Correlation of circulating betatrophin concentrations with insulin secretion capacity, evaluated by glucagon stimulation tests.

AIM: To investigate the relationship between plasma betatrophin concentrations and insulin secretion capacity in people with Type 2 diabetes. METHODS: Glucagon stimulation tests (1 mg) were performed in 70 people with Type 2 diabetes after an overnight fast. Plasma betatrophin concentrations were measured using an enzyme-linked immunosorbent assay. Insulin secretion capacity was evaluated by measuring increments of C-peptide concentration in response to glucagon stimulation, and creatinine clearance was determined by comparing creatinine concentrations in serum and 24-h urine samples. RESULTS: Plasma betatrophin concentrations were positively correlated with duration of Type 2 diabetes (r = 0.34, P = 0.003), and negatively correlated with increments of C-peptide concentration (r = 0.37, P = 0.001) and creatinine clearance (r = 0.37, P = 0.001). The correlation with increments of C-peptide concentration remained significant after adjustment for age and duration of Type 2 diabetes (r = 0.25, P = 0.037). Multivariate analysis identified age and increments of C-peptide concentration as independent factors associated with plasma betatrophin levels. CONCLUSION: Plasma betatrophin levels inversely correlate with insulin secretion capacity, suggesting that betatrophin levels are regulated by insulin secretion capacity in humans.

Aquagrams of raw milk for oestrus detection in dairy cows.

The purpose of this research was to develop rapid and cost-effective method for oestrus detection in dairy cows by means of near infrared spectroscopy and aquaphotomics, using raw milk from individual cows. We found that aquaphotomics approach showed consistent specific water spectral pattern of milk at the oestrus periods of the investigated Holstein cows. Characteristic changes were detected especially in foremilk collected at morning milking. They were reflected in calculated aquagrams of milk spectra where distinctive spectral pattern of oestrus showed increased light absorbance of strongly hydrogen-bonded water. Results showed that monitoring of raw milk near infrared spectra provides an opportunity for analysing hormone levels indirectly, through the changes of water spectral pattern caused by complex physiological changes related to fertile periods.

Prognostic value of nuclear DNA content and expression of the ras oncogene product in lung cancer.

We evaluated the prognostic significance of nuclear DNA content by flow cytometry and ras oncogene expression in paraffin-embedded sections of tumors obtained surgically from 112 non-small cell lung cancer patients. Sixty-five (77%) of the 84 tumors had DNA aneuploid patterns that were statistically higher in adenocarcinoma than in squamous cell carcinoma. Of the 91 patients analyzed immunohistochemically using anti-ras Mr 21,000 protein (p21) monoclonal antibody rp-35, positive reactions (weak and strong) were observed in 56% of squamous cell carcinomas and 68% of adenocarcinomas. A better 5-yr survival rate was observed in the DNA diploid group (61%) than in the DNA aneuploid group (35%) (P less than 0.01). Patients with p21-negative tumors survived significantly longer (5-yr survival rate of 64%) than did those with p21-weak tumors (38%, P less than 0.05) or those with p21-strong tumors (12%, P less than 0.01). Cox's multivariate analysis showed that DNA ploidy, ras p21 expression, and the stage of the disease were significant prognostic factors for survival. However, the DNA content was not a major independent prognostic factor in adenocarcinoma. The intensity of ras p21 expression was not correlated with nuclear DNA content. These results suggest that DNA content or enhanced ras p21 expression may be different biological markers indicating the malignant potential of lung tumors.

Two independent macrophage receptors for acetylated high-density lipoprotein.

We previously demonstrated that acetylated human high-density lipoprotein (acetyl-HDL) was recognized by a scavenger receptor of rat sinusoidal liver cells (Murakami, M., Horiuchi, S., Takata, K. and Morino, Y. (1987) J. Biochem. (Tokyo) 101, 729-741). The present study describes the interaction of acetyl-HDL with rat peritoneal macrophages in vitro. Acetylation of HDL enhanced its cell-association by 2-fold and cellular degradation by > 25-fold. The cell-association of [125I]acetyl-HDL was effectively inhibited by unlabeled acetyl-HDL (> 85%), whereas the inhibition by HDL or acetylated human low-density lipoprotein (acetyl-LDL) was partial (60% and 50%, respectively). However, when both HDL and acetyl-LDL were present, the cell-association of [125I]acetyl-HDL was effectively inhibited by > 80%, a level identical or closely similar to that by acetyl-HDL. The cellular degradation of [125I]acetyl-HDL was effectively suppressed by acetyl-LDL whereas the effect of HDL was much weaker. These findings indicate that acetyl-HDL is endocytosed by both the HDL receptor and the scavenger receptor for acetyl-LDL in which the ligands bound to the latter might be subjected to lysosomal degradation.

