[Gene fusion in studies on gene expression regulation and protein analysis. Use of lac fusion].
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Animals↗
Biomedical subjects
J Wild
Publications and source records attributed to J Wild.
Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K12.
Evidence is presented that alanine racemase activity in E. coli K12 is due to two distinct gene products. The predominant isozyme is inducible by either alanine stereoisomer and repressible by glucose. The gene dadX coding for its structure is located by the dadA gene determining the structure of D-amino acid dehydrogenase. The regulatory site for the expression of both genes, dadR, is located on the other side of dadA. The orientation of the dad operon established by multiple-point crosses and deletion mapping is as follows: fadR ...dadRAX ...hemA. The dadX alanine racemase activity is unusually refractory to changes of incubation temperature. It differs strikingly from that of the other isozyme, probably the product of the alr gene. The latter isozyme shows a typical dependence upon incubation temperature. The synthesis of alr alanine racemase is constitutive in respect of both alanine and glucose. In dadX mutants, in which alanine racemase activity equals only 15% of that in wild-type cells grown in the absence of an inducer or catabolite repressor, the dad operon cannot be induced by D-alanine. We presume, therefore, that L-alanine is involved more directly than D-alanine in dad operon regulation.
Alanine Racemase↗
Neural-tube defects and vitamins: the need for a randomized clinical trial.
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Clinical Trials as Topic↗
Sensitive solid phase radioimmunoassay for the detection of anti-DNA autoantibodies.
Serum from patients with systemic lupus erythematosus (SLE) and hybridoma culture fluids derived from the fusion of SLE lymphocytes contain antibodies to native DNA (nDNA) and denatured DNA (dDNA). A rapid, efficient solid phase radioimmunoassay (RIA) was developed to screen for minute quantities of these autoantibodies. The RIA, which utilized polystyrene tubes, required the addition of 0.1% bovine serum albumin and 0.01% Tween 20 detergent to decrease nonspecific immunoglobulin binding. Pretreatment of the polystyrene tubes with poly-L-lysine (PLL) prior to coating with DNA increased the binding of radiolabeled nDNA from 15 to 46% and of dDNA from 17 to 63%. This PLL precoating step resulted in a 3-fold increase in the specificity of the assay for nDNA but was not advantageous for dDNA. The method described is sensitive, specific, and can be applied to the screening of microgram quantities of anti-DNA autoantibodies in serum and hybridoma culture fluids.
Antibodies, Monoclonal↗
Arbovirus infections in humans in New South Wales. Seroepidemiology of the alphavirus group of togaviruses.
A seroepidemiological study of the prevalence of antibodies to alphaviruses (Ross River [RRV], Sindbis [SIN] and Getah [GET] viruses) was carried out on 16 842 specimens of sera collected during 1981 and 1982 from individuals of all ages living in all health regions of New South Wales. Prevalence rates were found to be low in the major coastal cities of Sydney, Newcastle and Wollongong, and in the whole of the Tablelands. In coastal populations, the rates were highest in the far and mid North Coast zones (about 16%), and slightly lower in the south. The ratio of RRV:SIN antibodies was 62:1 in coastal populations. Antibody prevalence rates varied from 10%-20% in the Western Slopes, rising to 30%-40% in centres in the Western Plains; the RRV:SIN antibody ratio here was 11:1. The unexpectedly high infection rates, especially in the Western Plains, suggest that Ross River virus, in particular, may constitute a public health problem of hitherto unsuspected significance in New South Wales.
Adolescent↗
Further experience of vitamin supplementation for prevention of neural tube defect recurrences.
In accordance with a previous protocol, a second cohort of 254 mothers with a history of previous neural tube defect (NTD) births was before a subsequent conception and continued until the time of the second missed menstrual period. There were 2 NTD recurrences (0.9% of 234 infants/fetuses examined), which is significantly fewer than the 11 NTD recurrences (5.1% of 215 infants/fetuses examined) born to 219 unsupplemented (US) mothers in the same centres over the same period. When the data for the two cohorts were combined, the overall recurrence rates were 0.7% for 454 fully supplemented (FS) mothers and 4.7% for 519 US mothers. The recurrence rates after 1 previous NTD were 0.5% for FS and 4.2% for US mothers: after 2 or more previous NTDs, 2.3% for FS and 9.6% for US. There were no recurrences among the offspring of a further 114 mothers whose duration of supplementation fell short of the full regimen (partially supplemented, PS).
