively large number of clones of primarily photoreceptor cells were observed in the peripheral retinas of both normal and rd mice. In a few cases in the rd, photoreceptor cells in a given clone consisted of both PNA- and Bluo-gal-labeled cells as well as of only Bluo-gal-labeled cells., Conclusion: These results suggest that during the period of intense cell death in the rd retina, a single dying photoreceptor cell can be surrounded by photoreceptors (either rods or cones) from the same clone that appear morphologically normal without evidence of degeneration.

Selective degeneration of rod photoreceptor cells in the retinal degenerative (rd) mouse prior to their complete maturation is thought to result from elevated cyclic guanosine monophosphate (cGMP) levels owing to the inherited defect in cGMP-phosphodiesterase. To investigate potential signaling pathways which might lead to apoptotic death of photoreceptors in the rd retina, the expression of immediate-early genes (IEG) of the activating protein-1 transcription factor (AP-1) family was examined. Increasing numbers of apoptotic photoreceptor nuclei were observed in the outer nuclear layer of the rd mouse beginning at postnatal day (P) 10. The peak incidence of apoptotic cells was observed at P13; by P16, almost the entire population of photoreceptors had been lost. Although c-Fos-like immunoreactivity was absent in photoreceptors of normal retinas, we observed that commencing at around P10, increasing numbers of rod photoreceptors in the rd retina exhibited nuclear staining for c-Fos protein. While no change in the distribution patterns of other members of the AP-1 family (c-Jun, JunB, and JunD) was observed in photoreceptors, Müller cell nuclei were transiently immunoreactive for c-Jun on P11. The incidence of c-Fos-positive photoreceptors peaked sharply at P12, 1 day earlier than the peak in apoptosis. Furthermore, the population of c-Fos-positive photoreceptors was distinct from apoptotic photoreceptors exhibiting chromatin condensation. The aberrant expression of c-Fos protein in rod photoreceptors immediately prior to their death in the rd mouse raises the possibility that c-Fos may be directly or indirectly involved in triggering the apoptotic cascade. Furthermore, the additional finding of c-Jun induction in Müller glia suggests that the IEG response to photoreceptor degeneration involves both intra- and intercellular signal transduction pathways.

Purpose: Viral-mediated gene transfer to retina, as well as to other tissues, is evolving rapidly. We have evaluated the potential of a retroviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells., Methods: Two recombinant retroviral vectors were prepared; in each Vector, a 1.4 kb fragment of the mouse opsin promoter was placed downstream from the neoR gene in the Moloney murine leukemia virus-based vector G1Na. The opsin promoter fragment was linked either to the cDNA for mouse rod photoreceptor phosphodiesterase (PDE) beta-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expression after transduction of 3T3 and Y79 cells, or of dissociated retinal cell cultures or retinal explants from neonatal mice., Results: As expected, PDE beta-subunit and beta-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. Northern blot analysis indicated that expression was derived from the viral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in which beta-galactosidase is driven by the viral LTR, resulted in expression in many cell types, while the opsin-lacZ vector mediated the expression of the lacZ reporter gene specifically in photoreceptor cells., Conclusions: The internal opsin promoter fragment appears capable of selectively directing gene expression to photoreceptor cells after retroviral-mediated gene transfer. These findings serve as a basis for future studies using the opsin promoter-beta PDE retroviral vector to rescue photoreceptor cells in the rd mutant mouse, in which the beta-PDE gene is mutated resulting in degeneration of photoreceptor cells during the early postnatal period.

Thrombospondin-1 (TSP) is a 450-KD glycoprotein that was initially discovered in the platelet alpha-granule. It now appears that TSP is intimately involved in the regulation of a variety of cellular functions and cell-to-cell interactions. Recently, it has been demonstrated that TSP functions as a p53-dependent inhibitor of angiogenesis in cultured fibroblasts from Li-Fraumeni patients and therefore may be an important factor involved with tumor invasion and metastasis. It has previously been demonstrated that TSP can be detected in frozen tissue sections by immunohistochemical methods. Our objective in this study was to determine the optimal antigen retrieval (AR) protocol for detection of TSP in formalin-fixed, paraffin-embedded tissue by using tissue sections from patients with invasive transitional cell carcinoma of the bladder. The optimal AR protocol was determined utilizing a variety of heating conditions and antigen retrieval buffers. Our results demonstrate that TSP can be reliably detected in paraffin-embedded tissue by immunohistochemical techniques that utilize AR with high-temperature microwave heating and a low-pH Tris-HCI buffer. The importance of this method is that it allows the reliable detection of TSP in archival tissue. This should facilitate further investigation into TSP's role in the regulation of cellular processes, including its influence on tumor angiogenesis and metastasis.

