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The liverwort Marchantia polymorpha has become one of the model organisms, since it has less genetic redundancy, sexual and asexual modes of reproduction and a range of genomic and molecular genetic resources. Cryopreservation of fertile spermatozoa eliminates time, space and labor for growing and maintaining male plants in reproductive phase, and also provides an optional way to backup lines. Here we report a protocol to cryopreserve spermatozoa of M. polymorpha in liquid nitrogen. A cryoprotective solution containing sucrose, glycerol and egg yolk and controlled cooling and warming processes led to successful recovery of motile M. polymorpha spermatozoa after the cryogenic process. The survival rate and average motility of spermatozoa after cryopreservation were maintained at 71 and 54% of those before cryopreservation, respectively. Cryopreserved spermatozoa were capable of fertilization to form normal spores. The technique presented here confers more versatility to experiments using M. polymorpha and could be applied to preservation of plant spermatozoa in general.

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase–derived reactive oxygen species (ROS) in PDGF-induced HSC proliferation. The human HSC line, LI-90 cells, murine primary-cultured HSCs, and PDGF-BB were used in this study. We examined the mechanism of PDGF-BB-induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF-BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF-BB–induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF-BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen-activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase–derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF-BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK. (HEPATOLOGY 2005;41:1272–1281.)

OBJECTIVES: Interleukin-10 (IL-10) has been implicated in immune deficiency in patients with cancer. The relationship of this cytokine as measured in serum to anti-tumor immunity and prognosis was investigated in unresectable hepatocellular carcinoma. METHODS: This study consisted of 74 consecutive patients with unresectable hepatocellular carcinoma (median age, 65 years). Forty-four healthy age-matched subjects and 32 patients with cirrhosis but no carcinoma served as controls. Patients with hepatocellular carcinoma were divided into those with serum IL-10 concentrations above (high group, n=39) or below (low group, n=35) 10pg/ml. RESULTS: Age, gender, Child-Pugh grade, and tumor stage distributions were similar in high and low groups. The patients of high group showed lower in lymphokine-activated killer (LAK) and natural killer (NK) activities than those of low group (P

Bone marrow (BM) stem cells have been shown to differentiate into liver cells. It remains difficult to sort and culture BM stem cells, and the gene expression of liver-specific proteins in these cells has not been fully investigated. We used a negative selective magnetic cell separation system to obtain stem cell-enriched BM cells. The cells obtained were cultured with hepatocytes or with hepatocyte growth factor (HGF), and the differentiation of BM cells into cells expressing liver-specific genes, hepatocyte nuclear factor (HNF) 1alpha, cytokeratin (CK) 8, alpha-fetoprotein (AFP), and albumin was investigated by the reverse transcription-polymerase chain reaction. We also investigated the gene expressions of Notch receptor-1 (Notch-1) and its ligand Jagged-1 in BM cell differentiation. Sorted BM cells showed positive for Sca-1 (Ataxin-1) by immunofluorescence staining. Fluorescence activated cell sorter analysis showed that 32.6% of sorted BM cells had a high level of expression of the hematopoietic stem cell marker CD90 (Thy-1). When cultured with hepatocytes, these cells expressed the liver-specific genes HNF1alpha and CK8 on culture day 3, AFP and albumin on culture day 7. When cultured with HGF (20ng/ml), the cells expressed HNF1alpha on day 3 and CK8 on day 7. Gene expressions of Notch-1 and Jagged-1 were detected in cultured BM cells on day 3. These results suggest that the negative selective magnetic cell separation system is useful for the rapid preparation of stem cell-enriched BM cells, and that the Notch signaling pathway plays a role in BM cell differentiation into a hepatocyte lineage in vitro.

