We have labeled recycling synaptic vesicles at the somatic Bufo marinus neuromuscular junction with the styryl dye FM2-10 and provide direct evidence for refractoriness of exocytosis associated with a muscle activity-dependent form of long-term depression (LTD) at this synapse. FM2-10 dye unloading experiments demonstrated that the rate of vesicle exocytosis from the release ready pool (RRP) of vesicles was more than halved in the LTD (induced by 20 min of low frequency stimulation). Recovery from LTD, observed as a partial recovery of nerve-evoked muscle twitch amplitude, was accompanied by partial recovery of the refractoriness of RRP exocytosis. Unexpectedly, paired pulse plasticity, another routinely used indicator of presynaptic forms of synaptic plasticity, was unchanged in the LTD. We conclude that the LTD induces refractoriness of the neuromuscular vesicle release machinery downstream of presynaptic calcium entry.

Background: Currently, no review has been completed regarding the information-gathering process for the provision of medicines for self-medication in community pharmacies in developing countries. Objective: To review the rate of information gathering and the types of information gathered when patients present for self-medication requests. Methods: Six databases were searched for studies that described the rate of information gathering and/or the types of information gathered in the provision of medicines for self-medication in community pharmacies in developing countries. The types of information reported were classified as: signs and symptoms, patient identity, action taken, medications, medical history, and others. Results: Twenty-two studies met the inclusion criteria. Variations in the study populations, types of scenarios, research methods, and data reporting were observed. The reported rate of information gathering varied from 18% to 97%, depending on the research methods used. Information on signs and symptoms and patient identity was more frequently reported to be gathered compared with information on action taken, medications, and medical history. Conclusion: Evidence showed that the information-gathering process for the provision of medicines for self-medication via community pharmacies in developing countries is inconsistent. There is a need to determine the barriers to appropriate information-gathering practice as well as to develop strategies to implement effective information-gathering processes. It is also recommended that international and national pharmacy organizations, including pharmacy academics and pharmacy researchers, develop a consensus on the types of information that should be reported in the original studies. This will facilitate comparison across studies so that areas that need improvement can be identified. (c) 2013 Elsevier Inc. All rights reserved.

BACKGROUND: Community pharmacies are at the forefront of primary care providers and have an important role in the referral of patients to a medical practitioner for review when necessary. Chronic cough is a common disorder in the community and requires medical assessment. The proficiency of community pharmacy staff to refer patients with chronic cough is currently unknown.OBJECTIVE: To assess the ability of community pharmacy staff to recognize and medically refer patients with a chronic nonproductive cough.METHODS: Following ethics approval, a simulated patient study of 156 community pharmacies in Perth, Western Australia, was conducted over a 3-month period. Simulated patients presented to the pharmacy requesting treatment for a cough. The simulated patient required a referral based on a designated scenario. Demographic details, assessment questions, and advice provided were recorded by the simulated patient immediately postvisit. A logistic regression analysis was performed, with referral for medical assessment as the dependent variable.RESULTS: Of the 155 community pharmacies included in the analysis, 38% provided appropriate medical referral. Cough suppressants were provided as therapy in 72% of all visits. Predictors of medical referral were assessment of symptom duration, medical history, current medications being taken, frequency of reliever use, and the position of the pharmacy staff member conducting the consultation. A third of community pharmacies provided appropriate primary care by recommending medical referral advice to patients with chronic cough. The majority of pharmacy staff members acquired information from the patient that suggested a need for medical referral, yet did not provide referral advice.CONCLUSIONS: Appropriate medical referral is more likely when adequate assessment is undertaken and when a pharmacist is directly involved in the consultation. This highlights the need for pharmacies to ensure that processes are in place for patients to access the pharmacist.

