Usefulness of serum procalcitonin levels in pulmonary tuberculosis.

There are very few data on serum procalcitonin (PCT) levels in pulmonary tuberculosis (PTB) patients who are negative for HIV. We assessed serum PCT in consecutive patients diagnosed with pulmonary tuberculosis or community-acquired pneumonia (CAP) on admission to discriminate between PTB and CAP, and examined the value of prognostic factors in PTB. 102 PTB patients, 62 CAP patients, and 34 healthy volunteers were enrolled. Serum PCT in PTB patients was significantly lower than in CAP patients (mean ± sd 0.21 ± 0.49 versus 4.10 ± 8.68 ng·mL⁻¹; p < 0.0001). By receiver-operating characteristic curve analysis, serum PCT was an appropriate discrimination marker for PTB and CAP (area under the curve 0.866). PTB patients with ≥ 0.5 ng·mL⁻¹ (normal cut-off) had significantly shorter survival than those with < 0.5 ng·mL⁻¹ (p < 0.0001). Serum PCT is not habitually elevated in HIV-negative PTB patients and is a useful biomarker for discriminating between PTB and CAP; however, when serum PCT is outside the normal range, it is a poor prognostic marker.

Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is characterized by periodontal tissue inflammation and alveolar bone loss. The intermittent administration of parathyroid hormone (PTH), a major regulator of bone remodeling, has been demonstrated to stimulate osteoblastic activity. Although the systemic administration of PTH has been reported to protect against periodontitis-associated bone loss, the effect of the topical administration of PTH is unclear. In this study, the effect of intermittent administration of PTH on osteoblastic differentiation was examined in cultured calvaria cells and then the effect of topical and intermittent administration of PTH was determined by measuring the recovery of alveolar bone loss after inducing experimental periodontitis in rats. MATERIAL AND METHODS: Alkaline phosphatase activity and bone nodule formation were measured in fetal rat calvaria cells. Experimental periodontitis was induced by placing nylon ligature around rat maxillary molars for 20 d. After ligature removal (day 0), PTH was topically injected into buccal gingiva three times a week for 10 wk. Micro-computed tomography analysis and histological examination were performed on days 35 and 70. RESULTS: Intermittent exposure of PTH in calvaria cells increased alkaline phosphatase activity and bone nodule formation by 1.4- and 2.4-fold, respectively. Ligature procedures induced marked alveolar bone loss around the molars on day 0 and greater bone recovery was observed in the PTH-treated rats on day 70. An increase in osteoid formation on the surface of alveolar bone was detected in the PTH-treated rats. CONCLUSION: Intermittent treatment with PTH stimulated osteoblastic differentiation in fetal rat calvaria cell cultures, and topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis.

Topical administration of simvastatin recovers alveolar bone loss in rats.

BACKGROUND AND OBJECTIVE: Simvastatin, a cholesterol-lowering drug, has been reported to show anabolic effects on bone metabolism. We examined the effects of simvastatin in vitro using cultured rat calvaria cells and in vivo using periodontitis-induced rats. MATERIAL AND METHODS: Alkaline phosphatase activity and bone nodule formation were measured in cultured rat calvaria cells. Nylon ligature was placed around the maxillary molars of Fischer male rats for 20 d to induce alveolar bone resorption. After ligature removal, simvastatin was topically injected into the buccal gingivae for 70 d and then microcomputed tomography and histological examinations were performed. RESULTS: Simvastatin maintained high alkaline phosphatase activity and increased bone nodule formation in rat calvaria cells in a dose-dependent manner, showing that simvastatin increased and maintained a high level of osteoblastic function. Microcomputed tomography images revealed that treatment with simvastatin recovered the ligature-induced alveolar bone resorption, showing a 46% reversal of bone height. Histological examination clarified that low-mineralized alveolar bone was formed in simvastatin-treated rats. CONCLUSION: These findings demonstrate that simvastatin has the potential to stimulate osteoblastic function and that topical administration of simvastatin may be effective for the recovery of alveolar bone loss in rats.

Polyunsaturated fatty acid deficiency during neurodevelopment in mice models the prodromal state of schizophrenia through epigenetic changes in nuclear receptor genes.

