Molecular cloning and tissue-specific expression of a new member of the regenerating protein family, islet neogenesis-associated protein-related protein.

Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.

The volume and compressibility changes of lysozyme associated with guanidinium chloride and pressure-assisted unfolding.

The apparent specific volumes and isentropic compressibilities of hen egg white lysozyme were measured in aqueous guanidinium chloride solutions at 25 degrees C by means of a vibrational densimeter and a sing-around ultrasonic velocimeter. Little transition attributable to a protein unfolding was detected in the partial specific volume, while the partial specific isentropic compressibility decreased slightly around the transition region. The pressure-assisted unfolding was also investigated in aqueous guanidinium chloride solutions by means of ultraviolet spectroscopy. Assuming a two-state transition model, it was found that the free energy change of unfolding depends almost linearly on pressure and the unfolding reaction is accompanied by a small decrease in volume. The compressibility behavior is in conflict with the notion that a protein structure is almost completely unfolded by guanidinium chloride and most of the amino acid residues in the protein interior are exposed to solvent. These results support the current view that globular proteins have some residual structures even in the unfolded state induced by a strong denaturant.

Pressure-induced unfolding of lysozyme in aqueous guanidinium chloride solution.

The pressure-induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two-state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 degrees C. Between 25 and 60 degrees C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 degrees C. These data were interpreted as suggesting that a two-state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 degrees C.

The lipid-lowering profile in rodents. AZ-1355, a new dibenzoxazepine derivative.

The lipid-lowering profile of ethyl 10,11-dihydro-4-methoxydibenz[b,f]-(1,4)oxazepine-8-carboxylate (AZ-1355) has been evaluated using clofibrate as a reference compound. This compound is structurally unrelated to any other hypolipidemic agent. AZ-1355 was selected not only for its effect in reducing serum lipids, but also because it inhibits platelet aggregation in vivo and elevates the prostaglandin I2/thromboxane A2 ratio in vitro. It lowers serum total cholesterol in Triton-treated hyperlipidemic mice, and also lowers serum total cholesterol and triglyceride in dietary hyperlipidemic rats. In golden hamsters chosen for further evaluation, AZ-1355 reduced serum, liver and cardiac lipids, improved the beta/alpha-lipoprotein ratio and increased the HDL cholesterol. Thus, it is apparent that the lipid-lowering profile of AZ-1355 differs from that of clofibrate.

Improved delivery through biological membranes. 4. Prodrugs of L-dopa.

Various classes of transient derivatives of L-Dopa have been synthesized, systematically protecting one or more of the main sites of metabolism in the molecule: the carboxy function, the amino, and/or the catechol system. The derivatives studied include carboxy esters, phenol esters, amides, peptides, and various combinations of these functions. A number of these derivatives effectively prevent the metabolism of L-Dopa prior to and/or during the absorption process, resulting in a significantly better bioavailability of the drug. In vivo studies using dogs showed up to 2.5-fold increase in L-Dopa blood levels. The metabolism as well as toxicity aspects of the prodrugs is also discussed.

Constant light housing attenuates circadian rhythms of mPer2 mRNA and mPER2 protein expression in the suprachiasmatic nucleus of mice.

Constant light (LL) or constant dark (DD) environmental lighting conditions cause a free-running period and activity reduction in the rodent behavioral circadian rhythm. In order to understand the molecular process underlying behavioral rhythms in LL or DD housing conditions, we examined the circadian profile of mPer2 mRNA and mPER2 in the suprachiasmatic nucleus (SCN), a main oscillator, of free-running mice. The circadian expression rhythm of mPer2 in the SCN was dampened under 7-day LL conditions, whereas that of mPER2 protein was moderately attenuated and its expression peak delayed. The circadian expression of mPer2 and its product was slightly attenuated and advanced by 7-day DD conditions. With arrhythmic behavioral activity caused by long-term LL housing, mPER2 protein lost its rhythmicity in the SCN. On the other hand, LL or DD housing did not affect the mPer2 gene and its product in the cerebral cortex. The present results suggest that mPER2 circadian expression in the SCN corresponds well with behavioral circadian oscillation under LL or DD conditions. Thus, the behavioral circadian rhythm seems to correlate with molecular clock works in the SCN.

Transplacental administration of diethylstilbestrol (DES) causes lesions in female reproductive organs of Donryu rats, including endometrial neoplasia.

