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Angiogenesis is the development of new blood vessels from pre-existing vasculature. Multiple factors control its course. Disorders of the distribution of angiogenic agents are responsible for development of solid tumors and its metastases. Understanding of the molecular interactions regulating pathological angiogenesis will allow for development of more effective, even personalized treatment. A simulation of angiogenesis under microflow conditions is a promising alternative to previous studies conducted on animals and on 2D cell cultures. In this review, we summarize what has been discovered so far in the field of vascularized tumor-on-a-chip platforms. For this purpose, we describe different vascularization techniques used in microfluidics, present various attempts to induce angiogenesis-on-a-chip and report some approaches to recapitulate vascularized tumor microenvironment under microflow conditions., 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 © 2024 Elsevier B.V. All rights reserved.)

Platelet-rich plasma (PRP) has garnered widespread and increasing attention in recent years. We aimed to characterize the most influential articles in PRP research while clarifying controversies surrounding its use and clinical efficacy and identifying important areas on which to focus future research efforts. The Science Citation Index Expanded subsection of the Web of Science Core Collection was systematically searched to identify the top 50 cited publications on orthopedic PRP research. Publication and study characteristics were extracted, and Spearman's correlations were calculated to assess the relationship between citation data and level of evidence. The top 50 articles were published between the years 2005 and 2016, with 68% published in the year 2010 or later. Of the 33 studies for which level of evidence was assessed, the majority were of level I or II (18, 54.5%). Seventeen articles (34%) were classified as basic science. All clinical studies were prospective, and most (12 studies, 60%) included a high number of metrics related to the PRP preparation protocol and composition. Knee osteoarthritis was the most common topic among clinical studies in the top 50 cited articles (11 studies, 34%). More recent articles were associated with higher citation rates ( ρ  = 0.46, p  < 0.001). The most influential articles on orthopaedic PRP research are recent and consist of high-level of evidence studies mostly. Randomized controlled trials were the most common study type, while basic science articles were relatively less common. The most influential clinical studies reported a high number of metrics related to their PRP preparation protocol and the final PRP composition. These results suggest a rapidly evolving field with the potential to better explain inconsistent clinical results with improved understanding and documentation of basic science concepts such as PRP composition, preparation, and combination techniques., Competing Interests: J.F.O.: none. D.A.L.: consulting with Vericel and Allosource; educational support, Evolution Surgical/Arthrex; research grant funding, American Orthopaedic Society for Sports Medicine, Arthroscopy Association of North America, Arthritis Foundation; committees, American Orthopaedic Society for Sports Medicine research committee, Arthroscopy Association of North America research committee, and Orthopaedic Research Society Knee Topic Co-Chair. P.L.: consulting, Stryker; Committees, American Academy of Orthopaedic Surgeons Committee on Devices, Technologies and Biologics, Orthopaedic Trauma Association research Committee, and Orthopaedic Research and Education Foundation Grant committee. J.A.B.: committees, Board of Directors of American Academy of Orthopaedic Surgeons. JK: none. CML: consulting, Intellijoint Surgical (2019), Fundamental Surgery Virtual Reality (unpaid consultant); committees, American Academy of Orthopaedic Surgeons Board of Directors, Executive Committee, American Academy of Orthopaedic Surgeons Political Action Committee; Speaker fee, Arthrex, Inc. (2018); other, spouse employed by Pfizer, Inc., and Global Engineering., (Thieme. All rights reserved.)

Angiogenesis is the development of new blood vessels from the existing vasculature. Its malfunction leads to the development of cancers and cardiovascular diseases qualified by the WHO as a leading cause of death worldwide. A better understanding of mechanisms regulating physiological and pathological angiogenesis will potentially contribute to developing more effective treatments for those urgent issues. Therefore, the main goal of the following study was to design and manufacture an angiogenesis-on-a-chip microplatform, including cylindrical microvessels created by Viscous Finger Patterning (VFP) technique and seeded with HUVECs. While optimizing the VFP procedure, we have observed that lumen's diameter decreases with a diminution of the droplet's volume. The influence of Vascular Endothelial Growth Factor (VEGF) with a concentration of 5, 25, 50, and 100 ng/mL on the migration of HUVECs was assessed. VEGF's solution with concentrations varying from 5 to 50 ng/mL reveals high angiogenic potential. The spatial arrangement of cells and their morphology were visualized by fluorescence and confocal microscopy. Migration of HUVECs toward loaded angiogenic stimuli has been initiated after overnight incubation. This research is the basis for developing more complex vascularized multi-organ-on-a-chip microsystems that could potentially be used for drug screening.

