Role of insulin receptor substrate-1 and pp60 in the regulation of insulin-induced glucose transport and GLUT4 translocation in primary adipocytes.

In muscle and fat, glucose transport occurs through the translocation of GLUT4 from an intracellular pool to the cell surface. Phosphatidylinositol (PI) 3-kinase has been shown to be required in this process. Insulin is thought to activate this enzyme by stimulating its association with tyrosine-phosphorylated proteins such as insulin receptor substrate (IRS)-1, IRS-2, Grb2-associated binder-1, and pp60. To study the role of these endogenous substrates in glucose transport, we analyzed adipocytes from IRS-1 null mice that we previously generated (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y. , Ikawa, Y., Kasuga, M., Yazaki Y., and Aizawa S. (1994) Nature 372, 182-186). In adipocytes from these mice, we showed that: 1) insulin-induced PI 3-kinase activity in the antiphosphotyrosine immunoprecipitates was 54% of wild-type; 2) pp60 was the major tyrosine-phosphorylated protein that associated with PI 3-kinase, whereas tyrosine phosphorylaion of IRS-2 as well as its association with this enzyme was almost undetectable; and 3) glucose transport and GLUT4 translocation at maximal insulin stimulation were decreased to 52 and 68% of those from wild-type. These data suggest that both IRS-1 and pp60 play a major role in insulin-induced glucose transport in adipocytes, and that pp60 is predominantly involved in regulating this process in the absence of IRS-1.

Elevation of circulating proadrenomedullin-N terminal 20-peptide in thyrotoxicosis.

BACKGROUND AND OBJECTIVE: Adrenomedullin (AM) is a recently discovered peptide which has potent vasodilatory activity. We have found that the plasma adrenomedullin level is elevated in hyperthyroidism, suggesting a potential role of AM in the decrease of vascular resistance in thyrotoxicosis. Proadrenomedullin, a precursor of adrenomedullin, yields another peptide termed proadrenomedullin-N terminal 20-peptide (PAMP). PAMP also has potent vasodilatory activity. Although the regulation of secretion of AM and PAMP is not fully understood and the mechanism by which the plasma AM level is elevated in hyperthyroidism remains unknown, it is of interest to determine the plasma concentration of PAMP in thyrotoxicosis. DESIGN AND PATIENTS: We measured the plasma concentration of immunoreactive AM and PAMP in newly recruited untreated thyrotoxic Graves' patients using specific antibodies to each peptide. RESULTS: Not only AM, but also the plasma concentration of PAMP in thyrotoxic patients was significantly (P < 0.01) elevated (4.7 +/- 0.9 pmol/l), compared to that in control subjects (2.6 +/- 0.8 pmol/l). The correlation was marginally significant between the plasma AM concentration and serum free thyroid hormone levels. The plasma PAMP level tended to be more elevated when thyrotoxicosis was severe but the correlation was not statistically significant. Correlation was not demonstrated between the AM and PAMP levels in thyrotoxic patients. CONCLUSIONS: Elevation of the plasma adrenomedullin and proadrenomedullin-N terminal 20-peptide levels raises the possibility of involvement of these vasodilatory peptides in the haemodynamic changes in thyrotoxicosis.

Plasma selectin levels in patients with Graves' disease.

Adhesion molecules relate to cell invasion of autoimmune thyroid disease. We studied plasma soluble P-Selectin (platelet activation-dependent granule-external membrane protein), E-Selectin (endothelial leukocyte adhesion molecule) and L-Selectin (leukocyte endothelial cell adhesion molecule-1) levels in patients with Graves' disease before and during methimazole treatment. Plasma P-, E- and L-Selectin levels in patients with untreated Graves' disease were significantly higher than those in normal subjects. Plasma P-Selectin levels decreased when their thyroid functions were normal for more than 6 months after the start of methimazole treatment. No significant change in plasma E- and L-Selectin levels in patients with Graves' disease was found between hyperthyroid state and euthyroid state after the start of methimazole treatment, but plasma L-Selectin levels in patients with untreated Graves' disease were significantly lower than those in the patients in the first euthyroid state. There was no significant correlation between plasma P-Selectin levels and serum FT4 levels, nor between plasma P-Selectin levels and serum FT3 levels. These results suggested that thyroid hormones might reflect expression of P-, L- and E-Selectin from endothelial cells, or lymphocytes, or platelets in patients with Graves' disease.

