Application of a glycinated bile acid biomarker for diagnosis and assessment of response to treatment in Niemann-pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative disease in which mutation of NPC1 or NPC2 gene leads to lysosomal accumulation of unesterified cholesterol and sphingolipids. Diagnosis of NPC disease is challenging due to non-specific early symptoms. Biomarker and genetic tests are used as first-line diagnostic tests for NPC. In this study, we developed a plasma test based on N-(3ß,5α,6ß-trihydroxy-cholan-24-oyl)glycine (TCG) that was markedly increased in the plasma of human NPC1 subjects. The test showed sensitivity of 0.9945 and specificity of 0.9982 to differentiate individuals with NPC1 from NPC1 carriers and controls. Compared to other commonly used biomarkers, cholestane-3ß,5α,6ß-triol (C-triol) and N-palmitoyl-O-phosphocholine (PPCS, also referred to as lysoSM-509), TCG was equally sensitive for identifying NPC1 but more specific. Unlike C-triol and PPCS, TCG showed excellent stability and no spurious generation of marker in the sample preparation or aging of samples. TCG was also elevated in lysosomal acid lipase deficiency (LALD) and acid sphingomyelinase deficiency (ASMD). Plasma TCG was significantly reduced after intravenous (IV) 2-hydroxypropyl-ß-cyclodextrin (HPßCD) treatment. These results demonstrate that plasma TCG was superior to C-triol and PPCS as NPC1 diagnostic biomarker and was able to evaluate the peripheral treatment efficacy of IV HPßCD treatment.

Loss-of-Function Variants in HOPS Complex Genes VPS16 and VPS41 Cause Early Onset Dystonia Associated with Lysosomal Abnormalities.

OBJECTIVES: The majority of people with suspected genetic dystonia remain undiagnosed after maximal investigation, implying that a number of causative genes have not yet been recognized. We aimed to investigate this paucity of diagnoses. METHODS: We undertook weighted burden analysis of whole-exome sequencing (WES) data from 138 individuals with unresolved generalized dystonia of suspected genetic etiology, followed by additional case-finding from international databases, first for the gene implicated by the burden analysis (VPS16), and then for other functionally related genes. Electron microscopy was performed on patient-derived cells. RESULTS: Analysis revealed a significant burden for VPS16 (Fisher's exact test p value, 6.9 × 109 ). VPS16 encodes a subunit of the homotypic fusion and vacuole protein sorting (HOPS) complex, which plays a key role in autophagosome-lysosome fusion. A total of 18 individuals harboring heterozygous loss-of-function VPS16 variants, and one with a microdeletion, were identified. These individuals experienced early onset progressive dystonia with predominant cervical, bulbar, orofacial, and upper limb involvement. Some patients had a more complex phenotype with additional neuropsychiatric and/or developmental comorbidities. We also identified biallelic loss-of-function variants in VPS41, another HOPS-complex encoding gene, in an individual with infantile-onset generalized dystonia. Electron microscopy of patient-derived lymphocytes and fibroblasts from both patients with VPS16 and VPS41 showed vacuolar abnormalities suggestive of impaired lysosomal function. INTERPRETATION: Our study strongly supports a role for HOPS complex dysfunction in the pathogenesis of dystonia, although variants in different subunits display different phenotypic and inheritance characteristics. ANN NEUROL 2020;88:867-877.

Integrating understanding of epidemiology and genomics in B-cell non-Hodgkin lymphoma as a pathway to novel management strategies.

Non-Hodgkin lymphomas include a biologically and clinically heterogeneous group of cancers distinguished by genetics, histology, and treatment outcomes. New discoveries regarding the genomic alterations and epidemiological exposures associated with these lymphomas have enhanced our understanding of factors that contribute to lymphomagenesis for specific subtypes. We explore the impact of normal B-cell biology engineered for recognizing a wide variety of antigens on the development of specific lymphoma subtypes, review lymphoma genetics, and examine the epidemiology of B-cell NHLs including recent investigations of risk factors for particular lymphoma subtypes based on large pooled analyses. Burkitt lymphoma, an aggressive form of B-cell NHL involving translocation of the MYC gene and an immunoglobulin gene has been associated with a history of eczema, hepatitis C, and occupation as a cleaner. Increased risk of diffuse large B-cell lymphoma has been associated with increased young adult body mass index, history of B-cell-activating autoimmune diseases, hepatitis C, and several single nucleotide variants involving the human leukocyte antigen (HLA) region of chromosome 6 and non-HLA loci near EXOC2, PVT1, MYC, and NCOA1. Tumor sequencing studies suggest that multiple pathways are involved in the development of DLBCL. Additional studies of epidemiological exposures, genome wide associations, and tumor sequencing in follicular, lymphoplasmacytic, marginal zone, and mantle cell lymphoma demonstrate overlapping areas of increased risk factors and unique factors for specific subtypes. Integrating these findings is important for constructing comprehensive models of NHL pathogenesis, which could yield novel targets for therapy and strategies for lymphoma prevention in certain populations.

