Rodent PSP94 gene expression is more specific to the dorsolateral prostate and less sensitive to androgen ablation than probasin.

To date, the rodent ventral prostate (VP) has been the focus of many studies on androgen action, less attention has been directed to the lateral prostate (LP) and the dorsal prostate (DP). The rodent VP has no clear homologous counterpart in the human prostate. The rodent LP and DP is the only prostate lobe comparable to the peripheral zone of the human prostate, where hormone-induced prostate cancer mainly occurs. To explore its utility for prostate targeting, we have studied the gene expression of PSP94 with rat probasin (rPB), a gene commonly used for prostate targeting in prostate cancer research and a gene typically responsive to androgen regulation. Firstly, we demonstrated PSP94 gene transcription being more specific to the LP and DP lobes than rPB, where rPB RNA was detected in the LP and DP and other lobes at different levels. Secondly, we found that PSP94 gene transcription decreased relatively slowly in response to androgen deprivation but recovered rapidly in response to testosterone replacement after complete ablation of PSP94 transcription. In the VP, gene transcripts of rPB were specifically responsive to androgen deprivation; however, they responded relatively slowly in the LP and DP. RNase protection experiments indicated that the slow response was not due to abnormal persistence of PSP94 messenger RNA specifically in the DP and LP lobes in comparison with rPB. Thirdly, Western blot analysis revealed that both PSP94 and rPB expression is specific to the LP and DP at the protein level, exhibiting slow responses to testosterone replacement after castration. We conclude that PSP94 gene expression at the transcriptional level is more specific to the LP and DP than rPB and thus less sensitive to androgen ablation. This may have clinical implications for strategies to target the prostate in cancer therapy.

PSP94 expression after androgen deprivation therapy: a comparative study with prostate specific antigen in benign prostate and prostate cancer.

PURPOSE: To examine the clinical use of PSP94 (prostate secretory protein of 94 amino acids) as an androgen independent marker, we conducted a comparative study of prostate samples including benign tissue and cancers which did and did not have androgen deprivation. MATERIALS AND METHODS: Among 163 radical prostatectomy cases 75 had androgen deprivation before operation, while surgery was performed in the remainder without prior hormone treatment. Considering the pathological up grading following hormone therapy, contiguous sections from radical prostatectomy samples were stained for PSP94 and prostate specific antigen (PSA) by immunohistochemistry, and equivalent tumor foci were evaluated by assessing the intensity and extent of the staining. RESULTS: In untreated benign prostate tissue PSP94 and PSA staining was positive and identical in all sections in the no pretreatment group. However, PSP94 expression in the androgen deprivation group was significantly higher than PSA in intensity (p = 0.0005) and extent (p = 0.034). In untreated cancer cases PSP94 intensity and extent demonstrated strong inverse association with Gleason grade (p <0.0001). In contrast, PSA expression was high in every grade, resulting in no statistical association with tumor grade. In the androgen deprivation group PSA staining was decreased in every grade compared to the no pretreatment group. On the other hand, PSP94 expression was decreased in grade 3 tumor foci but increased in grades 4 and 5 tumor foci compared with samples of the corresponding grade in the no pretreatment group (p = 0.0034). CONCLUSIONS: PSP94 expression in benign prostate persists under androgen deprivation compared to PSA. PSP94 synthesis in high grade tumor appears to be activated in the absence of androgen stimulation, indicating the possible alternative pathways in the regulation of PSP94.

Differential expression of PSP94 in rat prostate lobes as demonstrated by an antibody against recombinant GST-PSP94.

