Pharmacokinetics and pharmacodynamics of propofol: changes in patients with frontal brain tumours.

BACKGROUND: Models of propofol pharmacokinetics and pharmacodynamics developed in patients without brain pathology are widely used for target-controlled infusion (TCI) during brain tumour excision operations. The goal of this study was to determine if the presence of a frontal brain tumour influences propofol pharmacokinetics and pharmacodynamics and existing PK-PD model performance. METHODS: Twenty patients with a frontal brain tumour and 20 control patients received a propofol infusion to achieve an induction-emergence-induction anaesthetic sequence. Propofol plasma concentration was measured every 4 min and at each transition of the conscious state. Bispectral index (BIS) values were continuously recorded. We used non-linear mixed-effects modelling to analyse the effects of the presence of a brain tumour on the pharmacokinetics and pharmacodynamics of propofol. Subsequently we calculated the predictive performance of Marsh, Schnider, and Eleveld models in terms of median prediction error (MdPE) and median absolute prediction error (MdAPE). RESULTS: Patients with brain tumours showed 40% higher propofol clearance than control patients. Performance of the Schnider model (MdPEpk -20.0%, MdAPEpk 23.4%) and Eleveld volunteer model (MdPEpk -8.58%, MdAPEpk 21.6%) were good. The Marsh model performed less well (MdPEpk -14.3%, MdAPEpk 41.4%), as did the Eleveld patient model (MdPEpk -30.8%, MdAPEpk 32.1%). CONCLUSIONS: Brain tumours might alter the pharmacokinetics of propofol. Caution should be exerted when using propofol TCI in patients with frontal brain tumours due to higher clearance. TRIAL REGISTRY NUMBER: NCT01060631.

Prolonged occupational activity and 6-year changes in postural sway in elderly women.

OBJECTIVES: Prolonged occupational work such as farm work has been reported to adversely affect mobility in elderly women. The purpose of this study was to investigate possible relationships between prolonged occupational work and 6-year changes in postural sway in elderly women. METHODS: Subjects were 392 women aged ≥ 69 years who participated in a 6-year follow-up examination of the Muramatsu Cohort Study. Handgrip strength and postural sway, measured as gravity-center velocity (cm/s), were evaluated at baseline and 6-year follow-up. Interviews were conducted to determine the time spent on moderate occupational activity (3-5 metabolic equivalents) such as farm work. Activity levels were defined as: 1, no-activity; 2, 'short' (>0, ≤ 17.75 h/wk); and 3, 'long' ( ≥ 17.75 h/wk). RESULTS: At baseline, mean values for age, handgrip strength, and postural sway were 73.3 years (SD 3.7), 20.3 kg (SD 4.1), and 2.0 cm/s (SD 0.8), respectively, and 32.5% of participants engaged in occupational activity. The change in postural sway was significantly greater in the long-activity group (median, 35.0 h/wk) than the no-activity group (0.56 vs. 0.27 cm/s, P=0.021). CONCLUSIONS: Prolonged occupational work may be detrimental to the control of body balance. Accordingly, elderly individuals are not recommended to engage in prolonged occupational activity.

Evaluation of the predictive performance of a pharmacokinetic model for propofol in Japanese macaques (Macaca fuscata fuscata).

Propofol is a short-acting intravenous anesthetic used for induction/maintenance anesthesia. The objective of this study was to assess a population pharmacokinetic (PPK) model for Japanese macaques during a step-down infusion of propofol. Five male Japanese macaques were immobilized with ketamine (10 mg/kg) and atropine (0.02 mg/kg). A bolus dose of propofol (5 mg/kg) was administrated intravenously (360 mg/kg/h) followed by step-down infusion at 40 mg/kg/h for 10 min, 20 mg/kg/h for 10 min, and then 15 mg/kg/h for 100 min. Venous blood samples were repeatedly collected following the administration. The plasma concentration of propofol (Cp) was measured by high-speed LC-FL. PPK analyses were performed using NONMEM VII. Median absolute prediction error and median prediction error (MDPE), the indices of prediction inaccuracy and bias, respectively, were calculated, and PE - individual MDPE vs. time was depicted to show the variability of prediction errors. In addition, we developed another population pharmacokinetic model using previous and current datasets. The previous PK model achieved stable prediction of propofol Cp throughout the study period, although it underestimates Cp. The step-down infusion regimen described in this study would be feasible in macaques during noninvasive procedures.

AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1.

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers.

Vitamin D sufficiency is associated with low incidence of limb and vertebral fractures in community-dwelling elderly Japanese women: the Muramatsu Study.

UNLABELLED: Data on the association between vitamin D status and osteoporotic fracture in Asians are sparse. We conducted a 6-year cohort study of 773 community-dwelling elderly Japanese women and found that serum 25-hydroxyvitamin D (25(OH)D) ≥ 71 nmol/L was associated with a reduced risk of osteoporotic limb and vertebral fractures. INTRODUCTION: Data on the association between vitamin D status and osteoporotic fracture in Asians are sparse. This study aimed to clarify the association between vitamin D and other markers of nutritional status with the incidence of fracture in elderly Japanese women. METHODS: We conducted a cohort study with a 6-year follow-up of 773 community-dwelling women aged 69 years and older. The 6-year follow-up ended in 2009. We assessed serum 25-hydroxyvitamin D, undercarboxylated osteocalcin (an index of vitamin K status), and calcium intake. The primary outcome was incident limb and vertebral fractures. Covariates were forearm bone mineral density (BMD), age, body mass index, osteoporosis treatment, and physical activity. RESULTS: The mean serum 25(OH)D concentration was 60.0 nmol/L. Thirty-seven limb fractures and 14 vertebral fractures occurred in 4,392 person-years. Lower forearm BMD was significantly associated with increased incident fracture (P = 0.0242). The adjusted hazard ratios (HR) of fracture for the first quartile (<47.7 nmol/L) and the third quartile (59.2-70.9 nmol/L) of serum 25(OH)D, compared to the fourth quartile (≥71.0 nmol/L), were 2.82 (95% confidence interval (CI), 1.09-7.34) and 2.82 (95%CI, 1.09-7.27), respectively. The pooled adjusted HR was 0.42 (95%CI, 0.18-0.99) when the incidence in the fourth quartile (≥71.0 nmol/L) was compared to the other three quartiles combined (<71.0 nmol/L). Vitamin K status and calcium intake were not associated with incident fracture. CONCLUSIONS: Sufficient vitamin D status, i.e., serum 25(OH)D ≥ 71 nmol/L, is associated with low limb and vertebral fracture risk in community-dwelling elderly women.

C-reactive protein predicts incident fracture in community-dwelling elderly Japanese women: the Muramatsu study.

SUMMARY: In a 6-year cohort study of 751 community-dwelling elderly Japanese women, we found that C-reactive protein (CRP) is a significant predictor of osteoporotic fracture in elderly Asian women, who have significantly lower CRP levels than Caucasians. Mechanisms explaining such an association should be further studied. INTRODUCTION: While CRP, a systemic inflammation marker, is thought to be associated with osteoporosis, evidence supporting this claim has been limited. We aimed to assess the association between CRP levels and incident osteoporotic fracture in elderly women. METHODS: We conducted a cohort study with a follow-up period of 6 years. The study included 751 Japanese women aged 69 years or older. We measured serum high-sensitivity CRP (hs-CRP) levels as a major predictor. Covariates included age, body mass index, forearm bone mineral density, calcium intake, serum 25-hydroxyvitamin D, postural sway, osteoporosis medication, and physical activity. The primary outcome was incident limb and vertebral fractures. The Cox proportional hazards model was used to calculate the hazard ratio (HR) of fracture. RESULTS: Median hs-CRP values in study participants were 0.16 mg/L in the lowest tertile, 0.36 mg/L in the medium tertile, and 1.14 mg/L in the highest tertile. The hs-CRP values in these women were substantially lower than in their Caucasian counterparts. Limb or vertebral fractures occurred in 50 subjects during 4,250 person-years. Low CRP levels were associated with low incidence of limb or vertebral fractures (P for trend = 0.035). The adjusted HRs of fracture for the medium and highest quartiles of hs-CRP levels, compared to the lowest quartile, were 2.22 (95% CI, 1.02-4.84) and 2.40 (95% CI, 1.10-5.24), respectively. CONCLUSIONS: CRP is a significant predictor of osteoporotic fracture in elderly Asian women who have substantially lower CRP levels than Caucasians. Mechanisms explaining such an association should be further studied.