The risk of schizophrenia is increased in offspring whose mothers experience malnutrition during pregnancy. Polyunsaturated fatty acids (PUFAs) are dietary components that are crucial for the structural and functional integrity of neural cells, and PUFA deficiency has been shown to be a risk factor for schizophrenia. Here, we show that gestational and early postnatal dietary deprivation of two PUFAs-arachidonic acid (AA) and docosahexaenoic acid (DHA)-elicited schizophrenia-like phenotypes in mouse offspring at adulthood. In the PUFA-deprived mouse group, we observed lower motivation and higher sensitivity to a hallucinogenic drug resembling the prodromal symptoms in schizophrenia. Furthermore, a working-memory task-evoked hyper-neuronal activity in the medial prefrontal cortex was also observed, along with the downregulation of genes in the prefrontal cortex involved in oligodendrocyte integrity and the gamma-aminobutyric acid (GABA)-ergic system. Regulation of these genes was mediated by the nuclear receptor genes Rxr and Ppar, whose promoters were hyper-methylated by the deprivation of dietary AA and DHA. In addition, the RXR agonist bexarotene upregulated oligodendrocyte- and GABA-related gene expression and suppressed the sensitivity of mice to the hallucinogenic drug. Notably, the expression of these nuclear receptor genes were also downregulated in hair-follicle cells from schizophrenia patients. These results suggest that PUFA deficiency during the early neurodevelopmental period in mice could model the prodromal state of schizophrenia through changes in the epigenetic regulation of nuclear receptor genes.

Localization of epoxide-metabolizing enzymes in rat testis.

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much more intensely for epoxide hydrolase and glutathione S-transferases B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to epoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.

Generation of neutralizing antibody to the reverse transcriptase of human immunodeficiency virus type 1 by immunizing of mice with an infectious vaccinia virus recombinant.

Antibodies inhibiting the reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) were found to be generated in the serum of mice repeatedly infected with a vaccinia virus recombinant, WRRT, expressing the enzyme. A monoclonal antibody (mAb), 7C4, which specifically and almost completely inhibits the RNA-dependent DNA polymerase activity of HIV-1 RT was produced from a mouse repeatedly immunized with WRRT. 7C4 seems to be specific for HIV-1 among retroviruses: 7C4 inhibited RT activity of three strains of HIV-1 (IIIB, Bru, and IMS-1) but not of two strains of HIV-2 (GH-1 and LAV-2) or two strains of SIV (MAC and MND). The immunoglobulin isotype of three out of four mAbs produced from spleen cells of the immunized mouse were IgG2a. This immunization method that avoids protein denaturation may preferentially induce a T helper type-1 immune response and increase the chances of producing the only occasionally obtainable mAb capable of recognizing a conformational epitope and completely inhibiting enzyme activity.

Purification of normal lymphocytes from leukemic T-cells by lectin-affinity adsorbents - correlation with lectin-cell binding.

Utilization of leukemic T-cells from normal ones, using lectin-affinity adsorbents, is described. CNBr-activated Sepharose 6MB was covalently coupled to Soybean (SBA) or Dolichos Biflorus Agglutinins (DBA), then serves as an affinity probe for separation of leukemic T-cells from normal lymphocytes. The normal lymphocytes were removed almost completely by phosphate buffered saline (Ca(2+) and Mg(2+) free) (PBS(-)) from lectin-affinity column. More than 80% of the leukemic T-cells were retained on the lectin-affinity adsorbent, whereas another 10-15% were easily removed by PBS(-). There was a very good linear correlation between percent of cells, retained on the lectin-affinity adsorbent and percent of cells, interacting with the respective free lectin (r=0.97 for SBA, and r=0.93 for DBA). The viability of normal lymphocytes was not influenced after passing through the columns. In the case of leukemic T-cells - about 90% of the easily removed cells were dead, and another 10% were viable cells, non-interacting with DBA or SBA.

Fractionation of normal and leukemic T-cells by lectin-affinity column chromatography.

A method for rapid fractionation of normal and leukemic T-cells (Jurkat, RPMI-8402, MOLT-4), using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently conjugated with Dolichos biflorus agglutinin (DBA) over 24 h. The normal cells were eluted by phosphate buffered saline (Ca(2+) and Mg(2+) free), while the leukemic T-cells, interacting with DBA, were removed by N-acetyl-D-galactosamine or by low-concentrated acetic acid as a mobile phase. The cell fractions were detected spectrophotometrically at 600 nm. The rate of cell elution decreased in the order: normal>leukemic T-cells. The viability and the type of separated T-cell fractions were characterized by flow cytometry, using adequate fluorescent antibodies. The interactions between leukemic T-cells and DBA-saturated Sepharose beads were examined by fluorescent microscopy, using fluorescent isothiocyanate-DBA as a fluorescent marker.

