Hideki Ohtani

Molecular cloning of rat cerebellin-like protein cDNA which encodes a novel membrane-associated glycoprotein

Rat cerebellin and an apparent metabolite des-Ser1-cerebellin have been shown to be located in cerebellar Purkinje cells and the dorsal cochlear nucleus. A cDNA clone was isolated by screening a rat brain cDNA library using an oligonucleotide corresponding to rat cerebellins. The clone encodes 224 amino acid residues of a glycoprotein a part of whose sequence is virtually the same as that of cerebellins and which contains one putative transmembrane spanning domain. Neither an N-terminal cleavable signal peptide nor dibasic pair specific endopeptidase directly precedes or follows the cerebellin-like sequence. Expression of the cerebellin-like protein gene is developmentally regulated in rat cerebellum and shows tissue specific alternative splicing in the brain, adrenal gland and spleen of the rat. The results of Southern blot indicated the gene to possibly be a member of some gene family. Rat cerebellin-like protein, possibly a precursor of cerebellins, is a novel membrane-associated glycoprotein expressed in nervous, adrenal and immune systems and appears to synaptic functions in the central nervous system.

On-column enantiomerization of 3-hydroxybenzodiazepines during chiral liquid chromatography with optical rotation detection

Abstract Oxazepam and lorazepam, 3-hydroxybenzodiazepines that undergo reversible enantiomerization at ambient temperature, were eluted on a chiral HPLC column at various temperatures and various flow-rates. The profiles obtained by a UV detector were various, such as two peaks, a plateau sandwiched between two peaks, and one fused peak. The profiles obtained by an optical rotation detector were also various, such as two sharp peaks with opposite chirality (−, +), two peaks (−, +) with a great tailing or leading, two small peaks (−, +) next to each other. These peculiar profiles could be explained by on-column enantiomerization. Unexpectedly, the elution order of the (−)- and (+)-enantiomers was reversed for oxazepam and lorazepam. The simultaneous detection by UV and chiroptical measurements allowed us to plot HPLC profiles of the individual enantiomers.

Levels of interleukin‐6, CRP and α2 macroglobulin in cerebrospinal fluid (CSF) and serum as indicator of blood‐ CSF barrier damage

We measured the levels of interleukin‐6 (IL‐6), albumin, C‐reactive protein (CRP) and α2 macroglobulin (α2M), all of which have different spectrums of molecular weight, in the cerebrospinal fluid (CSF) and serum in 121 patients to evaluate damage to the blood‐cerebrospinal fluid barrier (BCB) in meningitis. There was an extraordinary high level of IL‐6 in the CSF when patients had bacterial or viral meningitis, but the level returned to a normal range within a week in almost all of these cases. There were no significant differences in CSF albumin levels among the different disease groups. The CRP level in CSF is considered to correlate with the serum level, and CSF CRP was higher in bacterial meningitis than in viral meningitis, however, CRP in CSF was increased in some of the infectious diseases without meningitis. The α2M in CSF, which tends to be at extraordinarily high levels when there is damage to the BCB, correlated highly with CSF cell counts. CSF IL‐6 seemed to be a useful indicator to identify the acute active phase of meningitis. CRP and α2M in CSF are considered to be useful to differentiate bacterial meningitis, bacterial infection without meningitis and viral meningitis. Extraordinarily high levels of α2M, which has a high molecular weight, in CSF is indicative of BCB damage.

On-column isomerization of sugars during high-performance liquid chromatography: analysis of the elution profile.

Four monosaccharides (glucose, galactose, mannose and fructose) and one disaccharide (maltose) were subjected to high-performance liquid chromatography with UV or refractive index detection. Various profiles such as broad, tailed and splitted peaks were produced, depending on column temperature and eluent flow-rate because these saccharides underwent isomerization. In contrast, alpha-methylglucoside, a non-converting derivative, always produced a sharp peak. By analyzing these profiles kinetic constants of the isomerization were obtained and compared with the literature data.

Kinetic Analysis of Molecular Interconversion of Immunosuppressant FK506 by High-Performance Liquid Chromatography

High-performance liquid chromatography of FK506, a macrolide immunosuppressant, was performed on a reversed-phase column. The peak was broad with the column kept at room temperature, which was accounted for by slow interconversion between the two forms of FK506. With the use of a heated column, a sharp peak was observed because of the rapid interconversion at high temperature. When the column was cooled to 0°C, two sharp peaks were observed because essentially no interconversion occurred at 0°C during elution. Analysis of the chromatograms obtained at various eluant flow rates indicated that the conversion of the two forms follows first-order kinetics, and the apparent activation energies for the conversions were calculated. The interconvertibility between two molecular forms may be related to the immunosuppressive activity.

Interconversion of cyclosporin molecular form inducing peak broadening, tailing and splitting during reversed-phase liquid chromatography

SummaryReversed-phase liquid chromatography of cyclosporin A, a peptide immunosuppressant, at various temperatures produced remarkably different chromatograms. At 60°C one sharp peak was obtained, at 23°C this became a single broad peak and between 15° and 0°C this became one high sharp peak followed by a tailing or low plateau. Remarkably different chromatograms were produced also by varying the mobile phase flow-rate. The effects of both temperature and flow-rate on the chromatogram could be accounted for by interconversion between two forms of the cyclosporin molecule. Kinetic analysis showed that one form was converted by first-order kinetics with a half-life of 2.0 min at 20°C and that the apparent activation energy for the conversion was about 18 kcal/mol. The other two immunosuppressants, cyclosporin C and D, were also shown to undergo interconversion. This kinetic analysis of the interconversion should be helpful in clarifying the relationship between molecular structure and activity of the immunosuppressants.