Embryonic lung maturation in the H-2 congenic pair, B10.A and B10, proceeds at different rates. The dependence of this heterochronic development on maternal haplotype suggests the involvement of a parentally imprinted gene. Since B10.A (H-2a) and B10 (H-2b) mice are genetically identical except for a 3-18 cM region of chromosome 17 that includes the H-2 complex, we sought a promising candidate gene(s) involved in regulating the rate of lung development from genes encoded in this region. The best candidate is the gene encoding the type II insulin-like growth factor receptor (IGF-IIR), whose ligand is the growth factor IGF-II. Only the maternal copy of this gene is expressed in postimplantation embryos. This receptor does not appear to transduce mitogenic signals; instead, IGF-IIR appears to regulate the levels of its ligand available to the growth-promoting type I IGF receptor (IGF-IR). Using in situ hybridization and indirect immunofluorescence, we demonstrate that IGF-IIR mRNA and protein are localized throughout the pulmonary mesenchyme, as well as in branching epithelia of the pseudoglandular and canalicular stages. We also examined the levels of IGF-IIR mRNA and protein expression by RNase protection assay and ligand blotting during the embryonic period of lung development in B10.A and B10 mice, and found that there is a highly significant positive correlation of IGF-IIR levels with progressive development in both strains. Further, slower-developing B10.A lungs contain significantly higher levels of IGF-IIR mRNA and protein than the more rapidly developing B10 lungs. These results suggest that haplotype-dependent elevation of IGF-IIR levels reduces the available concentration of IGF-II, resulting in a decreased rate of morphogenesis in B10.A mice. Heterochronic lung maturation, then, appears consequent to variable extracellular levels of this important growth factor. These results may be of clinical importance to predicting susceptibility to Respiratory Distress Syndrome in prenatal newborns.

Müller cells have been proposed to play an important role in photoreceptor cell development during the final stages of retinal maturation. The effect of disrupting Müller cells during mouse retinal development was investigated using the specific glial cell toxin, DL-alpha-aminoadipic acid (AAA). By giving multiple systemic injections over several days, impairment of Müller cell function was maintained during the period of photoreceptor migration and differentiation. Following three consecutive days of AAA treatment [commencing on post-natal (P) day 3, 5, 7 or 9, and examined at P8-P14], clumps of photoreceptor nuclei were displaced through the inner segments, lying immediately beneath the retinal pigment epithelium (RPE). Apart from the scalloped appearance of the outer retina, the overall lamination pattern of the retina was relatively well preserved. Even when AAA treatment commenced as early as P3, several days prior to the formation of the outer nuclear layer, the majority of photoreceptors migrated to their correct position and formed inner and outer segments. Therefore, the signals for photoreceptor migration are either provided by the Müller cells prior to P3, or, alternatively, are derived from different intrinsic or extrinsic cues. Disruption of Müller cell function was evidenced by decreased glutamine synthetase activity as well as by increased glial fibrillary acidic protein (GFAP) and decreased cellular retinaldehyde-binding protein (CRALBP) immunoreactivity. Immunocytochemistry with an antibody to CD44, which labels the microvilli of Müller cells at the outer limiting membrane, coupled with electron microscopic analysis, demonstrated that the zonulae adherentes between Müller cells and photoreceptors were either irregular or absent in areas adjacent to displaced clumps of photoreceptors. Thus AAA treatment of early post-natal mice results in localized disruption of the contacts between Müller cells and photoreceptors. These pathologic changes persist into adulthood since at P28, while short stretches of photoreceptors appeared relatively normal with fully developed outer segments, periodic clumps of displaced photoreceptor nuclei were still present adjacent to the RPE. In conclusion, Müller cell processes at the outer limiting membrane appear to play a critical role in providing a barrier to aberrant photoreceptor migration into the subretinal space.

