Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

The role of monocytes in rheumatoid synovitis

In non-specific and rheumatoid synovitis, the use of specific monoclonal antibodies against antigenic determinants of cells of the immune system showed that the characteristic changes of rheumatoid synovitis are located in the synovial internal layers. The monocytes were OKM1, OKM5, S100, OKDR positive, while the subintimal monocytes in non-specific synovitis were OKDR negative. We suggest that, in rheumatoid synovitis, the previously activated monocytes are transported by the bloodstream and pass through the so-called "sinovial barrier" to arrive in the subintimal layers ready to interact with T helper lymphocytes and initiate the immune response mechanisms responsible for lesions in rheumatoid synovitis.     

Familial porphyria cutanea tarda: hybridization analysis of the uroporphyrinogen decarboxylase locus

Familial porphyria cutanea tarda (PCT) results from a deficiency of uroporphyrinogen decarboxylase (URO-D) activity. Hybridization analysis of genomic DNA from unrelated normal individuals and PCT pedigree members failed to detect any major deletions, rearrangements or restriction fragment length polymorphisms at the URO-D locus.     

Nodular fasciitis initially diagnosed by aspiration cytology

A case of nodular fasciitis diagnosed by fine needle aspiration cytology is described. The cytologic findings included fusiform cells, mitoses, macrophages, multinucleated giant cells and mesenchymal elements in a characteristic granular background substance. The cytopathologic diagnosis was subsequently confirmed by the histopathologic study of the tumor and by electron microscopy.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.     

Fungitoxic effects of nonprotein imino acids on growth of saprophytic fungi isolated from the leaf surface of Calliandra haematocephala

Four saprophytic and pathogenic fungi were isolated from the leaf surface of Calliandra haematocephala, a tropical legume known to contain large amounts of rare nonprotein imino acids in its leaves and seeds. The fungi Aspergillus niger, Aspergillus sp., Curvularia sp., and Penicillium sp. were cultured in the laboratory and tested for susceptibility to leaf extracts of the host plant and to proline, pipecolic acid, cis-5-hydroxypipecolic acid, and 2,4-trans-4,5-cis-4,5-dihydroxypipecolic acid. Fungal spore germination and germ tube growth were measured. Aspergillus sp. was inhibited by plant extracts and by pipecolic acid and cis-5-hydroxypipecolic acid. Curvularia sp. growth was stimulated by plant extracts and by pipecolic acid. The other two fungi were unaffected by any of the treatments. The data indicate that imino acids may play a role in the specific resistance of Calliandra spp. to Aspergillus sp.     

Assignment of human uroporphyrinogen decarboxylase (URO-D) to the p34 band of chromosome 1

Summary A cDNA probe corresponding to mRNA encoding human uroporphyrinogen decarboxylase (URO-D) was used to determine the chromosomal localization of the URO-D gene in the human genome. In agreement with previous studies, we have found that the locus for URO-D is located on chromosome 1 in hybrid cell mapping panels. The use of in sity hybridization allowed us to map the URO-D locus to band 1p34.Part of this work was presented as an abstract entitled Localization of the uroporphyrinogen decarboxylase gene to 1p34 band, by in situ hybridization, by M. G. Mattei, A. Dubart, D. Beaupain, M. Goossens, and J. F. Mattei, for a poster presentation at the 8th International Conference on Human Gene Mapping, Helsinki, August 4–10, 1985     

Hints on the evolutionary design of a dimeric RNase with special bioactions.

Residues P19, L28, C31, and C32 have been implicated (Di Donato A, Cafaro V, D'Alessio G, 1994, J Biol Chem 269:17394-17396; Mazzarella L, Vitagliano L, Zagari A, 1995, Proc Natl Acad Sci USA: forthcoming) with key roles in determining the dimeric structure and the N-terminal domain swapping of seminal RNase. In an attempt to have a clearer understanding of the structural and functional significance of these residues in seminal RNase, a series of mutants of pancreatic RNase A was constructed in which one or more of the four residues were introduced into RNase A. The RNase mutants were examined for: (1) the ability to form dimers; (2) the capacity to exchange their N-terminal domains; (3) resistance to selective cleavage by subtilisin; and (4) antitumor activity. The experiments demonstrated that: (1) the presence of intersubunit disulfides is both necessary and sufficient for engendering a stably dimeric RNase; (2) all four residues play a role in determining the exchange of N-terminal domains; (3) the exchange is the molecular basis for the RNase antitumor action; and (4) this exchange is not a prerequisite in an evolutionary mechanism for the generation of dimeric RNases.     

Mutant hemoglobin stability depends upon location and nature of single point mutation

The temperature dependence of the rates of heme release from the beta subunits of methemoglobin A and 5 beta mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the alpha 1/beta 2 interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.     

Effect of temperature on the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases

A study on the response of the stability and activity of crystalline ox liver nuclear and mitochondrial glutamate dehydrogenases to temperature variations has been carried out. The thermodynamic properties of the heat inactivation process and of the reaction with the substrates glutamate and α-ketoglutarate have been investigated. The heat inactivation of nuclear glutamate dehydrogenase proceeds at a faster rate than that of the mitochondrial enzyme in the temperature range 40–51 °C; the enthalpy of activation of the inactivation process is higher and the entropy is almost double, compared to the values of mitochondrial glutamate dehydrogenase. The effect of temperature on the maximal velocity shows that, with both glutamate and α-ketoglutarate, the enthalpy of activation with nuclear glutamate dehydrogenase is double and the decrease in entropy almost half of the values of the mitochondrial enzyme. The variation of the apparent K_m with temperature shows a decrease of the affinity of both enzymes for glutamate, with no major difference in the thermodynamic properties of the reaction. With α-ketoglutarate, on the other hand, the affinity of nuclear glutamate dehydrogenase decreased, whereas that of the mitochondrial enzyme increased with temperature. The process is therefore exothermic with the former enzyme, endothermic with the latter; furthermore, it occurs with a decrease in enthropy with nuclear glutamate dehydrogenase, but with a large increase with the mitochondrial enzyme. The studies on the effect of temperature on the activity were carried out in the range 20–44 °C.