Electrophoretic and kinetic characterization of three variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase in human liver tissue

Three common variants of soluble cytoplasmic L-alanine:2-oxoglutarate aminotransferase (ALT, EC 2.6.1.2), sALT 1, 2-1 and 2, were isolated from normal human liver, and characterized by electrophoretic and kinetic analyses. The isoelectric point of sALT 1 was pH 6.45. sALT 2-1 was focused into three bands with pl 6.1, 6.2 and 6.45; sALT was focused into one band with pl 6.1. The electrophoretic mobilities of sALTs altered to the fast beta-globulin fraction after aging or papain treatment. Ammonia was produced during the latter, and the altered migration was considered to be caused by deamidation of sALT. The relative molecular mass of each of the enzymes was 110,000. Minor differences in the apparent Km values among the multiple forms for both L-alanine and 2-oxoglutarate were observed after incubation with 100 mumol/L of pyridoxal phosphate (PALP). PALP stimulation of the enzyme activities was also different. sALT 1 was more stable than sALT 2-1 and 2 after heat and urea treatments. In human sera from 1065 adult Japanese, sALT 2-1, a heterozygote form of sALT 1 and 2, was dominant.     

Hydrolytic activity is essential for aceclofenac to inhibit cyclooxygenase in rheumatoid synovial cells

To investigate the mechanisms of action underlying the anti-inflammatory effects of the nonsteroidal anti-inflammatory drug aceclofenac in humans, we studied the metabolism of aceclofenac in detail in primary cultured synovial cells of 10 patients with rheumatoid arthritis. Aceclofenac and 4'-hydroxyaceclofenac are the major compounds in human blood after the administration of aceclofenac, but they had no inhibitory effects on cyclooxygenase (COX) activity or COX expression in the rheumatoid synovial cells. In contrast, aceclofenac and 4'-hydroxyaceclofenac reduced prostaglandin E2 (PGE2) production by the rheumatoid synovial cells. We also observed that aceclofenac and 4'-hydroxyaceclofenac were hydrolyzed into the COX inhibitors diclofenac and 4'-hydroxydiclofenac, respectively, by the rheumatoid synovial cells. However, the hydrolytic activity differed markedly among the cell preparations. Because the suppressive potency of aceclofenac and 4'-hydroxyaceclofenac against the PGE2 production was proportionally correlated with the hydrolytic activity in rheumatoid synovial cell preparations, we suggest that the suppressive effects of aceclofenac and 4'-hydroxy aceclofenac on PGE2 production are facilitated by the hydrolytic activity in rheumatoid synovial cells.     

Application of nicotinamide-adenine dinucleotide analogs for clinical enzymology: a spectrophotometric method for clinical analysis of lactate dehydrogenase patterns with the use of a nicotinamide-adenine dinucleotide analog.

The activities of porcine lactate dehydrogenase (LHD) isoenzymes were analyzed using nicotinamide-adenine dinucleotide (NAD) or its analogs as a cofactor, with varying concentrations of L-lactate from 13 to 530 mM. The greatest differences between H4-type and M4-type isoenzymes in reaction rates were observed when their activities were compared in a reaction mixture containing 530 mM lactate and NAD, and also in a system of 13 mM lactate with thionicotinamide-hypoxanthine dinucleotide as a cofactor. The ratio of the LDH activity exerted in the former reaction mixture to that exerted in the latter was termed the N/T value. The N/T values of porcine H4 and M4 isoenzymes were 0.49 and 9.33, respectively. The N/T values of other three isoenzymes (H3M1, H2M2 and H1M3) were calculated by assuming that the single subunits H1 and M1 contribute one-fourth of the values of 0.49 and 9.33, respectively, and a given isoenzyme which is a combination of four subunits of H and M comprises the sum of their values. The calculated values agreed fairly well with the experimental results. The N/T value method was found to be applicable to human LDH isoenzymes, and sera from various patients were analyzed in comparison with hormonal sera. The method is particularly suitable for the numerical expression of LDH isoenzyme profiles.     