The influence of microbiota on the human body is currently the subject of many studies. The composition of bacteria colonizing the gastrointestinal tract varies depending on genetic make-up, lifestyle, use of antibiotics or the presence of diseases. The diet is also important in the species diversity of the microbiota. This study is an analysis of the relationships between physical activity, diet, and the microbiota of the gastrointestinal tract in athletes. This review shows the differences in the microbial composition in various sports disciplines, the influence of probiotics on the microbiome, the consequence of which may be achieved even better sports results. Physical activity increases the number of bacteria, mainly of the Clostridiales order and the genus: Lactobacillus, Prevotella, Bacteroides, and Veillonella, and their number varies depending on the sports discipline. These bacteria are present in athletes in sports that require a high VO 2 max. The players' diet also influences the composition of the microbiota. A diet rich in dietary fiber increases the amount of Lactobacillus or Bifidobacterium bacteria, probiotic microorganisms, which indicates the need to supplement the diet with probiotic preparations. It is impossible to suggest an unambiguous answer to how the microbiota of the gastrointestinal tract changes in athletes and requires further analyzes., (© 2022. The Author(s).)

Drought and heat events, such as the 2018 European drought, interact with the exchange of energy between the land surface and the atmosphere, potentially affecting albedo, sensible and latent heat fluxes, as well as CO 2 exchange. Each of these quantities may aggravate or mitigate the drought, heat, their side effects on productivity, water scarcity and global warming. We used measurements of 56 eddy covariance sites across Europe to examine the response of fluxes to extreme drought prevailing most of the year 2018 and how the response differed across various ecosystem types (forests, grasslands, croplands and peatlands). Each component of the surface radiation and energy balance observed in 2018 was compared to available data per site during a reference period 2004-2017. Based on anomalies in precipitation and reference evapotranspiration, we classified 46 sites as drought affected. These received on average 9% more solar radiation and released 32% more sensible heat to the atmosphere compared to the mean of the reference period. In general, drought decreased net CO 2 uptake by 17.8%, but did not significantly change net evapotranspiration. The response of these fluxes differed characteristically between ecosystems; in particular, the general increase in the evaporative index was strongest in peatlands and weakest in croplands. This article is part of the theme issue 'Impacts of the 2018 severe drought and heatwave in Europe: from site to continental scale'.

Objective: The monitoring of the populations' iodine status is an essential part of successful programmes of iodine deficiency elimination. The current study aimed at the evaluation of current iodine nutrition in school children, pregnant and lactating women as a marker of the effectiveness and sustainability of mandatory iodine prophylaxis in Poland., Design: The following iodine nutrition indicators were used: urinary iodine concentration (UIC) (all participants) and serum thyroglobulin (pregnant and lactating women)., Setting: The study was conducted in 2017 within the National Health Programme in five regions of Poland., Participants: The research included 300 pregnant women, 100 lactating women and 1000 school children (aged 6-12 years)., Results: In pregnant women, median UIC was 111·6 µg/l; there was no significant difference in median UIC according to the region of residence. In 8 % of pregnant women, thyroglobulin level was >40 ng/ml (median thyroglobulin 13·3 ng/ml). In lactating women, median UIC was 68·0 µg/l. A significant inter-regional difference was noted (P = 0·0143). In 18 % of breastfeeding women, thyroglobulin level was >40 ng/ml (median thyroglobulin 18·5 ng/ml). According to the WHO criteria, the investigated sample of pregnant and lactating women was iodine-deficient. Median UIC in school children was 119·8 µg/l (with significant inter-regional variation; P = 0·0000), which is consistent with iodine sufficiency. Ninety-four children (9·4 %) had UIC < 50 µg/l., Conclusions: Mandatory iodisation of household salt in Poland has led to a sustainable optimisation of iodine status in the general population. However, it has failed to assure adequate iodine nutrition during pregnancy and lactation.