Identification of two alpha-subunit species of GTP-binding proteins, Galpha15 and Galphaq, expressed in rat taste buds.

We cloned cDNAs for two G protein alpha-subunits belonging to the Galphaq family, each capable of activating PLCbeta, from rat tongue. One is a Galphaq in the narrow sense, and the other, termed rat Galpha15, is a rat counterpart of mouse Galpha15, sharing an amino acid sequence similarity of 94%. RT-PCR and Northern blot analysis demonstrated that rat Galpha15 and Galphaq were distinctly expressed in tongue epithelia containing taste buds. Immunostaining also showed that rat Galpha15, together with the Galphaq, was localized mainly in taste buds. These studies suggest the possibility that these two Galpha proteins function for taste signal transduction in sensory cells.

Characterization of the roles of the Saccharomyces cerevisiae RAD54 gene and a homologue of RAD54, RDH54/TID1, in mitosis and meiosis.

The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDH54/TID1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of R4D54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.

Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possible involvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases.

Expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) has been reported to be responsible for enhanced tumor growth and angiogenesis in various tumors. However, the relationships between tumor vascularity and COX-2 and iNOS expression have not been evaluated in hepatocellular carcinoma (HCC). In this study, we examined the expression of iNOS and COX-2 and microvessel density (MVD) by immunohistochemical staining in 100 tissue sections collected from HCC patients. iNOS expression was significantly higher in hepatitis C virus (HCV)-positive HCCs (P = 0.011). COX-2 expression was significantly correlated with iNOS expression (P = 0.046) and tumor MVD (P = 0.011) in HCV-positive HCCS: iNOS expression was neither correlated with MVD nor had any influence on patient survival; however, combined negative expression of iNOS and COX-2 had a significant impact on patient survival (P = 0.041 and 0.018, log-rank test for overall and recurrence-free survival rate, respectively). The present findings suggest that combined expression of iNOS and COX-2 may play an important role in prognosis of HCV-positive HCC patients and that this could be partially attributable to modulation of angiogenesis by COX-2.

Isolation of a Candida glabrata centromere and its use in construction of plasmid vectors.

A centromere has been isolated from Candida glabrata by functional selection based on the lethality of the SUP11 gene at high copy number. Nucleotide sequence analysis revealed a centromeric structure similar to that of Saccharomyces cerevisiae: the two highly conserved elements CDEI (8 bp) and CDEIII (26 bp) are separated by a 79-bp A+T-rich element, CDEII. Three centromere-bearing plasmid vectors with different selection markers have been constructed. These plasmids were highly stable in mitosis (< 1% loss rate per generation) and exist in one or two copies per cell.

Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation.

The Candida glabrata (Cg) TRP1 and HIS3 genes have been isolated by complementation of the Saccharomyces cerevisiae (Sc) trp1 and his3 mutants, respectively. Cg TRP1 encodes a polypeptide of 217 amino acids (aa), whose aa sequence is 58% identical to that of Sc TRP1. Cg HIS3 encodes a polypeptide of 210 aa, whose aa sequence is 73% identical to that of the Sc HIS3. Both Cg TRP1 and HIS3 were disrupted by sequential integrative transformation where the Sc URA3 was used as a selection marker for transformation. The resulting auxotrophic strain of his3- and trp1- was used to examine the ability of the Sc genes to complement the Cg mutations; Sc HIS3 and TRP1 complemented the Cg his3- and trp1- mutations, respectively.

Functional upper airway obstruction. Psychogenic pharyngeal constriction.

