Role of Melanin in Release of Extracellular Enzymes and Selection of Aggressive Isolates of Bipolaris sorokiniana in Barley

Eighteen barley isolates of Bipolaris sorokiniana belonging to wild and clonal type of black, mixed and white subpopulations were quantitatively assayed for their melanin content and aggressiveness with respect to production of some of the extracellular enzymes such as cellulase, pectinase, amylase and protease. Cellulase and pectinase constituted major portion of the enzymes recovered from the black, mixed and white isolates. Enzyme production and aggressiveness were relatively higher in melanin devoid or low melanin isolates. The melanin deficient isolates were also differentiated from black and mixed isolates on the basis of variation in internal transcribed spacer region of the ribosomal DNA. Higher enzyme productions positively correlated with area under disease progress curve (AUDPC) and lesion development. Melanin content was negatively correlated with extracellular enzymes and aggressiveness of the isolates. Based on melanin content, lesion size, AUDPC and extracellular enzymes, the isolates were grouped in two major clusters (I and II) with further division of cluster II into two sub-clusters (II-A and II-B). The results appears to indicate a possible role of melanin in release of extracellular enzymes and hence in evolution and selection of aggressive isolates of B. sorokiniana in barley.     

M89V Sialic Acid Acetyl Esterase (SIAE) and All Other Non-Synonymous Common Variants of This Gene Are Catalytically Normal

Catalytically defective rare variants of Sialic acid Acetyl Esterase (SIAE) have previously been linked to autoimmunity. Studies presented here confirm that the M89V SIAE protein and all other products of common variant alleles of SIAE are catalytically normal. Although overexpressing transfected non-lymphoid cells secrete small amounts of SIAE that can associate with the cell surface, normal human lymphocytes do not exhibit cell surface SIAE, supporting genetic evidence in mice that indicates that this protein functions in a lymphocyte intrinsic manner. Analyses of the plasma proteome also indicate that SIAE is not secreted in vivo. A re-analysis exclusively of catalytically defective rare variant alleles of SIAE in subjects in which this gene was completely sequenced confirmed an association of SIAE with autoimmunity. A subset of catalytically defective rare variant SIAE alleles has previously been typed in a large genotyping study comparing a diverse group of disease subjects and controls; our re-analysis of this data shows that catalytically defective alleles are enriched in disease subjects. These data suggest that SIAE may be associated with autoimmunity and that further study of catalytically defective rare variant SIAE alleles in terms of autoimmune disease susceptibility is strongly warranted.     

Osmoregulation in Dunaliella tertiolecta: effects of salt stress,and the external pH on the internal pH

The intracellular pH of the halotolerant green algae Dunaliella tertiolecta, was determined by the distribution of 5,5-dimethyl-2(¹⁴C)-oxalolidine-2,5-dione (DMO) between the cell and the surrounding medium. 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione was not metabolized by the algal cells. The intracellular pH of Dunaliella tertiolecta was 6.8 in the dark and 7.4 in the light. During a salt stress, after two hours, the intracellular pH was increased by 0.2 pH units in both light and dark. The salt stressed cells maintained a constant pH of about 7.5 over the pH range of 6.5 to 8.5. Because of the relatively low permeability coefficient of the plasma membrane for DMO, this technique does not permit rapid pH determinations during the induction period after a salt stress. The magnitude of the salt induced pH changes measured 2 h after the salt stress implies a minor importance of this alkalization in this time range, but does not exclude a larger importance of pH changes for osmoregulation during the induction period.Abbreviations Chl chlorophyll - DMO 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione - PCV packed cell volume - SDS sodium dodecyl sulfate     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Effect of mutation of cysteinyl residues in yeast Cu-metallothionein

