Govind S. Rao

Chemotaxis of human blood monocytes toward endothelin-1 and the influence of calcium channel blockers

The adherence of monocytes to the arterial endothelium followed by its migration into the arterial intima is the earliest event in atherogenesis. The vasoconstrictive peptide, Endothelin-1 (ET-1), is elevated in patients with atherosclerosis. We were interested to know whether ET-1 was a chemoattractant for blood monocytes. Using the modified membrane filter technique for chemotaxsis assessment, ET-1 increased monocyte chemotaxis in a dose-dependent manner. Ca2+ channel blockers, Nifedipine, Diltiazem and Verapamil (5 microM), reduced ET-1 chemotaxsis more than 60% (P < 0.001). Aspirin and Indomethacin (1 mM and 100 microM, respectively) reduced migration by 23% (P < 0.05). Alpha-Lipoic acid, Probucol and Neomycin (100 microM) were also migration inhibitory (37%, P < 0.01). These results suggest that ET-1 is a strong chemoattractant for blood monocytes; Ca2+ influx is probably the major stimulus for the accelerated migration induced by ET-1.

Serum selenium versus lymphocyte subsets and markers of disease progression and inflammatory response in human immunodeficiency virus-1 infection

Serum selenium levels were determined cross-sectionally in 57 HIV-infected patients who were classified according to the Centers for Disease Control (CDC) 1993 classification system. Mean serum selenium levels were lower in CDC stage II (58.7±12.2 μg/L;p<0.01;n=18) and stage III (47.6±11.3 μg/L;p<0.01;n=19) HIV-infected patients, than in healthy subjects (80.6±9.6 μg/L;n=48) and stage I patients (73.6±16.5 μg/L;n=20). Serum selenium levels were positively correlated with CD4 count, CD4/8 ratio, hematocrit, and serum albumin (r=0.42;r=0.39;r=0.48; andr=0.45;p<0.01, respectively) and inversely with serum levels of thymidine kinase (r=−0.49;p<0.01;n=49) and β2-microglobulin (r=−0.46;p<0.001;n=49). In addition, serum selenium levels in 20 randomly selected AIDS-free individuals (CDC I:n=10; CDC II:n=10) were inversely correlated with serum concentrations of interleukin-8 (IL-8) and soluble tumor necrosis factor receptors (sTNFR) types I and II. There was no correlation with serum immuneglobulin A and total serum protein levels. The results show that the progressive deprivation of serum selenium in HIV-infection is associated with loss of CD4+-cells and with increased levels of markers of disease progression and inflammatory response.

INTERFERON/ANTIOXIDANT COMBINATION THERAPY FOR CHRONIC HEPATITIS C--A CONTROLLED PILOT TRIAL

The effects of two forms of antioxidative co-therapy were analyzed in 24 interferon-alpha (IFN)-naive patients with chronic hepatitis C who were randomized to either receive IFN monotherapy (3 x 4.5 million units IFN-alpha 2a per week), (group A), or IFN and N-acetylcysteine (N-acetylcysteine (NAC) 1.800 mg/day) plus sodium selenite (400 microg/day) supplementation (group B), or treatment as in group B plus vitamin E (544 IU/day) (group C), over 24 weeks. Changes in histology, normalization of ALT, reduction of viral RNA, serum levels of glutathione, selenium, vitamin E, erythrocyte glutathione peroxidase, trolox equivalent antioxidative capacity (TEAC), thiobarbituric acid reactive substances (TBARS) and protein carbonyl groups were measured. Low baseline TEAC and elevated TBARS indicated increased oxidative stress before therapy, which was not affected by antioxidant supplementation. At the end of treatment complete responses were found in 3/8, 2/8 and 6/8 patients in groups A, B and C, respectively, but liver histology had not significantly improved. Vitamin E treated patients had a 2.4 greater chance (95% CI: 1.05-5.5) of obtaining a complete response and had significantly greater reduction in viral load (P = 0.028) than patients without vitamin E. Relapses, i.e. re-appearance of detectable hepatitis C virus (HCV) RNA and/or re-elevation of ALT-activity occurred in 7 out of the 11 responders within 6 months after termination of therapy (group A: 2/3, group B: 1/2 and group C: 4/6). Thus, no overall beneficial effect of antioxidant/IFN therapy was detected. However, the apparent trend towards a more favorable outcome with vitamin E supplementation warrants to further study this substance as an adjuvant to IFN therapy in chronic hepatitis C.

