Stylosanthes sp. aff.S. scabra: a putative diploid progenitor ofStylosanthes scabra (Fabaceae)

Stylosanthes sp. aff.S. scabra is an undescribed taxon showing affinities with the allotetraploid speciesS. scabra, but distinct in a number of attibutes. Several collections show potential as forage for clay soils in northern Australia. Twelve accessions have been analysed using STS (sequence-tagged-sites) as genetic markers, and they all displayed STS phenotypes of typical diploid species. Taking into account their morphological similarities, the STS analysis provides strong evidence thatStylosanthes sp. aff.S. scabra might be a diploid progenitor of the allotetraploidS. scabra. This speculation was supported by cytological examinations. Somatic chromosome numbers of two of these accessions were counted and both were found to be diploid (2n = 20). The level of polymorphism among the 12Stylosanthes sp. aff.S. scabra accessions, estimated using randomly amplified polymorphic DNA (RAPD) as markers, was 7.8%, and the dissimilarity value betweenStylosanthes sp. aff.S. scabra andS. viscosa (the other putative progenitor ofS. scabra) was 89%.     

Organization and functions of cells responsive to faces in the temporal cortex.

Cells selectively responsive to the face have been found in several visual sub-areas of temporal cortex in the macaque brain. These include the lateral and ventral surfaces of inferior temporal cortex and the upper bank, lower bank and fundus of the superior temporal sulcus (STS). Cells in the different regions may contribute in different ways to the processing of the facial image. Within the upper bank of the STS different populations of cells are selective for different views of the face and head. These cells occur in functionally discrete patches (3-5 mm across) within the STS cortex. Studies of output connections from the STS also reveal a modular anatomical organization of repeating 3-5 mm patches connected to the parietal cortex, an area thought to be involved in spatial awareness and in the control of attention. The properties of some cells suggest a role in the discrimination of heads from other objects, and in the recognition of familiar individuals. The selectivity for view suggests that the neural operations underlying face or head recognition rely on parallel analyses of different characteristic views of the head, the outputs of these view-specific analyses being subsequently combined to support view-independent (object-centred) recognition. An alternative functional interpretation of the sensitivity to head view is that the cells enable an analysis of 'social attention', i.e. they signal where other individuals are directing their attention. A cell maximally responsive to the left profile thus provides a signal that the attention (of another individual) is directed to the observer's left. Such information is useful for analysing social interactions between other individuals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a yeast artificial chromosome (YAC) contig around the human steroid sulfatase gene.

The region surrounding the steroid sulfatase (STS) locus on Xp22.3 is of particular interest since it represents a deletion hot spot, shares homology with the proximal long arm of the Y chromosome (Yq11.2), and contains genes for several well-described X-linked disorders. Here we describe yeast artificial chromosomes (YACs) covering 450 kb around the STS gene. Eight YAC clones were isolated from a human YAC library. Their STS exon content was determined and the overlap of the clones characterized. Two of the YAC clones were found to contain the entire STS gene. The most proximal and the most distal ends of the YAC contig were cloned but neither of them crossed the breakpoints in any of the previously described patients with entire STS gene deletions. This is consistent with deletions larger than 500 kb in all these patients. One of the YAC clones was found to contain sequences from the STS pseudogene on Yq11.2. Two anonymous DNA sequences, GMGXY19 and GMGXY3, previously mapped in the vicinity of the STS locus, were found within the YAC contig and their assignment with respect to the STS locus was thus possible. This contig is useful for the overlap cloning of the Xp22.3 region and for reverse genetic strategies for the isolation of disease genes in the region. Furthermore, it may provide insight into the molecular mechanisms of deletion and translocation events on Xp22.3 and in the evolution of sex chromosomes.     