Five modified numerical deconvolution methods for biopharmaceutics and pharmacokinetics studies.

Four improved finite-difference numerical deconvolution methods and one nonlinear regression numerical deconvolution method are proposed and implemented using IMSL/IDLTM. These five numerical deconvolution methods are evaluated using simulated data generated with and without added noise under six different dosing cases. Comparisons between these methods are made in terms of the superimposability of the calculated cumulative amount of drug released or absorbed-time profiles with the theoretical data. The results indicate that the proposed fixed step number equal step length numerical deconvolution method is simple and accurate and therefore is appropriate for pharmacokinetic and biopharmaceutic studies. When an analytic function is legitimate to represent the drug input rate, the nonlinear regression numerical deconvolution method will yield enhanced numerical accuracy and stability.

Theophylline controlled-release formulations: in vivo-in vitro correlations.

Four experimental controlled-release oral solid dosage formulations were developed and the in vitro dissolution characteristics of theophylline from these formulations were studied in USP apparatus I. Pharmacokinetic evaluation of these formulations was carried out in eight beagle dogs under fasting conditions. Theophylline in a 5% dextrose injection USP, oral solution, and Slo-Phyllin were used as controls to estimate the in vivo dissolution of these four formulations in the GI tract. The percentage cumulative amounts of drug absorbed and the percentage cumulative amounts of drug released into the GI tract from these four controlled-release formulations were obtained by numerical deconvolution methods. The in vivo and in vitro dissolution data demonstrated good correlation indicating that in vitro dissolution tests can be used to optimize the further design of controlled drug release oral solid dosage formulations for theophylline.

Anti-HBs kinetics after HBV vaccination in neonates.

BACKGROUND AND OBJECTIVES: We report the results of a study using a recombinant DNA HBV vaccine in newborns from an endemic area for HBV and compare the anti-HBs kinetics with observations in adults in order to make estimates about the need for booster vaccinations. STUDY DESIGN: One hundred and forty-eight neonates were vaccinated and followed for 62 months. Based on the presence or absence of hepatitis B surface antigen in the mother, cohorts of 'exposed' and of 'non-exposed' neonates were identified. RESULTS: A maximum concentration is normally observed after the booster vaccination followed by a rapid decline. According to Ambrosch et al. and Gesemann et al., titer calculations as a function of time, yielded 37 IU/1 and 47 IU/1 at month 60 respectively. The mean titer for the three groups of neonates investigated was at that time 74 IU/1. The prospective time intervals to arrive at an anti-HBs level of at least 10 IU/1 can be individually calculated from the individual titer after the booster vaccination. These calculated estimates show respectively: that 8.3% of the vaccinated neonates need a new booster vaccination within 14 months; that 26.7% will need a new booster within 50 months; and that only 65% need a new booster in 50 or more months. CONCLUSION: It can be concluded that anti-HBs kinetics in very young children and adults are comparable. The least expensive way of maintaining protection against HBV in neonates seems to be the determination of the individual titers after the first booster vaccination and calculation of the prospective time interval to arrive at a minimum titer of 10 IU/1 and the need for a new booster vaccination.

Determination of polyanionic macromolecules by size-exclusion chromatography.

This report presents an extension of a method developed for determination of dextran sulfate in rat serum. The drug is a negatively charged polysaccharide with a molecular mass of 8000. It is fractionated by molecular size and separated from serum components by high-performance size-exclusion chromatography. Sensitive detection is achieved by the post-column complexation of the analyte with 1,9-dimethylmethylene blue (DMMB). A metachromatic complex is formed; the absorbance maximum of the complex is shifted from that of the free dye. Various glycosaminoglycans and other macromolecular polyanions interact with DMMB. Several can be determined using the chromatographic conditions developed for dextran sulfate. The method provides a simple procedure for quantitation of these compounds. Compared to spectrophotometric assays, less sample preparation is required, selectivity is enhanced, and molecular mass information is provided. With modification of eluent composition, dye concentration, and detection wavelength, the method can be validated for determination of additional compounds.

Size-exclusion chromatographic determination of dextran sulfate in rat serum.

