A comparative morphological study of direct nerve implantation and neuromuscular pedicle methods in cross reinnervation of the rat skeletal muscle.

PURPOSE: The study was undertaken to evaluate the recovery of rat skeletal muscles reinnervated by crossed direct nerve implantation and crossed neuromuscular pedicle graft. MATERIAL AND METHODS: The animals (157) were divided into 3 groups. In the first group--direct nerve implantation --(DNI) 52 animals, the peroneal nerve was transected and implanted into the gastrocnemius muscle of the hind limb of the rats. In the second group (53 animals)--the neuromuscular pedicle (NMP) of the peroneal nerve was elevated and transferred to the gastrocnemius muscle. The third group consisted of 52 healthy animals. Muscle function was examined by electrophysiological methods (evoked electromyography and muscle isometric tetanic tension) and by morphological methods (reinnervated muscle weight, microscopic examinations, morphometric and histochemical examinations). RESULTS: The weight of reinnervated muscles in the first 3 weeks decreased. The lowest values of muscle weights were noted at 4 weeks. At the end of the experiment muscle weight in the first group was 64.3% of the control group and in the second group 65.2%. Morphometric, histological and histochemical analysis were performed after 12 and 36 weeks. At 12 weeks of the experiment the diameter of reinnervated muscle fibers in the second group was statistically higher than in the first group. At that time the process of reinnervation was more advanced than in the first group. At 36 weeks there were no statistical differences between the two groups. An increase in the number of muscle fibers was noted as the processes of reinnervations progressed. At the site of nerve (or pedicled nerve-muscle) implantation new motor end plates were formed. CONCLUSIONS: We consider that reinnervation of the experimentally paralyzed muscle is possible after crossed DNI and after NMP neurotization. The reinnervation with the NMP technique is quicker than with the DNI. Based on the morphological examinations--both methods guarantee only a partial recovery of the function of the paralyzed muscle.

An in vivo/in vitro comparison with a leuprolide osmotic implant for the treatment of prostate cancer.

An osmotically driven implantable system was designed and characterized for the delivery of leuprolide over a year-long duration. Leuprolide has been used in the treatment of prostate cancer since the 1980s. The DUROS implant consists of a titanium alloy cylinder, measures 4 mm in diameter by 45 mm in length and holds approximately 150 microl of formulation. Stability studies indicated that leuprolide was stable, as a solution formulation in DMSO, for several years at 37 degrees C. In vitro release rate testing, at weekly intervals, showed zero-order delivery for 1 year. DUROS implants demonstrated release rates that were reproducible and similar to one another after storage at 25 degrees C for 18 months prior to testing. In vivo studies, with implants placed subcutaneously, revealed delivery rates comparable to those observed under in vitro conditions. Leuprolide stability was also comparable between in vivo and in vitro conditions. Steady leuprolide serum levels produced by the implant resulted in the desired pharmacodynamic efficacy endpoint of testosterone suppression, both in canines and in humans. The good agreement between in vivo/in vitro delivery rates was as expected for a delivery system based on the principles of osmosis.

Effects of nandrolone decanoate therapy in borderline hypogonadal men with HIV-associated weight loss.

Serum testosterone concentrations are frequently in the low-normal range (lowest quartile, <500 ng/dl) in men with AIDS-wasting syndrome (AWS) and in other chronic wasting disorders. The response of patients in this group to androgen treatment has not been determined, however. Eighteen men with AWS (mean +/- standard error [SE]: 87% +/- 1% usual body weight; CD4 count 90 +/- 24) and borderline low serum testosterone concentrations (382 +/- 33 ng/dl) completed a 21-day placebo-controlled inpatient metabolic ward study comparing intramuscular (i.m.) placebo (n = 7) with low-dose (65 mg/week; n = 4) and high-dose (200 mg/week; n = 7) nandrolone decanoate, a testosterone analogue. Nitrogen balance, stable isotope-mass spectrometric measurement of de novo lipogenesis (DNL), resting energy expenditure, and gonadal hormone levels were measured. Both low-dose and high-dose nandrolone resulted in significant nitrogen retention (33-52 g nitrogen/14 days, representing gains of 0.5 to 0.9 kg lean tissue/week) compared with placebo (loss of 11 g nitrogen/week). This was reflected biochemically in a borderline significant reduction of high DNL (p < .06). Serum testosterone and gonadotropins were suppressed whereas resting energy expenditure was unchanged by nandrolone treatment. In 10 study subjects completing a 12-week open-label follow-up phase, body weight increased by 4.9 +/- 1.2 kg, including 3.1 +/- 0.5 kg lean body mass, and treadmill exercise performance also improved. In summary, nandrolone decanoate therapy in the absence of an exercise program in borderline hypogonadal men with AWS caused substantial nitrogen retention compared with placebo, similar in extent to the nitrogen retention previously achieved with recombinant growth hormone. It is reasonable to expand the criteria for androgen treatment in AWS to include at least patients in the lowest quartile of serum testosterone.

