Teratogenicity of 3-hydroxynorvaline in chicken and mouse embryos.

3-Hydroxynorvaline (HNV; 2-amino-3-hydroxypentanoic acid), a microbial L-threonine analogue, is toxic to mammalian cells and displays antiviral properties. In view of this, we investigated the toxicity and/or potential teratogenicity of HNV in developing chicken and mouse embryos. HNV was administered to chicken embryos (in ovo; dose 75-300 mumole/egg; 48 h post-incubation) and pregnant Hanover NMRI mice (per os; total dose 900-1800 mg/kg body mass; gestation days 7-9). Control animals received sterile saline solutions. Harvested embryos (chicken embryos, 10 days post-incubation; mouse embryos; gestation day 18) were fixed in glutaraldehyde and stereomicroscopically inspected for signs of dysmorphogenesis. Body mass, body and toe length and mortality of chicken embryos, and the body mass and mortality of mouse embryos were recorded. HNV exposure significantly increased the incidence of embryotoxic (growth retardation, toxic mortality) and congenital defects in both chicken and mouse embryos. All the observed effects were dose-dependent. In conclusion, HNV is an embryotoxic and teratogenic compound, which caused significant developmental delay and congenital defects in developing chicken and mouse embryos.

The effect of different apo(a) isoforms on plasminogen activation in black South African subjects.

OBJECTIVE: The present study evaluates the effect of different apolipoprotein(a) [apo(a)] isofroms on plasminogen activation. DESIGN: A cross-sectional study. SETTING: A rural village (Dikgale district) in the Northen Province of South Africa. SUBJECT: A total of 90 apparently healthy subjects (64 females and 36 males) aged 43 to 67 years participated in the study. RESULTS: The mean lipoprotein(a) [ Lp(a)] level in the subjects was 38.14 +/- 22.34 mg/dl. No association was found between Lp(a) isoforms. When the ratio of Lp(a):plasminogen was less then 1.3, a competitive inhibition was observed, but when the ratio exceeded 1.3, an uncompetitive inhibition was observed with all isoforms. CONCLUSION: The results of the present study suggest that the inhibition of plasminogen activation by Lp(a) is not dependent of apo(a) size.

Steroidogenic correlates of pregnancy in the rock hyrax (Procavia capensis).

In pregnant rock hyraxes isolated leucocytes metabolise both [3H]pregnenolone and [3H]progesterone while whole blood, erythrocytes and an erythrocyte/leucocyte mixture only metabolised [3H]progesterone. Plasma displayed no tendency to metabolically convert any one of these two steroids. In whole blood [3H]progesterone appears to be converted to 5alpha-pregnane-3,20-dione and a compound with chromatographic properties similar to that of 5alpha-pregnan-3alpha-ol-20-one. 5Alpha-pregnane-3,20-dione exhibited a high relative binding affinity for the uterine progesterone eceptor (94%), but 5alpha-pregnan-3alpha-ol-20-one displayed very little affinity for the same receptor (0.4%). 5Alpha-pregnane-3,20-dione may therefore aid in the maintenance of pregnancy. Corpora lutea metabolised progesterone to 17alpha-hydroxyprogesterone, a compound exhibiting no progestational function because of its low relative binding affinity for the uterine progesterone receptor (2%). Progesterone appears to be the main product of the corpus luteum. However, 5alpha-pregnane-3,20-dione circulated at concentrations approximately 8.5 times higher than progesterone, probably due to the metabolic conversion of progesterone to 5alpha-pregnane-3,20-dione by the blood. We conclude that in the hyrax progesterone, produced by the corpora lutea, enters the circulation, where it is reduced to 5alpha-pregnanes. 5Alpha-pregane-3,20-dione may then be transported to the uterus where it binds to the progesterone receptor to assist in the maintenance of pregnancy. This mechanism appears to be analogous to that of the African elephant which is phylogenetically related to the hyrax, except that in the elephant the 5alpha-reduced metabolites are produced by luteal tissue and not the blood.