The effects of transplacental administration of diethylstilbestrol (DES) on female reproductive organs were investigated using Donryu rats. The animals were given subcutaneous injections of DES dissolved in olive oil at doses of 0.01 or 0.1 mg/kg on days 17 and 19 of gestation. In female offspring, clinical signs, body weights and estrous cycles were continuously assessed until all survivors were killed at month 18. A low mean litter size and shortening of period of pregnancy were recognized in the 0.1 mg/kg group. Disorder and/or suspension of the estrous cycle (so called persistent estrus) also appeared very early in the 0.1 mg/kg group. Macroscopically, the incidences of hypoplasia of the oviduct, cystic dilatation of the uterus and small size of the uterine cervix were higher in the 0.1 mg/kg group than those in the control group. Histologically, in the ovary, the incidence and degree of atrophy were increased in both 0.01 and 0.1 mg/kg groups. In the uterus, total incidences of endometrial hyperplasias were about the same in all groups. However, endometrial adenocarcinomas were dose-dependently increased in the treated groups, the incidence in the 0.1 mg/kg group being significant, compared to that in the control. In the vagina, mucification was more prominent in the treated animals, especially at the higher dose, but no tumors were observed. The present results indicate that prenatal exposure to DES can produce uterine adenocarcinomas in rats, as reported earlier for mice, although its carcinogenic activity is not so strong. Increase of endometrial adenocarcinoma incidence might depend on hormonal imbalance resulting from the ovarian atrophy due to transplacental treatment of DES.

Time-dependent promotion activity of 17beta-estradiol on uterine carcinogenesis in mice initiated with N-ethyl-N-nitrosourea.

The time-dependent promotion activity of 17beta-estradiol (E2) by initiation with N-ethyl-N-nitrosourea (ENU) on induction of mouse uterine endometrial proliferative lesions was examined. Illumination-induced persistent estrous female CD-1 mice were divided into five groups at 9 weeks of age. At 10 weeks of age, mice in all groups (n=25) were given a single intra-uterine administration of ENU (50 mg/kg), dissolved in polyethylene glycol. Animals in Groups 2 to 5 were then implanted s.c. with an E2 pellet at 9, 11, 14 and 17 weeks of age. The implants were left in place for 8 weeks and then taken out. At the termination of the experiment (week 15 after the ENU-treatment), all surviving mice were killed and the development of uterine proliferative lesions were assessed. All groups demonstrated endometrial hyperplasias and adenocarcinomas and the incidences of the latter in ENU plus E2 treated animals (Groups 2 to 5; 36, 48, 35 and 36%, respectively) were significantly higher compared to 8% for Group 1, without any variation with the age at E2 treatment. However, the incidences of adenocarcinomas plus severe hyperplasias increased from Groups 1 to 5 (28, 40, 56; P<0.05, 61; P<0.05 and 80%; P<0.01, respectively), indicating that promotion effects of E2 on induction of uterine proliferative lesions in the uterine endometrium become more pronounced with the interval after ENU initiation.

Analysis of phospho- and phosphonosphingolipids by high-performance liquid chromatography.

A simple and efficient method for the separation of phosphosphingolipids including phosphonosphingolipids by high-performance liquid chromatography is described. A mixture of authentic lipids consisting of sphingomyelin, ceramide phosphorylethanolamine, ceramide 2-aminoethylphosphonate, and ceramide N-methylaminoethylphosphonate was completely separated using a silica gel (Zorbax SIL) column with acetonitrile-methanol-water 72:40:10 (v/v) as eluting solvent. The elution of these sphingolipids was monitored directly with an ultraviolet spectromonitor at 207 nm. The practical limit of detection of each sphingolipid was about 0.2 microgram or 0.3 nmol. Using this method, we found that from one to four different phosphono- and/or phosphosphingolipids in fresh-water shellfish can be routinely identified and reproducibly quantified.

Trandolaprilat, an angiotensin-converting enzyme inhibitor, is not excreted in bile via an ATP-dependent active transporter (cMOAT).

