The identification and distribution of gonadotropin-releasing hormone-like peptides in the central nervous system and ovary of the giant freshwater prawn, Macrobrachium rosenbergii

In the present study, we demonstrated the existence of GnRH-like peptides in the central nervous system (CNS) and ovary of the giant freshwater prawn, Macrobrachium rosenbergii using immunocytochemistry. The immunoreactivity (ir) of lamprey (l) GnRH-III was detected in the soma of medium-sized neurons located in neuronal cluster number 11 in the middle part of supraesophageal ganglion (deutocerebrum), whereas ir-octopus (oct) GnRH was observed in the soma of both medium-sized and large-sized neurons in thoracic ganglia, as well as in the fibers innervating the other medium-sized and large-sized neuronal cell bodies in the thoracic ganglia. In addition, ir-lGnRH-I was observed in the cytoplasm of late previtellogenic oocyte and early vitellogenic oocyte. These data suggest that M. rosenbergii contain at least three isoforms of GnRH: two GnRH isoforms closely related to lGnRH-III and octGnRH in the CNS, whereas another isoform, closely related to lGnRH-I, was localized in the ovary. This finding provides supporting data that ir-GnRH-like peptide(s) may exist in this decapod crustacean.     

Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.     

Characterization of mucus-associated proteins from abalone (Haliotis) - candidates for chemical signaling

Living in groups is a widespread phenomenon in the animal kingdom. For free-spawning aquatic animals, such as the abalone (Haliotis), being in the close proximity to potential mating partners enhances reproductive success. In this study, we investigated whether chemical cues could be present in abalone mucus that enable species-specific aggregation. A comparative MS analysis of mucus obtained from trailing or fixed stationary Haliotis asinina, and from seawater surrounding aggregations, indicated that water-soluble biomolecules are present and that these can stimulate sensory activity in conspecifics. Purified extracts of trail mucus contain at least three small proteins [termed H.?asinina mucus-associated proteins (Has-MAPs)-1-3], which readily diffuse into the surrounding seawater and evoke a robust cephalic tentacle response in conspecifics. Mature Has-MAP-1 is approximately 9.9 kDa in size, and has a glycine-rich N-terminal region. Has-MAP-2 is approximately 6.2 kDa in size, and has similarities to schistosomin, a protein that is known to play a role in mollusc reproduction. The mature Has-MAP-3 is approximately 12.5 kDa in size, and could only be identified within trail mucus of animals outside of the reproductive season. All three Has-MAP genes are expressed at high levels within secretory cells of the juvenile abalone posterior pedal gland, consistent with a role in scent marking. We infer from these results that abalone mucus-associated proteins are candidate chemical cues that could provide informational cues to conspecifics living in close proximity and, given their apparent stability and hydrophilicity, animals further afield.     

HLA-Associated Immune Pressure on Gag Protein in CRF01_AE-Infected Individuals and Its Association with Plasma Viral Load

Background

The human leukocyte antigen (HLA)-restricted cytotoxic T-lymphocyte (CTL) immune response is one of the major factors determining the genetic diversity of human immunodeficiency virus (HIV). There are few population-based analyses of the amino acid variations associated with the host HLA type and their clinical relevance for the Asian population. Here, we identified HLA-associated polymorphisms in the HIV-1 CRF01_AE Gag protein in infected married couples, and examined the consequences of these HLA-selected mutations after transmission to HLA-unmatched recipients.

Methodology/Principal Findings

One hundred sixteen HIV-1-infected couples were recruited at a government hospital in northern Thailand. The 1.7-kb gag gene was amplified and directly sequenced. We identified 56 associations between amino acid variations in Gag and HLA alleles. Of those amino acid variations, 35 (62.5%) were located within or adjacent to regions reported to be HIV-specific CTL epitopes restricted by the relevant HLA. Interestingly, a significant number of HLA-associated amino acid variations appear to be unique to the CRF01_AE-infected Thai population. Variations in the capsid protein (p24) had the strongest associations with the viral load and CD4 cell count. The mutation and reversion rates after transmission to a host with a different HLA environment varied considerably. The p24 T242N variant escape from B57/58 CTL had a significant impact on the HIV-1 viral load of CRF01_AE-infected patients.

Conclusions/Significance

HLA-associated amino acid mutations and the CTL selection pressures on the p24 antigen appear to have the most significant impact on HIV replication in a CRF01_AE-infected Asian population. HLA-associated mutations with a low reversion rate accumulated as a footprint in this Thai population. The novel HLA-associated mutations identified in this study encourage us to acquire more extensive information about the viral dynamics of HLA-associated amino acid polymorphisms in a given population as effective CTL vaccine targets.     