Influence of gestation on uterine endometrial steroid receptor concentrations in the African elephant, Loxodonta africana.

The modulatory effects of gestational age and circulating concentrations of progesterone, 5alpha-pregnane-3,20-dione, and estradiol-17beta on the uterine sex steroid hormone receptor levels of the African elephant were investigated. Uterine tissue biopsies and blood samples were obtained from animals culled in the Kruger National Park. Estrogen and progesterone receptor concentrations were determined in uterine biopsies from subadult, lactating, early-, mid-, and late-pregnant elephants, by equilibrium binding assays. Circulating estradiol-17beta and progesterone concentrations were measured by means of RIAs, while plasma concentrations of 5alpha-pregnane-3,20-dione were determined with an amplified ELISA. Significant inverse correlations of the concentrations of estrogen and progesterone receptors with the gestational stage of the elephants were observed. Pregnant uterine horns of individual animals contained lower levels of estrogen and progesterone receptors than the nonpregnant horns of the same animals. A strong positive correlation existed between uterine estrogen and progesterone receptors levels. Circulating concentrations of 5alpha-pregnane-3,20-dione and progesterone decreased with an increase in the concentrations of progesterone receptors as well as with fetal age. We conclude that the progesterone receptor concentrations are down-regulated with progressing gestation in the African elephant. This down-regulation appears to be linked to an increase in circulatory 5alpha-pregnane-3,20-dione concentration in the plasma of pregnant animals.

The effect of Simvastatin on the plasma antioxidant concentrations in patients with hypercholesterolaemia.

The aim of this study was to monitor the antioxidant status of patients with hypercholesterolaemia during treatment with Simvastatin. Forty-seven patients, of whom 25 had confirmed familial hypercholesterolaemia (FH), were treated with 10 or 20 mg of Simvastatin per day for 14 weeks. As expected, total cholesterol and LDL cholesterol concentrations decreased considerably, while HDL cholesterol concentrations increased during drug treatment. In neither FH nor non-FH patients were any significant changes observed for retinol status, while plasma vitamin C concentrations were also not adversely affected by the drug therapy. In both patient groups Simvastatin therapy led to a significant decrease in plasma alpha-tocopherol (P < 0.05) concentrations, however, the alpha-tocopherol/total cholesterol ratio increased by 9.1 (P < 0.01) and 12.1% (P < 0.01) in FH and non-FH patients, respectively, during the 14-week treatment period. The coenzyme Q10/total cholesterol ratio did not change significantly in non-FH patients, but was significantly lower (P < 0.05) than the baseline ratio after 4 and 14 weeks of Simvastatin treatment in FH patients. The alpha-tocopherol/total cholesterol ratio of FH patients remained consistently and significantly lower (P < 0.01) compared with non-FH patients, indicating that LDL from the former group may be more vulnerable to free radical-mediated damage and lipid peroxidation. Our results suggest that the significant decline in circulating alpha-tocopherol and coenzyme Q10 concentrations was mainly a function of the decrease in serum total cholesterol concentrations.

Spontaneous oxidation of methionine: effect on the quantification of plasma methionine levels.

Plasma methionine (Met), methionine sulfoxide (MSO), and total Met concentrations were determined by reversed-phase chromatography and fluorescence detection after automated precolumn derivatization with an o-phthalic aldehyde mercaptoethanol reagent. Addition of pure, MSO-free L-Met to plasma samples resulted in the anticipated linear increase in plasma Met concentrations, but simultaneously effected a dose-dependent, linear increase in MSO levels. In contrast, the addition of pure L-MSO to plasma samples rendered linear calibration curves for MSO, while the Met concentration remained constant. A strong buffering effect against the spontaneous or hydrogen peroxide induced oxidation of Met to MSO was observed in plasma samples. This protective effect could be neutralized by preincubating the plasma samples with sodium azide. The addition of relatively low concentrations of red cell lysates to plasma samples, prior to hydrogen peroxide oxidation, strongly inhibited the conversion of Met to MSO. Plasma samples from 127 healthy female volunteers were analyzed: MSO concentrations (mean, 3.6 +/- 2.1 microM) exhibited a weak positive correlation (r = 0.352) with Met levels (mean, 21.3 +/- 6.1 microM) but, after the exclusion of two probable outliers from the data set, no correlation was observed. Our results suggest that plasma Met concentrations should be corrected for oxidative losses incurred during storage, sample processing and because of the action of a variety of in situ oxidants, present in plasma, in order to obtain a reliable estimate of the methionine status of an individual.