Trandolapril is the prodrug of an angiotensin-converting enzyme (ACE) inhibitor. It has been proposed that its active metabolite, trandolaprilat, is mainly excreted in bile, but this has not been clearly demonstrated. Recently it has been reported that temocaprilat, an active metabolite of the ACE inhibitor temocapril, is effectively excreted in bile via an ATP-dependent active transporter (canalicular multispecific organic anion transporter: cMOAT). To investigate whether trandolaprilat has the pharmacological ability to affect the cMOAT system in a manner similar to temocaprilat. The lipophilicity of trandolaprilat and temocaprilat was measured to determine the n-octanol-water partition coefficients. The dose-dependent inhibition of the up-take of [3H]-estradiol-17beta-D-glucuronide and [3H]-2,4-dinitrophenyl-S-glutathione, which are good substrates for cMOAT, in canalicular membrane vesicles (CMVs) prepared from Sprague-Dawley rats was determined in the presence of trandolaprilat and temocaprilat. The partition coefficient of trandolaprilat (log Po/w - 1.1) was about 30 times higher than that of temocaprilat (log Po/w - 2.5). The uptake of [3H]-estradiol-17beta-D-glucuronide and [3H]-2,4-dinitrophenyl-S-glutathione was dose-dependently inhibited by the presence of temocaprilat, but trandolaprilat had no effect on the transport of [3H]-estradiol-17beta-D-glucuronide or [3H]-2,4-dinitrophenyl-S-glutathione into CMVs even at concentrations as high as 200 microM. It could be concluded that trandolaprilat has a higher lipophilicity than temocaprilat. But the hepatobiliary excretion system via cMOAT may not contribute to the excretion of trandolaprilat in bile.

Prediction of ACNU plasma concentration-time profiles in humans by animal scale-up.

Plasma concentration-time profiles of nimustine hydrochloride, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosour ea hydrochloride (ACNU), in the mouse, rat, rabbit, and dog were determined by high-performance liquid chromatographic analysis. The pharmacokinetic parameters for these four animal species and previously reported clinical data were analyzed for investigation of interspecies correlation. Log-log plots of body weight (W; kg) vs total plasma clearance (CLtot,p; ml/min) and steady-state distribution volume (Vd,ss; l) for the four animal species were linear, with high correlation coefficients (r 0.996 for both parameters), despite the fact that the nonrenal clearance was greater than 97% in these species. Linear regression on the plots excluding human data yielded allometric equations (CLtot,p = 50.6 W0.957; Vd, ss = 1.29 W1.03) that were extrapolated to predict ACNU pharmacokinetic parameters in humans. For both parameters, however, there were 3-fold differences between the predicted and observed parametric values. To investigate these discrepancies, we measured serum protein binding of ACNU in these animal species and in humans. The values of CLtot,p and Vd,ss were converted into those of CLutot,p and Vd,uss, which correspond to the parameters for unbound ACNU. In this case, correlation coefficients of the log-log plots excluding human data (CLutot,p = 71.7 W0.891; Vd,uss = 1.82 W0.966) were also high (r greater than or equal to 0.991). The extrapolated values vs those observed in a 70-kg human were the following: CLutot,p, 3,160 vs 2,290 ml/min; Vd,uss, 110 vs 106 l. Thus, the animal data were successfully extrapolated to yield better predictions of human pharmacokinetic parameters if the analysis was based on the unbound plasma concentration of ACNU. In addition, the predicted plasma concentration-time profile for humans also showed good agreement with the observed ones. These results suggest the importance of measuring unbound fractions of drugs for more accurate prediction of human pharmacokinetic parameters by extrapolation of animal data to the human situation.

Obstructive jaundice facilitates hepatic metastasis of B16F1 mouse melanoma cells: participation of increased VCAM-1 expression in the liver.

Obstructive jaundice facilitates experimental liver metastasis in the rat model, but the detailed mechanisms of this facilitation remain unclear. This study aimed to evaluate the contribution of vascular cell adhesive molecules-1 (VCAM-1) to augmented hepatic metastasis in cases of obstructive jaundice. Obstructive jaundice was induced in male C57BL/6 mice by common bile duct obstruction for 5 days using a Surgiclip. For the biliary decompression, obstructive jaundice was induced for 5 days, followed by removal of the Surgiclip. Liver specimens and blood samples were obtained from animals 5 days after biliary obstruction (OJ5) or sham operation and 2, 5, 11, 14 days after biliary decompression. The expression of VCAM-1 mRNA was increased in the livers from the OJ5 group. Western blot analysis demonstrated increased expression of VCAM-1 protein in the livers of the OJ5 group, in contrast with low VCAM-1 expression in the sham group. The expression of VCAM-1 protein was sustained at high levels at 2 days and decreased at 5 days after biliary decompression (BD5). For the induction of experimental hepatic metastasis, male C57BL/6 mice were randomized to three groups (sham, OJ5, BD5) of six animals each. B16F1 melanoma cells were introduced into the animals by an intraportal injection. Metastatic colonies in the livers were investigated 13 days after inoculation. The mean number of metastatic colonies was significantly increased in the OJ5 group (70.5+/-51.2) compared to that of the sham group (7.2+/-7.9) (p<0.05). This augmentation of hepatic metastasis was abrogated in the BD5 group (16.8+/-20.3). In conclusion, our results suggest that augmented hepatic metastasis in cases of obstructive jaundice are partly mediated through VCAM-1/VLA-4 interaction.

