Mnt1p and Mnt2p of Candida albicans are partially redundant alpha-1,2-mannosyltransferases that participate in O-linked mannosylation and are required for adhesion and virulence

The MNT1 gene of the human fungal pathogen Candida albicans is involved in O-glycosylation of cell wall and secreted proteins and is important for adherence of C. albicans to host surfaces and for virulence. Here we describe the molecular analysis of CaMNT2, a second member of the MNT1-like gene family in C. albicans. Mnt2p also functions in O-glycosylation. Mnt1p and Mnt2p encode partially redundant alpha-1,2-mannosyltransferases that catalyze the addition of the second and third mannose residues in an O-linked mannose pentamer. Deletion of both copies of MNT1 and MNT2 resulted in reduction in the level of in vitro mannosyltransferase activity and truncation of O-mannan. Both the mnt2Delta and mnt1Delta single mutants were significantly reduced in adherence to human buccal epithelial cells and Matrigel-coated surfaces, indicating a role for O-glycosylated cell wall proteins or O-mannan itself in adhesion to host surfaces. The double mnt1Deltamnt2Delta mutant formed aggregates of cells that appeared to be the result of abnormal cell separation. The double mutant was attenuated in virulence, underlining the importance of O-glycosylation in pathogenesis of C. albicans infections.     

Detection and frequency estimation of rare variants in pools of genomic DNA from large populations using mutational spectrometry

DNA variants underlying the inheritance of risk for common diseases are expected to have a wide range of population allele frequencies. The detection and scoring of the rare alleles (at frequencies of <0.01) presents significant practical problems, including the requirement for large sample sizes and the limitations inherent in current methodologies for allele discrimination. In the present report, we have applied mutational spectrometry based on constant denaturing capillary electrophoresis (CDCE) to DNA pools from large populations in order to improve the prospects of testing the role of rare variants in common diseases on a large scale. We conducted a pilot study of the cytotoxic T lymphocyte-associated antigen-4 gene (CTLA4) in type 1 diabetes (T1D). A total of 1228 bp, comprising 98% of the CTLA4 coding sequence, all adjacent intronic mRNA splice sites, and a 3′ UTR sequence were scanned for unknown point mutations in pools of genomic DNA from a control population of 10,464 young American adults and two T1D populations, one American (1799 individuals) and one from the United Kingdom (2102 individuals). The data suggest that it is unlikely that rare variants in the scanned regions of CTLA4 represent a significant proportion of T1D risk and illustrate that CDCE-based mutational spectrometry of DNA pools offers a feasible and cost-effective means of testing the role of rare variants in susceptibility to common diseases.     

The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase

In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN(1)-to-heme(2)) electron transfer supports oxygenase activity in the BM3 dimer.     

Histone deacetylase inhibitors induce a senescence-like state in human cells by a p16-dependent mechanism that is independent of a mitotic clock

We show here that histone deacetylase inhibitors (HDACIs) sodium dibutyrate (SDB) and trichostatin A (TSA) induce a phenotype that has similarities to replicative senescence in human fibroblasts. There was no evidence that SDB accelerated a constitutive cell division counting mechanism as previously suggested because cells pretreated with SDB for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn. SDB-treated cells upregulated the cell cycle inhibitors p21(WAF1) and p16(INK4A), but not p14(ARF), in the same sequential order as in senescence and the cells developed biochemical markers of senescence. However, the mechanism of senescence did not involve telomere dysfunction and there was no evidence for any posttranslational modification of p53. The expression of human papillomavirus (HPV) 16 E6 in human fibroblasts or targeted disruption of the p53 and p21(WAF) genes only weakly antagonized HDACI-induced senescence. However, expression of the E7 gene, which inhibits the function of pRb, cooperated with E6 to block SDB-induced senescence completely and human cells deficient in p16(INK4A) (but not p14(ARF)) were also resistant to SDB-induced senescence, suggesting that the p16(INK4A)/pRb pathway is the major mediator of HDACI-induced senescence in human cells. However, p53-/- mouse fibroblasts were resistant to HDACI-induced senescence, identifying p53 as the major pathway to senescence in this species.     