Simian virus 40-, but not human papillomavirus-, transformation of prostatic epithelial cells results in loss of growth-inhibition by 1,25-dihydroxyvitamin D-3.

In addition to its well known calcemic actions, 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D] exhibits differentiating and antiproliferative effects in several types of cancer cells. 1,25(OH)(2)D receptors (VDR) as well as 1,25(OH)(2)D-mediated growth-inhibition have been demonstrated in human prostate cancer cell lines. In order to further develop model systems for the study of 1,25(OH)(2)D action and to elucidate the mechanism of growth-inhibition, we studied several human prostate cell lines immortalized with either simian virus 40 (SV40) or human papillomavirus type 18 (HPV). The SV40-transformed cell lines P69SV40-T and P153SV40-T were not growth-inhibited by 1,25(OH)(2)D at concentrations as high as 100 nM, whereas the HPV-transformed cells PZ-HPV-7 and CA-HPV-10 were growth-inhibited. All cell lines expressed VDR, and VDR mRNA was demonstrated by Northern blot analysis. All cells exhibited induction of 24-hydroxylase mRNA, a 1,25(OH)(2)D responsive gene, after 1,25(OH)(2)D treatment. In an attempt to understand the apparent dissociation of 1,25(OH)(2)D actions in the SV40-transformed cells, we turned to the human prostate cancer cell line DU 145. These cells, like the SV40-transformed cells, are not growth-inhibited but demonstrate induction of 24-hydroxylase mRNA after 1,25(OH)(2)D treatment. DU 145 cells contain a mutated retinoblastoma gene (Rb) which contributes to their uncontrolled growth, analogous to the disruption of Rb by SV40 and HPV. We compared DU,145 cells to DU 145 cells transfected with normal Rb (DU 145/Rb). Similar to DU 145, DU 145/Rb cells were not growth-inhibited by 1,25(OH)(2)D, while 24-hydroxylase mRNA was induced. These results suggest that divergent pathways mediate the growth-inhibitory effect of 1,25(OH)(2)D and its induction of 24-hydroxylase. It also appears that the antiproliferative effect of 1,25(OH)(2)D is mediated by an Rb-independent mechanism.

Vitamin D and prostate cancer.

Our findings demonstrate the presence of VDR in various human prostate cancer cell lines and in primary cultures derived from normal, BPH and prostate cancer. In addition, 1,25-D induced several bioresponses in these cells including growth inhibition and PSA stimulation. Based on examples in many different malignant cells as well as our data in prostate cells, that vitamin D is anti-proliferative and promotes cellular maturation, it seem clear that vitamin D must be viewed as an important cellular modulator of growth and differentiation if addition to its classical role as regulator of calcium homeostasis. In this respect, vitamin D has the potential to have beneficial actions on various malignancies including prostate cancer. Its ultimate role in prostate cancer remains to be determined, but 1,25-D may prove useful in chemoprevention and/or differentiation therapy. We believe the data currently available provide the basis for an optimistic view on the possible use of vitamin D to treat prostate cancer in patients and that further investigation is clearly warranted to better define its potential therapeutic utility.

Actions of vitamin D3, analogs on human prostate cancer cell lines: comparison with 1,25-dihydroxyvitamin D3.

