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Biomedical subjects

K Raja

Publications and source records attributed to K Raja.

14 recordsLinked to original sources
PubMed2001

Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung.

p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to T:A transversions at codon 248, G:C to T:A transversions and G:C to A:T transitions at codon 249, and C:G to T:A transitions at codon 250 when compared with the DMSO-treated controls. These data are consistent with the hypothesis that chemical carcinogens such as BP in cigarette smoke cause G:C to T:A transversions at p53 codons 157, 248, and 249 and that nontumorous lung tissues from smokers with lung cancer carry a high p53 mutational load at these codons.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2000

Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases.

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 (P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 (P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 (P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen/nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.

Aldehydes↗
PubMed2000

Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease.

Ulcerative colitis (UC) is a chronic inflammatory disease that produces reactive oxygen and nitrogen species and increases the risk of colorectal cancer (CRC). The p53 tumor suppressor gene is frequently mutated in UC-associated dysplastic lesions and CRC. We are exploring the hypothesis that p53 mutations in the nontumorous colonic tissue in noncancerous UC cases indicate genetic damage from exposure to exogenous and endogenous carcinogens and may identify individuals at increased cancer risk. We are reporting, for the first time, the frequency of specific p53 mutated alleles in nontumorous colon tissue from donors either with or without UC by using a highly sensitive genotypic mutation assay. Higher p53 mutation frequencies of both G:C to A:T transitions at the CpG site of codon 248 and C:G to T:A transitions at codon 247 were observed in colon from UC cases when compared with normal adult controls (P = 0.001 and P = 0.001, respectively). In the UC cases, higher p53 codon 247 and 248 mutation frequencies were observed in the inflamed lesional regions when compared with the nonlesional regions of their colon (P < 0.001 and P = 0.001). The colonic nitric oxide synthase-2 activity was higher in UC cases than in non-UC adult controls (P = 0.02). Our data are consistent with the hypothesis that a higher frequency of p53 mutant cells can be generated under oxidative stress in people with UC. The increased frequency of specific p53 mutated alleles in noncancerous UC colon tissue may confer susceptibility to the development of CRC in an inflammatory microenvironment.

Adult↗
PubMed2000

Genetic variation of basal iron status, ferritin and iron regulatory protein in mice: potential for modulation of oxidative stress.

Toxic and carcinogenic free radical processes induced by drugs and other chemicals are probably modulated by the participation of available iron. To see whether endogenous iron was genetically variable in normal mice, the common strains C57BL/10ScSn, C57BL/6J, BALB/c, DBA/2, and SWR were examined for major differences in their hepatic non-heme iron contents. Levels in SWR mice were 3- to 5-fold higher than in the two C57BL strains, with intermediate levels in DBA/2 and BALB/c mice. Concentrations in kidney, lung, and especially spleen of SWR mice were also greater than those in C57BL mice. Non-denaturing PAGE of hepatic ferritin from all strains showed a major holoferritin band at approximately 600 kDa, with SWR mice having > 3-fold higher levels than C57BL strains. SDS PAGE showed a band of 22 kDa, mainly representing L-ferritin subunits. A trace of a subunit at 18 kDa was also detected in ferritin from SWR mice. The 18 kDa subunit and a 500 kDa holoferritin from which it originates were observed in all strains after parenteral iron overload, and there was no major variation in ferritin patterns. Although iron uptake studies showed no evidence for differential duodenal absorption between strains to explain the variation in basal iron levels, acquisition of absorbed iron by the liver was significantly higher in SWR mice than C57BL/6J. As with iron and ferritin contents, total iron regulatory protein (IRP-1) binding capacity for mRNA iron responsive element (IRE) and actual IRE/IRP binding in the liver were significantly greater in SWR than C57BL/6J mice. Cytosolic aconitase activity, representing unbound IRP-1, tended to be lower in the former strain. SWR mice were more susceptible than C57BL/10ScSn mice to the toxic action of diquat, which is thought to involve iron catalysis. If extrapolated to humans, the findings could suggest that some people might have the propensity for greater basal hepatic iron stores than others, which might make them more susceptible to iron-catalysed toxicity caused by oxidants.

Absorption↗
PubMed1999

Nonliposomal approach--a study of preparation of egg albumin nanospheres containing amphotericin-B.

The stability of liposomes after introduction into the body is presently being discussed and needs thorough understanding. Hence, as a nonliposomal approach, egg albumin nanospheres were prepared by the pH-coacervation method, and a preliminary study was carried out of the influence of process variables on the size and shape of nanospheres by changing the pH of the albumin solution, concentration of albumin solution, and volume of cross-linking agent. The batch prepared with an albumin medium of pH 9, 2% concentration, and 100 microliters of 4% glutaraldehyde-ethanol solution was found to have a spherical uniform shape with an average size of 497.6 nm. The ideal batch was loaded with the systemic antifungal drug amphotericin-B. Drug-loaded nanospheres were evaluated to study their in vitro release. They were found to exhibit a biphasic pattern with a cumulative percentage release of 97.7%.

Albumins↗
PubMed1999

Trans-vaginal oocyte retrieval and subsequent in vitro production of embryos from a cow involuntarily culled.

