Evaluation of different cooling management strategies for lactating Holstein × Gir dairy cows.

Heat stress negatively impacts production, reproduction, and health of ruminants and strategies to alleviate these losses are warranted. Therefore, four experiments evaluated different cooling strategies on vaginal temperature (VT) of Holstein × Gir cows. Experiment 1 compared different amounts of water (2- or 4-L) over a 1-hour period from 1000 to 1100 h and 1600 to 1700 h. Experiment 2 evaluated the effects of sprinkling duration (in hours; 1- or 2-H), whereas Experiment 3 evaluated the effects of water amount (4- or 8-L) applied for 1- or 2-H. Lastly, the effects of a cooling strategy on specific hours of the day, starting at either 0700 (T-1) or 1100 h (T-2; Experiment 4), were evaluated. In all experiments, 12 Holstein × Gir cows were used in a 2 × 2 Latin Square Design containing two periods of 6 days each. Temperature and humidity index (THI) were recorded hourly and VT was recorded every 10-min throughout the experiments. As expected, an hour effect was observed for THI (P < 0.0001), which peaked early in the afternoon. In Experiment 1, a treatment × hour interaction was observed (P < 0.0001) for VT, as animals assigned to receive 4-L had a reduced VT at 1100, 1600, 1700, and 2300 h (P ≤ 0.03). During the cooling applications, cows receiving 4-L for 1 h had a reduced VT from 60 to 150 min (P ≤ 0.04). In Experiment 2, a treatment × hour interaction was observed (P < 0.0001) for VT, as animals assigned to receive 4-L of water for 2-H had a reduced VT at 1200 h (P = 0.05). Moreover, during the cooling process, VT was reduced for 2-H cows from 140 to 170 min after the beginning of the cooling process (P ≤ 0.05). In Experiment 3, animals assigned to receive 4-L + 2H had a reduced VT at 1200, 1700, 1800, and 1900 h (P < 0.001). A treatment × hour interaction was observed (P < 0.0001), as VT was reduced for 4-L + 2-H cows from 130 to 180 min after the beginning of the cooling process (P ≤ 0.05). In Experiment 4, by the time when the first cooling cycle of T-1 was applied (0700 h), T-1 cows consistently had (P ≤ 0.05) a reduced VT up to the hottest hours and greatest THI of the day (1400 and 1500 h). This pattern was maintained until the end of the last cooling cycle, whereas T-2 cows had a reduced VT. In summary, 4 L of water over a 5-min cycle for a period of 2 hours twice a day maintained VT of Holstein × Gir cows at lower levels. Moreover, the hour at which the first cooling cycle starts also should be considered when evaluating the efficacy of a cooling strategy for an entire day.

Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle.

Despite half a century of research, the biology of dinoflagellates remains enigmatic: they defy many functional and genetic traits attributed to typical eukaryotic cells. Genomic approaches to study dinoflagellates are often stymied due to their large, multi-gigabase genomes. Members of the genus Symbiodinium are photosynthetic endosymbionts of stony corals that provide the foundation of coral reef ecosystems. Their smaller genome sizes provide an opportunity to interrogate evolution and functionality of dinoflagellate genomes and endosymbiosis. We sequenced the genome of the ancestral Symbiodinium microadriaticum and compared it to the genomes of the more derived Symbiodinium minutum and Symbiodinium kawagutii and eukaryote model systems as well as transcriptomes from other dinoflagellates. Comparative analyses of genome and transcriptome protein sets show that all dinoflagellates, not only Symbiodinium, possess significantly more transmembrane transporters involved in the exchange of amino acids, lipids, and glycerol than other eukaryotes. Importantly, we find that only Symbiodinium harbor an extensive transporter repertoire associated with the provisioning of carbon and nitrogen. Analyses of these transporters show species-specific expansions, which provides a genomic basis to explain differential compatibilities to an array of hosts and environments, and highlights the putative importance of gene duplications as an evolutionary mechanism in dinoflagellates and Symbiodinium.

Mixing and internal dynamics of droplets impacting and coalescing on a solid surface.