A sensitive and selective method for the determination of dextran sulfate in rat serum has been developed. The analysis is suitable for quantitation of the drug and for monitoring molecular mass changes occurring during biotransformation. Dextran sulfate is resolved from higher molecular mass serum components by high-performance aqueous size-exclusion chromatography. The method has been validated for the direct injection of serum. Sensitive detection is achieved by post-column reaction of the polyanionic drug with the dye 1,9-dimethylmethylene blue. Components of serum which inhibit complex formation are separated chromatographically from dextran sulfate. Absorbance of the metachromatic complex is monitored at 525 nm.

Effect of surfactant phase in perfluorocarbon emulsification efficiency.

The effect of preparation temperature on the emulsification efficiency of perfluoro-3-butyltetrahydrofuran (FC-75) was investigated. Polyoxyethylene (POE) oleyl ether surfactants were used as the emulsifier(s) in a range of HLB values of 7.5 to 9.5. The emulsions were prepared by paddle mixing as a method of low-shear emulsification. After centrifugation of the resulting O/W emulsions, the volume of FC-75 which separated was utilized as a measure of the emulsification efficiency. In general, emulsions prepared at temperatures where the surfactant was in a lamellar-to-isotropic surfactant solution transition, L alpha----L3, displayed a better emulsification efficiency than those prepared with other surfactant phases.

The relationship of salicylate lipophilicity to rectal insulin absorption enhancement and relative lymphatic uptake.

The sodium salts of the 3,5-dichloro, 3,5-dibromo-, 3,5-diiodo-, and 5-methoxy- analogs of salicylic acid have been evaluated as enhancers of rectal insulin absorption. A relationship was found between adjuvant potency and relative lipophilicity. Maximal adjuvant activity was obtained with 0.1 M 3,5-dichlorosalicylate. Higher concentrations (0.15 M) of 3,5-diiodosalicylate produced a decline in adjuvant activity, which may be associated with extraction of specific cellular proteins. This may indicate the existence of an optimal salicylate lipophilicity for adjuvant efficacy. Relative adjuvant activity was found to be related to the lymph:plasma absorption ratio of [125I]insulin. Lymphatic uptake of insulin was not related to lymph flow rate.

Connection mechanism of physical-contact optical fiber connectors with spherical convex polished ends.

We present an experimental and analytical investigation of the connection mechanism of physical-contact optical-fiber connectors with spherical convex polished ends and confirm that reducing the curvature radius of the spherical convex ferrule end face is effective for establishing a stable connection with slight axial compressive force on the ferrules.

Predicting coordinated lipid biosynthesis: application to the surfactant-accommodated epidermis.

Factorialized correlation analysis is proposed as a method for predicting the coordination of multiple enzyme pathways. The approach can be used potentially to find new relationships and to predict relationships that have been established in other tissues. However, careful tracer studies are needed to verify the cause-and-effect relationships between precursor and products. In this study, guinea pigs that were chronically treated with an anionic, a nonionic and a cationic surfactant passed through an irritation stage to a clinical state that appeared normal. The method was used to examine binary coordination of lipid biosynthesis in the epidermis by using a factorialized table of regression coefficients. Coordinated lipid relationships that have been reported in other tissues were predicted between sphingomyelin and cholesterol, as well as between phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. A new inverse relationship was found between triglycerides and both sphingomyelin and cholesterol, using this method. These data are discussed with respect to a membrane fluidization model for the accommodated state.

Prevalence of anti-delta antibodies in pregnant women in Bandung, Indonesia.

Knowledge of the epidemiology of hepatitis D virus (HDV)-infection is very scarce in many parts of the world. The endemicity of delta-infection is believed to be maintained and spread through the network of hepatitis B surface antigen (HBsAg) carriers in the community. In the Far East and the Pacific area, the prevalence of chronic HBsAg carriers is mostly high. Markers of HDV infection are very frequent in some regions (e.g. parts of China, Fiji Isles, Samoa), in other regions they are almost absent (e.g. Taiwan, Thailand, parts of China). In the Bandung region (West Java, Indonesia) we found 26 (2.8%) HBsAg carriers among 926 pregnant women. Most of them are chronic carriers (anti hepatitis B core (HBc) IgM negative). Although HBsAg is frequent in this Indonesian population, we could not find any anti-HD positive. This data warrants the conclusion that HDV infection has not yet been introduced in that densely populated area of Indonesia.

Prevalence and determinants of hepatitis B virus markers in pregnant women in West Java, Indonesia.