Data from epidemiological studies has suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. We have previously demonstrated the presence of vitamin D receptors (VDR) in three human prostate carcinoma cell lines (LNCaP, PC-3, and DU-145) as well as in primary cultures of stromal and epithelial cells derived from normal and malignant prostate tissues. We have also shown that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can elicit an antiproliferative action in these cells. In the present study we compared the biological actions of 1,25-(OH)2D3 to those of a series of natural vitamin D3 metabolites and several synthetic analogs of vitamin D3 known to exhibit less hypercalcemic activity in vivo. In ligand binding competition experiments, we demonstrated the following order of potency in displacing [3H]1,25-(OH)2D3 from VDR: EB-1089 > 1,25-(OH)2D3 > MC-903 > 1,24,25-(OH)3D3 > 22-oxacalcitriol (OCT) > 1 alpha,25-dihydroxy-16-enecholecalciferol (Ro24-2637) > 25-hydroxyvitamin D3, with EB-1089 being approximately 2-fold more potent than the native hormone. No competitive activity was found for 25-hydroxy-16,23-diene-cholecalciferol. When compared for ability to inhibit proliferation of LNCaP cells, MC-903, EB-1089, OCT, and Ro24-2637 exhibited 4-, 3-, and 2-fold greater inhibitory activity than 1,25-(OH)2D3. Interestingly, although OCT and Ro24-2637 exhibit, respectively, 10 and 14 times lower affinity for VDR than 1,25-(OH)2D3, both compounds inhibited the proliferation of LNCaP cells with a potency greater than that of the native hormone. The relative potency of vitamin D3 metabolites and analogs to inhibit cell proliferation correlated well with the ability of these compounds to stimulate prostate-specific antigen secretion by LNCaP cells as well as with their potency to induce the 25-hydroxyvitamin D3-24-hydroxylase messenger RNA transcript in PC-3 cells. In conclusion, these results demonstrate that synthetic analogs of vitamin D3, known to exhibit reduced calcemic activity, can elicit antiproliferative effects and other biological actions in LNCaP and PC-3 cell lines. It is noteworthy that although binding to VDR is critical for 1,25-(OH)2D3 action, the analog data indicate that additional factors significantly contribute to the magnitude of the biological response. Finally, the strong antiproliferative effects of several synthetic analogs known to exhibit less calcemic activity than 1,25-(OH)2D3 suggest that these compounds potentially may be useful as an additional therapeutic option for the treatment of prostate cancer.

Inhibition of aldosterone synthesis in rat adrenal cells by nicotine and related constituents of tobacco smoke.

Previous work has shown that nicotine and related constituents of tobacco smoke inhibit selected P450 enzymes in the glucocorticoid and sex steroid synthetic pathways. Because aldosterone synthesis is also cytochrome P450 dependent, we hypothesized a similar inhibitory action on aldosterone production. In this study we examined the effects of nicotine, anabasine (a related alkaloid), and cotinine (the major metabolite of nicotine) on in vitro aldosterone synthesis. Freshly isolated rat adrenal cells were assayed for corticosterone and aldosterone production in the basal state and after stimulation with ACTH or angiotensin-II (ANG-II). The addition of nicotine, anabasine, and cotinine in concentrations up to 100 microM did not inhibit stimulated corticosterone production. However, there was a potent dose-dependent inhibitory action of all three tobacco compounds on aldosterone production. The relative inhibitory potency was: cotinine > anabasine > nicotine. When employed at a concentration of 100 microM, the three compounds inhibited ACTH-stimulated aldosterone synthesis by 75%, 44%, and 21%, respectively. ANG-II-stimulated aldosterone synthesis was inhibited by 92%, 78%, and 62%, respectively. The plasma cotinine concentration range attained in tobacco smokers is between 1-10 microM. When tested with [3H]corticosterone and [3H]progesterone as exogenous substrates, 1-10 microM cotinine caused a significant dose-dependent inhibition of ACTH- and ANG-II-stimulated aldosterone synthesis. Cotinine substantially blocked the conversion of corticosterone to 18-hydroxycorticosterone, implicating the 18-hydroxylase or corticosterone 18-methyloxidase-I (CMO-I) step as the major site of inhibition. In summary, our results indicate that tobacco compounds cause direct and specific inhibition of aldosterone synthesis, primarily at the CMO-I step. This enzymatic blockade would be expected to result in activation of the renin-angiotensin system in vivo. We postulate that chronic stimulation of the renin-angiotensin system by this mechanism might contribute to the cardiovascular damage that occurs with long term tobacco use.

Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells.

