The growth of many breast cancers is stimulated by the action of the hormone estrogen. Hormonal therapy used to treat these estrogen-dependent breast cancers acts by interfering with the action of estrogen. Current treatments, such as tamoxifen, are not consistently useful due to development of resistance to these drugs. Tamoxifen treatment can also lead to the development of other gynecological cancers, therefore the discovery of novel treatment options for breast cancer is critical. Vitamin B6 is well documented for its role as a modulator of steroid hormones. Pyridoxal phosphate (PLP), the active form of Vitamin B6, may interfere with the action of the estrogen receptor (ER) by blocking the hormone-binding and/or DNA-binding site of the ER. The objective of this study was to examine the effects of Vitamin B6 supplementation on cell proliferation and estrogen-dependent gene expression in breast cancer cells. To accomplish this, estrogen-dependent (MCF-7 and T-47D) and estrogen-independent (BT-20) breast cancer cells were grown in medium supplemented with 0,100, or 300 µM pyridoxal (PL) in the absence or presence of 0.01µM estradiol. Cell counts and [3H]-thymidine incorporation into DNA were assessed in all cell lines. The expression of pS2, an estrogen-sensitive gene, was performed using RNA extracted from MCF-7 cells. PL supplementation was found to significantly decrease total cell numbers and DNA synthesis in both the estrogen-dependent (ER+) and -independent (ER-) breast cancer cells, but did not alter the expression of pS2. These results indicate that PL significantly impairs growth of breast cancer cells and may be exerting its effects via a steroid-independent mechanism. / Master of Science

Vitamin B-6 status was examined in a group of 46 elderly subjects who were selected from nursing home residents, hospital patients, and free living individuals in Denton County. Subjects were limited to men and women over 60 years of age. Erythrocyte aspartate aminotransferase stimulation with pyridoxal phosphate (in-vitro) was studied as the biochemical criterion of vitamin B-6 status. The pyridoxine status of these 46 subjects (the reference group) was measured in order to be able to identify people with a relatively poor B-6 status. A sub-group of the reference group was composed of 4 subjects who took B-6 supplements (supplemented group). There was no significant difference (0.05 level) in the basal activity, stimulated activity, percentage stimulation, or body weight, after treatment with 10 mg pyridoxine hydrochloride for 4 weeks, even though all 4 subjects had an improved B-6 status (based on percentage stimulation) after taking the supplement. The data indicated that of the 4 subjects tested, 2 showed a large change in the basal activity, stimulated activity, and percentage stimulation. The lack of significant difference (0.05 level) was probably due to a small sample size. One subject reported an increased appetite and body weight after treatment with pyridoxine.

Pyridoxal 5'-phosphate (PLP) is the cofactor for over 140 vitamin B6 (PLP)-dependent enzymes that are involved in various metabolic and biosynthetic pathways. Pyridoxal kinase (PL kinase) and pyridoxine 5’-phosphate oxidase (PNP oxidase) are the two key enzymes that metabolize nutritional forms of vitamin B6, including pyridoxal (PL), pyridoxine (PN), and pyridoxamine (PM) to the active cofactor form, PLP. Disruption of the PLP metabolic pathway due to mutations in PNP oxidase or PL kinase result in PLP deficiency, which is implicated in several neurological pathologies. Several ingested compounds are also known to result in PLP deficiency with concomitant neurotoxic effects. How these mutations and compounds affect B6 metabolism is not clearly understood. On the other hand, an emerging health problem is the intake of too much vitamin B6 as high doses of the reactive PLP in the cell exhibits toxic effects, including sensory and motor neuropathies. The overall aim of this research is to understand the catalytic function of PL kinase and the regulatory pathway of PLP metabolism. Using site-directed mutagenesis (Asp235Asn, Asp235Ala), kinetic and structural studies, we have shown that Asp235 may play a catalytic role in PL kinase phosphorylation activity. We also show that human PL kinase binds its substrates, PL and MgATP synergistically, and that the enzyme requires Na+ (or K+) and Mg2+ for its activity. Using kinetic study, we show severe induced MgATP substrate inhibition of PL kinase in the presence of its product, PLP, and we postulate this to be due to the formation of a non-productive ternary complex (Enzyme•PLP•MgATP). Consistently, our crystal structure of human PL kinase (2.1 Å) co-crystallized with MgATP and PLP showed both MgATP and PLP trapped at the active site. Our hypothesis is that this abortive ternary complex might be a physiological process, and that PL kinase uses this mechanism to self-regulate its activity. Our inhibition studies show theophylline, a bronchodilator as a mixed competitive inhibitor of human PL kinase with Ki of 71 μM. Our structural study (2.1 Å) shows theophylline bound at the substrate, PL binding site of human PL kinase. We also identified several potential PL kinase inhibitors from the DrugBank Chemical Compound database. Some of these compounds, including enprofylline, theobromine, caffeine, and lamotrigine, which incidentally exhibit similar neurotoxic effects as theophylline, show significant inhibitory effect on human PL kinase. Further studies are also planned to investigate the effect of these drugs on vitamin B6 metabolism in vivo.