Ligand specificity of uterine oestrogen and progesterone receptors in the subadult African elephant, Loxodonta africana.

The ligand specificity of progesterone and oestrogen receptors in the uteri of four nonpregnant, nonlactating African elephants, killed during routine culling in the Kruger National Park, were determined. The mean (+/-SEM) Kd values of the oestrogen (0.18 +/- 0.019 x 10(-9) mol l-1, n = 12) and progesterone (0.22 +/- 0.025 x 10(-9) mol l-1, n = 12) receptors were essentially similar when [3H]promegestone was used as radioligand in the progesterone receptor assays. However, when [3H]progesterone was used as radioligand, the progesterone receptor exhibited a significantly higher Kd value (1.03 +/- 0.132 x 10(-9) mol l-1, n = 12) than that of the oestrogen receptor. The use of the different radioligands did not significantly affect the quantitative values obtained for the progesterone receptor. Both the oestrogen and the progesterone receptors displayed a high ligand specificity. The 5 alpha-reduced metabolites of progesterone exhibited a high relative binding affinity for the progesterone receptor (5 alpha-pregnane-3,20-dione: relative binding affinity = 43%; 5 alpha-pregnane-3 alpha-ol-20-one: relative binding affinity = 20%) but the synthetic antiprogestin RU 486 did not compete successfully with progesterone in competitive binding studies. However, norethindrone (relative binding affinity = 293%) competed successfully for binding to the progesterone receptor, and may have some potential in the future development of a technique to control reproductive output in the African elephant.

Correlation between essential fatty acids and parameters of bone formation and degradation.

There are two types of essential fatty acids (EFAs), the n-6 derived from linoleic acid (LA) and the n-3, derived from alpha-linolenic acid (ALA). Most of the functions of the EFAs require the conversion of LA and ALA to their metabolites including, gammalinolenic (GLA), dihomogammalinolenic (DGLA), arachidonic (AA) (n-6) and eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids (n-3). Supplementing specific GLA:EPA ratios has effects on bone formation and degradation. A study was designed to investigate the effect of various dietary ratios of n-6:n-3 on calcium homeostasis. Female Sprague Dawley rats were ovariectomised (OVX) at age =11 weeks, and were supplemented from age 12 weeks for six weeks with different ratios (9:1; 3:1; 1:3; 1:9) of GLA:EPA. Bone parameters and red blood cell (RBC) fatty acid profiles were measured at age=18 weeks. RBC GLA and DGLA increased in groups 9:1 and 3:1(p<0.05). EPA and DGLA increased in 1:3 and 1:9 while AA decreased (p<0.05). Correlations were calculated between bone calcium, deoxypyridinoline (Dpyd) and specific fatty acids. DGLA was positively correlated with femur calcium and negatively with Dpyd excretion while DHA and EPA were correlated with femur calcium.

Oestrogen and progesterone receptor status and PgR/ER ratios in normal and myomatous human myometrium.

The concentration and molecular properties of the oestrogen (ER) and the progesterone (PgR), present in normal myometria and uterine leiomyometria, obtained from a group of age-matched, pre-menopausal, negroid female patients were investigated. Serum oestrogen and progesterone levels did not differ significantly in the two groups. Significant differences were detected in ER and PgR levels between normal and leiomyomatous myometria. Both ER (154%; p < 0.0001) and PgR (33%; p < 0.05) were significantly increased in uterine leiomyomas. PgR levels were less affected than the ER levels, causing a significant decrease (44%; p < 0.05) in the PgR/ER ratio in myomatous myometria. Dissociation and sedimentation constants, as well as iso-electric points of ER and PgR were essentially similar in normal and in myomatous myometria. According to our results, tissue pathology does not appear to be associated with defects in the molecular properties of ER and PgR, but with differential changes in the ER and PgR levels, subsequently affecting the PgR/ER ratio.