Background Context: Traditionally, cervical radiculopathy is thought to present with symptoms and signs in a standard, textbook, reproducible pattern as seen in a "Netter diagram." To date, no study has directly examined cervical radicular patterns attributable to single level pathology in patients undergoing ACDF., Purpose: The purpose of this study is to examine cervical radiculopathy patterns in a surgical population and determine how often patients present with the standard textbook (ie, Netter diagram) versus nonstandard patterns., Study Design/setting: A retrospective study., Patient Sample: Patients who had single-level radiculopathy with at least 75% improvement of preoperative symptoms following ACDF were included., Outcome Measures: Epidemiologic variables were collected including age, sex, weight, body mass index, laterality of symptoms, duration of symptoms prior to operative intervention, and the presence of diabetes mellitus. The observed pattern of radiculopathy at presentation, including associated neck, shoulder, upper arm, forearm, and hand pain and/or numbness, was determined from chart review and patient-derived pain diagrams., Methods: We identified all patients with single level cervical radiculopathy operated on between March 2011 and March 2016 by six surgeons. The observed pattern of radiculopathy was compared to a standard textbook pattern of radiculopathy that strictly adheres to a dermatomal map Fisher exact test was used to analyze categorical data and Student t test was used for continuous variables. A one-way ANOVA was used to determine differences in the observed versus expected radicular pattern. A logistic regression model assessed the effect of demographic variables on presentation with a nonstandard radicular pattern., Results: Overall, 239 cervical levels were identified. The observed pattern of pain and numbness followed the standard pattern in only 54% (129 of 239; p=.35). When a nonstandard radicular pattern was present, it differed by 1.68 dermatomal levels from the standard (p<.0001). Neck pain on the radiculopathy side was the most prevalent symptom; it was found in 81% (193 of 239) of patients and did not differ by cervical level (p=.72). In a logistic regression model, none of the demographic variables of interest were found to significantly impact the likelihood of presenting with a nonstandard radicular pattern., Conclusions: Observed patterns of cervical radiculopathy only followed the standard pattern in 54% of patients and did not differ by the cervical level involved. Cervical radiculopathy often presents with a nonstandard pattern. Surgeons should think broadly when identifying causative levels because they frequently may not adhere to textbook descriptions in actual clinical practice. We observed III level of evidence., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The establishment of invasive Halyomorpha halys (Stål) outside of its native range may impact native species assemblages, including other pentatomids and their scelionid parasitoids. This has generated interest in defining species diversity and host-parasitoid associations in this system to better understand the impact of invasive alien species on trophic interactions in invaded regions. Information on scelionid-pentatomid associations in natural habitats is lacking, and species-level identification of these associations can be tenuous using rearing and dissection techniques. Naturally occurring pentatomid eggs were collected in areas where H. halys has established in Canada and were analysed using a modified DNA barcoding approach to define species-level trophic interactions. Identification was possible for >90% of egg masses. Eleven pentatomid and five scelionid species were identified, and trophic links were established. Approximately 70% of egg masses were parasitized; parasitism and parasitoid species composition were described for each species. Telenomus podisi Ashmead was the dominant parasitoid and was detected in all host species. Trissolcus euschisti Ashmead was detected in several host species, but was significantly more prevalent in Chinavia hilaris (Say) and Brochymena quadripustulata (Fabricius). Trissolcus brochymenae Ashmead and Tr. thyantae Ashmead were recorded sporadically. Parasitism of H. halys was 55%, and this species was significantly less likely to be parasitized than native pentatomids. The scelionid species composition of H. halys consisted of Te. podisi, Tr. euschisti and Tr. thyantae. Although these species cannot develop in fresh H. halys eggs, we demonstrate that parasitoids attempt to exploit this host under field conditions., (© 2018 John Wiley & Sons Ltd.)

