Shared role for differentially methylated domains of imprinted genes

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.     

A new enzymatic activity from elicitor‐treated pear cell cultures converting trans‐cinnamic acid to benzaldehyde

Cell cultures of Asian pear (Pyrus pyrifolia) are known to produce benzoate‐derived biphenyl phytoalexins upon elicitor treatment. Although the downstream pathway for biphenyl phytoalexin biosynthesis is almost known, the upstream route of benzoic acid biosynthesis in pear has not been completely elucidated. In the present work, we report benzaldehyde synthase (BS) activity from yeast extract‐treated cell suspension cultures of P. pyrifolia. BS catalyzes the in vitro conversion of trans‐cinnamic acid to benzaldehyde using a non‐oxidative C₂‐side chain cleavage mechanism. The enzyme activity was strictly dependent on the presence of a reducing agent, dithiothreitol being preferred. C₂‐side chain shortening of the cinnamic acid backbone resembled the mechanisms catalyzed by 4‐hydroxybenzaldehyde synthase (HBS) activity in Vanilla planifolia and salicylaldehyde synthase (SAS) activity in tobacco and apple cell cultures. A basal BS activity was also observed in the non‐elicited cell cultures. Upon yeast extract‐treatment, a 13‐fold increase in BS activity was observed when compared to the non‐treated control cells. Moreover, feeding of the cell cultures with trans‐cinnamic acid, the substrate for BS, resulted in an enhanced level of noraucuparin, a biphenyl phytoalexin. Comparable accumulation of noraucuparin was observed upon feeding of benzaldehyde, the BS product. The preferred substrate for BS was found to be trans‐cinnamic acid, for which the apparent K_m and V_max values were 0.5 mM and 50.7 pkat mg^?1 protein, respectively. Our observations indicate the contribution of BS to benzoic acid biosynthesis in Asian pear via the CoA‐independent and non‐β‐oxidative route.     

Expression of human p140trk receptors in p140trk-deficient,PC12/endothelial cells results in nerve growth factor-induced signal transduction and DNA synthesis

Nerve growth factor (NGF) regulates proliferation, differentiation, and survival of sympathetic and sensory neurons through the tyrosine kinase activity of its receptor, p140^trk. These biological effects of NGF depend upon the signal-mediating function of p140^trk substrates which are likely to differ from cell to cell. To define p140^trk receptor substrates and the details of signalling by NGF in the hybrid cell PC12EN, we stably transfected cultures with a vector encoding a full-length human p140^trk cDNA sequence. Two stably transfected clones, one expressing p140^trk with higher affinity (PC12EN-trk3; K_d 57.4 pM, B_max 9.7 pmole/mg) and one expressing p140^trk with a lower affinity (PC12EN-trk1; K_d 392.4 pM, B_max 5.7 pmole/mg) were generated. Radioreceptor assays indicate that transfected p140^trk receptors show slow NGF-dissociation kinetics, are resistant to trypsin or Triton X-100 treatment, are specific for NGF compared to other neurotrophins, and are internalized or downregulated as are native PC12 p140^trk receptors. NGF stimulates p140^trk tyrosine phosphorylation in a dose- (0.01-10 ng/ml) and time- (5-120 min) dependent manner, and tyrosine phosphorylation was inhibited by 200-1,000 nM K-252a. NGF-induced Erk stimulation for 60 min was assessed using myelin basic protein as a substrate. NGF treatment also led to an increased phosphorylation of p70^S6k, SNT, and phospholipase Cγ, demonstrating that the major NGF-stimulated signalling pathways found in other cells are activated in PC12EN-trk cells. Staurosporine (5-50 nM) rapidly and dBcAMP (1 mM) more slowly, but not NGF induced morphological differentiation in PC12EN-trk cells. Rather, NGF treatment in low-serum medium stimulated a 1.3- and 2.3-fold increase in DNA synthesis measured by [³H]thymidine incorporation in PC12EN-trk1 and PC12EN-trk3, respectively. These data highlight the functionality of the transfected p140^trk receptors and indicate that these transfected cells may serve as a novel cellular model facilitating the study of the mitogenic properties of NGF signalling and the transducing role of the p140^trk receptor substrates. J. Cell. Biochem. 66:229-244. © 1997 Wiley-Liss, Inc. This article is a U.S. Government work and, as such, is in the public domain in the United States of America.     

Membranotropic effects of peptides from the V3 loop of HIV-1

The V3 loop from HIV-1 envelope glycoprotein gp120 is involved in viral entry and determines the cellular tropism and HIV-1-induced cell–cell fusion. Earlier we have shown that V3 loop peptides representing the sequences of syncytia-inducing HIV strains have high membranotropic activity. These peptides caused the lysis of liposomes of various lipid compositions, could fuse negatively charged liposomes and induced hemolysis of erythrocytes. In contrast, peptides mimicking the sequences of non-syncytia-inducing viruses showed no lytic or fusion activities at the same concentrations. Now we have found that the V3 loop synthetic peptides containing the conserved GPGR region, derived from T-lymphotropic strains (BRU and MN), as opposed to peptides containing the GPGQ region, are able to cause a pronounced membrane permeabilization (dissipation of the pH and the of human peripheral blood lymphocytes, erythrocytes and plasma membrane vesicles at micromolar concentrations with a dose-dependent kinetics. Analysis of the secondary structures of the peptides by circular dichroism revealed conformational changes in V3 loop peptides depending on solvent hydrophobicity: from random coil in water to an -helix/-sheet conformation in trifluoroethanol. Such structural changes of the V3 loop together with the membrane insertion of the gp41 N-terminal fusion peptide may promote the formation of the fusion pore during virus–cell fusion.     

