Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M _r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M _r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with ⁵⁵Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with ⁹⁹Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Comparative evaluation of macrophage stimulation and depression on tumor growth and macrophage content and function in mice

Summary In a syngeneic murine adenocarcinoma model, the administration of glucan, an RE stimulant, inhibited tumor growth and increased tumor macrophage populations. Conversely, the administration of methyl palmitate, an RE depressant, potentiated tumor growth and decreased the number of tumor macrophages. Glucan and methyl palmitate also produced diverse alterations in serum lysozyme levels that reflected their contrasting influences on RE functional status, thus supporting the role of serum lysozyme as an index of macrophage function. The diverse results produced by macrophage stimulation or depression in regard to tumor growth, tumor macrophage population, and serum lysozyme concentration indicate that a relationship may exist between macrophage functional activity and host resistance to neoplasia.     

Post-partum release of prostaglandin F(2alpha) and uterine involution in the cow

Peripheral plasma levels of the main blood plasma metabolite of PGF(2alpha) (15-keto-13,14-dihydro-PGF(2alpha)) and progesterone were investigated during the immediate, post-partum period in 59 normally calving cows. Uterine involution was monitored by weekly rectal palpations. The levels of the prostaglandin metabolite were high at parturition and remained thereafter elevated for periods varying up to 7-23 days. Uterine involution was completed during periods ranging from 16-53 days. According to the clinical findings, the animals were divided into three groups. Group A comprises 46 animals which had an uncomplicated, puerperal period. A significant (p<0.001) correlation between the duration of elevated prostaglandin levels and the time for completed uterine involution (Y=29.6 - 1.3 (X - 13.5)) was found for these animals. Group B animals (n=8) had periods of varying length with uterine discharge during the first 30 days post-partum. When compared to group A animals, the animals in group B had comparatively longer periods of prostaglandin release and also longer periods for completion of uterine involution. Group C animals (n=5) at times had palpable, thin-walled, cystlike structures in the ovaries during the first 30 days post-partum. In this group of animals, the periods of high prostaglandin levels, as well as for the completion of uterine involution, were similar to those for the animals in group A. Progesterone levels remained low during the immediate post-partum period and in no case were elevated levels found until the prostaglandin release had ceased.     

Waterborne transmission ofCampylobacter enteritis

Campylobacter jejuni is an important cause of human diarrheal disease throughout the world and likeSalmonella enteritidis, has a large animal reservoir which includes most of man's domestic animals. Until recently, it has been difficult to trace the chain of transmission from animals to man because of inadequate environmental sampling techniques and means to distinguish strains. Recent improvements in these techniques have made environmental studies more feasible in 2 water-related out-breaks.In 1 study,C. jejuni was found to be an important cause of sporadic, summertime diarrheal disease among hikers in national wilderness areas of Wyoming. In this setting, illness was significantly associated with drinking untreated surface water. SubsequentlyC. jejuni was isolated from surface water, including mountian streams, and from animals in the area. Some of the environmental isolates were serotypically identical to strains isolated from humans.A second study occurred as a result of an outbreak of Campylobacter enteritis in a community in northern Illinois which was epidemiologically associated with the community water system.Campylobacter jejuni was isolated from several surface water sources and from the implicated water system. These studies demonstrate that environmental isolation ofC. jejuni is now possible and may add to our understanding of disease transmission.     

Diurnal patterns of testosterone, dihydrotestosterone, estradiol, and cortisol in serum of rhesus males: Relationship to sexual behavior in aging males

This study was carried out to determine differences between old and young rhesus males in levels and diurnal patterns of testosterone, dihydrotestosterone, cortisol, and estradiol, and to determine correlations between these hormones and sexual behavior of the old males. Blood was drawn from old (n = 9) and young (n = 9) rhesus males over 5 consecutive days at 0900, 1300, and 2100 hr. The two groups of males did not differ in mean serum levels of testosterone, dihydrotestosterone, or estradiol at any time. Although the old and young did not differ in cortisol levels at 0900 and 1300 hr, the cortisol levels at 2100 hr were lower in the old males. Diurnal variations in testosterone, dihydrotestosterone, and cortisol were comparable in old and young males. Mean serum levels of estradiol were significantly higher at 0900 hr than at 1300 hr in the old males, whereas in the young males estradiol levels did not differ with time of day. There was a significant positive correlation between testosterone and yawning rate, and cortisol levels were correlated positively with rate of contacting, rate of mounting, and percentage of tests with erections. The decline in sexual performance of old rhesus males cannot be attributed to changes in the levels or diurnal patterns of testosterone, dihydrotestosterone, or estradiol, but lower cortisol levels in old males may contribute to the decline in sexual behavior.     

Purification and properties of four species of lysyl oxidase from bovine aorta

Lysyl oxidase of bovine aorta was resolved into four enzymically active species by elution from DEAE-cellulose with a salt gradient in 6m-urea, consistent with purification results obtained with enzyme of other tissues [Stassen (1976) Biochim. Biophys. Acta438, 49-60]. In the present study, each of the four peaks of activity was purified to apparent homogeneity by subsequent chromatography on gel-filtration media in 6m-urea. Each enzyme is eluted as a species with mol.wt. approx. 30000 under these conditions, although lysyl oxidase polymerizes to a series of multimers with molecular weights ranging up to 1000000 in the absence of urea. The apparent subunit molecular weight of each enzyme species determined by electrophoresis in sodium dodecyl sulphate and 8m-urea is approx. 32000-33000. The amino acid compositions of the purified forms of lysyl oxidase are similar to each other, although sufficient differences exist to conclude that each is a unique molecular species. Incorporation of alpha-toluenesulphonyl fluoride into the purification scheme does not alter the resolution of enzyme into four species, suggesting that proteolysis during isolation is not the basis of the heterogeneity. The similar sensitivities of each form of enzyme to chelating agents and to semicarbazide and isoniazid indicate that each requires the participation of a metal ion, presumably Cu(2+), and of a carbonyl compound for enzyme function. The present study describes a method for the purification of multiple species of lysyl oxidase and reveals that significant chemical differences exist between the different enzyme forms.