Differential effects of hyperoxia and hydrogen peroxide on DNA damage, polyadenosine diphosphate-ribose polymerase activity, and nicotinamide adenine dinucleotide and adenosine triphosphate contents in cultured endothelial cells and fibroblasts

The effects of oxidative stress on DNA damage and associated reactions, increased polyadenosine diphosphate-ribose polymerase (PARP) activity and decreased nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) contents, have been tested in primary cultures of porcine aortic endothelial cells. The cells were treated with 50-500 microM H2O2 for 20 min or 100 microM paraquat for 3 days or were exposed to 95% O2 for 2 and 5 days. The administration of 250-500 microM H2O2 resulted in a marked increase in PARP activity and a profound depletion of ATP and NAD. Although hyperoxia had no effect on PARP activity and reduced only slightly the ATP and NAD stores, it markedly reduced the ability of endothelial cells to increase PARP activity upon exposure to DNase. Paraquat had a similar effect. Human dermal fibroblasts were also exposed to 50-500 microM H2O2 for 20 min or 95% O2 for 5 days. Their response to H2O2 differed from that of endothelial cells by their ability to maintain the ATP content at a normal level. Fibroblasts were also insensitive to the effect of hyperoxia. These results suggest that the oxidant-related DNA damage is a function of the type of oxidative stress used and may be cell-specific.     

Metabolic engineering of Clostridium acetobutylicum ATCC 824 for increased solvent production by enhancement of acetone formation enzyme activities using a synthetic acetone operon

The ability to genetically alter the product-formation capabilities of Clostridium acetobutylicum is necessary for continued progress toward industrial production of the solvents butanol and acetone by fermentation. Batch fermentations at pH 4.5, 5.5, or 6.5 were conducted using C. acetobutylicum ATCC 824 (pFNK6). Plasmid pFNK6 contains a synthetic operon (the "ace operon") in which the three homologous acetone-formation genas (adc, ctfA, and ctfB) are transcribed from the adc promoter. The corresponding enzymes (acetoacetate decarboxylase and CoA-transferase) were best expressed in pH 4.5 fermentations. However, the highest levels of solvents were attained at pH 5.5. Relative to the plasmid-free control strain at pH 5.5, ATCC 824 (pFNK6) produced 95%, 37%, and 90% higher final concentrations of acetone, butanol, and ethanol, respectively; a 50% higher yield (g/g) of solvents on glucose; and a 22-fold lower mass of residual carboxylic acids. At all pH values, the acetone-formation enzymes were expressed earlier with ATCC 824 (pFNK6) than in control fermentations, leading to earlier induction of acetone formation. Furthermore, strain ATCC 824 (pFNK6) produced butanol significantly earlier in the fermentation and produced significant levels of solvents at pH 6.5. Only trace levels of solvents were produced by strain ATCC 824 at pH 6.5. Compared with ATCC 824, a plasmid-control strain containing a vector without the ace operon also produced higher levels of solvents [although lower than those of strain ATCC 824 (pFNK6)] and lower levels of acids. Strains containing plasmid-borne derivatives of the ace operon, in which either the acetoacetate decarboxylase or CoA-transferase alone were expressed at elevated levels, produced acids and solvents at levels similar to those of the plasmid-control strain. (c) 1993 John Wiley & Sons, Inc.     

Presymptomatic direct detection of adenomatous polyposis coli (APC) gene mutations in familial adenomatous polyposis

The recent identification of the familial adenomatous polyposis (FAP) gene (designated as APC) enables conclusive genetic testing of at-risk family members for the specific mutation in families in which the germline gene mutation has been characterized. Presymptomatic molecular diagnosis of FAP was performed by direct direction of mutations in lymphocyte DNA in four families. Each of the families has a different mutation of the APC gene. Twenty-seven offspring of affected individuals (a priori risk of 50%) were tested. Ten of the 27 had already developed clinical features of FAP. Of the remaining seventeen, two had had a negative colon exam at an early age, and nine had never had colon exams (mean age, 12.1±3.1 SD years). Six children from this group (54%) were found to carry their affected parent's mutation. No change in the conventional FAP colon screening regimen is recommended for these children. In contrast, when direct tests indicate that an individual does not have the FAP mutation, we recommended that screening be decreased. Reduction of uncertainty for at-risk FAP family members is an important benefit of genetic testing.     

Posttranslational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-glycosylation sites.

Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Genetic polymorphism of serum complement components in the chimpanzee

Significant polymorphism of serum complement components Bf, C2, C3, C6, and C8 in the chimpanzee has been demonstrated. The data are consistent with the hypothesis thatC2 andBf are closely linked toChLA and argue against close linkage ofChLA toC3 or toC8, as in man. In addition, a blank allele for C6 and C6 deficiency was detected in several chimps.     

Ionic currents across pancreatic acinar cell membranes and their role in fluid secretion

Fluid and enzyme secretion from a number of mammalian exocrine glands is controlled by the action of neurotransmitters and hormones on acinar cell membranes. Sustained stimulation evoking sustained fluid and enzyme secretion also evokes sustained membrane depolarization and increase in conductance. Mouse and rat pancreatic fluid and enzyme secretion, as well as membrane depolarization and conductance increase evoked by sustained stimulation with acetylcholine or cholecystokinin-gastrin peptides, are acutely dependent on extracellular calcium. However, the initial stimulant-evoked conductance increase and secretion appear to be triggered by calcium released from inside the cells. Direct measurement of membrane current during sustained stimulation in voltage-clamp experiments with resolution of the total current into its Na, Cl and K components has allowed calculations of stimulant-evoked Na and Cl uptake into the acinar cells. The NaCl uptake is quantitatively sufficient to account for the stimulant-evoked fluid secretion. The role of the stimulant-evoked transmembrane ionic current appears to be the supply of salt for the fluid secretion. Calcium derived from intracellular sources in the initial phase of secretion, and from the extracellular fluid in the sustained phase, couples fluid and enzyme secretion to hormone-receptor interaction.     

Drilomermis leioderma n. gen., n. sp. (Mermithidae:Nematoda) parasitizing Cybister fimbriolatus (Say) (Dystiscidae-Coleoptera)

The nematode Drilomermis leioderma n. gen., n. sp. (Merrnithidae) is described from larvae of Cybister fimbriolatus (Say) (Dytiscidae: Coleoptera) in Louisiana. Diagnostic characters of the genus Drilomermis are: medium-sized nematodes with the cuticle appearing smooth (lacking cross fibers) under the light microscope, six cephalic papillae, without mouth papillae, six hypodermal cords at midbody, 2 extremely long spicules (longer than 10 times body width at anus) which are separate and parallel (not twisted), an S-shaped vagina, medium-sized amphids located near head papillae, and postparasitic juvenile with a tail appendage. D. leioderma possesses a ventrally displaced mouth, very long vagina, and male genital papillae arranged in 3 double rows in the vicinity of the cloacal opening. Even when containing multiple parasites, about 40% of the hosts sulwived emergence of the memithids and lived several more days. In nature, some of these hosts may be able to continue their development, which is unusual since most mermithid-parasitized hosts die soon after the nematode emerges.