Uma metodologia rápida e seletiva foi desenvolvida para a quantificação da piridoxina em medicamentos. O método foi padronizado para aplicação da espectrofotometria derivada no ultravioleta na análise direta da vitamina em preparações multivitamínicas sólidas (cápsulas) e líquidas (solução oral e injetável). As interferências do espectro UV convencional devidas aos excipientes (veículos) e demais fármacos presentes foram eliminados. As retas de calibração foram calculadas, obtendo-se, para a derivada de 1ª ordem, o coeficiente de correlação linear de 0.99997. Os resultados foram estatisticamente estudados e determinaram-se o desvio padrão, coeficiente de variação e intervalo de confiança. O método foi empregado na análise de amostras comerciais e simuladas e os resultados, quando comparados com aqueles provenientes da aplicação do método da Farmacopéia Americana XXII rev., evidenciaram nítidas vantagens quanto à exatidão e precisão, além da facilidade operacional. / A rapid and selecrive method for rhe dererminarion of pyridoxine in pharmaceuticals has been described. The procedure has been developed using direct UV first-derivative spectrofotometry in solid and liquid preparations (tablets, oral solution and injection). Spectral inrerferences from formulation excipienrs and other drugs in simple UV spectrophotometric methods have been eliminated by the application of the proposed method. Calibration curves have been made and the correlation coefficienr for. the first-order derivative was 0,99997. Standard deviation, coefficient of variation and confidence interval were calculated. The method was applied in the analysis of commercial and simulated samples. The results when compared with those obtained by using the USP 22nd. ed. official method shows clear advanrages related to accuracy, precision and practical application.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Uma metodologia rápida e seletiva foi desenvolvida para a quantificação da piridoxina em medicamentos. O método foi padronizado para aplicação da espectrofotometria derivada no ultravioleta na análise direta da vitamina em preparações multivitamínicas sólidas (cápsulas) e líquidas (solução oral e injetável). As interferências do espectro UV convencional devidas aos excipientes (veículos) e demais fármacos presentes foram eliminados. As retas de calibração foram calculadas, obtendo-se, para a derivada de 1ª ordem, o coeficiente de correlação linear de 0.99997. Os resultados foram estatisticamente estudados e determinaram-se o desvio padrão, coeficiente de variação e intervalo de confiança. O método foi empregado na análise de amostras comerciais e simuladas e os resultados, quando comparados com aqueles provenientes da aplicação do método da Farmacopéia Americana XXII rev., evidenciaram nítidas vantagens quanto à exatidão e precisão, além da facilidade operacional. / A rapid and selecrive method for rhe dererminarion of pyridoxine in pharmaceuticals has been described. The procedure has been developed using direct UV first-derivative spectrofotometry in solid and liquid preparations (tablets, oral solution and injection). Spectral inrerferences from formulation excipienrs and other drugs in simple UV spectrophotometric methods have been eliminated by the application of the proposed method. Calibration curves have been made and the correlation coefficienr for. the first-order derivative was 0,99997. Standard deviation, coefficient of variation and confidence interval were calculated. The method was applied in the analysis of commercial and simulated samples. The results when compared with those obtained by using the USP 22nd. ed. official method shows clear advanrages related to accuracy, precision and practical application.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.