Mode of entry of steroid and thyroid hormones into cells

Characteristics of the processes by which steroid and thyroid hormones enter tissues, cells and membrane vesicles are reviewed. Several authors suggest that entry is by passive diffusion: the accumulation within cells is attributed to cytoplasmic binding proteins. Other authors, however, propose a membrane-mediated process of entry. The involvement of saturability, high specificity, sensitivity to temperature, sulfhydryl and cell-surface-perturbing reagents and hydrolytic enzymes support the latter view. Purified plasma-membrane vesicle preparations retain several characteristics of entry shown by intact cells. Intracellular hormone-binding protein would not contribute to processes observed with these preparations.

Uptake of thyroid hormone by isolated rat liver cells.

L-Triiodothyronine is taken up by isolated rat liver cells by a process which is saturable and exhibits sigmoidity. Two uptake systems make themselves evident: A system with high affinity with an apparent Kt value of 52±22 nM and a system with low affinity with an apparent Kt value of 1446±764 nM. Cells heated at 60°C or after freezing do not show saturability of uptake. KCN inhibits the uptake by the low affinity system. In the presence of L-thyroxine and L-tyrosine the uptake of L-triiodothyronine is increased. The results suggest transport of L-triiodothyronine by proteins in the plasma membrane of the liver cell.

An improved assay for steroid glucuronyltransferase in rat liver microsomes.

Abstract A simple and economical method of assaying rat liver microsomal estrone and testosterone glucuronyltransferase activity has been developed. Liver microsomes were activated by pretreatment with Lubrol WX. The incubation was carried out at 37°C for 30 min and contained 30–600 μ m steroid, 1–2 m m UDP-glucuronic acid, 10 m m MgCl 2 , and 80–150 μg of microsomal protein. Enzyme activities showed a maximum at pH 8.8 with Tris-HCl buffer. After incubation the unreacted substrate was quantitatively removed by a single extraction with dichloromethane. The glucuronide was estimated by counting an aliquot of the aqueous phase in a liquid scintillation counter. The variation coefficients with estrone and testosterone as substrates were 6.0 and 4.0%, respectively.

Sodium selenite and N-acetylcysteine in antiretroviral-naive HIV-1-infected patients: a randomized, controlled pilot study.

The aim of this work was to study the effects of combined oral administration of N‐acetylcysteine (NAC) and sodium selenite (Se) on plasma glutathione (GSH), lymphocyte subpopulations and viral load in asymptomatic human immunodeficiency virus (HIV)‐infected patients.

Kinetics of steroid transport through cell membranes: Comparison of the uptake of cortisol by isolated rat liver cells with binding of cortisol to rat liver cytosol

Abstract Viable rat liver cells suspended in a medium containing cortisol take up the steroid rapidly; the uptake is temperature dependent. After incubation for 1 min at 27°C the uptake is 20 times higher than that observed at 5°C. The activation energy calculated for the uptake of cortisol was 18 kcal between 5° to 20°C and 6 kcal from 20° to 32°C. With increasing concentrations of cortisol two saturable systems were observed, one with an apparent K M value of 0.2 μM and the other with 2.0 μM. The uptake after 1 min of incubation at 27°C was 9 pmol per mg cell protein. Diffusion of cortisol into the cell was also observed at the steroid concentrations tested. Metabolic inhibitor KCN inhibited the saturable uptake system; pCMB decreased the uptake of cortisol. Binding of cortisol to liver cytosol, carried out under conditions of uptake of cortisol by rat liver cells was more rapid; however, the binding after 1 min of incubation at 27°C was twice that observed at 5°C. The Arrhenius plot was linear from 5° to 32°C; the activation energy was calculated to be 1.2 kcal. The binding was linear from 5 to 10,000 nM cortisol. After incubation for 1 min at 27°C the binding of cortisol to cytosol proteins was 5% of the uptake by liver cells. KCN did not inhibit the binding, whereas pCMB decreased the binding. The results suggest that the uptake of cortisol by liver cells is mediated by carrier proteins in the plasma membrane and that cytosol proteins are not directly involved in this initial uptake process.

