Search

Terence Chau, 1,* Merlyn Joseph, 2,* Jesus Ledesma Jr, 3 David Wei Hau Hsu 3 1Department of Pharmacy, Cooper University Hospital, Camden, NJ, 08103, USA; 2Texas A&M University, Irma Lerma Rangel College of Pharmacy, Houston, TX 77030, USA; 3Emergency Department, Memorial Hermann Memorial City Medical Center, Houston, TX, 77024, USA*These authors contributed equally to this workCorrespondence: Terence ChauCooper University Hospital, One Cooper Plaza, Camden, NJ 08103, USATel +1 856 342 2975Fax +1 856 968 8408Email chau-terence@cooperhealth.eduAbstract: A patient was brought to the hospital with severe accidental hypothermia due to cold exposure associated with acute alcohol intoxication. Initial bladder core temperature was 21°C (70°F). The patient was agitated and combative with altered mental status and suffered rescue collapse during transport. Initial rhythm was ventricular fibrillation and we initiated a standard advanced cardiac life support (ACLS) protocol with rewarming measures. The patient received 28 mg of epinephrine and 13 shocks. Active and passive rewarming were initiated without extracorporeal rewarming. The patient achieved return of spontaneous circulation (ROSC) at a core temperature of 23.8°C (74.8°F). Patient was discharged 15 days later neurologically intact with no organ damage. The clinical management and implications for further research in severe accidental hypothermia management are discussed. In patients with severe accidental hypothermia (defined as < 30°C or < 86°F) in cardiac arrest, the optimal rewarming technique, use of epinephrine, and time when defibrillation should be attempted remain controversial. In our patient, the patient achieved ROSC in less than 2 hours with standard ACLS procedures despite a minimal increase in core temperature (21°C to 23.8°C or 70°F to 73.9°F).Keywords: accidental severe hypothermia, cardiac arrest, ACLS

Colon cancer is a common human cancer worldwide. The survival rate of late staged or metastatic colon cancer patients remains low even though the effectiveness of treatment in colon cancer has greatly improved. Research on tumorigenesis mechanisms and discovery of novel molecular target for treating colon cancer is critical. The promotion roles of WHSC1 in multiple myeloma have been demonstrated previously, yet, the regulation of WHSC1 in other cancers is largely unknown, especially in colon cancer. Here, in this study, we analyzed and identified WHSC1 while studying the genetic regulations of HMGA2 in colon cancer cells by microarray analysis, and investigated the HMGA2-WHSC1 interaction. We then applied CRISPR technology to establish stable WHSC1 knockout cells, to address the functional regulation of WHSC1 in colon cancer. In summary, our results for the first time identified the HMGA2-WHSC1 interaction in colon cancer. Moreover, we discovered that WHSC1 promotes cancer proliferation, facilitates resistance of chemotherapy agent, and promotes metastatic capacity of colon cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known. We address this utilizing Dictyostelium discoideum strains lacking H3K4me3 due to disruption of the gene encoding the Set1 methyltransferase or mutations in endogenous H3 genes. Loss of H3K4me3 confers resistance to TSA-induced developmental inhibition and delays accumulation of H3K9Ac and H3K14Ac. H3K4me3-directed H3Ac is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that interacts with H3K4me3 via a tandem tudor domain (TTD). We identify an Sgf29 orthologue in Dictyostelium with a TTD that specifically recognizes the H3K4me3 modification. Disruption of the gene encoding Sgf29 delays accumulation of H3K9Ac and abrogates H3K4me3-directed H3Ac. Either loss or overexpression of Sgf29 confers developmental resistance to TSA. Our results demonstrate that rapid acetylation of H3K4me3 histones regulates developmental sensitivity to TSA. Levels of H3K4me3 or Sgf29 will provide useful biomarkers for sensitivity to this class of chemotherapeutic drug., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

BACKGROUND Minimally invasive surgery (MIS) has rapidly advanced, but its use in transplant patients has lagged. We share our experience of MIS for patients after kidney and liver transplantation and compare our results with similar studies in the literature. MATERIAL AND METHODS This study included 14 MIS (12 laparoscopic, 2 transvaginal) procedures for 13 transplant cases (6 liver and 7 kidney) done from May 2006 to May 2018. Gastrointestinal surgery was performed in 6 cases: appendectomy performed 8 months after liver transplant and 16 months after kidney transplant in 2 cases, radical right hemi-colectomy performed 6 weeks after liver transplant in 1 case; exploration for chylous ascites 6 months after liver transplant in 1 case, sleeve gastrectomy performed 3 years after kidney transplant in 1 case, and partial hepatectomy performed 12 years after kidney transplant in 1 case. For urological problems, 2 patients received ipsilateral right-side nephroureterectomy performed 10 and 12 years after kidney transplant, and 1 patient received contralateral left-side nephroureterectomy performed 12 years after kidney transplant. The 2 liver transplant patients with huge incisional hernias received repair approximately 3 and 2 years after liver transplant. Three patients underwent gynecological surgery: 2 transvaginal for pelvic floor reconstruction in 1 patient with liver transplant and 1 hysterectomy in a kidney transplant patient, and 1 laparoscopic-assisted hysterectomy in a kidney transplant patient. We retrospectively analyzed the clinical presentation, operative findings, operation time, postoperative complications, and length of stay. RESULTS The postoperative course was uneventful, with early resumption of oral intake, including immunosuppressants administered the same as in the non-transplant patients. All surgical procedures in these transplant patients were achieved without conversion, showed stable kidney and liver function, had better surgical outcomes in comparison with traditional surgery, and most of them were discharged within 1 week. CONCLUSIONS Laparoscopic and non-laparoscopic MIS surgery are feasible and safe for abdominal organ transplant patients and are helpful for timely intervention in cases with acute abdomen. No adjustment of immunosuppressant is usually needed, as oral intake can be resumed very soon after surgery.

Plant pathogens secrete proteins called effectors into the cells of their host to modulate the host immune response against colonization. Effectors can either modify or arrest host target proteins to sabotage the signaling pathway, and therefore are considered potential drug targets for crop disease control. In earlier research, the Xanthomonas type III effector XopAI was predicted to be a member of the arginine-specific mono-ADP-ribosyltransferase family. However, the crystal structure of XopAI revealed an altered active site that is unsuitable to bind the cofactor NAD+, but with the capability to capture an arginine-containing peptide from XopAI itself. The arginine peptide consists of residues 60 through 69 of XopAI, and residue 62 (R62) is key to determining the protein-peptide interaction. The crystal structure and the molecular dynamics simulation results indicate that specific arginine recognition is mediated by hydrogen bonds provided by the backbone oxygen atoms from residues W154, T155, and T156, and a salt bridge provided by the E265 sidechain. In addition, a protruding loop of XopAI adopts dynamic conformations in response to arginine peptide binding and is probably involved in target protein recognition. These data suggest that XopAI binds to its target protein by the peptide-binding ability, and therefore, it promotes disease progression. Our findings reveal an unexpected and intriguing function of XopAI and pave the way for further investigation on the role of XopAI in pathogen invasion.