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. However, regional expression and clinical significance of the ASGPR in acute hepatic damage is presently unknown. Our aim was to clarify the clinical significance of the regional expression of ASGPR in human livers with acute hepatitis (AH) and fulminant hepatic failure (FHF). Eighteen volunteers, 42 patients with AH and 10 with FHF were studied using a newly developed, conventional 99mTc-GSA SPECT analysis. Using Cantlie's line as a guide, ASGPR expression was analyzed separately in the right and left hepatic lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased in accordance with the severity of acute hepatic damage. In the FHF group, the reduction in LUR and LUD values in the right lobes was more significant than in the left lobes. The LUR and LUD values for the whole liver correlated well with hepatic functional reserve and total bilirubin levels. The right LUR and LUD values in particular correlated well with these parameters. A time-course observation of 13 patients with either AH or FHF revealed that the expression of ASGPRs in the right lobe recovered faster than in the left. We first evaluated the regional expression of AGSPRs by 99mTc-GSA SPECT analysis in both AH and FHF patients, which is a clinically useful and reliable indicator for assessing the severity of regional hepatic damage and evaluating regional liver regeneration.

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.

The asialoglycoprotein receptor (ASGPR) is abundantly expressed on the sinusoidal surfaces of hepatocytes. We aimed to clarify the clinical significance of the regional distribution of ASGPRs in the human liver, especially in chronic viral hepatitis. Eighteen volunteers, 34 patients with chronic hepatitis, and 33 patients with cirrhosis (11/Child-Pugh A, 11/Child-Pugh B, 11/Child-Pugh C) were studied using a newly developed, conventional technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin (99mTc-GSA), single photon emission computed tomography (SPECT) method. Using Cantlie's line as a guide, ASGPR dynamics were analyzed separately in the right and left lobes, as well as in the whole liver, using novel indices (the liver uptake ratio [LUR] and the liver uptake density [LUD], which reflect the amount and density of ASGPRs in the liver, respectively). Mean LUR and LUD values for the whole liver and the right and left lobes decreased with increasing progression of chronic viral hepatitis. The LUR for the whole liver correlated well with parameters measuring the hepatic functional reserve and the platelet count. The right LUR correlated particularly well with conventional liver function tests, and comparison of the right LUD with histologic findings showed that it was a good indicator of periportal and/or bridging necrosis and fibrosis. In conclusion, our 99mTc-GSA SPECT method was clinically useful in evaluating regional hepatic function and the progression of chronic viral hepatitis using dynamic changes in ASGPRs. (Hepatology 2003;38:1401-1409.)

A systematic method for determination of 6 kinds of pesticides (thiabendazole, benomyl, imazalil, diphenyl, o-phenylphenol and 2, 4-D) in citrus fruits was developed. The pesticides were extracted from the citrus fruits with acetone and the extract was concentrated. After addition of ethyl acetate to the concentrated solution, 2, 4-D and the basic pesticides (thiabendazole, benomyl and imazalil) were partitioned into an alkaline aqueous layer (pH 9) and then an acid layer (0.1N HCl), respectively.The basic pesticides were re-extracted from the acid layer with ethyl acetate at pH 10, and the extract was subjected to high performance liquid chromatography (HPLC) with either UV detection or fluorescence detection. The ethyl acetate layer was concentrated for the determination of diphenyl and o-phenylphenol, and subjected to HPLC with fluorescence detection. 2, 4-D was re-extracted from the alkaline aqueous layer with ethyl ether and methylated, and the resulting derivative was determined by gas chromatography-mass spectrometry with selected ion monitoring after clean-up.The recoveries of 6 pesticides added to citrus fruits at 0.2-1.0μg/g were in the range of 80.5-97.3%. The detection limits were 0.02-0.2μg/g.

A systematic method for determination of 42 kinds of pesticides (organophosphorus, organonitrogen and pyrethroid pesticide) in citrus fruits was developed. The pesticides were extracted from the citrus fruits with acetone, and the extract was concentrated. After addition of ethyl acetate to the concentrated solution, both acid and basic compounds were removed. The ethyl acetate layer was then subjected to capillary GC with NPD detection after clean-up with a Sep-pak Florisil cartridge.The recoveries of 42 pesticides added to lemon at 0.010-1.00μg/g were in the range of 59.2-100.0%. The detection limits were 0.01-0.2μg/g.

