Lack of pulse and surge modes and glutamatergic stimulation of luteinising hormone release in Kiss1 knockout rats.

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin-releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin-GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge-mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle-stimulating hormone surge and an oestrogen-induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild-type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.

[Antimicrobial activities of carbapenems and fourth generation cephems against clinically isolated strains].

We determined the antibacterial activities of 4 carbapenems (imipenem, panipenem, meropenem and biapenem) and 2 forth generation cephems (cefozopran and cefepime) against 850 bacterial strains (18 species) isolated during the period from January 1998 through September 2000. The 4 carbapenems showed excellent antibacterial activities against methicillin-susceptible S. aureus (MSSA), S. pneumoniae, E. faecalis, P. aeruginosa, M. (B). catarrhalis, B. fragilis and Peptostreptococcus spp. as compared with the cephems except the activity of panipenem against P. aeruginosa. Meropenem showed the highest antibacterial activity against 10 species of all Gram-negative strains determined. The antibacterial activities of 2 forth generation cephems against 6 species of Enterobacteriaceae were equal to or higher than those of carbapenems except meropenem. All drugs showed low antibacterial activities against methicillin-resistant S. aureus (MRSA).

[Antimicrobial activities of meropenem against clinically isolated strains in 1999].

We determined the antibacterial activity of meropenem (MEPM) and control drugs against clinical isolates of 310 strains of Gram-positive bacteria (14 species), 590 strains of Gram-positive bacteria (21 species), and 120 strains of anaerobic bacteria (10 species) in 1999. We compared the results thus obtained with those in 1993 and 1997. The results were as follows; 1. MEPM showed excellent antibacterial activities against most of the species tested, except for MRSA, E. faecium, E. avium, and methicillin-resistant S. epidermidis (MRSE). 2. MEPM had much higher activity than IPM and PAPM against Gram-negative bacteria including S. marcescens and P. aeruginosa, part of which have been reported to produce metallo-beta-lactamase. 3. There was little difference in the susceptibility of clinical isolates to MEPM between 1993 and 1999. Thus MEPM was shown to retain its potent and broad antibacterial activity now at the same level as before available for use in 1995.

Immunocytochemical localization of histamine in enterochromaffin-like (ECL) cells in rat oxyntic mucosa: a transmission electron microscopy study using monoclonal antibodies and conventional glutaraldehyde-based fixation.

Histamine (HA), contained in the enterochromaffin-like (ECL) cells of the gastric mucosa in animals, plays an important role in gastric acid secretion, although methods for its exact morphological localization are still lacking. We used a pre-embedding indirect immunoperoxidase approach to define the fine structural localization of HA in rat oxyntic mucosa that was fixed with a glutaraldehyde-based fixative and HA monoclonal antibodies (MAbs AHA-1 and 2). Transmission electron microscopy showed that the peroxidase endproduct not only was concentrated in the cores of cytoplasmic granules but also was distributed to a high degree in the cytoplasm peripheral to the granules of the ECL cells. These results suggest that in ECL cells HA is enzymatically synthesized in the cytoplasm, then is transported and stored in the cores of the granules before its release from the basal lamina. The present HA immunoelectron microscopic method with MAbs would be applicable more generally to the ultrastructural identification of HA-containing cells.

Reversed circadian blood pressure rhythm independently predicts endstage renal failure in non-insulin-dependent diabetes mellitus subjects.

To investigate the significance of reversed circadian blood pressure (BP) rhythm as a predictor for diabetic endstage renal failure, introduction of hemodialysis (HD) was determined as an end point in 325 noninsulin-dependent diabetes mellitus (NIDDM) subjects, in whom 24-h BPs had been monitored during their first admissions between 1988 and 1996. Circadian BP rhythm was analyzed by the COSINOR method, as previously reported. After exclusion of 68 dropout subjects, 257 were recruited for further analyses, in which 194 had normal circadian BP rhythms (N), and the remaining 63 had reversed rhythms (R). During this follow-up period, the numbers of HD-introduced subjects in N and R were 6 and 16, respectively, showing a higher prevalence in the latter (p < 0.001, chi2 test). Follow-up periods were significantly shorter in HD-introduced diabetic subjects of N and R than those in HD-free subjects of each group. In baseline characteristics, there were no differences in age, gender, or serum creatinine between HD-free and HD-introduced subjects of N or R. With regard to microvascular complications, the degree of retinopathy and nephropathy in N and R tended to be more pronounced in HD-introduced subjects than in HD-free subjects. Further, mean levels of circadian mean BP rhythms in HD-introduced subjects of N or R were similarly high, compared with those in HD-free subjects of each group, irrespective of circadian BP pattern. Unadjusted HD-free times were estimated by the Kaplan-Meier method, with a significant difference noted between N and R (p < 0.001; log-rank test). The Cox proportional-hazards model adjusted for circadian BP pattern, age, gender, blood pressure level, glycemic control, duration of diabetes, serum total protein, and serum creatinine demonstrated that circadian BP pattern, age, gender (female), blood pressure level (hypertension), and serum creatinine exhibited significant high relative risks. Thus, our data suggest that reversed circadian BP rhythm is an independent predictor of endstage renal failure in NIDDM subjects.