Background: Inverse associations have been reported between homocysteine concentrations and poor cognitive performance in several cross-sectional studies of healthy elderly subjects. Folate supplementation with or without vitamins B-12 and B-6 is an effective means of lowering homocysteine concentrations. Mood disturbances, from mild mood changes to clinical depression, are common in older populations. Several studies have shown that depressed people have lower levels of folate and vitamin B-12 and higher levels of homocysteine than non-depressed people. Improvement of mood has been reported in depressed people following supplementation with folic acid. Clinical trials are required to determine if lowering homocysteine concentration with vitamins improves cognitive function and/or mood in healthy elderly participants. Objective: The primary aim of this research project was to carry-out a 2 year randomised, double-blind, placebo-controlled trial to determine if a supplement containing folate (1mg L-Mefolinic acid), vitamin B-12 (500(mu)g) and vitamin B-6 (10mg) improves scores or prevents decline on tests of cognition in a group of healthy older people ([greater than or equal to]̲ 65 years) with a plasma homocysteine concentration [greater than or equal to]̲13 (mu)mol/L. A second aim of this study was to determine if homocysteine lowering vitamins improved scores on tests of mood in this group. Methods: Four hundred and sixty-five individuals, aged 65 and over, were recruited from Dunedin and surrounds, and asked to attend a screening clinic and provide a fasting blood sample. Two-hundred and seventy-six volunteers with a plasma homocysteine concentration [greater than or equal to]13(mu)mol/L were randomised to take either a combination of 1mg L-Mefolinic acid, 500(mu)g vitamin B-12 and 10mg vitamin B-6 or placebo for 2 years. A battery of cognitive tests and indices of mood was administered at baseline, one year, and two years. A fasting blood sample was collected at baseline and every six months thereafter. Results: From baseline to 6 months of the intervention, homocysteine concentrations decreased by 37.5%, from 16.7 to 10.5 (mu)mol/L in the vitamin supplemented group and then plateaued. In the vitamin supplemented group there was a 181% increase in red blood cell folate concentration from a mean of 977 to 2752 nmol/L, and a 90.1% increase in plasma vitamin B-12 (from a mean 283 to 538 (mu)mol/L) over the study period of two years. In the vitamin supplemented group there was a trend to poorer performance on almost all tests of cognition compared to placebo group. The vitamin group was 8% slower on Part B of the Reitan Trail Making Test, a test of speeded attention, mental tracking, visual search and mental flexibility (p=0.009). The vitamin group scored significantly lower on tests of short-term recall, Weschler Paragraphs (p=0.03) after 2 years, and the Rey Auditory Verbal Learning Test ((p=0.04) after one year, than the placebo group. There was no difference in mood score by treatment in this largely non-depressed group. Conclusion: These results suggest a detrimental effect of high dose homocysteine lowering vitamin supplements on cognitive function in healthy older people. These results need to be confirmed in other randomised controlled trials.