Influence of modes of ACNU administration on tissue and blood drug concentration in malignant brain tumors.

The water-soluble nitrosourea compound ACNU is also lipid-soluble at normal physiological pH levels. It lacks toxic effects on vision that nitrosoureas occasionally produce following intra-arterial administration. In 28 cases of both primary and secondary malignant brain tumors, ACNU was administered at surgery or angiography by three different modes: intravenous injection in Group I (10 cases), intra-arterial injection via the carotid artery in Group II (11 cases), and intra-arterial injection via the carotid artery after opening the blood-brain barrier (BBB) by means of mannitol in Group III (seven cases). Tumor tissue and blood samples were taken serially at various time intervals after ACNU injection, and ACNU was measured by high-performance liquid chromatography. The time-concentration curve for ACNU was calculated in each case by the two- and one-compartment open models for determination of ACNU levels in blood and tissue, respectively. Pharmacokinetic parameters including biological half-life, blood and tissue levels (0-t minutes and 0-infinity minutes), total plasma clearance, and distribution volume of the beta phase were compared. Statistical analysis of tissue ACNU levels at 0-t minutes revealed higher concentrations in Group III patients than in Groups II and I: levels in Group II were significantly higher than in Group I. Mean biological half-life was 30.3, 23.0, and 38.5 minutes in Groups I, II, and III, respectively. Levels of ACNU were significantly increased in tumor tissue as well as in peritumoral tissue in one Group III patient with multiple metastatic anaplastic adenocarcinoma. In this series, treatment of malignant brain tumor by intra-arterial administration of ACNU produced significantly higher tissue levels of ACNU than did the systemic intravenous route.

The pharmacokinetic profile of RS-8359.

RS-8359 is very rapidly absorbed, metabolized and distributed following oral dosing, with significant concentrations of both parent drug and its principal metabolite appearing in plasma within 15 min. Plasma levels were linearly related to doses of up to 300 mg, beyond which absorption was diminished, perhaps due to a saturation effect. Clearance of RS-8359 is mainly through the renal system as the metabolite, and is virtually complete within 24 h. The kinetic profile of the drug best fits a two-compartment model, and mean residence time and half-life (beta) of the drug support a twice a day regimen for extended use. During multiple dosing twice a day for up to 6 weeks, steady state plasma drug levels were achieved within 48 h and there was no evidence of significant accumulation.

Microbial biofilms in the food processing industry--should they be a concern?

Biofilm formation will occur on solid surfaces in contact with a liquid. Organic and inorganic material in the liquid sediment onto the solid material. Subsequently, biologically active microorganisms will be attracted to this conditioned surface and adhere to it. The microbial cells will initiate growth, form an attachment matrix and develop into a complex community forming a microbial biofilm. Such microbial biofilms are common on solid surfaces in contact with many different kinds of liquids, fresh water, sea water, oil, milk and so on. These biofilms may be of benefit or be detrimental to the environment where they form. The goal of this review has been to summarize the literature on the development of microbial biofilms in these different environments with particular emphasis on what occurs in the environment of a food processing plant. Methods to control adherent microorganisms and subsequent biofilms in the food processing plant are discussed. It is apparent from the data that has been reviewed that the potential for the development of microbial biofilms in the environment of the food processing plant exists. However, the cleaning and sanitizing practices carried out in the food industry have been shown to control biofilm formation on food contact surfaces. Microbial attachment has been shown to occur on non-food contact surfaces and these attached microbes, if left undisturbed, will form biofilms. The potential for contamination of food with undesirable spoilage and pathogenic bacteria from attached microbes and biofilms exists in these food processing systems. Biofilm formation on non-food contact surfaces needs to be studied further and methods developed to prevent and control these biofilms.

Dosage form design for improvement of bioavailability of levodopa III: Influence of dose on pharmacokinetic behavior of levodopa in dogs and Parkinsonian patients.