In this study, 29 formalin-fixed, paraffin-embedded astrocytic tumors were analyzed immunocytochemically with the antikeratin monoclonal antibodies Mak-6 and Cam 5.2 and a polyclonal antibody against glial fibrillary acidic protein (GFAP). Immunoreactivity for Mak-6 was present in 29 cases (100%) including six well-differentiated astrocytomas, nine anaplastic astrocytomas, and 14 glioblastomas multiforme. Cam 5.2 immunoreactivity was focally present in one case of GBM (4%) but was absent in the remaining 28 cases. All cases were immunoreactive with an antibody against GFAP. Cytokeratin (CK) expression was examined in extracts of four separate well-characterized astrocytoma cell lines by Western blotting with the monoclonal antibodies Mak-6, Cam 5.2, and anti-CK 18 and by Northern analysis using a cDNA probe for the human CK 18 gene. The Western blots revealed the presence of immunoreactive bands corresponding to CK numbers 14/15, 16, and 18 in extracts from all four cell lines and additional bands corresponding to CK 8 in 3/4 lines and CK 19 in 1/4 lines. Northern analysis detected CK 18 mRNA in extracts from 2/4 astrocytic cell lines. These findings demonstrate that CK immunoreactivity is frequent in astrocytic tumors and confirm through the molecular and biochemical analysis of CK 18 gene expression and of keratin intermediate filament proteins that the basis for CK immunoreactivity in astrocytic tumors is bona fide CK expression, not cross-reactivity with other antigens or artifact. The demonstration of CK expression by astrocytic neoplasms has important implications for pathologists involved in the diagnosis of poorly differentiated tumors.

The segment of the malarial circumsporozoite (CS) protein designated Region II is highly conserved among different malarial species. A similar sequence is also present in several other proteins, including thrombospondin, properdin, and a blood-stage antigen of Plasmodium falciparum. By means of peptides synthesized from sequences of the Plasmodium vivax CS protein in the vicinity of Region II, it was found that two overlapping 18- to 20-amino acid peptides promoted the adhesion of a variety of human hematopoietic cell lines. The amino acid sequence valine-threonine-cysteineglycine (VTCG), contained within this common motif, was shown to be the critical sequence for the observed cell-adhesive properties.

A series of overlapping peptides, representing sequences in the vicinity of region II on the Plasmodium vivax circumsporozoite protein, was synthesized. One of the peptides (PV-23), a 20-mer containing the 6 C-terminal amino acids of region II, was found to evoke an in vitro T-cell proliferative response in spleen cells from C3Hf (H-2km2) mice immunized with the peptide. These results demonstrate that PV-23 contains a T-cell epitope. To our knowledge, this is the first report of a T-cell epitope on the circumsporozoite protein of P. vivax.

The hepatic glucagon receptor was covalently labeled with [125I-Tyr10]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccinimidyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradiogram of the gel showed one band at Mr = 63,000 that was sensitive to excess unlabeled glucagon and GTP. The labeled receptor was solubilized with Lubrol-PX and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f degrees = 1.8 and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees for 15 min prior to the addition of [125I-Tyr10] permitted us to identify the high molecular weight form (Mr approximately equal to 113,000) by direct SDS-gel electrophoretic analysis. Limited elastase treatment of the hormone-occupied receptor results in the appearance of a Mr = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a Mr = 24,000 fragment that binds hormone in a GTP-sensitive manner. The Mr = 24,000 fragment is contained within the Mr = 33,000 fragment. The Mr = 63,000 receptor upon treatment with endo-beta-N-acetylglucosamine F for 4 h yields four fragments of apparent Mr = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither Mr = 33,000 and 24,000 fragment appear to be substrates for endo-beta-N-acetylglucosaminidase F. These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of approximately 60,000 dalton hormone binding subunit which is a glycoprotein containing at least four N-linked glycans accounting for 18,000 daltons of its mass. Both the hormone binding function and the capacity for the interaction with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans.

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.

