Functional consequences of the excision of an omega loop, residues 40-55, from mitochondrial cytochrome c

A novel technique for protein semisynthesis, enzymic activation, has been used to create a mitochondrial cytochrome c analogue in which the conventional bottom loop has been deleted. The resulting structure resembles that of cytochrome c555 from a primitive photosynthetic sulfur bacteria. Comparisons of this analogue with natural cytochromes show which of the functional differences between cytochromes c and c555 may be related directly to the incorporation of the loop. The structure is an example of an omega loop, recently defined as a discrete category of protein secondary structure. The analogue maintains the overall structure of the parent protein, but a significant change in redox potential has been engineered. It provides support for the prediction that omega loops act as independent modules in folding, function, and evolution. The rapidity of the synthesis and the high yield of product show that this technique for protein engineering is both competitive with, and complementary to, genetic methods.     

Demographic Trends of Aging — Application to Gerodontology1

Aging of the global world population both in developed and developing countries, an unbalanced male-female ratio within this elderly population, increasing populations – including elderly – within urban areas, all will have a marked effect on the practice of dentistry. Furthermore, major epidemiological changes occurring in the dental field, including an increasing dentition life expectancy, increasing root caries prevalence, decreasing coronal caries rates in children, and the relative increase in the prevalence of periodontal problems will have further impact on the dental care system. Major changes need to be anticipated both at the level of the care provider, including the dentist, the hygienist and the dental assistant, and at the level of the potential patient, especially among the geriatric population, if the profession is to be prepared to adequately address these issues.     

Enhanced Survival of the Cyanobacterium Oscillatoria terebriformis in Darkness under Anaerobic Conditions

Oscillatoria terebriformis, a thermophilic cyanobacterium, maintained viability in darkness under anaerobic conditions by fermenting exogenous glucose or fructose to lactic acid. The time period of survival was greatly extended when the environmental redox potential was lowered by the addition of sodium thioglycolate or titanium(III) citrate. When exposed to aerobic conditions in darkness, many trichomes underwent lysis in 6 h, and death of all cells occurred in 2 to 3 days. The endogenous aerobic respiration rate was high, and the limited dark aerobic survival period appeared to be due to depletion of stored glycogen. Fructose or glucose did not support or increase aerobic respiration in darkness or lengthen aerobic survival time. Enhanced survival of O. terebriformis in darkness under anaerobic, reducing conditions correlates well with the natural nighttime position of this species within sulfide-rich microbial mats associated with hot springs of western North America.     

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Regression of blood vessels precedes cartilage differentiation during chick limb development

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.     

Purification and separation of two soluble fucosyltransferase activities of small intestinal mucosa

1. Rat small intestinal soluble fucosyltransferase is purified more than 2000-fold using chromatographic procedures with DEAE-cellulose, CM-cellulose, GDP-Sepharose and Concanavalin A-Sepharose. 2. Chromatography on Sephadex G15 of the final enzymatic fraction clearly separates two activities: a first peak incorporates fucose on asialoserotransferrin and a second peak on asialofetuin. 3. The use of small saccharidic acceptors (phenylgalactose, lactose, lacto-N-fucopentaose I) and the analysis of fucosylated asialoglycoproteins indicate that the first activity corresponds to an alpha-(3/4)-fucosyltransferase and the second one to an alpha-(1-2)-fucosyltransferase. 4. Protein analysis by polyacrylamide gel electrophoresis in the presence of SDS for each enzyme shows two bands corresponding to a mol. wt of about 65,000 and 70,000. The two enzymes have the same sensitivity to the action of N-ethylmaleimide.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

A toxin fromPasteurella multocida serogroup D enhances swine herpesvirus 1 replication/lethality in vitro and in vivo

In swine, the nasal turbinate epithelium is both a site of swine herpesvirus 1 (pseudorabies virus, PRV) replication and a tissue affected by toxin fromPasteurella multocida serogroup D. We examined the effects of exposure to PRV and exposure to toxin in mice, swine, and nasal turbinate cell cultures. Increased mortality in mice was observed when nonlethal doses of PRV (1000 or 100 plaque-forming units, PFU) were administered along with nonlethal doses (60–200 ng/kg) of toxin. In swine, clinical disease and death in adult pigs was observed after an intradermal injection of toxin (20 ng/kg) and intranasal exposure to 1000 PFU/kg of PRV. Nasal turbinate cell cultures incubated with toxin and PRV had increased protein synthesis, DNA synthesis, and increased recovery of virus particles. These findings indicate that a toxin fromP. multocida serogroup D enhances swine herpesvirus 1 replication and lethality in cell cultures and animal models.