Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.     

Childhood leukemia in Belarus before and after the Chernobyl accident

Childhood leukemia (ICD 204-208 [1]) incidence rates in the different regions of Belarus are reported for a period before and after the Chernobyl accident (1982-1994). There are, at this point, no recognizable trends towards higher rates.     

Experience in the study of the effects of lead on the health status of children in Belovo]

Lead releases in Belovo town containing metallurgy enterprise had reached 120 tons/year earlier, but in recent years have decreased to 9 tons/year. Reduction of the production induced decrease of lead levels in the ambient air from 0.7-2.3 mg/m3 in 1994 to 0.001-0.24 mg/m3. Lead concentration in the soil ranges from 30 to 3000 mg/kg. Lead levels were measured in serum of 91 children, in hair of 67 ones and in teeth of 15 children. Serum lead levels in children aged 7-8 years varied from 0.5 to 39 mg/dl, with an average of 9.9 mg/dl (SD is 5.2 mg/dl), geometric mean is 8.5 mg/dl and error of geometric mean is 3.3. 46% of the children had serum lead levels exceeding the normal one (10 mg/dl). Average lead level in the hair equaled 4.5 mg/g (SD is 4.9 mg/g). The children living in towns with higher environmental lead levels demonstrated more frequent anxiety and changes in higher psychic functions. The major points influencing the serum lead level are proximity to highway, dietary load of goods grown near the residence, mother's smoking. Biokinetic model describing lead transfer into the blood helped to evaluate various modes of the enterprise functioning and efficiency of some environmental protection measures. The most efficient are measures aimed to lower dietary intake of lead, less efficiency is associated with measures reducing lead levels in air, dust and soil.     

Characterization of benzo[a]pyrene-initiated mouse skin papillomas for Ha-ras mutations and protein kinase C levels

The frequency and spectrum of Ha-ras mutations in benzo[a]pyrene (B[a]P)-initiated/12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted CD-1 mouse skin papillomas were characterized by amplifying high molecular weight papilloma DNA using the polymerase chain reaction (PCR) followed by direct DNA sequencing. Analysis of 10 individual B[a]P-initiated early emergence papillomas indicated that 90% contained a Ha-ras mutation. Twenty percent of these papillomas contained a GGA-->GTA transversion in the 12th codon, 50% contained a GGC-->GTC transversion in the 13th codon and 20% contained a CAA-->CTA transversion in the 61st codon. A characteristic of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated papillomas, which contain an A-->T mutation in the 61st codon of Ha-ras, is that they exhibit a constitutive decrease in both protein kinase C (PKC) activity and PKC alpha and beta 2 isozyme levels when compared to epidermis. In the present study we found that total PKC activity, as well as PKC alpha and beta 2 isoforms, were markedly decreased in B[a]P-initiated early emergence papillomas and that this decrease was also accompanied by an altered subcellular distribution of PKC activity. The particulate/cytosolic (P/C) ratio of PKC activity in the epidermis was 0.39, whereas the P/C ratio in the papillomas was 0.77. These results demonstrate that B[a]P-initiated/TPA-promoted papillomas exhibit a high incidence of specific ras mutations and that PKC levels are constitutively decreased in these papillomas, indicating that an activated ras gene is associated with and may contribute to the observed decrease in PKC levels.     

The crystallographic structure of the protease from human immunodeficiency virus type 2 with two synthetic peptidic transition state analog inhibitors

The crystal structure of human immunodeficiency virus (HIV) type 2 protease has been determined in complexes with peptidic inhibitors Noa-His-Cha psi [CH(OH)CH(OH)]Val-Ile-Amp (U75875) and Qnc-Asn-Cha psi [CH(OH)CH2]Val-Npt(U92163) (where Noa is naphthyloxyacetyl, Cha is cyclohexylalanine, Amp is 2-aminomethylpyridine, Qnc is quinoline-2-carbonyl, and Npt is neopentylamine), which have dihydroxyethylene and hydroxyethylene moieties, respectively, in place of the normal scissile bond of the natural ligand. The complexes crystallize in space group P2(1)2(1)2(1), with one dimer-inhibitor complex per asymmetric unit and average cell dimensions of a = 33.28 A, b = 45.35 A, c = 135.84 A. Data were collected to approximately 2.5-A resolution. The model structures were refined with resulting R-factors of around 0.19. As expected, the HIV-2 protease structure is approximately C2-symmetric with a gross structure very similar to that of the HIV-1 enzyme. The inhibitors bind in an extended conformation positioned lengthwise in the binding cleft in a manner similar to that found in the HIV-1 protease-inhibitor complexes previously reported. The substitution of the bulkier Ile82 side chain in the HIV-2 protease may help explain the better ability of HIV-2 protease to bind and hydrolyze ligands with small P1 and P1' side groups. It appears that differences in specificity between the proteases of HIV-1 and HIV-2 are not merely a result of simple side chain substitutions, but may be complicated by differences in main chain flexibility as well.     