Supplemented gamma-linolenic acid and eicosapentaenoic acid influence bone status in young male rats: effects on free urinary collagen crosslinks, total urinary hydroxyproline, and bone calcium content.

The effect of different ratios of the prostaglandin precursors gamma-linolenic (GLA) and eicosapentaenoic (EPA) acids on bone status in growing rats measured as a function of free urinary pyridinium crosslinks and hydroxyproline levels was investigated. Male Sprague-Dawley rats were weaned onto an essential fatty acid deficient diet and from their fifth week, different groups of rats received a balanced, semisynthetic diet, supplemented with different ratios of GLA:EPA supplied as a mixture of evening primrose oil (EPO) and fish oil (FO). Controls were supplemented with linoleic (LA; sunflower oil) and alpha-linolenic (ALA; linseed oil) acids (3:1) or a commercially available rat chow. Animals were terminated at 84 days and femur length, ash weight, calcium content, free urinary pyridinium crosslinks (Pyd and Dpyd), total hydroxyproline (Hyp), and creatinine levels measured. Free urinary Pyd and Dpyd are good indicators of bone status and they correlated well with Hyp. Pyd and Dpyd excretion were significantly decreased in the higher GLA:EPA dietary groups and correlated well (r = 0.7) with Hyp levels. Concomitantly, bone calcium content increased significantly in the same dietary groups. These results suggest that diet supplementation with relatively high GLA:EPA ratios are more effective in inhibiting bone resorption than LA:ALA.

Cholate and pH reduce interference by sodium dodecyl sulfate in the determination of DNA with Hoechst.

The use of the fluorescent dye 33258 Hoechst (Hoe) to quantitatively determine DNA in cell culture in the presence of lysing agents like sodium dodecyl sulfate (SDS) is limited by the masking effect of high levels of nonspecific fluorescence, caused by the binding of Hoe to micelles. The masking effect can be reduced substantially by increasing the concentration of the counterion, the addition of cholate, or the pH of the buffer. An optimized method was developed, combining the antimasking effects of sodium chloride, cholate, and pH to accurately determine DNA concentrations as low as 15 ng/ml in the presence of up to 6.9 mM (0.2%) SDS. The effectiveness of SDS in cell dissolution can now be combined with the specificity and sensitivity of Hoe to determine cellular DNA.

Vitamin requirements for the treatment of hyperhomocysteinemia in humans.

We have previously shown that a modest vitamin supplement containing folic acid, vitamin B-12 and vitamin B-6 is effective in reducing elevated plasma homocysteine concentrations. The effect of supplementation of the individual vitamins on moderate hyperhomocysteinemia has now been investigated in a placebo-controlled study. One hundred men with hyperhomocysteinemia were randomly assigned to five groups and treated with a daily dose of placebo, folic acid (0.65 mg), vitamin B-12 (0.4 mg), vitamin B-6 (10 mg) or a combination of the three vitamins for 6 wk. Folic acid supplementation reduced plasma homocysteine concentrations by 41.7% (P < 0.001), whereas the daily vitamin B-12 supplement lowered homocysteine concentrations by 14.8% (P < 0.01). The daily pyridoxine dose did not reduce significantly plasma homocysteine concentrations. The combination of the three vitamins reduced circulating homocysteine concentrations by 49.8%, which was not significantly different (P = 0.48) from the reduction achieved by folate supplementation alone. Our results indicate that folate deficiency may be an important cause of hyperhomocysteinemia in the general population.

Suramin in combination with mitomycin C in hormone-resistant prostate cancer. A phase II clinical study.

