Maturation-specific type II cyclic AMP-dependent protein kinase in goat sperm plasma membrane

Highly purified plasma membranes (PMs) isolated by aqueous two-phase polymer methods from goat sperm undergoing epididymal maturation, have been analyzed for the isoenzymes of cyclic AMP-dependent protein kinase (RC). The mature and the immature spermatozoa showed profound differences in the profile of the isoenzymes of RC solubilized from the isolated PMs with 0.1% Triton X-100. The immature sperm PM consists of only type I RC in contrast to the mature sperm membrane which possesses both the type I and II isoenzymes. The type II kinase represents nearly 30% of the total membrane-bound RC of the mature cells. The analysis of the surface topography of these isoenzymes of the maturing spermatozoa by using diazonium salt of sulfanilic acid as the surface probe shows that the PM-bound RC(s) are oriented primarily on the external surface of these intact cells. The data demonstrate that type II RC is a maturation-specific ecto-kinase as it appears on the sperm surface specifically during the maturation of spermatozoa in the epididymis.     

Dephosphorylation of cell-surface phosphoproteins of goat spermatozoa

Multiple ecto-phosphoproteins of the goat cauda-epididymal intact spermatozoa have been shown to undergo dephosphorylationin vitro by endogenous phosphoprotein phosphatase(s) located on the sperm outer surface. The major ecto-phosphoproteins that are dephosphorylated have molecular masses of 27, 40, 70, 116 and 205 kDa. The cell surface dephosphorylation reaction is not dependent on bivalent metal ions. Mg²⁺ (5 mM), Mn²⁺ (5 mM), orthovanadate (200ΜM) and cAMP (5 ΜM) have no effect on this surface reaction whereas it is inhibited nearly 50% by Co²⁺ or Zn²⁺ (1 mM). Spermidine (5 mM), or Ca²⁺ (1mM) inhibited to a small extent (approx. 25%) the cell surface dephosphorylation of proteins.     

Occurrence of an ecto-phosphoprotein phosphatase in goat epididymal spermatozoa

Intact spermatozoa from goat cauda epididymis possess phosphoprotein phosphatase activity that causes dephosphorylation of externally added [32p]histones. The enzymic reaction was linear with time for at least 15 min and there was little uptake of [32p]histones by these cells. The activity of the enzyme of the whole spermatozoa was not due to contamination of the broken cells or epididymal plasma and leakage of the intracellular enzymic activity during incubation. The activity of the phosphoprotein phosphatase was strongly inhibited by the thiol reagent: p-chloromercuribenzenesulfonic acid, which is believed not to enter the cells. There was no appreciable loss of the enzymic activity from the cells when washed with EDTA (2.0 mM) or a hyperosmotic medium. These data are consistent with the view that the observed activity of the enzyme is located on the spermatozoal external surface. Studies with unlabelled p-nitrophenyl phosphate and beta-glycerophosphate indicate that the sperm ecto-enzyme is not a non-specific phosphatase.     

Hypermethylation of metallothionein-I promoter and suppression of its induction in cell lines overexpressing the large subunit of Ku protein.

We have shown previously that the heavy metal-induced metallothionein-I (MT-I) gene expression is specifically repressed in a rat fibroblast cell line (Ku-80) overexpressing the 80-kDa subunit of Ku autoantigen but not in cell lines overexpressing the 70-kDa subunit or Ku heterodimer. Here, we explored the molecular mechanism of silencing of MT-I gene in Ku-80 cells. Genomic footprinting analysis revealed both basal and heavy metal-inducible binding at specific cis elements in the parental cell line (Rat-1). By contrast, MT-I promoter in Ku-80 cells was refractory to any transactivating factors, implying alteration of chromatin structure. Treatment of two clonal lines of Ku-80 cells with 5-azacytidine, a potent DNA demethylating agent, rendered MT-I gene inducible by heavy metals, suggesting that the gene is methylated in these cells. Bisulfite genomic sequencing revealed that all 21 CpG dinucleotides in MT-I immediate promoter were methylated in Ku-80 cells, whereas only four CpG dinucleotides were methylated in Rat-1 cells. Almost all methylated CpG dinucleotides were demethylated in Ku-80 cells after 5-azacytidine treatment. To our knowledge, this is the first report that describes hypermethylation of a specific gene promoter and its resultant silencing in response to overexpression of a cellular protein.     

Effect of interleukin-1α on the growth of Mycobacterium microti within J774A.1 cells

Abstract The effect of mouse recombinant interleukin-1 α on the intracellular growth of Mycobacterium microti in a murine macrophage cell line J774A.1 was investigated. Interleukin-1 α added after infection to the M. microti -infected macrophage monolayers enhanced the growth of M. microti in a concentration-dependent manner and this growth enhancement was abrogated by neutralization of interleukin-1 α with anti-interleukin-1 α antibody. Cyclic adenosine monophosphate level in J774A.1 cells was increased by the addition of interleukin-1 α . Addition of dibutyryl cyclic adenosine monophosphate to infected J774A.1 cells increased the number of intracellular bacteria in a concentration-dependent manner. These results suggest that interleukin-1 α acts as a growth enhancer for intracellular M. microti and the growth enhancing effect of interleukin-1 α may be due to enhanced cellular cyclic adenosine monophosphate level.