Burkholderia pseudomallei is a selective agent that causes septic melioidosis and exhibits a broad range of lethal doses in animals. Host cellular virulence and phagocytic resistance are pathologic keys of B. pseudomallei . We first proposed Caenorhabditis elegans as the host cellular virulence model to mimic bacterial virulence against mammals and second established the resistance of B. pseudomallei to predation by Dictyostelium discoideum as the phagocytosis model. The saprophytic sepsis-causing Burkholderia sp. ( B. pseudomallei , Burkholderia thailandensis , Burkholderia cenocepacia , and Burkholderia multivorans ) exhibited different virulence patterns in both simple models, but B. pseudomallei was the most toxic. Using both models, attenuated isolates of B. pseudomallei were selected from a transposon-mutant library and a panel of environmental isolates and reconfirmed by in vitro mouse peritoneal exudate cell association and invasion assays. The distinct pathological patterns of melioidosis were inducted by different selected B. pseudomallei isolates. Fatal melioidosis was induced by the isolates with high virulence in both simple models within 4-5 day, whereas the low-virulence isolates resulted in prolonged survival greater than 30 day. Infection with the isolates having high resistance to D. discoideum predation but a low C. elegans killing effect led to 83% of mice with neurologic melioidosis. By contrast, infection with the isolates having low resistance to D. discoideum predation but high C. elegans killing effect led to 20% cases with inflammation in the salivary glands. Our results indicated that individual B. pseudomallei isolates selected from simple biological models contribute differently to disease progression and/or tissue tropism.

Although laccase is involved in the biotransformation of 2,4,6-trinitrotoluene (TNT), little is known regarding the effect of E. coli laccase on TNT biotransformation. In this study, E. coli K12 served as the parental strain to construct a laccase deletion strain and two laccase-overexpressing strains. These E. coli strains were used to investigate the effect of laccase together with copper ions on the efficiency of TNT biotransformation, the variety of TNT biotransformation products generated and the toxicity of the TNT metabolites. The results showed that the laccase level was not relevant to TNT biotransformation in the soluble fraction of the culture medium. Conversely, TNT metabolites varied in the insoluble fraction analyzed by thin-layer chromatography (TLC). The insoluble fraction from the laccase-null strain showed fewer and relatively fainter spots than those detected in the wild-type and laccase-overexpressing strains, indicating that laccase expression levels were interrelated determinants of the varieties and amounts of TNT metabolites produced. In addition, the aquatic invertebrate Tigriopus japonicus was used to assess the toxicity of the TNT metabolites. The toxicity of the TNT metabolite mixture increased when the intracellular laccase level in strains increased or when purified E. coli recombinant Laccase (rLaccase) was added to the culture medium. Thus, our results suggest that laccase activity must be considered when performing microbial TNT remediation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nasal obstruction is a common presenting symptom to clinicians and affects up to one-third of the population. There are several factors that cause nasal obstruction, including anatomic, physiologic, and pathophysiologic factors. The anatomy and physiology of nasal obstruction is complicated and is influenced by patency of nasal passages, mucociliary function, airflow receptors, autonomic function, and degree of mucosal inflammation. Common anatomic causes include internal nasal valve stenosis/collapse, septal deviation, and turbinate hypertrophy. Common physiologic causes include sinonasal inflammatory disorders and iatrogenic causes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Salicylic acid (SA) is a key phytohormone that mediates a broad spectrum of resistance against a diverse range of viruses; however, the downstream pathway of SA governed antiviral immune response remains largely to be explored. Here, we identified an orchid protein containing A20 and AN1 zinc finger domains, designated Pha13. Pha13 is up-regulated upon virus infection, and the transgenic monocot orchid and dicot Arabidopsis overexpressing orchid Pha13 conferred greater resistance to different viruses. In addition, our data showed that Arabidopsis homolog of Pha13, AtSAP5, is also involved in virus resistance. Pha13 and AtSAP5 are early induced by exogenous SA treatment, and participate in the expression of SA-mediated immune responsive genes, including the master regulator gene of plant immunity, NPR1, as well as NPR1-independent virus defense genes. SA also induced the proteasome degradation of Pha13. Functional domain analysis revealed that AN1 domain of Pha13 is involved in expression of orchid NPR1 through its AN1 domain, whereas dual A20/AN1 domains orchestrated the overall virus resistance. Subcellular localization analysis suggested that Pha13 can be found localized in the nucleus. Self-ubiquitination assay revealed that Pha13 confer E3 ligase activity, and the main E3 ligase activity was mapped to the A20 domain. Identification of Pha13 interacting proteins and substrate by yeast two-hybrid screening revealed mainly ubiquitin proteins. Further detailed biochemical analysis revealed that A20 domain of Pha13 binds to various polyubiquitin chains, suggesting that Pha13 may interact with multiple ubiquitinated proteins. Our findings revealed that Pha13 serves as an important regulatory hub in plant antiviral immunity, and uncover a delicate mode of immune regulation through the coordination of A20 and/or AN1 domains, as well as through the modulation of E3 ligase and ubiquitin chain binding activity of Pha13., Competing Interests: The authors have declared that no competing interests exist.

Rhinitis and sinusitis are common medical conditions that affect the geriatric population and have a significant impact on their quality of life. Because few studies examine differences in the clinical management between the geriatric and general adult population, therapies should be based on current guidelines. Special considerations should be made when treating these patients in regards to multiple comorbidities and the potential for drug interactions from polypharmacy. Further research on the pathogenesis of sinusitis in the geriatric population may provide specific differences in the clinical management in this population., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins. The genetic tractability of Dictyostelium and its relatively simple genome structure makes it possible to disrupt PARP gene combinations, in addition to specific ADP-ribosylation sites at endogenous loci. Together, this makes Dictyostelium an attractive model to assess how ADP-ribosylation regulates a variety of cellular processes including DNA repair, transcription, and cell-type specification. Here we describe a range of techniques to study ADP-ribosylation in Dictyostelium, including analysis of ADP-ribosylation events in vitro and in vivo, in addition to approaches to assess the functional roles of this modification in vivo.

The alternative transcription factor σ B in Bacillus cereus governs the transcription of a number of genes that confer protection against general stress. This transcription factor is regulated by protein-protein interactions among RsbV, RsbW, σ B , RsbY, RsbM and RsbK, all encoded in the sigB cluster. Among these regulatory proteins, RsbV, RsbW and σ B comprise a partner-switching mechanism. Under normal conditions, σ B remains inactive by associating with anti-sigma factor RsbW, which prevents σ B from binding to the core RNA polymerase. During environmental stress, RsbK activates RsbY to hydrolyze phosphorylated RsbV, and the dephosphorylated RsbV then sequesters RsbW to liberate σ B from RsbW. Although the σ B partner-switching module is thought to be the core mechanism for σ B regulation, the actual protein-protein interactions among these three proteins in the cell remain to be investigated. In the current study, we show that RsbW and RsbV form a long-lived complex under transient stress treatment, resulting in high persistent expression of RsbV, RsbW and σ B from mid-log phase to stationary phase. Full sequestration of RsbW by excess RsbV and increased RsbW:RsbV complex stability afforded by cellular ADP contribute to the prolonged activation of σ B . Interestingly, the high expression levels of RsbV, RsbW and σ B were dramatically decreased beginning from the transition stage to the stationary phase. Thus, protein interactions among σ B partner-switching components are required for the continued induction of σ B during environmental stress in the log phase and significant down-regulation of σ B is observed in the stationary phase. Our data show that σ B is temporally regulated in B. cereus., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

ADP-ribosyltransferases (ARTs) modify proteins with single units or polymers of ADP-ribose to regulate DNA repair. However, the substrates for these enzymes are ill-defined. For example, although histones are modified by ARTs, the sites on these proteins ADP-ribosylated following DNA damage and the ARTs that catalyse these events are unknown. This, in part, is due to the lack of a eukaryotic model that contains ARTs, in addition to histone genes that can be manipulated to assess ADP-ribosylation events in vivo. Here we exploit the model Dictyostelium to identify site-specific histone ADP-ribosylation events in vivo and define the ARTs that mediate these modifications. Dictyostelium histones are modified in response to DNA double strand breaks (DSBs) in vivo by the ARTs Adprt1a and Adprt2. Adprt1a is a mono-ART that modifies H2BE18 in vitro, although disruption of this site allows ADP-ribosylation at H2BE19. Although redundancy between H2BE18 and H2BE19 ADP-ribosylation is also apparent following DSBs in vivo, by generating a strain with mutations at E18/E19 in the h2b locus we demonstrate these are the principal sites modified by Adprt1a/Adprt2. This identifies DNA damage induced histone mono-ADP-ribosylation sites by specific ARTs in vivo, providing a unique platform to assess how histone ADP-ribosylation regulates DNA repair.