A Holstein cow of high genetic merit, in late lactation (205 days) and diagnosed with salpingitis (after 4 infertile services and veterinary consultation), was subjected to 1 trans-vaginal oocyte collection attempt, prior to slaughter. Of an estimated 10 follicles punctured, a total of 4 cumulus-oocyte complexes were retrieved. These were matured in vitro in a maturation medium for 24 hours. After 24 hours maturation, the oocytes were fertilised in vitro with Percoll-processed frozen/thawed imported semen, of the owner's choice. Fertilisation was achieved in a modified Tyrode's medium. At 18 hours post-insemination, the presumptive zygotes were transferred into culture in vitro in Charles Rosenkran's amino-acid medium and supplemented on Day 4 post-insemination with 10% foetal calf serum. All in vitro procedures were conducted in 50 microl medium droplets, under oil, in a humidified incubator at 38.5 degrees C in 5% CO2 in air. Three of the potential zygotes cleaved and, by Day 7 of culture, these had developed to the morula stage. The embryos were frozen in 1.5 M ethylene glycol and later transferred non-surgically to synchronised Holstein recipient heifers. One morula resulted in the only pregnancy and subsequent birth of a healthy heifer calf. An independent commercial company confirmed parentage through standard blood-typing assays. The genetic salvage of oocytes, for in vitro production of embryos, has potential benefits to the producer.

Animals↗
PubMed1998

Genetic and acquired predisposing factors and treatment of osteoporosis in thalassaemia major.

We have previously shown a high incidence of osteopenia and osteoporosis in patients with thalassaemia major. These bone changes, were more severe in males than females, in those with diabetes mellitus and with hypogonadal-hypogonadism. Our recent studies concern the relationship of erythroid activity, assessed by serum transferrin receptors as an overall measure of anaemia, to osteoporosis. Serum transferrin receptor levels correlated with the mean pre-transfusion haemoglobin level, but there was no correlation with the incidence of osteopenia and osteoporosis. As osteoporosis has a strong genetic component we have also studied the COLIA1 and COLIA2 genes which code for the major protein of bone (type 1 collagen). Studies by others have shown in non-thalassaemic patients that a polymorphism G-->T or TT in a regulatory region of COLIA1 at the recognition site for transcription factor Sp1 is associated with the presence of osteoporosis. Our studies suggest that Sp1 polymorphism is not specific to any one ethnic group; the polymorphism occurs more commonly in females (female to male ratio 2:1). In male thalassaemia major patients the presence of the Sp1 mutation was associated with more severe osteoporosis of the spine and the hip compared with female patients. There is failure of improvement in spinal osteoporosis with bisphosphonate therapy (intravenous Pamidronate) in male patients with the Sp1 mutation.

Adolescent↗
PubMed1994

Contrasting uptakes of 59Fe into spleen, liver, kidney and some other soft tissues in normal and hypotransferrinaemic mice. Influence of an antibody against the transferrin receptor.

Uptake of iron-59 from blood into various soft tissues of anaesthetized mice was investigated by continuous intravenous infusion of the radiotracer during 2 hr. The 59Fe was given either as ferrous chloride with ascorbate or as 59Fe-transferrin. Infusions were made into adult mice with and without pretreatment with a monoclonal antibody against transferrin receptors, and into hypotransferrinaemic mice and appropriate controls. In normal mice, 59Fe uptake into spleen was much higher than into other tissues and was 94-96% inhibited by the antibody. Inhibitions due to the antibody were less complete in liver and renal cortex, and there was evidence of some non-transferrin-mediated transport during infusion of 59Fe/ascorbate. In the hypotransferrinaemic mice, tissue uptakes of 59Fe during infusion of 59Fe/ascorbate were enormous, being two to three orders of magnitude greater than in the normal controls. The rank order for size of uptake was liver > renal cortex > pancreas > spleen > other tissues. All tissues examined have a considerable potential capacity for uptake of non-transferrin-bound iron, this being greatest in liver and renal cortex.

Animals↗
PubMed1989

Effect of insulin induced hypoglycaemia on in vitro uptake of 3-O-methylglucose by rat jejunum.

Acute hypoglycaemia (less than 2.0 mmol/l) selectively increased in vitro 3-O-methylglucose uptake by rat jejunum. In contrast, uptake of 3H-phenylalanine or 59Fe (III) was unchanged in the hypoglycaemic animals. The hypoglycaemia was accompanied by decreased tissue adenosine nucleotide energy charge. The increased uptake was phlorrhidzin-inhibitable and therefore reflected increased Na+ dependent glucose carrier activity. Cycloheximide did not block the increased 3-O-methylglucose uptake, implying that the mechanism is not the result of increased de novo carrier protein synthesis.

3-O-Methylglucose↗
PubMed1984

Granulocytic cryopreservation: further studies on the pathogenesis of impaired cellular function.

The effects of different methods of purifying peripheral blood granulocytes on their functional capacity after cryopreservation have been studied. The standard method of isolating granulocytes by centrifugation through Lymphoprep sensitizes cells to damage induced by subsequent cryopreservation. Some of this damage occurs during addition of cryoprotectant and we describe how to add and remove dimethyl sulphoxide without impairing cell function. The granulocytic function that suffers most during cryopreservation is granulocyte migration. This impairment may be partially due to uncontrolled entry into the cell of calcium. In contrast the bactericidal capacity in vitro is not so greatly reduced and ability to reduce nitroblue tetrazolium redox dye even less so.

Blood Bactericidal Activity↗