Metallothioneins have been isolated from Saccharomyces cerevisiae CUP1 mutants generated by Wright et al. (Wright, C. F., Hamer, D. H., and McKenney, K. (1986) Nucleic Acids Res. 14, 8489-8499). In the mutant metallothioneins, pairs of cysteinyl residues have been converted to seryl residues. The mutant proteins differ only in the positions of the double substitutions; each mutant molecule contains 10 cysteinyl residues. Each mutant protein lacks the first 8 residues at the amino terminus from the decoded gene sequence of the CUP1 locus. Mutant molecules consist of 53 residues analogous to the wild-type metallothionein and are designated 9/11, 24/26, 36/38, and 49/50 (in reference to the sequence positions of the Cys----Ser conversions). The properties of the mutant metallothioneins are vastly different, and host cells harboring the different plasmid-encoded mutant molecules show marked differences in sensitivity to CuSO4. Growth inhibition was observed at CuSO4 concentrations up to mM in cells containing the 9/11, 24/26, and 36/38 molecules, but not for cells containing protein 49/50. A CuSO4 concentration of 5 mM was required to inhibit the growth of yeast containing either 49/50 or the wild-type metallothionein. In the purified proteins the copper binding stoichiometry of each molecule, except protein 24/26, was nearly 8 mol eq. Protein 24/26 bound 5.5 copper ions/molecule. The Cu(I) chelator bathocuproine disulfonate reacted with over 50% of the copper ions in proteins 9/11, 24/26, and 36/38, but less than 10% of the copper ions in proteins 49/50 and wild-type metallothionein were reactive. The thiolates in 9/11, 24/26, and 36/38 were also more reactive in a disulfide exchange reaction with dithiodipyridine compared with the sulfhydryls in 49/50 and the wild-type molecules. The four mutant copper proteins are luminescent and exhibit a similar quantum yield. The cluster structures contributing to the particular electronic transitions are markedly more sensitive to oxygen in proteins 9/11, 24/26, and 36/38 compared with 49/50 and the wild-type molecules. The air-sensitive proteins exhibit a tertiary fold not recognized by polyclonal antibodies directed to a conformational epitope on yeast Cu-metallothionein. Protein 49/50 cross-reacts with the antibody in a concentration-dependent fashion similar to the wild-type protein. Mutation of 2 cysteinyl residues in the carboxyl portion of metallothionein does not significantly alter properties of the molecule, whereas mutation of several cysteines in the amino-terminal portion of the molecule yields a different conformation.     

HLA and autoimmunity in North Indian type I (insulin-dependent) diabetic multiplex families

The HLA haplotype segregation and autoantibody spectrum in 7 type I (insulin-dependent) diabetic multiplex families of North Indian origin were determined. Of the total of 17 diabetic sibs, 7 shared both haplotypes and 3 shared one haplotype with the proband. No HLA-non-identical sibs were observed. This distribution of haplotypes was non-random (P approximately equal to 0.005). The mode of inheritance was compatible with an autosomal recessive model, while a dominant model was unlikely. Pancreatic islet-cell antibodies were found in 23.5% of affected sibs, but in no healthy family member. A high incidence of other autoantibodies (parietal-cell and thyroglobulin/thyroid microsomal antibodies) was detected in both the diabetic patients (26.3%), and in healthy first-degree relatives (22.2%). These findings emphasize the role of HLA-linked genes and autoimmunity in the pathogenesis of type I diabetes in North India.     

Suppressor T lymphocytes from lepromatous leprosy skin lesions

The immune response in leprosy forms a spectrum with lepromatous leprosy patients exhibiting specific unresponsiveness to antigens of Mycobacterium leprae. This unresponsiveness is thought to be related to the prevalence of T8-positive lymphocyte in these lepromatous lesions. To analyze the immunoregulatory function of these T8 cells, we developed simple procedures to extract lymphocytes from skin biopsy specimens of patients with leprosy. These lymphocytes were sorted for T8 and T4 positive cells, and cell lines were established by expansion with interleukin 2 (IL 2) and irradiated feeder cells. All T8 positive lines tested were positive for IL 2 receptors and HLA-DR determinants. These lines were additionally assayed for lepromin-induced suppression of the normal peripheral blood lymphocyte Con A proliferative response. Thirteen of 32 lines from six lepromatous patients showed significant suppressor activity, whereas nine lines from six tuberculoid patients and one line from normal peripheral blood failed to show suppression (p less than 0.001). Taken together, the finding of M. leprae-triggered suppressor cells within lepromatous skin lesions may in part explain the M. leprae unresponsiveness of lepromatous leprosy patients.