A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the QcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex.

Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its E(m, 7) was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3'-downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe-S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b(6) show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b(6) and green sulfur-low G+C Gram-positive bacteria.

Establishment and usefulness of an anti-human CD57 IgG1 monoclonal antibody.

In the present study we established a new monoclonal antibody, JNK-1, which recognizes all cells recognized by CD57/HNK-1 mAb. JNK-1 and CD57 mAbs inhibited the binding of each other, suggesting that the molecules they recognize are either identical or sufficiently close to cause steric hindrance in the binding assay. JNK-1 mAb detected the 110-kDa protein, which is identical to the protein recognized by CD57/HNK-1 mAb in Western immunoblot analysis combined with immunoprecipitation. Therefore, JNK-1 mAb appears to recognize homogeneous molecules identified by the currently available CD57 mAb. Notably, JNK-1 mAb is composed of mouse IgG1 heavy chains, and thus can be used easily in immunoprecipitation, which cannot easily be performed with the available CD57 mAb because it is an IgM isotype. Thus, JNK-1, which is an IgG isotype, may present a useful tool to elucidate the CD57 protein.

Pulmonary hypertension and antiphospholipid antibody in a patient with Sjögren's syndrome.

A 57-year-old female patient with Sjögren's syndrome was complicated with pulmonary hypertension (PH) and antiphospholipid antibody (aPL). She had a history of fetal losses, deep vein thrombosis and chronic thyroiditis. On admission, severe pulmonary hypertension, thrombocytopenia, lupus anticoagulant and a decreased level of protein C were found. Pulmonary artery perfusion scintigram revealed multiple defects. She died suddenly despite an intensive therapy. Intimal proliferation with angiomatoid lesions in small pulmonary arteries was observed by autopsy. Since a close relationship between PH and aPL in connective tissue disease is found, it is important to carefully analyze the antiphospholipid antibodies in patients with PH.

[A trial of combination chemotherapy. Cis-platinum, peplomycin and adriamycin for advanced prostatic cancer].

Between September 1982 and May 1984, combination chemotherapy with cis-platinum, peplomycin and adriamycin was administered to 12 patients with histologically confirmed adenocarcinoma of the prostate and progressive disease with evaluable parameters. Cis-platinum (CDDP) 20 mg/sqm was administered intravenously on Days 1-5, peplomycin 5 mg/sqm by 24-hour continuous drip infusion on day 1-5 and adriamycin 25 mg/sqm on Day 1. This course was repeated every 28 days. The dose and schedule were modified by hematologic toxicity or other side effects. One patient refused therapy because of severe nausea and vomiting; therefore 11 patients were eligible for response evaluation. Of the 11 patients, two had a documented PR, five had SD and four had PD. Ten patients whose disease eventually progressed received a second line of therapy consisting either of estramustine or estrogen. Of these ten patients, 6 had a documented PR, one had SD and three had PD. It is concluded that this combination chemotherapy regimen may prime advanced prostatic cancer to respond to hormonal therapy, even though it has only a limited effect on advanced prostatic cancer.

[Ultrastructural study of interlobular bile ductal disorganization in autoimmune liver diseases].

Ultrastructures of interlobular bile ductules were examined in 7 cases of lupoid hepatitis and related disease (LH) and 8 cases of primary biliary cirrhosis (PBC). Mononuclear cell, especially lymphocyte infiltrations into bile ducts cross basement membrane were common findings found in 57.1% of LH and in 50% of PBC patients, thus, statistically, the occurrence of lymphocyte infiltration was similar for both groups. Cell contact between bile duct epithelia and infiltrated cells differed in LH and PBC. In LH, 86.7% of the cells contacted at small point, but 92.3% PBC cells had broad contact with each other. Stratification of bile duct epithelia and other visible changes in PBC epithelial cells was statistically more extensive compared with LH. Dilatation of intercellular space was often observed in basal region of LH. Destruction and degeneration of LH and PBC bile duct epithelial cells was mainly observed in basal and luminal regions, respectively. Rupture and thickening of basement membrane was seen in PBC, but rarely in LH. The average diameter of interlobular bile ducts were larger and oval shaped in patients with PBC compared to smaller circular ducts observed in LH patients. These results not only revealed that several changes in interlobular bile ducts occur but that similar changes with LH and PBC. This suggests that both distinctions and similarities exist between LH and PBC as autoimmune hepatic disease.

