Clathrin-Coated Vesicle Subtypes in Mammalian Brain Tissue: Detection of Polypeptide Heterogeneity by Immunoprecipitation with Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radioiodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A-7C11 recognized a 40-kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one-quarter of the total brain CVs. The mAb S-11D9 reacted with a 44-kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S-11D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C-10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa greater than LCb). Each of the mAbs yielded different immunofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor-differentiated PC12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.     

Brain clathrin and clathrin-associated proteins.

The assembly of clathrin into baskets or cages in vitro may depend on formation of complex between clathrin and a polypeptide doublet migrating in the 30000-mol.wt. region. Clathrin with several associated proteins was isolated from coated-vesicle fractions of bovine cerebral cortex. Most associated proteins were separated by Sepharose 4B column chromatograhy. The eluted clathrin retained only the 30000-mol.wt. doublet and assembled into baskets at pH 6.5. Limited proteolysis of coated vesicles or clathrin assembled as baskets removed these clathrin-associated proteins (CAPs) without detectably altering clathrin. Enzyme-treated clathrin assembled into open-lattice structures but no longer formed baskets in vitro. Latex particles with bound enzyme cleaved the CAPs from coated vesicles and clathrin baskets, suggesting that the CAPs protrude from the exterior of the clathrin lattice.     

Partial purification and characterization of thiosulfate oxidase from Pseudomonas aeruginosa.

Soluble thiosulfate oxidase from Pseudomonas aeruginosa was purified 85-fold and coverted thiosulfate to tetrathionate by using either ferricyanide or cytochrome c as an electron acceptor.     

alpha-Actinin and tropomyosin interactions with a hybrid complex of erythrocyte-actin and muscle-myosin.

alpha-Actinin isolated from dog muscle was used to incite antibodies in rabbits, Antibodies, purified by affinity chromatography on CNBr-Sepharose coupled with alpha-actinin and then ferritin-labeled were found to localize on the Z disc of muscle sarcomeres. Molecules of alpha-actinin as an adsorbed monolayer on the surface of polystyrene Lytron particles could bind muscle-actin and tropomyosin from solution. Both the ATPase activity and superprecipitation of an erythrocyte-actin and muscle-myosin hybrid actomyosin complex were altered by alpha-actinin, while tropomyosin diminished these alpha-actinin effects. The binding properties of alpha-actinin are consistent with those of an anchoring protein for microfilaments in nonmuscle cells.     

西藏墨脱格当乡野生虎捕食家畜现状与保护建议

曾经在西藏东南的阔叶林中广泛分布的孟加拉虎,目前仅有一个小种群残留在墨脱县境内。2000年5－6月间,在大型家畜遭受野生虎捕食最严重的墨脱县格当乡展开调查,试图寻找减轻虎害的方法。结果表明：1994－1995年虎的捕食率达到最高,对牛和骡马的捕食率分别是17．9％和9．4％;但1996年后,捕食率分别降低到7．8％和1．8％,这可能与1996年当地曾捕杀过一头虎有关。1993－1999年间,全乡牛的数量下降了11％,但骡马数量上升了23％,这是因为当地为增加运输能力而从别处购得骡马。据反映,目前格当乡境内大约有4－5头虎。1997年和1999年,均见母虎和小虎同行,说明该种群尚有繁殖。在抽样的21户居民家中,1999年4月到2000年5月间,66．6％的人家有大型牲畜遭虎捕杀,共损失牛27头,马12匹,而自1993年以来,21户中共有18户（85．8％）,有牲畜被杀记录,共计损失117头。其中对牛的捕食率达19．7％,对马达11．9％,平均每户损失牛1．2头,骡马0．5匹。非法狩猎减少了虎的猎物如野猪、羚牛等的数量,是老虎转向家畜的主要原因之一。虎害已对格当乡群众的经济造成较大负面影响。但格当乡以及周边地区保护着中国最后的野生孟加拉虎种群,为确保虎的长期生存同时减少人－虎冲突,建议改变目前放牧方式,尽可能联合放牧、增加看护;改善放牧地条件,清除牧场周围蕨草丛;减少对羚牛等有蹄类的猎杀,以减少对老虎猎物种群的破坏;对部分家畜移入棚内试行圈养,既保护家畜,又提高乳制品产量和增加农家肥料;实行多种经营方法,建议养一些山羊和家禽;政府应该帮助安置好部分愿意外迁的居民,这样既满足这些居民的需要,同时也减轻对当地野生动物种群的压力。     

