Mutational analysis of conserved residues in HhaI DNA methyltransferase

HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase.     

Cytotoxic cell granule-mediated apoptosis. Characterization of the macromolecular complex of granzyme B with serglycin

We have recently shown that the physiological mediator of granule-mediated apoptosis is a macromolecular complex of granzymes and perforin complexed with the chondroitin-sulfate proteoglycan, serglycin (Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) Immunity 16, 417-428). We now report our biophysical studies establishing the nature of granzyme B-serglycin (GrB.SG) complex. Dynamic laser light scattering studies establish that SG has a hydrodynamic radius of approximately 140 +/- 23 nm, comparable to some viral particles. Agarose mobility shift gels and surface plasmon resonance (SPR), show that SG binds tightly to GrB and has the capacity to hold 30-60 GrB molecules. SPR studies also indicate equivalent binding affinities (K(d) approximately 0.8 microm), under acidic (granule pH) and neutral isotonic conditions (extra-cytoplasmic pH), for GrB.SG interaction. Finally, characterization of GrB.SG interactions within granules revealed complexes of two distinct molecular sizes, one held approximately 4-8 molecules of GrB, whereas the other contained as many as 32 molecules of GrB or other granule proteins. These studies provide a firm biophysical basis for our earlier reported observations that the proapoptotic granzyme is exocytosed predominantly as a macromolecular complex with SG.     

Requirement of Skp1-Bub1 interaction for kinetochore-mediated activation of the spindle checkpoint

The spindle checkpoint transiently prevents cell cycle progression of cells that have incurred errors or failed to complete steps during mitosis, including those involving kinetochore function. The molecular nature of the primary signal transmitted from defective kinetochores and how it is detected by the spindle checkpoint are unknown. We report biochemical evidence that Bub1, a component of the spindle checkpoint, associates with centromere (CEN) DNA via Skp1, a core kinetochore component in budding yeast. The Skp1's interaction with Bub1 is required for the mitotic delay induced by kinetochore tension defects, but not for the arrest induced by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. We propose that the Skp1-Bub1 interaction is important for transmitting a signal to the spindle checkpoint pathway when insufficient tension is present at kinetochores.     

Antifertility,antibacterial, antifungal and percent disease incidence aspects of macrocyclic complexes of manganese(II)

Macrocyclic complexes of Mn(II) were synthesized by template condensation using 2,6-diaminopyridine and diethylenetriamine with malonic, succinic, glutaric and adipic acids. The reaction proceeded smoothly to completion. These 16- to 24-membered N(6), but behaving as tetradentate, macrocyclic complexes were characterized by elemental analyses, molecular weight determinations, infrared, electronic, mass and X-ray spectral analyses. The elemental analyses are consistent with the formation of complexes [Mn(N(6)L(n))Cl(2)]. All the complexes are stable and monomeric in nature, as indicated by the molecular weight determinations. The spectral studies confirmed the proposed framework of the new macrocyclic complexes and indicated an octahedral geometry around the central metal atom. The complexes were screened in vitro against a number of pathogenic fungi and bacteria to assess their growth-inhibiting potential. The testicular sperm density, sperm morphology, sperm motility, density of cauda epididymis, spermatozoa and fertility in mating trials and the biochemical parameters of the reproductive organs of the rat were examined and are discussed.     

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.     

Metastatic sweat gland adenocarcinoma: A clinico-pathological dilemma

Background  

Sweat gland adenocarcinoma is a rare malignancy with high metastatic potential seen more commonly in later years of life. Scalp is the most common site of occurrence and it usually spreads to lymph nodes. Liver, lung and bones are the distant sites of metastasis with fatal results. The differentiation between apocrine and eccrine metastatic sweat gland carcinoma is often difficult. The criteria's are inadequate to be of any practical utility.     

Regulation of glucose utilization and lipogenesis in adipose tissue of diabetic and fat fed animals: Effects of insulin and manganese

In order to evaluate the modulatory effects of manganese, high fat diet fed and alloxan diabetic rats were taken and the changes in the glucose oxidation, glycerol release and effects of manganese on these parameters were measured from adipose tissue. An insulin-mimetic effect of manganese was observed in the adipose tissue in the controls and an additive effect of insulin and manganese on glucose oxidation was seen when Mn²⁺ was addedin vitro. The flux of glucose through the pentose phosphate pathway and glycolysis was significantly decreased in high fat fed animals. Although thein vitro addition of Mn²⁺ was additive with insulin when¹⁴CO2 was measured from control animals, it was found neither in young diabetic animals (6–8 weeks old) nor in the old (16 weeks old). Both insulin and manganese caused an increased oxidation of carbon-1 of glucose and an increase of its incorporation into¹⁴C-lipids in the young control animals; the additive effect of insulin and manganese suggests separate site of action. This effect was decreased in fat fed animals, diabetic animals and old animals. Manganese alone was found to decrease glycerol in both the control and diabetic adipose tissue inin vitro incubations. The results of the effects of glucose oxidation, lipogenesis, and glycerol release in adipose tissue of control and diabetic animals of different ages are presented together with the effect of manganese on adipose tissue from high fat milk diet fed animals.     