Rapid, sensitive and specific diagnosis of Bordetella pertussis using the polymerase chain reaction.

Use of a repetitive DNA sequence of Bordetella pertussis allowed successful detection of the organism by the polymerase chain reaction (PCR). The method was highly sensitive, being able to detect B. pertussis in specimens containing only a few cells. It was also highly specific, with no amplification of specimens containing other organisms, for example Haemophilus influenzae or Neisseria, being observed. A diagnosis could be made within 1 day. The PCR assay was also evaluated in clinical specimens. Among 47 nasopharyngeal specimens obtained from 24 patients with laboratory-confirmed pertussis, 27 were positive by PCR and 19 by culture. In particular, all three bronchial aspirates from one patient with pertussis were positive by PCR, but only one showed positive on culture. Eleven specimens from parapertussis patients and 65 specimens from patients without pertussis tested negative. It was concluded that this newly developed PCR method for the diagnosis of pertussis was more rapid and sensitive than the usual culture method. Polymerase chain reaction could have a major impact on the treatment and control of this infection and would be a useful tool for studying the pathogenesis of B. pertussis infection.

Intraintestinal capsaicin transiently reduces CGRP-like immunoreactivity in rat submucosal plexus.

Intraintestinal infusion of the sensory neurotoxin, capsaicin, transiently abolishes behavioral responses to chemical stimulation of the intestine. This desensitizing action of capsaicin may be due to an action on CGRP-containing nerve terminals, which are postulated to serve a sensory function in the enteric plexuses. To determine whether intraintestinal capsaicin treatment alters CGRP-like immunoreactivity (CGRP-li) in the enteric plexuses, we performed immunohistochemical analyses of the small intestinal submucosal and myenteric plexuses of rats at various times after intestinal infusion of capsaicin (5 mg) or its vehicle. Intestinal capsaicin treatment, but not vehicle treatment, reduced CGRP-li, but not substance-P-like immunoreactivity (SP-li), in nerve fibers of the submucosal plexus. CGRP-li was reduced in submucosal interganglionic connectives and in nerve fibers associated with submucosal blood vessels. CGRP-li of submucosal connectives was reduced by 1 h post-infusion. Reduction of CGRP-li in the submucosal fibers also was pronounced 24 h after intraintestinal capsaicin treatment. By 48 h after intestinal capsaicin infusion, CGRP-li was not distinguishable from vehicle-treated animals. There were no consistent immunohistochemical changes in CGRP-li or SP-li in the myenteric plexus at any time. These results indicate that intestinal capsaicin selectively induces transient reduction of CGRP-li in nerve fibers of the submucosal plexus. The chronology of depletion and reappearance of CGRP-li is congruent with previously reported, transient impairment of sensory function observed following intestinal capsaicin infusion.

A causal relationship between Bordetella pertussis and Bordetella parapertussis infections.

Bordetella parapertussis is isolated during the late stages of pertussis outbreaks and occasionally from patients infected with pertussis. The relationship between Bordetella pertussis and B. parapertussis was investigated in mice with monoinfections and mixed infections. Four groups of 10 2-week-old suckling mice were studied: mice born to mice vaccinated against pertussis during pregnancy and unvaccinated controls consequently did and did not receive antipertussis toxin (PT) antibody transcolostrally. The mice were infected transnasally with B. parapertussis strain 422 and 2 identical groups were infected transnasally with B. parapertussis strain 422 and B. pertussis strain 18-323. Bacterial colonization of the lungs and trachea was studied at 1, 2, and 3 weeks after challenge. No persistent colonization by B. parapertussis of the lungs or trachea of monoinfected suckling mice were observed. Persistent colonization by B. parapertussis was observed when suckling mice that received anti-PT antibody transcolostrally were infected with both species. These findings are consistent with the clinical characteristics of B. parapertussis. The results of this study demonstrate that B. pertussis infection facilitates B. parapertussis infection.

Intestinal capsaicin transiently attenuates suppression of sham feeding by oleate.