Female↗
Isolation of mannose-binding proteins from human and rat liver.
A binding assay for the detection of mannose-binding proteins was developed, which uses a ligand of mammalian origin, 125I-labelled bovine pancreatic ribonuclease B. The binding assay was validated by using the recognized mannose-binding protein, concanavalin A. Microgram quantities of concanavalin A or mannose-binding proteins could be assayed. A mannose-binding protein was isolated from rat liver by affinity chromatography on mannose-Sepharose 6B. It has a Mr of approx. 900000 under non-dissociating conditions and contains a subunit of approx. 34000 Mr. When ribonuclease B-Sepharose was used as a ligand for affinity chromatography, the predominant mannose-binding material isolated from rat liver had a native Mr of 205000-225000 and consisted largely of a subunit of Mr 70000, which yielded subunits of Mr 28500 and 34000 on reduction. It is suggested that different mannose-binding proteins are isolated by the two affinity-chromatography ligands. A mannose-binding protein was also purified from human liver by affinity chromatography on mannose-Sepharose 6B. It has a native Mr of over 1000000 and consists of subunits with Mr 28000 and 30500. Its isolation suggests that mannose-mediated endocytosis or intracellular transport of glycoproteins occurs in human liver.
Animals↗
The effect of periconceptional supplementation on blood vitamin concentrations in women at recurrence risk for neural tube defect.
1. We measured erythrocyte folic acid and riboflavin, serum folic acid and leucocyte vitamin C in women at high risk for neural tube defect (NTD) recurrence who were receiving periconceptional vitamin supplementation, before they received extra vitamins, after 28 d of supplementation and at the 8th week of pregnancy. Blood vitamin concentrations in unsupplemented high-risk women were also compared with the values found in unsupplemented low-risk women. 2. Vitamin supplementation with Pregnavite Forte F (Bencard) raised the mean values for all vitamins measured by the 8th week of pregnancy. Mean erythrocyte folic acid rose from 250 to 478 ng/ml; plasma folic acid from 8.4 to 26.1 ng/ml; leucocyte vitamin C from 1.82 to 3.21 micrograms/ml blood; erythrocyte riboflavin (glutathione reductase (EC 1.6.4.2) activation ratio) from 1.08 to 1.04. All women receiving supplements had increased their serum and erythrocyte folic acid levels above the highest values found in women in an earlier study, who subsequently gave birth to children with NTD. Not all women, however, increased their leucocyte ascorbic acid or erythrocyte riboflavin levels above the highest values. 3. When vitamin concentrations in unsupplemented high-risk women compared with levels in unsupplemented women at low risk for NTD, no significant differences were found in the mean values. However, a significantly higher proportion of high-risk compared with low-risk women had erythrocyte folic acid and leucocyte vitamin C values on or below the 5th percentile of the adult normal range. 4. The effectiveness of Pregnavite Forte E (Bencard) for increasing maternal vitamin reserves is discussed with a view to preventing NTD and the possibility of identifying groups at risk for NTD because of low blood vitamin levels is considered.
Ascorbic Acid↗
Equine herpesvirus abortion in Australia 1977 to 1982.
Until 1977 no case of abortion caused by equine herpesvirus 1 (EHV1) had been recorded in Australia although the virus, called equine rhinopneumonitis virus, had been known to have been present at least since 1962. Outbreaks of EHV1 abortion occurred in New South Wales in 1977 and in 1981. Sporadic cases of EHV1 abortion had been confirmed in some parts of Australia each year since 1975. It was concluded that an abortigenic subtype of EHV1 had been introduced to Australia in 1977 and that the previously endemic respiratory subtype occasionally caused abortion. Virus isolation in a variety of cell cultures and histopathological examination of tissue were shown to be satisfactory methods of diagnosis of EHV1 abortion. Lung proved to be the specimen of choice. Slight serological differences between "abortigenic" and "respiratory" subtypes of EHV1 were found in cross neutralisation tests. A serological survey of 219 Sydney horses of various ages revealed that most yearlings had already acquired neutralising antibody to both subtypes.