The main objective of this proposal was to investigate the effect of vitamin B6 on colon carcinogenesis in vivo. Two in vivo studies were conducted to determine the role of vitamin B6 as a biological modifier of colon carcinogenesis. It is hypothesized that vitamin B6 may serve as an antioxidant in vivo and will modulate colon carcinogenesis. In the first study, a 2X3 factorial experimental design was used to determine if three different levels of vitamin B6, classified as low, normal and high in conjunction with two different levels of protein intake, classified as normal or high, will affect post-initiation stages of colon carcinogenesis, in Sprague-Dawley rats. Male Sprague-Dawley male were injected with azoxymethane for two weeks (15mg/kg/week) and then one week later they were allocated to different dietary treatment groups. After eight weeks, the effects of dietary treatment on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of preneoplastic lesions, aberrant crypt foci (ACF), were evaluated. The lowest level of vitamin B6 intake with a high protein diet reduced the growth and development of ACF. Vitamin B6 had no significant effect on the oxidative stress markers. The level of protein was an important variable in modulating the levels of 3-nitrotyrosine and 8-OH-DG which were lower in high protein groups than normal protein counterparts. The objective of the second study was to investigate if a supraphysiological (5 fold higher than normal level) dosage of vitamin B6 could have an antioxidant effect in a metabolically compromised state like obesity and thereby lower the risk of colon cancer. Female Zucker obese (Zk-OB) rats received normal (Zk-OBN, 7 mg/kg) or high (Zk-OBH, 35 mg/kg) vitamin B6 (Pyridoxine-HCl) diets two weeks prior to, during and six weeks following injection with colon carcinogen AOM. The effects of supplemental vitamin B6 on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of ACF were carried out. High intake of vitamin B6 significantly lowered liver weights and plasma cholesterol compared to the normal intake (p≤0.05). Zk-OBH rats had significantly reduced number of ACF compared to Zk-OBN (p≤0.05). Hepatic GSH increased in the ZK-OBH group with a concomitant decrease in GPx activity. The findings demonstrate that in Zucker Obese rats, a high B6 intake augmented the antioxidant potential and decreased sensitivity to colon carcinogenesis. These findings suggest that high vitamin B6 plays an important therapeutic role in the compromised state of obesity.

The main objective of this proposal was to investigate the effect of vitamin B6 on colon carcinogenesis in vivo. Two in vivo studies were conducted to determine the role of vitamin B6 as a biological modifier of colon carcinogenesis. It is hypothesized that vitamin B6 may serve as an antioxidant in vivo and will modulate colon carcinogenesis. In the first study, a 2X3 factorial experimental design was used to determine if three different levels of vitamin B6, classified as low, normal and high in conjunction with two different levels of protein intake, classified as normal or high, will affect post-initiation stages of colon carcinogenesis, in Sprague-Dawley rats. Male Sprague-Dawley male were injected with azoxymethane for two weeks (15mg/kg/week) and then one week later they were allocated to different dietary treatment groups. After eight weeks, the effects of dietary treatment on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of preneoplastic lesions, aberrant crypt foci (ACF), were evaluated. The lowest level of vitamin B6 intake with a high protein diet reduced the growth and development of ACF. Vitamin B6 had no significant effect on the oxidative stress markers. The level of protein was an important variable in modulating the levels of 3-nitrotyrosine and 8-OH-DG which were lower in high protein groups than normal protein counterparts. The objective of the second study was to investigate if a supraphysiological (5 fold higher than normal level) dosage of vitamin B6 could have an antioxidant effect in a metabolically compromised state like obesity and thereby lower the risk of colon cancer. Female Zucker obese (Zk-OB) rats received normal (Zk-OBN, 7 mg/kg) or high (Zk-OBH, 35 mg/kg) vitamin B6 (Pyridoxine-HCl) diets two weeks prior to, during and six weeks following injection with colon carcinogen AOM. The effects of supplemental vitamin B6 on hematological status, oxidative stress markers and antioxidant enzymes, as well as enumeration of ACF were carried out. High intake of vitamin B6 significantly lowered liver weights and plasma cholesterol compared to the normal intake (p≤0.05). Zk-OBH rats had significantly reduced number of ACF compared to Zk-OBN (p≤0.05). Hepatic GSH increased in the ZK-OBH group with a concomitant decrease in GPx activity. The findings demonstrate that in Zucker Obese rats, a high B6 intake augmented the antioxidant potential and decreased sensitivity to colon carcinogenesis. These findings suggest that high vitamin B6 plays an important therapeutic role in the compromised state of obesity.