Propofol infusions using a human target controlled infusion (TCI) pump in chimpanzees (Pan troglodytes).

Chimpanzees are genetically and physiologically similar to humans. Several pharmacokinetic models of propofol are available and target controlled infusion (TCI) of propofol is established in humans, but not in chimpanzees. The purpose of this study was to investigate if human pharmacokinetic models can accurately predict propofol plasma concentration (Cp) in chimpanzees and if it is feasible to perform TCI in chimpanzees. Ten chimpanzees were anaesthetized for regular veterinary examinations. Propofol was used as an induction or maintenance agent. Blood samples were collected from a catheter in a cephalic vein at 3-7 time points between 1 and 100 min following the propofol bolus and/or infusion in five chimpanzees, or TCI in six chimpanzees. Cp was measured using high-performance liquid chromatography. The Marsh, Schnider and Eleveld human pharmacokinetic models were used to predict Cp for each case and we examined the predictive performances of these models using the Varvel criteria Median PE and Median APE. Median PE and Median APE for Marsh, Schnider and Eleveld models were within or close to the acceptable range. A human TCI pump was successfully maintained propofol Cp during general anesthesia in six chimpanzees. Human propofol pharmacokinetic models and TCI pumps can be applied in chimpanzees.

Tetraparesis resembling acute transverse myelitis in a captive chimpanzee (Pan troglodytes): long-term care and recovery.

BACKGROUND: A 24-year-old, male chimpanzee (Pan troglodytes) developed acute tetraparesis. Magnetic resonance imaging showed a diffuse T2-weighted hyperintensive lesion, indicating inflammation at the C1-2 level. All infective, autoimmune, and vascular investigations were unremarkable. RESULTS AND CONCLUSIONS: The chimpanzee's condition most resembled acute transverse myelitis (ATM) in humans. The chimpanzee was in severe incapacitated neurological condition with bedridden status and required 24-hour attention for 2 months followed by special care for over a year. Initially, corticosteroid therapy was performed, and his neurological symptoms improved to some extent; however, the general condition of the chimpanzee deteriorated in the first 6 months after onset. Pressure ulcers had developed at various areas on the animal's body, as the bedridden status was protracted. Supportive therapy was continued, and the general condition, appetite, mobility, and pressure ulcers have slowly but synergistically recovered over the course of 2 years.

Minimally cultured bone marrow mesenchymal stem cells ameliorate fibrotic lung injury.