Cultures of adult human prostatic epithelial and fibroblastic cells were established from normal, benign hyperplastic, and malignant tissues. Vitamin D receptors were detected by ligand binding of [3H]1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in cytosolic extracts prepared from all types of cell cultures as well as from fresh prostatic tissues. Vitamin D receptor transcripts were demonstrated by Northern blot analysis. 1,25-(OH)2D3 inhibited the growth of epithelial cells with half-maximal inhibition at approximately 1 nM. The growth of fibroblasts was also inhibited by 1,25(OH)2D3 but to a lesser extent. This is consistent with the apparently lower level of vitamin D receptors in fibroblasts compared to epithelial cells determined by ligand binding and Northern analysis of RNA transcripts. The growth inhibition of epithelial cells by 1,25(OH)2D3 was irreversible even after a short 2-h exposure, but morphology and keratin expression were not appreciably altered by long-term exposure to the hormone. A physiological role for 1,25(OH)2D3 in the prostate is postulated, and the inhibitory effect of 1,25(OH)2D3 on cancer-derived prostate cells may provide a basis for new preventive or therapeutic strategies.

Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines.

It has been suggested that vitamin D deficiency may promote prostate cancer, although the mechanism is not understood. In this study three human prostate carcinoma cell lines, LNCaP, DU-145, and PC-3, were examined both for the presence of specific 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] receptors (VDRs) and also employed to study the effects of hormone on cell proliferation and differentiation. Ligand binding experiments demonstrated classical VDR in all three cell lines examined with an apparent dissociation constant of 7.5, 5.4, and 6.3 x 10(-11) M for LNCaP, DU-145, and PC-3 cells, respectively. Corresponding binding capacity for the three prostate carcinoma cell lines were 27, 31, and 78 fmol/mg protein, respectively. The presence of VDR in the three cell lines was also confirmed by immunocytochemistry. In addition, one major 4.6-kilobase messenger RNA transcript hybridizing with a specific human VDR complementary DNA probe was identified in all three cell lines. Interestingly, both DU-145 and PC-3 but not LNCaP cell lines exhibited 1,25(OH)2D3-stimulated induction of 24-hydroxylase messenger RNA employed as a marker of 1,25(OH)2D3 action. Physiological levels of 1,25(OH)2D3 dramatically inhibited proliferation of the LNCaP and PC-3 cell lines. However, in spite of the presence of high affinity VDR, proliferation of DU-145 cells was not inhibited by 1,25(OH)2D3 at the doses tested. Treatment with 1,25(OH)2D3 caused a dose-dependent stimulation of prostate-specific antigen secretion by LNCaP cells. In conclusion, these results demonstrate that these three human prostate carcinoma cell lines all possess specific VDR and that 1,25(OH)2D3 treatment can elicit both an antiproliferative and a differentiating action on these cancer cells. The findings lend support to the hypothesis that vitamin D might exert beneficial actions on prostate cancer risk.

Effect of variations in dietary fat and carbohydrate intake on postprandial lipemia in patients with noninsulin dependent diabetes mellitus.

The effect of dietary composition on concentrations of postprandial lipoproteins was studied in eight sulfonylurea-treated patients with noninsulin dependent diabetes mellitus. Two diets were consumed by each patient for 2 weeks in random order, one contained (as percent of total calories) 15% protein, 40% fat, and 45% carbohydrate (CHO), whereas the other consisted of 15% protein, 25% fat, and 60% CHO. At the end of each dietary period, patients were given Vitamin A (60,000 U/m2) with their noon meal, and the concentration of triglyceride (TG) and retinyl esters in plasma and two lipoprotein fractions (Sf > 400 and Sf 20-400) determined over the next 12 h. The results indicated that both postprandial TG and retinyl ester concentrations were higher in plasma (Sf > 400, and Sf 20-400 lipoproteins), when patients ate the 25% fat/60% CHO diet. Thus, replacing saturated fat with CHO accentuates the magnitude of postprandial lipemia. Since TG-rich lipoproteins may be atherogenic, appropriate dietary advice for patients with type 2 diabetes may deserve reappraisal.

Differences in postprandial lipemia between patients with normal glucose tolerance and noninsulin-dependent diabetes mellitus.