A 15-year-old boy, known to have asthma, developed acute inspiratory airway obstruction with marked stridor. Spirometry indicated extrathoracic airway obstruction and a bronchofiberoptic examination disclosed narrowing in the hypopharynx. After administration of sedatives, the stridor suddenly disappeared. Psychotherapy decreased the frequency of subsequent stridor attacks. It is suggested that psychogenic pharyngeal constriction may have caused the upper airway obstruction with respiratory distress.

The gene polymorphism of tumor necrosis factor-beta, but not that of tumor necrosis factor-alpha, is associated with the prognosis of sarcoidosis.

OBJECTIVES: Few genetic markers for the prognosis of sarcoidosis have been found. Tumor necrosis factor (TNF)-alpha has been implicated in the pathogenesis of sarcoidosis. Induced TNF-alpha or TNF-beta levels have been shown to be associated with the polymorphisms of the TNF genes. We investigated the roles of such polymorphisms in the development and prolongation of sarcoidosis. SUBJECTS AND MEASUREMENTS: One hundred ten Japanese patients with sarcoidosis and 161 control subjects were genotyped for three biallelic polymorphisms in the promoter region of TNF-alpha gene by direct sequencing of polymerase chain reaction (PCR) products. A polymorphism of the TNF-beta gene (TNFB*1/TNFB*2) was detected by NCO: I restriction fragment length polymorphism analysis of PCR products spanning intron 1 and exon 2 of the TNF-beta gene. RESULTS: None of the polymorphisms conferred susceptibility to sarcoidosis. However, our study identified the allele TNFB*1, detected by the presence of a NCO: I restriction site, as a marker of prolonged clinical course, with the resolution of sarcoidosis being defined as the disappearance of all clinical symptoms, physical signs of active lesions, abnormal chest radiograph findings, and abnormal results of pulmonary function and biochemical tests. When the probability of remission in patients homozygous for TNFB*2 was defined as 1.00, it was 0.48 (95% confidence interval, 0.26 to 0.88; p < 0.05) in patients with TNFB*1 (genotypes TNFB*1/1 and TNFB*1/2). CONCLUSIONS: The TNFB*1 allele is a marker for prolonged clinical course in patients with sarcoidosis. Our study is the first to link a cytokine gene polymorphism to the prognosis of sarcoidosis.

Plasma cells in the bronchoalveolar lavage fluid of a patient with eosinophilic pneumonia. Morphologic proof of local production of antibodies.

We describe a case of eosinophilic pneumonia in which plasma cells appeared in bronchoalveolar lavage fluid (BALF). A 49-year-old man presented with wheezing, lung infiltrates, peripheral eosinophilia, and extremely high IgE levels in serum and BALF. A differential count of BALF revealed 56.6 percent lymphocytes and 1.3 percent plasma cells. The appearance of plasma cells suggests local maturation of B cells and represents a morphologic proof of local production of immunoglobulins. The increased number of lymphocytes suggests their role in B cell differentiation via the lymphokine network.

Acute rise in serum immunoglobulin E concentration in pulmonary thromboembolism.

Events mediated by immunoglobulin E (IgE) may be related to platelet activation and aggregation, and there may be an association between IgE and pulmonary thromboembolism (PTE). Fourteen patients with PTE were studied with regard to serum concentrations of IgE, IgA, IgG, IgM, fibrinogen, and D-dimer and with regard to blood neutrophil, lymphocyte, platelet, and eosinophil counts during acute and recovery phases. The serum IgE concentration increased during the acute phase to 402 +/- 310 IU/ml and decreased afterwards in all patients. The increase in serum IgE concentration lagged a few days behind that of the serum D-dimer concentration, indicating later IgE production than thrombus formation and lysis. Infarction and pleural fluid accumulation developed in patients with a high initial serum IgE concentration. These results indicated a relationship between serum IgE concentration and the pathophysiology of PTE. Serum IgE may be an indicator of the severity of PTE and provide insight into its pathogenesis, thereby facilitating the diagnosis of PTE.

Modulation of accessory molecules on lung T cells.