A balanced immune response to infection is essential to prevent the pathology and tissue damage that can occur from an unregulated or hyperactive host defense. Interferons (IFNs) are critical mediators of the innate defense to infection, and in this study we evaluated the contribution of a specific gene coding for IFIT2 induced by type I IFNs in a murine model of disseminated Candida albicans Invasive candidiasis is a frequent challenge during immunosuppression or surgical medical interventions, and C. albicans is a common culprit that leads to high rates of mortality. When IFIT2 knockout mice were infected systemically with C. albicans , they were found to have improved survival and reduced fungal burden compared to wild-type mice. One of the mechanisms by which IFIT2 increases the pathological effects of invasive C. albicans appears to be suppression of NADPH oxidase activation. Loss of IFIT2 increases production of reactive oxygen species by leukocytes, and we demonstrate that IFIT2 is a binding partner of a critical regulatory subunit of NADPH oxidase, p67 phox Since the administration of IFN has been used therapeutically to combat viral infections, cancer, and multiple sclerosis, we evaluated administration of IFN-β to mice prior to C. albicans infection. IFN-β treatment promoted pathology and death from C. albicans infection. We provide evidence that IFIT2 increases the pathological effects of invasive C. albicans and that administration of IFN-β has deleterious effects during infection. IMPORTANCE The attributable mortality associated with systemic C. albicans infections in health care settings is significant, with estimates greater than 40%. This life-threatening disease is common in patients with weakened immune systems, either due to disease or as a result of therapies. Type I interferons (IFN) are cytokines of the innate defense response that are used as immune modulators in the treatment of specific cancers, viral infections, and multiple sclerosis. In this study, we show using a murine model that the loss of a specific IFN-stimulated gene coding for IFIT2 improves survival following systemic C. albicans infection. This result infers a harmful effect of IFN during C. albicans infection and is supported by our finding that administration of IFN-β prior to invasive infection promotes fatal pathology. The findings contribute to our understanding of the innate immune response to C. albicans , and they suggest that IFN therapies present a risk factor for disseminated candidiasis., (Copyright © 2018 Stawowczyk et al.)

Posterior stabilized constrained (PSC) inserts are intended to provide greater varus-valgus and rotational constraint than conventional PS inserts. We determined whether the added constraint resulted in more damage to the post in PSC compared to PS inserts. Retrieved PSC inserts were matched to retrieved PS inserts from the same manufacturer according to patient age, body mass index, and length of implantation. Surface damage was visually assessed, and 3-D surface deviation from pristine was measured. Damage scores for the PSC posts were significantly greater than those of the PS posts. Surface deviation was significantly greater in the posterior and medial post regions of the PSC inserts. Based on short-term follow-up, our results suggest that added constraint is accompanied by greater polyethylene surface damage.

The talus is the second most common fractured tarsal bone. While their incidence may be low, talus fractures are severe injuries that can lead to long-term disability and pain. Displaced talar body fractures are typically treated through an open approach with the aim of obtaining anatomic reduction and stable fixation. There are several case reports in the literature demonstrating successful management of talus fractures arthroscopically. An arthroscopic approach minimizes soft tissue trauma, which can help decrease postoperative wound complications and infections. In this article, the authors describe a surgical technique of an arthroscopic reduction and internal fixation of a comminuted posterior talar body fracture. Compared with an open posterior approach with or without osteotomies, an arthroscopic technique improved visualization and allowed precise reduction and fixation., Levels of Evidence: Level V: Case report.

Progranulin (PGRN) is a growth factor that has been implicated in wound healing, inflammation, infection, tumorigenesis, and is most known for its neuroprotective and proliferative properties in neurodegenerative disease. This pleiotropic growth factor has been found to be a key player and regulator of a diverse spectrum of multi-systemic functions. Its critical anti-inflammatory role in rheumatoid arthritis and other inflammatory disease models has allowed for the propulsion of research to establish its significance in musculoskeletal diseases, including inflammatory conditions involving bone and cartilage pathology. In this review, we aim to elaborate on the emerging role of PGRN in the musculoskeletal system, reviewing its particular mechanisms described in various musculoskeletal diseases, with special focus on osteoarthritis and inflammatory joint disease patho-mechanisms and potential therapeutic applications of PGRN and its derivatives in these and other musculoskeletal diseases.