Prostate secretory protein (PSP94, 94 amino acids) is one of the most abundant proteins secreted from the prostate. Its biological role is unknown and still controversial, although it is assumed to have the potential to be a biomarker and a suppressor of prostate cancer. In order to establish an animal model to further elucidate its biological role, we expressed the mature form of rat PSP94 in Escherichia coli, using a glutathione S-transferase (GST) fusion expression vector; we generated a polyclonal rabbit antibody against the recombinant protein. The antibody specifically recognized recombinant rat PSP94 and cross-reacted only very weakly with its human homologue. Using the characterized anti-rat PSP94 antibody, we found that PSP94 was located primarily in rat prostate. Furthermore, PSP94 is present at different levels in different lobes of rat prostate, with significant levels detectable only in the lateral lobe (LP). In addition, the most abundant PSP94 expression was found in the prostate lobe secretions, and PSP94 levels in LP secretions were at least seven times higher than in secretions from the dorsal prostate (DP). The rat ventral prostate (VP) and other regions of the male accessory glands were found to be almost completely devoid of PSP94. Since most rat prostate dysplasia induced by steroid hormone treatment occurs only in dorsolateral prostate, prostate tissue-specific expression and the expression of PSP94 in dorsolateral, but not other, lobes of the prostate suggest a potential role in prostate targeting and prostate cancer development.

cDNA, genomic cloning, and gene expression analysis of mouse PSP94 (prostate secretory protein of 94 amino acids).

The potential use of prostate secretory protein of 94 amino acids (PSP94) as a diagnostic biomarker or a therapeutic agent for prostate cancer has been reported. In order to establish an animal model to further elucidate on its biological role, we cloned the mouse PSP94 cDNA (approximately 500 bp) by reverse transcriptase-polymerase chain reaction (RT-PCR) and disclosed its genomic structure. The whole mouse PSP94 gene (approximately 23 kb) was amplified by long and accurate-PCR and also cloned by screening of a mouse embryo stem-cell genomic library. Computational and statistical analyses have demonstrated several highly conserved characteristics of PSP94 among different species. Comparison of PSP94 from human, two primates, pig, and rodents revealed that the most significant feature is that PSP94 is rich in cysteines (10% of the total sequence) and their positions are highly conserved. The three intron-four exon structure of the human PSP94 gene and the consensus sequence (....GT-intron-AG...) for mRNA splicing are also strongly conserved. A high divergence in cDNA sequence in the protein-coding region and also in the genomic sequence of PSP94 was also observed among these species. Comparing with alpha-globin, a typical evolutionally conserved gene, with the PSP94 gene, the rate of nonsynonymous changes per site per year (kN) is 2 to 6 times higher, indicating that PSP94 gene has been under far fewer evolutionary constraints than other genes and has a potential role as a species barrier in reproductive biology. In order to test this hypothesis, we investigated the gene expression of PSP94 and its tissue distribution in various rodent tissues by RT-PCR and in situ hybridization (ISH). Gene expression was found only in the prostate, suggesting that PSP94 is probably more tissue specific in the prostate of rodents than in mammals. The ISH analysis also revealed a prostate lobe-specific expression of the PSP94 gene in both mice and rats. It was strongly expressed in the lateral prostate, but the findings were negative in the dorsal and ventral lobe. Therefore, it is hypothesized that one of the primary functions of rodent PSP94, as a major prostate secretory protein, is related to reproductive biology.

Lymphatic transport of alpha-, gamma- and delta-tocotrienols and alpha-tocopherol in rats.

Lymphatic transport of alpha-, gamma- and delta-tocotrienols and alpha-tocopherol was measured in thoracic duct-cannulated rats. Animals were administered 3 ml of a test emulsion containing 200 mg sodium taurocholate, 50 mg fatty acid free-albumin, 200 mg fat and 100 mg of a mixture of tocotrienols and alpha-tocopherol (Exp. 1) or 10 mg of purified alpha-, gamma- or delta-tocotrienol or alpha-tocopherol (Exp. 2) through a gastric tube. Quantitative lymphatic recovery of oleic acid given as triolein was obtained in these experimental conditions. The 24-hours recovery of tocotrienols and alpha-tocopherol were 10-20% of the administered dose in Exp. 1. The recovery of alpha-tocotrienol was about 2-times higher than that of alpha-tocopherol, while that of gamma- and delta-tocotrienols was intermediate between these two alpha-forms. In Exp. 2, where these compounds were administered individually, the 24 hours recovery ranged from 22 to 37% of the administered dose. Again, the recovery of alpha-tocotrienol was significantly higher than that of the other tocotrienols and alpha-tocopherol, while that of gamma- and delta-tocotrienols and alpha-tocopherol was comparable. Thus, the results show the preferential absorption of alpha-tocotrienol compared to gamma- and delta-tocotrienols and alpha-tocopherol.

