Comparison of marker types and map assumptions using Markov chain Monte Carlo-based linkage analysis of COGA data

We performed multipoint linkage analysis of the electrophysiological trait ECB21 on chromosome 4 in the full pedigrees provided by the Collaborative Study on the Genetics of Alcoholism (COGA). Three Markov chain Monte Carlo (MCMC)-based approaches were applied to the provided and re-estimated genetic maps and to five different marker panels consisting of microsatellite (STRP) and/or SNP markers at various densities. We found evidence of linkage near the GABRB1 STRP using all methods, maps, and marker panels. Difficulties encountered with SNP panels included convergence problems and demanding computations.     

Downregulation of cGMP-dependent protein kinase-1 activity in the corpus cavernosum smooth muscle of diabetic rabbits

Increased guanosine 3',5'-cyclic monophosphate (cGMP), induced by nitric oxide release, is crucial for corpus cavernosum smooth muscle (CCSM) relaxation within the penis. This CCSM relaxation (necessary for penile erection) is impaired in men with erectile dysfunction (ED), especially those men with diabetes. One of the effector proteins for cGMP is cGMP-dependent protein kinase-1 (PKG-1). PKG-1 knockout mice exhibit detrusor overactivity (Am J Physiol Regul Integr Comp Physiol 279: R1112-R1120, 2000) and, more relevant to this study, ED (Proc Natl Acad Sci USA 97: 2349-2354, 2000), suggesting an in vivo role for PKG-1 in urogenital smooth muscle relaxation. In the current study, using normal rabbit CCSM, Western blot analysis revealed high expression of PKG-1 at levels almost equivalent to aorta (previously shown to have high PKG-1 expression) and that the two known alternatively spliced isoforms of PKG-1 (alpha and beta) are expressed in nearly equal amounts in the CCSM. However, in response to alloxan-induced diabetes, there was a decrease in expression of both PKG-1 isoforms at the mRNA and protein levels as determined by real-time RT-PCR and Western blotting, respectively, but with the PKG-1alpha isoform expression decreased to a greater extent. Moreover, diabetes was associated with significantly decreased PKG-1 activity of CCSM in vitro, correlating with decreased CCSM relaxation. Immunofluorescence microscopy revealed a diabetes-associated decrease in PKG-1 in the CCSM cells. In conclusion, our results demonstrate for the first time a significant downregulation of PKG-1 expression associated with decreased PKG-1 activity in the CCSM in response to diabetes. Furthermore, these results suggest a mechanistic basis for the decreased efficacy of phosphodiesterase V inhibitors in treating diabetic patients with ED.     

Landscape genetics of an endangered lemur (Propithecus tattersalli) within its entire fragmented range

Habitat fragmentation may strongly reduce individuals’ dispersal among resource patches and hence influence population distribution and persistence. We studied the impact of landscape heterogeneity on the dispersal of the golden‐crowned sifaka (Propithecus tattersalli), an endangered social lemur species living in a restricted and highly fragmented landscape. We combined spatial analysis and population genetics methods to describe population units and identify the environmental factors which best predict the rates and patterns of genetic differentiation within and between populations. We used non‐invasive methods to genotype 230 individuals at 13 microsatellites in all the main forest fragments of its entire distribution area. Our analyses suggest that the Manankolana River and geographical distance are the primary structuring factors, while a national road crossing the region does not seem to impede gene flow. Altogether, our results are in agreement with a limited influence of forest habitat connectivity on gene flow patterns (except for North of the species’ range), suggesting that dispersal is still possible today among most forest patches for this species. Within forest patches, we find that dispersal is mainly among neighbouring social groups, hence confirming previous behavioural observations.     

The histological structure of the calcified lung of the fossil coelacanth Axelrodichthys araripensis (Actinistia: Mawsoniidae)

Abstract: The palaeohistological study of the calcified internal organ of Axelrodichthys araripensis Maisey, 1986, a coelacanthiform from the Lower Cretaceous of Brazil (Crato (Aptian) and Santana (Albian) formations of the Araripe Basin), shows that the walls of this organ consist of osseous blades of variable thickness separated from each other by the matrix. This indicates that, in the living individuals, the walls were reinforced by ossified plates, probably separated by conjunctive tissue. This calcified sheath present in Axelrodichthys, as well as in other fossil coelacanths, lies in ventral position relative to the gut and its single anterior opening is located under the opercle, suggesting a direct connection with the pharynx or the oesophagus. The calcified organ of Axelrodichthys, like that of other fossil coelacanths, is here regarded as an ‘ossified lung’ and compared with the ‘fatty lung’ of the extant coelacanth Latimeria. The reinforcement of the pulmonary walls by the overlying osseous blades could be interpreted as a means of adapting volumetric changes in the manner of bellows, a necessary function for ventilation in pulmonary respiration. Other functional hypotheses such as hydrostatic and/or acoustic functions are also discussed.     

