The DNA sequence of chromosome I of an African trypanosome: gene content,chromosome organisation,recombination and polymorphism

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99–100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.     

Functional interactions between the estrogen receptor coactivator PELP1/MNAR and retinoblastoma protein

PELP1 (proline-, glutamic acid-, and leucine-rich protein-1 (also referred to as MNAR, or modulator of nongenomic activity of estrogen receptor)), a recently identified novel coactivator of estrogen receptors, is widely expressed in a variety of 17 beta-estradiol (E2)-responsive reproductive tissues and is developmentally regulated in mammary glands. pRb (retinoblastoma protein), a cell cycle switch protein, plays a fundamental role in the proliferation, development, and differentiation of eukaryotic cells. To study the putative function of PELP1, we established stable MCF-7 breast cancer cell lines overexpressing PELP1. PELP1 overexpression hypersensitized breast cancer cells to E2 signaling, enhanced progression of breast cancer cells to S phase, and led to persistent hyperphosphorylation of pRb in an E2-dependent manner. Using phosphorylation site-specific pRb antibodies, we identified Ser-807/Ser-811 of pRb as a potential target site of PELP1. Interestingly, PELP1 was discovered to be physiologically associated with pRb and interacted via its C-terminal pocket domain, and PELP1/pRb interaction could be modulated by antiestrogen agents. Using mutant pRb cells, we demonstrated an essential role for PELP1/pRb interactions in the maximal coactivation functions of PELP1 using cyclin D1 as one of the targets. Taken together, these findings suggest that PELP1, a steroid coactivator, plays a permissive role in E2-mediated cell cycle progression, presumably via its regulatory interaction with the pRb pathway.     

Potential involvement of extracellular signal-regulated kinase 1 and 2 in encystation of a primitive eukaryote,Giardia lamblia. Stage-specific activation and intracellular localization

Mitogen-activated protein kinase (MAPK) pathways are major signaling systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified Giardia lamblia homologues of two members of the MAPK family ERK1 and ERK2. Functional characterization of giardial ERK1 and ERK2 revealed that both kinases were expressed in trophozoites and encysting cells as 44- and 41-kDa polypeptides, respectively, and were catalytically active. Analysis of the kinetic parameters of the recombinant proteins showed that ERK2 is approximately 5 times more efficient than ERK1 in phosphorylating myelin basic protein as a substrate, although the phosphorylating efficiency of the native ERK1 and ERK2 appeared to be the same. Immunofluorescence analysis of the subcellular localization of ERK1 and ERK2 in trophozoites showed ERK1 staining mostly in the median body and in the outer edges of the adhesive disc and ERK2 staining in the nuclei and in the caudal flagella. Our study also showed a noticeable change in the subcellular distribution of ERK2 during encystation, which became more punctate and mostly cytoplasmic, but no significant change in the ERK1 localization at any time during encystation. Interestingly, both ERK1 and ERK2 enzymes exhibited a significantly reduced kinase activity during encystation reaching a minimum at 24 h, except for an initial approximately 2.5-fold increase in the ERK1 activity at 2 h, which resumed back to the normal levels at 48 h despite no apparent change in the expression level of either one of these kinases in encysting cells. A reduced concentration of the phosphorylated ERK1 and ERK2 was also evident in these cells at 24 h. Our study suggests a functional distinction between ERK1 and ERK2 and that these kinases may play a critical role in trophozoite differentiation into cysts.     

Cytotoxic factor-autoantibodies: possible role in the pathogenesis of dengue haemorrhagic fever