The coalescence and mixing of a sessile and an impacting liquid droplet on a solid surface are studied experimentally and numerically in terms of lateral separation and droplet speed. Two droplet generators are used to produce differently colored droplets. Two high-speed imaging systems are used to investigate the impact and coalescence of the droplets in color from a side view with a simultaneous gray-scale view from below. Millimeter-sized droplets were used with dynamical conditions, based on the Reynolds and Weber numbers, relevant to microfluidics and commercial inkjet printing. Experimental measurements of advancing and receding static contact angles are used to calibrate a contact angle hysteresis model within a lattice Boltzmann framework, which is shown to capture the observed dynamics qualitatively and the final droplet configuration quantitatively. Our results show that no detectable mixing occurs during impact and coalescence of similar-sized droplets, but when the sessile droplet is sufficiently larger than the impacting droplet vortex ring generation can be observed. Finally we show how a gradient of wettability on the substrate can potentially enhance mixing.

Dynamics of contact line motion during the wetting of rough surfaces and correlation with topographical surface parameters.

Dynamics of contact line motion and wettability is essential in many industrial applications such as liquid coating, lubrication, printing, painting, condensation, etc. However, the wettability of surfaces depends not only on liquid-solid chemical properties but also can be strongly affected by surface roughness. As a practical application of controlled wettability, we can mention the self-cleaning surfaces, protective clothing, microfluidics devices, electro wetting, etc. In this article, we experimentally investigate the spreading of droplets deposited onto rough surfaces. Anisotropic surfaces were prepared by abrasive polishing on the following materials: aluminium alloy AA7064, titanium alloy Ti-6Al-4V, steel AISI 8630, copper alloy UNS C17000, machinable glass ceramic, and poly-methylmethacrylate. Topographical 2D parameters were calculated according to the following standards, defining Geometrical Product Specifications (GPS): ISO 4287, ISO 12085, ISO 13565, ISO 12780, and ISO 12181. The influence of topographical parameters on wettability and spreading phenomenon has been evaluated by statistical covariance analysis. The following parameters have strong influence on fluid spreading on rough surfaces: R(mr) is the relative material ratio of the roughness profile, T(rc) is the microgeometric material ratio, P(mr) is the relative material ratio of the raw profile, K(r) is the mean slope of the roughness motifs, RON(t) is the peak to valley roundness deviation, and P(sk) is the Skewness of the raw profile. The physical meaning of selected parameters is discussed, and K(r) (the mean slope of the roughness motifs) is selected as the most important and physically meaningful parameter. It has been found that for all tested materials, fluid spreading shows increasing tendency when mean slope of the roughness motifs (K(r) ) increases.

High efficiency nano-focusing kinoform optics for synchrotron radiation.

Modern synchrotron sources have provided for decades intense beams of photons over a large energy spectrum. The availability of improved optics and detectors has opened up new opportunities for the study of matter at the micrometre and nanometre scale in many disciplines. Whilst exploitation of micro-focused beams occurs almost daily in many beamlines, the production of beams of 100 nm is achieved on few instruments which use specialised optics. Refractive lenses, zone plates, curved mirrors, multilayers, and multilayer Laue lenses, can all focus x-rays to less than 50 nm under strict beam stability conditions. Focusing the synchrotron radiation to beam sizes smaller than 10 nm is considered the ultimate goal for the current decade. Silicon micro-technology has so far provided some of the most advanced x-ray refractive lenses; we report on design and characterisation of a novel silicon kinoform lens that is capable of delivering nano-beams with high efficiency.

The dynamics of the impact and coalescence of droplets on a solid surface.

A simple experimental setup to study the impact and coalescence of deposited droplets is described. Droplet impact and coalescence have been investigated by high-speed particle image velocimetry. Velocity fields near the liquid-substrate interface have been observed for the impact and coalescence of 2.4 mm diameter droplets of glycerol∕water striking a flat transparent substrate in air. The experimental arrangement images the internal flow in the droplets from below the substrate with a high-speed camera and continuous laser illumination. Experimental results are in the form of digital images that are processed by particle image velocimetry and image processing algorithms to obtain velocity fields, droplet geometries, and contact line positions. Experimental results are compared with numerical simulations by the lattice Boltzmann method.

Displacement of liquid droplets on a surface by a shearing air flow.

