RAB2A: a major subacrosomal protein of bovine spermatozoa implicated in acrosomal biogenesis

The perinuclear theca (PT) of mammalian sperm is a unique subcellular structure encapsulating the nucleus. Compositionally, the PT is made up of at least six prominent polypeptides (60, 36, 31, 28, 24, and 15 kDa), of which only two have been sequence identified, as well as many less prominent ones. As an ongoing process in unveiling the protein composition of the PT, we have uncovered the sequence identity of the prominent 24-kDa polypeptide (PT24). Initial N-terminal sequence analysis obtained by Edman degradation suggested that PT24 is a RAB2 protein. This was corroborated by mass spectrometric analyses of trypsin-digested fragments of PT24, identifying RAB2A of the RAB2 subfamily as the best sequence match. Quadrapole/time-of-flight analysis identified 72%% sequence coverage between PT24 and bull, human, mouse, or rabbit RAB2A. Since a genome search only identified two RAB2 subfamily members, RAB2A and RAB2B, the 72%% sequence coverage of PT24 provides assurance that this protein is RAB2A and not a new RAB2 subfamily member. Furthermore, commercial RAB2A antibodies, raised against oligopeptide fragments in the unique C-terminal region of RAB2A, specifically labeled PT24 on Western blot analysis of PT extracts. These anti-RAB2A antibodies, along with immune serum that we raised and affinity purified against isolated PT24, demonstrated at both light and electron microscope levels that RAB2 is associated with the periphery of the growing proacrosomic and acrosomic vesicles in the Golgi and cap phases of spermiogenesis and consequently assembled as part of the PT. This pattern of subacrosomal assembly is reminiscent of the pathway used by SubH2Bv (PT15), another prominent and exclusive subacrosomal protein, indicating a common route for subacrosomal-PT assembly. Traditionally somatic RAB2 proteins are involved in vesicular transport between the endoplasmic reticulum and the cis-side of the Golgi apparatus. Our study suggests an unprecedented direction of RAB2A-mediated vesicular transport in spermatids during acrosomal biogenesis, from the trans-side of the Golgi apparatus to the nuclear envelope.     

东亚飞蝗hunchback基因在卵子形成和胚胎发育过程中的原位表达

hb（hunchback）基因是昆虫胚胎前后轴模式形成的关键基因．对东亚飞蝗（Locusta migratoria manilensis,Meyen）hb基因的功能已有报道,但其表达模式还不清楚．为了研究胁基因在东亚飞蝗卵子形成和胚胎发育过程中的时空表达情况,本研究采用免疫组化方法在蛋白质水平上检测了hb基因的时空表达模式．在卵子形成过程中,hb基因局限在卵细胞核区中表达,随着卵子的发育逐渐移至卵细胞的后端;卵受精后,核区里的Hb蛋白向外扩散,在卵后端形成浓度梯度;胚盘期,hb基因在胚盘中央呈带状表达;胚盘分化为原头和原躯干后,表达条带变宽,并呈现出梯度表达,该表达区域将形成颌、胸部的部分体节;随着腹节开始形成,hb基因在颌胸部的表达逐渐减弱,而在腹部后端的“生长区”表达,并呈现出不连续性．经比较,hb易基因在昆虫颌胸部的表达较为保守,而在卵子形成过程中和腹部的表达具有较大的变异性．与黑腹果蝇等长胚带昆虫相比,东亚飞蝗hb基因在体节形成的基因级联调控中具有更重要、更直接的调控作用．     

心脏特异表达LMNA^E82K转基因小鼠的建立

目的建立心脏特异表达LMNAE82K转基因小鼠,为研究LMNAE82K与心肌病发病机制的关系提供工具动物。方法把LMNAE82K基因插入α-MHC启动子下游,构建转基因表达载体,显微注射法建立C57BL/6JLMNAE82K转基因小鼠,PCR鉴定转基因小鼠的基因型,采用Western Blot鉴定LMNAE82K在心脏组织中的表达,H＆E染色和超声检测转基因小鼠心脏的病理改变。结果建立了2个心脏组织特异表达LMNAE82K的转基因小鼠品系。超声检查显示转基因小鼠心室壁变薄,收缩期容积和舒张期容积增加,射血分数及短轴缩短率降低。结论LMNAE82K转基因小鼠具有LMNAE82K引起的家族性扩心病有类似的病理变化,为研究LMNAE82K与心肌病发病机制的关系的研究提供了有价值的疾病动物模型。     

Global transcriptional response of pig brain and lung to natural infection by Pseudorabies virus

Background  

Pseudorabies virus (PRV) is an alphaherpesviruses whose native host is pig. PRV infection mainly causes signs of central nervous system disorder in young pigs, and respiratory system diseases in the adult.     