PGRN, a pleiotrophic growth factor, is known to play an important role in the maintenance and regulation of the homeostatic dynamics of normal tissue development, proliferation, regeneration, and the host-defense response and therefore, has been widely studied in the fields of infectious diseases, wound healing, tumorigenesis, and neuroproliferative and degenerative diseases. PGRN has also emerged as a multifaceted immune-regulatory molecule through regulating the signaling pathways known to be critical for immunology, especially TNF/TNFR signaling. In this review, we start with updates about the interplays of PGRN with ECM proteins, proteolytic enzymes, inflammatory cytokines, and cell-surface receptors, as well as various pathophysiological processes involved. We then review the data supporting an emerging role of PGRN in the fields of the "Cubic of I", namely, immunity, infection, and inflammation, with special focus on its regulation of autoimmune syndromes. We conclude with insights into the immunomodulating, anti-inflammatory, therapeutic potential of PGRN in treating diseases with an inflammatory etiology in a vast range of medical specialties.

The alpha-factor mating pheromone receptor (encoded by STE2) activates a G protein signaling pathway that stimulates the conjugation of Saccharomyces cerevisiae yeast cells. The alpha-factor receptor is known to undergo several forms of post-translational modification, including phosphorylation, mono-ubiquitination, and N-linked glycosylation. Since phosphorylation and mono-ubiquitination have been shown previously to play key roles in regulating the signaling activity and membrane trafficking of the alpha-factor receptors, the role of N-linked glycosylation was investigated in this study. The Asn residues in the five consensus sites for N-linked glycosylation present in the extracellular regions of the receptor protein were mutated to prevent carbohydrate attachment at these sites. Mutation of two sites near the receptor N-terminus (N25Q and N32Q) diminished the degree of receptor glycosylation, and the corresponding double mutant was not detectably N-glycosylated. The nonglycosylated receptors displayed normal function and subcellular localization, indicating that glycosylation is not important for wild-type receptor activity. However, mutation of the glycosylation sites resulted in improved plasma membrane localization for the Ste2-3 mutant receptors that are normally retained intracellularly at elevated temperatures. These results suggest that N-glycosylation may be involved in the sorting process for misfolded Ste2 proteins, and may similarly affect certain mutant receptors whose altered trafficking is implicated in human diseases.

The transcriptional activator Ste12p is required for the expression of genes induced by mating pheromone in the yeast Saccharomyces cerevisiae. We identified mutations in the amino-terminal DNA-binding domain of Ste12p that lead to constitutively high-level transcription of pheromone-induced genes. The behaviour of these mutant proteins is consistent with an enhanced DNA-binding ability. Cells carrying these hyperactive proteins retain their sensitivity to pheromone treatment, and their phenotype is largely dependent on the presence of at least one of the MAP kinases (Fus3p or Kss1p) and the scaffold protein Ste5p. Deletion of either FUS3 or KSS1 leads to a marked increase in Ste12p activity, consistent with a negative regulatory role for Fus3p, similar to that described for Kss1p. The properties of the constitutive mutants support the idea that the pheromone response pathway plays a role in basal as well as pheromone-induced transcription., (Copyright 2000 John Wiley & Sons, Ltd.)

Binding of the alpha-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway in MATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Galpha subunits in an alpha-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress the GPA1-A345T mutant Galpha subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the alpha-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for alpha-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the alpha-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor-G-protein preactivation complexes.

Mating pheromone receptors activate a G protein signal pathway that leads to the conjugation of the yeast Saccharomyces cerevisiae. This pathway also induces the production of Afr1p, a protein that negatively regulates pheromone receptor signaling and is required to form pointed projections of new growth that become the site of cell fusion during mating. Afr1p lacks strong similarity to any well-characterized proteins to help predict how it acts. Therefore, we investigated the relationship between the different functions of Afr1p by isolating and characterizing seven mutants that were defective in regulating pheromone signaling. The AFR1 mutants were also defective when expressed as fusions to STE2, the alpha-factor receptor, indicating that the mutant Afr1 proteins are defective in function and not in co-localizing with receptors. The mutant genes contained four distinct point mutations that all occurred between codons 254 and 263, identifying a region that is critical for AFR1 function. Consistent with this, we found that the corresponding region is very highly conserved in the Afr1p homologs from the yeasts S. uvarum and S. douglasii. In contrast, there were no detectable effects on pheromone signaling caused by deletion or overexpression of YER158c, an open reading frame with overall sequence similarity to Afr1p that lacks this essential region. Interestingly, all of the AFR1 mutants showed a defect in their ability to form mating projections that was proportional to their defect in regulating pheromone signaling. This suggests that both functions may be due to the same action of Afr1p. Thus, these studies identify a specific region of Afr1p that is critical for its function in both signaling and morphogenesis.