In Bandung, West Java, 300 consecutive pregnant women were screened for hepatitis B virus (HBV) markers at a prenatal consultation. The prevalence of HBsAg and of anti-HBs/anti-HBc was 4.7% (14/300) and 35.6% (107/300) respectively, while 59.7% (179/300) was sero-negative. Prevalence of HBV markers increased significantly with both age and parity. Women with less schooling and a low socioeconomic class seemed to be at higher risk for HBV infection (HBV-markers prevalence of 49.3% (35/71) and 58.3% (21/36) respectively). Among employed women, the subgroup of school-teachers had a significantly higher HBV-markers prevalence of 54.8% (23/42), with a HBsAg carrier rate of 11.9% (5/42). This could indicate an important nonparenteral transmission of HBV in schools. The prevalence of HBeAg in HBsAg positive women was 64.3% (9/14). Based on historical data on perinatal HBV transmission, this would lead to a HBV carriership in 2.4 to 3.5% of all newborns. Possible strategies of prevention of HBV infection in newborns are briefly discussed.

Immunogenicity of a recombinant DNA hepatitis B vaccine in neonates.

Infants of HBsAg-positive mothers (Group I) as well as those born to women without HBV markers (Group II) were vaccinated with a 10 micrograms dose of a recombinant DNA hepatitis B vaccine within 24 hours after birth according to a 0, 1, and 2 month schedule, with a booster dose planned 12 months later. Vaccination results in 14 (Group I) and 47 (Group II) neonates showed that at two months after the third dose of vaccine, 86% (6/7) and 100% (37/37), respectively, seroconverted, with anti-HBs geometric mean titres of 80 IU/l and 266 IU/l in the respective groups. No adverse reactions to the vaccine were observed. These preliminary results indicate that the recombinant DNA hepatitis B vaccine is safe and highly immunogenic in newborns.

The pharmacodynamic response of the basal skin potential to quinidine gluconate.

The basal skin potential (BSP) was explored as an indirect means of continuously monitoring the cardiac response of quinidine gluconate. A method was developed to follow the BSP using a high impedance, recording polygraph and nonpolarizing calomel electrodes. Intravenous administration of quinidine gluconate caused time dependent changes in the BSP. Further studies showed that blood levels during the elimination phase and the QT interval of the electrocardiogram (ECG) over their entire time period were highly correlated with the BSP. Optimal correlation with the QT interval occurred when the BSP curve was shifted to earlier times by approximately 10 minutes, reflecting possible differences in the accessibility or mechanism of the respective pharmacologic compartments. Further application of the BSP for pharmacodynamic monitoring will require electrode refinements and an increased understanding of its mechanism of action.

Percutaneous absorption: a new physicochemical predictive model for maximum human in vivo penetration rates.

A diffusion model for stratum corneum-limited percutaneous absorption based on the interaction of the diffusate with the stratum corneum was derived. Two types of interactions were proposed, ion-dipole and lipid-lipid, based on current knowledge of the stratum corneum and on irreversible thermodynamic arguments. The resulting flux equations predict a linear dependence of flux on the dipole moment and ln X of the diffusates , where X is the mole fraction solubility. These flux equations were tested on 21 different diffusates whose human percutaneous absorption rates in vivo had been previously determined. A solubility method was used to classify the interaction pathway for each diffusate . Correlation of the maximum absorption rate for the lipid and polar pathways give correlation coefficients of 0.946 and 0.998, respectively. It is believed that these studies provide a starting point for the ultimate goal of percutaneous absorption research: to be able to bypass in vivo and in vitro studies and to predict absorption solely on the basis of the physicochemical properties of the diffusates .

Guinea pig ear as a new model for in vivo percutaneous absorption.

A new animal model for in vivo percutaneous absorption utilizing the hairless, relatively thick skin of the guinea pig ear is proposed. Topical absorption studies were carried out with [14C]hydrocortisone and [14C]testosterone. Systemic studies were also conducted to correct for incomplete urinary excretion. In addition, a single stratum corneum correction factor was developed from published data to enable the guinea pig ear skin to be directly compared with human forearm skin. A comparison of human percutaneous absorption with the corrected guinea pig ear absorption shows a high correlation for both hydrocortisone and testosterone. The effects of ambient changes in relative humidity are also discussed with respect to in vivo percutaneous absorption.