Kenichi Imagawa

Production of rabbit antibody specific for amino-terminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response

Antibody specific for the amino-terminal region of cholecystokinin octapeptide (CCK-8) was generated in a highly reproducible way in New Zealand white rabbits by a novel immunization procedure which involves immunization with CCK-8 peptide conjugate coupled with keyhole limpet hemocyanin (KLH) and inhibiting cross-reacting antibody formation by treatment of the animals with a potent tolerogenic conjugate of beta-alanyl-tetragastrin and a copolymer of D-glutamic acid and D-lysine (D-GL). The antisera thus produced specifically react with an amino-terminal region of CCK-8 but not with the non-sulfate form of CCK-8, nor with the carboxy-terminal region which shares a cross-reactive determinant among gastrin and cholecystokinin-related peptides (caerulein, CCK-4, CCK-8, CCK-33 and CCK-39). The antisera produced by this method allowed us to measure specifically CCK in extracts from tissue such as duodenum containing gastrin and CCK at comparable levels.

A retinoic acid responsive gene, MK, produces a secreted protein with heparin binding activity

MK is a gene whose expression increases transiently during retinoic acid-induced differentiation of embryonal carcinoma cells. MK polypeptide was secreted by differentiating HM-1 embryonal carcinoma cells and by L-cells transfected with an MK cDNA under the control of the beta-actin promoter and Rous sarcoma virus enhancer. MK polypeptide was found to have heparin binding activity. Conditioned medium of the transfected L-cells promoted growth of PC-12 pheochromocytoma cells. These findings support the view that MK polypeptide is a secreted factor involved in regulation of growth and differentiation.

The location of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence

SummaryThe location of the perikarya of LH-RH neurons in the rat hypothalamus and their pathways to the median eminence were studied by immunohistochemistry and radioimmunoassay after placing stereotaxic electrolytic lesions in several parts of the hypothalamus. The principal location of the cell somata was found to be in the ventral part of the medial preoptic area; their pathways were classified into a main baso-lateral pathway and an accessory descending pathway branching off from the former. The main pathway was found to cross in the vicinity of the corresponding neuronal perikarya. The central median eminence and the dorsal and ventral walls of the tubero-infundibular sulcus of the caudal part of the median eminence are innervated mainly by the baso-lateral pathway. On the other hand, the rostral and most caudal portions of the median eminence are innervated principally by the descending pathway and have a subsidiary dual innervation. The projection of LH-RH neurons to the OVLT is believed to originate from perikarya adjacent to this circumventricular organ.

Delayed gastric emptying by Helicobacter pylori lipopolysaccharide in conscious rats

The present study was carried out to investigatethe possibility that lipopolysaccharide deprived fromHelicobacter pylori may alter gastric motility. Toaddress the question, we examined the effect of H. pylori lipopolysaccharide on gastricemptying in conscious rats. Gastric emptying wasevaluated by the phenol red method. Time-course anddose-related effects of intraperitoneal administrationof H. pylori lipopolysaccharide were investigated.Intraperitoneal injection of H. pylorilipopolysaccharide significantly suppressed gastricemptying of a liquid meal in a dose-dependent manner.The inhibitory action of H. pylori lipopolysaccharide wasobserved 2, 4, 8, or 12 hr after the injection. Theseresults suggest for the first time that H. pylorilipopolysaccharide may suppress gastric emptying in along-lasting fashion. It is also suggested that H. pylorimay influence gastric function through its cell wallstructure named lipopolysaccharide.

Synergistic antiviral effect of a combination of mouse interferon-α and interferon-γ on mouse hepatitis virus

Although interferon (IFN)‐α and IFN‐γ have been reported to exhibit a synergistic antiviral effect through the different signaling pathways in vitro, their therapeutic efficacy is not well defined in vivo. The current study was carried out to investigate the combined antiviral effect in a model of mouse hepatitis virus Type 2 (MHV‐2) infection, in which fulminant hepatitis is developed. MHV‐2 was injected intraperitoneally into 4‐week‐old ICR mice, IFN or the vehicle was administered intramuscularly for 5 days, and the antiviral effect was evaluated based on survival periods, liver histology, serum alanine transaminase (ALT) levels, and MHV‐2 virus titers in the liver tissues. The animals in the group treated with a combination of IFN‐α and IFN‐γ survived for longer periods than the groups treated with IFN‐α alone and IFN‐γ alone (IFN‐α 103 (IU/mouse)/‐γ 103 vs. IFN‐α 103, P < 0.005; IFN‐α 103/‐γ 103 vs. IFN‐γ 103, P < 0.001). This is consistent with the lower levels of hepatocellular necrosis and serum ALT and the decreased titers of MHV‐2 virus in the liver tissues (48 hr, P < 0.001; 72 hr, P < 0.001). These findings indicate that a combination of IFN‐α and IFN‐γ exhibits a synergistic antiviral effect on MHV‐2 infection. The biology of MHV‐2 is quite different from that of human hepatitis viruses; however, these results suggest the beneficial combined therapy of IFN‐α and IFN‐γ for the treatment of human viral hepatitis. J. Med. Virol. 69:188–194, 2003.