BACKGROUND: Hormone-resistant prostate cancer can respond to mitomycin C or to suramin. This trial was undertaken to investigate the value of mitomycin C given with low dose suramin. PATIENTS AND METHODS: Thirty-two patients with hormone-refractory prostate cancer were given suramin 350 mg/m2 daily for 5 days, followed by 350 mg/m2 weekly starting on day 14. Mitomycin C 12 mg/m2 was given every 5 weeks starting on day 14. RESULTS: Toxicity included maculo-papular skin rash in 8 patients, haematological toxicity in 16 (anaemia 13, leucopenia 11 and thrombocytopenia 9, bleeding 8), infection in 4 and fatigue in 11. Ten patients developed neurotoxicity, (temporary sensory peripheral neuropathy in 8, upper limb motor neuropathy in 1, and restless legs syndrome in 1) and 9 developed proteinuria. Other toxicities were mild nausea and vomiting, oedema, transient elevation of liver enzymes, stomatitis, upper gastrointestinal symptoms, and alopecia. During induction treatment the median trough suramin level was 140 micrograms/ml (range 100-273) and during maintenance treatment the median suramin level was 93 micrograms/ml. The median overall trough level was 93 micrograms/ml. There were one complete and 6 partial responses. Fifteen patients had disease stabilization. The median time to treatment failure was 103 days, and the median survival 209 days. CONCLUSION: The combination of suramin and mitomycin C has therapeutic activity, but causes significant toxicity in patients with hormone-resistant prostatic carcinoma.

Hepatic estrogen-binding proteins in male and female vervet monkeys.

Hepatic cytosols from male and female vervet monkeys (Cercopithecus pygerythrus) were found to contain the same levels of high affinity estrogen-binding proteins. Multipoint saturation analyses revealed that male liver cytosols contain two distinctly different binding components: a high affinity (HAEB) and a low affinity estrogen binder (LAEB). Female livers appeared to contain only the HAEB. Sucrose density gradient (SDG) analyses, however, clearly established the presence of a 3.8 S as well as an 8.1 S estrogen-binding component in the hepatic cytosols of both sexes. The 3.8 S binding component appeared to be more prominent in male SDG profiles. Cytosols, prepared in the presence of sodium molybdate (cyt +) exhibited significantly lower (50%) levels of specific estrogen-binding than cytosols prepared in the absence of the oxyanion (cyt-). SDG analyses, however, indicated that in cyt+ the 8.1 S binding component was stabilized at the cost of the 3.8 S binder. This phenomenon was observed in both sexes. Large excess levels of cortisol did not have any effect on specific estrogen binding by hepatic cytosols. The hepatic estrogen-binding proteins displayed a lower relative binding affinity for diethylstilbestrol than for its native ligand and higher affinities for estriol and estrone than expected.

Molybdate and the molecular properties of the vervet monkey estrogen receptor.

The Vervet monkey (Cercopithecus aethiops pygerythrus) uterine estrogen receptor was partially characterised. The effect of the molybdate oxyanion on various molecular properties of the receptor was investigated. Molybdate appeared to affect the subunit structure and apparent heterogeneity of the receptor. Anion exchange chromatography of uterine cytosols yielded two ligand binding subunits in a 1:1 ratio in the absence of sodium molybdate, while only a single labelled complex could be demonstrated in cytosols prepared in molybdate containing buffers. Chromatofocussing of the nonstabilized cytosols revealed substantial receptor heterogeneity (7 peaks) while a much simpler pattern (2 peaks) could be observed in the presence of the molybdate. Likewise, iso-electric focussing of labelled cytosols on agarose gels yielded at least 3 high affinity binding components (pI:6.8, 6.2, 5.9) in the absence and only one major band in the presence of sodium molybdate (pI 5.9).

Circulating progesterone, progesterone-binding proteins and oestradiol-17 beta concentrations in the pregnant Cape porcupine, Hystrix africaeaustralis.