Clinical use of bone marrow mesenchymal stem cells (BMMSCs) holds great promise for regenerative medicine in intractable lung diseases, such as lung fibrosis or acute respiratory distress syndrome. However, a severe obstacle to the clinical application of BMMSC transplantation is the time-consuming, laborious processes required for cell culture. In order to evaluate the clinical applicability of BMMSC transplantation, we tested whether engraftment of minimally cultured BMMSCs ameliorates progressive fibrotic lung injury. Differences between murine BMMSCs cultured for 2 h (2-h adherent BMMSCs) and conventionally (9-day) cultured BMMSCs were examined in vitro. The effects of grafting either type of BMMSCs on fibrotic lung injury were then assessed by transfer experiments in a murine bleomycin-induced lung fibrosis model, in which donor cells were administered 3 days after challenge. 2-h adherent BMMSCs were smaller, less granular, possessed higher proliferative capacity and expressed higher levels of several stem cell markers and chemokine receptors than 9-day cultured BMMSCs, but lower type I procollagen, alpha-smooth muscle actin, tumour necrosis factor-beta and oncogenic transcription factor c-Myc, suggesting that they may be advantageous for cell-based therapy compared with 9-day cultured BMMSCs. Grafting 2-h adherent BMMSCs ameliorated inflammatory and fibrotic lung disorders, and reduced mortality equally well or better than 9-day cultured BMMSCs. Minimally cultured BMMSCs can substitute for conventionally cultured BMMSCs and will be a promising cell source for the treatment of acute fibrotic lung injury.

T-cell/Histiocyte-rich Large B-cell Lymphoma of the Larynx in a Juvenile Japanese Macaque (Macaca fuscata).

An 11-month-old female Japanese macaque (Macaca fuscata), born in captivity in a research institute, suddenly died without clinical signs. Necropsy examination revealed a nodular mass protruding from the left ventral aspect of the larynx, compressing the epiglottis anteriorly. Histopathologically, the laryngeal mass was comprised of medium- to large-sized atypical cells. Immunohistochemically, these were positive for CD20 and partially positive for CD79α. Among the atypical cells were CD3+ T cells and CD68+ histiocytes. Based on the findings, this case was diagnosed as T-cell/histiocyte-rich large B-cell lymphoma. Epstein-Barr virus (EBV)-encoded small RNAs were frequently detected in the atypical cells by in-situ hybridization, which was consistent with the finding that the macaque was seropositive for EBV antigen. This is the first report showing the potential association of simian lymphocryptovirus, the simian homologue of EBV, with lymphoma in a juvenile non-human primate.

Superconductivity and magnetic fluctuations developing in the vicinity of strong first-order magnetic transition in CrAs.

We report single crystal preparation, resistivity, and nuclear quadrupole resonance (NQR) measurements for new pressure-induced superconductor CrAs. In the first part, we present the difference between crystals made by different thermal sequences and methods, and show the sample dependence of superconductivity in CrAs. In the latter part, we show NQR data focusing the microscopic electronic state at the phase boundary between the helimagnetic and the paramagnetic phases. They suggest strongly that a quantum critical point is absent on the pressure-temperature phase diagram of CrAs, because of the strong first-order character of the magnetic transition; however, the spin fluctuations are observed in the paramagnetic phase. The close relationship between the spin fluctuations and superconductivity can be seen even in the vicinity of the first-order magnetic transition in CrAs.

Propofol-fentanyl interaction in Beagles - Apnea, response to mechanical ventilation, endotracheal tube, and tetanic stimulation.

The objective of this study was to characterize the propofol-fentanyl interaction in Beagles for four pharmacodynamic endpoints: apnea, response to mechanical ventilation, endotracheal tube, and tetanic stimulation. After anesthesia was induced with varying combinations of propofol and fentanyl, the pharmacodynamic endpoints were assessed in intubated dogs (n=6) using the cross-over design. Effective concentrations of propofol plasma concentration (Cp) and fentanyl Cp were assessed using additive, reduced Greco, Minto, and hierarchical interaction models. The interaction was best described as synergistic by the hierarchical model. A 1ng/mL fentanyl Cp reduced the effective propofol Cp to half or less of that without fentanyl for all endpoints. An additional increment of fentanyl Cp to 5ng/mL or higher hardly reduced effective propofol Cp for all endpoints except response to tetanic stimulation. Additionally, the effective propofol Cp in 50% dogs for response to tetanic stimulation (15% increase of heart rate) was lower than that for the other endpoints at fentanyl Cp >7ng/mL. Peripheral oxygen saturation decreased below 90% after extubation in five treatments in which fentanyl Cps were ≥5ng/mL. Propofol and fentanyl interacted synergistically. To avoid patient-ventilator dyssynchrony and hypoxemia after extubation, fentanyl Cp at 1-5ng/mL may be appropriate in intubated dogs. When a dog responds to mechanical ventilation or endotracheal tube at a high fentanyl Cp >5ng/mL under propofol anesthesia even if the dog tolerate to tetanic stimulation, it may be necessary to increase propofol Cp to eliminate the responses because an additional fentanyl may be little impact.

