Therapy and clinical trials.
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Anticholesteremic Agents↗
Biomedical subjects
D Bhatnagar
Publications and source records attributed to D Bhatnagar.
avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus.
Recent studies have shown that at least 17 genes involved in the aflatoxin biosynthetic pathway are clustered within a 75-kb DNA fragment in the genome of Aspergillus parasiticus. Several additional transcripts have also been mapped to this gene cluster. A gene, avnA (previously named ord-1), corresponding to one of the two transcripts identified earlier between the ver-1 and omtA genes on the gene cluster was sequenced. The nucleotide sequence of the avnA gene contains a coding region for a protein of 495 amino acids with a calculated molecular mass of 56.3 kDa. The gene consists of three exons and two introns. Disruption of the avnA gene in the wild-type aflatoxigenic A. parasiticus strain (SU1-N3) resulted in a nonaflatoxigenic mutant which accumulated a bright yellow pigment. Thin-layer chromatographic studies with six different solvent systems showed that the migration patterns of the accumulated metabolite were identical to those of averantin, a known aflatoxin precursor. Precursor feeding studies with this mutant showed that norsolorinic acid and averantin were not converted to aflatoxin whereas 5'-hydroxyaverantin, averufanin, averufin, versicolorin A. sterigmatocystin, and O-methylsterigmatocystin were converted to aflatoxins. Southern blot analysis of the wild-type strain and avnA-disrupted mutant strain indicated that the avnA gene was disrupted in the mutant strain. A search of the GenBank database for similarity indicated that the avnA gene encodes a cytochrome P-450-type monooxygenase, and it has been assigned to a new P-450 gene family named CYP60A1. We have therefore concluded that the avnA gene encodes a fungal cytochrome P-450-type enzyme which is involved in the conversion of averantin to averufin in the aflatoxin biosynthetic pathway in A. parasiticus.
Aflatoxins↗
Influence of immunosuppressive therapy on lipoprotein(a) and other lipoproteins following renal transplantation.
Coronary heart disease (CHD) is more common in patients with chronic renal failure and is a major cause of death after renal transplantation. Elevated serum levels of lipoprotein(a) (Lp(a)) are a known risk factor for CHD in the general population and levels have been reported to be increased in renal transplant recipients. It has been suggested that cyclosporin may elevate Lp(a) levels. We therefore measured the serum concentration of Lp(a) in 50 renal transplant recipients who were receiving cyclosporin alone as immunosuppressive therapy and 50 who were treated with azathioprine and prednisolone, but not cyclosporin. The patients attended two renal transplant centres, one where cyclosporin alone was used as immunosuppressive treatment when possible and another where many patients commenced on azathioprine and prednisolone remain on this medication rather than cyclosporin. Patients in each group were matched for age and sex, but the time since transplantation was greater in those not receiving cyclosporin. Transplant function, obesity and the underlying cause of renal disease were similar in both groups of patients. Median Lp(a) concentration in the cyclosporin monotherapy group was 32.0 (range <0.8-140.3) mg/dl and was significantly (p < 0.05) greater than that of the azathioprine and prednisolone group which was 18.3 (range <0.8-167.7) mg/dl. The serum high-density lipoprotein (HDL) cholesterol concentration, which was 1.24 +/- 0.39 mmol/l (mean +/- SD) in patients receiving cyclosporin, was significantly (p < 0.05) less than that of those treated with azathioprine and prednisolone in whom it was 1.41 +/- 0.40 mmol/l. The lower level in those on cyclosporin was due to a decrease in the HDL2 subfraction. Serum lipid and lipoprotein concentrations were otherwise similar in the two groups of patients. The serum level of Lp(a) after renal transplantation may be influenced by the choice of immunosuppressive therapy.
Adolescent↗
The pattern of clinical advice sought by general practitioners from a medical consultant in clinical biochemistry.