To determine differential tissue antigens in the same section immunocytochemically using the electron microscope, the neurohypophysis was examined following the application of a freeze-drying tissue preparation and staining with the protein A-colloidal gold-antibody complex method (Hisano S, Adachi T, Daikoku S: J Histochem Cytochem 32:705, 1984). At the light microscopic level, colocalized immunostaining for methionine-enkephalin (ENK) and oxytocin (OXT) was found in the rat neurohypophysis under different physiological states. Small pieces of the neurohypophysial tissue were frozen and dried. The dried tissue was fixed with paraformaldehyde vapor and embedded. The ultrathin sections were stained with the antibody for ENK coupled with protein A-small colloidal gold, and antibody for OXT or vasopressin (VP) conjugated with protein A-large colloidal gold. The ultrastructures of the nerve terminals were well preserved and showed many membrane-limited secretory granules. It was possible to identify both OXT- and VP-containing nerve terminals as their secretory granules were differentially labeled with protein A-colloidal gold anti-OXT or anti-VP complex, respectively. The secretory granules, which were labeled with large gold particles for OXT, also carry small gold particles. It is evident that ENK coexists with OXT in the same granules.

Rathke's pouch of 12.5-day-old fetal rats was cultured alone or with various fetal organs. After 10 days of culture with the anterior or posterior wall of the basal diencephalon, the size of the pouch was about eight times larger than that in single culture or in combined culture with the telencephalon or liver anlage. There was no chronological difference in the appearance of immunoreactive ACTH and prolactin cells between single and combined cultures. These results suggest that fetal diencephalon promotes the growth of Rathke's pouch but has no potency to promote differentiation of the pituitary cells.

The ontogenesis of immunoreactive (ir) ACTH cells and ir α-MSH cells in rat hypothalamus was studied in vivo and in vitro. Ir ACTH cells first appeared in the neuroepithelial cell layer lining the floor of the third ventricle on Day 13.5 of gestation, whereas ir α-MSH first appeared in the cytoplasm of several ir ACTH cells in the basal part of the arcuate nucleus of the hypothalamus on Day 19.5. When the medial-basal hypothalamus of 12.5-day embryos was cultured alone, a few ir ACTH cells were found after culture for 10 days, but not 3 days, and no ir α-MSH cells were observed in the cultures. When the hypothalamus was cultured with Rathke's pouch (intact or without the intermediate lobe anlage), ir ACTH cells appeared within 3 days. In these cultures on Days 6 and 10, long beaded fibers were seen projecting from cells in the neuronal tissue, and some cells showed immunolabeling for α-MSH. When the hypothalamus was cocultured with oral epithelium instead of Rathke's pouch, the appearance of neuronal ir ACTH cells was like that in cultures of hypothalamus alone. These in vitro findings suggest that stimulus from the anterior lobe anlage of the pituitary is necessary for normal development of ir ACTH α - MSH cells in the hypothalamus.

Postembedding immunocytochemical labeling was performed on sections of rat neurohypophysis prepared by either freeze-drying, vapor fixation and Spurr resin embedding, or conventional aqueous fixation and Spurr resin embedding. Arginine vasopressin (AVP) and oxytocin (OXT) were immunolabeled with protein A-gold-anti-AVP and protein A-gold-anti-OXT complexes, respectively. The freeze-drying procedure (FD) resulted in excellent preservation of ultrastructure and greater antigenicity than the conventional procedure (Con). More gold particles were seen over secretory granules in FD sections than in Con sections. In addition, in FD sections, the gold label was restricted to secretory granules while in Con sections, both the granules and the extragranular axoplasm exhibited label. The two antigens in FD sections could be labeled simultaneously with protein A-small gold particle-anti-OXT complex and protein A-large gold particles-anti-AVP complex. In this way the two antigens were seen to be present in secretory granules within different axon terminals. Thus FD preparations should be useful for demonstrating the presence of multiple antigens in the same granules of nerve terminals.

