Recovery of rat mast cells after secretion: a morphometric study

Granule reconstitution in rat peritoneal mast cells following massive secretion was studied by morphometric techniques. Immediately following secretion, the earliest identifiable mast cells showed a substantial decrease in cell volume associated with granule loss. Cell volume then increased almost to the original level over a period of a month. The size of the Golgi apparatus increased markedly in the week following secretion and then returned to its original size. The total volume of granules increased slowly after the secretory depletion and by 34 days had not returned to the original value although the number of granules had recovered fully. The reconstitution of mast cells after secretion is a prolonged process with several phases resulting in mast cells of varying appearance and content. This heterogeneity generated by reconstitution post secretion must be considered in studies of populations of mast cells in vivo.     

Non-C-G recognition sequences of DNA cytosine-5-methyltransferase from rat liver

The eukaryotic DNA cytosine-5-methyltransferase (E.C.2.1.1.37) is known to methylate cytosine in DNA mainly, but not exclusively in C-G. In the present study the minor, non-C-G recognition sequences of a rat DNA methyltransferase were analyzed by Maxam-Gilbert sequencing of in vitro methylated SV40 DNA. The enzyme methylates C-A and C-T at a 50-fold lower initial rate than C-G. Methylation of C-C at the 5'C was not observed in the piece of DNA sequenced. The methylation of C-A is very low in the trinucleotides ACA and CAC, the other C-A containing trinucleotides in DNA are much better methylacceptors. C-T was found methylated predominantly in the sequences CCTAA, ACTAA, and ACTGT. A comparison of the activity with different substrates is in favour of the enzyme making its recognition in the major groove of the DNA.     

Protein kinase C inhibition by calmodulin and its fragments

Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10^–5 M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC₅₀ of 6 µ M compared to 1 µ M of intact calmodulin. They were identified as Ser³⁸-Arg⁷⁴ and His¹⁰⁷-Lys¹⁴⁸. Each of the inhibiting fragments contains an intact Ca²⁺-binding domain with complete helix-loop-helix structure (EF hand). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.     

An improved method for the detection of Dcm methylation in DNA molecules

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.     

Ultrastructural study of cholecystokinin-like immunoreactivity in endocrine cells of the insect midgut

Summary Electron-microscopic immunocytochemistry for the demonstration of CCK-like material in basal endocrine cells of the midgut of Aeschna cyanea, Locusta migratoria, Carausius morosus and Periplaneta americana was performed by use of the peroxidase-antiperoxidase procedure and the colloidal gold method. Immunoreactive cells appeared scattered among digestive and regenerative cells of the epithelium. Immunoreactivity was specifically detected over round to oval electron-dense granules whose size appeared rather different from species to species. Thus, the average size of 30% (d30) of the largest granules ranged from 312 nm in Periplaneta americana to 159 nm in Carausius morosus with intermediate values in Aeschna cyanea (d30=195 nm) and Locusta migratoria (d30=225 nm).     

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.     

Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.     

Characterization of poliovirus 2A proteinase by mutational analysis: residues required for autocatalytic activity are essential for induction of cleavage of eukaryotic initiation factor 4F polypeptide p220.

The poliovirus proteinase 2A is autocatalytically released from the poliovirus polyprotein by cotranslational cleavage at its own amino terminus, resulting in separation of structural and nonstructural protein precursors. Cleavage is a prerequisite for further processing of the structural protein precursor and consequently for poliovirus encapsidation. A second function of 2Apro is in the rapid shutoff of host cell protein synthesis that occurs upon infection with poliovirus. This is associated with proteolytic cleavage of the p220 component of eukaryotic initiation factor eIF-4F, which is induced but not directly catalyzed by 2Apro. We introduced single-amino-acid substitutions in the 2Apro-coding region of larger poliovirus precursors that were subsequently translated in vitro and thus demonstrated that His-20, Asp-38, and Cys-109 (which constitute the putative catalytic triad) are essential for, and that His-117 is an important determinant of, the autocatalytic activity of 2Apro. This is consistent with the proposal that 2Apro is structurally related to a subclass of trypsinlike serine proteinases. Moreover, 2Apro containing a Cys109Ser substitution retained a small but significant autocatalytic activity. Cleavage of p220 was not induced by those mutants that had reduced proteolytic activity, indicating that the cellular factor that cleaves p220 is probably activated by 2Apro-catalyzed proteolytic cleavage.