Molecular cloning of a candidate tumor suppressor gene, DLC1, from chromosome 3p21.3.

The short arm of chromosome 3 is thought to contain multiple tumor suppressor genes, because one copy of this chromosomal arm frequently is missing in carcinomas that have arisen in a variety of tissues. We have isolated a novel gene encoding a 1755-amino acid polypeptide, through large-scale sequencing of genomic DNA at 3p21.3. Mutational analysis of this gene by reverse transcription-PCR revealed the lack of functional transcripts and an increase of nonfunctional RNA transcripts in a significant proportion (33%) of cancer cell lines and primary cancers (4 of 14 esophageal cancer cell lines, 2 of 2 renal cancer cell lines, 11 of 30 primary non-small cell lung cancers, and 3 of 10 primary squamous cell carcinomas of the esophagus). However, no alterations of the gene itself were detected in any of the cancers examined. Introduction of the cDNA significantly suppressed the growth of four different cancer cell lines, two of which produced no normal transcript on their own. No such effect occurred when antisense cDNA, cDNA corresponding to an aberrant transcript, or the vector DNA alone were transfected. These data suggest that aberrant transcription of this gene, designated DLC1 (deleted in lung cancer 1), may be involved in carcinogenesis of the lung, esophagus, and kidney.

Isolation and characterization of a novel human gene, DRCTNNB1A, the expression of which is down-regulated by beta-catenin.

Beta-catenin plays significant roles in cell-to-cell adhesion and the Wnt/Wg signal transduction pathway. Accumulation of this protein in the cytoplasm and nucleus as a result of mutations of the adenomatous polyposis coli tumor suppressor gene or of the beta-catenin gene itself is often seen in a wide variety of tumors including carcinomas of the colon, liver, uterus, and brain. Interaction of accumulated beta-catenin with Tcf/Lef transcription factors is known to deregulate expression of some downstream genes, but the precise mechanisms whereby beta-catenin contributes to carcinogenesis remain to be disclosed. Here we report isolation of a novel murine gene, Drctnnb1a (down-regulated by Ctnnb1, a), the expression of which was experimentally down-regulated in response to the activated form of beta-catenin. To investigate a possible role of DRCTNNB1A in cancers, we also isolated the human homologue, DRCTNNB1A, the deduced product of which was 91% identical to the murine protein. The transcript was expressed in all human tissues examined, and we assigned the genomic location of DRCTNNB1A to chromosomal band 7p15.3 by in situ hybridization. Expression of DRCTNNB1A in SW480 colon cancer cells was significantly increased in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of the beta-catenin-binding domain of the adenomatous polyposis coli gene into the cells. Furthermore, we documented reduced expression of DRCTNNB1A in 12 of 15 primary colorectal cancers examined, compared with corresponding adjacent noncancerous mucosae. Our results implied that DRCTNNB1A is one of the genes involved in the beta-catenin-Tcf/Lef signaling pathway, and that reduced expression of DRCTNNB1A may have some role in colorectal carcinogenesis.

Significant differences in the frequency of transcriptional units, types and numbers of repetitive elements, GC content, and the number of CpG islands between a 1010-kb G-band genomic segment on chromosome 9q31.3 and a 1200-kb R-band genomic segment on chromosome 3p21.3.