Promoting collaborations between biomedical scholars in the U.S. and sub-Saharan Africa

The premise of this piece is that a priority of international health should be to increase the number of investigators in the US and other developed countries who engage in research and other kinds of scholarly work in underdeveloped parts of the world, particularly sub-Saharan Africa where the overall disease burden is the highest and the gap in biomedical research infrastructure is the widest. The author's aim is to encourage medical students, resident doctors, and medical school faculty to devote a part of their career to teach, acquire clinical skills, or participate in research with health professionals at teaching hospitals in Africa. After briefly describing the thinking that led the author to Nigeria 30 years ago to teach and study biochemical aspects of health problems in rural and urban areas, he discusses some of the factors one needs to consider before entering into an international partnership, including identifying the right foreign collaborators, selecting a suitable research site, setting realistic goals, learning the local culture and indigenous language, and defining a theme for your program. Lastly, the piece points out potential pitfalls and problems that are often overlooked or underestimated in the early phases of planning an international partnership, including lukewarm institutional support at home, inflexible institutional review boards, dominance of the program by the US partner, maintaining continuity, and striking the right balance between scholarly work and humanitarian efforts. My hope is that US students and faculty in the health professions who read this piece will be stimulated and encouraged to consider how they might integrate into their curriculum or academic life visits lasting several months or more each year during which they would teach or train others or engage in research at a teaching hospital in some country in Africa.     

Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution

Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to simulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20–25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same K_m for the substrate 4-coumaroyl-CoA but a lower V_max than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.Abbreviations AA amino acid - CHS chalcone synthase - STS stilbene synthase Correspondence to: J. Schröder 0592The data are discussed on the level of the presently available CHS and STS sequences although many were published after beginning the experiments several years ago. The new information changed the CHS consensus in some details but otherwise confirmed the deductions on the potential significance of amino acid differences between CHS and STS     

The nature of inhibition of steroid sulphatase activity by tibolone and its metabolites

Tibolone is used for hormone replacement therapy and acts in a tissue-specific manner being oestrogenic on CNS and bone but not on breast tissues or endometrium. The ability of tibolone and its metabolites to inhibit steroid sulphatase (STS) activity has a crucial role in regulating its tissue-specific effects. In this study, we have examined the ability of tibolone and its non-sulphated and sulphated metabolites to inhibit STS activity in different enzyme preparations and in intact cells. For this, we have used an 'extracellular' method, which measures the amount of product released into culture medium, and an 'intracellular' method, which assesses the extent of product formation within cells. In addition, the nature by which tibolone and some of its metabolites inhibit STS activity was investigated using intact cells and an enzyme kinetic method. In MCF-7 and T47D breast cancer cells and JEG-3 choriocarcinoma cells, which have high STS activity, tibolone and its metabolites were relatively potent inhibitors of STS activity (33-57% inhibition at 10 microM) using the extracellular assay method. In HOS-TE-85 osteoblast-like cells, tibolone and its Delta-4 metabolite were relatively inactive whereas the 3alpha/3beta-hydroxy metabolites and their sulphated conjugates inhibited activity by 39-55%. When STS activity was assessed in HOS-TE-85 cells using an 'intracellular' method tibolone and its 3beta-hydroxy metabolite were inactive. Pre-treatment of breast cancer cells and JEG-3 cells, and removal of drugs prior to assaying for STS activity, revealed that in these cells tibolone and its metabolites were acting mainly as reversible inhibitors. This finding was confirmed in an enzyme kinetic study to measure concentration-dependent STS inhibition. In HOS-TE-85 cells, pre-treatment of cells and removal of compounds before assaying for remaining STS activity indicated that some tibolone metabolites appeared to stimulate STS activity. Possible mechanisms by which this might occur are discussed but, if confirmed, this could contribute to the positive oestrogenic effects that tibolone has on bone.     

Steroid sulfatase gene in XX males.

The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)     

AFLP-derived STS markers for the identification of sex in Asparagus officinalis L.