Clinical biochemistry departments can be a valuable source of clinical advice for further investigations and the need for referral to specialist clinics. This paper outlines the pattern of clinical advice sought by general practitioners in a district hospital setting, and addresses some of the issues regarding seeking such advice and the implications for continuing medical education and training.
Biochemical Phenomena↗
Lipid peroxidation and antioxidant enzymes in erythrocytes and tissues in aged diabetic rats.
Antidiabetic treatment with powdered fruit of Capparis decidua decreased alloxan induced lipid peroxidation (LPO) significantly in erythrocytes, kidney and heart. Erythrocyte superoxide dismutase (SOD) activity decreased while the kidney and heart SOD increased in diabetic animals. These alterations in SOD were counteracted by insulin as well as with powdered fruit of C. decidua. Increased catalase (CAT) activity in erythrocytes, liver, kidney and heart with C. decidua treatment indicate that the treatment may neutralize H2O2 toxicity by its increased decomposition by CAT. Result shows that treatment with C. decidua lowers alloxan induced LPO and alters SOD and CAT enzymes to reduce oxidative stress.
Aging↗
Plasma lipoprotein composition and cholesteryl ester transfer from high density lipoproteins to very low density and low density lipoproteins in patients with non-insulin-dependent diabetes mellitus.
We have examined cholesteryl ester transfer (CET) from HDL to low density and very low density lipoproteins (LDL and VLDL) and lecithin: cholesterol acyl transferase (LCAT) activity in plasma from 28 men with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet alone or diet and sulphonylurea drugs and in 27 healthy non-diabetic controls. Patients and healthy subjects had similar LCAT activity, but CET was significantly higher in NIDDM 26.1 +/- 11.5 mumol l-1 h-1) than in healthy men (17.8 +/- 6.5 mumol l-1 h-1) (p = 0.001). Diabetic men also had higher CET compared to 15 healthy non-diabetic men (18.7 +/- 5.6 mumol l-1 h-1) (p = 0.001) with similar serum lipids. CET activity was similar in patients treated with diet alone (24.8 +/- mumol l-1 h-1) or with sulphonylureas (27.7 +/- 15.8 mumol l-1 h-1). The Sf 0-12 fraction was significantly enriched with total cholesterol (p = 0.0001) and free cholesterol (p = 0.0006) in diabetic subjects whether treated with diet alone or on sulphonylureas compared to the 15 non-diabetic controls matched for serum triglycerides. The free cholesterol/phospholipid, the free cholesterol/total protein and the free cholesterol/mass ratios were increased in the Sf 0-12 fraction in diabetic subjects (p < 0.01). These findings indicate that CET is accelerated in patients with NIDDM and that this may be due to the altered composition of acceptor lipoproteins.
Adult↗
Characterization of the Aspergillus parasiticus niaD and niiA gene cluster.
The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus.
Amino Acid Sequence↗
Structure and function of fas-1A, a gene encoding a putative fatty acid synthetase directly involved in aflatoxin biosynthesis in Aspergillus parasiticus.
A novel gene, fas-1A, directly involved in aflatoxin B1 (AFB1) biosynthesis, was cloned by genetic complementation of an Aspergillus parasiticus mutant strain, UVM8, blocked at two unique sites in the AFB1 biosynthetic pathway. Metabolite conversion studies localized the two genetic blocks to early steps in the AFB1 pathway (nor-1 and fas-1A) and confirmed that fas-1A is blocked prior to nor-1. Transformation of UVM8 with cosmids NorA and NorB restored function in nor-1 and fas-1A, resulting in synthesis of AFB1. An 8-kb SacI subclone of cosmid NorA complemented fas-1A only, resulting in accumulation of norsolorinic acid. Gene disruption of the fas-1A locus blocked norsolorinic acid accumulation in A. parasiticus B62 (nor-1), which normally accumulates this intermediate. These data confirmed that fas-1A is directly involved in AFB1 synthesis. The predicted amino acid sequence of fas-1A showed a high level of identity with extensive regions in the enoyl reductase and malonyl/palmityl transferase functional domains in the beta subunit of yeast fatty acid synthetase. Together, these data suggest that fas-1A encodes a novel fatty acid synthetase which synthesizes part of the polyketide backbone of AFB1. Additional data support an interaction between AFB1 synthesis and sclerotium development.