Effect of the viscosity or deacetylation degree of chitosan on fecal fat excreted from rats fed on a high-fat diet.

Several chitosan preparations, either with a comparable degree of deacetylation but differing viscosity or with comparable viscosity but a differing degree of deacetylation, were examined for their effect on the fecal fat excreted from rats fed on a high-fat diet. As the viscosity or deacetylation degree of a chitosan preparation increased, the more its effect on the apparent fat digestibility by rats became conspicuous. A supplement of ascorbic acid to each chitosan diets resulted in a significant depression of fat digestion and absorption in the lumen. The chitosan intake caused a higher level of fat to be excreted in the feces of the corn oil-receiving rats than the lard-receiving ones, although the effect was strong with both diet groups.

Mechanism for the inhibition of fat digestion by chitosan and for the synergistic effect of ascorbate.

We investigated the mechanism for the inhibition of fat digestion by chitosan, and the synergistic effect of ascorbate. The important inhibition characteristics of fat digestion by chitosan from observations of the ileal contents were that it dissolved in the stomach and then changed to a gelled form, entrapping fat in the intestine. The synergistic effect of ascorbate (AsA) on the inhibition of fat digestion by chitosan is thought not to be acid-dependent but due to the specificity of AsA itself, according to the data resulting from using preparations supplemented with sodium ascorbate (AsN). The mechanism for the synergistic effect is considered to be 1) viscosity reduction in the stomach, which implies that chitosan mixed with a lipid is better than chitosan alone, 2) an increase in the oil-holding capacity of the chitosan gel, and 3) the chitosan-fat gel being more flexible and less likely to leak entrapped fat in the intestinal tract.

Lymphatic transport of stearic acid and its effect on cholesterol transport in rats.

Lymphatic transport of stearic acid, given as completely hydrogenated rapeseed oil (R10), 9 to 1 (R9) and 5 to 5 (R5) mixtures of R10, and soybean oil and completely hydrogenated tallow (T) was examined in the rat cannulated thoracic duct. R10, R9, R5, and T contained 91.4, 81.5, 46.5, and 63.6% stearic acid, respectively. A large portion of the remaining fatty acids in T was palmitic acid (31%). These fats were emulsified with bile salt and albumin, and administered via a stomach tube. Lymphatic recovery of stearic acid at 24 h was lowest in R10 and highest in R5, and intermediate in R9 and T. Recovery of oleic and linoleic acids in rats given R5 was almost complete and significantly higher than that of stearic acid. When T was given, the 24 h recovery of stearic acid was significantly lower than that of palmitic acid. A highly inverse correlation between the recovery and the content of stearic acid in administered fats was observed in R10, R9, and R5. Lymphatic recovery of cholesterol was almost parallel with that of stearic acid. Although the content of stearic acid in T was lower than that in R9, the recovery of stearic acid and cholesterol was almost similar. The results indicate that the rate of lymphatic recovery of stearic acid is affected by the quantity and quality of coexisting fatty acids.

Lymphatic transport of eicosapentaenoic and docosahexaenoic acids as triglyceride, ethyl ester and free acid, and their effect on cholesterol transport in rats.