Intraintestinal infusion of oleic acid reduces food intake in rats and other mammals. The neural mechanisms that mediate this behavioral response to intestinal stimulation are incompletely appreciated. We have found that intraintestinal infusion of capsaicin reduces sham feeding. In addition, 24 h after a single intestinal capsaicin infusion, reduction of sham feeding by intestinal oleate infusion was attenuated. However, by 48 h post-capsaicin, suppression of sham feeding by oleate had returned to pre-capsaicin levels. Repeated intestinal administration of capsaicin produced less attenuation of oleate-induced suppression of sham feeding, suggesting the development of tolerance to capsaicin. Unlike systemic capsaicin, intestinal capsaicin does not impair cholecystokinin-induced reduction of feeding or the corneal chemosensory reflex. Furthermore, there are no histochemical signs of vagal sensory degeneration in the hindbrain after intraintestinal capsaicin. Our results suggest that a capsaicin-sensitive substrate in or near the intestine is responsible for mediating the reduction of sham feeding by intestinal oleate infusion.

Effects of oleic acid/propylene glycol on rat abdominal stratum corneum: lipid extraction and appearance of propylene glycol in the dermis measured by Fourier transform infrared/attenuated total reflectance (FT-IR/ATR) spectroscopy.

Fourie transform infrared/attenuated total reflection analysis demonstrated that the absorbance intensity of C = O stretching bands, which reflect the amounts of lipids in the stratum corneum, decreased with an increase in the duration of skin treatment with 0.15 M oleic acid/propylene glycol (PG) system, suggesting that the oleic acid/PG system induced the lipid extraction, which was followed by a reorganization of the stratum corneum structures. The spectral peaks which originated from the PG molecule were detected in dermal tissues after 30 min of treatment of the stratum corneum with the same system. This observation suggested that the reorganization of the lipid domains due to the lipid extraction by the oleic acid/PG system helped the PG molecules enter the dermal tissues. It was also suggested that an effective volume within the stratum corneum for solutes and/or solvents which could penetrate through the inter-, and/or intracellular routes could be altered in conjunction with the structural changes of the lipids.

[The effects of position and ventilation during thoracotomy on oxygen uptake and carbon dioxide elimination in humans].

The effects of position and ventilation were investigated in ten adult patients undergoing scheduled thoracotomy. Oxygen uptake (VO2) and carbon dioxide elimination (VCO2) in gas phase were measured continuously, while gas exchange ratio (R) and dead space ventilation ratio (VD/VT) were calculated with these results. VO2 and VCO2 during general anesthesia decreased about 60% from the normal values due to reduction of metabolism. Position and ventilation had no obvious effect on VO2 during surgery. VCO2 decreased slightly during bilateral ventilation with lateral decubitus position, because of mismatch of ventilation-perfusion distributions, and/or reduction of metabolism without surgical stress. The results suggest that carbohydrate calories are mainly metabolized under surgical stress, resulting in an R equal to 1.0. Continuous non-invasive monitoring of VO2 and VCO2 is one of the most effective parameters to evaluate the oxygen balance and systemic metabolism.

Melibiose transport system in Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.

Hypoxic ventilatory response in cats lightly anesthetized with ketamine: effects of halothane and sevoflurane in low concentrations.

The effect of low concentration sevoflurane and halothane on the ventilatory response to isocapnic hypoxia was studied in sixteen cats. The cats were divided into two groups, sevoflurane group and halothane group, of eight subjects each. As parameters of the hypoxic ventilatory response, A value [the slope of the hyperbolic curve, V(E) = V(0) + A/(Pa(O)(2)-32)] and ratio of V(50) (the minute volume obtained from the hyperbolic equation when Pa(O)(2) = 50 mmHg) to V(0) were studied. These two parameters were examined at three states, sedative state with ketamine as the control, ketamine plus 0.1 MAC inhalation anesthetic, and ketamine plus 0.5 MAC inhalation anesthetic. In the sevoflurane group, the A values were 4789 +/- 1518, 2187 +/- 1214, 1730 +/- 880 (mean +/- SE. ml.min(-1).mmHg) at the control state, 0.1 MAC and 0.5 MAC, respectively. In the halothane group, the A values were 6411 +/- 2368, 2529 +/- 842 and 2372 +/- 545, respectively. The ratios of V(50) to V(0) were 1.32 +/- 0.09, 1.22 +/- 0.09, 1.25 +/- 0.08 in the sevoflurane group, 1.47 +/- 0.18, 1.32 +/- 0.11, 1.54 +/- 0.18 in the halothane group, respectively. The A value at 0.1 MAC of the halothane group was less than the control value significantly. This proved that even low concentration halothane depressed the hypoxic ventilatory responses. The depression of hypoxic ventilatory response could cause postanesthetic hypoventilation. On the other hand, we could not find significant depression on the hypoxic ventilatory response in the sevoflurane group, but we should notice that variances of the hypoxic ventilatory response were large.

Further evaluation of the pertussis component vaccine produced by apoceruloplasmin affinity chromatography.

