Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n = 5) or 150% (Fat; n = 6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater (P < 0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater (P < 0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected (P = 0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) β tended (P = 0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-β was greater in female fetuses than male fetuses from Control ewes (P = 0.05). Although amount of ER-β mRNA did not differ among male fetuses (P = 0.7), amounts tended to be less (P = 0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ (P ≥ 0.8) from male fetuses. Hypothalamic ER-α mRNA tended (P = 0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced (P = 0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended (P = 0.06) to be greater in male fetuses than female fetuses and tended to be less (P = 0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction (P = 0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ (P ≥ 0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-α, and decrease amount of ER-β in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.     

Design and characterization of a compact dual channel virus counter.

BACKGROUND: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. METHODS: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. RESULTS: The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. CONCLUSIONS: The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).     

The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region

The gene-silencing effect of short interfering RNA (siRNA) is known to vary strongly with the targeted position of the mRNA. A number of hypotheses have been suggested to explain this phenomenon. We would like to test if this positional effect is mainly due to the secondary structure of the mRNA at the target site. We proposed that this structural factor can be characterized by a single parameter called "the hydrogen bond (H-b) index," which represents the average number of hydrogen bonds formed between nucleotides in the target region and the rest of the mRNA. This index can be determined using a computational approach. We tested the correlation between the H-b index and the gene-silencing effects on three genes (Bcl-2, hTF, and cyclin B1) using a variety of siRNAs. We found that the gene-silencing effect is inversely dependent on the H-b index, indicating that the local mRNA structure at the targeted site is the main cause of the positional effect. Based on this finding, we suggest that the H-b index can be a useful guideline for future siRNA design.     

Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is widespread in cultured Penaeus monodon and P. vannamei in Thailand. It causes runt-deformity syndrome that is characterized by physical abnormalities and stunted growth in P. vannamei, but causes no apparent disease in P. monodon. In both species, the virus may produce Cowdry Type A inclusions in tissues of ectodermal and mesodermal origin, but these are common in P. vannamei and rare in P. monodon. The virus can be more easily detected in both species by IHHNV-specific PCR primers. By in situ hybridization (ISH) using specific IHHNV probes, fixed phagocytes associated with myocardial cells tended to show strong positive reactions in both shrimp species. Ovarian and neural tissue (neurons in the nerve ganglia and glial cells in the nerve cord) were ISH positive for IHHNV only in P. vannamei. By transmission electron microscopy, necrotic cells were found in the gills of IHHNV-infected P. vannamei, while paracrystalline arrays of virions and apoptotic cells rather than necrotic cells were found in the lymphoid organ of IHHNV-infected P. monodon. Thus, it is possible that apoptosis in P. monodon contributes to the absence of clinical disease from IHHNV. These findings reveal different responses to IHHNV infection by the 2 shrimp species. A curious feature of IHHNV infection in P. monodon was inconsistency in the comparative viral load amongst tissues of different specimens, as detected by both ISH and real-time PCR. This inconsistency in apparent tissue preference and the reasons for different cellular responses between the 2 shrimp species remain unexplained.     

(3R)-4-[(3R)-3-Amino-4-(2,4,5-trifluorophenyl)butanoyl]-3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one, a selective dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Replacement of the triazolopiperazine ring of sitagliptin (DPP-4 IC(50)=18nM) with 3-(2,2,2-trifluoroethyl)-1,4-diazepan-2-one gave dipeptidyl peptidase IV (DPP-4) inhibitor 1 which is potent (DPP-4 IC(50)=2.6nM), selective, and efficacious in an oral glucose tolerance test in mice. It was selected for extensive preclinical development as a potential back-up candidate to sitagliptin.     

Reconstructing asymmetrical reproductive character displacement in a periodical cicada contact zone

Selection against costly reproductive interactions can lead to reproductive character displacement (RCD). We use information from patterns of displacement and inferences about predisplacement character states to investigate causes of RCD in periodical cicadas. The 13-year periodical cicada Magicicada neotredecim exhibits RCD and strong reproductive isolation in sympatry with a closely related 13-year species, Magicicada tredecim. Displacement is asymmetrical, because no corresponding pattern of character displacement exists within M. tredecim. Results from playback and hybridization experiments strongly suggest that sexual interactions between members of these species were possible at initial contact. Given these patterns, we evaluate potential sources of selection for displacement. One possible source is 'acoustical interference', or mate-location inefficiencies caused by the presence of heterospecifics. Acoustical interference combined with the species-specificity of song pitch and preference appears to predict the observed asymmetrical pattern of RCD in Magicicada. However, acoustical interference does not appear to be a complete explanation for displacement in Magicicada, because our experiments suggest a significant potential for direct sexual interactions between these species before displacement. Another possible source of selection for displacement is hybrid failure. We evaluate the attractiveness of inferred hybrid mating signals, and we examine the viability of hybrid eggs. Neither of these shows strong evidence of hybrid inferiority. We conclude by presenting a model of hybrid failure related to life cycle differences in Magicicada.     

Field studies and the IACUC: protocol review, oversight, and occupational health and safety considerations

Field studies involving animals, like other institutional research endeavors, are regulated and evaluated by an IACUC to insure that the protocols employed in those studies are consistent with the most humane care and use of animals. Unlike many other on-site research projects, however, field studies present unique logistical and other challenges to IACUC evaluation. The authors explore many of these challenges, including the special protocol considerations and idiosyncratic occupational health and safety concerns characteristic of field-study reviews.     

Species-Independent Inhibition of Abnormal Prion Protein (PrP) Formation by a Peptide Containing a Conserved PrP Sequence

Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.     

HLS5, a novel RBCC (ring finger, B box, coiled-coil) family member isolated from a hemopoietic lineage switch, is a candidate tumor suppressor

Hemopoietic cells, apparently committed to one lineage, can be reprogrammed to display the phenotype of another lineage. The J2E erythroleukemic cell line has on rare occasions developed the features of monocytic cells. Subtractive hybridization was used in an attempt to identify genes that were up-regulated during this erythroid to myeloid transition. We report here on the isolation of hemopoietic lineage switch 5 (Hls5), a gene expressed by the monocytoid variant cells, but not the parental J2E cells. Hls5 is a novel member of the RBCC (Ring finger, B box, coiled-coil) family of genes, which includes Pml, Herf1, Tif-1alpha, and Rfp. Hls5 was expressed in a wide range of adult tissues; however, at different stages during embryogenesis, Hls5 was detected in the branchial arches, spinal cord, dorsal root ganglia, limb buds, and brain. The protein was present in cytoplasmic granules and punctate nuclear bodies. Isolation of the human cDNA and genomic DNA revealed that the gene was located on chromosome 8p21, a region implicated in numerous leukemias and solid tumors. Enforced expression of Hls5 in HeLa cells inhibited cell growth, clonogenicity, and tumorigenicity. It is conceivable that HLS5 is one of the tumor suppressor genes thought to reside at the 8p21 locus.