Lymphatic transport of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids given in the forms of triglyceride, ethyl ester of free acid and their effect on cholesterol transport was compared in lymph-cannulated rats. Lymphatic recovery of DHA and EPA given by stomach tube in the form of triglyceride in which they were mainly located at the 2-position was significantly higher than that of the ethyl ester or free acid during the first 6 hr after the administration and the tendency continued until 9 hr. In contrast, the 9 to 24 hr recovery of DHA and EPA in the forms of ethyl ester and free acid was considerably higher than that of triglyceride. Consequently, cumulative 24 hr recovery of EPA was comparable among the three forms. However, the 24 hr recovery of DHA was highest in free acid, lowest in ethyl ester and intermediate in triglyceride. Recovery of the free acid between 9 and 24 hr after administration was significantly higher than that given in the forms of triglyceride or ethyl ester. Cholesterol recovery in lymph of rats given with ethyl ester or free acid was lower than that given with triglyceride at an early stage after the administration in both EPA and DHA. Cumulative 24 hr recovery of cholesterol in rats given these fatty acids as ethyl ester was significantly lower than in those given as the other two forms.

Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats.

A(-)-epicatechin (EC) and (-)-epigallocatechin (EGC) mixture and a mixture of their gallates (ECG and EGCG, respectively) markedly lowered lymphatic cholesterol absorption in rats with a cannulated thoracic duct. A mixture of ECG and EGCG was more effective in reducing cholesterol absorption than the EC and EGC mixture. These catechins also tended to decrease lymphatic absorption of triacylglycerols, although not so pronounced as in cholesterol absorption. An in vitro study on micellar solubility of cholesterol showed that these catechin mixtures precipitated cholesterol solubilized in mixed bile salt micelles in a dose-dependent manner. A mixture of ECG and EGCG more effectively precipitated micellar cholesterol than a mixture of EC and EGC. When purified EC, EGC, ECG and EGCG were used, EGCG was more effective in precipitating micellar cholesterol than ECG. The effect of EC and EGC was comparable and weaker than their gallate esters. The bile acid concentration in the micelles was not affected by these catechins. A positive correlation was observed between the amount of coprecipitated EGCG and cholesterol. These results clearly show that tea catechins, in particular their gallate esters, effectively reduce cholesterol absorption from the intestine by reducing solubility of cholesterol in mixed micelles. The observation accounts for the hypocholesterolemic effect of tea catechins.

Prolongation of antipyretic action and reduction of gastric ulcerogenicity in the rat by controlled-release granules of bermoprofen, a new nonsteroidal anti-inflammatory drug.

While bermoprofen [(+-)-10,11-dihydro-alpha,8-dimethyl-11- oxodibenz[b,f]oxepin-2-acetic acid], a new nonsteroidal antiinflammatory drug (NSAID), has potent antipyretic and analgesic activities with a short biological half-life, it shows ulcerogenic activity as a side-effect like other nonsteroidal anti-inflammatory drugs. A bermoprofen preparation was specially designed to prolong its duration of action and to reduce its side effect. Immediate-release granules (IRGs) were prepared by coating particles of Nonpareil 103 with bermoprofen. The IRGs were also coated by spraying a film solution composed of ethylcellulose and hydroxypropylmethylcellulose (3:2), and, thereby, three kinds of retard-release granules (RRGs) were obtained with the coating amounts of 1.5, 2.5, and 4%. Peak plasma bermoprofen levels were seen 0.5, 1, 2, and 3 h after single oral administrations of IRG, RRG(1.5%), RRG (2.5%), and RRG(4%), respectively, in rats. The potency order in ulcerogenic activity was RRG(1.5%) greater than IRG greater than RRG(2.5%) greater than RRG(4%) after a single oral dosage in rats. Then, IRG and RRG(4%) were mixed (in the ratio 3:7, which was calculated from their simulated plasma bermoprofen profiles) in order to get fast onset and long duration of antipyretic action. These mixed granules [i.e., the sustained-release granules (SRGs)] exhibited a falt plasma bermoprofen profile, and longer lasting antipyretic and lower ulcerogenic activities in rats in comparison with IRG or bermoprofen. From these results, it is suggested that a bermoprofen SRG preparation has more durable therapeutic activity and lower side-effects as a NSAID.