An assessment of the frequency of mutations in the GBA and VPS35 genes in Hungarian patients with sporadic Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, with cases of either familial or sporadic origin. Several polymorphisms in a number of genes have been proved to have an important role in the development of PD. Particular attention has recently been paid to genes of the glucocerebrosidase (GBA) and the vacuolar protein sorting-associated protein 35 (VPS35). In this study, the three most common mutations (L444P, N370S and R120W) of the GBA gene and the D620N mutation of the VPS35 gene were examined in 124 Hungarian patients diagnosed with sporadic PD (SPD) and 122 control subjects. The frequency of the L444P mutation of the GBA gene proved to be higher in the PD patients (2.4%) than in the controls (0%), although the difference was not statistically significant. All the patients who carried the mutant allele were in the early-onset PD (EOPD) group. However, neither the R120W nor the N370S variant of the GBA gene nor D620N mutation of the VPS35 gene were detected among the PD cases or the controls. Even though these results suggest that the studied mutations are quite rare in SPD patients, the most frequent L444P mutation of the GBA gene may be associated with the development of EOPD in the Hungarian population.

El uso de placebo en ensayos clínicos de fase III en Brasil / The use of placebos in phase III clinical trials in Brazil

El Consejo Federal de Medicina de Brasil (CFM) -órgano normativo y fiscalizador del ejercicio ético de la medicina- prohibió, en 2008, la participación de médicos brasileños en investigaciones que utilizaran placebo para enfermedades con tratamiento eficaz y efectivo, en contraposición a la Declaración de Helsinki, que permite su uso en condiciones metodológicamente justificadas. Con el objetivo de verificar si la normativa ética del CFM modificó el uso de placebo en ensayos clínicos de fase III en Brasil, se analizaron varias características de sus registros en el ClinicalTrials.gov, en los períodos de 2003 a 2007 y de 2009 a 2013. Se concluye que: a) la normativa promulgada por el CFM en 2008 fue ineficaz y prevaleció la posición adoptada por la Declaración de Helsinki; b) el patrocinio de ensayos con placebo por parte de la industria farmacéutica multinacional fue significativo; c) predominaron las investigaciones de fármacos para enfermedades crónicas, y fueron poco significativas para las enfermedades postergadas, de importancia para Brasil.

In 2008, Brazil's Federal Council of Medicine [Conselho Federal de Medicina] (CFM) - regulatory and supervisory agency on the ethical practice of medicine - banned the participation of Brazilian doctors in studies using placebos for diseases with efficient and effective treatment. This position differs with the Helsinki Declaration, which allows the use of placebos in methodologically justified conditions. To ascertain whether the CMF's ethical regulation modified the use of placebos in phase III clinical trials in Brazil, characteristics of the records in ClinicalTrials.gov were researched in the periods from 2003 to 2007 and from 2009 to 2013. The conclusions reached were: a) the regulations issued by the CFM in 2008 were ineffective and the position adopted by the Helsinki Declaration prevails; b) there was significant sponsorship by the multinational pharmaceutical industry of trials with placebos; c) the research was predominantly on new drugs for chronic diseases, with little study done of the neglected diseases which are of great importance to Brazil.

No-reflow reversibility: a study based on serial assessment of multiple biomarkers.