Targeting and anti-tumor efficacy of liposomal 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine in mice lung bearing B16BL6 melanoma.

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a potent anti-cancer agent, and we previously observed that liposomal formulation of 5'-O-dipalmitoylphosphatidyl derivative of CNDAC (DPP-CNDAC) is desirable for targeting. For targeting to pulmonary cancer, we investigated the in vivo behavior of liposomes containing DPP-CNDAC by a non-invasive method using positron emission tomography. Liposomes composed of DPP-CNDAC and cholesterol (DPP-CNDAC/CH liposomes) were markedly accumulated in mice lung bearing B16BL6 melanoma. In metastatic pulmonary cancer model, DPP-CNDAC/CH liposomes significantly reduced the lung colonization in a dose-dependent manner. The activity was significantly superior to conventional liposomal formulation or soluble CNDAC. These results suggest that DPP-CNDAC/CH liposomes are useful for metastatic pulmonary cancer.

Intracellular and intramembranous localization of a protein disulfide isomerase in rat liver.

The intracellular distribution of a membrane-bound protein disulfide isomerase (PDI) in rat liver was studied by quantitative immunoprecipitation, and its microsomal localization was confirmed. The content of the enzyme was 1 to 2% of total microsomal protein, and it was almost equally distributed between rough and smooth microsomes. The enzyme was not solubilized from microsomes by high concentrations of KCl, but was readily solubilized by detergents. Since PDI in microsomes was susceptible to digestion by trypsin, at least some parts of the enzyme molecule are exposed on the outside surface of microsomal vesicles. However, the binding of antibodies to microsomal PDI and the modification of the glutamine residues of PDI molecules by transglutaminase suggested that the molecules are not extensively exposed on the surface. Solubilized PDI was unable to rebind to microsomes or to become incorporated into reconstituted membrane of detergent-solubilized microsomes, showing that the association of this enzyme with the membrane is not simply mediated by hydrophobic interaction.

Biosynthesis and turnover of a microsomal protein disulfide isomerase in rat liver.

The biosynthesis and turnover of one type of microsomal protein disulfide isomerase (PDI) in rat liver were studied. The enzyme is predominantly synthesized by the membrane-bound ribosomes of rough endoplasmic reticulum, as judged from the results of immunological analyses of puromycin-released nascent peptides and in vitro translation products of isolated free and bound polyribosomes. Nascent peptides of PDI were released from rough microsomes by treatment with puromycin in the presence of 0.5 M KCI, which indicates that the nascent peptides of this enzyme are released on the outer surface of microsomal vesicles. This enzyme accounts for about 0.1 to 0.2% of the total protein synthesis by membrane-bound ribosomes. The half-life of PDI was about 7 days, which was significantly longer than the average half-life of microsomal proteins.

Purification and immunofluorescence localization of the mutant gene product of a Tetrahymena cdaA1 mutant affecting cell division.

A division arrest mutant, cdaA, of Tetrahymena thermophila is known to have a ts-defect in the formation of the fission zone which determines the position of the fission plane. A protein (Mr = 85,000; pI = 4.7, designated as p85) has recently been identified in our laboratory as a possible gene product of the cdaA locus by two-dimensional gel electrophoresis and genetic experiments (Ohba et al., submitted). In the present research, we have isolated p85, prepared its antibody, and demonstrated that in wild-type cells or in cdaA cells at permissive temperature, immunofluorescence for p85 appears on the equatorial basal bodies at the predicted fission zone just before formation of the zone. In such a case, the fission zone appears to be formed just anterior to the fluorescence-associated basal bodies, and then constriction of the division furrow occurs at the zone. However, in cdaA cells at the restrictive temperature, the equatorial p85 deposit and subsequent fission zone formation and furrowing do not occur at all. Thus, we conclude that p85 plays a key role in the formation of the fission zone and in the positioning of the equatorial fission line.

Binding of saccharides to ricin E isolated from small castor beans.