The motion of droplets on surfaces is crucial to the performance of a wide range of processes; this study examines the initiation of droplet motion through a shearing mechanism generated here by a controlled air flow. Systematic experiments are carried out for a range of fluids and well defined surfaces. A model is postulated that balances surface tension forces at the contact line and the drag force due to the air motion. Experiments reveal that the critical velocity at which droplet motion is initiated depends on the contact angle and the droplet size. Visualizations highlight three modes of motion: (I) the droplet retains a footprint similar to that at the point of motion; (II) a tail exists at the rear of the droplet; (III) a trail remains behind the droplet (that can shed smaller droplets). The predictions of droplet initiation velocity are good for type I motion, in accordance with the assumptions inherent within the model. This model confirms the dominant physics associated with the initiation of droplet motion and provides a useful predictive expression.

Characterization of germanium linear kinoform lenses at Diamond Light Source.

The unprecedented brilliance achieved by third-generation synchrotron sources and the availability of improved optics have opened up new opportunities for the study of materials at the micrometre and nanometre scale. Focusing the synchrotron radiation to smaller and smaller beams is having a huge impact on a wide research area at synchrotrons. The key to the exploitation of the improved sources is the development of novel optics that deliver narrow beams without loss of brilliance and coherence. Several types of synchrotron focusing optics are successfully fabricated using advanced miniaturization techniques. Kinoform refractive lenses are being developed for hard X-ray beamlines, and the first test results at Diamond are discussed in this paper.

Equine amnionitis and fetal loss: the case definition for an unrecognised cause of abortion in mares.

A series of abortions occurred in mares in New South Wales during 2004 that involved similar and unusual findings on post mortem examination of aborted fetuses and fetal membranes. The term Equine Amnionitis and Fetal Loss (EAFL) was developed to describe the condition. This form of abortion had not been previously recognised in Australia. The pathology alone is not specific for EAFL and diagnosis requires demonstration of a combination of certain pathological and bacteriological features. The purpose of this paper is to describe patterns considered consistent with EAFL cases as a working case definition for use by veterinarians and veterinary pathologists in identifying future cases of EAFL. More detailed papers are in preparation to fully describe the epidemiological, histopathological, and microbiological aspects of EAFL.

Evaluation of rayon swab surface sample collection method for Bacillus spores from nonporous surfaces.

AIM: To evaluate US Centers for Disease Control and Prevention recommended swab surface sample collection method for recovery efficiency and limit of detection for powdered Bacillus spores from nonporous surfaces. METHODS AND RESULTS: Stainless steel and painted wallboard surface coupons were seeded with dry aerosolized Bacillus atrophaeus spores and surface concentrations determined. The observed mean rayon swab recovery efficiency from stainless steel was 0.41 with a standard deviation (SD) of +/-0.17 and for painted wallboard was 0.41 with an SD of +/-0.23. Evaluation of a sonication extraction method for the rayon swabs produced a mean extraction efficiency of 0.76 with an SD of +/-0.12. Swab recovery quantitative limits of detection were estimated at 25 colony forming units (CFU) per sample area for both stainless steel and painted wallboard. CONCLUSIONS: The swab sample collection method may be appropriate for small area sampling (10 -25 cm2) with a high agent concentration, but has limited value for large surface areas with a low agent concentration. The results of this study provide information necessary for the interpretation of swab environmental sample collection data, that is, positive swab samples are indicative of high surface concentrations and may imply a potential for exposure, whereas negative swab samples do not assure that organisms are absent from the surfaces sampled and may not assure the absence of the potential for exposure. SIGNIFICANCE AND IMPACT OF THE STUDY: It is critical from a public health perspective that the information obtained is accurate and reproducible. The consequence of an inappropriate public health response founded on information gathered using an ineffective or unreliable sample collection method has the potential for undesired social and economic impact.

Expert consensus (SBC/SBHCI) on the use of drug-eluting stents: recommendations of the Brazilian society of interventional cardiology/ Brazilian society of cardiology for the Brazilian public single healthcare system.