Abortion, Veterinary↗
Immunologic characteristics of the leukocyte adherence inhibition assay in rheumatoid arthritis.
Peripheral blood leukocytes (PBL) from patients with rheumatoid arthritis (RA) when used in the leukocyte adherence inhibition (LAI) assay were capable of distinguishing antigenic differences between RA and osteoarthritic (OA) synovial membrane extracts. Normal PBL from control subjects with nonadherence index (NAI) values of 7 +/- 2 were able to respond positively (NAI values of 31 +/- 6) in the LAI assay if briefly preincubated with IgG obtained from LAI-position RA subjects. The LAI-positive response of PBL from RA patients was negated by preincubating the cells with sera obtained from LAI-nonreactive RA patients. Preincubation of reactive LAI-positive RA cells with urinary protein from 4 LAI-nonreactive RA patients blocked the response of reactive RA leukocytes in the LAI assay (42 +/- 2 to 14 +/- 2). In contrast, preincubation of reactive LAI-positive RA cells with urinary protein from LAI-reactive RA patients or patients with gout had no effect on subsequent LAI reactivity. This study suggests that LAI-nonreactive RA subjects have a 'rheumatoid neoantigen-like material' in their circulation which is excreted in their urine and is capable of being recognized by PBL of reactive, LAI-positive RA patients.
Antigens↗
Folate nutrition in early pregnancy.
Mean daily folate intakes of 195 women in the first trimester of pregnancy were assessed by 5-7 day weighed dietary records. Intakes for total folate ranged from 64.7 to 302.0 micrograms/day with a mean intake of 148.0 micrograms/day. The lowest intakes were associated with social classes III, IV and V, maternal age less than 20 years and vomiting on three or more days per week.
Adolescent↗
Regulation of expression of the dadA gene encoding D-amino acid dehydrogenase in Escherichia coli: analysis of dadA-lac fusions and direction of dadA transcription.
The catabolism of most d-amino acids is carried out by d-amino dehydrogenase, coded by the dadA gene. Employing Mud(Aprlac) phage (Casadaban and Cohen 1979) the lac structural gene were fused to the control region of the dadA locus. Expression the of the lacZ gene in the dad-lac fusions was shown to be inducible by alanine and catabolite-repressible, thus responding to regulatory signals known to affect expression of the dadA gene. The method of MacNeil et al. (1980) was adopted to transfer the chromosomal dadA-lac fusion into lambda phage. By use of isolated lambda ddadA-lac1 phage, the promoter for the dadA gene was located, which enabled determination of the direction of dadA transcription as being counter-clockwise.
Bacteriophage lambda↗
Immune reactivity to native type II collagen in rheumatoid arthritis assessed by the leukocyte adherence inhibition assay and radioimmunoassay.
Cellular immunity to native type II collagen as well as to rheumatoid and osteoarthritic synovial membrane homogenates was assessed in a leukocyte adherence assay (LAI) in patients with rheumatoid arthritis (RA). Eleven out of 14 RA patients (79%) were positive in the LAI assay, demonstrating increased reactivity to rheumatoid over osteoarthritic synovial extracts. Increased reactivity to native type II collagen was not noted in the LAI assay. Radioimmunoassay studies demonstrated that 3 out of 14 RA patients (22%) had circulating IgG rheumatoid factor. None of the 11 patients with osteoarthritic or seronegative arthritis had antibodies to native type II collagen or circulating IgG rheumatoid factor, although 2 subjects with ankylosing spondylitis yielded positive LAI results. Our results suggest that type II collagen is not the important constituent in rheumatoid synovial membrane extract responsible for the positive LAI results observed in rheumatoid arthritis patients.
Arthritis, Rheumatoid↗
D-Amino acid dehydrogenase of Escherichia coli K12: positive selection of mutants defective in enzyme activity and localization of the structural gene.