For a simple, rapid and PCR-based screening of sex in the cultivated asparagus (Asparagus officinalis L.), we developed five STS markers from previously mapped, low-copy, sex-linked AFLP markers. A male/female PCR assay was feasible with these STS markers either by direct amplification or by digestion with restriction enzymes. Similar to the AFLP markers from which they were derived, STS4150.1, STS4150.2, STS4150.3 and STS3156 did not give recombinants in five different populations. STS3660 could be scored codominantly, enabling the differentiation of XY from YY males in the screened F₂ mapping population. The use of the sex-linked STS markers should allow early identification of sex, thus accelerating the breeding process for new asparagus varieties. Further, 10 additional AFLP markers obtained with PstI/MseI primer combinations have been mapped on the L5 chromosome, bringing the total number of known AFLP and STS markers flanking the sex locus to 24. These markers can be utilized for fine mapping of the sex gene in asparagus, which will pave the way for a map-based cloning approach. Received: 31 May 1999 / Accepted: 22 June 1999     

Cross-reaction of chalcone synthase and stilbene synthase overexpressed in Escherichia coli.

Chalcone synthase (CHS) and stilbene synthase (STS) are related plant polyketide synthases belonging to the CHS superfamily. CHS and STS catalyze common condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA but different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Using purified Pueraria lobata CHS and Arachis hypogaea STS overexpressed in Escherichia coli, bisnoryangonin (BNY, the derailed lactone after two condensations) and p-coumaroyltriacetic acid lactone (the derailed lactone after three condensations) were detected from the reaction products. More importantly, we found a cross-reaction between CHS and STS, i.e. resveratrol production by CHS (2.7-4.2% of naringenin) and naringenin production by STS (1.4-2.3% of resveratrol), possibly due to the conformational flexibility of their active sites.     

Evidence for a dosage effect at the X-linked steroid sulfatase locus in human tissues.

Steroid sulfatase (STS) is an X-linked enzyme whose locus escapes X inactivation in human somatic cells. STS activity was determined in human fibroblasts varying in X-chromosome number from one to four. Greater STS activity was detected in 2X cell strains when compared to 1X cell strains; however, increased STS activity was not found in 3 and 4X strains as compared with the 2X strains. Greater STS activity was also observed in female hair follicles when compared with male hair follicles. These data provide evidence of a gene dosage effect at the STS locus in human tissues.     

Fine mapping of the distal short arm of the human X chromosome using X/Y translocations.

The loci for steroid sulfatase (STS), the deficiency of which causes X-linked ichthyosis, the cell surface antigen 12E7 (MIC2X), and the blood group antigen Xg (Xg) have been mapped to Xp22.3. These loci are of particular interest since they do not appear to undergo X-chromosome inactivation. In an attempt to establish the relative order of STS and MIC2X, fibroblasts from carriers of four different X/Y translocations and an X/10 translocation were obtained and fused with mouse cell lines deficient in hypoxanthine phosphoribosyltransferase. The breakpoints on the X chromosome in these five translocations are in Xp22. Several independent clones from each fusion were isolated in HAT medium. The clones were examined cytogenetically, and in each case at least two independent clones were identified that have an active X/Y or X/10 translocation chromosome in the absence of other X or Y material. These clones were then tested for STS and 12E7 expression. In two of the X/Y translocations, the markers, STS and 12E7, were both absent. In the X/10 and a third X/Y translocation, both markers were retained. In each of three clones containing the fourth X/Y translocation, STS activity was retained but 12E7 antigenicity was lost. Assuming that this is a simple translocation and does not represent a more complex rearrangement, these results suggest that MIC2X is distal to STS.     

Tissue-specific expression of human arylsulfatase-C isozymes and steroid sulfatase.