In this paper we have compared the postprandial increase in triglyceride (TG) rich lipoproteins of intestinal origin in 10 patients with noninsulin-dependent diabetes mellitus (NIDDM) and 10 subjects with normal glucose tolerance. The two groups were matched for age, sex distribution, body mass index, and plasma TG concentration. Breakfast was consumed at 0800 h and lunch at 1200 h, at which time vitamin A was also administered. Blood was sampled frequently from 1200 h to 2400 h, and measurements made of glucose, insulin, and TG concentrations. Furthermore, the retinyl palmitate (RP) content of plasma, the Sf > 400 lipoprotein fraction, and the Sf 20-400 lipoprotein fraction was also determined, and differences compared by two-way analysis of variance. Fasting and postprandial (from 1200 h to 2400 h) TG concentrations in the plasma and the two lipoprotein fractions were not significantly different in normal subjects and patients with NIDDM. In addition, the postprandial RP concentration of the two groups was not different in the chylomicron containing Sf > 400 lipoprotein fraction. However, the postprandial Sf 20-400 RP concentration was significantly higher (P < 0.001) in patients with NIDDM, estimated as hourly values over time, peak value, or total integrated response area. Significant correlation coefficients (r = 0.60-0.75, P < 0.08 < 0.02) were seen in patients with NIDDM between the total integrated insulin response and both the TG and RP responses in the Sf > 400 and Sf 20-400 fractions. In addition, fasting high density lipoprotein-cholesterol concentration in patients with NIDDM was significantly correlated with the postprandial TG response in the Sf > 400 (r = -0.64, P < 0.05) and the Sf 20-400 (r = -0.68, P < 0.05) lipoprotein fractions. In summary, the postprandial RP concentration in the Sf 20-400 lipoprotein fraction was higher than normal in patients with NIDDM. In addition, associations have been defined in patients with NIDDM between postprandial insulin response, fasting TG and high density lipoprotein-cholesterol concentrations, and magnitude of postprandial increase in TG-rich lipoproteins of intestinal origin.

Effect of nicotinic acid on plasma glucose concentration in normal individuals.

In order to assess the ability of nicotinic acid to decrease plasma glucose concentration, normal individuals were given continuous four hour infusions of either nicotinic acid (NA), somatostatin (SRIF), NA + SRIF, or 0.9% NaCl (Saline). Plasma non-esterified fatty acid (NEFA) concentration decreased to about one-fourth of the basal value in response to either NA or NA + SRIF, associated with statistically significant decreases in plasma glucose concentration. The ability of NA and NA + SRIF to decrease plasma glucose concentration was seen despite the fact that plasma insulin concentrations also fell significantly during both infusions. Although plasma glucose concentration fell significantly in response to both NA and NA + SRIF, the effect of NA + SRIF was approximately twice as great as that seen with NA alone. The augmented hypoglycaemic effect of NA + SRIF as compared to NA alone was associated with a concomitant fall in plasma glucagon concentration. In contrast, plasma glucose concentration did not change following Saline, and was actually higher than baseline after the infusion of SRIF alone. These results provide evidence that NA can lower plasma glucose concentration in normal volunteers, and suggests that this is mediated by the NA-associated decrease in plasma NEFA concentration.

Adenosine receptors in basolateral membranes of rat renal cortex.

High affinity binding sites for adenosine were identified in rat kidney cortex basolateral membranes. Kinetic analysis indicates two sets of [3H]adenosine, [3H]ADO, binding sites, one with high affinity and Kd = 0.84 +/- 0.25 microM, one with low affinity and Kd = 4.74 +/- 0.37 microM. The ADO receptors were further characterized using ADO analogs as binding inhibitors. The most potent inhibitor of [3H]ADO binding was N-methyl-adenosine with a Kd of 5 microM, whereas 2-deoxyadenosine was about 50 times less potent. The binding of [3H]phenylisopropyladenosine, [3H]PIA, and [3H]-N-ethylcarboxamidoadenosine, [3H]NECA, to basolateral membranes was rapid and reversible. The Scatchard plot of [3H]PIA binding showed monophasic curves for experiments performed at 0 degrees C and 37 degrees C. The apparent Kd of [3H]PIA binding at 0 degrees C was 0.19 +/- 0.05 nM and 0.34 +/- 0.07 nM at 37 degrees C. The binding of [3H]NECA to basolateral membranes was found with an apparent affinity Kd of 110 +/- 50 nM at 0 degrees C. Pretreatment of membranes with N-ethylmaleimide (NEM) inhibited the [3H]PIA binding and did not affect the [3H]NECA binding. These results demonstrate that both A1 and A2 adenosine receptors are present in basolatertal membranes of rat kidney.