Bioavailability of cyclandelate from capsules in beagle dogs and dissolution rate: correlations with bioavailability in humans.

The bioavailability in beagle dogs and the dissolution rates of cyclandelate from five capsule preparations commercially available in Japan were measured. One of the capsules that showed an extremely low bioavailability in humans also showed the lowest bioavailability in beagle dogs, although the difference in bioavailability with the highest preparation was smaller than in humans. A significant correlation was obtained between the results of the studies in humans and beagles. However, the power of the test using beagles was extremely low in comparison with that in the human study. Food enhanced the bioavailability of cyclandelate from the capsules having the highest and lowest bioavailability in the fasted state in beagles as observed in the human study previously. The bioinequivalence of the cyclandelate capsules detected in the fasted state disappeared in the fed state in the beagle dog study, while the bioinequivalence still remained in the non-fasted state in human subjects. Thus bioequivalence testing in the fed state led to different results in both species. The most poorly bioavailable capsule in both species in the fasted state showed a slow dissolution rate by several dissolution methods with moderate stirring. In order to obtain a good correlation with in vivo bioavailability, a large volume of test solution and addition of Tween 80 were required. Extensive growth of whiskers (needle-like crystals) was observed in the entire capsule mass having the lowest bioavailability.

Influence of specific gravity and food on movement of granules in the gastrointestinal tract of rats.

The suitability of rats as an animal model for estimating the bioavailability of controlled-release granules in humans was investigated. Non-disintegrating granules (diameter of 0.8 mm; specific gravity of 0.9-1.85) were used as a model preparation. Twenty granules were administered to fed rats, fasted rats and rats given soft food, and the number of granules remaining in the gastrointestinal tract was counted at suitable intervals. Granules with a specific gravity of 1.25 administered to fasted rats were rapidly emptied from the stomach with a 50% gastric emptying time of 1 h as compared with granules with a specific gravity of less than 1.0 or with a high specific gravity such as 1.85. The presence of food in the stomach reduced the emptying rate of granules. The mean transit time of granules through the small intestinal tract was not influenced by the specific gravity or the presence of food. The mean transit time was about 3 h. It was found that the transit profile of granules through the gastrointestinal tract in rats was similar to that of granules in humans. Accordingly, it is possible to use rats at the preformulation stage for estimating the bioavailability of controlled-release granules in humans.

Characteristics of the gastrointestinal absorption of morphine in rats.

The absorption characteristics of morphine were investigated by using rat gastrointestine. Absorption and transport experiments were carried out by the in situ loop and the in vitro everted sac methods, respectively. Brush border membrane vesicles (BBMVs) were used for uptake experiments. Morphine and its metabolites, morphine-3-glucuronide (M-3-G), and morphine-6-glucuronide (M-6-G), in biological samples were simultaneously determined by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection and electrochemical detection. In the in situ loop method, morphine was well absorbed in the order of jejunal site greater than duodenal site greater than ileal site greater than middle intestinal site greater than rectal site, but it was poorly absorbed from the stomach. In each of the everted duodenal and jejunal sacs, 2,4-dinitrophenol, a metabolic inhibitor, inhibited the transport of morphine from the mucosal side to the serosal side. Further, HgCl2 pretreatment reduced the absorption of morphine from the duodenal and the jejunal loops. The initial uptake of morphine by BBMVs was stimulated in the presence of an H+ gradient (inner pH 7.5 and outer pH 5.0) and an overshoot phenomenon was observed. The initial uptake showed concentration dependence, i.e., it was saturable. Results obtained in this study indicate that carrier-mediated transport stimulated by the H+ gradient is partly involved in the duodeno-jejunal absorption of morphine, although morphine is passively absorbed from other sites.