A single intraarticular injection of carrageenin into the rabbit knee joint initiates an inflammatory reaction in the synovial tissues. the exudate from the joint was able to degrade proteoglycan at pH 5.2 and pH 7.2. Further characterization of proteolytic enzymes in the inflamed synovial tissues showed the presence of cathepsin D, a neutral protease, and cathepsin B1. Maximum activities of two lysosomal enzymes, acid phosphatase and cathepsin D, were observed within 7 days of injection. Most of this activity was found to be associated with cells in the synovial fluid.

The pattern and hormonal control of rat androgen-binding protein (rABP) secretion in vitro was investigated using animals of different ages to initiate primary cultures of Sertoli cells. Sertoli cells were isolated from testes of 7- to 31-day-old rats and cultured for periods of up to 30 days in serum-free medium, medium supplemented with insulin, transferrin, and epidermal growth factor (3F), or 3F plus FSH, testosterone, progesterone, hydrocortisone, and vitamin E (8F). The amount of rABP secreted by Sertoli cells during the first 24 h in culture (initial rate) exhibited an age-dependent pattern which reflected the apparent in vivo activity of these cells. Between 7 and 25 days of age, the initial rate of rABP secretion per Sertoli cell increased 20-fold; a further 2-fold increase occurred between 25 and 35 days of age. The pattern of rABP secretion exhibited by Sertoli cells cultured for several weeks was dependent not only on added factors (3F or 8F), but also on Sertoli cell age, expressed as the total of animal age plus time in culture (total age). In cultures of Sertoli cells isolated from very young animals (7-10 days old), the rate of rABP secretion increased until 20 days (total age), but declined thereafter. This early increase in rABP secretion was augmented by, but not dependent on, hormone additions. In contrast, Sertoli cells isolated from older animals always showed decreasing rates of secretion with time in culture. Furthermore, Sertoli cells from very young animals retained the capacity to respond to hormones in vitro with increased secretion of rABP and maintenance of cell viability. This responsiveness decreased with age, similar to the loss of hormone response seen in vivo after puberty. In conclusion, culture conditions were established which permitted the study of FSH-dependent and independent regulation of rABP secretion and of the acquisition of hormone resistance at the time of puberty. The initial rate of rABP secretion in culture (first 24 h) correlates with the age of the animal from which the cultures are obtained. The pattern of rABP secretion during subsequent long term culture is determined by the total age (animal and culture age), with increasing rates of secretion up to 20 days and decreasing rates thereafter. This inherent pattern of rABP secretion as well as loss of responsiveness of the Sertoli cell to hormonal stimulation appear to be programmed early in development.

To study the effects of seminiferous tubule damage on Leydig cell function and morphology, rats were treated by fetal irradiation (to induce Sertoli cell-only syndrome, SCO), 3 months administration of hydroxyurea (HU), or chronic feeding of a vitamin A-deficient diet (VAD). Leydig cell function was assessed by the measurement of serum LH and testosterone and the response of serum testosterone to hCG stimulation, while morphology was studied by electron microscopy after perfusion fixation. Serum LH was significantly elevated in each experimental group, while basal serum testosterone was significantly lower only in SCO rats. In all treatment groups, the serum testosterone response to hCG was significantly decreased when measureed as the area under the response curve. Despite a decreased response to hCG, the Leydig cells were larger than normal and showed striking increases in quantities of smooth endoplasmic reticulum, mitochondria and Golgi complex. Leydig cell dysfunction has been demonstrated in animals with varying degrees of seminiferous tubule damage, but paradoxically the cytological features of the Leydig cells were indicative of hypertrophy.

Chronic feeding of rats with camostate results in pancreatic hypertrophy or hyperplasia, or both. Previous studies suggest that this effect of camostate occurs via an increase in endogenous cholecystokinin due to an enteral feedback mechanism involving the inhibition of trypsin in the duodenum. Studies employing proglumide, a weak and relatively nonspecific cholecystokinin antagonist, have failed to fully abolish camostate-induced pancreas growth. We examined the effects of L-364,718, a new and highly potent cholecystokinin receptor antagonist, on camostate-induced pancreas growth in rats. The pancreas weights and the concentrations of ribonucleic acid, protein, and chymotrypsinogen in the pancreas of rats treated with camostate alone were significantly elevated over those of controls. These effects of camostate were completely abolished in rats treated with camostate + L-364,718. The pancreas weights and the concentrations of deoxyribonucleic acid and ribonucleic acid in the pancreas of rats treated with L-364,718 alone were significantly lower than values in control rats. These data indicate that camostate-induced pancreas growth in rats appears to be dependent on the actions of endogenous cholecystokinin and that cholecystokinin may play a role in the maintenance of pancreatic growth in normal rats.