Purpose: To determine the rate of persistent tympanic membrane perforation after intratympanic steroid injection. To determine which comorbid conditions and risk factors are associated with prolonged time to perforation closure following intratympanic steroid injection., Materials and Methods: Clinical data were gathered for patients who had undergone intratympanic steroid injection to treat sudden sensorineural hearing loss or Ménière's disease. Primary outcomes analysis included rate of persistent tympanic membrane perforation, defined as perforation at least 90days following last injection, and time to perforation healing. Age, sex, number of injections, smoking status, diabetes mellitus, previous head and neck irradiation, and concurrent oral steroids, were analyzed as potential predictors of persistent perforation., Results: One hundred ninety two patients were included in this study. Three patients (1.6%) had persistent tympanic membrane perforations. All three patients received multiple injections. One patient underwent tympanoplasty for repair of persistent perforation. The median time to perforation healing was 18days. There was no statistically significant variable associated with time to perforation healing. However, patients with prior history of head and neck radiation averaged 36.5days for perforation healing compared to 17.5days with no prior history of radiation and this approached statistical significance (p=0.078)., Conclusions: The rate of persistent tympanic membrane perforation following intratympanic steroid injection is low. Patients with a history of radiation to the head and neck may be at increased risk for prolonged time for closure of perforation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We describe our experience of cervical lymphadenectomy with microvascular anastomoses involving levels I to V through a minimally-invasive neck dissection. We retrospectively studied 12 patients who had levels I to IV neck dissection with free flap reconstruction between July 2013 and April 2015 at Poole Hospital (male:female ratio 8:4, mean (range) age 66 (49 - 83) years). The mean (range) operating time was 7 (5 - 8) hours, and the total volume drained from the neck was 105 (60-300) ml. The mean (range) number of harvested lymph nodes was 26 (13-39) from levels I to III, and 33 (20-42) from levels I to IV. Four patients developed weakness of the marginal mandibular nerve, but there were no serious complications. All flaps were successful, there was no regional recurrence, and most patients were discharged on postoperative day 15. This technique provides adequate exposure for lymphadenectomy and anastomosis, and we think that head and neck surgeons should include it in the armamentarium of reconstruction., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Reconstruction of a composite defect of the lower lip after oncological resection is challenging, and it is essential to consider both functional and aesthetic components when repairing lips. We report a technique that can be used to repair anything ranging from 30% to the whole of the lower lip with a bilateral commissurotomy and advancement of skin, muscle, and mucosal flaps. This technique helps to achieve good oral function, excellent lip function, and a pleasant aesthetic appearance. It also prevents microstomia and allows patients to maintain normal sensory innervation., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

σB, an alternative transcription factor, controls the response of the cell to a variety of environmental stresses in Bacillus cereus. Previously, we reported that RsbM negatively regulates σB through the methylation of RsbK, a hybrid sensor kinase, on a signaling helix (S-helix). However, RsbK comprises a C-terminal receiver (REC) domain whose function remains unclear. In this study, deletion of the C-terminal REC domain of RsbK resulted in high constitutive σB expression independent of environmental stimuli. Thus, the REC domain may serve as an inhibitory element. Mutagenic substitution was employed to modify the putative phospho-acceptor residue D827 in the REC domain of RsbK. The expression of RsbKD827N and RsbKD827E exhibited high constitutive σB, indicating that D827, if phosphorylatable, possibly participates in σB regulation. Bacterial two-hybrid analyses demonstrated that RsbK forms a homodimer and the REC domain interacts mainly with the histidine kinase (HK) domain and partly with the S-helix. In particular, co-expression of RsbM strengthens the interaction between the REC domain and the S-helix. Consistently, our structural model predicts a significant interaction between the HK and REC domains of the RsbK intradimer. Here, we demonstrated that coordinated the methylatable S-helix and the REC domain of RsbK is functionally required to modulate σB-mediated stress response in B. cereus and maybe ubiquitous in microorganisms encoded RsbK-type sensor kinases.

ADP-ribosylation of proteins at DNA lesions by ADP-ribosyltransferases (ARTs) is an early response to DNA damage. The best defined role of ADP-ribosylation in the DNA damage response is in repair of single strand breaks (SSBs). Recently, we initiated a study of how ADP-ribosylation regulates DNA repair in Dictyostelium and found that two ARTs (Adprt1b and Adprt2) are required for tolerance of cells to SSBs, and a third ART (Adprt1a) promotes nonhomologous end-joining (NHEJ). Here we report that disruption of adprt2 results in accumulation of DNA damage throughout the cell cycle following exposure to agents that induce base damage and DNA SSBs. Although ADP-ribosylation is evident in adprt2(-) cells exposed to methylmethanesulfonate (MMS), disruption of adprt1a and adprt2 in combination abolishes this response and further sensitises cells to this agent, indicating that in the absence of Adprt2, Adprt1a signals MMS-induced DNA lesions to promote resistance of cells to DNA damage. As a consequence of defective signalling of SSBs by Adprt2, Adprt1a is required to assemble NHEJ factors in chromatin, and disruption of the NHEJ pathway in combination with adprt2 increases sensitivity of cells to MMS. Taken together, these data indicate overlapping functions of different ARTs in signalling DNA damage, and illustrate a critical requirement for NHEJ in maintaining cell viability in the absence of an effective SSB response.

Dynamic acetylation of all lysine-4-trimethylated histone H3 is a complex phenomenon involved in Immediate-early gene induction in metazoan eukaryotes. Higher eukaryotes express repeated copies of three closely related H3 variants, inaccessible to genetic analysis. We demonstrate conservation of these phenomena in Dictyostelium which has three single-copy H3 variant genes. Although dynamic acetylation is targeted to two H3 variants which are K4-trimethylated, K9-acetylation is preferentially targeted to one. In cells lacking Set1 methyltransferase and any detectable K4-trimethylation, dynamic acetylation is lost demonstrating a direct link between the two. Gene replacement to express mutated H3 variants reveals a novel interaction between K4-trimethylation on different variants. Cells expressing only one variant show defects in growth, and in induction of a UV-inducible gene, demonstrating the functional importance of variant expression. These studies confirm that dynamic acetylation targeted to H3K4me3 arose early in evolution and reveal a very high level of specificity of histone variant utilization in a simple multicellular eukaryote.