Short-term low-salinity tolerance by the longhorn sculpin, Myoxocephalus octodecimspinosus

The bottom-dwelling, longhorn sculpin, Myoxocephalus octodecimspinosus, is traditionally viewed as a stenohaline marine fish, but fishermen have described finding this sculpin in estuaries during high tide. Little is known about the salinity tolerance of the longhorn sculpin; thus, the purposes of these experiments were to explore the effects of low environmental salinity on ion transporter expression and distribution in the longhorn sculpin gill. Longhorn sculpin were acclimated to either 100% seawater (SW, sham), 20% SW, or 10% SW for 24 or 72 hr. Plasma osmolality, sodium, potassium, and chloride concentrations were not different between the 20 and 100% treatments; however, they were 20-25% lower with exposure to 10% SW at 24 and 72 hr. In the teleost gill, regulation of Na(+), K(+)-ATPase (NKA), Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), and the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR) are necessary for ion homeostasis. We immunolocalized these proteins to the mitochondrion-rich cell of the gill and determined that acclimation to low salinity does not affect their localization. Also, there was not a downregulation of gill NKA, NKCC1, and CFTR mRNA or protein during acclimation to low salinities. Collectively, these results suggest that down to 20% SW longhorn sculpin are capable of completely regulating ion levels over a 72-hr period, whereas 10% SW exposure results in a significant loss of ions and no change in ion transporter density or localization in the gill. We conclude that longhorn sculpin can tolerate low-salinity environments for days but, because they cannot regulate ion transporter density, they are unable to tolerate low salinity for longer periods or enter freshwater (FW). The genus Myoxocephalus has three FW species, making this group an excellent model to test evolutionary and physiological mechanisms that allow teleosts to invade new low salinities successfully.     

Dynamin activates NO production in rat renal inner medullary collecting ducts via protein-protein interaction with NOS1

We hypothesized that nitric oxide synthase (NOS) isoforms may be regulated by dynamin (DNM) in the inner medullary collecting duct (IMCD). The aims of this study were to determine which DNM isoforms (DNM1, DNM2, DNM3) are expressed in renal IMCDs, whether DNM interacts with NOS, whether a high-salt diet alters the interaction of DNM and NOS, and whether DNM activates NO production. DNM2 and DNM3 are highly expressed in the rat IMCD, while DNM1 is localized outside of the IMCD. We found that DNM1 interacts with NOS1α, NOS1β, and NOS3 in the inner medulla of male Sprague-Dawley rats on a 0.4% salt diet. DNM2 interacts with NOS1α, while DNM3 interacts with both NOS1α and NOS1β. DNM2 and DNM3 do not interact with NOS3 in the rat inner medulla. We did not observe any change in the DNM/NOS interactions with rats on a 4% salt diet after 7 days. Furthermore, NOS1α interacts with DNM2 in mIMCD3 and COS7 cells transfected with NOS1α and DNM2-GFP constructs and the NOS1 reductase domain is necessary for the interaction. Finally, COS7 cells expressing NOS1α or NOS1α/DNM2-GFP had significantly higher nitrite production compared with DNM2-GFP only. Nitrite production was blocked by the DNM inhibitor dynasore or the dominant negative DNM2K44A. Ionomycin stimulation further increased nitrite production in the NOS1α/DNM2-GFP cells compared with NOS1α only. In conclusion, DNM and NOS1 interact in the rat renal IMCD and this interaction leads to increased NO production, which may influence NO production in the renal medulla.     

The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species

Background

The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations.

Results

We produced a draft de novo genome assembly of Australia’s major tephritid pest species, Bactrocera tryoni. The male genome (650 -700 Mbp) includes approximately 150Mb of interspersed repetitive DNA sequences and 60Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions.

Conclusions

This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1153) contains supplementary material, which is available to authorized users.