Background: Over one quarter of asthma reliever medications are provided without prescription by community pharmacies in Australia. Evidence that community pharmacies provide these medications with sufficient patient assessment and medication counseling to ensure compliance with the government's Quality Use of Medicines principles is currently tacking. Objective: To assess current practice when asthma reliever medication is provided in the community pharmacy setting and to identify factors that correlate with assessment of asthma control. Methods: Researchers posing as patients visited a sample of Perth metropolitan community pharmacies in May 2007. During the visit, the simulated patient enacted a standardized scenario of someone with moderately controlled asthma who wished to purchase a salbutamol (albuterol) inhaler without prescription. Results of the encounter were recorded immediately after the visit. Regression analysis was performed, with medication use frequency (a marker of asthma control) as the dependent variable. Results: One hundred sixty community pharmacies in the Perth metropolitan area were visited in May 2007. Pharmacists and/or pharmacy assistants provided some form of assessment in 84% of the visits. Counseling was provided to the simulated patients in 24% of the visits. Only 4 pharmacy staff members asked whether the simulated patient knew how to use the inhaler. Significant correlation was found between assessment and/or counseling of reliever use frequency and 3 independent variables: visit length (p < 0.001), number of assessment questions asked (p < 0.001), and the simulated patient who conducted the visit (p < 0.02). Conclusions: Both patient assessment and medication counseling were suboptimal compared with recommended practice when nonprescription asthma reliever medication was supplied in the community pharmacy setting. Pharmacy and pharmacist demographic variables do not appear to affect assessment of asthma control. This research indicates the need for substantial improvements in practice in order to provide reliever medication in line with Quality Use of Medication principles of ensuring safe and effective use of medication.

The cellular localization of voltage-gated calcium channels (VGCCs) and synaptic vesicle-associated proteins, SV2, synapsin I, and vesicle-associated membrane protein (VAMP) (synaptobrevin), was investigated in the guinea pig cochlea using immunocytochemistry and confocal laser scanning microscopy. Reactivity, in guinea pig, of antibodies to the α1 subunits of L-type, α1C [Cav1.2] and α1D [Cav1.3]; P/Q-type, α1A [Cav2.1]; and R-type, α1E [Cav2.3] high voltage-activated calcium channels, was determined by Western blotting and immunolabeling of cerebellum. In the cochlea the sensory inner hair cells of the organ of Corti displayed strong intracellular staining, predominantly localized to their basolateral poles, with an antibody directed against the α1C subunit. Some α1C labeling was also observed in the inner pillar cells, in cell bodies of afferent neurons in the spiral ganglion, and in the inferior region of the spiral ligament. The supporting pillar cells were strongly immunoreactive throughout for α1D, but no α1D labeling of the inner hair cells was seen. The α1A subunit showed a cytoplasmic distribution in all three rows of outer hair cells. α1E labeling localized to the outer hair cells, predominantly in the subcuticular plate region, and also to nerve fiber bundles beneath these hair cells. Strong immunoreactivity was consistently seen with antibodies directed against SV2 and synapsin I in neuronal structures surrounding the basolateral surfaces of both the inner and outer hair cells but was absent from the sensory cells themselves. VAMP labeling was found throughout the cytoplasm of the inner hair cells and in neuronal structures beneath the hair cells. These results reveal a differential distribution of VGCC-types in the sensory and nonsensory elements of the guinea pig cochlea, with the inner hair cells expressing α1C L-type channels and VAMP but not synapsin I or SV2.

Structural relationships between cardiac isomyosins were analyzed in 10 species using native-gel electrophoresis and radioimmunoassay. In the rat and rabbit, three types of ventricular isomyosin, V1, V2, and V3, were identified by electrophoresis. Monoclonal antibodies specific for the heavy chains of either type V1 or type V3 isomyosin in the rat and rabbit were used for comparison of immunological relationships between atrial and ventricular myosins in other species. Normal guinea pig ventricular myosin reacted with both anti-V1 and anti-V3 antibodies, but only a single myosin band was detected in this species by electrophoresis. When thyrotoxic cardiac hypertrophy was induced in guinea pigs, there was a decrease in myosin reactivity with the anti-V3 antibody and an increase in anti-V1 reactivity. This change in immunological reactivity indicated a change in proportions of two cardiac isomyosins in the guinea pig ventricle even though no myosin heterogeneity was detected by electrophoresis. In six other species including Xenopus, chicken, dog, pig, beef, and human, only a single band of myosin was detected by electrophoresis, and each myosin reacted only with the anti-V3 antibody. In the mouse, three types of ventricular myosin were also detected by electrophoresis. However, unlike V1 isomyosin of the rat and rabbit, mouse V1 isomyosin reacted equally with both anti-V1 and anti-V3 antibodies. In conclusion, we have identified highly conserved epitopes in cardiac myosin, which were found to specifically occur on either the high Ca2+-ATPase type V1 isomyosin or the lower ATPase type V3 ventricular isomyosin in most of the species examined.