Circulating concentrations of progesterone, progesterone-binding plasma proteins (PBPP) and oestradiol-17 beta in pregnant porcupines remained relatively low until Days 25-30 post coitum. Progesterone values peaked (102-180 ng/ml; N = 3) 42-60 days post coitum and the rapid increase in oestradiol-17 beta concentrations approximated that of progesterone with peak values (170-210 pg/ml) being attained 60-85 days post coitum. The pattern of PBPP synthesis, as suggested by circulating concentrations, was closely related to that of plasma progesterone, with values remaining low (less than 20 pmol/ml) until Day 31 post coitum, reaching peak levels at Days 50-56 and Days 73-77 post coitum. The production of PBPP during pregnancy is, as in related New World hystricomorph species, considered to be a mechanism which facilitates a reduction in the rate of progesterone metabolism during pregnancy.

Value of steroid hormone receptor analysis in predicting time to treatment failure in operable breast cancer.

An analysis of prognostic factors for predicting time to treatment failure (TTF) was performed on 246 patients who were referred for adjuvant chemotherapy after mastectomy. Oestrogen receptor (ER) and progesterone receptor (PgR) analyses were carried out on 172 and 102 patients respectively. Prognostic factors predicting for TTF were, in decreasing order of significance: nodal status, tumour size, ER status and age of the patient. Comparison of TTF for ER+ and ER- (without categories of other prognostic factors) showed that ER status is prognostic irrespective of nodal status. ER is prognostically significant within both categories of PgR. PgR predicted significantly for TTF only in patients without nodal involvement.

Reversibility of the stabilization effect of sodium molybdate on uterine estrogen and progesterone receptors of the vervet monkey.

Sodium molybdate affected the stability of vervet monkey (Cercopithecus aethiops pygerythrus) uterine estrogen (ER) and progesterone (PR) receptors. Yields of receptors were invariably higher (20-40%) when cytosols were prepared in the presence of 10mM sodium molybdate. No changes were observed in the binding affinities for the natural ligands as reflected in dissociation constants. Receptor-ligand association at 0 degrees C and 20 degrees C was not affected in the presence or absence of molybdate. Stability studies at 37 degrees C indicated both receptors to be more resistant to inactivation in the presence of molybdate. Dissociation of ER and PR was biphasic, indicating the existence of slow (SDC), as well as fast dissociating (FDC) complexes. Rate constants of dissociation were significantly affected by the presence of sodium molybdate. Although no significant changes in the sedimentation coefficients were observed, marked differences in the actual gradient profiles could be illustrated in the presence or absence of sodium molybdate. Observed effects could only be partially reversed in sedimentation dialysis experiments. Proteolytic inhibitors phenylmethylsulfonylfluoride (PMSF) and leupeptin had no inhibitive effect on the molybdate stabilization of ER and PR.

Estrogen and progesterone receptors in the uterus of the vervet monkey.

Experimental conditions for the optimal measurement of estrogen (ER) and progesterone (PR) receptors in normal vervet monkey (Cercopithecus aethiops pygerythrus) uteri are described. The uteri of this primate were found to contain relatively high concentrations of both ER and PR. Levels of ER ranged from 151 to 822 femtomoles per mg protein (mean for group assayed is 327 +/- 165 femtomoles per mg protein). PR assays were performed on the same cytosols and the levels ranged from 444 to 2267 femtomoles per mg protein (mean of 1285 +/- 511 femtomoles per mg protein). Mean Kd values for the ER- and PR-ligand complexes were found to be 3.15 +/- 1.4 X 10(-10)M and 2.38 +/- 0.2 X 10(-9)M respectively, within the group analysed (n = 21). The ratio of PR to ER varied between 1.1 and 13.1 with a mean of 4.5 +/- 2.4. Ligand specificity studies revealed that [3H]-17 beta-estradiol binding to the ER could only be inhibited by estrogens or estrogen analogues. The PR however exhibited an affinity for a wider range of ligand types. In low ionic strength buffers both ER and PR sedimented as approximately 8S type molecules in the presence or absence of 10mM sodium molybdate. Both receptors dissociated into smaller components, following a short exposure to 0.4 M KCl and subsequent centrifugation in a gradient containing 0.4 M KCl.