Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Regional histochemical aspects of xanthine oxidase activity in ischemic and reperfused small intestine of the rat

The study describes regional changes of xanthine oxidase and succinate dehydrogenase activities as shown by the ischemic and reperfused small intestine of the rat. The results are obtained with enzyme histochemical methods, including densitometrical verifications, and are substantiated with biochemical enzyme determinations. The decrease of xanthine oxidase activity was best visible in the anoxic duodenum and jejunum, where the findings of histochemical enzyme determinations agreed with those achieved biochemically. The activities of succinate dehydrogenase as measured densitometrically may serve as a further control, considering also the typical intracellular distribution of the reaction products.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Mutation in phenol-type herbicide resistance maps within the psbA gene in Synechocystis 6714

A Synechocytis 6714 mutant resistant to the phenol-type herbicide ioxynil was isolated and characterized. Sensitivity to DCMU and atrazine was tf measured in whole cells and isolated thylakoids. The mutant presents the same sensitivity to atrazine as the wild type and a slightly increased sensitivity to DCMU. A point mutation has been found at codon 266 in the psbAI coding locus (AAC to ACC) resulting in an amino acid change from asparagine to threonine in the D1 protein.     

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.     

The CYP2D gene subfamily: analysis of the molecular basis of the debrisoquine 4-hydroxylase deficiency in DA rats

The DA rat has been proposed as an animal model for the human debrisoquine 4-hydroxylase/bufuralol 1'-hydroxylase genetic deficiency. To determine the mechanism of this deficiency, we isolated and sequenced five cDNAs in the CYP2D gene subfamily including a new IID1 allele and two cDNAs of novel P450s, designated IID3 and IID5. IID3 and IID5 cDNA-deduced amino acid sequences contained 500 and 504 residues with calculated molecular weights of 56,683 and 57,081, respectively. IID5 displayed 20 amino acid differences with the IID1, yet bore only 72% and 76% similarity to IID2 and IID3. Despite an overall nucleotide similarity of 80-98% between the 4 cDNAs, a region of 134 nucleotides of sequence exists that contains only 1 base difference. This region is probably the result of gene conversion events between the P450 IID genes. Although all IID cDNAs were expressed into immunodetectable proteins using the COS cell SV40-based expression system, only IID1 could effectively catalyze the oxidation of the prototype substrate bufuralol. Expression of a cDNA isolated in an earlier study [Gonzalez, F. J., Matsunaga, T., Nagata, K., Meyer, U. A., Nebert, D. W., Pastewka, J., Kozak, C. A., Gillette, J., Gelboin, H. V., & Hardwick, J. P. (1987) DNA 6, 149-161], previously called db1 and now designated IID1v, produced a protein with a drastically reduced activity as compared to cDNA-expressed IID1 despite only four amino acid differences between the two cDNA-deduced protein sequences.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.