The relationship between the dose of levodopa and its pharmacokinetic behavior following intravenous and oral administration was investigated in dogs and parkinsonian patients. Six beagle dogs received single doses of 2.4, 4.8, and 9.6 mg of levodopa/kg iv and single doses of 4.8, 9.6, and 19.2 mg of levodopa/kg po in a crossover fashion on separate occasions. Three parkinsonian patients received single oral doses of approximately 3.8, 7.7, and 15.4 mg of levodopa/kg in a crossover test. Plasma samples were analyzed for intact levodopa and total dopamine. The relationship between the area under the plasma concentration-time curve (AUC) of levodopa and the intravenous dose to dogs was linear. However, in both dogs and patients, the relationship after oral dosing was nonlinear, with the relative AUC increasing with increasing dose. Therefore, the pharmacokinetic behavior of levodopa after oral administration to dogs and patients was dose dependent.

Dosage form design for improvement of bioavailability of levodopa II: bioavailability of marketed levodopa preparations in dogs and parkinsonian patients.

To estimate the absolute bioavailability of oral levodopa, plasma concentrations and urinary excretion of levodopa and its metabolites were determined in beagle dogs and in parkinsonian patients after intravenous and oral drug administration. The absolute bioavailability of orally administered levodopa was estimated to be about 35% in both dogs and patients; however, the total amount absorbed of intact drug and levodopa metabolites was estimated to be 80--90% of the administered dose. Due to the similarities of the pharmacokinetic characteristics of levodopa found in beagle dogs and in humans, beagle dogs can serve as a model to study bioavailability, absorption, and metabolic mechanisms.

Dosage form design for improvement of bioavailability of levodopa V: Absorption and metabolism of levodopa in intestinal segments of dogs.

Plasma levels of levodopa, total dopamine, and residual amounts of levodopa and its metabolites at the administered site were analyzed following administration of single 100-mg doses of levodopa in solution into isolated segments of the duodenum, jejunum, and ileum of the dog. The largest area under the plasma concentration-time curve (AUC) of levodopa during the 1.0-hr study was obtained following administration in the duodenum, followed by the jejunum and ileum. In addition, the residual amounts of levodopa and its metabolites detected at the administration sites were: ileum, 23%; jejunum, 7% and duodenum, less than 1%. The largest AUC of total dopamine was obtained following administration in the jejunum, followed by the ileum and duodenum. This order was consistent with the order of levodopa decarboxylase enzyme activity reported previously. Therefore, it can be concluded that the major absorption site of levodopa in the intestine resides in the upper small intestine. Levodopa in 10-, 50-, and 100-mg doses was administered into isolated duodenal segments. The AUC of levodopa increased nonlinearly with increasing dose. Negligible amounts of both levodopa and its metabolites were observed in the segment at 1.0 hr after administration, indicating that the duodenal absorption of levodopa was not saturable within the dose range tested.

Dosage form design for improvement of bioavailability of levodopa VI: formulation of effervescent enteric-coated tablets.

A new dosage form of levodopa, which has the characteristics of loading high concentrations of levodopa at the upper part of the intestine, has been developed to improve its bioavailability. It is shown that an effervescent tablet formulation, coated with hydroxypropyl methylcellulose phthalate (carboxybenzoyl radical content: 20-24%) as the enteric material, is suitable for the purpose of dissolution. This was confirmed from animal experiments, which showed that tablets of this composition disintegrate instantly on reaching the upper part of the intestine. This tablet was considered appropriate for the bioavailability tests described in this paper.

Dosage form design for improvement of bioavailability of levodopa IV: Possible causes of low bioavailability of oral levodopa in dogs.

Several potential mechanisms for reduced levodopa bioavailability following oral administration to dogs and humans were investigated by studying the influence of the administration route on plasma levodopa levels after intravenous, hepatoportal, and duodenal administrations to dogs. The observed average areas under the plasma concentration-time curves (AUC) of levodopa following hepatoportal injection and intravenous injection were virtually identical; but following duodenal administration a decrease in the AUC of levodopa was observed with a concomitant increase in the AUC of total dopamine. The possible involvement of intestinal microorganisms in levodopa metabolism was explored in dogs that had been administered a combination of paromomycin and kanamycin to reduce intestinal microflora. Similar patterns of plasma level profiles and urinary excretion were observed between control and treated dogs. As measured by the release of [14C]carbon dioxide from [14C]levodopa, the distribution of levodopa decarboxylase enzyme activity in various parts of the intestine was studied in homogenates prepared from isolated intestinal segments of the duodenum and upper, middle, and lower parts of the jejunum and ileum. The jejunum showed the highest decarboxylase activity followed by the ileum and duodenum. These data indicate that the reduced bioavailability of orally administered levodopa occurs as a result of metabolism by levodopa decarboxylase enzyme in the gut wall.