Mating pheromones stimulate Saccharomyces cerevisiae yeast cells to form a pointed projection that becomes the site of cell fusion during conjugation. To investigate the role of mating projections, we screened for mutations that enhanced the weak mating defect of MATa ste2-T326 cells that are defective in forming pointed projections. These cells are also 10-fold more sensitive to alpha-factor pheromone because ste2-T326 encodes truncated alpha-factor receptors that are not regulated properly. Mutations in AXL1, STE6 and FUS3 were identified in the screen. AXL1 was studied further because it is required for efficient a-factor pheromone production and for selecting the site for bud morphogenesis. Mutation of AXL1 did not enhance the morphogenesis or pheromone sensitivity defects of ste2-T326. Instead, the synergistic mating defect was apparently due to decreased a-factor production because the axl1Delta ste2-T326 cells mated well with a sst2 alpha mating partner that is supersensitive to a-factor. When combined with a wild-type mating partner, the ste2-T326 axl1Delta cells failed to mate because they did not lock cell walls, one of the earliest steps in conjugation. Analysis of axl1Delta in combination with other mutations that cause defects in morphogenesis or pheromone sensitivity (e.g. bar1, sst2, afr1) indicated that both phenotypes of ste2-T326 cells, supersensitivity to alpha-factor and the defect in forming pointed projections, contributed to the synergistic mating defect. We suggest a model that the synergistic mating defect is caused by the combined effects of ste2-T326 and axl1Delta on the presentation of a-factor to partner cells. Altogether, these results demonstrate an important linkage between the incoming and outgoing pheromone signals during the intercellular communication that promotes yeast mating., (Copyright 1999 John Wiley & Sons, Ltd.)

We studied the ligand-induced endocytosis of the yeast alpha-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to alpha-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Delta. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to alpha-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes.

The alpha-factor pheromone receptor (Ste2p) of the yeast Saccharomyces cerevisiae belongs to the family of G protein-coupled receptors that contain seven transmembrane domains (TMDs). Because polar residues can influence receptor structure by forming intramolecular contacts between TMDs, we tested the role of the five polar amino acids in TMD6 of the alpha-factor receptor by mutating these residues to nonpolar leucine. Interestingly, a subset of these mutants showed increased affinity for ligand and constitutive receptor activity. The mutation of the most polar residue, Q253L, resulted in 25-fold increased affinity and a 5-fold-higher basal level of signaling that was equal to about 19% of the alpha-factor induced maximum signal. Mutation of the adjacent residue, S254L, caused weaker constitutive activity and a 5-fold increase in affinity. Comparison of nine different mutations affecting Ser254 showed that an S254F mutation caused higher constitutive activity, suggesting that a large hydrophobic amino acid residue at position 254 alters transmembrane helix packing. Thus, these studies indicate that Gln253 and Ser254 are likely to be involved in intramolecular interactions with other TMDs. Furthermore, Gln253 and Ser254 fall on one side of the transmembrane helix that is on the opposite side from residues that do not cause constitutive activity when mutated. These results suggest that Gln253 and Ser254 face inward toward the other TMDs and thus provide the first experimental evidence to suggest the orientation of a TMD in this receptor. Consistent with this, we identified two residues in TMD7 (Ser288 and Ser292) that are potential contact residues for Gln253 because mutations affecting these residues also cause constitutive activity. Altogether, these results identify a new domain of the alpha-factor receptor that regulates its ability to enter the activated conformation.