A method for the positive selection of dadA mutants defective in D-amino acid dehydrogenase has been devised. It consists in isolating mutants resistant to beta-chloro-D-alanine and screening for mutant colony color on a special agar medium. All 70 Escherichia coli K12 dadA mutants isolated either by this method or by other selection procedures map at a locus which is near to hemA and closely linked with dadR. Since some of the dadA mutants are thermosensitive in D-methionine utilization in vivo and have thermolabile D-amino acid dehydrogenase in vitro, it is proposed that the dadA gene codes for the enzyme structure. The broad substrate specificity, apparent membrane localization, inducibility by alanine, and repressibility by glucose strongly suggest that the D-amino acid dehydrogenase coded by the dadA gene is a species variant of the enzyme described under the same name in Salmonella typhimurium. It may be identical or homologous with the enzymes described under the names alaninase, D-alanine oxidase or D-alanine dehydrogenase in E. coli K12 or B.
D-Amino-Acid Oxidase↗
The effect of estradiol on a 25-hydroxycholecalciferol binding protein in the uterus of the mouse.
When ovariectomized mice are treated with estradiol-17 beta, their uterine tissue shows an increase in 25-hydroxycholecalciferol binding protein as well as an increase in estradiol- and progesterone-binding proteins.
Animals↗
Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12.
Three temperature-sensitive mutant strains for RNA polymerase beta or beta' subunits (carrying mutations tsx, A2R7 and R120) were used in order to investigate the dependence of the induced lac expression on stimulation by cyclic AMP after the shift to non-permissive temperature. High temperature lowered the rate of beta-galactosidase synthesis. However, the low rate of synthesis could be strongly increased by cyclic AMP (30, 2.4 and 5.7-fold increases for tsX, A2R7 and R120 mutants, respectively). At the permissive temperature stimulation by cyclic AMP was less than 1.4-fold (minimal medium supplemented with glycerol). The results suggest that the maximal expression of the lac operon is saturated, that is, a hypothetical increase in RNA polymerase or cAMP-CRP concentration in the cell with not enhance the expression. The concept of saturation explains why it was possible to increase the beta-galactosidase synthesis in conditions of limited promoter binding activity of RNA polymerase through increase in concentration of cyclic AMP-CRP complex in the cell (addition of cyclic AMP) to the values higher than that observed on glycerol.
Cyclic AMP↗
25-Hydroxycholecalciferol receptors in human breast cancer.
Cytosol receptors for 25-hydroxycholecalciferol, oestradiol and progesterone were measured in human mammary carcinomas. Significant positive correlations were found between the concentrations of all three receptors.
Breast Neoplasms↗
Effects of progesterone on the oestrogen-stimulated uterus: a comparative study of the mouse, guinea pig, rabbit and sheep.
To examine more closely the anti-oestrogenic action of progesterone (P), its effect on various parameters in the 17 beta-oestradiol (E2)-primed uterus of the mouse, guinea pig, rabbit and ewe was studied. Changes in uterine wet weight, rate of in vitro protein synthesis, protein : DNA and RNA : DNA ratios, peroxidase activity and the level of cytosol receptors for E2 and P were measured. Considerable between-species differences in the effect of P on these parameters were observed. The anti-uterotrophic action was greater in the mouse than in the guinea pig and was not seen in the rabbit or ewe. P inhibited protein synthesis in the mouse, was without significant effect in the guinea pig and was mildly stimulatory in the rabbit and ewe. Inhibitory effects on protein : DNA and RNA : DNA ratios were substantial in the mouse, minor in the guinea pig and absent in the rabbit and ewe. Peroxidase activity was decrease in the mouse and guinea pig, essentially lacking in the rabbit and not detectable in the ewe. In all species the level of both oestrogen and progesterone cytosol receptors was decreased, although the effect on the E2 receptor was less marked in the ewe. The results indicate that in the species studied an effect of P on the replenishment of the E2 receptor is not necessarily associated with other anti-oestrogenic actions of P and argue against the concept that initial anti-oestrogenic actions of P are mediated via a specific effect on such replenishment.
Animals↗