The authors review percutaneous coronary intervention (PCI) evolution and its growing application in myocardial revascularization for patients with coronary heart disease in Brazil and worldwide. PCI was introduced in 1977 using only the catheter balloon. Limitations of this method (acute occlusion and coronary restenosis) led to the adoption of coronary stents and more recently the advent of drug-eluting stents2, which were developed to drastically reduce restenosis rates. These developments allowed the exponential growth of percutaneous coronary intervention (PCI) procedures in Brazil which have replaced many bypass surgery procedures and have become the gold standard for the majority of symptomatic patients suffering from coronary artery disease. The preference for this procedure gained new dimensions in 2000 when the Brazilian Public Healthcare System (SUS) began reimbursing for stent procedures. This measure exemplified the importance of the Public Healthcare System's participation in incorporating medical advances and offering a high standard of cardiovascular treatment to a large portion of the Brazilian population. It is emphasized that prevention of in-stent restenosis is complex due to its unpredictable and ubiquitous occurrence. Control of this condition improves quality of life and reduces the recurrence of angina pectoris, the need to perform new revascularization procedures and hospital readmissions. The overall success of the drug-eluting stents has proven to be reliable and consistent in overcoming restenosis and has some beneficial impact for all clinical and angiographic conditions. This paper discusses the adoption and criteria for the use of drug-eluting stents in other countries as well as the recommendations established by the Brazilian Society of Interventional Cardiology for their reimbursement by SUS. The incorporation of new healthcare technology involves two distinct stages. During the first stage, the product is registered with the National Health Surveillance Agency (ANVISA). During this stage the interested company submits to the regulatory agency, results from clinical studies that demonstrate the efficacy and safety of the new device or pharmaceutical product. Frequently, in addition to clinical studies, approval records for clinical use from the regulatory agencies of other countries, mainly the United States of America and the European Community are also submitted. The successful completion of this stage means that the medication or device may be prescribed or used by the physicians in Brazil. The second stage in the incorporation of new healthcare technology involves the reimbursement or financing of the treatment that was approved in the previous stage based on its efficacy and safety. This stage can be more complex than the first one since the new technology, whether a substitution for established treatment methods or the introduction of a new treatment concept, are usually more expensive. The incorporation of new technology requires a cost-effectiveness analysis so that fund administrators can make decisions based on the universal scenario of limited resources to finance healthcare with treatments that are more and more burdensome. The difficulties of funding management are aggravated by medical and social ethical implications that arise when a treatment is approved based on its efficacy and safety but is not made available to patients who could benefit greatly from it. In Brazil, assessment methods for the incorporation of new technology based on reimbursement or financing have not been fully developed for either the private healthcare plans or the Brazilian Public Healthcare System (SUS). The implementation of new technology in both healthcare systems is a slow process and frequently the implementation is a result of the requirements of patients or the organizations that represent them and at times is the result of legal proceedings or political pressure imposed by physicians and their respective scientific societies. Our objective is to review the evolution of percutaneous coronary intervention (PCI) in Brazil and its current status in view of the advent of drug-eluting stents, the growing participation of drug-eluting stents in myocardial revascularization to treat patients with coronary heart disease, as well as, to compare the regulatory standards from Brazil and other countries regarding the incorporation and recommendations for the use of this new technology.

Consenso de especialistas (SBC/SBHCI) sobre o uso de stents farmacológicos: recomendações da sociedade brasileira de cardiologia/sociedade brasileira de hemodinâmica e cardiologia intervencionista ao sistema único de saúde / Expert consensus (SBC/SBHCI) on the use of drug-eluting stents: recommendations of the Brazilian society of interventional cardiology/ Brazilian society of cardiology for the Brazilian public single healthcare system / Expert consensus (SBC/SBHCI) on the use of drug-eluting stents: recommendations of the Brazilian society of interventional cardiology/ Brazilian society of cardiology for the Brazilian public single healthcare system