No-reflow after primary percutaneous coronary intervention (pPCI) may be reversible. 40 patients undergoing pPCI were evaluated by assessing either improvement or lack of changes regarding angiographic and electrocardiographic indexes of no-reflow between admission and pre-discharge. Myeloperoxidase (MPO; in nanograms per milliliter), C-reactive protein (CRP; in milligrams per liter), endothelin-1 (ET-1; in nanograms per milliliter), angiopoietin-2 (Ang-2, in picograms per milliliter), and their pre-discharge/basal values variations (Δ) were related to no-reflow evolution. ΔMPO and ΔCRP were greater in patients with sustained no-reflow or lack of ST-segment resolution (STR) as compared with those with reversible no-reflow or lack of STR (p = 0.033, p = 0.04, p < 0.001, and p = 0.001, respectively), whereas ΔET-1 was similar in the two groups. ΔAng-2 was greater in patients with sustained no-reflow or lack of STR as compared with those with reversible no-reflow or lack of STR (p = 0.01 and 0.044, respectively). Bigger ΔMPO, ΔCRP (increasing levels), and ΔAng-2 (decreasing levels) are associated with sustained no-reflow, thus they might have a role in no-reflow evolution.

Protein-coat dynamics and cluster phases in intracellular trafficking.

Clustering of membrane proteins is a hallmark of biological membranes' lateral organization and crucial to their function. However, the physical properties of these protein aggregates remain poorly understood. Ensembles of coat proteins, the example considered here, are necessary for intracellular transport in eukaryotic cells. Assembly and disassembly rates for coat proteins involved in intracellular vesicular trafficking must be carefully controlled: their assembly deforms the membrane patch and drives vesicle formation, yet the protein coat must rapidly disassemble after vesiculation. Motivated by recent experimental findings for protein-coat dynamics, we study a dynamical Ising-type model for coat assembly and disassembly, and demonstrate how simple dynamical rules generate a robust, steady-state distribution of protein clusters (corresponding to intermediate budded shapes) and how cluster sizes are controlled by the kinetics. We interpret the results in terms of both vesiculation and the coupling to cargo proteins.

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients.

BACKGROUND: The presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients. METHODS: Gene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection. RESULTS: Six genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients. CONCLUSIONS: The panel of six genes was found superior to EpCAM and hMAM for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.

Event ordering in live-cell imaging determined from temporal cross-correlation asymmetry.

We use the temporal asymmetry of the cross-correlation function to determine the temporal ordering of spatially localized cellular events in live-cell multichannel fluorescence imaging. The analysis is well suited to noisy, stochastic systems where the temporal order may not be apparent in the raw data. The approach is applicable to any biochemical reaction not in chemical equilibrium, including protein complex assembly, sequential enzymatic processes, gene regulation, and other cellular signaling events. As an automated quantitative measure, this approach allows the data to be readily interpreted statistically with minimal subjective biases. We first test the technique using simulations of simple biophysical models with a definite temporal ordering. We then demonstrate the approach by extracting the temporal ordering of three proteins-actin, sorting nexin 9, and clathrin-in the endocytic pathway.

A quantitative assessment of gene expression (QAGE) reveals differential overexpression of DOPEY2, a candidate gene for mental retardation, in Down syndrome brain regions.

The brain alterations and mental retardation in Down syndrome are associated with overdosage of chromosome 21 genes. To shed light on the understanding of the molecular effect of this genetic overdosage, gene expression studies have crucial importance to quantify expression variations in Down syndrome tissues compared to normal ones. Herein, an in situ Quantitative Assessment of Gene Expression (QAGE) was used to quantify and statistically analyze, for the first time, DOPEY2 expression variations in different regions of the Down syndrome human fetal brains and to compare them to corresponding normal brains. DOPEY2, which is localized in the Down Syndrome Critical Region (DSCR) and is a candidate gene for neurological alterations in Down syndrome, showed a delimited regional and cellular expression pattern in the cortex, hippocampus and cerebellum, characterized by different transcriptional intensities in both normal and trisomic brains. DOPEY2 is overexpressed more than 50% (1.79-, 1.97- and 2.12-folds in the cortex, cerebellum and hippocampus, respectively), and showed statistically significant differences in the overexpression ratios in the three brain regions expressing DOPEY2. The demonstration of differential DOPEY2 expression and overexpression in human fetal brains suggests that this gene is submitted to a complex transcriptional control and could depend from other human chromosome 21 genes. Moreover, DOPEY2 overexpression in the brain regions, that are altered in Down syndrome patients and involved in learning and memory processes, is in agreement to the hypothesis that this gene plays a potential role in functional brain alterations and in the pathogenesis of mental retardation in Down syndrome. This new in situ QAGE approach allowed quantitative measurements of transcriptional changes and statistical evaluations of the expression and overexpression patterns of DOPEY2 at specific regions of the brain, which is a complementary approach to qRT-PCR and microarray for transcriptome study. Moreover, this approach could be a powerful tool to study the candidate chromosome 21 genes for Down syndrome and other pathologies caused by regionalized quantitative transcriptional alterations, for greater interpretation of functional processes driving gene expression.