G-protein-coupled receptors (GPCRs) transduce the signals for a wide range of hormonal and sensory stimuli by activating a heterotrimeric guanine nucleotide-binding protein (G protein). The analysis of loss-of-function and constitutively active receptor mutants has helped to reveal the functional properties of GPCRs and their role in human diseases. Here we describe the identification of a new class of mutants, dominant-negative mutants, for the yeast G-protein-coupled alpha-factor receptor (Ste2p). Sixteen dominant-negative receptor mutants were isolated based on their ability to inhibit the response to mating pheromone in cells that also express wild-type receptors. Detailed analysis of two of the strongest mutant receptors showed that, unlike other GPCR interfering mutants, they were properly localized at the plasma membrane and did not alter the stability or localization of wild-type receptors. Furthermore, their dominant-negative effect was inversely proportional to the relative amount of wild-type receptors and was reversed by overexpressing the G-protein subunits, suggesting that these mutants compete with the wild-type receptors for the G protein. Interestingly, the dominant-negative mutations are all located at the extracellular ends of the transmembrane segments, defining a novel region of the receptor that is important for receptor signaling. Altogether, our results identify residues of the alpha-factor receptor specifically involved in ligand binding and receptor activation and define a new mechanism by which GPCRs can be inactivated that has important implications for the evaluation of receptor mutations in other G-protein-coupled receptors.

The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.

Two new yeast genes, ASF1 (Anti-Silencing Function) and ASF2, as well as a C-terminal fragment of SIR3, were identified as genes that derepressed the silent mating type loci when overexpressed. ASF2 overexpression caused a greater derepression than did ASF1. ASF1 overexpression also weakened repression of genes near telomeres, but, interestingly, ASF2 had no effect on telomeric silencing. Sequences of these two genes revealed open reading frames of 279 and 525 amino acids for ASF1 and ASF2, respectively. The ASF1 protein was evolutionarily conserved, MCB motifs, sequences commonly present upstream of genes transcribed specifically in S phase, were found in front of both genes, and, indeed, both genes were transcribed specifically in the S phase of the cell cycle. While an asf2 mutant was viable and had no obvious phenotypes, an asf1 mutant grew poorly. Neither mutant exhibited derepression of the silent mating type loci. The asf1 mutant was sensitive to methyl methane sulfonate, slightly UV-sensitive and somewhat deficient in minichromosome maintenance. It also lowered the restrictive temperature of a cdc13ts mutant. These phenotypes suggested a role for ASF1 in DNA repair and chromosome maintenance.

Haploid cells of the yeast Saccharomyces cerevisiae normally undergo a budding life cycle, but after binding the appropriate mating pheromone they undergo a different developmental pathway that leads to conjugation. This intercellular communication between the two mating types activates a signal transduction pathway that stimulates the diverse physiological changes required for conjugation, such as induction of cell surface agglutinins, cell division arrest in G1, morphogenesis to form a conjugation tube, and cell fusion. The components of this pathway include a G protein-coupled receptor, several protein kinases, and a pheromone-responsive transcription factor. The molecular mechanisms that transduce the pheromone signal are remarkably similar to the mechanisms of hormone signaling used in multicellular organisms. Thus, the analysis of the pheromone signal pathway in yeast directly contributes to the study of cell growth and development in other eukaryotic organisms.

Wild-type S. cerevisiae cells of both mating types prefer partners producing high levels of pheromone and mate very infrequently to cells producing no pheromone. However, some mutants that are supersensitive to pheromone lack this ability to discriminate. In this study, we provide evidence for a novel role of alpha pheromone receptors in mating partner discrimination that is independent of the known G protein-mediated signal transduction pathway. Furthermore, in response to pheromone, receptors become localized to the emerging region of morphogenesis that is positioned adjacent to the nucleus, suggesting that receptor localization may be involved in mating partner discrimination. Actin, myosin 2, and clathrin heavy chain are involved in mating partner discrimination, since strains carrying mutations in the genes encoding these proteins result in a small but significant defect in mating partner discrimination.

Chronic myelogenous leukaemia (CML) is a clonal disease arising from malignant transformation of pluripotent hematopoietic stem cells. In most cases, it is characterized by the presence of the Philadelphia (Ph1) chromosome (22q-) which results from a reciprocal translocation between chromosomes 9 and 22 (refs 1-3). In this translocation, the human homologue of the Abelson virus oncogene, c-abl, normally on chromosome 9, is moved to chromosome 22, while c-sis, the cellular homologue of the simian sarcoma virus oncogene, is moved from chromosome 22 to chromosome 9 (refs 4-6). CML cells carrying the t(9;22) chromosomal translocation are known to produce an 8-kilobase (kb) c-abl transcript in addition to the normal 6- and 7-kb transcripts and to express the normal p145 abl protein and a p210 c-abl protein possessing a tyrosine kinase activity not detected in the p145 species. Results of our analyses using somatic cell hybrids between a mouse fibroblast line and two human CML-derived cell lines which carry the Ph1 chromosome and are phenotypically identical to the fibroblast parent indicate that only the hybrid cells containing Ph1 chromosome express both the 8-kb c-abl RNA and the p210 protein. Thus, expression of the altered c-abl transcripts and protein depends on the presence of the Ph1 chromosome and is not myeloid-specific.

Rat liver cytosolic phosphoenolpyruvate carboxykinase (PEPCK) utilizes inosine 5'-(3-thiotriphosphate) (ITP gamma S) as an excellent substrate, with Km and V values of 0.08 mM and 37 mumol min-1 (mg of protein)-1, respectively, compared with the corresponding values of 0.168 mM and 76 mumol min-1 (mg of protein)-1 for ITP. Thus, the V/Km values for the two substrates are the same. Reaction of (RP)-[gamma-18O2]ITP gamma S with oxalacetate catalyzed by cytosolic PEPCK produces (SP)-thio[18O]phosphoenolpyruvate. Therefore, thiophosphoryl transfer catalyzed by this enzyme proceeds with overall inversion of configuration at P. The reaction mechanism involves an uneven number of phosphotransfer steps, most likely a single step transfer between bound substrates. The results do not support the involvement of a phosphoryl enzyme intermediate in the mechanism.

STE2 encodes a component of the S. cerevisiae alpha-pheromone receptor that is essential for induction of physiological changes associated with mating. Analysis of C-terminal truncation mutants of STE2 demonstrated that the essential sequences for ligand binding and signal transduction are included within a region containing seven putative transmembrane domains. However, truncation of the C-terminal 105 amino acids of the receptor resulted in a 4- to 5-fold increase in cell-surface pheromone binding sites, a 10-fold increase in pheromone sensitivity, a defect in recovery of cell division after pheromone treatment, and a defect in pheromone-induced morphogenesis. Overproduction of STE2 resulted in about a 6-fold increase in alpha-pheromone binding capacity but did not produce the other phenotypes associated with the ste2-T326 mutant receptor. We conclude that the C-terminus of the receptor is responsible for one aspect of cellular adaptation to pheromone that is distinct from adaptation controlled by the SST2 gene, for decreasing the stability of the receptor, and for some aspect of cellular morphogenesis.

The phosphorus atoms of NAD+ bound within the active site of UDP-galactose 4-epimerase from Escherichia coli exhibit two NMR signals, one at delta = -9.60 +/- 0.05 ppm and one at delta = -12.15 +/- 0.01 ppm (mean +/- standard deviation of four experiments) relative to 85% H3PO4 as an external standard. Titration of epimerase.NAD+ with UMP causes a UMP-dependent alteration in the chemical shifts of the resulting exchange-averaged spectra, which extrapolate to delta = -10.51 ppm and delta = -11.06 ppm, respectively, for the fully liganded enzyme, with an interconversion rate between epimerase.NAD+ and epimerase.NAD+.UMP of at least 490 s-1. Conversely, the binding of 8-anilinonaphthalene-1-sulfonate, which is competitive with UMP, causes a significant sharpening of the epimerase.NAD+ resonances but very little alteration in their chemical shifts, to delta = -9.38 ppm and delta = -12.16 ppm, respectively. UMP-dependent reductive inactivation by glucose results in the convergence of the two resonances into a single signal of delta = -10.57 ppm, with an off-rate constant for UMP dissociation from the epimerase.NADH.UMP complex estimated at 8 s-1. Reductive inactivation by borohydride under anaerobic conditions yields a single, broad resonance centered at about delta = -10.2 ppm. The data are consistent with, and may reflect, the activation of NAD+ via a protein conformational change, which is known from chemical studies to be driven by uridine nucleotide binding. Incubation of epimerase.NAD+ with UMP in the absence of additional reducing agents causes a very slow reductive inactivation of the enzyme with an apparent pseudo-first-order rate constant of 0.013 +/- 0.001 h-1, which appears to be associated with liberation of inorganic phosphate from UMP.