Specific interaction of corticosteroids with components of the cell membrane which are involved in the translocation of the hormone into the intravesicular space of purified rat liver plasma membrane vesicles

Abstract The uptake of [ 3 H]-corticosterone was studied by a highly purified rat liver plasma membrane vesicle fraction. At 23°C, [ 3 H]-corticosterone is taken up very rapidly; equilibrium is reached as early as 5 s. At high concentrations of corticosterone (30–8100 nM) uptake occurs predominantly by a non-saturable process, but the presence of a saturable process is also detectable. When the intravesicular space is successively decreased by increasing the osmolarity of the external medium, uptake of [ 3 H]-corticosterone decreased. About 45% of the total steroid taken up is translocated into the lumen of the vesicle. In the presence of a 200-fold molar excess of non-labeled corticosterone about 40% of the total uptake of [ 3 H]-corticosterone is displaceable. At low concentrations of corticosterone (2—230 nM) and considering only the specific portion of uptake the presence of a high and a low affinity uptake system is observable. The K D of the two systems were 7.2 ± 2.0 nM and 234 ± 67 nM, respectively. The K D of the high affinity system corresponds to the concentration of free corticosterone (about 8 nM) in the plasma of the female rat. The maximal binding capacity was about 180 fmol/(mg membrane protein). Structural analogues of corticosterone reduce the displaceable portion of the uptake of [ 3 H]corticosterone; cortisol, progesterone. dexamethasone and 5α-dihydrocorticosterone are strong inhibitors. Among the sex hormones 5α-dihydrotestosterone and diethylstilbestrol are the most efficient inhibitors. These results suggest that corticosterone reacts in a specific manner with components of the plasma membrane which may function to translocate the steroid into the cell.

Vitamin E status in patients with liver cirrhosis: Normal or deficient?

The study aim was to compare the ratio of vitamin E to serum cholesterol with the serum vitamin E level alone as a measure of vitamin E status in patients with different degrees of liver dysfunction. Assessment of serum vitamin E and total serum cholesterol was performed in 85 patients with liver cirrhosis at Childs stage A (n = 26), B (n = 26), and C (n = 33) and 50 patients with noncirrhotic liver disease. As surrogate markers of liver function, 7alpha-hydroxycholesterol and prealbumin concentrations and the plasma prothrombin time were determined. Mean serum vitamin E concentrations in Child A, B, and C patients were 27.4%, 36.9%, and 37.3% lower, respectively, than in healthy controls (P<.01). Twelve of 26 Child A, 14 of 26 Child B, and 14 of 33 Child C patients had vitamin E deficiency with respect to the absolute values, i.e., serum levels less than 13.76 micromol/L (5% percentile of healthy controls). In contrast, only two of 26 Child A, five of 26 Child B, and five of 33 Child C patients (P<.01 for Child A/B and P<.05 for Child C) were vitamin E-deficient according to the serum vitamin E to cholesterol ratio, i.e., less than 2.86 micromol/mmol. Serum vitamin E was correlated significantly with prealbumin, 7alpha-hydroxycholesterol, and the plasma prothrombin time, but the vitamin E to cholesterol ratio was not. Correcting serum vitamin E for total serum cholesterol in patients with liver cirrhosis leads to the phenomenon of reduced serum vitamin E levels inadvertently shifted toward normal values. In patients with liver cirrhosis, the absolute vitamin E concentration correlates better with the typical clinical and biochemical findings of the disease than the vitamin E to cholesterol ratio. Therefore, a considerable number of patients with advanced liver cirrhosis might actually be vitamin E-deficient.