Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Maturation of adult rat peritoneal and mesenteric mast cells

Summary Repopulation and maturation of rat mesenteric and peritoneal mast cells were studied after mast cell depletion by intraperitoneal injection of distilled water. Immature mast cells were first identified in the mesentery and peritoneal fluid 5 and 6 days, respectively, after water injection. The most immature mast cells that could be identified contained a few orthochromatic granules. Upon maturation, the granules became metachromatic and increased in size and number. Heparin, revealed by toluidine blue staining and berberine sulfate fluorescence, appeared simultaneously with orthophthaldialdehyde (OPT)-induced histamine fluorescence. Paraformaldehyde-induced serotonin fluorescence appeared somewhat later. Repopulation of mesentery and peritoneal fluid by mast cells seemed to be independent of each other and to occur from undifferentiated precursor cells.     

A photolyase-like protein from Agrobacterium tumefaciens with an iron-sulfur cluster

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.     

Characterization of the Bactericidal Effects of Sodium Nitroprusside and Other Pentacyanonitrosyl Complexes on the Food Spoilage Bacterium Clostridium sporogenes

The potent bactericidal activity of sodium nitroprusside {SNP; Na₂[Fe(CN)₅(NO)]} towards Clostridium sporogenes has been investigated. SNP inhibited cell growth in the concentration range of 10 to 40 μM. Concentrations above 80 μM caused irreversible loss of cell viability and cell lysis. Inhibition of cell growth was similar in complex and in defined media. SNP was found to be unreactive towards individual components of the defined medium, with the exception of cysteine. The chemical characteristics responsible for the potency of SNP were investigated by synthesizing analogs of SNP in which the Fe was replaced by different metals. The inhibitory potency of the pentacyanonitrosyl complexes decreased in the order Fe > Cr > V, which correlates with N-O stretching frequency (vNO). In contrast, the Ru complex which had a vNO comparable to that of Fe was a poor inhibitor. Electron paramagnetic resonance spectroscopy showed that SNP was rapidly reduced to the paramagnetic Fe(I) compound [Fe(CN)₄(NO)]²⁻ on contact with cells. Analysis of fractions from SNP-treated cells showed 90% oxidation of thiols in the cell walls compared with those in control cells. The toxicity of SNP involves S-nitrosation and reduction, the lack of toxicity of the Ru analog being consistent with the fact that it has poor reactivity towards thiols. When C. sporogenes cells were exposed to sublethal concentrations of SNP and viewed under the electron microscope, they showed blisters on the surface. These results point to the cell wall surface as a primary point of attack of the nitrosyl complex.