Alpha-2-Macroglobulin Deficiency in Patients with Advanced Prostate Cancer

Four out of approximately 36,000 specimens, examined by immunoelectrophoresis, showed no precipitin line of alpha-2-macroglobulin (alpha 2M) with anti-whole human serum and antiserum to alpha 2M. Serum alpha 2M levels were below 10 mg/100 ml with single radial immunodiffusion. These cases were diagnosed as having prostate cancer based on prostate biopsy. After estrogen or anti-androgen therapy, the initially severe deficiency of alpha 2M changed toward normal limits. Phytohemagglutinin (PHA)-induced lymphocytes from patients with severe alpha 2M deficiency revealed normal response. Sera from patients with alpha 2M deficiency and healthy subjects (serum alpha 2M concentration of 8-208 mg/100 ml) did not suppress lymphocyte response to PHA, while purified alpha 2M from fetal cord and healthy sera, and serum from a patient with advanced urinary cancer (serum alpha 2M concentration above 360 mg/100 ml) to PHA-induced lymphocytes demonstrated a dose-dependent suppression. Thus, these results indicate that alpha 2M in elevated concentration results in decreased PHA-induced lymphocyte response and suggest that alpha 2M may have, principally suppressive, immunoregulatory properties in vivo.

Levels of acute inflammatory biomarkers in advanced prostate cancer patients with α2-macroglobulin deficiency

C-reactive protein (CRP), serum amyloid A (SAA), interleukin-6 (IL-6), α1-antitrypsin (α1AT), α1-acid glycoprotein (α1AG) and ceruloplasmin (CP) are acute inflammatory biomarkers that increase in various conditions including infection, inflammation, malignancy and tissue disturbance. In contrast, α2-macroglobulin (α2M) is involved in inflammation through its function as a carrier protein of IL-6. We had previously reported on advanced prostate cancer (PCa) patients with multiple distant bone metastases in whom serum α2M levels were markedly decreased (α2M deficiency). However, the relationship between serum levels of α2M and acute inflammatory biomarkers in PCa patients with or without α2M deficiency has not been demonstrated. In the present study, we examined serum levels of CRP, SAA, IL-6, α1AT, α1AG and CP in PCa patients with or without α2M deficiency to establish clinical significance and changes in these biomarkers during PCa disease progression. We found that upon addition of recombinant IL-6 (rIL-6) to serum from PCa patients with α2M deficiency, since a function of α2M is to bind and stabilize IL-6, the α2M-IL-6 complex and free endogenous IL-6 were not detectable. Serum levels of the α2M-independent markers, α1AT, α1AG and CP, in all PCa patients regardless of α2M deficiency were significantly higher than in healthy controls, but those of the α2M-dependent molecules, CRP, SAA and IL-6, were not increased in PCa patients with α2M deficiency. Therefore, quantitation of both α2M-dependent (CRP, SAA and IL-6) and α2M-independent (α1AT, α1AG and CP) acute inflammatory biomarkers in advanced PCa patients may be an auxiliary indicator, together with prostate-specific antigen (PSA), to monitor PCa disease progression.

Radioreceptor assay of clonazepam and diazepam in blood for therapeutic drug monitoring.

Rat cerebral cortex was used to prepare a suspension of benzodiazepine receptors. The suspension was mixed with an extract of specimen and [3H]flunitrazepam. The mixture was filtered through a membrane, and radioactivity on the membrane was measured. Clonazepam concentrations in patient plasma specimens determined by radioreceptor assay and gas-liquid chromatography agreed well. However, diazepam equivalent concentrations determined by radioreceptor assay were close to the sum of diazepam and desmethyldiazepam concentrations determined by high-performance liquid chromatography, as desmethyldiazepam had binding activity for the receptor. This receptor assay method is accurate, simple, requires less than 0.5 ml of plasma, and is therefore suitable for analyzing numerous samples in a short time.

Clinicopathological characteristics of androgen-dependent advanced prostate cancer patients with α2-macroglobulin deficiency

α2-Macroglobulin (α2M) is thought to be involved in cancer metastasis and inflammatory reaction through its functions as a proteinase inhibitor and carrier protein for interleukin-6 (IL-6). We previously reported that advanced prostate cancer (PCa) patients with multiple distant bone metastases had markedly decreased serum α2M levels (<20 mg/dl) and no detection of α2M by immunoelectrophoresis (defined as α2M deficiency). We also showed a relationship between serum α2M levels and acute inflammatory biomarkers in PCa patients with or without α2M deficiency. In this study, we analyzed in detail the clinicopathological characteristics and pathogenesis of α2M deficiency in androgen-dependent advanced PCa patients. In this study, 15 PCa patients were diagnosed at the Kitasato University Hospital. α2M levels were determined by laser-nephelometry and immunoelectrophoresis, and PSA levels were determined by enzyme immunoassay. IL-6 levels were measured by a specific luminescence sandwich-type enzyme-linked immunosorbent assay, and CRP levels were determined by latex nephelometry. Immunohistochemical staining for PSA in PCa specimens was also performed. The binding assay for purified α2M and PSA was analyzed by western blotting. α2M deficiency was specific for advanced PCa patients with multiple distant bone metastases. PSA was markedly detected in sera and prostate specimens of advanced PCa patients with α2M deficiency, and there was a negative correlation between serum α2M and PSA levels during the course of clinical treatment. Acute inflammatory biomarkers such as IL-6 and CRP were within reference range in α2M-deficient patients. The binding assay showed that PSA easily bound to α2M, which was detected as an approximately 800-kDa complex by western blotting. Further, genetic analysis of a α2M-deficient patient showed no mutations in the α2M gene. These results suggested that α2M deficiency develops from catabolism of α2M in androgen-dependent advanced PCa patients, and serum α2M level may be an indicator of PCa disease progression in addition to PSA level.