During dengue virus infection a unique cytokine, cytotoxic factor (hCF), is produced that is pathogenesis-related and plays a key role in the development of dengue haemorrhagic fever (DHF). However, what regulates the adverse effects of hCF is not known. We have previously shown that anti-hCF antibodies raised in mice, neutralise the pathogenic effects of hCF. In this study we have investigated the presence and levels of hCF-autoantibodies in sera of patients with various severity of dengue illness (n=136) and normal healthy controls (n=50). The highest levels of hCF-autoantibodies (mean+/-S.D.=36+/-20 U ml(-1)) were seen in patients with mild illness, the dengue fever (DF), and 48 out of 50 (96%) of the sera were positive. On the other hand the hCF-autoantibody levels declined sharply with the development of DHF and the levels were lowest in patients with DHF grade IV (mean+/-S.D.=5+/-2 U ml(-1); P=<0.001 as compared to DF). Only one of the 13 DHF grade IV patients had an antibody level above the 'cut-off' value (mean plus 3 S.D. of the control sera). The analysis of data with respect to different days of illness further showed that the highest levels of hCF-autoantibodies were present in DF patients at >9 days of illness. Moreover, the DF patients at all time points, i.e. 1-4, 5-8 and >9 days of illness had significantly higher levels of hCF-autoantibodies (P<0.001) than patients with DHF grade I, II, III and IV. In addition DHF grade I and grade II patients had significantly more positive specimens than DHF grade III and grade IV patients at all time points. These results suggest that elevated levels of hCF-autoantibodies protect the patients against the development of severe forms of DHF and, therefore, it may be useful as a prognostic indicator.     

Proteinase suppression by E-cadherin-mediated cell-cell attachment in premalignant oral keratinocytes

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novo E-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3'-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.     

Effects of chromium on the immune system

The performance of integral membrane antigens (IMAs) of Mycobacterium habana TMC 5135 in detecting antimycobacterial antibodies in serum and body fluids of patients mainly of extrapulmonary tuberculosis was evaluated. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana. Antimycobacterial antibodies were detected by ELISA using IMAs in serum and body fluids of 42 patients and 62 control subjects. As authentic adjunct Mycobacterium tuberculosis antigens were also detected (by ELISA) in body fluids and circulating immune complexes using anti-M. tuberculosis H37Ra antibodies. Anti-M. habana IMA antibody detection increased the positivity rate from 26.% (11/42) and 10% (4/42) obtained by culture and smear microscopy, respectively, to 86% (36/42). M. tuberculosis antigens were also found in 29 out of 36 anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also positive for anti-M. habana IMA antibodies. The mean antigen titres in 23 cases, positive for antigens in body fluids, were 2.34 times higher in those who were also positive for anti-IMA antibodies in serum than in those negative for these antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.     

Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma

Establishing an effective epidermal barrier requires a series of coordinated molecular events involving keratinocytes (KCs) within a stratified epithelium. Epidermal maturation depends on convergence of pathways involving components of NF-kappaB and peroxisome proliferator activated receptor (PPAR) signaling systems that promote terminal differentiation and production of a stratum corneum. The Notch-1 receptor and its ligand Delta-1 have been proposed by others to participate in early events in KC differentiation. Here, we establish differential expression patterns for several Notch receptors and ligands in normal human skin. These immunolocalization findings, together with functional studies demonstrating increased levels of Notch ligand/receptors occurring during the onset of differentiation, prompted use of a soluble Notch ligand, a peptide derived from the most conspicuously expressed ligand in skin, Jagged-1. Exposing submerged KC monolayers to this peptide (JAG-1) in co-presence of elevated calcium ion concentration, produced stratification with loricrin expression. Using a living human epidermal equivalent (EE) model system, when submerged cultures were raised to an air/liquid interface to generate a fully mature epidermis, activation of Notch signaling was detected. Addition of JAG-1 peptide to submerged EEs was sufficient to induce epidermal maturation. Moreover, a soluble decoy Notch inhibitor prevented such differentiation and corneogenesis in human EEs exposed to either an air/liquid interface or to the JAG-1 peptide. In KC monolayers, addition of JAG-1 peptide induced IKKalpha mediated NF-kappaB activation, as well as increased PPARgamma expression. Immunoprecipitation/Western blot analysis revealed a physical association between the p65 subunit of NF-kappaB and PPARgamma. These results indicate that activation of Notch signaling is necessary for maturation of human epidermis, and activation by a soluble Notch ligand is sufficient to trigger complete KC differentiation including cornification.     

Baylis-Hillman reaction: convenient ascending syntheses and biological evaluation of acyclic deoxy monosaccharides as potential antimycobacterial agents

A series of acyclic deoxy carbohydrate derivatives from easily available carbohydrate enals 1, 2, 3 or 5 were prepared involving the Baylis-Hillman reaction. These newly formed carbohydrate based Baylis-Hillman adducts and their amino derivatives were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H(37)R(v). Among the compounds evaluated for their antimycobacterial activity, compound (10) showed the desired activity in the range of 3.125 microg/mL.     

Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.