Nutritional status and age at secondary sterility in rural Bangladesh

In a prospective study of 2324 women in Matlab, Bangladesh, the occurrence of primary and 2ndary sterility by age groups was examined. The results were related to the nutritional status of the women, as assessed by measurements of height, weight, arm circumference and ponderal index. Approximately 98% of the women who were in the age group 15-19 were found to be fertile. This proportion decreases gradually up to the age group 30-34 years and thereafter declines sharply, reaching only 31% in the age group 45-49. The height data suggest no significant difference in the age pattern of sterility among the 3 groups of women, although there is a slight tendency that women who were less tall reached menopause earlier than the other 2 groups. Variations in weight are more conspicuous than in height. There is the suggestion that thinner women may experience an earlier menopause. Women having an arm circumference less than 21 cm, between 21-22 cm, and 23 cm and above, and currently aged 17 years, have an expected fertile life estimated at 25.0, 25.8, and 26.6 years respectively. The median ages at sterility were 42.8, 44.0, and 44.3 years respectively with a difference of about 1 year between the 1st 2 groups. This suggests that sterility occurs earlier among the thinner women. Since detailed investigation of nutritional status was not possible, it was assessed by anthropometry. There was strong evidence that nutritional status is an important factor in the estimated age at sterility, with thinner women experiencing an earlier menopause. Although it is impossible to measure the onset of sterility, one can obtain a minimum estimate of it from the age-specific distribution of the proportion of women who have not produced a child for 5 years of being at risk.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.     

Comparative analysis of genetic structure of 2 species of marine fish Dicentrarchus labrax and Dicentrarchus punctatus

We present here the genetic structure existing among five samples of the spotted sea bass Dicentrarchus punctatus, and we compare it to what prevails in the common sea bass D. labrax, a congeneric species sampled on almost the same geographical range. A genetic distance tree inferred from the polymorphism at six microsatellite loci shows a distinct pattern for the two species. D. labrax samples appears to be genetically more homogeneous with a global Fst of 3% as compared to the 10% observed at D. punctatus, indicating a lesser level of gene flow in the latter species. While appearing more differentiated, D. punctatus presents no clear geographical organisation of its genetic variability in opposition to D. labrax samples. This allows us to propose this pair of closely relative species as a good candidate for the study by comparative analysis of the biological and/or historical factors affecting genetic differentiation in marine environment.     

Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

CXCR2-Driven Ovarian Cancer Progression Involves Upregulation of Proinflammatory Chemokines by Potentiating NF-κB Activation via EGFR-Transactivated Akt Signaling

Ovarian cancer is an inflammation-associated malignancy with a high mortality rate. CXCR2 expressing ovarian cancers are aggressive with poorer outcomes. We therefore investigated molecular mechanisms involved in CXCR2-driven cancer progression by comparing CXCR2 positive and negative ovarian cancer cell lines. Stably CXCR2 transfected SKOV-3 cells had a faster growth rate as compared to control cells transfected with empty vector. Particularly, tumor necrosis factor (TNF), abundantly expressed in ovarian cancer, enhanced cell proliferation by decreasing the G0-G1 phase in CXCR2 transfected cells. TNF increased nuclear factor-κB (NF-κB) activity to a greater degree in CXCR2 transfected cells than control cells as well as provided a greater activation of IκB. CXCR2 transfected cells expressed higher levels of its proinflammatory ligands, CXCL1/2 and enhanced more proliferation, migration, invasion and colony formation. CXCR2 positive cells also activated more EGFR, which led to higher Akt activation. Enhanced NF-κB activity in CXCR2 positive cells was reduced by a PI3K/Akt inhibitor rather than an Erk inhibitor. CXCL1 added to CXCR2 positive cells led to an increased activation of IκB. CXCL1 also led to a significantly greater number of invasive cells in CXCR2 transfected cells, which was blocked by the NF-κB inhibitor, Bay 11-7082. In addition, enhanced cell proliferation in CXCR2 positive cells was more sensitive to CXCL1 antibody or an NF-κB inhibitor. Finally, CXCR2 transfection of parental cells increased CXCL1 promoter activity via an NF-κB site. Thus augmentation of proinflammatory chemokines CXCL1/2, by potentiating NF-κB activation through EGFR-transactivated Akt, contributes to CXCR2-driven ovarian cancer progression.     

Transmission dynamics of methicillin-resistant Staphylococcus aureus in a medical intensive care unit in India

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a global pathogen and an important but seldom investigated cause of morbidity and mortality in lower and middle-income countries where it can place a major burden on limited resources. Quantifying nosocomial transmission in resource-poor settings is difficult because molecular typing methods are prohibitively expensive. Mechanistic statistical models can overcome this problem with minimal cost. We analyse the transmission dynamics of MRSA in a hospital in south India using one such approach and provide conservative estimates of the organism''s economic burden.

Methods and Findings

Fifty months of MRSA infection data were collected retrospectively from a Medical Intensive Care Unit (MICU) in a tertiary hospital in Vellore, south India. Data were analysed using a previously described structured hidden Markov model.Seventy-two patients developed MRSA infections and, of these, 49 (68%) died in the MICU. We estimated that 4.2% (95%CI 1.0, 19.0) of patients were MRSA-positive when admitted, that there were 0.39 MRSA infections per colonized patient month (0.06, 0.73), and that the ward-level reproduction number for MRSA was 0.42 (0.08, 2.04). Anti-MRSA antibiotic treatment costs alone averaged $124/patient, over three times the monthly income of more than 40% of the Indian population.

Conclusions

Our analysis of routine data provides the first estimate of the nosocomial transmission potential of MRSA in India. The high levels of transmission estimated underline the need for cost-effective interventions to reduce MRSA transmission in hospital settings in low and middle income countries.