Objectives: To evaluate current trends in the management of idiopathic sudden sensorineural hearing loss (ISSNHL), determine if variance in diagnostic and treatment protocols exists, and compare diagnostic and treatment strategies of ISSNHL between nonotologists/neurotologists (NONs) and otologists/neurotologists (ONs)., Study Design: Cross-sectional survey of practicing otolaryngologists., Setting: Otolaryngology practices within the United States., Subjects and Methods: In January 2010, a survey was mailed to 500 NONs and 500 ONs. Data were collected and analyzed using χ(2), standard deviations, and variance., Results: A variety and distribution of responses were seen in the definition of ISSNHL, including dB loss necessary for diagnosis, number of consecutive frequencies involved, and maximum duration of hearing loss. Differences in diagnostic tools were also seen, with 50.4% of respondents (NON 34.2%, ON 66.7%; P = .0001) always using magnetic resonance imaging in their workup. Of the total respondents, 26.7% (NON 35.0%, ON 18.3%; P < .0001) preferred oral steroids alone; 22.1% (NON 11.7%, ON 32.5%; P < .0001) preferred a combination of oral and intratympanic steroids. Of the respondents, 68.6% (NON 57.5%, ON 80.0%; P = .0003) would continue with additional treatment after partial response; 20.8% (NON 33.3%, ON 8.3%; P < .005) would retreat with oral steroids alone and 46.6% (NON 35.8%, ON 57.3%; P < .05) with intratympanic injections. Overall, 69.2% (NON 45.8%, ON 92.5%; P = .0001) were very comfortable managing ISSNHL., Conclusion: Significant differences exist in the diagnosis and treatment of ISSNHL. Such lack of uniformity highlights the need for strong evidence-based research-ultimately leading to formalized practice guidelines and educational outreach.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

Background: Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores. Cells in which the gene encoding Cdk8 has been deleted fail to enter aggregates due to a failure of early gene expression., Principal Findings: We have monitored the expression levels of cyclin C protein during development and find levels decrease after the multicellular mound is formed. This decrease is triggered by extracellular cAMP that, in turn, is working in part through an increase in intracellular cAMP. The loss of cyclin C is coincident with a reduction in the association of Cdk8 with a high molecular weight complex in the nucleus. Overexpression of cyclin C and Cdk8 lead to an increased rate of early development, consistent with the levels being rate limiting., Conclusions: Overall these results show that both cyclin C and Cdk8 are regulated during development in response to extracellular signals and the levels of these proteins are important in controlling the timing of developmental processes. These findings have important implications for the role of these proteins in controlling development, suggesting that they are targets for developmental signals to regulate gene expression.

Repair of DNA double strand breaks (DSBs) is critical for the maintenance of genome integrity. DNA DSBs can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). Whilst HR requires sequences homologous to the damaged DNA template in order to facilitate repair, NHEJ occurs through recognition of DNA DSBs by a variety of proteins that process and rejoin DNA termini by direct ligation. Here we review two recent reports that NHEJ is conserved in the social amoeba Dictyostelium discoideum. Certain components of the mammalian NHEJ pathway that are absent in genetically tractable organisms such as yeast are present in Dictyostelium and we discuss potential directions for future research, in addition to considering this organism as a genetic model system for the study of NHEJ in vivo.

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). In vertebrates, the first step in NHEJ is recruitment of the DNA-dependent protein kinase (DNA-PK) to DNA termini. DNA-PK consists of a catalytic subunit (DNA-PKcs) that is recruited to DNA ends by the Ku70/Ku80 heterodimer. Although Ku has been identified in a wide variety of organisms, to date DNA-PKcs has only been identified experimentally in vertebrates. Here, we report the identification of DNA-PK in the nonvertebrate Dictyostelium. Dictyostelium Ku80 contains a conserved domain previously implicated in recruiting DNA-PKcs to DNA and consistent with this observation, we have identified DNA-PKcs in the Dictyostelium genome. Disruption of the gene encoding Dictyostelium DNA-PKcs results in sensitivity to DNA DSBs and defective H2AX phosphorylation in response to this form of DNA damage. However, these phenotypes are only apparent when DNA damage is administered in G(1) phase of the cell cycle. These data illustrate a cell cycle-dependent requirement for Dictyostelium DNA-PK in signaling and combating DNA DSBs and represent the first experimental verification of DNA-PKcs in a nonvertebrate organism.

Abnormalities of the p53 tumor-suppressor gene are found in a significant proportion of astrocytic brain tumours. We studied tumour specimens from 74 patients evaluated over 20 years at the Massachusetts General Hospital, where clinical outcome could be determined and sufficient pathologic material was available for immunostaining. p53 expression studies employed an affinity-purified p53 monoclonal antibody, whose specificity was verified in absorption studies and, in a minority of cases, a second antibody recognising a different epitope of p53. Significant overexpression of p53 protein was found in 48% of the 74 tumours included in this series and high levels of expression were associated with higher mortality from astrocytic tumours (P<0.001, log rank). Multivariate analyses revealed that immunohistochemically detected p53 was an independent marker of shortened progression-free and overall actuarial survival in patients with astrocytic tumours, suggesting that increased expression of p53 plays an important role in the pathobiology of these tumours. In a subset of 36 cases, coding regions of the p53 gene were completely sequenced via SSCP and direct DNA sequencing, revealing that overexpression of p53 protein is not always associated with point mutations in conserved exons of the p53 gene. Finally, we confirmed p53 protein expression in early-passage human glioma cell lines of known p53 mutational status and immunostaining scores. Although grade continues to be the strongest prognostic variable, the use of p53 staining as a prognostic indicator, in contrast to mutational DNA analyses, may be a useful adjunct in identifying patients at higher risk of treatment failure.

The Cdk8 proteins are kinases which phosphorylate the carboxy terminal domain (CTD) of RNA polymerase II (Pol II) as well as some transcription factors and, therefore, are involved in the regulation of transcription. Here, we report that a Cdk8 homologue from Dictyostelium discoideum is localized in the nucleus where it forms part of a high molecular weight complex that has CTD kinase activity. Insertional mutagenesis was used to abrogate gene function, and analysis of the null strain revealed that the DdCdk8 protein plays an important role in spore formation during late development. As previously reported [Dev. Growth Differ. 44 (2002) 213] Ddcdk8- cells also exhibit impaired aggregation, although we report that the severity of the defect depends upon experimental conditions. When aggregation occurs, Ddcdk8- cells form abnormal terminally differentiated structures within which the Ddcdk8- cells differentiate into stalk cells but fail to form spores, indicating a role for DdCdk8 in cell differentiation. When Ddcdk8 is expressed from its own promoter, the protein is able to rescue both the late developmental defect and the impaired aggregation. However, when expressed from an heterologous promoter, only the impaired aggregation is rescued. This result demonstrates that the defect during late development is not a consequence of impaired aggregation and indicates a direct role for DdCdk8 in spore formation.

The purpose of this investigation was to elucidate the association between microvascular blood volume and glucose uptake and to link these measures with tumor angiogenesis. We demonstrate a regionally specific correlation between tumor relative microvascular blood volume (CBV), determined in vivo with functional magnetic resonance imaging techniques, and tumor glucose uptake determined with fluorodeoxyglucose positron emission tomography. Regions of maximum glucose uptake were well matched with maximum CBV across all patients (n = 21; r = 0.572; P = 0.023). High-grade gliomas showed significantly elevated CBV and glucose uptake compared with low-grade gliomas, (P = 0.009 and 0.008, respectively). Correlations between CBV and glucose uptake were then determined on a voxel-by-voxel basis within each patient's glioma. Correlation indices varied widely, but in 16 of 21 cases of human glioma, CBV and glucose uptake were correlated (r > 0.150). These measures were well correlated in all cases when comparing healthy brain tissue in these same patients. Tumor vascularity, as determined immunohistochemically and morphometrically on clinical samples, revealed statistically significant relationships with functional imaging characteristics in vivo. Regional heterogeneities in glucose uptake were well matched with functional magnetic resonance imaging CBV maps. Our findings support the concept that there is an association of microvascular density and tumor energy metabolism in most human gliomas. In addition, the findings are likely to have important clinical applications in the initial evaluation, treatment, and longitudinal monitoring of patients with malignant gliomas.

The genomic methylation patterns in the mammalian somatic cells are presumably maintained by a single enzyme, dnmt1. In mouse, this DNA (cytosine-5)-methyltransferase, or CpG MTase, is encoded by the Dnmt1 gene. We now present evidence that in different tissues and cell types, the primary transcript of mouse dnmt1 is alternatively spliced to generate two poly-(A) RNAs of approximately similar abundance. This alternative splicing most likely originates from the existence of two tandemly arranged acceptor sites separated by only 3 nt. The two Dnmt1 mRNAs thus encode two CpG MTases differing by two amino acids. We discuss the implications of the discovery of two dnmt1 isozymes, instead of one enzyme as previously thought, in the somatic cells of both mouse and human.