The role of serum KL-6 measurement in common pediatric respiratory infections

KL-6 is a useful marker for interstitial pneumonia of various origins. However, the role of KL-6 in common pediatric respiratory infections is largely unknown. In order to determine whether the KL-6 level is elevated during respiratory infection, and whether KL-6 is a useful biomarker for the disease activity, we evaluated serum KL-6 levels in 132 children with various respiratory infections. KL-6 levels were significantly higher in patients with measles, influenza, or respiratory syncytial virus infection than in the control subjects. On the other hand, KL-6 levels in patients with bacterial infections such as mycoplasma, chlamydia, or pertussis were comparable to the control values. In patients with viral infections, high KL-6 levels, as defined by the mean plus 2 standard deviations of the control group, significantly correlated with low SpO₂ or days of O₂ administration, but did not correlate with C-reactive protein or white blood cell counts. These results indicate that measurement of serum KL-6 levels is helpful for the management of common pediatric respiratory infections.     

Neutrophil alkaline phosphatase activity in respiratory viral infection

Neutrophil alkaline phosphatase (NAP) is used as a diagnostic marker in several hematological disorders. In regard to the role of NAP in infectious diseases, previous investigators have presented the hypothesis that NAP activity is useful to distinguish viral infections from bacterial infections. Because the numbers of patients enrolled in previous studies of viral infections were limited, we intended to evaluate the hypothesis by measuring NAP activity in a large number of pediatric patients with respiratory viral infections. A cytochemical analysis of NAP was performed in 160 patients with various types of respiratory infections. In patients with adenovirus or respiratory syncytial (RS) virus infection, NAP activity was significantly higher than the control value newly established at our department, while in patients with Epstein-Barr virus, measles, or influenza infection, it was comparable to the control value. On an individual basis, NAP scores (determined from NAP cytochemical activity) in 22 of 26 patients (84.6%) with adenovirus infection, and 31 of 42 patients (73.8%) with RS virus infection were found to exceed the 95% confidence upper limit of the control group. In conclusion, NAP activity is quite varied among different respiratory viral infections. When NAP activity is high in respiratory infections, adenovirus or RS virus infection, as well as bacterial infections, should be taken into consideration.     

Transient receptor potential 1 regulates capacitative Ca(2+) entry and Ca(2+) release from endoplasmic reticulum in B lymphocytes

Capacitative Ca(2+) entry (CCE) activated by release/depletion of Ca(2+) from internal stores represents a major Ca(2+) influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca(2+) release-activated Ca(2+) currents and inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor-mediated Ca(2+) oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP(3) receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca(2+) signaling in B lymphocytes.     

Primary colorectal signet-ring cell carcinoma: Clinicopathological features and postoperative survival

Purpose

The objective of this study was to investigate the clinicopathological features and postoperative survival of primary colorectal signet-ring cell carcinoma.

Methods

Nineteen patients with primary colorectal signet-ring cell carcinoma were identified from a database of 5884 surgical patients with colorectal cancers treated surgically at Osaka University Hospital and affiliated hospitals between 1993 and 2007. The clinicopathological data of those patients were compared with those of 5792 patients with non-signet-ring cell colorectal carcinoma (5417 with well or moderately differentiated adenocarcinoma and 375 with poorly differentiated adenocarcinoma or mucinous carcinoma).

Results

All patients showed a tumor depth of over T3. Lymph node involvement occurred in 14 patients. Seven of 19 patients presented with distant metastasis at the time of diagnosis. The overall 5-year survival rate in primary signet-ring cell carcinoma was significantly lower at 24.1%, in comparison to 77.5% in well or moderately differentiated adenocarcinoma and 57.7% in poorly differentiated adenocarcinoma or mucinous carcinoma. Likewise, the postoperative survival in Stage III was also significantly worse. On the other hand, no significant difference was observed in Stage II or IV.

Conclusion

The most important feature of primary colorectal signet-ring cell carcinoma is the advanced stage at the time of diagnosis. In addition, the postoperative survival is worse than for other types of colorectal cancer.