Os autores revisam a evolução da intervenção coronariana percutânea, a sua crescente aplicação na revascularização miocárdica de pacientes portadores de doença arterial coronariana, seja no Brasil, seja no âmbito mundial. Desde a introdução do método, em 1977, com a utilização isolada do cateter-balão, a constatação de que o mesmo tinha limitações (oclusão aguda e reestenose), a adoção dos stents coronarianos e, mais recentemente, o advento dos stents farmacológicos, idealizados para reduzir ainda mais as taxas de reestenose, possibilitaram o crescimento exponencial da aplicação da intervenção coronariana percutânea (ICP) no Brasil, superando a cirurgia de revascularização e tornando-se o tratamento majoritário para enfermos sintomáticos, acometidos de aterosclerose obstrutiva coronariana. Esta preferência se salienta, a partir do ano 2000, após o início do reembolso dos stents pelo Sistema de Unico de Saúde Brasileiro. Este fato demonstra a importância do Sistema Público de Saúde, quando este incorpora os avanços médicos, e passa a oferecer bons padrões de tratamento cardiovascular a grande número de brasileiros. Destaca-se a complexidade da profilaxia da reestenose intra-stent, por sua ocorrência imprevisível e ubíqua. O controle deste fenômeno melhora a qualidade de vida, reduzindo o retorno da angina do peito, a realização de novos procedimentos de revascularização e a re-internação hospitalar. Os stents farmacológicos lograram êxito sólido e consistente na conquista deste objetivo de forma abrangente, beneficiando todas as apresentações clínicas e angiográficas, em maior ou menor grau. Sua adoção e critérios para sua utilização em outros países são discutidos, assim como a formalização das indicações preconizadas pela Sociedade Brasileira de Hemodinâmica e Cardiologia Intervencionista, para o seu reembolso pelo SUS. A incorporação de novas tecnologias em saúde é um processo que compreende duas etapas distintas: na primeira, o registro do produto é efetivado na Agência Nacional de Vigilância Sanitária (ANVISA)...

The authors review percutaneous coronary intervention (PCI) evolution and its growing application in myocardial revascularization for patients with coronary heart disease in Brazil and worldwide. PCI was introduced in 1977 using only the catheter balloon. Limitations of this method (acute occlusion and coronary restenosis) led to the adoption of coronary stents and more recently the advent of drug-eluting stents², which were developed to drastically reduce restenosis rates. These developments allowed the exponential growth of percutaneous coronary intervention (PCI) procedures in Brazil which have replaced many bypass surgery procedures and have become the gold standard for the majority of symptomatic patients suffering from coronary artery disease. The preference for this procedure gained new dimensions in 2000 when the Brazilian Public Healthcare System (SUS) began reimbursing for stent procedures. This measure exemplified the importance of the Public Healthcare System's participation in incorporating medical advances and offering a high standard of cardiovascular treatment to a large portion of the Brazilian population. It is emphasized that prevention of in-stent restenosis is complex due to its unpredictable and ubiquitous occurrence. Control of this condition improves quality of life and reduces the recurrence of angina pectoris, the need to perform new revascularization procedures and hospital readmissions. The overall success of the drug-eluting stents has proven to be reliable and consistent in overcoming restenosis and has some beneficial impact for all clinical and angiographic conditions. This paper discusses the adoption and criteria for the use of drug-eluting stents in other countries as well as the recommendations established by the Brazilian Society of Interventional Cardiology for their reimbursement by SUS. The incorporation of new healthcare technology involves two distinct stages. During the first stage, the product is registered with the National Health Surveillance Agency (ANVISA)...

Double-masked, placebo-controlled safety and efficacy trial of diquafosol tetrasodium (INS365) ophthalmic solution for the treatment of dry eye.

PURPOSE: To investigate the safety and efficacy of diquafosol tetrasodium, a P2Y2 receptor agonist that stimulates fluid and mucin secretion on the ocular surface, as a novel topical treatment of dry eye disease. METHODS: Subjects with dry eye (n=527) were evaluated in a randomized, double-masked, parallel-group trial comparing 24 weeks of treatment with 2 concentrations of diquafosol (1% and 2%) versus placebo instilled 4 times daily. Corneal staining, conjunctival staining, Schirmer tests, and subjective symptoms of dry eye were evaluated. Use of artificial tears was permitted as necessary. RESULTS: Subjects treated with 2% diquafosol had significantly lower corneal staining scores compared with placebo at the 6-week, primary efficacy time point (P<0.001), and superiority continued throughout the 24-week study. Reductions in corneal staining were observed as early as after 2 weeks of treatment, were maintained throughout the 24-week study, and were observed to worsen slightly (toward baseline) when diquafosol treatment was discontinued (week 25). Results for conjunctival staining were consistent with those observed for corneal staining. Schirmer scores at week 6 were significantly higher with diquafosol treatment than with placebo (P

Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting.