The heat-treated apoceruloplasmin (Apocp) is a useful protein as an affinity ligand for the purification of pertussis toxin (PT). The amounts of Apocp in the purified antigens or the pertussis component vaccine were determined. Anti-Apocp antibodies were not detected by the passive cutaneous anaphylaxis (PCA) test in rats. No anti-Apocp antibody was detected after hyperimmunization of rabbits with the vaccine. Apocp was not detected in PT and filamentous hemagglutinin (FHA) by ELISA using rabbit anti-Apocp IgG. In the experiments using 125I-labelled Apocp, 125I-Apocp was not detected in either PT or FHA which were purified by 125I-labeled Apocp-Sepharose, DEAE Sepharose, and cellulose sulfate chromatography. The contents of human DNA were also determined to be less than 10 pg per 1 mg of Apocp, by the dot-blot hybridization method using the 32P-labeled DNA probe of Alu sequence. In the tests for the presence of inapparent viruses, HBs antigen and HTLV-III antibody, no contamination was found in either the Apocp or in the vaccine. Large amounts of various viruses, which were intentionally added to the Apocp (spiking test), were completely inactivated by heating at 65 degrees C for 18 hr. Both the Apocp and the vaccine passed the general pharmacology and acute toxicity tests. From these results, the heat-treated Apocp was considered to be a suitable affinity ligand for the purification of the antigens for the pertussis component vaccine.

Characterization of mutant strains producing pertussis toxin cross reacting materials.

We have isolated 120 mutant strains producing pertussis toxin (PT) cross reacting materials (CRMs) from B. pertussis, strain Tohama, phase I by nitrosoguanidine treatment. Strains producing higher PT tend to show higher virulence in mice. No direct correlation between the virulence and other factors, such as filamentous hemagglutinin, adenylate cyclase or dermonecrotic heat labile toxin, was found. Most CRMs were less reactive to the anti-S1 monoclonal antibody, 1B7. When the PT CRMs produced by strains 69D, 74E or 79G, which were less or non-toxic, were mixed with A protomer purified from native PT, the PT activity assayed by clustering of CHO-cells increased significantly, but not when they were mixed with B oligomer. These CRMs may be composed of defective S1 and intact S2, S3, S4 and S5. Molecular sizes of PT CRMs outside and inside the cells were analysed by sucrose density gradient centrifugation. The sizes of the CRMs were in the range of 10K to 210K, but the biological activity of PT was detected at only the same molecular size, 106 K, as native PT. The majority of the CRM was released into culture medium if all five subunits were assembled; otherwise they accumulated inside the cell without completion of assembly to form the hexamer in the PT-form. One of the non-toxic mutants named 79G showed one point mutation from G to A at the 730th base from the Eco R1 site of the PT gene. Replacement of Cys-41 with Tyr-41 in S1 must have resulted from this mutation. 79G PT composed of S234 (5) was accumulated both inside and outside the cells because the mutant S1 could not form the disulfide bond in the molecule to form the hexamer with the B oligomer, and also S1 must be degraded because of its instability in the cells. Nevertheless 79 GPT showed high immunoprotectivity in mice by active or passive immunization against ic or aerosol challenge with B. pertussis, strain 18323, respectively. It may have a proper conformational structure for protective immunogenicity and could become a good candidate strain for production of a safer and effective pertussis vaccine in the future.

Molecular analysis of 5' J region of immunoglobulin heavy chain gene in human acute leukemias.

Ig heavy chain (IgH) J (JH) region organization in 83 human acute leukemias was studied. In 31 of the 35 precursor B acute lymphocytic leukemia (ALL) the rearrangements of the IgH gene involved sites between D regions 5' to DQ52 and JH. The study of the IgH gene organization in 43 T lineage ALL showed nine cases with the rearrangement at the IgJH region, one of which involved the D region 5' to DQ52. When analyzed with another restriction enzyme (BglII), three of the remaining eight showed rearrangement between DQ52 and JH. In the remaining five samples rearrangement of the IgJH region was not shown, but the restriction fragment length polymorphism (RFLP) between MspI sites in 5'DQ52 loci was observed. Thus the RFLP in 5'DQ52 locus was identified and was distinguished from the recombination between DQ52 and JH in non-B lineage leukemias.

Effects of angiotensin II on phosphatidylinositide hydrolysis in rat brain.

Angiotensin II has been coupled to the hydrolysis of phosphatidylinositide in various peripheral tissues; however, little is yet known about its association in the central nervous system. Intracellular phosphatidylinositide was prelabeled by incubation with [(3)H]myo-inositol and the rate of hydrolysis determined by monitoring the rate of formation of [(3)H]inositol monophosphate. In hypothalamus, thalamus, septum and midbrain tissues, angiotensin II reduced the formation of [(3)H]inositol monophosphate and this effect was partially reversed by the presence of an angiotensin II antagonist, sarcosine, isoleucine angiotensin II. These data suggest that angiotensin II receptor stimulation may inhibit the hydrolysis of phosphatidylinositide in the brain.