Rat sperm AS-A: subcellular localization in testis and epididymis and surface distribution in epididymal sperm

In this study, we investigated the subcellular compartmentalization of arylsulfatase-A (AS-A) in the testis and epididymis as well as the surface distribution in rat epididymal sperm. Testicular AS-A was compartmentalized specifically to the area underneath the outer acrosomal membrane of the acrosomal granule and to the dorsal aspect of the sperm acrosome. Epididymal AS-A was synthesized in the endoplasmic reticular (ER) network of principal cells and secreted into epididymal lumen as evident by its reactivity in the apical cytoplasm and vesicles therein underneath stereocilia. In clear cells, AS-A reactivity was found throughout the cytoplasmic machineries involved in endocytosis. Surface distribution of AS-A was initially detectable at the concave ridge as early as in sperm of the initial segment (IS). AS-A was additionally localized to the post-acrosomal region in caput (CP), corpus (CO) and cauda (CD) epididymal sperm. The expression levels of surface AS-A gradually increased during sperm transit from IS to CD epididymidis. These results favored the adsorption of AS-A from epididymal fluid onto the sperm surface, rather than shunting from the acrosome as a consequence of capacitation-associated membrane priming.This work was supported by Research Initiate Grant funded by Faculty of Science, Mahidol University to W.W.     

Changes in the histological organization and spheroid formation in lymphoid organ of Penaeus monodon infected with yellow head virus

The changes in the histological and three dimensional organizations of lymphoid organ and characteristics of lymphoid cells after chronic infection with yellow head virus (YHV) in Penaeus monodon were investigated. The vascular cast of infected lymphoid organ showed less branching and dramatically shortened terminal capillaries that formed the lymphoid tubules, and only large stumps of these lymphoid tubules remained. This might occur because the terminal ends of the tubules were damaged from YHV infection, as stromal cells and hemocytes in the LT wall were infected and formed foci that could give rise to lymphoid spheroids that broke away from the original lymphoid tubules. Histologically, there was a decrease of PAS-stained connective tissue materials in lymphoid spheroids as well as a decrease of stromal cells as marked by anti-vimentin antibody. This indicated that stromal cells together with type 1 fibers and associated extracellular matrix degenerated in lymphoid spheroids, while type 2 or reticular fibers proliferated and encapsulated the spheroids.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Chemical modifications at the 22-hydroxyl group of ecdysteroids: alternative structural requirements for high moulting activity.

A number of 22-O-alkyl ether and acyl ester derivatives have been prepared and their moulting activity determined, using the Musca assay. It was found that a free 22-hydroxyl group is not an essential structural requirement for an ecdysteroid to exhibit high moulting activity. The activity of a 22-O-substituted ecdysteroid may even be higher than that of the parent compound, providing that the substituent constitutes a functional group that can enhance biological activity. The moulting activity is not sensitive to steric factors at the 22-position. Ecdysteroid without a 22-hydroxyl group may exhibit high moulting activity if a functional group that can enhance activity is present at an appropriate position.     

Larvae and juveniles of two engraulid species, <Emphasis Type="Italic">Thryssa setirostris</Emphasis> and <Emphasis Type="Italic">Thryssa hamiltoni</Emphasis>i,occurring in the surf zone at Trang,southern Thailand

 Engraulid larvae and juveniles collected from the surf zone off the Sikao River, Trang, southern Thailand, in the period between August 1999 and September 2000 were identified as Thryssa setirostris (n = 249, 14.1–25.7 mm SL) and T. hamiltonii (n = 731, 15.2–22.2 mm SL), based on the number of lower gillrakers and maxilla morphology. The two species were easily distinguishable by the melanophore pattern distributed on the anterior part of the striated hindgut, T. setirostris possessing three to five melanophores along the ventral midline and T. hamiltonii lacking such melanophores. The ratios of head length and eye diameter to SL also differed between the species, both being higher in T. hamiltonii. The distribution of the two species was limited to the surf zone outside the river mouth, that region being characterized by a higher salinity than the river and its tributaries.     

Comparative distribution of a putative egg-laying hormone in neural and reproductive tissues of four Decapoda crustaceans

Evidence for the presence of a putative egg-laying (ELH) hormone has been previously described in the black tiger shrimp, Penaeus monodon, so a further investigation was carried out to detect its presence in a range of Decapoda crustaceans prior to a full molecular analysis. The crustaceans were represented by the Australian fresh water yabbie, Cherax destructor, the Australian southern rock lobster, Jasus edwardsii, the snow crab, Chionoecetes opilio, and the blue swimmer crab, Portunus pelagicus. Female cerebral ganglia, ventral nerve cords and gonads were investigated in a comparative study of the distribution of the immunoreactive hormone using immunoenzyme and immunofluorescence techniques. Immunoreactivity was detected in all tissues of interest, and the distribution patterns showed similarity within the four species, as well as that of P. monodon reported in the earlier study. There were minor variations. These data indicate that a putative ELH-like neuropeptide is widespread in crustaceans, and supports its previous identification in a range of molluscs and other invertebrates. Elucidation of the molecular structure of the peptide hormone and its encoding gene, as well as its involvement in spawning behaviour of crustaceans, is now fully under investigation.