The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7 S and 20 S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.

A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with delta-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses.

Confocal immunofluorescence microscopy showed strong monocarboxylate transporter 2 (MCT2) labeling of Purkinje cell bodies and punctate labeling in the molecular layer. By immunogold cytochemistry, it could be demonstrated that the MCT2 immunosignal was concentrated at postsynaptic densities of parallel fiber-Purkinje cell synapses. The distribution of MCT2 transporters within the individual postsynaptic densities mimicked that of the delta2 glutamate receptor, as shown by use of two different gold-particle sizes. The MCT2 distribution was also compared with the distributions of other monocarboxylate transporters (MCT1 and MCT4). The MCT1 immunolabeling was localized in the endothelial cells, while MCT4 immunogold particles were associated with glial profiles, including those abutting the synaptic cleft of the parallel fiber-spine synapses. The postsynaptic density (PSD) molecules identified so far can be divided into five classes: receptors, their anchoring molecules, molecules involved in signal transduction, ion channels, and attachment proteins. Here, we provide evidence that this list of molecules must now be extended to comprise an organic molecule transporter: the monocarboxylate transporter MCT2. The present data suggest that MCT2 has specific transport functions related to the synaptic cleft and that this transporter may allow an influx of lactate derived from perisynaptic glial processes. The expression of MCT2 in synaptic membranes may allow energy supply to be tuned to the excitatory drive.

Expression and distribution of lactate/monocarboxylate transporter isoforms in pancreatic islets and the exocrine pancreas.

Transport of lactate across the plasma membrane of pancreatic islet beta-cells is slow, as described by Sekine et al. (J Biol Chem 269:4895-4902, 1994), which is a feature that may be important for normal nutrient-induced insulin secretion. Although eight members of the monocarboxylate transporter (MCT) family have now been identified, the expression of these isoforms within the exocrine and endocrine pancreas has not been explored in detail. Using immunocytochemical analysis of pancreatic sections fixed in situ, we demonstrated three phenomena. First, immunoreactivity of the commonly expressed lactate transporter isoform MCT1 is near zero in both alpha- and beta-cells but is abundant in the pancreatic acinar cell plasma membrane. No MCT2 or MCT4 was detected in any pancreatic cell type. Second, Western analysis of purified beta- and non-beta-cell membranes revealed undetectable levels of MCT1 and MCT4. In derived beta-cell lines, MCT1 was absent from MIN6 cells and present in low amounts in INS-1 cell membranes and at high levels in RINm5F cells. MCT4 was weakly expressed in MIN6 beta-cells. Third, CD147, an MCT-associated chaperone protein, which is closely colocalized with MCT1 on acinar cell membranes, was absent from islet cell membranes. CD147 was also largely absent from MIN6 and INS-1 cells but abundant in RINm5F cells. Low expression of MCT1, MCT2, and MCT4 contributes to the enzymatic configuration of beta-cells, which is poised to ensure glucose oxidation and the generation of metabolic signals and may also be important for glucose sensing in islet non-beta-cells. MCT overexpression throughout the islet could contribute to deranged hormone secretion in some forms of type 2 diabetes.

SNAP-25 with mutations in the zero layer supports normal membrane fusion kinetics.

Considerable data support the idea that intracellular membrane fusion involves a conserved machinery containing the SNARE proteins. SNAREs assembled in vitro form a stable 4-helix bundle and it has been suggested that formation of this complex provides the driving force for bilayer fusion. We have tested this possibility in assays of exocytosis in cells expressing a botulinum neurotoxin E (BoNT/E)-resistant mutant of SNAP-25 in which additional disruptive mutations have been introduced. Single or double mutations of glutamine to glutamate or to arginine in the central zero layer residues of SNAP-25 did not impair the extent, time course or Ca2+-dependency of exocytosis in PC12 cells. Using adrenal chromaffin cells, we found that exocytosis could be reconstituted in cells transfected to express BoNT/E. A double Q-->E mutation did not prevent reconstitution and the kinetics of single granule release events were indistinguishable from control cells. This shows a high level of tolerance of changes in the zero layer indicating that the conservation of these residues is not due to an essential requirement in vesicle docking or fusion and suggests that formation of a fully stable SNARE complex may not be required to drive membrane fusion.