Thus far, only one major form of vertebrate DNA (cytosine-5) methyltransferase (CpG MTase, EC 2.1.1.37) has been identified, cloned, and extensively studied. This enzyme, dnmt1, has been hypothesized to be responsible for most of the maintenance as well as the de novo methylation activities occurring in the somatic cells of vertebrates. We now report the discovery of another abundant species of CpG MTase in various types of human cell lines and somatic tissues. Interestingly, the mRNA encoding this CpG MTase results from alternative splicing of the primary transcript from the Dnmt1 gene, which incorporates in-frame an additional 48 nt between exons 4 and 5. Furthermore, this 48-nt exon sequence is derived from the first, or the most upstream, copy of a set of seven different Alu repeats located in intron 4. The ratios of expression of this mRNA to the expression of the previously known, shorter Dnmt1 mRNA species, as estimated by semiquantitative reverse transcription-PCR analysis, range from two-thirds to three-sevenths. This alternative splicing scheme of the Dnmt1 transcript seems to be conserved in the higher primates. We suggest that the originally described and the recently discovered forms of CpG MTase be named dnmt1-a and dnmt1-b, respectively. The evolutionary and biological implications of this finding are discussed in relation to the cellular functions of the CpG residues and the CpG MTases.

Mitotic index > 6, proliferating cell nuclear antigen (PCNA) index > 5%, high tumour grade and absence of progesterone receptors (PR) are significant predictors for poor outcome in meningiomas. Since MIB-1 (Ki-67) is a more specific cell proliferation marker, and overexpression of TGF-alpha is also associated with tumour progression, we compared the prognostic significance of these factors with the other indices. Intracranial meningiomas from 21 men and 36 women (age 54.5 +/- 1.7, mean +/- SEM) were classified as 24 benign, 24 atypical and nine malignant. Twenty-one of the 57 tumours recurred (mean interval to recurrence was 57.3 +/- 13.1 months). The mean follow-up period for patients without tumour recurrence was 81.9 +/- 8.7 months. MIB-1 labelling index (LI) was expressed as percentage of labelled nuclei to total tumour nuclei counted in the most densely labelled areas. Analysis of variance revealed significant differences between tumour grades for MIB-1 labelling indices (0.75 +/- 0.21 for benign, 3.2 +/- 0.57 for atypical 6.04 +/- 1.48 for malignant; P < or = 0.0066), and between malignant and non-malignant meningiomas for TGF alpha staining scores (P < or = 0.029). MIB-1 LI also correlated with mitotic and PCNA indices (P < or = 0.0001), but not with age of the patients. Male patients had higher tumour MIB-1 LI than females (P < or = 0.0128). Univariate analysis indicated that MIB-1 LI > 3%, TGF alpha score > 4 (scoring scale 0-5), mitotic index > 6, and negative PR status were significant factors for worse outcome. Higher MIB-1 LI, TGF alpha score and mitotic index as continuous variables were also significant negative predictors. With multivariate analysis, both MIB-1 LI and TGF alpha score remained significant factors when paired with all other variables: TGF alpha or MIB-1 LI, respectively, mitosis, PCNA, tumour grade, PR status, age, sex, postoperative radiation therapy. We conclude that MIB-1 LI and TGF alpha score are important independent prognostic indicators for patients with meningiomas.

Objective: These studies evaluated the ability of transplanted pituitary cells to restore pituitary function in hypophysectomized rats., Methods: The pituitary glands of neonatal Lewis rats were rapidly removed, enzymatically dispersed, and stereotactically introduced into the third ventricle of hypophysectomized adult male Lewis rats. Four weeks after implantation, plasma levels of anterior pituitary hormones in implanted animals were compared with those of sham-transplanted control animals., Results: Plasma levels of prolactin, growth hormone, thyroid-stimulating hormone, and beta-endorphin were below the range of detection in 14 sham-operated animals. In implanted animals, restitution of serum prolactin occurred in 100% of the animals tested, with levels of 2.6 +/- 1.0 ng/ml (mean +/- standard error of the mean; normal, 2-4 ng/ml). Growth hormone was assayable in 71% of the animals, with a mean value of 29 +/- 13 ng/ml over all animals (normal, 1-100 ng/ml); thyroid-stimulating hormone was restored in 68%, with mean resting levels of 79 +/- 13 ng/ml (normal, 100-400 ng/ml); luteinizing hormone levels were found in 53%, with mean levels over all animals of 0.2 +/- 0.1 ng/ml (normal, 0.5-1.0 ng/ml); and beta-endorphin was restored in 45% to high resting levels of 163 +/- 31 pg/ml (normal, 20-30 pg/ml). A challenge with hypothalamic releasing factor and a cold stress test were performed on the animals that had received transplants. Positive hormone responses to both of these tests suggested sensitivity of the pituitary grafts to both endogenous and exogenous sources of stimulation. Histological sections of paraformaldehyde-fixed brains from implanted animals clearly demonstrated survival of clusters of grafted pituitary cells. Positive immunohistochemical staining for adrenocorticotropic hormone and thyroid-stimulating hormone was demonstrated in sections of the grafted tissue., Conclusion: These data suggest survival of neonatal pituitary transplants in the third ventricle of adult hypophysectomized rats with concomitant restoration of anterior pituitary hormone function.

The majority of cases of Cushing's disease are due to an underlying pituitary corticotroph microadenoma (< or = 10 mm). Corticotroph macroadenomas (> 10 mm) are a less common cause of Cushing's disease, and little is known about specific clinical and biochemical findings in such patients. To define further the clinical characteristics of patients with corticotroph macroadenomas, we performed a retrospective review of Cushing's disease due to macroadenomas seen at Massachusetts General Hospital between 1979 and 1995. Of 531 patients identified with a diagnostic code of Cushing's syndrome, 20 were determined to have Cushing's disease due to a macroadenoma based on radiographic evidence of pituitary adenoma greater than 10 mm and pathological confirmation of a pituitary adenoma. A comparison review of charts of 24 patients with Cushing's disease due to corticotroph microadenomas identified on the basis of radiographic evidence of a normal pituitary gland or a pituitary adenoma 10 mm or less in diameter was also performed. The mean ages of the patients (+/- SD) with macroadenomas and microadenomas were similar (39 +/- 12 and 38 +/- 14 yr, respectively). The baseline median 24-h urine free cortisol (UFC) excretion was 1341 nmol/day (range, 304-69,033 nmol/day) and 877 nmol/day (range, 293-2,558 nmol/day) for macroadenoma and microadenoma patients, respectively (P = 0.058). After the 48-h high dose dexamethasone suppression test, UFC decreased by 77 +/- 19% (mean +/- SD) and 91 +/- 7% in macroadenoma and microadenoma subjects, respectively (P = 0.04). Fifty-six percent of macroadenoma patients and 92% of microadenoma patients had greater than 80% suppression of UFC after high dose dexamethasone administration (P = 0.03). The baseline median 24-h urinary 17-hydroxysteroid (17-OHCS) excretion was 52 mumol/day (range, 25-786 mumol/day) and 44 mumol/day (range, 17-86 mumol/day) for macroadenoma and microadenoma subjects, respectively (P = 0.09). After the standard high dose dexamethasone suppression test, 17-OHCS excretion decreased by 46 +/- 33% and 72 +/- 22% for macroadenoma and microadenoma subjects, respectively (P = 0.02). Fifty-three percent of patients with macroadenomas and 86% of patients with microadenomas had greater than 50% suppression of 17-OHCS after high dose dexamethasone administration (P = 0.02). Baseline plasma ACTH values were above the normal range in 83.3% of macroadenoma patients and in 45% of microadenoma subjects (P = 0.05). Tumors were immunostained with the MIB-1 antibody for Ki-67 to investigate proliferation in the adenomas. There was a trend for a higher Ki-67 labeling index in corticotroph macroadenomas, and seven (44%) macroadenomas vs. three (18%) microadenomas had labeling indexes greater than 3%, but this was not statistically significant. In summary, corticotroph macroadenomas are often associated with less glucocorticoid suppressibility than the more frequently occurring microadenomas. Therefore, the lack of suppression of UFC or 17-OHCS after the administration of high dose dexamethasone in a patient with Cushing's disease does not necessarily imply the presence of ACTH-independent Cushing's syndrome and is more commonly seen in patients with corticotroph macroadenomas than in those with microadenomas. Increased plasma ACTH concentrations are typical of patients with corticotroph macroadenomas and may be a more sensitive indicator of neoplastic corticotrophs than the UFC or 17-OHCS response to standard high dose dexamethasone testing.

The grading of glial tumors has traditionally relied on histological assessment, but the distinction between grade II and grade III gliomas is still a subject of debate. We examined the value of the monoclonal antibody MIB-1 (Ki-67) labeling index (LI) in the differentiation between grade II and grade III gliomas by either the 1993 WHO grading scheme or the St. Anne-Mayo grading scale. The MIB-1 Li in the most densely labeled areas from 80 diffuse cerebral hemispheric gliomas was determined. The tumors included 16 grade II, 31 grade III and 33 grade IV gliomas by the WHO scale. The mean LIs (%) were 0.88 +/- 0.29 for grade II, 8.75 +/- 1.71 for grade III, and 9.12 +/- 1.55 for grade IV gliomas. Analysis of variance indicated a significant difference in mean LIs between grades II and III and grades II and IV (p < or = 0.0001), but not between grades III and IV. Seven tumors were classified differently by the 2 systems (grade III by WHO, but grade 2 by St. Anne-Mayo), and all had MIB-1 LI over 3%. Univariate analysis showed that MIB-1 LI with a cut-off point at 1.5% was a significant prognostic factor (p < or = 0.0005). High tumor grade (WHO, p < or = 0.0002; St. Anne-Mayo, p < or = 0.0006) and patient age > 50 (p < or = 0.0001) were also significant factors for shorter survival. Using Cox Regression Multivariate Analysis, MIB-1 LI > 1.5% was a significant independent predictor of shorter disease survival when paired with tumor grade (p < or = 0.032), patient age (p < or = 0.0065), or gender (p < or = 0.0007). We conclude that the MIB-1 immunoreactivity is useful in distinguishing grade II from grade III gliomas, and maybe more sensitive in assigning aggressive gliomas to grade III than the St. Anne-Mayo grading system.

Purpose: Meningiomas are common intracranial tumors, often well controlled with surgical resection alone. While the efficacy of radiation therapy in improving local control and progression-free survival is well documented, prognostic data substantiate factors that are predictive of poor local control following definitive radiation therapy. PCNA is a DNA polymerase expressed at the highest levels in the S-phase, the most resistant portion of the cell cycle to ionizing radiation in vitro. We investigated the possible correlation between the levels of PCNA expression and the clinical outcome of patients treated with definitive radiation therapy., Methods and Materials: Archival tissue was collected from 33 cases of meningioma treated at our institution for definitive radiation therapy between 1970 and 1990. Age-matched normal meningeal tissue and asymptomatic meningiomas removed at autopsy served as tissue controls. A standard ABC immumoperoxidase technique employing antibodies to PCNA, PC-10 (Dako, California) was used to stain specimen slides for PCNA. PCNA index was defined as the number of positive nuclei per 10 high-power fields at 400x magnification. Two independent observers scored the slides without prior knowledge of the cases at hand., Results: Patients with high PCNA index were less likely to be controlled by therapeutic radiation (p < 0.001, Kaplan-Meier). All patients with a PCNA index greater that 25 failed radiation therapy. Using multivariate analyses, malignant (but not atypical), histology and PCNA index were significant predictors of progression following radiation therapy (p < 0.05, log rank)., Conclusion: PCNA index may be a useful adjunct to more standard histopathologic criteria in the determination of meningioma local control and progression-free survival following therapeutic irradiation. Data on a more expanded population evaluated on a prospective basis will be needed before such criteria are routinely employed in the clinical setting.

Meningiomas often contain steroid hormone receptors, but the correlation of receptor presence with patient outcome or mitotic index is unclear. Intracranial meningiomas from 70 patients (27 males and 43 females, mean age 52.9 + 1.7 years [mean +/- standard error of the mean], range 15-78 years) were evaluated immunocytochemically for female sex hormone receptors using specific monoclonal antibodies. Prognostic correlations were determined using statistical analyses that included clinical and histological variables. Twenty-eight tumors were benign, 27 had atypical features, and 15 were malignant. Thirty tumors were meningotheliomatous, 11 were fibroblastic, 28 were transitional, and one was secretory. Twenty-nine of the 70 primary tumors recurred (mean interval to recurrence 50.1 +/- 10 months). The mean progression-free follow-up period for patients without recurrence was 82.1 +/- 7.7 months. Nuclear staining for the progesterone receptor (PR) was found in 58 cases (83%) and PR status was scored as 0 (0% nuclei positive), 1 (< 1%), 2 (1-9%), 3 (10-49%), or 4 (> 50%). Only six tumors (8.6%) contained nuclear estrogen receptor (ER) staining, which was limited to a small number of nuclei (< 1%). Fisher's exact test (two-tailed) showed an inverse correlation between tumor grade and PR staining score (p < or = 0.001), with 96% of benign and 40% of malignant meningiomas containing PR-positive nuclei. No correlation between age or histological subtype and PR score was detected. Meningiomas from female patients had more PRs (p < or = 0.05). Analysis of variance revealed that the mitotic index (total counts of mitoses per 10 high-power fields) for tumors with 0 PR staining (18 +/- 4.4) was higher (p < or = 0.0001) than for those with PR scores of 1 to 4 (4.3 +/- 1.9, 5.1 +/- 2, 2.2 +/- 0.8, and 1.7 +/- 0.9, respectively). Univariate analysis indicated that the absence of PR, high mitotic index, and higher tumor grade were significant factors for shorter disease-free intervals. Multivariate analysis showed that a three-factor interaction model, with a PR score of 0, mitotic index greater than 6, and malignant tumor grade, was a highly significant predictor (p < or = 0.0001) for worse outcome in patients harboring meningiomas. These data indicate that the presence of PRs, even in a small number of tumor cells, is a favorable prognostic factor for meningiomas.

Flow cytometric analysis was applied to embedded tissue to measure the proliferative activity and the DNA ploidy of 16 recurrent and 17 nonrecurrent pituitary adenomas. The results were compared with data from a previous study which demonstrated that proliferating cell nuclear antigen (PCNA) labeling index was higher in recurrent adenomas than in nonrecurrent adenomas. Flow cytometric analysis as a tool for predicting aggressive behavior has been useful in a variety of human tumors; however, its prognostic value in pituitary adenomas is controversial. Therefore, we decided to explore the relationship of the results of flow cytometry and proliferating cell nuclear antigen labeling indices with the prognosis of pituitary adenomas. Three out of 16 recurrent adenomas and five out of 17 nonrecurrent adenomas demonstrated a DNA aneuploid pattern. All the nonfunctional recurrent adenomas had a diploid pattern, while only 40% of the functional recurrent adenomas had a diploid pattern. The GO/G1 phase fraction was higher in the recurrent adenomas, than in the nonrecurrent ones (p = 0.0005). In contrast, the S-phase fraction and the coefficient of variation were higher in the nonrecurrent adenomas (5.9 +/- 1.0%, 7.0 +/- 0.75, respectively) than in the recurrent ones (2.5 +/- 0.6%, 4.0 +/- 0.2%, respectively) (p = 0.003 and p = 0.001, respectively). The proliferating cell nuclear antigen labeling indices were higher in the recurrent adenomas (18.9 +/- 4.5%) than in the nonrecurrent adenomas (2.6 +/- 1.6%) (p = 0.003). The S-phase of flow cytometry correlated weakly with the proliferating cell nuclear antigen labeling indices when the recurrent and the nonrecurrent adenomas were considered as one group. (r = -0.356, p = 0.033). But no significant correlations were observed when the groups of recurrent (r = -0.311, p = 0.195) and nonrecurrent tumors (r = -0.019, p = 0.942) were compared separately. The results of flow cytometric analysis suggest that recurrent adenomas may have a higher proportion of cells in the presynthetic phase than the nonrecurrent adenomas. This study suggests that flow cytometric analysis is of limited value in predicting recurrence of pituitary adenomas.

Purpose: Do tumor cells which survive high dose fractionated irradiation exhibit modified metastasis activity, proliferation kinetics, and/or radiation sensitivity? To address this question experimentally, we have studied three recurrent human tumor xenograft systems., Methods and Materials: Three models were derived from a soft tissue sarcoma (HSTS26T), a colon adenocarcinoma (HCT15), and a glioblastoma (HGL21) which had recurred after 90 Gy, 109 Gy, or 77.4 Gy administered in 30 equal doses, respectively. Their production of spontaneous metastasis and cell proliferation characteristics were studied in early generation xenografts in SCID mice, and were compared to those in their previously unirradiated counterparts. As a control, we have also studied each tumor as a post-surgical recurrence. Specimens from the irradiated recurrent and their unirradiated primary tumors were cultured in vitro and their radiation sensitivity determined by clonogenic assay., Results: The three irradiated recurrent tumor systems retained the individual histological features of their unirradiated primary xenografts. A lower metastatic incidence was observed in two of the three irradiated recurrent tumor lines in comparison with their unirradiated control tumors and their surgical recurrent counterparts. No significant differences were found between the irradiated recurrent tumors and their unirradiated counterparts with respect to: volume doubling time, growth time, potential doubling time, mitotic index, PCNA index, and SF2 values., Conclusions: High dose irradiation given in 30 fractions did not increase the metastatic activity in the three human tumor xenograft systems. Furthermore, the fractionated irradiation did not significantly change their proliferation characteristics and cellular radiation sensitivity.

Little is known of the molecular genetic mechanisms contributing to meningioma tumor progression. We evaluated a total of 26 clinical cases of meningioma: twenty three patients with meningioma treated at our institution between 1978 and 1990 and three asymptomatic cases found initially at autopsy. In addition, histologically normal meninges obtained at post-mortem examination from 5 cases were evaluated. There were 13 men and 10 women in the patient group with a median age of 48.7 years, treated by surgery and/or irradiation. Median follow-up was 46 months (range 16-152 months). Archival cases and age-matched normal meningeal tissue obtained at autopsy during the same time period were obtained for study. Patients with TGF alpha scores greater than 3.0 were more likely to fail treatment and had lower overall survival times than those with immunostaining scores of 1 or 2. Three autopsy cases where meningioma had been silent clinically had overall staining scores of 0.75, while 10 samples of normal meninges harvested from 5 cases at autopsy had staining scores of 0. Two patients each underwent 3 surgeries for recurrent tumor, serial specimens showed increased TGF alpha expression over time, though all material from these procedures was consistent with the diagnosis of histologically benign meningioma.

GAP-43 is a growth-associated phosphoprotein expressed at high levels in neurons during development, axonal regeneration, and neuritic sprouting. GAP-43 gene expression in mature neurons is probably functionally important for the structural remodeling of synapses as required for learning and establishing new memory. The widespread aberrant neuritic growth accompanied by impaired synaptic plasticity in Alzheimer's disease (AD) suggests that abnormal GAP-43 gene expression may contribute to the cascade of neurodegeneration. In the present study, end-stage AD brains exhibited reduced neuronal expression but increased glial cell levels of GAP-43 mRNA and protein. Glial cell localization of GAP-43 gene expression was confirmed by in situ hybridization of cerebral tissue, Northern blot analysis of microdissected cerebral white matter, and independent analysis of astrocytoma cell lines and primary malignant astrocytomas. In addition, in AD, GAP-43 immunoreactivity was translocated from the cytosol to membranes of swollen neuritic (dendritic) and glial cell processes throughout cerebral cortex and white matter. Downregulated and aberrant neuronal GAP-43 gene expression appears to reflect an important molecular lesion that precedes and progresses with the widespread synaptic disconnection and dementia in AD. At the same time, the presence of similar neuronal abnormalities in Pick's disease, diffuse Lewy body disease, Parkinson's disease, and Down syndrome suggests common mechanisms in the respective cascades of neurodegeneration. Finally, the finding of aberrantly increased glial cell GAP-43 gene expression in AD exposes a previously unrecognized neurodegenerative change that may account for the axonal loss and white matter atrophy detected early in the course of disease.

Urokinase-type plasminogen activator (u-PA) is a 54-kd enzyme shown to participate in tissue degradation under certain normal and pathological conditions, including cancer invasion and metastasis. Increased u-PA expression has been found in cancers of the breast, lung, colon, and prostate, and correlated with worse outcome in patients with lung and breast cancer. We examined the correlation between u-PA expression in gliomas and patient survival. Seventy-seven gliomas from 41 men and 36 women (ages 2 to 73) were immunostained for u-PA using monoclonal antibody 394 directed against human urokinase. The tumors included 32 grade 4, 16 grade 3, and 20 grade 2 astrocytomas (Daumas-Duport scale), and 9 pilocytic astrocytomas. Strong cytoplasmic staining was found in tumor cells of all grade 4, most of the grade 3, and a few of the lower grade tumors. Adjacent normal brain tissue showed faint staining associated with subpial cell processes and white matter fibers. The fiber staining was stronger in brain tissue infiltrated by tumor cells. Cytoplasmic u-PA staining in tumor cells was scored from 0 (no staining) to 6 (strong and widespread staining). The mean u-PA scores were 5.08 +/- 0.19 (mean +/- SEM) for grade 4, 3.97 +/- 0.46 for grade 3, 1.65 +/- 0.39 for grade 2, and 1.22 +/- 0.60 for pilocytic astrocytomas. The statistical analysis was based on cytoplasmic staining only. Analysis of variance revealed significant differences between the mean u-PA scores of different grades (P < 0.02 between grades 4 and 3, and P = 0.0001 between grades 4 or 3 and 2, and between grades 4 or 3 and pilocytic), except between grade 2 and pilocytic astrocytomas. Univariate analysis indicated that u-PA score > or = 4 (P = 0.0001), tumor grade 4 (P = 0.01), and age > 50 (P < 0.001) were all significant predictors for shorter disease survival. A three-way interaction model by multivariate analysis indicated that u-PA score > or = 4, tumor grade 4, and age > 50, taken together, were significant factors for shorter patient survival (P < 0.02). We conclude that u-PA may be used as a prognostic tool in conjunction with tumor grade and patients' age in predicting survival for patients with gliomas.