Aflatoxin B1↗
Molecular characterization of an Aspergillus parasiticus dehydrogenase gene, norA, located on the aflatoxin biosynthesis gene cluster.
An Aspergillus parasiticus cDNA library was screened with monoclonal antibody raised against a purified A. parasiticus 43-kDa protein demonstrating norsolorinic acid reductase (NOR) activity. One immunopositive clone contained a cDNA insert of 1,418 bp. DNA sequence analysis of this cDNA identified an open reading frame of 1,167 bp that represented the norA gene. The deduced amino acid sequence of the norA coding region consisted of 388 residues capable of encoding a polypeptide of 43.7 kDa. Southern blot analysis of genomic DNA from A. parasiticus indicated that there may be an additional copy of norA. Western blot (immunoblot) analysis of crude protein extracts of A. parasiticus mycelia demonstrated a band of reactivity at 43 kDa only when the fungus was grown in a medium conducive to aflatoxin biosynthesis. Northern (RNA) blot analysis of total RNA from the fungus demonstrated a band of hybridization at about 1.5 kb. As observed with the fungal NORA protein, the norA transcript was present only when the fungus was grown in medium conducive to aflatoxin biosynthesis. Hybridization of the norA cDNA with cosmid DNAs known to encompass a major portion of the A. parasiticus and Aspergillus flavus aflatoxin biosynthetic pathway gene cluster placed the norA gene coding region just upstream of the ver-1 gene. The deduced amino acid sequence of norA had 49% amino acid identity with that of an aryl-alcohol dehydrogenase (aad) gene from Phanerochaete chrysosporium.
Aflatoxins↗
Characterization of experimentally induced, nonaflatoxigenic variant strains of Aspergillus parasiticus.
Six previously isolated, nonaflatoxigenic variants of Aspergillus parasiticus, designated sec mutants, were characterized morphologically by electron microscopy, biochemically by biotransformation studies with an aflatoxin precursor, and genetically by Northern (RNA) hybridization analysis of aflatoxin biosynthetic gene transcripts. Scanning electron micrographs clearly demonstrated that compared with the parental sec+ forms, the variant sec forms had an abundance of vegetative mycelia, orders of magnitude reduced number of conidiophores and conidia, and abnormal metulae. Conidiospores were detected in sec cultures only at higher magnifications (x 500), in contrast to the sec+ (wild-type) strain, in which abundant conidiospores (masking the vegetative mycelia) were observed at even lower magnifications (x 300). All sec+ forms, but none of the sec forms, showed bioconversion of sterigmatocystin to aflatoxins. Northern blots probed with pathway genes demonstrated lack of expression of both the aflatoxin biosynthetic pathway structural (nor-1 and omtA) and regulatory (aflR) genes in the sec forms; PCR and Southern hybridization analysis confirmed the presence of the genes in the sec genomes. Thus, the loss of aflatoxigenic capabilities in the sec form is correlated with alterations in the conidial morphology of the fungus, suggesting that the regulation of aflatoxin synthesis and conidiogenesis may be interlinked.
Aflatoxins↗
A comparative study of six commercial lipoprotein(a) assays in seventeen laboratories within the British Isles.