Gas chromatographic methods with 63Ni electron capture detection (GC-ECD) or mass spectrometry (selected ion monitoring, SIM) was investigated for the determination of tributyltin compounds in fish. Tributyltin compounds in a homogenized flesh sample of fish were extracted as tributyltin chloride (TBTC) with a hydrochloric acid-methanol mixture and transferred to a hexane solution. The extract was charged to an alumina column and eluted stepwise with ethyl acetate followed by hexane-ethanol (4 : 1, v/v) mixture. TBTC eluted with the latter solvent was transferred to a hexane layer by liquid-liquid extraction, and the hexane layer was concentrated by a rotary evaporator. The concentrate was subjected to gas chromatographic separation on a 10% KOCL-Sn column. The calibration curves were linear in the range of 0.1-2 μg/ml with GC-ECD and in the range of 0.05-1 μg/ml with SIM, respectively. The detection limit of TBTC was 0.005 μg/g in sample by the SIM method. Eleven samples of fish were analyzed by the GC-ECD and SIM methods, and the determined values by both the methods were in good agreement.

Although S. Ishii (1977, “Proc. First Int. Symp. Avian Endocrinol.,” Calcutta, Jan. 1977, 50–53) as well as P. Licht and A. R. Midgley, Jr. (1976a, Biol. Reprod., 15, 195–205; reported that specific FSH receptors were not detectable in the gonad of the bullfrog, in this study we found that the testis of the frog, Xenopus laevis, contains specific receptors for FSH at a high concentration. The specific binding of radioiodinated rat FSH to a particulate fraction of homogenate of the Xenopus testis showed the maximum at 37° and pH 7.4. The binding was competitively and quantitatively inhibited by mammalian FSH. Rat LH showed slight competition only at extremely high concentrations. The apparent equilibrium constant of dissociation of the specific binding obtained by a saturation experiment was 0.50 nM. The 95% confidence interval of this value was 0.38 to 0.72 nM. The capacity of the binding was 18.5 fmol/mg of testicular tissue. These properties of the Xenopus FSH receptors are similar to those of mammals and birds, except that Xenopus FSH receptors showed a several-fold higher binding level at 0° than mammalian or reptilian receptors. It is noteworthy that rat thyrotropin showed a significant competitive binding potency, although it was not as high as the potency of mammalian FSH. The radioautographic study revealed that the binding of rat FSH was restricted mostly to the interstitial tissue. This suggests that specific receptors for FSH locate mainly in interstitial cells of the testis in Xenopus laevis.

The application of freeze-substitution (FS) and freeze-drying (FD) techniques and the protein A-gold-antibody complex immunocytochemical methods are described. The two tissue-preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue-preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A-gold-antibody complexes are easily prepared and label the antigens clearly. If the protein A-coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50-nm gold particles, and immunoreactive methionine-enkephalin, labeled with 15-nm gold particles, in the axonal terminals of the FD-prepared rat neurohypophysis. This study demonstrates the value of the use of gold-antibody complexes for immunocytochemical labeling on FS- or FD-treated tissues.

1,2,4-Trichlorobenzene (TCB) labeled with C-14 was given perorally to rats at a dosage of 50 mg/kg for excretion and distribution studies. About 66% and 17% of the oral dose was excreted in the urine and feces, respectively, within 7 days. Trapped radioactivity in the expired air amounted to 2.1% of the dose, but production of labeled carbon dioxide was negligible. Tissue residues were evenly distributed throughout the organs and tissues examined, except for the adipose tissue which consistently had a little higher concentration. The urinary, fecal and expiratory metabolites were identified. Free 2,4,5- and 2,3,5-trichlorophenol (TCP) and their conjugates were mainly detected in the urine. 5- or 6-Sulfhydryl, methylthio, methylsulfoxide and methylsulfone derivatives of TCB were also detected as minor metabolites. Dichlorobenzenes and unchanged TCB were confirmed in the expired air. Reductive dechlroination seems to be catalysed by intestinal microflora enzymes.