Lack of geographic variation in anonymous nuclear polymorphisms in the American oyster, Crassostrea virginica

Comparing geographic variation of noncoding nuclear DNA polymorphisms, which presumably are neutral to natural selection, with geographic variation of allozymes is potentially a good way to detect the effects of selection on allozyme polymorphisms. A previous study of four anonymous nuclear markers in the American oyster, Crassostrea virginica, found dramatic differences in allele frequency between the Gulf of Mexico and the Atlantic Ocean. In contrast, 14 allozyme polymorphisms were fairly uniform in frequency between the two areas. This led to the conclusion that all of the allozyme polymorphisms were kept uniform in frequency by balancing selection. To test the robustness of this pattern, six additional anonymous nuclear DNA polymorphisms were surveyed in oysters from Panacea, Fla, and Charleston, S.C. on the Gulf and Atlantic coasts, respectively. Unlike the previously studied DNA markers, the six DNA polymorphisms examined here show geographic variation that is not significantly greater than that of allozymes. The reason for the discrepancy between the two sets of DNA polymorphisms is unclear.      

The mitogenome of Onchocerca volvulus from the Brazilian Amazonia focus

We report here the first complete mitochondria genome of Onchocerca volvulus from a focus outside of Africa. An O. volvulus mitogenome from the Brazilian Amazonia focus was obtained using a combination of high-throughput and Sanger sequencing technologies. Comparisons made between this mitochondrial genome and publicly available mitochondrial sequences identified 46 variant nucleotide positions and suggested that our Brazilian mitogenome is more closely related to Cameroon-origin mitochondria than West African-origin mitochondria. As well as providing insights into the origins of Latin American onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an epidemiological resource.     

Validating the use of a commercial enzyme immunoassay to measure oxytocin in unextracted urine and saliva of the western lowland gorilla (<Emphasis Type="Italic">Gorilla gorilla gorilla</Emphasis>)

The neuroendocrine hormone oxytocin, which is an important physiological driver of social behavior and bonding, is increasingly being measured in conjunction with behavior to better understand primate sociality. To date no data are available on oxytocin concentrations within the genus Gorilla; however, as a result of their close genetic relatedness to humans, and tolerance-based social system, Gorilla represents an important group of study. The purpose of this study was to validate the measurement of urinary and salivary oxytocin in western lowland gorillas (Gorilla gorilla gorilla) to help facilitate future study of the interaction between oxytocin and behavior within the subspecies. The primary validation procedure was an intranasal challenge. Elevated oxytocin concentrations were observed in saliva samples taken 15–120 min post challenge. Urine levels remained within baseline range approximately 30 and 90 min following the challenge; however, elevated levels were observed 24 h post challenge. No diurnal variation was found in salivary samples taken at regular intervals throughout the day; however, morning urine samples had higher concentrations than afternoon samples. In addition, samples were collected opportunistically following three social events: play, breeding, and the death of a conspecific. Following the play bouts, salivary oxytocin was almost three times greater than baseline. Salivary oxytocin was also significantly higher 15 min post breeding compared to match-control samples. Following the death of a conspecific, the group mate’s urinary oxytocin concentrations decreased by half compared to a baseline period when the group was intact. This study provides a biological validation of the measurement of urinary and salivary oxytocin in western lowland gorillas. These results suggest that urinary oxytocin measurements are suitable for establishing baseline levels, as they represent the build up of the previous day’s concentrations, and salivary oxytocin measurements are suitable for assessing changes following specific events.