Low-resolution structural studies of human Stanniocalcin-1

Background  

Stanniocalcins (STCs) represent small glycoprotein hormones, found in all vertebrates, which have been functionally implicated in Calcium homeostasis. However, recent data from mammalian systems indicated that they may be also involved in embryogenesis, tumorigenesis and in the context of the latter especially in angiogenesis. Human STC1 is a 247 amino acids protein with a predicted molecular mass of 27 kDa, but preliminary data suggested its di- or multimerization. The latter in conjunction with alternative splicing and/or post-translational modification gives rise to forms described as STC₅₀ and "big STC", which molecular weights range from 56 to 135 kDa.     

1H NMR metabolomics reveals increased glutaminolysis upon overexpression of NSD3s or Pdp3 in Saccharomyces cerevisiae

NSD3s, the proline-tryptophan-tryptophan-proline (PWWP) domain-containing, short isoform of the human oncoprotein NSD3, displays high transforming properties. Overexpression of human NSD3s or the yeast protein Pdp3 in Saccharomyces cerevisiae induces similar metabolic changes, including increased growth rate and sensitivity to oxidative stress, accompanied by decreased oxygen consumption. Here, we set out to elucidate the biochemical pathways leading to the observed metabolic phenotype by analyzing the alterations in yeast metabolome in response to NSD3s or Pdp3 overexpression using ¹H nuclear magnetic resonance (NMR) metabolomics. We observed an increase in aspartate and alanine, together with a decrease in arginine levels, on overexpression of NSD3s or Pdp3, suggesting an increase in the rate of glutaminolysis. In addition, certain metabolites, including glutamate, valine, and phosphocholine were either NSD3s or Pdp3 specific, indicating that additional metabolic pathways are adapted in a protein-dependent manner. The observation that certain metabolic pathways are differentially regulated by NSD3s and Pdp3 suggests that, despite the structural similarity between their PWWP domains, the two proteins act by unique mechanisms and may recruit different downstream signaling complexes. This study establishes for the first time a functional link between the human oncoprotein NSD3s and cancer metabolic reprogramming.     

Increased corpus cavernosum smooth muscle tone associated with partial bladder outlet obstruction is mediated via Rho-kinase

Numerous studies have now demonstrated that lower urinary tract symptoms (LUTS) are associated with erectile dysfunction (ED) in men independent of age or general health. Because one-third of men over the age of 50 will develop LUTS and a recent study showed ED in 62% of patients presenting for LUTS, the importance of determining the mechanistic link between these two pathologies is clear. Using a rabbit model of partial bladder outlet obstruction (PBOO), a primary cause of LUTS, we have identified an increased basal corpus cavernosum smooth muscle (CCSM) tone associated with an elevated level of smooth muscle myosin (SMM) phosphorylation in PBOO compared with sham-operated control rabbits (sham). Results from in vitro kinase and phosphatase assays using purified smooth muscle myosin showed increased kinase and decreased phosphatase activities in cellular extracts from corpora cavernosa isolated from PBOO compared with sham rabbits. Increased Rho-kinase expression in the CCSM of PBOO rabbits was suggested by the observations that Rho-kinase inhibitors attenuated the increased kinase activity and were less effective in relaxing CCSM strips from PBOO vs. sham rabbits. This hypothesis was then confirmed by RT-PCR and Western blotting, which demonstrated increased expression of both isoforms of Rho-kinase (ROKalpha and ROKbeta). Increased SMM basal phosphorylation (necessary for SM contraction) in the CCSM of PBOO rabbits, mediated via an increase in Rho-kinase expression/activity, would be expected to make the CCSM more difficult to relax (necessary for erection), which suggests that the RhoA/Rho-kinase pathway as being involved in the mechanism for LUTS-associated ED.     

Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2‐1 knockout and asn2‐2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO₂ assimilation was not significantly different between lines under both 21 and 2% O₂. ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered ¹⁵N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant.     

Alteration in expression of myosin isoforms in detrusor smooth muscle following bladder outlet obstruction

Partial urinary bladder outlet obstruction (PBOO) in men, secondary to benign prostatic hyperplasia, induces detrusor smooth muscle (DSM) hypertrophy. However, despite DSM hypertrophy, some bladders become severely dysfunctional (decompensated). Using a rabbit model of PBOO, we found that although DSM from sham-operated bladders expressed nearly 100% of both the smooth muscle myosin heavy chain isoform SM-B and essential light chain isoform LC_17a, DSM from severely dysfunctional bladders expressed as much as 75% SM-A and 40% LC_17b (both associated with decreased maximum velocity of shortening). DSM from dysfunctional bladder also exhibited tonic-type contractions, characterized by slow force generation and high force maintenance. Immunofluorescence microscopy showed that decreased SM-B expression in dysfunctional bladders was not due to generation of a new cell population lacking SM-B. Metabolic cage monitoring revealed decreased void volume and increased voiding frequency correlated with overexpression of SM-A and LC_17b. Myosin isoform expression and bladder function returned toward normal upon removal of the obstruction, indicating that the levels of expression of these isoforms are markers of the PBOO-induced dysfunctional bladders. bladder remodeling; bladder dysfunction; SM-A; LC_17a; benign prostatic hyperplasia