Molecular-Based Haplotype Analysis of the β 2-Adrenergic Receptor Gene (ADRB2) in Japanese Asthmatic and Non-Asthmatic Subjects

BACKGROUND The β2-adrenergic receptor gene (ADRB2) is a target molecule of β2-agonists. Single nucleotide polymorphisms (SNPs) in the ADRB2 are related to the effectiveness of β2-agonists. However, there are some discrepancies in the results of pharmacogenetic studies of ADRB2 among different ethnic groups. The aims of this study were to determine the ADRB2 haplotypes and diplotypes in Japanese asthmatic and non-asthmatic subjects and to examine their relation to asthma and to compare these results with previous studies done in other ethnic groups. METHODS Complete sequences for 3 kb promoter and 1.2 kb structural regions of ADRB2 were analyzed in 48 Japanese asthmatics and 100 controls, and haplotypes and diplotypes of SNPs were analyzed. RESULTS Fifteen SNPs including a novel one in -839 were observed. Allele frequencies for all SNPs were similar between asthmatics and controls. We also identified 42 haplotypes and 54 diplotypes of ADRB2 in a Japanese population. The frequencies were similar between the two groups. They were classified into 17 and 23 types, respectively, according to Drysdales haplotype-organization system, and a significant ethnic difference was observed between the Japanese and Caucasian populations. CONCLUSIONS The frequencies of SNPs and ADBR2 haplotypes in Japanese are different from those in Caucasians and African Americans. These divergences might imply the need for independent pharmacogenetic studies for ADBR2 in each ethnic group.

Postnatal ontogeny of catecholamine and somatostatin neuron systems in the median eminence of the rat as revealed by a colocalization technique

The ontogenetic development of catecholamine (CA) and somatostatin containing nerve terminals in the rostral, central and caudal median eminence (ME) of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. Somatostatin terminals were detected earlier in development than CA terminals and had already appeared in the lateral part of the external layer of the central ME by the 1st postnatal day. CA nerve terminals were first observed in the same region of the ME on the 7th postnatal day. At about this stage both types of terminals seemed to show early signs of a correlation in their distribution which became progressively closer as maturation proceeded. Their distribution reached a stable condition in density and pattern on the 21st postnatal day when the majority were found in the lateral part of the external layer of the central ME, in the basal part of the brain just dorsal to the tuberoinfundibular sulcus and in the upper and lower labia of the tuberoinfundibular stalk. They subsequently matured to the adult pattern of stabilized distribution and anatomical relationship. It was also established that on the 28th postnatal day the somatostatin immunoreactive terminals reached a greater concentration than at any other stage of development including that of the adult.

Detection of catecholamine and luteinizing hormone-releasing hormone (LH-RH) containing nerve endings in the median eminence and the organon vasculosum laminae terminalis by fluorescence histochemistry and immunohistochemistry on the same microscopic sections ☆

Distribution of catecholamine (CA) and LH-RH nerve endings in the median eminence (ME) and the organon vasculosum laminae terminalis (OVLT) of the rat was investigated by application of fluorescence histochemistry and immunohistochemistry on the same sections of the tissue. In the ME, those two kinds of endings coexisted in the lateral portion of the middle part of ME, and in the wall of tuberoinfundibular sulcus, where they might be considered to have functional correlation. In the OVLT they were also distributed in fairly near distance, but they were not so closely associated as observed in the ME.

Glucose as regulator of glucose transport activity and glucose-transporter mRNA in hamster β-cell line

To investigate the role of glucose in regulating glucose transporters in pancreatic β-cells, we studied the hamster clonal β-cell line HIT-T15, which retains responsiveness to glucose. Northern blot analysis demonstrates that GLUT2 and GLUT1 mRNA are abundant in HIT cells. After a 24-h culture with various concentrations of glucose (0–22.2 mM [0–400 mg/dl]), the GLUT2 mRNA level in HIT cells increased by 40% at 22.2 mM (400 mg/dl) glucose compared with 11.1 mM (200 mg/dl) without a change in mRNA stability. It also decreased proportionally to the reduction of glucose concentration. Glucose deprivation resulted in a decrease of GLUT2 mRNA to an almost undetectable level, with a marked increase in the degradation rate of mRNA. In contrast, the GLUT1 mRNA was not affected by glucose. We show that glucose uptake is highest in HIT cells incubated at 2.8–5.5 mM (50–99 mg/dl) glucose for 24 h, and that levels in cells cultured at 0 mM (0 mg/dl) and 22.2 mM (400 mg/dl) glucose decrease to ∼ 20% of the maximum level. This decrease is consistent with the effects of glucose on glucose-stimulated insulin secretion in HIT cells. Our results indicate that glucose is involved in regulating GLUT2 mRNA and glucose uptake activity and that the glucose responsiveness of the insulin secretion correlates with the glucose-induced change in glucose uptake activity in HIT cells.

Correlative ontogenetic development of catecholamine- and LHRH-containing nerve endings in the median eminence of the rat

SummaryThe ontogenetic development of catecholamine (CA)-and LHRH-containing nerve endings in the median eminence of the rat was investigated by combining fluorescence histochemistry and immunohistochemistry in the same tissue section. LHRH-terminals appeared earlier than CA-terminals and were already detectable in the lateral part of the external layer of the central ME on the first day after birth. CA-nerve endings were first seen in a corresponding region of the ME on the seventh postnatal day. At this stage both types of terminals showed the earliest manifestation of a correlative pattern of their distribution. Subsequently the development of both types of nerve endings proceeded rapidly, and at 14 days their distribution pattern corresponded to that in adult animals. The authors conclude that at this stage the CA-neurons play a constant and significant role in the release of LHRH into the portal capillaries. The correlation between both types of nerve endings and the ontogenetic development of the capillary plexuses of the hypophysial portal system is discussed.