A soluble fatty acyl-acyl carrier protein synthetase from the bioluminescent bacterium Vibrio harveyi

An enzyme catalyzing the ligation of long chain fatty acids to bacterial acyl carrier protein (ACP) has been detected and partially characterized in cell extracts of the bioluminescent bacterium Vibrio harveyi. Acyl-ACP synthetase activity (optimal pH 7.5-8.0) required millimolar concentrations of ATP and Mg2+ and was slightly activated by Ca2+, but was inhibited at high ionic strength and by Triton X-100. ACP from either Escherichia coli (apparent Km = 20 microM) or V. harveyi was used as a substrate. Of the [14C]fatty acids tested as substrates (8-18 carbons), a preference for fatty acids less than or equal to 14 carbons in length was observed. Vibrio harveyi acyl-ACP synthetase appears to be a soluble hydrophilic enzyme on the basis of subcellular fractionation and Triton X-114 phase partition assay. The enzyme was not coinduced with luciferase activity or light emission in vivo during the late exponential growth phase in liquid culture. Acyl-ACP synthetase activity was also detected in extracts from the luminescent bacterium Vibrio fischeri, but not Photobacterium phosphoreum. The cytosolic nature and enzymatic properties of V. harveyi acyl-ACP synthetase indicate that it may have a different physiological role than the membrane-bound activity of E. coli, which has been implicated in phosphatidylethanolamine turnover. Acyl-ACP synthetase activity in V. harveyi could be involved in the intracellular activation and elongation of exogenous fatty acids that occurs in this species or in the reactivation of free myristic acid generated by luciferase.     

Human collagen gene COL5A1 maps to the q34.2----q34.3 region of chromosome 9, near the locus for nail-patella syndrome.

Type V collagen is a fibrillar collagen that is widely distributed in tissues as a minor component of extracellular matrix and is usually composed of one pro alpha 2 (V) and two pro alpha 1 (V) chains. In this report, recently isolated cDNA and genomic clones, which encode the pro alpha 1 (V) chain, are used as probes for hybridization to filter-bound DNA from a panel of human-mouse hybrid cell lines and for in situ hybridization to metaphase chromosomes. These studies establish the chromosomal location of the COL5A1 gene, which encodes the pro alpha 1 (V) chain, within segment 9q34.2----q34.3. These findings add to the previously characterized dispersion of collagen genes in the human genome, as this is the first example of a collagen locus on chromosome 9. In addition, these studies place COL5A1 near the locus for the genetic disorder, nail-patella syndrome (hereditary osteo-onychodysplasia), which also maps to 9q34.     

Novel heme-binding component in the serum of the channel catfish (Ictalurus punctatus)

The serum of the channel catfish (Ictalurus punctatus) was examined for heme- and hemoglobin-binding proteins. Electrophoretic mobility retardation assays failed to detect a hemoglobin-binding material similar to mammalian haptoglobin; however, a heme-binding component (not previously described) was identified in catfish seru. The heme-binding component was purified by gel filtration chromatography; electrophoretic analyses suggested it to be composed of two polypeptide subunits of molecular masses about 115 and 98 kDa. This composition is inconsistent with hemopexin, the known heme-binding serum protein of mammals. Although it was not fully saturated with heme, the catfish component contained detectable heme in normal sera. When complexed by the binding material, heme was used as an iron source by isolates of the bacterial Gram-negative genusAeromonas; the capacity of other bacteria to use the complex was not tested. The physiological function of the catfish heme-binding serum protein is presently not clear.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Isolation of plasma membranes from cultured glioma cells and application to evaluation of membrane sphingomyelin turnover

A rapid and reliable method for the isolation of plasma membranes and microsomes of high purity and yield from cultured glioma cells is described. The procedure involves disruption by N2 cavitation, preliminary separation by centrifugation in Tricine buffer, and final separation on a gradient formed from 40% Percoll at pH 9.3. Enzyme and chemical markers indicated greater than 60% yield with six- to eightfold enrichment for plasma membranes and greater than 25% yield with three- to fourfold enrichment for a microsomal fraction consisting mainly of endoplasmic reticulum. The final fractions were obtained with high reproducibility in less than 1 h from the time of cell harvesting. Application of this procedure to human fibroblasts in culture is assessed. The isolation procedure was applied to investigations of synthesis and turnover of sphingomyelin and phosphatidylcholine in plasma membranes of glioma cells following incubation for 4-24 h with [methyl-3H]choline. These studies indicated that radioactivity from phosphatidylcholine synthesized in microsomes from exogenous choline may serve as a precursor of the head-group of sphingomyelin accumulating in the plasma membrane.     