We determined the nucleotide sequence of the entire 1,010,525-bp insert contained in CEPH YAC clone 867e8. This human genomic segment was derived from chromosome 9q31.3 and corresponds to a G-band region. We compared this segment, in terms of structure, with a previously characterized 1,201,033-bp sequence in CEPH YAC936c1 that had come from a portion of human chromosome 3p21.3 corresponding to an R-band region. The two segments were significantly different with respect to the frequency of transcriptional units, the types and numbers of repetitive elements present, their GC content, and the number of CpG islands. Alu elements, GC content, and CpG islands all showed positive correlations with the abundance of exons, but the distribution of LINE1s did not. These observations might reflect an influence of the first three of these features on the functions or expression of genes in the respective regions. In addition to a novel gene (F36) lying at the centromeric end of the 9q segment, we found a cluster of placenta-specific genes within a small section (about 400 kb) on the telomeric side of YAC867e8. This cluster consisted of four apparently unrelated ESTs and two genes, pregnancy-associated plasma protein-A (PAPP-A) and a novel gene (tentatively named EST-YD1). Our characterization of the two chromosomal regions provided evidence that genes are not evenly distributed throughout the human genome, and that gene richness is correlated with the GC content and with the frequency of either Alu elements or CpG islands.

Characterization of a 1200-kb genomic segment of chromosome 3p22-p21.3.

We previously determined the nucleotide sequence and characterized the 685-kb proximal half of CEPH YAC936c1, which corresponds to a portion of human chromosome 3p21.3. In the study reported here, we characterized the remaining 515-kb of this YAC clone corresponding to the telomeric half of its human insert. The newly sequenced region contained a total of ten genes including six reported previously: phospholipase C delta 1 (PLCD1), human activin receptor type IIB (hActR-IIB), organic cation transporter-like 1 (OCTL1), organic cation transporter-like 2 (OCTL2), oxidative stress response 1 (OSR1), and human xylulokinase-like protein (XYLB). The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes. One (designated ENGL) of the novel sequences was found to encode an amino-acid sequence homologous to the family of DNA/RNA endonucleases, especially endonuclease G. The other gene F56 revealed no significant homology to any known genes. These results disclosed complete physical and transcriptional maps of the 1200-kb region of 3p present in YAC 936c1.

Multiple receptor-like protein tyrosine phosphatases in the form of chondroitin sulfate proteoglycan.

The possibility that some of the brain proteoglycans are receptor-like protein tyrosine phosphatases (PTPases) was investigated. Membrane-bound proteoglycan fractions were prepared from the postnuclear membrane fraction of 8-day-old rat brain by DEAE ion-exchange chromatography and CsCl density gradient centrifugation. The isolated proteoglycan fractions showed high PTPase specific activities together with the typical PTPase characteristics. Renaturation experiments indicated that chondroitin sulfate proteoglycans with 380- and 170-kDa core proteins carried the PTPase activity. The proteoglycan with 380-kDa core protein was identified as RPTP beta/zeta bearing HNK-1 carbohydrate.

Clarithromycin increases the release of heat shock protein B from Helicobacter pylori.

BACKGROUND: Clarithromycin (CAM) may have certain indirect effects on Helicobacter pylori (H. pylori) other than its inhibitory activity on bacterial growth, as indicated in other infections with Gram-negative micro-organisms. In the present study, we examined the effects of lower concentrations of CAM on the release of heat shock protein B (HspB), one of the major antigenic proteins from H. pylori cells, as well as the changes in humoral immune response and histological degree of antral gastritis in patients who received eradication therapy with CAM. METHODS: The H. pylori strain 26695 and three CAM-resistant clinical isolates were cultured in broth with and without CAM (2-500 ng/mL). Expression of H. pylori proteins was examined by two-dimensional (2D)-electrophoresis followed by N-terminal amino acid sequencing. Changes in host immune response and histological degree of antral gastritis were monitored in patients with peptic ulcer disease who received H. pylori eradication therapy. RESULTS: 2D electrophoresis showed 26 spots in extracellularly released proteins with different profiles from those in cytoplasmic proteins. The release of HspB increased after incubation with CAM (30-500 ng/mL) in all three H. pylori clinical isolates tested. Patients with failed H. pylori eradication after triple therapy with CAM, but not those with failed eradication after dual therapy without CAM, showed an increase in serum IgG1 and IgG2 antibodies against HspB along with a decrease in the degree of neutrophil and H. pylori colonization density in tissue sections. CONCLUSIONS: CAM may induce a humoral immune response against H. pylori and a decrease in gastric mucosal inflammation through up-regulation of the release of HspB from the bacteria in infected patients.