Stimulation of T cells via the CD3/TCR complex is associated with the reduced expression of accessory molecules, a phenomenon called modulation. To investigate whether the modulation is implicated in the pathogenic mechanism of pulmonary disease, we determined the MFI of CD3, CD4 and CD8 on lung and blood lymphocytes in ten normal subjects and 54 patients with various pulmonary and extrapulmonary diseases. Although there were no significant differences in MFI of these accessory molecules on lung lymphocytes among the groups, MFI of CD3 on lung lymphocytes was significantly decreased compared with that on blood lymphocytes in all groups, demonstrating the modulation of CD3 on lung lymphocytes. Since it has been shown that T cells whose CD3/TCR is modulated are hyporesponsive to various mitogenic signals, our observation seems to represent another aspect of the mechanism whereby the local pulmonary milieu maintains the autoregulation of immune response.

Association of HLA-DR with sarcoidosis. Correlation with clinical course.

HLA-DR typing was performed in 58 Japanese patients with sarcoidosis. We found a significantly increased frequency of HLA-DRw52 (p less than 0.0007; pc less than 0.009) and DR5J (p less than 0.004; pc less than 0.05). (Corrected probability value is given as pc.) In 34 of 58 patients the disease resolved spontaneously. There was no significant difference between the frequency of DRw52 and the resolution rate of disease. The frequency of DR5J was increased significantly in the unresolved cases. Our results also suggest that DRw52 is concerned with onset of sarcoidosis and DR5J antigen has an effect on the clinical course of this disease.

CD8+ cell-dominant alveolitis in pulmonary sarcoidosis.

A 55-year-old woman had bilateral hilar lymphadenopathy and retinouveitis. Microscopic examination of a biopsy specimen from a mediastinal lymph node revealed noncaseating epithelioid cell granulomas. Since granulomatous diseases of infectious origin and sarcoid reaction were reasonably excluded, she was diagnosed as having pulmonary sarcoidosis. Bronchoalveolar lavage findings were atypical in that the ratio of helper T cells to suppressor T cells (CD4/8 ratio) was 0.5 in two separate examinations, despite a moderately increased proportion of lymphocytes. Immunohistochemical examination of the lymph node also showed a predominancy of suppressor T cells. The inversion of the CD4/8 ratio suggests the presence of heterogeneity in the immunoregulatory mechanism in pulmonary sarcoidosis and may have a prognostic significance.

Release of tumor necrosis factor-alpha from human alveolar macrophages is decreased in smokers.

It is known that smoking affects the development and maintenance of certain types of granulomatous lung diseases. To explore this mechanism(s), we measured tumor necrosis factor (TNF)-alpha concentrations in the culture supernatants of lipopolysaccharide (LPS)-stimulated alveolar macrophages (AMs) in 13 healthy nonsmokers, 13 healthy smokers, 13 nonsmoking sarcoid patients, and 16 smoking sarcoid patients. We found that the capacity of smokers' AMs to release TNF-alpha was significantly decreased both in the normal and sarcoid groups. We also confirmed the previous observation that there was an exaggerated TNF release in patients with pulmonary sarcoidosis. These results indicate a significant role of TNF-alpha in the pathogenetic mechanisms of pulmonary sarcoidosis and suggest the possible involvement of TNF in the mechanisms by which smoking modulates local immune phenomena.

Possible release of nitric oxide from cholinergic axons in the thalamus by stimulation of the rat laterodorsal tegmental nucleus as measured with voltammetry.

By means of the differential direct current voltammetry technique with carbon fiber electrodes in urethane-anesthetized rats, we monitored nitric oxide (NO) concentrations in the thalamus in the basal condition and following electrical stimulation of the laterodorsal tegmental nucleus (LDT), whose neurons have the strongest activity of NADPH-diaphorase, or NO synthase, together with acetylcholine. NO levels, measured as the height of the peak at +970-1000 mV in the voltammetry (NO was soon oxidized in vivo to be detected at the voltage of this peak, so that NO levels in this report are, in the strict sense, levels of the oxidized metabolites reflecting very possibly those of NO in physiological conditions; see Section 2, Methods), increased just after repetitive stimulation of the LDT. Stimulation of the surrounding areas or the cerebellum produced virtually no change in NO levels. An intravenous injection of L-nitroarginine methyl ester reduced the basal level of NO, but stimulation of the LDT still increased NO levels, which may be due to very strong activity of NO synthase in the LDT neurons. These results are consistent with the notion that NO can be released from axons of the LDT neurons by their excitation.