Combined metformin-sulfonylurea treatment of patients with noninsulin-dependent diabetes in fair to poor glycemic control.

The effect of metformin treatment was studied in 13 patients with noninsulin-dependent diabetes mellitus (NIDDM), whose fasting plasma glucose concentration was greater than 10 mmol/L with maximal sulfonylurea doses. Patients were studied before and 3 months after receiving 2.5 g/day metformin. The fasting plasma glucose concentration (12.4 +/- 0.8 vs. 8.8 +/- 0.7 mmol/L), mean hourly postprandial plasma glucose concentration from 0800-1600 h (14.0 +/- 1 vs. 9.4 +/- 0.9 mmol/L), and glycosylated hemoglobin level (12.3 +/- 0.6% vs. 9.0 +/- 0.6%) were all significantly (P less than 0.005-0.001) lower after the administration of metformin. The improvement in glycemic control was associated with a 24% increase (P less than 0.05) in insulin-stimulated glucose uptake during glucose clamp studies and a 16% decrease in basal hepatic glucose production (P less than 0.05). Mean hourly concentrations of plasma insulin (411 +/- 73 vs. 364 +/- 73 pmol/L) and FFA concentrations (440 +/- 31 vs. 390 +/- 40 mumol/L) were also lower after 3 months of metformin treatment. However, neither insulin binding nor insulin internalization by isolated monocytes changed in response to metformin. Finally, plasma triglyceride, very low density lipoprotein triglyceride, and very low density lipoprotein cholesterol were significantly decreased (P less than 0.01-0.001), and high density lipoprotein cholesterol was significantly increased (P less than 0.001) after metformin treatment. Thus, the addition of metformin to sulfonylurea-treated patients with NIDDM not in good glycemic control significantly lowered fasting and postprandial plasma glucose concentrations, presumably due to the combination of enhanced glucose uptake and decreased hepatic glucose production. Since the dyslipidemia present in these patients also improved, the results suggest that metformin may be of significant clinical utility in patients with NIDDM not well controlled with sulfonylurea compounds.

Effect of acute variations in dietary fat and carbohydrate intake on retinyl ester content of intestinally derived lipoproteins.

Vitamin A was administered to eight patients with noninsulin-dependent diabetes mellitus in conjunction with the two different test meals containing (as percentage of total calories) either 15% protein, 60% carbohydrate (CHO), and 25% fat or 15% protein, 40% CHO, and 45% fat. The vitamin A and test meals were given at noon (4 h after a standard breakfast), and blood was obtained hourly from noon to midnight for measurement of plasma glucose, insulin, triglyceride (TG), and cholesterol concentrations; concentrations of TG and cholesterol in Sverdberg floatation (Sf) unit above 400 and Sf 20-400 lipoproteins; retinyl ester concentration in plasma; and both Sf more than 400 and Sf 20-400 lipoproteins. The postprandial TG response in plasma, Sf more than 400 lipoproteins, and Sf 20-400 lipoproteins from noon to midnight was only slightly higher than values seen after consumption of the 60% CHO diet, which contained much less fat (25% vs. 45%) and the retinyl ester concentration was actually higher in both lipoprotein fractions after the diet containing the smallest amount of fat (60% CHO). Furthermore, the cholesterol concentration in the plasma and two lipoprotein fractions was identical after the two diets, despite the great difference in fat content. These data indicate that the acute ingestion of high CHO (60%), low fat (25%) diets by patients with noninsulin-dependent diabetes mellitus led to little or no decrease in postprandial plasma or lipoprotein TG or cholesterol concentrations and an actual increase in concentration of potentially atherogenic small chylomicron and/or chylomicron remnants.

Regulation of non-esterified fatty acid and glycerol concentration by insulin in normal individuals and patients with type 2 diabetes.

