Manganese Regulation of Veratryl Alcohol in White Rot Fungi and Its Indirect Effect on Lignin Peroxidase

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.     

Biochemical variation of Cryptococcus neoformans

Ninety-seven strains of Cryptococcus neoformans and C. bacillisporus were examined for 44 biochemical characters and the results were analyzed numerically. One phenon emerged at the 86% level of similarity when strains were clustered according to their M-similarity values. All strains grew in ten carbon sources (D-glucose, D-galactose, arbutin, maltose, sucrose, D-melezitose, D-xylose, D-mannitol, D-glucitol, and meso-inositol), and also grew at 37 ° C and produced urease and phenoloxidase. None of them grew in melibiose, lactose, nor valine, and none reduced nitrate to nitrite. Comparison of selected biochemical characters, creatinine utilization, and serotypes of 49 aberrant strains is presented. Forty-eight of the 97 strains produced the Filobasidiella state either alone or when paired with a strain of compatible mating-type. Filobasidiella neoformans serotypes A and D were interfertile with compatible mating-types of F. bacillispora serotypes B and C. The 44 biochemical characters and 4 serotypes did not predict barriers to mating competence. The present study further substantiates that Filobasidiella neoformans and F. bacillispora are one species.     

Dissociation between Ca2+-ATPase and alkaline phosphatase activities in plasma membranes of rat duodenum

The presence of Ca²⁺-ATPase activities with high-affinity sites for Ca²⁺ in brush border as well as basolateral plasma membranes of rat duodenal epithelium has been reported previously (Ghijsen, W.E.J.M. and van Os, C.H. (1979) Nature 279, 802–803). Since both plasma membranes contain alkaline phosphatase (EC 3.1.3.1), which also can be stimulated by Ca²⁺, the substrate specificity of Ca²⁺-induced ATP-hydrolysis has been studied to determine whether or not alkaline phosphatase and Ca²⁺-ATPase are two distinct enzymes. In basolateral fragments, the rate of Ca²⁺-dependent ATP-hydrolysis was greater than that of ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ at Ca²⁺ concentrations below 25 μM. At 0.2 mM Ca²⁺ the rates of ATP, ADP, AMP and $\text{p-}\text{nitrophenylphosphate}$ hydrolysis were not significantly different. In brush border fragments the rates of ATP, ADP and AMP hydrolysis were identical at low Ca²⁺, but at 0.2 mM Ca²⁺, Ca²⁺-induced hydrolysis of ADP and AMP was greater than either ATP or $\text{p-}\text{nitrophenylphosphate}$. Alkaline phosphatase in brush border and basolateral membranes was inhibited by 75% after addition of 2.5 mM theophylline. Ca²⁺-stimulated ATP hydrolysis at 1 μM Ca²⁺ was not sensitive to theophylline in basolateral fragments while the same activity in brush border fragments was totally inhibited. At 0.2 mM Ca²⁺, Ca²⁺-induced ATP hydrolysis in both basolateral and brush border membranes was sensitive to theophylline. Oligomycin and azide had no effect on Ca²⁺-stimulated ATP hydrolysis, either at low or at high Ca²⁺ concentrations. Chlorpromazine fully inhibited Ca²⁺-stimulated ATP hydrolysis in basolateral fragments at 5 μM Ca²⁺, while it had no effect in brush border fragments. From these results we conclude that, (i) Ca²⁺-ATPase and alkaline phosphatase are two distinct enzymes, (ii) high-affinity Ca²⁺-ATPase is exclusively located in basolateral plasma membranes, (iii) alkaline phosphatase activity, present on both sides of duodenal epithelium, is stimulated slightly by low Ca²⁺ concentrations, but this Ca²⁺-induced activity is inhibited by theophylline and shows no specificity with respect to ATP, ADP or AMP.     

The biogenesis of rat mitochondrial ATPase. Two subunits are synthesized inside the mitochondria

Isolated mitochondria from regenerating rat liver synthesize at least five different polypeptides with molecular weights ranging from 19 000 to 43 000. Among these, two polypeptides with molecular weights of 22 000 and 25 ooo could be identified as ATPase subunits. It has previously been shown that these subunits, designated 6 and 7, are lacking in the ATPase complex that is formed in vivo when mitochondrial protein synthesis is blocked by thiamphenicol treatment. The 22 000 Mr protein is enriched in the fraction containing the fully assembled ATPase complex, whereas the 25 000 Mr protein is not.     

The location of eggs retained in the oviducts of mares

The oviducts of 24 mares were examined to determine the site of retention of unfertilized eggs. The ampullary-isthmic junction regions of 42 of the 48 oviducts were serially sectioned and examined histologically. The remaining parts of the oviducts were flushed and the flushings searched microscopically. Of 45 eggs located, 40 were in the sectioned segments of 24 oviducts and only 5 were in the flushings. All but one of the sectioned segments contained prominent masses of material obstructing the lumen, but these were apparently not the direct cause of egg retention since eggs were found on both sides of them.     

Differential behavior of cytotoxic effector cells against HLA antigens in strong genetic linkage disequilibrium

Five sets of cytotoxic effector cells were generated, using haploidentical, first degree relatives in five different families, against the HLA-A3; B7 serological determinants combined with different DR antigens. When tested against a panel of cells bearing combinations of the HLA-A, -B and -DR antigens it was shown that the HLA-B7 antigen was as strong a CML target determinant alone as it was in the presence of HLA-A3. The strength of the HLA-A3 antigen as target determinant varied. With effector cells primed to the HLA-A3; B7; DR2 haplotype, the A3 antigen alone behaved as a weak target determinant. When the same target cells were tested with the effector cells generated against HLA-A3; B7 without DR2, the A3 antigen behaved as a strong target determinant. A number of target cells lacking the serologically detectable HLA determinants present on the sensitizing HLA haplotype were identified as being killed by specific effector cells. These data suggest either a number of new CML target determinants controlled by different loci or the presence of a single, new locus with multiple alleles controlling CML targets.     

Modification by simetryn sulphoxide of a specific thiol group in rat haemoglobin.

Native rat haemoglobins were found to bind simetryn sulphoxide to an extent 40-fold greater than human haemoglobin. This specific behaviour was studied by using only high-pressure ('performance') liquid chromatography for the preparative separation of globin chains and the isolation of peptides resulting from chemical and enzymic degradation. High recoveries (greater than 80%) of peptides throughout the procedures in combination with microsequence techniques, allow a definitive assignment of the residue undergoing modification. The haemoglobin beta-chain cystine-125 residue, with a stoichiometry of one per tetramer of rat haemoglobin, was found to be modified. Stereochemical implications of this finding are discussed. Simetryn sulphoxide would appear to be useful as a specific reagent for the mapping of exposed thiol residues in proteins.     

O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.