Seventeen laboratories in the British Isles participated in a study to compare six different commercially available immunoassays for serum lipoprotein(a) (Lp(a)) and to establish reasons for the variations in the measurement of serum Lp(a) concentrations. Pooled serum was distributed neat and after dilution at a central laboratory. In addition, the central laboratory sent unpooled serum sampled monthly from six healthy volunteers to each of the participating laboratories for 12 months. The assays all gave linear dilution curves which were parallel, although the reported values varied twofold. There were major differences in the values assigned to different manufacturers' calibrants which was not explained by whether the units employed were whole Lp(a), the protein moiety of Lp(a) or simply apolipoprotein(a). The coefficient of variation for the reported value of Lp(a) over 12 months was 33%. The component variation was 10% after adjustment for inter-laboratory and intra-laboratory variation. Some individuals clearly had a greater tendency to variable serum Lp(a) concentrations than others, but all the assays responded to this in the same way. Thus, the assays tested probably measured the same analyte. The problem of calibration could largely be addressed if agreement were reached by the manufacturers. Even with improvements in analytical precision it should be realized that multiple measurements of serum Lp(a) levels are necessary if the true mean value is to be appreciated. Individuals showing wide variation in serum Lp(a) may reward further study if its role is to be established.
Clinical Laboratory Techniques↗
Severe insulin resistance, diabetes mellitus, hypertriglyceridemia, and pseudoacromegaly.
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Acromegaly↗
Comparison of the omtA genes encoding O-methyltransferases involved in aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus.
O-methyltransferase (OMT) is one of the key enzymes in aflatoxin (AF) biosynthesis in the fungi, Aspergillus flavus (Af) and A. parasiticus (Ap). Genomic DNA clones containing the omtA genes from Ap strain SRRC 143 and Af strain CRA01-2B were sequenced. Comparison of the genomic DNA sequences with the cDNA of this Ap gene revealed the presence of four introns ranging from 52 to 60 bp in length in both species; the region encoding the putative S-adenosylmethionine-binding motif was located between the third and fourth introns. The coding sequence of omtA from Ap strain SRRC 143 demonstrated a greater than 97% sequence identity with that from Af strain CRA01-2B, within the coding region.
Aflatoxins↗
The Aspergillus parasiticus polyketide synthase gene pksA, a homolog of Aspergillus nidulans wA, is required for aflatoxin B1 biosynthesis.
Aflatoxins comprise a group of polyketide-derived carcinogenic mycotoxins produced by Aspergillus parasiticus and Aspergillus flavus. By transformation with a disruption construct, pXX, we disrupted the aflatoxin pathway in A. parasiticus SRRC 2043, resulting in the inability of this strain to produce aflatoxin intermediates as well as a major yellow pigment in the transformants. The disruption was attributed to a single-crossover, homologous integration event between pXX and the recipient A. parasiticus genome at a specific locus, designated pksA. Sequence analysis suggest that pksA is a homolog of the Aspergillus nidulans wA gene, a polyketide synthase gene involved in conidial wall pigment biosynthesis. The conserved beta-ketoacyl synthase, acyltransferase and acyl carrier-protein domains were present in the deduced amino acid sequence of the pksA product. No beta-ketoacyl reductase and enoyl reductase domains were found, suggesting that pksA does not encode catalytic activities for processing beta-carbon similar to those required for long chain fatty acid synthesis. The pksA gene is located in the aflatoxin pathway gene cluster and is linked to the nor-1 gene, an aflatoxin pathway gene required for converting norsolorinic acid to averantin. These two genes are divergently transcribed from a 1.5 kb intergenic region. We propose that pksA is a polyketide synthase gene required for the early steps of aflatoxin biosynthesis.
Aflatoxin B1↗
Nucleotide sequence of a Aspergillus parasiticus gene strongly repressed by thiamine.
A cDNA clone demonstrating a high degree of homology to the thiamine repressed nmt1 gene of Schizosaccharomyces pombe was isolated from the aflatoxigenic fungus, Aspergillus parasiticus. The deduced polypeptide of a cDNA clone from A. parasiticus had an amino acid sequence identity of 60% with that of the nmt1 gene of S. pombe. Transcription of the nmt1 gene homolog in the fungus was strongly inhibited by concentrations of thiamine of 2.0 microM or higher.