The prognostic value of tumor proliferative indices in meningiomas was assessed by mitotic counts and by immunocytochemistry using a monoclonal antibody against the proliferating cell nuclear antigen (PCNA) (clone 19A2), a 36-kd nuclear protein involved in DNA synthesis. Sixty-three intracranial meningiomas were classified as benign (26), with atypical features (24) or as malignant (13). The patients included 24 men and 39 women, mean age 54.2 +/- 1.7 (mean +/- SEM) (range 15-78) at initial presentation. Twenty-four of the 63 primary tumors recurred locally, including 23.1% (6/26) of the benign, 37.5% (9/24) of the atypical, and 69.2% (9/13) of the malignant meningiomas. Among tumors that recurred, 1/9 (11%) of the atypical and 5/9 (55.5%) of the malignant tumors had had macroscopical total excision at the initial surgery. The mean interval to recurrence was 52 +/- 11.8 months. The mean progression-free follow-up period for patients without tumor recurrence was 82 +/- 8.5 months. Analysis of variance revealed that significant differences existed between tumor grades for both PCNA indices (1.16 +/- 0.29% for benign; 14.14 +/- 2.07% for atypical and 21.37 +/- 5.47% for malignant) and mitotic indices (total counts per ten high power fields) (0.08 +/- 0.05 for benign, 4.75 +/- 0.91 for atypical and 19.00 +/- 4.07 for malignant). Multivariate regression analysis indicated that mitotic index > 6 was the single most important factor (p < 0.05) for shorter disease-free interval. Age, sex and total surgical excision were not significant factors. PCNA index was a significant factor in the univariate model, but not in the multivariate model.(ABSTRACT TRUNCATED AT 250 WORDS)

The recurrence rate of pituitary adenomas has been reported to be as high as 10% to 35% despite their generally benign nature. A monoclonal antibody directed against proliferating cell nuclear antigen (PCNA) was used to investigate whether the proliferative index might help to predict adenoma recurrence. This antigen is a nuclear protein identified as the auxiliary protein of deoxyribonucleic acid polymerase delta, and its gene expression correlates with cell proliferation. The authors studied 30 patients with recurrent pituitary adenomas, 32 with nonrecurrent adenomas, and seven normal pituitary tissue samples. The mean interval to recurrence ( +/- standard error of the mean) was 5.3 +/- 0.7 years. The age- and sex-matched nonrecurrent group had a mean follow-up period of 6.6 +/- 0.3 years without clinical recurrence. Mean percentages of PCNA-positive tumor nuclei in both the initial and the second surgical specimens of the recurrent adenomas (13.45% +/- 3.02% and 19.56% +/- 3.66%, respectively) were significantly higher than that of the nonrecurrent group (2.49% +/- 1.21%). In addition, recurrent tumors had a higher PCNA index than the initial tumors in the same patients. Normal anterior pituitary gland tissue had a significantly lower mean PCNA index (0.12% +/- 0.11%) than either patient group. Stepwise multivariate regression analysis indicated that factors which collectively correlated significantly with recurrence were: high PCNA index, large tumor size, extrasellar extension, and incomplete surgical excision. The PCNA nuclear count was not associated with age, sex, or hormone hypersecretion, but was higher in macro- than in microadenomas, in tumors with extrasellar extension, and in those incompletely excised. A higher PCNA index also correlated with a shorter disease-free interval. The authors conclude that evaluation of the PCNA index assists in predicting the likelihood of pituitary adenoma recurrence.

Activating mutations of the Gs alpha subunit have been identified in a subset of somatotroph adenomas. The mutant form of the Gs alpha subunit causes persistent activation of adenylyl cyclase and consequently results in high intracellular levels of cAMP. Because cAMP is known to stimulate the synthesis of the glycoprotein hormone (GPH) alpha-subunit as well as GH, we examined somatotroph tumors with and without Gs alpha mutations for GPH alpha-subunit production. GPH alpha-subunit production was assessed in vivo by measuring serum hormone levels and in vitro by analyzing hormone secretion by cultured pituitary tumor cells. DNA was extracted from the pituitary tumors of 26 acromegalic patients. The Gs alpha gene was amplified by the polymerase chain reaction and screened for mutations at codons 201 and 227 using oligonucleotide specific hybridization. Nine of the 26 tumors (35%) had point mutations at Arg 201. Seven of these tumors contained a CGT to TGT mutation (Arg to Cys) and 2 contained a CGT to CAT mutation (Arg to His). No mutations were detected at codon 227. There were no significant differences in age, sex distribution, tumor size, or serum levels of GH or insulin-like growth factor-1 between the groups of patients with or was Gs alpha mutations. The mean serum level of the free GPH alpha-subunit was 1.9-fold higher in the group with Gs alpha mutations (0.48 +/- 0.37 micrograms/L) than in patients without mutations (0.25 +/- 0.17) (P less than 0.05). In pituitary tumor cell culture, 75% of somatotroph tumors with Gs alpha mutations secreted free GPH alpha-subunit into the media compared with 45% of tumors without Gs alpha mutations. The amount of GPH alpha-subunit secretion was 12-fold greater in the group of tumors containing the Gs alpha mutation (P less than 0.05). Immunocytochemical detection of the free GPH alpha-subunit was similar in the two groups of patients with 75% positive for the GPH alpha-subunit in tumors with Gs alpha mutations and 67% positive in tumors without mutations (P = 0.69). We conclude that GPH alpha-subunit production occurs in somatotroph tumors with and without Gs alpha mutations. The increased levels of GPH alpha-subunit secretion in vivo and in vitro suggest that the Gs alpha mutation may increase the amount of preexisting GPH alpha-subunit biosynthesis in the tumors, perhaps via activation of the cAMP pathway.

Clinically nonfunctioning pituitary adenomas represent approximately 25% of all pituitary tumors. Recent studies using a number of in vitro techniques show that the majority of such tumors produce gonadotropins. Hypersecretion of uncombined gonadotropin subunits by these tumors has also been identified raising the possibility that gonadotropin biosynthetic alterations may occur in neoplastic pituitary tissue. To determine whether underlying intracellular biosynthetic alterations lead to imbalanced secretion of the gonadotropin subunits by such tumors, we investigated 1) steady state gonadotropin-subunit messenger ribonucleic acid (mRNA) levels in tumor tissue from 49 patients with clinically nonfunctioning adenomas, 2) secretion of gonadotropins in dispersed pituitary tumor cultures, and 3) serum concentrations of gonadotropins and free subunits. Northern blots of RNA extracted from surgically obtained pituitary tumor tissue were hybridized with complementary DNA probes for FSH beta, LH beta, and alpha-subunit, and quantitative analysis was done to compare alpha- and beta-subunit biosynthesis in individual tumors. Of these tumors, 47 contained sufficient RNA for Northern analysis and 77% of these tumors contained one or more of the gonadotropin-subunit mRNAs. Steady state alpha-subunit mRNA was detected in 57% of tumors, FSH beta mRNA in 49%, and LH beta in 1 (2%). We found FSH beta mRNA in excess of alpha-subunit mRNA in one-third of tumors, including 9 tumors where alpha-subunit mRNA was undetectable. In cultured cells, FSH beta was secreted in excess of alpha-subunit in 41% of tumors. For those tumors in which both mRNA and culture data were available, FSH beta mRNA and secreted subunit levels were in excess of alpha-subunit in 64% of tumors. We conclude that clinically nonfunctioning pituitary adenomas frequently synthesize excess FSH beta subunit relative to alpha-subunit. This finding is in contrast to previous data in normal pituitary or placental tissue where alpha-subunit is present in excess of beta-subunits at both the mRNA and protein levels. The free-beta-subunit hypersecretion identified in pituitary adenomas may be due to biosynthetic abnormalities intrinsic to neoplastic gonadotrophs.