N-glycolylneuraminic acid conjugates: implications of their absence in mammalian biochemistry

N-glycolylneuraminic acid (Neu5Gc) is one of the two most common forms of sialic acids present in glycoproteins and glycolipids of mammalian tissues. It is synthesized from the most ubiquitous sialic acid, N-acetylneuraminic acid (Neu5Ac) in a hydroxylation reaction catalysed by the enzyme Neu5Ac hydroxylase. Though Neu5Gc conjugates are prevalent in many tissues of mammals, they are absent in glycolipids and only trace amounts are present in glycoproteins of the brain and central nervous system. In humans Neu5Ac is the main sialic acid as Neu5Ac hydroxylase is inactive due to mutation of its gene. The importance of sialic acids in biochemical phenomena and the distinct roles played by specific forms of these amino sugars is adequately reflected in functional studies of selectin and sialoadhesin families of adhesion molecules. The absence of Neu5Gc, therefore, in tissues of humans and brain of mammals has raised interest, especially with regard to its impact on biochemical differences evident between humans and other mammals. It is suggested that though Neu5Gc conjugates are important in cellular interactions, their presence in brain and the central nervous system is deleterious to the latter's normal functions. Their interaction with other cellular components to form supramolecular associations is indicated that may have a bearing on major biochemical differences, a few of which are presently evident between humans and other mammals.     

Protective role of selenium status on T3/T4 kinetics in rats under hyperlipidemia

The effect of high fat diet (HFD) on thyroid hormones (T3/T4) and protective role of selenium (Se) were studied in rats. Se levels in serum and liver decreased significantly, whereas glutathione peroxidase (GSH-Px) in liver and lipid levels (cholesterol and triglycerides) in serum increased after 1, 2 and 3 months of HFD feeding in comparison to controls in all the three Se status i.e. deficient (0.02 ppm), adequate (0.2 ppm) and excess (1 ppm) groups. Levels of T3/T4 decreased significantly on HFD feeding, as compared to respective controls in all the groups. Within the deficient group, as Se deficiency progressed, T3/T4 levels decreased after 2 and 3 months in comparison to 1 month. A significant increase was observed in T3/T4 concentration on feeding 1 ppm (excess) Se supplemented diet, in comparison to adequate group. Also, in 1 ppm Se supplemented group as the Se deposition increased i.e. after 2 and 3 months, levels of T3/T4 increased significantly. So, the present study indicates that Se supplementation up to 1 ppm normalizes the T3 and T4 concentrations or regulates the hypothyroidism induced by hyperlipidemia.     

Identification,purification and characterization of a receptor for dengue virus-induced macrophage cytotoxin (CF2) from murine T cells

Dengue type-2 virus infection in mice induces a subpopulation of T lymphocytes to produce a cytokine cytotoxic factor, which induces macrophages (Mphi) to produce a biologically active cytotoxic cytokine, the Mphi cytotoxin (CF2). Previously we have identified the presence of intermediate-affinity receptors for CF2 on mouse peritoneal Mphi. The present study was undertaken to identify the CF2-receptors (CF2-R) on murine T cells followed by their purification and characterization. Receptor binding assay and Scatchard analysis revealed single, high-affinity (1.0309 nM) receptors for CF2 on T cells (22000 receptors per cell). The binding of [125I]CF2 on murine T cells was saturable and specific. Furthermore, CF2-R was purified from normal mouse T cell plasma membrane by affinity chromatography followed by reversed-phase high-pressure liquid chromatography. The presence of CF2-R was confirmed by indirect dot-blot assay and its binding with [125I]CF2. The purified CF2-R is a 90-95-kDa protein as characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. The chemical crosslinking of [125I]CF2 and its receptor complex showed a product of 100-110 kDa on different subpopulations of murine T cells. The pretreatment of target cells with anti-CF2-R antisera inhibited the cytotoxic activity of CF2 in a dose-dependent manner and thus confirmed the biological significance of CF2-R. Moreover, the presence of CF2-R was also identified on normal human peripheral blood mononuclear cells and T and B cells by crosslinking with [125I]CF2, thus revealing the possible role of CF2 and CF2-R in the immunopathogenesis of dengue virus disease.