Conservation of helical bundle structure between the exocyst subunits.

BACKGROUND: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies. METHODOLOGY/PRINCIPAL FINDINGS: Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% alpha-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners. CONCLUSIONS/SIGNIFICANCE: Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded domain of Sec10p.

Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding.

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K(d) >1 microM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In approximately 70% of the complexes, the distance root-mean-square is less than 5 A from the experimental structures. In approximately 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the approximately 300 microM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.

Clinical and molecular genetic assessment of a chorea-acanthocytosis pedigree.

BACKGROUND: Chorea-acanthocytosis (ChAc) is an autosomal recessive hereditary disease characterized by neurodegeneration in the striatum and acanthocytosis that is caused by mutations in the VPS13A gene. There are only few reports that studied clinical status of the obligate carriers of ChAc. Clinical courses with follow-up neuroradiological and neuropsychological evaluations in individuals with ChAc have been rarely reported. METHODS: We followed an index patient with ChAc and evaluated the clinical features of the pedigree members. Genetic analyses of VPS13A and genes responsible for other neuroacanthocytotic and neurodegenerative diseases were performed. CONCLUSIONS: The index patient was homozygous for a 3889C>T nonsense mutation in the VPS13A gene and presented with a typical ChAc phenotype. Neuropsychological evaluation with brain imaging in the patient over 3 years revealed atrophy and a decrease in blood flow at the basal ganglia and frontal lobe, and impairment in cognitive function reflecting frontal lobe dysfunction in progressive manners. Four out of five heterozygous mutation carriers in the pedigree showed signs or symptoms potentially attributable to a heterozygous VPS13A mutation.

Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis.

Lymphangiogenesis is a novel prognostic parameter for several cancers that is preferentially quantified by immunohistochemistry of the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Recently, the specificity of LYVE-1 was challenged by serendipitous observations of LYVE-1 expression in rare tissue macrophages. As expression of the hyaluronan receptor-like molecule stabilin-1 is shared by sinusoidal endothelium and macrophages, a thorough analysis of LYVE-1 expression was performed using macrophage-specific markers in vivo and in vitro. In murine tumour models and excisional wound healing, LYVE-1 expression occurred in a subset of CD11b(+), F4/80(+) tissue macrophages that preferentially co-expressed stabilin-1. Upon comparison of single- and double-labelling immunofluorescence, it became apparent that LYVE-1(+) macrophages mimic sprouting and collapsed lymphatic vessels. In vitro, LYVE-1 expression was induced in 25-40% of murine bone marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived macrophages were LYVE-1(+), stabilin-1(+) double-positive, while 9.9% were LYVE-1(+), stabilin-1(-) and 33.5% were LYVE-1(-), stabilin-1(+). Northern and western analyses confirmed expression of LYVE-1 mRNA and protein in bone marrow-derived macrophages. In the light of the current debate about true endothelial trans-differentiation versus endothelial mimicry of monocytes/macrophages, LYVE-1(+), stabilin-1(+) non-continuous endothelial-like macrophages will require further developmental and functional analyses. In conclusion, the findings imply that LYVE-1 staining must be supplemented by double labelling with macrophage markers in order to differentiate clearly between LYVE-1(+) lymphatics and LYVE-1(+) tumour-infiltrating macrophages. This improved approach will help to clarify the prognostic significance of lymphangiogenesis in malignant tumours.

LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer.