Myogenic cells migrate into the limb from the semitic mesoderm, taking up positions in the dorsal and ventral compartments before differentiating into the limb musculature. This process is influenced by the tissues of the limb in a number of ways. Cells of the lateral part of the semitic dermomyotome are induced to express a migratory myogenic phenotype by a diffusible signal originating in the limb bud mesoderm. The limb and body wall musculature are entirely derived from cells of this migratory myogenic lineage. Components of the extracellular matrix of the limb mesoderm, such as fibronectin, facilitate myogenic cell migration by providing sites for adhesion and directed migration. Differences in the composition of the matrix along the proximo-distal axis make the distal mesoderm more attractive for migrating myogenic cells. Anterior-posterior movement of myogenic cells within the limb bud is highly restricted, resulting in a regionalised contribution of cells to the limb musculature based on semitic origin. Lastly, there is a process that excludes myogenic cells from the most distal region of the limb bud mesoderm; a similar process is involved in the aggregation of myogenic cells into the dorsal and ventral premuscle masses. These findings illustrate the high degree of control over myogenic cell migration mediated by both diffusible factors and the extracellular matrix in the developing limb mesenchyme.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.

The relative proportion of the two putative heavy chains of smooth muscle myosin (MHC1 and MHC2) was determined in the caudal and femoral arteries of spontaneously hypertensive rats (SHR) and normotensive (WKY) rats at 16 weeks of age. The heavy chain polypeptides with Mr 204000 and 200000 were resolved electrophoretically under denaturing conditions in porous polyacrylamide gels. Both proteins reacted strongly with a monoclonal antibody (2C4) to smooth muscle MHC. In caudal arteries the ratio of MHC1/MHC2 was 3.1∶1 in SHR rats compared with 1.8∶1 in WKY rats (p<0.005) and similarly in femoral arteries, 2.8∶1 vs 1.5∶1 (p<0.001). In the portal vein there was no significant difference, 1.7∶1 vs 1.5∶1. The possibility that the higher MHC ratio in the SHR is the genetically mediated defect in arterial smooth muscle cells leading to the hypertension is discussed as an alternative to the elevated systemic blood pressure causing the altered MHC ratio.

Myosin expression during hypertrophy of the chicken anterior latissimus dorsi (ALD) muscle was investigated by immunocytochemical procedures using monoclonal antibodies to the fast and slow isoforms of the myosin heavy chain (myosin HC). Antifast antibody 1F9 bound to the adult fast HC of pectoralis muscle and cross-reacted with the HC found in early developing muscle. Antislow antibody 3D1 bound exclusively to the HC of slow myosin 2 (SM2). Stretch hypertrophy of the ALD was produced by attaching a weight to the wing; there was no evidence for a change in fibre number in the muscle. Between 4 and 6 days of stretch there appeared a dramatic increase in the number of fibres staining with the antifast antibody which reached a peak between 12 and 19 days. By this time between 28 and 52% of the fibres in the stretched ALD stained to varying degrees with the antifast antibody compared with ≪ 1% in the unstretched control ALD. Most antifast-stained fibres in the stretched muscle also stained with the antislow antibody; the contralateral control muscle showed mostly antislow staining except for the very small number of strongly antifast-stained fibres. By 50 days in some birds and by 80 days in all birds antifast staining had returned to normal. Analysis of the isomyosin composition of the ALD by native gel electrophoresis did not reveal a significant increase in fast myosin content of the hypertrophied muscle even though immunocytochemical staining may have suggested otherwise.

Many avian muscles contain a characteristic topographical distribution of fibre types. In order to study the role of nerves in the establishment and distribution of these fibre types, monoclonal antibodies (McAb) to the heavy chain subunit of myosin (MHC) were produced. The anti-fast McAb (2B12) bound to adult fast MHC and cross-reacted with the embryonic isoform of MHC. The anti-slow McAb (3D1) bound specifically to the heavy chain of slow myosin 2. By indirect immunofluorescence, anti-fast (2B12) stained all myotubes in the anterior latissimus dorsi and triceps and biceps muscles at stage 37 (11 days embryonic), whilst anti-slow (3D1) staining was largely restricted to the future slow fibres of these muscles. Brachial levels of the neural tube were surgically removed at stage 12 (2 days embryonic) so that muscles developed aneurally. Muscles at aneural stage 37 were smaller than normal, but the distribution of myotube types was not altered; all myotubes present still stained with anti-fast antibody while anti-slow staining was restricted to the anterior latissimus dorsi and myotubes in the deep parts of the triceps brachii pars scapularis, triceps brachii par humeralis and biceps brachii muscles (the future slow fibres of normal muscles). The results suggest that despite an overall reduction in MHC in aneural muscles, specialized fast and slow primary myotubes arise independently of the nerve in appropriate regions of the muscle.