Toxicity of L-ascorbic acid to L929 fibroblast cultures: relevance to biocompatibility testing of materials for use in wound management

Fibroblast cultures are often used to evaluate materials intended for medical use, cytotoxicity being taken as an indicator of bioincompatibility. Such an approach has previously been taken with ascorbic acid in determining its value in wound healing. We have now reexamined the toxicity of L-ascorbic acid to L929 fibroblast cells in culture. Concentrations of ascorbic acid between 0.5 mM and 11 mM were tested. At concentrations above 2 mM, ascorbic acid was found to inhibit cell proliferation, with cell viability decreasing as the concentration was increased. This effect could be prevented by the addition of either superoxide dismutase or catalase to the culture medium. Assays of glutathione and glutathione disulfide were carried out on 8 day old cultures exposed for 24 h to the same concentrations of ascorbic acid. A dose-related depletion of glutathione occurred whilst glutathione disulfide levels remained essentially constant. Lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities were induced by ascorbic acid at all concentrations tested but the ratio of NADP to NADPH nevertheless increased as the concentration of ascorbic acid increased. Finally, ATP in cells from 8-day-old cultures became depleted in the presence of ascorbic acid at concentrations in excess of about 5 mM when assayed after 24 h incubation. These biochemical changes and the concomitant cytostatic/cytotoxic effects may be ascribed to the reactive oxygen species produced by the autoxidation of ascorbic acid in the culture medium. Ascorbic acid breakdown products appeared not to be directly involved. In addition, our results suggested that superoxide acted cooperatively with hydroxyl to elicit these effects on the fibroblasts. It is evident from this study that the microenvironment surrounding fibroblasts in culture may differ fundamentally from that surrounding fibroblasts in a healing wound, making it impossible to extrapolate directly to an in vivo situation and hence to make any recommendations from these results concerning the use of ascorbic acid in wound healing.     

A new enzyme superfamily - the phosphopantetheinyl transferases

BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active. The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain. The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase. Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS. RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs. This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases. Three of these proteins, E. coli EntD and o195, and B. subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function. Three P-pant transferases are now known to be present in E. coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively. The apo-protein substrate for o195 has not yet been identified. Sfp is responsible for the activation of the surfactin synthetase. CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains. This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.     

Use of trans-retinoic acid in the treatment of acute promyelocytic leukemia

OBJECTIVE: To discuss acute promyelocytic leukemia (APL) and review the literature concerning differentiation treatment of APL with trans-retinoic acid (t-RA). DATA SOURCES: English-language articles concerning APL or its treatment with t-RA were identified with a MEDLINE search. STUDY SELECTION: All studies available at the time of article preparation, which addressed t-RA treatment in APL, were selected. DATA EXTRACTION: Data extraction and assessment were performed subjectively by the authors. An extensive discussion of specific study details is included in the article. DATA SYNTHESIS: APL is a unique subset of acute myelogenous leukemia and is typified by an accumulation of malignant promyelocytes in the bone marrow. Within the granulocyte cell cycle of a patient with APL, differentiation has been halted at the level of the promyelocyte, preventing formation of mature granulocytes. Upon treatment with traditional cytotoxic chemotherapy, complete remission rates of approximately 70 percent, with a five-year survival ranging from 25 to 40 percent have been achieved. In most patients with APL, a characteristic chromosomal t(15q+;17q-) translocation has been found, which may be responsible for the production of an aberrant retinoic acid receptor-alpha. Therefore, t-RA induction therapy has been investigated and has produced promising results. Administration of t-RA in dosages of 45-100 mg/m2/d has induced complete remissions. The apparent mechanism of t-RA is the induction of promyelocyte differentiation and maturation. The most common adverse effects noted have been dry skin, cheilitis, and headaches. CONCLUSIONS: Upon consideration of the initial trials, t-RA appears to be a promising and unique treatment for APL.     

Systemic leakage during isolated limb perfusion for melanoma

A hazard of regional perfusion for melanoma is incomplete isolation, resulting in leakage of the cytostatic drug into the systemic circulation. Data were analysed retrospectively on 438 melphalan perfusions performed for melanoma of the extremities during the period 1978-1990; continuous isotopic measurement of systemic leakage was carried out. The cumulative systemic leakage after 60 min perfusion was 0.9 per cent (95 per cent confidence interval 0.7-1.1 per cent). Systemic leakage of > or = 1 per cent was detected in 12.6 per cent of perfusions, > or = 5 per cent in 6.2 per cent and > or = 10 per cent in 1.4 per cent. In 2.3 per cent of patients, systemic side-effects in the form of mild transient bone marrow depression occurred. Six variables related to the perfusion technique were assessed by multivariate analysis for their influence on systemic leakage. The level of isolation and diameter of the venous cannula emerged as significant factors. In addition, ligation of the internal iliac vein provided optimal isolation during iliac perfusion.