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.

Specific RIAs for rat transferrin (rTF) and androgen-binding protein (rABP) were used to determine whether the secretion of these proteins was coordinately regulated in the Sertoli cell under a variety of conditions. Sertoli cell-enriched primary cultures were prepared from the testes of 20-day-old rats, and rTF and rABP were assayed in medium from the same culture. There was a strong effect of cell density on both rABP and rTF secretion per cell, with increased secretion per cell at high densities. Human TF (hTF), FeSO4, and desferrioxamine had little or no effect on rTF secretion. The age of the animal at the time of preparation of cells for culture had a strong effect on the pattern of rTF and rABP secretion in vitro; however, the effects of animal age, time in culture, and medium supplementation differed for the two proteins. In cultures prepared from 20-day-old animals, insulin, epidermal growth factor, and testosterone stimulated both rTF and rABP secretion, although to different extents. Retinoic acid was required for the stimulation and maintenance of rTF secretion, but had no effect on rABP secretion in the presence of insulin, hTF, and epidermal growth factor. Conversely, FSH and isoproterenol stimulated rABP, but not rTF, secretion. These data suggest that the secretion of rABP and rTF by Sertoli cells is under differential control.

Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.

The effect of prolonged exposure of S49 murine lymphoma cells to (-) isoprenaline on the levels of beta-adrenergic receptors and the stimulatory regulatory component of adenylyl cyclase (Gs) was investigated. Exposure of wild-type S49 cells (WT cells) to isoprenaline for 16 h resulted in an 84% decrease in beta-adrenergic receptors and a 78% decrease in catecholamine-stimulated adenylyl cyclase activity. The desensitization of adenylyl cyclase was homologous since no change in stimulation by any other effectors was observed. Similar treatment of cyc- S49 cells, a mutant that lacks Gs, resulted in only a 10-15% receptor loss despite a 50% decrease in catecholamine stimulation of reconstituted adenylyl cyclase activity. By quantitative extraction of Gs from control and isoprenaline-treated WT cell membranes and reconstitution into untreated cyc- cell membranes, it was demonstrated that the levels and behaviour of Gs in WT cell membranes were unchanged after isoprenaline treatment, despite a substantial loss of receptors from the cell surface. The beta-adrenergic receptors lost from the cell surface could not be identified in any intracellular membrane fraction. Addition of cycloheximide to the culture medium of WT cells previously treated with isoprenaline resulted in a complete blockade of the restoration of beta-adrenergic receptors which was otherwise observed within 16 h after removal of the agonist. It is concluded that, in the S49 cell line, prolonged exposure to agonists results in a loss of beta-adrenergic receptors by a process dependent on the presence of Gs. (ABSTRACT TRUNCATED AT 250 WORDS)

Adult rats were made bilaterally cryptorchid and studied at intervals of 3, 7, 14 or 21 days to study temporal changes in Leydig cell function. Serum FSH and LH levels were measured and the cross-sectional area of the Leydig cells assessed by morphometry. The function of the Leydig cells was judged by the binding of 125I-labelled hCG to testicular tissue in vitro and the testosterone response of the testis to hCG stimulation in vitro. By 3 days after cryptorchidism, the binding of labelled hCG to testicular tissue was significantly decreased compared to that of controls, but the testes were able to respond to hCG stimulation in vitro. At 7, 14 and 21 days after cryptorchidism, an enhanced testosterone response was observed and the size of the Leydig cells was significantly greater than that of the controls, which indicated increased secretory activity by the cryptorchid testis. Although serum FSH levels were significantly elevated after 3 days of cryptorchidism, serum LH levels did not rise until 7 days, thereby suggesting that the loss of receptors is unlikely to result from down-regulation by LH. The reduced testosterone response of the cryptorchid testis in vivo to low doses of hCG and the enhanced response at high doses are probably related to the reduced blood flow to the cryptorchid testis and the decreased sensitivity of the Leydig cells induced by LH/hCG receptor loss.

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.