The binding of saccharides to ricin E isolated from small castor beans was studied by equilibrium dialysis and spectroscopy. Equilibrium dialysis data indicate that ricin E has two galactose-binding sites, a high affinity site (HA-site) and a low affinity site (LA-site). The binding of specific saccharides to ricin E induces a shift of the fluorescence spectrum to shorter wavelength by 3 nm and UV-difference spectra with a maximum at 290 nm and a negative intensity around 300 nm. The interaction of ricin E with its specific saccharides was analyzed in terms of the variation of the intensity at 320 nm in the fluorescence spectrum and the magnitude of the negative intensity at 300 nm in the UV-difference spectra as functions of saccharide concentration. The results indicate that these spectroscopic changes are representative of the binding of saccharides to the LA-site, which contains a tryptophan residue. By comparing the association constants of saccharides for ricin E with those for ricin D, isolated from the large castor beans, it was found that the HA of ricin E binds saccharides with an affinity of less than one-half that of ricin D, while the saccharide-binding abilities of the LA-site of the two ricins were about the same.

Tetrahymena actin: localization and possible biological roles of actin in Tetrahymena cells.

Though actin is ubiquitous in eukaryotes, its existence has not been clearly proven in Tetrahymena. Recently, we have succeeded in cloning and sequencing the Tetrahymena actin gene using a Dictyostelium actin probe (Hirono, M. et al. (1987) J. Mol. Biol. 194, 181-192). The primary structure of the Tetrahymena actin deduced from the nucleotide sequence of its gene is greatly divergent from those of other known actins, making it necessary to ascertain whether the predicted Tetrahymena actin is indeed an actin. In this paper, we investigated the localization of the predicted Tetrahymena actin by an immunofluorescence technique using antibody against its synthetic N-terminal peptide, in order to elucidate its possible biological roles. The results showed that immunofluorescence was localized in the division furrow of the dividing cell, and in the intranuclear filament bundles formed in cells exposed to heat shock or DMSO. In addition, the oral apparatus and the proximity of the cytoproct, which are organelles involved in endocytosis and exocytosis, respectively, also fluoresced. Thus, we conclude that the Tetrahymena actin we identified is indeed an actin and plays the same biological roles as ubiquitous actins do, although it is considerably divergent in its amino acid sequence.

Contribution of cytochrome b5 to androgen synthesis in rat testicular microsomes.

Cytochrome b5 was purified from porcine testicular microsomes, and its amino acid composition was determined. Rabbit antibody against the purified cytochrome b5 was prepared in order to study the contribution of cytochrome b5 to testicular microsomal oxygenases related to androgen production. In the presence of NADPH alone as the electron donor, the antibody against cytochrome b5 inhibited the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase of rat testicular microsomal fraction. Addition of NADH to the NADPH-supported oxygenase assay system enhanced both steroid oxygenase activities, and addition of the antibody against cytochrome b5 decreased the NADH-caused stimulation of steroid 17 alpha-hydroxylase and C-17-C-20 lyase activities. When dehydroepiandrosterone and NAD+ were added as substrates for 3 beta-hydroxy-delta 5-steroid dehydrogenase in order to synthesize NADH by enzymatic reaction, the NADPH-supported activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase were further stimulated as compared with the addition of NADH, and this stimulation was suppressed by the antibody against cytochrome b5. These results suggest that cytochrome b5, together with 3 beta-hydroxy-delta 5-steroid dehydrogenase, contributes to the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase in the testicular microsomal fraction.

Steroid hormone production in testis, ovary, and adrenal gland of immature rats irradiated in utero with 60Co.

Pregnant rats received whole-body irradiation at 20 days of gestation with 2.6 Gy lambda rays from a 60Co source. Endocrinological effects before maturation were studied using testes and adrenal glands obtained from male offspring and ovaries from female offspring irradiated in utero. Seminiferous tubules of the irradiated male offspring were remarkably atrophied with free germinal epithelium and containing only Sertoli cells. Female offspring also had atrophied ovaries. Testicular tissue obtained from intact and 60Co-irradiated rats was incubated with 14C-labeled pregnenolone, progesterone, 17 alpha-hydroxyprogesterone, and androstenedione as a substrate. Intermediates for androgen production and catabolic metabolites were isolated after the incubation. The amounts of these metabolites produced by the irradiated testes were low in comparison with the control. The activities of delta 5-3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, C17,20-lyase, and delta 4-5 alpha-reductase in the irradiated testes were 30-40% of those in nonirradiated testes. Also, the activities of 17 beta- and 20 alpha-hydroxysteroid dehydrogenases were 72 and 52% of the control, respectively. In adrenal glands, the 21-hydroxylase activity of the irradiated animals was 38% of the control, but the delta 5-3 beta-hydroxysteroid dehydrogenase activity was comparable to that of the control. On the other hand, the activity of delta 5-3 beta-hydroxysteroid dehydrogenase of the irradiated ovary was only 19% of the control. These results suggest that 60Co irradiation of the fetus in utero markedly affects the production of steroid hormones in testes, ovaries, and adrenal glands after birth.