Circadian change of VIP mRNA in the rat suprachiasmatic nucleus following p-chlorophenylalanine (PCPA) treatment in constant darkness.

Neuronal activity of the suprachiasmatic nucleus (SCN) is known to be regulated by two major extrinsic factors conveyed by three anatomically distinct pathways to the SCN: photic stimulus by the direct retinohypothalamic tract (RHT) and the indirect geniculohypothalamic tract (GHT), and information from the brainstem by ascending forebrain serotonergic (5-hydroxytryptamine: 5-HT) tract. It has been shown that VIP mRNA level in neurons of the SCN is altered by external light, but remains stable in constant darkness. In the present study, by using the in situ hybridization technique combined with computer-assisted image analysis, we examined VIP mRNA expression in the SCN of rats in which the two major factors were eliminated, i.e. photic stimulus by exposing animals in total darkness and 5-HT transmission by three-day successive administration of p-chlorophenyl-alanine methylester (an inhibitor of tryptophan hydroxylase, 200 mg/kg, daily). In saline-treated controls, VIP mRNA levels remained almost constant throughout the day. In contrast, in PCPA-treated rats, a significant rhythm of VIP mRNA was observed with a peak at CT 4 and a trough at CT 20. These observations suggest that the removal of photic and 5-HT influence induces VIP mRNA rhythm in the SCN, indicating that VIP mRNA is controlled not only by photic information but also by the circadian clock.

Characterization and developmental regulation of proteoglycan-type protein tyrosine phosphatase zeta/RPTPbeta isoforms.

Protein tyrosine phosphatase zeta (PTPzeta/RPTPbeta) is a receptor-like protein tyrosine phosphatase specifically expressed in the brain. Alternative splicing produces three isoforms of this molecule: PTPzeta-A, the full-length form of PTPzeta; PTPzeta-B, the short form of PTPzeta; and PTPzeta-S, an extracellular variant. Here, we identified all these isoforms, including PTPzeta-B, as chondroitin sulfate proteoglycans, and characterized their carbohydrate modification and expression profiles in the rat brain. The level of PTPzeta-A expression was maintained during the prenatal period and decreased rapidly after birth. PTPzeta-S was expressed in a similar manner, although the postnatal decrease was gradual. In contrast, relatively constant amounts of PTPzeta-B were observed from embryonic day 13 (E13) through adulthood. PTPzeta-A and -S were constantly expressed only as proteoglycans during development, but a substantial amount of PTPzeta-B was detected in a non-proteoglycan form at E13-15. Moreover, PTPzeta-B did not contain LeX, HNK-1 carbohydrate, or keratan sulfate, although PTPzeta-A and -S were generally modified with these carbohydrates. L cells transfected with PTPzeta-A and -B cDNAs expressed these proteins as enzymatically active chondroitin sulfate proteoglycans. The PTPzeta-A and -B in L cells showed essentially similar localizations in cell cortical structures on immunofluorescence microscopy, although immature or processed forms of PTPzeta-A were accumulated additively in intracellular patchy structures. These results show that the three isoforms of PTPzeta are differentially regulated during development, and that the extracellular deleted region in PTPzeta-B is important for determination of carbohydrate modification.