The specific binding site of follicle stimulating hormone (FSH) in homogenates of the testes of birds was characterized with respect to its affinity, capacity, and specificity. The usefulness of this system for the radioreceptor assay of lower vertebrate FSH was also studied. The apparent equilibrium constant of dissociation was obtained for the specific binding of rat FSH to homogenates of testis of the Japanese quail. It was 4.1 × 10 −9 M . The binding capacity was 6 fmol/mg of tissue. The former and latter parameters obtained with the fowl testis homogenate were 1.5 × 10 −9 M and 11 fmol/mg of tissue, respectively. These values are in good agreement with those previously reported in testis homogenates of white-crowned sparrows, rats, and cattle. The binding was competitively inhibited at various rates by mammalian gonadotropin preparations. The rates of competition were fully explained by the concentration of FSH contained in the preparations. The binding was also inhibited by adenohypophyseal extracts of the Japanese quail and a chromatographic fraction of bullfrog adenohypophysis but not by a hypophyseal extract of salmon. The FSH activity of acetone-dried adenohypophysis of the Japanese quail estimated from the competition curve was 0.14 × NIAMD-rat FSH-RP-1, and that of the bullfrog adenohypophyseal fraction was 3.0 × FSH-RP-1. The precision index (λ) of the assay was 0.09 ± 0.04 (mean ± SD).

Nonresonant lepton-pair production in pion-nucleon collisions is analyzed in terms of a two-component model. Results are compared with experimental data.

This paper proposes a process/device optimization technique which is well suited for determining optimum process conditions for MOSFETs. After linking process simulator SUPREM II and device simulator FEDAS, process conditions which realize desirable device performance can be obtained by introducing the optimization procedure. The Fletcher-Powell method, one of the most efficient optimization techniques now available, is applied to the present simulator. The validity of the algorithm is successfully demonstrated by the determination of channel doping conditions, such as implanted boron dose and annealing temperature. The present simulator is expected to be an important tool for device design.

The development of immunoreactive (ir) somatostatin-containing nerve terminals in the rat median eminence (ME) has been examined electron-microscopically. Nerve fibers containing ir particles scattered throughout the axoplasm are first seen in the external layer of the ME on day 18.5 of gestation, and, on day 21.5 appear to terminate on the basement membrane of the perivascular space of the portal vessels. After birth, the fiber terminals contain several membrane-limited granules, which are labeled with ir PAP particles. Ultrathin, Epon-embedded sections of ME, treated by the protein A gold-labeling method for somatostatin, demonstrate positively labeled granules in the nerve fibers in the postnatal ME, but in the prenatal tissue, no specific gold-labeling is found. These findings show that, in the external layer of the ME, somatostatin storing occurs in the granules in the axonal terminals after birth.

Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.

Pituitary responsiveness to LHRH and anti-LHRH serum was investigated in fetal rats aged 18.5–22.5 days. Synthetic LHRH injection in utero into fetuses brought about a remarkable depletion of pituitary-LH with a corresponding increase of serum-LH on day 18.5. On the contrary, anti-LHRH serum administration to day-20.5 fetuses caused a significant augmentation of pituitary-LH 1 day later. These data indicate that LH-gonadotrophs respond to LHRH even on day 18.5, and that endogenous LHRH begins to affect LH-gonadotrophs on day 20.5.

A specific binding site for follicle-stimulating hormone (FSH) was demonstrated in the turtle ovary. The binding of 125 I-labeled rat FSH to ovarian homogenate of the turtle ( Geoclemys reevesii ) was maximum at about 37° and had a pH optimum at 7.4. Analysis by Scatchard plot for the binding of rat FSH revealed that the apparent equilibrium constant of dissociation was 10.6 n M and that the number of binding sites was 471 fmol/mg of tissue. These properties of the turtle ovarian FSH receptor are similar to those of mammalian and avian gonadal FSH receptors reported previously, although the affinity is lower in the turtle. The binding of rat FSH to the turtle ovary was competitively inhibited by gonadotropin preparations obtained from hypophyses of the rat, sheep, domestic fowl, bullfrog, and tuna fish. The slope of the inhibition curve was common among all of these preparations. Relative binding potencies determined from the distances between the inhibition curves were 0.25, 0.43, 0.33, and 0.000061 × NIH-FSH-S10 in NIAMD rat LH-RP-1, chromatographic fractions obtained from fowl and bullfrog adenohypophyses, and a glycoprotein fraction of tuna hypophyses, respectively.