RNA trafficking signals in human immunodeficiency virus type 1

Intracellular trafficking of retroviral RNAs is a potential mechanism to target viral gene expression to specific regions of infected cells. Here we show that the human immunodeficiency virus type 1 (HIV-1) genome contains two sequences similar to the hnRNP A2 response element (A2RE), a cis-acting RNA trafficking sequence that binds to the trans-acting trafficking factor, hnRNP A2, and mediates a specific RNA trafficking pathway characterized extensively in oligodendrocytes. The two HIV-1 sequences, designated A2RE-1, within the major homology region of the gag gene, and A2RE-2, in a region of overlap between the vpr and tat genes, both bind to hnRNP A2 in vitro and are necessary and sufficient for RNA transport in oligodendrocytes in vivo. A single base change (A8G) in either sequence reduces hnRNP A2 binding and, in the case of A2RE-2, inhibits RNA transport. A2RE-mediated RNA transport is microtubule and hnRNP A2 dependent. Differentially labelled gag and vpr RNAs, containing A2RE-1 and A2RE-2, respectively, coassemble into the same RNA trafficking granules and are cotransported to the periphery of the cell. tat RNA, although it contains A2RE-2, is not transported as efficiently as vpr RNA. An A2RE/hnRNP A2-mediated trafficking pathway for HIV RNA is proposed, and the role of RNA trafficking in targeting HIV gene expression is discussed.     

Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 A reveals novel features of cytochrome P450

The first structure of a P450 to an atomic resolution of 1.06 A has been solved for CYP121 from Mycobacterium tuberculosis. A comparison with P450 EryF (CYP107A1) reveals a remarkable overall similarity in fold with major differences residing in active site structural elements. The high resolution obtained allows visualization of several unusual aspects. The heme cofactor is bound in two distinct conformations while being notably kinked in one pyrrole group due to close interaction with the proline residue (Pro(346)) immediately following the heme iron-ligating cysteine (Cys(345)). The active site is remarkably rigid in comparison with the remainder of the structure, notwithstanding the large cavity volume of 1350 A(3). The region immediately surrounding the distal water ligand is remarkable in several aspects. Unlike other bacterial P450s, the I helix shows no deformation, similar to mammalian CYP2C5. In addition, the positively charged Arg(386) is located immediately above the heme plane, dominating the local structure. Putative proton relay pathways from protein surface to heme (converging at Ser(279)) are identified. Most interestingly, the electron density indicates weak binding of a dioxygen molecule to the P450. This structure provides a basis for rational design of putative antimycobacterial agents.     

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.     

Kinetics of the hydrolysis of lean meat protein by alcalase: Derivation of two alternative rate equations and their fit to experimental data

Two rate equations have been developed to model the hydrolysis of ground lean meat protein by Alcalase. The first equation was based on classical Michaelis-Menten kinetics and the second on the adsorption of enzyme to the protein prior to reaction. It was assumed that this adsorption could be modelled by a Langmuir-type adsorption isotherm. Each equation considered the enzyme to be competitively inhibited by reaction product, and considered enzyme inactivation to be first order. Both rate equations have been fitted to experimental data obtained from the hydrolysis of meat protein by Alcalase. Initial rate data indicated that the adsorption model was more appropriate. However, both equations gave satisfactory fits to 11 reaction progress curves determined over a wide range of enzyme and substrate concentrations.     

Steroid hormones and agonistic behavior in a cichlid teleost, Aequidens pulcher

The effects of three steroid hormones on the agonistic behavior of female Aequidens pulcher have been evaluated. Testosterone, estradiol, and cortisol were tested using an immersion technique to minimize trauma, and we also examined metyrapone, a blocker of cortisol biosynthesis. Two different experimental protocols were employed, the first investigating agonistic interactions within groups of fish, and the second examining the responses of isolated fish to models and mirrors. Differences between replicates were small, and both protocols supported similar conclusions. Each of the three hormones produced a characteristically different spectrum of behaviors when compared to the controls. Testosterone increased agonistic behavior in all experimental situations, while estradiol had a generally opposite effect; this may reflect the natural modulation of behavior by hormones during the reproductive cycle of A. pulcher. Cortisol also had distinct behavioral effects; available evidence suggests that this steroid increases submissive components of agonistic behavior, and that observed increases in some aggressive components are an indirect consequence, dependent upon the feedback of social information received by each fish. Metyrapone treatment greatly reduced all agonistic behaviors, groups of fish forming shoals typical of juveniles. This was not reversed by replacement therapy with cortisol, which suggests that metyrapone affects behavior by an alternative, possibly toxic, mechanism.