AIMS/METHODS: Normal and malignant pulmonary and endometrial tissues were analysed for lymphatic vessels to assess the process of lymphangiogenesis and its role at these sites, using specific immunostaining for LYVE-1 and the panendothelial marker CD31. RESULTS: Lymphatics were clearly demonstrated in some normal tissues (myometrium, bronchial submucosa, and intestinal submucosa), but not in others (endometrium and alveolar tissue). LYVE-1 positive lymphatic vessels were detected at the tumour periphery of endometrial and lung carcinomas, but not within the main tumour mass. Double staining for LYVE-1 and the MIB1 proliferation marker revealed a higher proliferation index in lymphatic endothelial cells at the invading front of endometrial carcinomas, compared with myometrial areas distal to the tumour. Lung and endometrial carcinomas did not have an intratumorous lymphatic network. CONCLUSIONS: Although lymphangiogenesis may occur at the invading tumour front, incorporated lymphatics do not survive. Therefore, the dissemination of cancer cells through the lymphatics may occur by invasion of peripheral cancer cells into the adjacent normal lymphatics, or through shunts eventually produced at the invading tumour front as a consequence of active angiogenesis and lymphangiogenesis.

Chondroitin sulfate proteoglycans of the endothelia of human umbilical vein and arteries and assessment for the adherence of Plasmodium falciparum-infected erythrocytes.

Infection with Plasmodium falciparum during pregnancy leads to chondroitin 4-sulfate-mediated adhesion of the infected red blood cells (IRBCs) in the placenta, causing severe health complications to fetus and the mother. The IRBCs are also frequently found in low density in the umbilical cord of infected placentas. In this study, the CSPGs of umbilical vein and arteries were purified, characterized, and their localization and IRBC-binding abilities were studied. While a versican type CSPG was found both in the vein and arteries, a serglycin type CSPG was present exclusively in the vein. The CSPGs were present at significant level on the endothelial surface of the umbilical vein but not on that of arteries. Although the purified versican and serglycin type CSPGs could bind IRBCs, their binding abilities were significantly less compared to the low sulfated CSPGs of the placenta because of the predominance of 6-sulfated disaccharide moieties in the CS chains. Therefore, IRBCs were unable to bind efficiently onto the umbilical cord endothelial surface. Unexpectedly, however, the IRBCs adhered densely in the blood vessels of fetal villi in the placental tissue sections and sparingly in the blood spaces of the umbilical cord vein, presumably because the CSPG that can efficiently bind IRBCs is present at high levels in the fetal blood vessels and at very low levels in the umbilical cord blood vessels. Since the C4S-adherent IRBCs that enter the fetal blood vessels cannot adhere to the cord endothelial surface and parasites cannot efficiently grow due to fetal hemoglobin toxicity and protection by maternal antibodies, transplacental infection may be quickly cleared without clinical episodes.

Activation of store-operated calcium channels: assessment of the role of snare-mediated vesicular transport.

Store-operated calcium channels (SOC) play a central role in cellular calcium homeostasis. Although it is well established that SOC are activated by depletion of the endoplasmic reticulum calcium stores, the molecular mechanism underlying this effect remains ill defined. It has been suggested that SOC activation requires fusion of endomembrane vesicles with the plasmalemma. In this model, SNARE-dependent exocytosis is proposed to deliver channels or their activators to the surface membrane to initiate calcium influx. To test this hypothesis, we studied the requirement for membrane fusion events in SOC activation, using a variety of dominant-negative constructs and toxins that interfere with SNARE function. Botulinum neurotoxin A (BotA), which cleaves SNAP-25, did not prevent SOC activation. Moreover, SNAP-25 was not detectable in the cells where BotA was reported earlier to inhibit SOC. Instead, the BotA-insensitive SNAP-23 was present. Impairment of VAMP function was similarly without effect on SOC opening. We also tested the role of N-ethylmaleimide-sensitive factor, a global regulator of SNARE-mediated membrane fusion. Expression of a mutated N-ethylmaleimide-sensitive factor construct inhibited all aspects of membrane traffic tested, including recycling of transferrin receptors to the plasma membrane, fusion of endosomes with lysosomes, and retrograde traffic to the Golgi complex. Despite this global inhibition of vesicular fusion, which was accompanied by gross alterations in cell morphology, SOC activation persisted. These observations cannot be easily reconciled with the vesicle-mediated coupling hypothesis of SOC activation. Our findings imply that the SOC and the machinery necessary to activate them exist in the plasma membrane or are associated with it prior to activation.

Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy.

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.

Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family.

The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices. A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion. Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif. In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes.