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The retinoblastoma protein (Rb) and its homologs p107 and p130 are critical regulators of gene expression during the cell cycle and are commonly inactivated in cancer. Rb proteins use their "pocket domain" to bind an LxCxE sequence motif in other proteins, many of which function with Rb proteins to co-regulate transcription. Here, we present binding data and crystal structures of the p107 pocket domain in complex with LxCxE peptides from the transcriptional co-repressor proteins HDAC1, ARID4A, and EID1. Our results explain why Rb and p107 have weaker affinity for cellular LxCxE proteins compared with the E7 protein from human papillomavirus, which has been used as the primary model for understanding LxCxE motif interactions. Our structural and mutagenesis data also identify and explain differences in Rb and p107 affinities for some LxCxE-containing sequences. Our study provides new insights into how Rb proteins bind their cell partners with varying affinity and specificity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%); other affected genes included PMS1 , KMT2C , BRCA2 , PALB2 , MUTYH , MEN1 , MSH2 , CHEK2 , NCOR1 , PIK3CA , ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AF tissue ≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.

Objective: To investigate the 1) socio-demographic characteristics, 2) working environment, 3) tasks and responsibilities and 4) work experiences of intercultural mediators (IMs) working in Belgian hospitals., Methods: Cross-sectional quantitative survey among all IMs working in Flemish and Brussels hospitals (n = 66). Data were descriptively analyzed. Meaningful associations between variables were also studied., Results: Most IMs are young women from first- and second-generation migrant groups with different levels of education. They work under different superiors and most IMs are not employed full-time. They work mainly with patients from their own ethnic group. Mostly they intervene directly in daily intercultural communication, as per their official task description, but they also perform other tasks, such as offering support to patients/families/staff/management. IMs would prefer more of the tasks they perform to be formalized. Furthermore, they want to have policy-making responsibilities. IMs have positive and negative work experiences, e.g. working overtime., Conclusions: IMs' socio-demographic characteristics (ethnic origin - sex - education) and official task description is only adapted to needs in the workplace to a limited extent. Furthermore, intercultural mediation is poorly integrated into hospitals' organizational structure., Practice Implications: Different measures are needed, including tailored education and offering IMs enough organizational support and policy responsibilities., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: Dual antiplatelet therapy (DAPT) after percutaneous coronary intervention (PCI) reduces the rate of ischemic events but increases bleeding risk. DAPT score helps identify patients who benefit from prolonged DAPT. Nevertheless, its accuracy in patients with acute myocardial infarction (AMI) remains uncertain. The aim of this study was to validate the use of DAPT score to predict ischemic and bleeding events in patients undergoing PCI for AMI and who received prolonged DAPT., Material and Methods: This study included a cohort of patients with AMI who underwent PCI with stent placement and were treated with DAPT for more than 12 months., Results: Two hundred thirty subjects were included in the final analysis (age: 64 ± 12 years, 78% men, median follow-up: 31 months). Ischemic event (reinfarction or revascularization of target vessel or lesion) occurred in 17% and bleeding occurred in 5% of patients. DAPT score demonstrated modest prediction performance for ischemic events (C-statistic: 0.59, 95% confidence interval [CI]: 0.50-0.68, p<0.001) and a good prediction performance for bleeding events (C-statistic: 0.79, 95% CI: 0.66-0.92, p<0.001). Subjects with a DAPT score ≥2 had a greater risk of ischemic events (hazard risk [HR]: 3.1, 95% CI: 1.2-7.8, p = 0.019) and a lower risk of bleeding (HR: 0.23, 95% CI: 0.07-0.79, p = 0.019). Kaplan-Meier curves at 4 years showed that patients with a DAPT score ≥2 had lower ischemic-free survival rates (79% ± 4 vs. 90% ± 5, p = 0.0137) and higher bleeding-free survival rates (97% ± 2 vs. 90% ± 4, p = 0.0106)., Conclusions: DAPT score is useful in patients with AMI, and a cut-off value of 2 identifies patients with a higher risk of ischemic events who might benefit from prolonged DAPT., (Copyright © 2018 Hellenic Society of Cardiology. Published by Elsevier B.V. All rights reserved.)

Modifications of plant immune complexes by secreted pathogen effectors can trigger strong immune responses mediated by the action of nucleotide binding-leucine-rich repeat immune receptors. Although some strains of the pathogen Pseudomonas syringae harbor effectors that individually can trigger immunity, the plant's response may be suppressed by other virulence factors. This work reveals a robust strategy for immune suppression mediated by HopZ3, an effector in the YopJ family of acetyltransferases. The suppressing HopZ3 effector binds to and can acetylate multiple members of the RPM1 immune complex, as well as two P. syringae effectors that together activate the RPM1 complex. These acetylations modify serine, threonine, lysine, and/or histidine residues in the targets. Through HopZ3-mediated acetylation, it is possible that the whole effector-immune complex is inactivated, leading to increased growth of the pathogen., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that modulates key signaling molecules involved in synaptic plasticity and neuronal function. Targets include extracellular-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase p38 (p38), the Src family tyrosine kinase Fyn, N-methyl-D-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). STEP-mediated dephosphorylation of ERK1/2, p38, and Fyn leads to inactivation of these enzymes, whereas STEP-mediated dephosphorylation of surface NMDARs and AMPARs promotes their endocytosis. Accordingly, the current model of STEP function posits that it opposes long-term potentiation and promotes long-term depression. Phosphorylation, cleavage, dimerization, ubiquitination, and local translation all converge to maintain an appropriate balance of STEP in the central nervous system. Accumulating evidence over the past decade indicates that STEP dysregulation contributes to the pathophysiology of several neuropsychiatric disorders, including Alzheimer's disease, schizophrenia, fragile X syndrome, epileptogenesis, alcohol-induced memory loss, Huntington's disease, drug abuse, stroke/ischemia, and inflammatory pain. This comprehensive review discusses STEP expression and regulation and highlights how disrupted STEP function contributes to the pathophysiology of diverse neuropsychiatric disorders.

4-Vinylcyclohexene diepoxide (VCD), an occupational chemical that specifically destroys primordial and small primary follicles in the ovaries of rats and mice, is thought to target an oocyte-expressed tyrosine kinase receptor, Kit. This study compared the temporal effect of VCD on protein distribution of KIT and its downstream PIK3-activated proteins, AKT and FOXO3. Postnatal Day 4 Fischer 344 rat ovaries were cultured in control media ± VCD (30 μM) for 2-8 days (d2-d8). KIT, AKT, phosphorylated AKT, FOXO3, and pFOXO3 protein levels were assessed by Western blotting and/or immunofluorescence staining with confocal microscopy. Phosphorylated AKT was decreased (P < 0.05) in oocyte nuclei in primordial (39% decrease) and small primary (37% decrease) follicles within 2 days of VCD exposure. After d4, VCD reduced (P < 0.05) oocyte staining for KIT (primordial, 44% decrease; small primary, 39% decrease) and FOXO3 (primordial, 40% decrease; small primary, 36% decrease) protein. Total AKT and pFOXO3 were not affected by VCD at any time. Akt1 mRNA, as measured by quantitative RT-PCR, was reduced (P < 0.05) by 23% on d4 of VCD exposure, but returned to control levels on d6 and d8. VCD exposure reduced Foxo3a mRNA by 26% on d6 (P < 0.05) and by 23% on d8 (P < 0.1). These results demonstrate that the earliest observed effect of VCD is an inhibition of phosphorylation and nuclear localization of AKT in the oocyte of primordial and small primary follicles. This event is followed by reductions in KIT and FOXO3 protein subcellular distribution prior to changes in mRNA. Thus, these findings further support that VCD induces ovotoxicity by directly targeting the oocyte through posttranslational inhibition of KIT-mediated signaling components.

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder. Early in the pathophysiology of AD, synaptic function is disrupted by soluble Aβ oligomers, possibly through Aβ-mediated internalization of NMDA receptors. Striatal-enriched phosphatase (STEP) is a tyrosine phosphatase that regulates the internalization of NMDA receptors. Recent work shows that STEP is elevated in the prefrontal cortex of human AD patients and in animal models of AD. Here, we use genetic manipulations to reduce STEP activity in a triple transgenic AD mouse model and show that a decrease in STEP levels reverses cognitive and cellular deficits observed in these mice. Our results suggest that STEP inhibitors may prove therapeutic for this devastating disorder.

Traditional approaches to the study of hormones and cognition have been primarily observational or correlational in nature. Because this work does not permit causal relationships to be identified, very little is known about the specific molecules and cellular events through which hormones affect cognitive function. In this review, we propose a new approach to study hormones and memory, where the systematic blocking of cellular events can reveal which such events are necessary for hormones to influence memory consolidation. The discussion will focus on the modulation of the hippocampus and hippocampal memory by estrogens, given the extensive literature on this subject, and will illustrate how the application of this approach is beginning to reveal important new information about the molecular mechanisms through which estrogens modulate memory consolidation. The clinical relevance of this work will also be discussed., (Copyright © 2009 Elsevier B.V. All rights reserved.)

In its natural environment, the plant cuticle, which is composed of the biopolymer cutin and a mixture of surface and embedded cuticular waxes, experiences a wide variety of temperatures and hydration states. Consequently, a complete understanding of cuticular function requires study of its thermal and mechanical properties as a function of hydration. Herein, we report the results of a comprehensive 13C nuclear magnetic resonance (NMR) relaxation study of hydrated tomato fruit cuticle. Cross-polarization and direct-polarization experiments serve to measure the solid-like and liquid-like components, respectively, of hydrated cuticle. Localized, high-frequency motions are probed by T1(C) spin relaxation measurements, whereas T1rho(H) and T1rho(C) experiments reflect low-frequency, lower amplitude polymer-chain motions. In addition, variable-temperature measurements of T1(C) and T1rho(C) for dry tomato cuticles are used to evaluate the impact of temperature stress. Results of these experiments are interpreted in terms of changes occurring in individual polymer motions of the cutin/wax components of tomato cuticle and in the interaction of these components within intact cuticle, both of which are expected to influence the functional integrity of this protective plant covering.

The extracellular signal-regulated kinase (ERK) pathway is critical for various forms of learning and memory, and is activated by the potent estrogen 17beta-estradiol (E(2)). Here, we asked whether E(2) modulates memory via ERK activation and putative membrane-bound estrogen receptors (ERs). Using ovariectomized mice, we first demonstrate that intraperitoneal injection of 0.2 mg/kg E(2) significantly increases dorsal hippocampal levels of phosphorylated ERK protein 1 h after injection. Second, we show that E(2) administered intraperitoneally (0.2 mg/kg) or via intrahippocampal infusion (5.0 microg/side) immediately after training in an object recognition task significantly enhances memory retention, and that the beneficial effect of intraperitoneal E(2) is blocked by dorsal hippocampal inhibition of ERK activation. Third, using bovine serum albumin-conjugated 17beta-estradiol (BSA-E(2)), we demonstrate that E(2) binding at membrane-bound ERs can increase dorsal hippocampal ERK activation and enhance object memory consolidation in an ERK-dependent manner. Fourth, we show that this effect is independent of nuclear ERs, but is dependent on the dorsal hippocampus. By demonstrating that E(2) enhances memory consolidation via dorsal hippocampal ERK activation, this study is the first to identify a specific molecular pathway by which E(2) modulates memory and to demonstrate a novel role for membrane-bound ERs in mediating E(2)-induced improvements in hippocampal memory consolidation.

Repeated daily dosing of rats with the occupational chemical 4-vinylcyclohexene diepoxide (VCD) depletes the ovary of primordial and primary follicles through an increase in the natural process of atresia. Additionally, in vitro exposure of Postnatal Day 4 (PND 4) rat ovaries to VCD causes similar follicular depletion. This study was designed to investigate survival signaling pathways that may be associated with VCD-induced ovotoxicity in small preantral follicles. Female Fischer 344 rats (PND 28) were dosed daily (80 mg/kg/day VCD i.p.; 12 days in vivo), and PND 4 ovaries were cultured (VCD 20 or 30 microM; 8 days in vitro). Microarray analysis identified a subset of 14 genes whose expression was increased or decreased by VCD in both experiments (i.e., via both exposure routes). Particularly, the analysis showed that relative to controls, VCD did not affect mRNA expression of growth and differentiation factor 9 (Gdf9), whereas there were decreases in mRNA encoding bone morphogenic protein receptor 1a (Bmpr1a) and Kit. To confirm findings from microarray, the genes Gdf9, Bmpr1a, and Kit were further examined. When growth factors associated with these pathways were added to ovarian cultures during VCD exposure, GDF9 and BMP4 had no effect on VCD-induced ovotoxicity; however, KITL attenuated this follicle loss. Additionally, there was a decrease in Kit and an increase in Kitl expression (mRNA and protein) following VCD exposure, relative to control. These results support that VCD compromises KIT/KITL signaling, which is critical for follicular survival in primordial and primary follicles.

Grating-coupled surface plasmon resonance imaging (GCSPRI) is a method for the accurate assessment of both cell phenotype and function. In GCSPRI, cells and/or proteins of interest are flowed across antibodies immobilized on a gold-coated sensor chip. The surface of the chip is illuminated with monochromatic light that couples with surface plasmons in the gold. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs. Shifts in the GCSPR angle can be correlated with refractive index changes following cell or analyte capture by the immobilized antibodies. In addition, GCSPRI can image the cells as they are being captured. GCSPRI's multiplexed format allows for the parallel assessment of up to 400 individual antibody regions. In this paper, we demonstrate GCSPRI's ability to identify cells and proteins of interest and compare results to a traditional flow cytometry system. This technology represents a fast and powerful method for the simultaneous assessment of cell phenotype and function.

Protein kinase C (PKC)-mediated signal transduction pathways convert extracellular stimulation into a variety of cellular functions. However, the role of various PKC isoforms in sphingosine 1-phosphate (S1P)-stimulated endothelial cells is not well understood. PKCalpha and PKCepsilon activity are increased in endothelial cell cultures, and S1P receptor transfection studies indicate S1P(3) stimulates PKCalpha and S1P1 leads to PKCepsilon activity. Infection of endothelial cells with dominant negative (DN)PKCalpha adenovirus reduces cell migration and greatly inhibits morphogenesis in cells stimulated with S1P. This effect is specific to PKCalpha, as infection with DN PKCepsilon does not alter either migration or morphogenesis. The PKC-specific chemical inhibitor GF109203X also inhibits these two responses. Infection of endothelial cells with dominant negative PKCalpha reduces S1P-induced calcium rise. This maximal rise requires calcium uptake, but it does not require enzymatic activity of the kinase. Pretreatment of these cells with the PKC-specific inhibitor GF109203X does not inhibit S1P-induced calcium rise. S1P-induced morphogenesis but not cell migration is critically dependent on extracellular calcium. Pretreatment of endothelial cells with phorbol 12-myristate 13-acetate for 5min abolishes S1P-stimulated rise in calcium but had little or no effect on migration. The PMA-inhibited calcium rise can be prevented by PKC inhibitor or infection with dominant negative PKCalpha.

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of analyte in a multiplexed format using small amounts of sample. In GCSPR, the analyte is flowed across specific receptors (e.g. antibodies or other proteins) that have been immobilized on a sensor chip. The chip surface is illuminated with p-polarized light that couples to the gold surface's electrons to form a surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs, thus reducing the intensity of reflected light. Shifts in the GCSPR angle can be correlated with refractive index increases following analyte capture by chip-bound receptors. Because regions of the chip can be independently analyzed, this system can assess 400 interactions between analyte and receptor on a single chip. We have used this label-free system to assess a number of molecules of immunological interest. GCSPR can simultaneously detect an array of cytokines and other proteins using the same chip. Moreover, GCSPR is also compatible with assessments of antigen expression by intact cells, detecting cellular apoptosis and identifying T cells and B cells. This technology represents a powerful new approach to the analysis of cells and molecular constituents of biological samples.

Environmental enrichment paradigms that incorporate cognitive stimulation, exercise, and motor learning benefit memory and synaptic plasticity across the rodent lifespan. However, the contribution each individual element of the enriched environment makes to enhancing memory and synaptic plasticity has yet to be delineated. Therefore, the current study tested the effects of three of these elements on memory and synaptic protein levels. Young female C57BL/6 mice were given 3h of daily exposure to either rodent toys (cognitive stimulation) or running wheels (exercise), or daily acrobatic training for 6 weeks prior to and throughout behavioral testing. Controls were group housed, but did not receive enrichment. Spatial working and reference memory were tested in a water-escape motivated radial arm maze. Levels of the presynaptic protein synaptophysin were then measured in frontoparietal cortex, hippocampus, striatum, and cerebellum. Exercise, but not cognitive stimulation or acrobat training, improved spatial working memory relative to controls, despite the fact that both exercise and cognitive stimulation increased synaptophysin levels in the neocortex and hippocampus. These data suggest that exercise alone is sufficient to improve working memory, and that enrichment-induced increases in synaptophysin levels may not be sufficient to improve working memory in young females. Spatial reference memory was unaffected by enrichment. Acrobat training had no effect on memory or synaptophysin levels, suggesting a minimal contribution of motor learning to the mnemonic and neuronal benefits of enrichment. These results provide the first evidence that different elements of the enriched environment have markedly distinct effects on spatial memory and synaptic alterations.

This study tested whether chronic oral estrogen could improve memory and alter neural plasticity in the hippocampus and neocortex of middle-aged female mice. Ovariectomized C57BL/6 mice were administered 1,000, 1,500, or 2,500 nM 17beta-estradiol in drinking water for 5 weeks prior to and during spatial and object memory testing. Synaptophysin, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) levels were then measured in hippocampus and neocortex. The medium dose impaired spatial reference memory in the radial-arm maze, whereas all doses improved object recognition. The high dose increased hippocampal synaptophysin and NGF levels, whereas the medium dose decreased these neocortical levels. The high dose decreased neocortical BDNF levels. These data suggest that chronic oral estrogen selectively affects memory and neural function in middle-aged female mice.

Estradiol benzoate (EB) has repeatedly been shown to increase hippocampal CA1 spine synapse density in ovariectomized female rats. Although this increase has been assumed to enhance memory, a direct link between increased spine synapse density and memory has not been demonstrated. Furthermore, while androgens, such as testosterone propionate (TP) and dihydrotestosterone (DHT), also increase spine synapse density in females, their effects on memory have yet to be investigated. In the present study, ovariectomized female rats were given two injections, 24 h apart, of sesame oil (control), 10 microg EB, 500 microg TP or 500 microg DHT. Forty-eight hours after the second injection, rats were tested in a 1-day spatial Morris water maze task and then immediately perfused for analysis of CA1 spine synapse density (using electron microscopy and unbiased stereology). In the spatial acquisition phase of testing, EB, but not TP or DHT, significantly impaired memory relative to controls. Hormone treatment did not affect spatial retention or performance in the non-spatial phase of testing. In contrast to previous work, spine synapse density was not increased by EB, TP or DHT. We therefore examined a new set of EB-treated females, only half of which were water maze tested. Consistent with previous work, EB significantly increased spine synapse density among behaviorally naïve females. In contrast, spine synapse densities did not differ among behaviorally tested control and EB females, although they were higher than behaviorally naïve controls. These data indicate that 1-day water maze testing can eliminate the hormone-induced increases in CA1 spine synapse density typically observed in behaviorally naïve females.

The present study tested whether environmental enrichment can reduce age-related spatial reference memory deficits and alter synaptic protein levels in aged female mice. Female C57BL/6 mice, (4 or 27-28 months), were tested in spatial and cued Morris water maze tasks. Prior to (14 days) and during testing, a subset of aged females was exposed to rodent toys and running wheels for 3h per day. The remaining aged females were group housed but were not exposed to enriching objects. At the conclusion of testing, levels of the presynaptic protein synaptophysin were measured in hippocampus and frontoparietal cortex. Enrichment improved spatial memory acquisition; relative to young controls, aged enriched females performed similarly, whereas aged control females were impaired. Enrichment also accelerated the development of a spatial bias in spatial probe trials. In contrast, the cued task was not significantly affected by enrichment. Hippocampal and cortical synaptophysin levels were increased in aged enriched females relative to young and aged controls. These data suggest that environmental enrichment can be a potent cognitive enhancer for aged females and suggests a potential neurobiological mechanism of this effect.

Estrogen deficiency during menopause is often associated with memory dysfunction. However, inconsistencies regarding the ability of estrogen to improve memory in menopausal women highlight the need to evaluate, in a controlled animal model, the potential for estrogen to alleviate age-related mnemonic decline. The current study tested whether estrogen could ameliorate spatial reference memory decline in aged female mice. At the conclusion of testing, levels of the presynaptic protein synaptophysin, and activities of the synthetic enzymes for acetylcholine and GABA, were measured in the hippocampus and neocortex. Aged (27-28-month-old) female C57BL/6 mice were given daily subcutaneous injections of 1 microg or 5 microg of beta-estradiol-3-benzoate dissolved in sesame oil. Control mice received daily injections of sesame oil or no injections. Estradiol treatment began 5 days prior to behavioral testing and continued throughout testing. Spatial and non-spatial memory were assessed in the Morris water maze. The 5 microg dose of estradiol significantly improved spatial learning and memory in aged females. The performance of 5 microg females improved significantly more rapidly than that of control females; estradiol-treated females performed at asymptotic levels by session 2. Furthermore, 5 microg females exhibited a more robust spatial bias than controls during probe trials. In contrast, 1 microg of estradiol did not improve spatial task performance. Neither dose affected performance of the non-spatial task. In the hippocampus, synaptophysin was increased in 5 microg females relative to controls. Estrogen did not affect enzyme activities in either brain region. This study is the first to examine the effects of estrogen replacement on spatial reference memory and synaptophysin expression in aged post-estropausal female rodents. The results suggest that: (1) estrogen can profoundly improve spatial reference memory in aged females, and (2) this improvement may be related to increased hippocampal synaptic plasticity, but not modulation of the synthetic enzymes for acetylcholine and GABA.

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.

Changes in membrane dynamic properties associated with membrane fusion are studied employing in vitro myoblast fusion as a model system. We utilize a microscopic fluorescence relaxation approach which makes feasible the study of local variations in membrane dynamics within surface subdomains of single intact cells. Studies of the average rotational mobility of the fluorescent probe-1-anilino-naphthalene-8-sulfonate by this technique indicate that myoblast fusion activity is preceded by a generalized increased in membrane fluidity and that areas of cell contact between fusing cells exhibit higher fluidity and polarity, locally, than non-fusion regions.

The surface topography of concanavalin A (con A) bound to normal and transformed murine fibroblasts has been studied by a new technique involving fluorescence resonance energy transfer (RET), RET can provide a high resolution "map" of the distances separating con A-receptor complexes in single living cells. The distribution of con A is non-random in both normal and transformed cells, but sites are more closely approximated in the transformed. Approximation is induced by the con A but occurs at extremely slow rates indicating that the topography is not primarily determined by simple diffusion of complexes.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.

We have investigated changes in topography and lateral translational mobility of concanavalin A (Con A) receptors on the surface of cultured chick muscle cells during the period of myoblast fusion. A temporal correlation between these phenomena and the alteration in membrane fluidity known to occur during this time period is established. Receptor topography and mobility are studied by means of a resonance energy transfer technique employing pyrene- and FITC-Con A conjugates. All measurements are performed through a microscope on single cells. Our results reveal that during the period of myoblast fusion Con A receptors undergo a dramatic redistribution on the cell surface. Furthermore, our data suggest that the changes in membrane fluidity observed during muscle differentiation serve to modulate the lateral mobility of these receptors.