The buccopharyngeal mucosa of the turtles (testudines)

Gross and histological examination of all extant families of turtles revealed that the buccopharyngeal mucosa is morphologically highly varied. The tongues of aquatic species have small lingual papillae or lack them entirely, while terrestrial species have tongues with numerous glandular papillae. The pharynx and the esophagus also have papillae in some species. These either facilitate swallowing in which case they are long, pointed, keratinized, and occur commonly in marine turtles, or they are vascular and nonkeratinized, facilitate respiratory gas exchange and are found in the Trionychidae, Dermatemyidae, and Carettochelyidae. The morphology of the buccopharyngeal mucosa of turtles reflects their diet, feeding behavior, habitat, and relationships. Convergence in the morphology of the buccopharyngeal mucosa occurs among families, especially among the Emydidae and other familes of turtles. Intergeneric parallelism is also seen within the Emydidae.     

Intraventricular 5,7-Dihydroxytryptamine Increases Thyrotropin-Releasing Hormone Content in Regions of Rat Brain

Rats received intraventricular (i.v.t.) injections of 5,7-dihydroxytryptamine (5,7-DHT) (100-600 micrograms). Some animals also received intraperitoneal injections of the 5-hydroxytryptamine uptake blocker fluoxetine (FX) (20 mg/kg) or the norepinephrine uptake blocker desmethylimipramine (DMI) (48 mg/kg) 30-90 min prior to i.v.t. 5,7-DHT. Rats were killed between 2 and 35 days following i.v.t. 5,7-DHT, brains were dissected, and regions were assayed for thyrotropin-releasing hormone (TRH) by radioimmunoassay. Dose-dependent increases in TRH content following i.v.t. 5,7-DHT were noted in the brainstem and hippocampus. DMI pretreatment blocked the increase in hippocampal TRH, but not in brainstem TRH. FX pretreatment was ineffective in blocking any increases in TRH content. These results suggest differential regulation of regional TRH content by interactions with specific neurotransmitter systems.     

Rostral pores in turtles

Rostral pores are epidermal invaginations which occur on the internarial region of most chelonians. Representatives of all Recent chelonian families and 67 of the 74 extant genera were examined grossly and/or histologically. Pores are absent only in the families Carettochelyidae, Cheloniidae, and Dermochelyidae. The number and microscopic structure of pores vary markedly within and between taxa. Morphological data suggest that rostral pores could function in mechanoreception. The possible origin and evolution of rostral pores are discussed in the context of other chelonian integumentary speializations.     

Application of a semi-automated vocal fingerprinting approach to monitor Bornean gibbon females in an experimentally fragmented landscape in Sabah,Malaysia

Vocal individuality has been documented in a variety of mammalian species and it has been proposed that this individuality can be used as a vocal fingerprint to monitor individuals. Here we provide and test a classification method using Mel-frequency cepstral coefficients (MFCCs) to extract features from Bornean gibbon female calls. Our method is semi-automated as it requires manual pre-processing to identify and extract calls from the original recordings. We compared two methods of MFCC feature extraction: (1) averaging across all time windows and (2) creating a standardized number of time windows for each call. We analysed 376 calls from 33 gibbon females and, using linear discriminant analysis, found that we were able to improve female identification accuracy from 95.7% with spectrogram features to 98.4% accuracy when averaging MFCCs across time windows, and 98.9% accuracy when using a standardized number of windows. We divided our data randomly into training and test data-sets, and tested the accuracy of support vector machine (SVM) predictions over 100 iterations. We found that we could predict female identity in the test data-set with a 98.8% accuracy. Using SVM on our entire data-set, we were able to predict female identity with 99.5% accuracy (validated by leave-one-out cross-validation). Lastly, we used the method presented here to classify four females recorded during three or more recording seasons using SVM with limited success. We provide evidence that MFCC feature extraction is effective for distinguishing between female Bornean gibbons, and make suggestions for future vocal fingerprinting applications.     

Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans

We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1^Δ18/Δ18), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5′ part of the ITPR1 gene, encompassing exons 1–10, 1–40, and 1–44 in three studied families, underlies SCA15 in humans.     

Development of sex differences in the principal nucleus of the bed nucleus of the stria terminalis of mice: role of Bax-dependent cell death

Neuron number in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is greater in adult male mice than in females. Deletion of the proapoptotic gene, Bax, increases the number of BNSTp cells in adulthood and eliminates the sex difference in cell number. Here, we map the ontogeny of sex differences in nuclear volume and cell number in the BNSTp of neonatal mice, and evaluate the role of cell death in the development of these differences. We find that BNSTp volume and cell number do not differ between male and female wild-type mice on postnatal days P3, P5, or P7. Sex differences emerge after the first postnatal week and both measures are significantly greater in males than in females on P9 and P11. Cell death, assessed by TUNEL staining, was observed in the BNSTp of both sexes from P1-P8. Females had more TUNEL-positive cells than males from approximately P3-P6, with the maximum number of dying cells observed on P5/P6. To test whether the Bax gene is required for sexually dimorphic cell death in the BNSTp, TUNEL cells were counted on P6 in Bax -/- mice and their Bax +/+ siblings. Bax gene deletion nearly abolished TUNEL-positive cells in the BNSTp of both sexes. Together, these findings support the interpretation that the sex difference in BNSTp cell number seen in adulthood is due to Bax-dependent, sexually dimorphic cell death during the first week of life.     

Effect of 17β-estradiol and flavonoids on the regulation of expression of newly identified oestrogen responsive genes in a rat raphe nuclei-derived cell line

Due to the health risks attributed to perimenopausal hormone therapy, phytoestrogens such as flavonoids are receiving widespread attention to help alleviate menopausal symptoms, including hormone-driven mood disorders. Based on our previous reporter gene study regarding their transactivational activity in raphe nuclei cells from a brain region involved in regulation of mood disturbances, we herein study their effects on the regulation of expression of 17β-estradiol (E2)-regulated genes. DNA microarray was used to globally assess E2-induced gene expression in RNDA cells, a rat raphe nuclei-derived cellular model expressing oestrogen receptor β. Out of 212 regulated genes, six were selected for verification and as endpoints for the effect of flavonoids on the regulation of mRNA expression in proliferating as well as differentiating RNDA cells. Under proliferative conditions, E2 up-regulated mRNA expression of Cml-5, Sox-18 and Krt-19. Similar effects were observed in response to 8-prenylnaringenin (8-PN), genistein (GEN), daidzein (DAI) and equol (EQ). In line with E2, mRNA expression of Nefm and Zdhhc-2 was down-regulated following 8-PN, GEN, DAI, EQ and naringenin treatment. No regulation was observed on Slc6a4 mRNA expression in response to E2 or the flavonoids in proliferating RNDA cells. When cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards E2 and the tested flavonoids were noticed. These expression studies additionally highlighted some of the genes as markers for RNDA cellular differentiation. RNDA cells should prove useful to elucidate molecular and cellular mechanisms of exogenous oestrogen receptor ligands with neural cell populations.     

High speed detection of circulating tumor cells

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.     

Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed “phenotypic” FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4–specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)–treated cells. We found that SUV39H1–mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1γ and cohesin are co-recruited to D4Z4 in an H3K9me3–dependent and cell type–specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type–specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1γ/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.     

Synthesis and antibacterial activity of novel bifunctional macrolides

We report the discovery of a novel class of macrolide antibiotics that have improved antibacterial activity against Ery-resistant organisms.