Plasma glycerol and non-esterified fatty acid (NEFA) concentrations were determined in the basal state and in response to physiological hyperinsulinaemia in 30 non-obese individuals, 15 with Type 2 diabetes and 15 with normal glucose tolerance. Patients with Type 2 diabetes had higher basal concentrations of both glycerol (81 +/- 7 (+/- SE) vs 61 +/- 7 mumol l-1, p less than 0.05) and NEFA (842 +/- 40 vs 630 +/- 46 mumol l-1, p less than 0.002). Plasma NEFA and glycerol concentrations fell in both groups when steady-state plasma insulin concentrations were raised to approximately 450 pmol l-1 by an infusion of exogenous insulin, but plasma concentrations of glycerol (28 +/- 3 vs 13 +/- 3 mumol l-1, p less than 0.002) and NEFA (186 +/- 15 vs 109 +/- 14 mumol l-1, p less than 0.001) were still higher in patients with Type 2 diabetes. Percentage decrease in glycerol from basal levels in response to insulin was significantly less in patients with Type 2 diabetes than in control subjects (64 +/- 3 vs 80 +/- 3%, p less than 0.005); percentage decrease in plasma NEFA concentration was similar in the two groups (78 +/- 3 vs 80 +/- 4%). These results suggest that both plasma glycerol and NEFA concentrations are higher than normal in patients with Type 2 diabetes when measured at the same insulin concentration, both under basal conditions and in response to physiological hyperinsulinaemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of an estrogen-binding protein in the yeast Candida albicans.

An estrogen-binding protein (EBP) has been identified and characterized in the cytosol of the pathogenic yeast Candida albicans. Binding of [3H]estradiol was found to be optimal at pH 7.4 in the presence of 0.3 M KCl and was linearly related to protein concentration. Binding was very rapid, reaching maximal levels in about 30 min, and was reversible with a dissociation rate constant of 13.2 +/- 1.7 x 10(-4) sec-1. EBP binding was destroyed by treatment with proteolytic enzymes and by high temperatures. Scatchard analysis of the [3H]estradiol equilibrium binding data of C. albicans (strain stn-1) yielded an apparent dissociation constant of 12.3 +/- 2.1 nM and a maximal binding capacity of 753 +/- 145 fmol/mg protein. Binding competition experiments showed very high specificity and stereoselectivity of EBP, demonstrating the following order of potency in displacing [3H]estradiol: 17 beta-estradiol greater than estrone greater than estriol greater than 17 alpha-estradiol. Negligible competitive potency was found for other mammalian steroid hormones, diethylstilbestrol, tamoxifen, or fungal hormones. The abundance of EBP was 4- to 10-fold higher during the early logarithmic growth phase of yeast cells than during the stationary phase. The molecular size of EBP, measured by Sephacryl S-200 gel exclusion chromatography, yielded a Stokes radius of approximately 29 A. Sucrose density gradient sedimentation showed a sedimentation coefficient (S2020,W) of 4, with no ionic dependent aggregation of the [3H]estradiol-EBP complex. The apparent mol wt of the EBP is approximately 46,000, with an axial ratio of 1, indicating the symmetrical shape of the molecule. In summary, in addition to the previously described corticosterone-binding protein, a separate high affinity, stereospecific, estrogen-selective binder has been demonstrated in the cytosol of C. albicans.

A new method for the iodometric determination of sulphoxides.

A method is described for determination of sulphoxides through their reaction with iodide in a trifluoroacetic acid/acetone medium to produce iodine, which is then titrated with thiosulphate.

Photoaffinity labeling of peripheral-type benzodiazepine receptors in rat kidney mitochondria with [3H]PK 14105.

The peripheral-type benzodiazepine receptor in rat kidney has been identified by photoaffinity labeling. PK 14105, a derivative of the selective peripheral-type ligand PK 11195, was used to covalently label peripheral-type benzodiazepine receptors. In the absence of UV light PK 14105 demonstrated reversible, high affinity (KD = 4.8 nM) binding to rat kidney mitochondrial membranes. Inhibition of the reversible binding of [3H]PK 14105 by various benzodiazepine and other ligands demonstrated that this ligand bound with all the characteristics expected of a ligand interacting specifically with peripheral-type benzodiazepine receptors. A similar order of relative potencies was obtained for inhibition of photolabeling, indicating that reversible binding and photolabeling occurred at the same class of binding sites. Examination of photolabeled binding sites from kidney, heart, brain and adrenal membranes using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the probe is photoincorporated into a single peptide of Mr = 18,500. The results indicate that [3H]PK 14105 identifies the ligand binding domain of the peripheral-type benzodiazepine receptor, which is a peptide with Mr = 18,500, that is of similar size in kidney, heart, brain and adrenals.