Absence of linkage between radiosensitivity and the predisposing atp7b gene mutation for heritable hepatitis in the LEC rat.

The LEC rat is known to be a mutant strain that spontaneously develops heritable hepatitis due to copper accumulation, caused by mutation of the copper-transporting ATPase gene (Atp7b). Immunodeficiency and radiosensitivity have also been observed. Hayashi et al. extensively examined the radiosensitivity of the LEC rat and concluded that its hypersensitivity is controlled by a single autosomal gene. Furthermore, they suggested the possibility that it correlates to copper accumulation due to the Atp7b gene mutation, because ionizing radiation-induced hydroxyl radicals might act in concert with copper-induced hydroxyl radicals. In the present experiment, we analyzed linkage between radiosensitivity and the mutation responsible for hepatitis in F(1) animals of a cross with the F344 rat. Our results clearly demonstrated an absence of any significant association. In addition, partial dominance for radiosensitivity was observed, and radiosensitive (F(1) x LEC) backcross rats were twice as numerous as their radioresistant counterparts, suggesting the possibility of control by two or more recessive genes.

Chemoprevention by dietary dehydroepiandrosterone against promotion/progression phase of radiation-induced mammary tumorigenesis in rats.

When pregnant rats received whole body irradiations with 260 cGy gamma-ray at day 20 of pregnancy, and were then implanted with a diethylstilbestrol (DES) pellet for an experimental period of 1 year under feeding of a control diet, a high incidence (96.2%) of mammary tumors was observed. Administration of dietary 0.6% dehydroepiandrosterone (DHEA) together with DES implantation significantly decreased the incidence (35.0%) of mammary tumors. The first appearance of palpable tumors in the DHEA-fed group was 4.5 months later than that in the control group. For clarification of the mechanism of the chemopreventive action, we measured hormone levels in the serum of DHEA-fed rats. In the DHEA diet rats, the concentration of estradiol-17 beta exceeded, by approximately 6-fold, that in the control rats, while the levels of progesterone and prolactin were decreased by 30 and 45%, respectively. Interestingly, DHEA feeding prevented DES-induced hypertrophy of pituitary glands and DES-induced high level of prolactin in pituitary glands detected by immunohistochemical studies, but stimulated the development of mammary glands more than that in control rats treated with DES alone. These findings suggest that DHEA has a potent preventive activity against the promotion/progression phase of radiation-induced mammary tumorigenesis. The mechanism of chemoprevention by change of endocrinological environment is discussed.

Dose-response and duration effects of acute administrations of cocaine and GBR12909 on dopamine synthesis and transporter in the conscious monkey brain: PET studies combined with microdialysis.

The dose-response and duration effects of acute administration of the dopamine transporter (DAT) blocker cocaine and GBR12909 on dopamine synthesis and transporter availability were evaluated in the brains of conscious monkeys using high-resolution positron emission tomography (PET) in combination with microdialysis. Rate of dopamine synthesis and DAT availability were evaluated using L-[beta-11C]DOPA and [11C]beta-CFT (WIN35,428), respectively. Administration of cocaine (0.5, 2 and 5 mg/kg) resulted in dose-dependent elevation of dopamine level in the striatal extracellular fluid (ECF) at 0.5 h after injection, and returned to the baseline level within 1.5 h post-injection. At 0.5 post-injection, cocaine reduced dopamine synthesis rate and DAT availability in a dose-dependent manner. The reduction of DAT availability by cocaine (2 mg/kg) returned to baseline level at 3 h post-injection and thereafter. Interestingly, dopamine synthesis rate was significantly higher at 3 h than baseline level and returned to baseline level 5.5 h post-injection. When GBR12909 (0.5, 2 and 5 mg/kg) was administered 0.5 h before tracer injection, dopamine synthesis rate and DAT availability were significantly decreased in a dose-dependent manner. These reductions induced by GBR12909 (2 mg/kg) lasted at least until 5.5 h post-injection. GBR12909 induced dose-dependent elevation of dopamine level in ECF, and the elevation lasted up to 7 h. The present results indicated that cocaine and GBR12909 affect dopamine synthesis rate and DAT availability in the striatum with difference time courses as measured by PET in the conscious monkey brains.