Evidence implicating heterozygous deletion of chromosome 7 in the pathogenesis of familial leukemia associated with monosomy 7.

Complete or partial monosomy 7 is a recurring cytogenetic abnormality in acute myelogenous leukemia (AML) and myeloproliferative syndromes (MPS) and is particularly common in patients with Fanconi's anemia and in secondary AML. A familial form of monosomy 7 has been recognized in which two or more siblings develop MPS or AML before age 20. We tested the hypothesis that a recessive cancer susceptibility locus on chromosome 7 was important in the pathogenesis of leukemia in familial monosomy 7 by determining the parental origins of the chromosome 7 retained in the bone marrows of three pairs of affected siblings. We found no overlapping region where all three pairs retained DNA derived from the same paternal or maternal chromosome. These data suggest that inactivation of a single allele of a putative tumor-suppressor gene may be sufficient to contribute to leukemic transformation in familial monosomy 7.     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Antibiotic production by the marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: localization of activity to the chromatophores

Abstract Over 200 strains of marine purple photosynthetic bacteria were isolated. Two strains showed antibiotic activity towards Saccharomyces cerevisiae and were tentatively identified as Chromatium purpuratum . Crude antibiotic, prepared by solvent extraction, showed a broad antimicrobial spectrum. The highest activity was found in the chromatophore fraction. Chromatographic separation of purified light harvesting complex from one strain, NKPB 031704, showed the presence of two separate pigmented compounds which were responsible for antimicrobial activity. Our findings reveal the unexpected ability of photosynthetic bacteria to produce broad spectrum antibiotics. In addition, this is the first example of intracellular localization of antibiotic activity in a marine bacterium.     

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.     

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.     

The use of the rat hepatoma cell line MH1C1 to investigate the stimulus-secretion response of hepatic acute phase proteins

The rat hepatoma cell line MH1C1 has been characterized to show a stimulated secretion of C-reactive protein in response to both leukocyte supernatant and a purified human interleukin-1 preparation. The time-dependency and dose-response relationship of CRP secretion were comparable to and somewhat more sensitive than the effects of leukocyte supernatant and purified human interleukin-1 on the proliferative rate of murine thymocytes; the proliferative rate of the hepatoma cell line MH1C1 was unchanged under these conditions. Agents which affect the thymocyte bioassay response to interleukin-1 namely interleukin-2, lipopolysaccharide, concanavalin A and phytohemagglutinin showed no effect on the C-reactive protein release of the MH1C1 cell line. These data strongly support the suitability of this cell line for the in vitro study of the hepatic acute phase stimulus-secretion response.     

Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of hydrocortisone and cyclic AMP

We have examined the effect of hydrocortisone and cyclic AMP on the maintenance of lipid synthesis in primary cultures of adult rat alveolar type II cells. These hormones were tested in the presence of either 1% or 5% charcoal-stripped rat serum (CS-rat serum). The effect of substratum on responsiveness to these hormones was evaluated by comparing cells cultured for 4 days on tissue culture plastic, on floating type I collagen gels, on rat lung fibroblast feeder layers on floating collagen gels (floating feeder layers), and on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. Type II cells cultured on floating feeder layers in medium containing 1% CS-rat serum and 10(-5) M hydrocortisone plus 0.5 mM dibutyryl cyclic AMP exhibited significantly increased incorporation of [14C]acetate into total lipids (238% of control). The hormone combination also increased the relative percentage of acetate incorporated into phosphatidylglycerol (PG; 7.3% versus 1.9%) and saturated phosphatidylcholine (PC; 43.6% versus 37.6%). The percentage of acetate incorporated into neutral lipids was significantly decreased by the addition of hormones (28.6% versus 70.0%). The addition of hydrocortisone and cyclic AMP to medium containing 5% CS-rat serum resulted in an increase in the relative incorporation of acetate into saturated PC (51.2% versus 46.4%), but had no effect on the relative incorporation of acetate into PG or on the incorporation of acetate into total lipids. Type II cells cultured on EHS gels in medium containing 1% CS-rat serum plus hydrocortisone and cyclic AMP showed increased acetate incorporation into total lipids (204% of control) and a relative decrease in the percentage of acetate incorporated into neutral lipids (16.9% versus 47.0%). The hormone combination also increased the relative incorporation of acetate into PG (4.4% versus 2.5%) and saturated PC (49.9% versus 42.1%). Hydrocortisone and cyclic AMP added to medium containing 5% CS-rat serum concentration increased the relative incorporation of acetate into saturated PC by type II cells on EHS gels, but these additions had no effect on acetate incorporation into PG. No responses to these soluble factors were seen when type II cells were cultured on floating type I collagen gels without feeder layers or on tissue culture plastic. These data indicate that there are positive interactions between substratum, soluble factors and serum in the maintenance of differentiated function of adult rat alveolar type II cells in vitro.     

Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.     

Purification and characterization of an inhibitor of plasminogen activator released by rat mammary adenocarcinoma cells

An inhibitor of plasminogen activator (PA) secreted by a tumorigenic, but non-metastatic, rat mammary adenocarcinoma cell line has been purified to apparent homogeneity and characterized. It strongly inhibited human urokinase, but was 100 times less potent in inhibiting bovine trypsin and had no effect on plasmin or thrombin. A secreted, urokinase-type PA (Mr 48 000) and a cell-associated PA from a metastatic rat adenocarcinoma cell line were also strongly inhibited. In contrast, a tissue-type PA (Mr 66 000), secreted by human melanoma cells, was only slightly inhibited. Purified inhibitor showed a band of Mr 66 000 in sodium dodecyl sulphate/polyacrylamide gel electrophoresis and an isoelectric point of 4.5 after chromatofocusing. The inhibition of human urokinase was non-competitive.     

Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5-Hydroxytryptamine in the Rat Hypothalamus

The effects of tryptophan administration on neurochemical estimates of synthesis [5-hydroxytryptophan (5-HTP) accumulation following administration of a decarboxylase inhibitor], storage [5-hydroxytryptamine (5-HT) concentrations], and metabolism [5-hydroxyindoleacetic acid (5-HIAA) concentrations] of 5-HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30-300 mg/kg i.p.) produced a dose-dependent increase in the rate of 5-HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4-dihydroxyphenylalanine. Peak 5-HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose-dependent increases in 5-HT and 5-HIAA concentrations in these same regions without changes in the 5-HIAA/5-HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5-HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5-HT uptake with chlorimipramine or fluoxetine produced modest (10-40%) reductions in 5-HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5-HIAA is derived from released and recaptured 5-HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5-HT. In both chlorimipramine- and fluoxetine-treated rats, 5-HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5-HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5-HT.