Steroid sulfatase (STS; E.C.3.1.6.2), which acts on 3-hydroxysteroid sulfates, and arylsulfatase-C (ARC; E.C.3.1.6.1), assayed with aromatic artificial substrates, are both membrane-bound, microsomal enzymes with alkaline pH optima. Although they copurify during preparation and their gene loci are mapped to the short arm of the human X chromosome where they appear to have escaped from X inactivation, it has not been settled whether STS and ARC are the same enzyme or not. Recent work from our laboratory has shown that ARC exists in two electrophoretically distinct forms in human fibroblasts. We now report that these two forms--the faster migrating (F) and more slowly migrating (S)--occur in human tissues. Each of 11 human tissue types from 10 subjects showed a consistent pattern of ARC isozymes. Thyroid, heart, spleen, skeletal muscle, and adrenal tissue mainly had the S form. In contrast, kidney, liver, and pancreas tissue had mainly the F form, while gonadal, lung, and intestinal tissue had both the S and the F forms. The question of escape of their gene locus from X-chromosome inactivation was examined by comparing the specific activities of ARC and STS in male-derived vis-à-vis female-derived tissues. The majority of the tissues did not show any significant difference in these activities between the sexes, the exceptions being heart muscle, gonadal, and kidney tissue. None showed the 1:2 ratio between male- and female-derived tissues expected of a locus that had escaped X inactivation. The question of identity between ARC and STS was examined by comparing the ratios of their activities in these tissue types: if the enzymes were identical, the ratios of their activities should have remained constant across the different tissue types. It was thus shown that ARC activity varied by as much as 100-fold, depending on the ARC isozymic pattern of the tissue. STS, measured as estrone sulfatase and dehydroepiandrosterone sulfatase, did not show similar variations. This provides further evidence that ARC activity is not necessarily identical to that of STS.     

Development of STS markers for Verticillium wilt resistance in cotton based on RGA–AFLP analysis

Verticillium wilt (VW) is one of the most destructive diseases of cotton that decreases yield and quality. Identification of resistance gene analogs (RGAs) can provide potential candidate gene markers for marker-assisted selection (MAS) for VW resistance and cloning of VW resistance genes. The objective of this study was to convert RGA-targeted RGA–AFLP (amplified fragment length polymorphism) markers into sequence-tagged site (STS) markers. A total of 54 RGA–AFLP markers, including 28 from a backcross inbred line (BIL) population and 26 from a recombinant inbred line (RIL) population, were cloned and sequenced. Of the resulted 86 unique sequences, the majority were found to be homologous to genes, some of which showed similarity to genes encoding resistance-related products. A total of 163 STS primer pairs were designed and screened in the BIL population, 72 of which were also screened in the RIL population, resulting in 21 polymorphic STS markers in the BIL population and seven in the RIL population. Twelve STS markers were mapped onto eight chromosomes or linkage groups in the BIL population, six of which were mapped on the same chromosomes with their original RGA–AFLP markers including two STS markers on c4 flanking two VW resistance quantitative trait loci. These STS markers will be useful in MAS in breeding cotton for VW resistance. This study represents the first attempt to successfully convert RGA–AFLP markers into STS for mapping resistance genes in cotton.     

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.     

X-inactivation of the Sts locus in the mouse: an anomaly of the dosage compensation mechanism

The behaviour of the X- and Y-borne Sts locus has been studied in male and female mice. There was considerable heterogeneity in STS activity between inbred mouse strains, with a four fold difference in activity between the highest (101/H) and lowest (Ju/Ct) activity strains, which can be interpreted in terms of allelic differences. In all inbred strains male STS levels were higher than those of female STS levels and in the majority of strains tested male STS levels were nearly twice as high as female levels. Reciprocal crosses between C3H/HeH and the STS-deficient substrain, C3H/An, demonstrated that activities of the X- and Y-borne genes in males are essentially the same and this suggested that the lower STS level in females derives from X-inactivation of the locus. The possibility that hormonal differences could instead be responsible for the lower activity in females was ruled out by the findings that (a) castration of males did not reduce their STS levels and (b) sex-reversed males, X/X Sxr, had STS levels typical of females. Final proof that the mouse Sts locus can be subject to the X-inactivation process was provided by the observation that XX females had STS levels that were only slightly (20%) higher than those of XO females. The difference may indicate incomplete inactivation of the locus. Linkage data verifying the location of Sts on the distal end of the X chromosome are provided.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sino-U.S. partnerships in research,education, and patient care: The experience of the University of Pittsburgh and UPMC