Influence of chemical reactivity of urushiol-type haptens on sensitization and the induction of tolerance

Contact sensitization to components of the urushiol oils of poison oak and poison ivy appears to require covalent bond formation between the o-quinones derived from urushiol catechols and nucleophilic groups on proteins. Previous studies using a murine delayed hypersensitivity model demonstrated that 5-methyl-3-pentadecylcatechol (5-Me-PDC) is an epicutaneous tolerogen to the parent compound and a weak sensitizer to itself. To investigate further the structural requirements for sensitization vs suppression, 5,6-dimethyl-3-pentadecylcatechol (5,6-di-Me-PDC) and 4,5,6-trimethylpentadecylcatechol (4,5,6-tri-Me-PDC) were synthesized. The former compound is blocked at both preferred sites for covalent bond formation and the latter is completely blocked towards conjugate addition reactions. These compounds were tested for sensitizing and suppressive ability. Epicutaneous application of both analogs suppressed subsequent induction of sensitization to 3-pentadecylcatechol (PDC) and 3-heptadecylcatechol (HDC). Lymph node cells from animals treated with 5,6-di-Me-PDC could transfer suppression. The dimethyl analog, 5,6-di-Me-PDC, but not the trimethyl analog also exhibited weak sensitizing capacity. The urushiol analogs 5-pentadecylresorcinol (PDR) and 3-heptadecylveratrole (HDV) which cannot form o-quinones were found to be ineffective sensitizers as well. HDV in addition produced no blastogenesis in draining lymph nodes whereas lymph node cell proliferation induced by 4,5,6-tri-Me-PDC followed the same kinetics as previously observed for HDC. PDR elicited weak proliferation with a different time course. These and previous studies indicate that blocking the C5-position on the catechol ring favors the induction of suppression, although some sensitizing capacity may be retained. Covalent bond formation may not be necessary for the induction of active suppressor cell populations.     

Human genes for insulin-like growth factors I and II and epidermal growth factor are located on 12q22----q24.1, 11p15, and 4q25----q27, respectively

The genes coding for insulin-like growth factors I and II and epidermal growth factor have been localized to human chromosomes 12q22----q24.1, 11p15, and 4q25----q27, respectively.     

Osteogenesis imperfecta type IV: evidence of abnormal triple helical structure of type I collagen

Summary Skin fibroblasts from a patient with mild osteogenesis imperfecta (OI) type IV synthesize two populations of type I procollagen molecules. One population contains pro1(I) and pro2(I) chains that migrate normally in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a second population contains only slower migrating pro1(I) and pro2(I) chains. The total amount of type I procollagen made by OI cells and the ratio of pro1(I): pro2(I) is normal. When labeled under conditions that inhibit post-translational modification of pro chains, the OI cells produce only single populations of pro1(I) and pro2(I) chains indicating that the apparent increased molecular weight of some OI pro chains is due to excessive post-translational modification rather than peptidyl insertions. Peptide maps indicate that excessive post-translational modification occurs along the entire triple helical segment of some 1(I) and 2(I) chains produced by OI cells. The effect of the mutation is to lower the melting temperature of the molecules containing slow migrating 1(I) and 2(I) chains to 39.5°C (compared to 41.5°C for control), and to delay secretion of the overmodified type I procollagen from OI cells. These data are consistent with a mutation near the carboxyl-terminal end of the triple helical domain which delays triple helical formation and renders all chains available for further post-translational modification amino-terminal to the mutation. Such alterations in triple helical structure, thermal stability, and secretion previously associated only with the lethal OI type II phenotype are thus also seen in the mild OI type IV phenotype.     

MORPHOLOGICAL CHANGES AND THE REQUIREMENTS FOR MACROMOLECULE SYNTHESIS DURING EXCYSTMENT OF ACANTHAMOEBA CASTELLANII

Light and phase-contrast microscopic observations of excystment in Acanthamoeba castellanii have been used to classify cells in excysting populations as free trophozoites, or mature, activated, or preemergent cysts. These categories have been used to describe the kinetics of excystment. A pH of 7 and a temperature of 30°C have been found to be optimal for the activation of mature cysts. Both activation and emergence are inhibited by cycloheximide and actinomycin D, but neither process is much affected by hydroxyurea. Cell-free extracts of high molecular weight components of cyst cytoplasm can support protein synthesis in vitro, although less efficiently than similar extracts from trophozoites. Evidence indicates that some of the functional RNA in the cyst extracts is synthesized before excystment.