The value of the freeze-substitution (FS) method for preparing tissues for electron microscopic immunohistochemistry was studied by comparing anterior pituitary cells prepared by this method and by a conventional method. Ultrathin sections of tissues embedded in Epon were subjected to immunostaining. The antigens adrenocorticotropin (ACTH) and prolactin (PRL) in a single ultrathin section were demonstrated by a simple double-labeling technique using a protein A-colloidal gold-antibody (pAG-Ab) complex. The preservation of cellular ultrastructure was superior in preparations obtained by FS. Gold-labeling was seen over secretory granules, and in ACTH cells also over the cytoplasmic matrix. The labeling was more intense in preparations obtained by FS, judging from the numbers of gold particles. In the double-labeling procedure, in which the pA-small colloidal gold-anti-PRL complex and pA-large colloidal gold-anti-ACTH complex were applied sequentially to sections, no cross-labeling with small and large gold particles was observed. It is concluded that if the antisera are sufficiently specific, the use of FS and the pAG-Ab complex is very effective in peptide immunohistochemistry. However, in the double-labeling procedure it is essential that the Fc-binding sites of pAG are saturated by the use of excess amounts of antibodies.

The localization of neurons containing immunoreactive thyrotropin-releasing hormone (TRH) was examined in the hypothalamus of intact, propylthiouracil (PTU)-treated, and colchicine-treated adult rats. In intact animals, immunoreactive TRH neurons were occasionally found in the paraventricular and dorsomedial nuclei, and in the anterior and lateral hypothalamic areas. In PTU-treated animals, the cellular appearance of the hypothalamus with the exception of the paraventricular nucleus was almost similar to that of intact animals. In the paraventricular nucleus, only the cells localized in the periventricular and medial parvocellular subdivisions significantly increased in number and became hypertrophic in comparison with intact animals. The distribution of immunoreactive fibers in the hypothalamus was almost equal among the 3 animal groups with the exception of that in the median eminence, in which the fibers were most densely concentrated in intact animals, and most sparse in PTU-treated rats. The fibers projecting into the median eminence were distinguished into the periventricular and lateral pathways, which are derived from the neurons in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus, respectively. Thus, among immunoreactive TRH neurons in the hypothalamus, only those in the periventricular and medial parvocellular subdivisions of the paraventricular nucleus may be involved in the hypothalamic-hypophysial-thyroid axis.

Absorption, distribution, metabolism and excretion were studied in rats following a single oral administration of N-cyclohexyl-2-benzothiazyl sulfenamide (CBS) at a dose of 250 mg/kg. About 65% and 24% of the dose were excreted into urine and feces, respectively, for 3 days after administration of labeled CBS (cyclohexyl-14C) Biliary excretion amounted to about 5 % of the dose for 3 days. While about 92% of the dose was recovered in urine and feces at a ratio of 1: 1 within 3 days when 14C-2CBS was given.No specific organ-affinity was observed in distribution study. Cyclohexylamine and 2-mer-captobenzothiazole were identified as urinary metabolites.

The inhibitory effects of different coal tar dyes, most of which are used for food additives at present time, on pancreatic lipase were examined in vitro by the titration method using a pH stat.Xanthene dyes except acid red showed the significant inhibition of lipase activity in order of eosine, erythrosine, phloxine and rose bengale. The inhibition was gradually enhanced during preincubation of dye with enzyme or substrate, almost independent of pH in preincubation. On the other hand, other dyes tested did not inhibit the enzyme activity at neutral pH, but most of them showed remarkable inhibition at pH 2.The inhibitory effect of rose bengale was considerably enhanced by exposure of preincubation mixture under a fluorescent lamp, suggesting a photo-inactivation of the enzyme by halogen atom in the excited molecule of dye.The inhibition was also influenced by the co-exsistence of other substances such as protein, sodium chloride and sucrose. From the kinetic study, the type of inhibition of pancreatic lipase by rose bengale was non-competitive.