In 2011, the University of Pittsburgh School of Medicine (UPSOM) and Tsinghua University formed a partnership to further the education of Tsinghua medical students. These students come to UPSOM as visiting research scholars for two years of their eight-year MD curriculum. During this time, the students, who have completed four years at Tsinghua, work full-time in medical school laboratories and research programs of their choice, essentially functioning as graduate students. In their first two months in Pittsburgh, the scholars have a one-week orientation to biomedical research, followed by two-week rotations in four labs selected on the basis of the scholars’ scientific interests, after which they choose one of these labs for the remainder of the two years. Selected labs may be in basic science departments, basic science divisions of clinical departments, or specialized centers that focus on approaches like simulation and modeling. The Tsinghua students also have a brief exposure to clinical medicine. UPSOM has also formed a similar partnership with Central South University Xiangya School of Medicine in Changsha, Hunan Province. The Xiangya students come to UPSOM for two years of research training after their sixth year and, thus, unlike the Tsinghua students, have already completed their clinical rotations. UPSOM faculty members have also paved the way for UPMC (University of Pittsburgh Medical Center), UPSOM’s clinical partner, to engage with clinical centers in China. Major relationships involving advisory, training, managerial, and/or equity roles exist with Xiangya International Medical Center, KingMED Diagnostics, First Chengmei Medical Industry Group, and Macare Women’s Hospital. Both UPSOM and UPMC are actively exploring other clinical and academic opportunities in China.     

Determinants of sit-to-stand capability in the motor impaired elderly.

Among the healthy elderly, sit-to-stand (STS) movement largely depends on: (a) trunk bending momentum, (b) centre of gravity (CG) position before the body rises and (c) lower limb extensor muscle strength. Because determining whether (c) improvement would affect STS capability in the motor impaired elderly (MIE) has been recommended, we studied the relative importance of (a), (b) and (c) in determining a successful fast STS movement comparing the healthy elderly with MIE with orthopaedic disorders studied before and after a rehabilitation program. Force platform was used to measure body's posture and kinematics during a STS test and therefore to assess (a), (b) and maximum vertical velocity (VVpeak), assumed as outcome measurement. Knee extensor maximal isometric voluntary contraction normalized by body mass (nMVC) was an indicator of (c). A multiple regression model was built to predict VVpeak from the three determinants of STS movement. In both groups, the model significantly determined VVpeak, with (a) and (c) being significant predictors of VVpeak and (a) being the major predictor. Rehabilitation was effective in improving nMVC. This process resulted in a change of the relative importance of (a) and (c), strength becoming the major predictor of VVpeak. In conclusion the present study demonstrates that a rehabilitative intervention aimed at increasing strength is effective in improving STS capability in MIE.     

N-Acyl arylsulfonamides as novel, reversible inhibitors of human steroid sulfatase

Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of novel chemotypes of STS inhibitors, we had previously identified nortropinyl-arylsulfonylureas 1; however, while these compounds were good inhibitors of purified STS (lowest K(i)=76 nM), they showed only weak inhibition of STS activity in cells (lowest IC(50) around 2 microM). Extended structure-activity relationship studies involving modification of the phenylacetyl side chain and replacement of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piperidine-4-carbonyl)-benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC(50)=270 nM).     

Cytological research on the action of sodium thiosulfate on the process of induced acute pancreatitis in rats

The influence of sodium thiosulfate (STS) on the process of experimental acute pancreatitis (EAP) in rats was studied by cytomorphology, morphometry, autoradiography and cytophotometry. The influence was shown to vary at different stages of disease development. At the first stage ("primary effect" state) STS leads to the increase in the stability of exocrine pancreacytes (EP) against the toxins and to the decrease in the activity of proteases formed during necrobiosis. This results in the drop of the number of degrading EP and of the degree of inter- and intracellular oedema, and brings about shifts towards the normal values of the nucleus cytoplasm shapes, the nucleus/cytoplasm ratio, the EP population structure and their RNA and protein content. At the second stage STS stimulates DNA synthesis in EP and their proliferation leading to accelerated restoration of the number of viable cells. STS also stimulates the regeneration process hence preventing pancreatitis from passage to its chronic form. The mechanism of STS action of EP functions in normal cells and during pathogenesis is discussed.