Amino Acid Sequence↗
Coronary risk factors in people from the Indian subcontinent living in west London and their siblings in India.
Several reports have shown that migrants from southeast Asia tend to have an increased risk of coronary heart disease when settled in their new country. We compared coronary risk factors in a randomly selected group of 247 migrants from the Indian subcontinent of Punjabi origin living in West London and 117 of their siblings living in the Punjab in India. The West London cohort had a greater body mass index (p < 0.001), systolic blood pressure (p = 0.0087), serum cholesterol (p < 0.001), apolipoprotein B (p < 0.001), lower high-density lipoprotein cholesterol (p < 0.05) and higher fasting blood glucose (p < 0.05) than their siblings in the Punjab. Insulin sensitivity, derived from the homoeostatic assessment mathematical model, was lower in men in West London than in their counterparts in India (p < 0.05). Indians in West London had lower beta cell function than those in the Punjab (p < 0.001). Serum lipoprotein (a) concentrations were similar in both the West London and Punjab population, but were significantly higher (p = 0.01) than those of white European populations in the UK. Increases in serum cholesterol after migration from India lead to increased coronary risk conferred by high serum lipoprotein (a) concentrations and greater insulin resistance. Such between-country comparisons are an important means of establishing the importance of coronary risk factors.
Adult↗
Spontaneous platelet aggregation in whole blood is increased in non-insulin-dependent diabetes mellitus and in female but not male patients with primary dyslipidemia.
Increased platelet aggregability has been shown in hypercholesterolemia, and stirring-induced spontaneous aggregation in whole blood is increased in insulin-dependent diabetes mellitus (DM). We have determined spontaneous aggregation in citrated (10 mM) whole blood, from 27 primary dyslipidemic patients (DYS; 14F, 13M), 16 male non-insulin-dependent DM (NIDDM) patients, and 17 normolipidemic controls (N; 6F, 11M), using platelet counting to quantify aggregation. Spontaneous aggregation was significantly higher, both in the female DYS group (median 30% [interquartile range 25,50], P < 0.005) and the NIDDM group (33% [25,41], P < 0.005), than in the N group (17% [12,27]), but did not differ significantly in the male DYS group (23% [10,33]). Similar results were obtained in the presence of indomethacin (25 mumol/l) to prevent artefactual thromboxane (TX) A2 formation, indicating that increased spontaneous aggregation was TXA2-independent. Interestingly, increased spontaneous aggregation appeared to be independent of serum cholesterol and triglyceride concentrations, as well as age and sex per se. We conclude that spontaneous platelet aggregation was increased both in female primary dyslipidemic patients and NIDDM patients, but not in male DYS patients. The clinical significance of increased spontaneous platelet aggregability is that it may favour shear-induced aggregation which may occur at critical arterial stenoses in vivo leading to thrombus formation.
Adult↗
Hybridization of genes involved in aflatoxin biosynthesis to DNA of aflatoxigenic and non-aflatoxigenic aspergilli.
Southern blots of DNA from a number of aspergilli belonging to Aspergillus section Flavi, including aflatoxin-producing and non-aflatoxigenic isolates of A. flavus and A. parasiticus, were probed with the aflatoxin pathway genes aflR and omt-1. DNA of all A. flavus, A. parasiticus and A. sojae isolates examined hybridized with both genes. None of the A. oryzae isolates examined hybridized to the aflR probe and one of the three did not hybridize to the omt-1 probe. None of the A. tamarii isolates examined hybridized to either gene. Our results suggest that some isolates in this section do not produce aflatoxin because they lack at least one of the genes necessary for biosynthesis, and that non-producing A. flavus, A. parasiticus and A. sojae strains either lack a gene we did not examine or have genes that are not being expressed.
Aflatoxins↗