Pericanalicular microfilaments of hepatocytes in patients with familial non-hemolytic hyperbilirubinemia.

We observed pericanalicular webs (PCW) of liver cells in cases with familial non-hemolytic hyperbilirubinemia using electron microscopy. The area and width of PCW were determined by morphometric methods as a way of quantitating this feature. The mean PCW width was 0.175 +/- 0.003 micron (mean +/- SE) in Dubin-Johnson syndrome and 0.184 +/- 0.005 micron in Rotor's syndrome. In both of these syndromes PCW width was significantly larger than that in Gilbert's syndrome (0.124 +/- 0.003 micron) (p less than 0.01). The mean PCW area was 0.585 +/- 0.017 micron 2 in Dubin-Johnson syndrome and 0.582 +/- 0.030 micron 2 in Rotor's syndrome. Values in these two syndromes were significantly larger than that in Gilbert's syndrome (0.382 +/- 0.014 micron 2) (p less than 0.01). Widths and areas of PCW in these three syndromes were not significantly different between central, intermediate, and peripheral zones of the hepatic lobules. There was a positive correlation between serum direct bilirubin levels and widths or areas of PCW in these syndromes. These results suggested that disturbances of bile flow caused by the dysfunction of pericanalicular microfilaments are partly involved in the pathogenesis of Dubin-Johnson syndrome and Rotor's syndrome.

Reticular meshwork of the spleen in rats studied by electron microscopy.

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.

Renal prostaglandin E in the hypotensive mechanism of MK-421 in conscious rats.

To assess the role of renal prostaglandin E in the hypotensive mechanism of MK-421, we evaluated the effects of chronic infusion of MK-421 (6 mg/kg/day i.p.) on systolic blood pressure and urinary prostaglandin E excretion in conscious rats in states of sodium repletion or depletion and also during chronic infusion of norepinephrine (1.8 mg/kg/day i.p.) or vasopressin (7.2 U/kg/day i.p.). The hypotensive effect of MK-421 was greater in sodium depleted than in sodium repleted rats. The hypertensive effect of norepinephrine or vasopressin was inhibited by the simultaneous administration of MK-421. MK-421 induced an increase in the excretion of urinary prostaglandin E, in both sodium repleted and depleted rats. However, simultaneous administration of MK-421 had no influence on the increase in urinary prostaglandin E excretion induced by norepinephrine or vasopressin. In addition, the combined administration of MK-421 with indomethacin (10 mg/kg/day s.c.) still abolished the hypertensive effect of norepinephrine or vasopressin. The disparate effect of MK-421 on urinary prostaglandin E excretion suggests that the renal prostaglandin system is not essential for the mechanism of the hypotensive effect of MK-421.

Antihypertensive effect of MK-421 in rats. Role of the renal kallikrein-kinin system.

To study the hypotensive mechanism of the new oral converting-enzyme inhibitor, MK-421, we evaluated the antihypertensive effect of MK-421 in rats with hypertension induced by chronic administration of norepinephrine (NE) or vasopressin and measured urinary kallikrein and kinin excretions as indices of the renal kallikrein-kinin system. When 6 mg/kg/day of MK-421 was administered simultaneously with 1.8 mg/kg/day of NE, the systolic blood pressure of conscious rats rose on Day 1 to only 122.6 +/- 3.4 mm Hg compared with the rise to 146.3 +/- 1.6 mm Hg when NE alone was infused (p less than 0.001). Similarly, when the same dose of MK-421 was administered simultaneously with 7.2 U/kg/day of vasopressin, the systolic blood pressure of conscious rats rose on Day 1 to only 117.4 +/- 3.8 mm Hg compared with the rise to 141.6 +/- 3.4 mm Hg when vasopressin alone was infused (p less than 0.01). The antihypertensive effect of MK-421 was sustained for 6 days in rats infused with NE or vasopressin. Infusion of NE alone resulted in a small but significant increase in urinary kallikrein excretion and no change in urinary kinin excretion. The combined administration of NE with MK-421 induced additional increases in urinary kallikrein and kinin excretions. Vasopressin alone resulted in marked decreases in urinary kallikrein and kinin excretions. The combined administration of vasopressin with MK-421 induced no additional changes in urinary kallikrein and kinin excretion. These results indicate that the hypotensive effect of MK-421 may depend on a reduced sensitivity of the peripheral arteries to vasoconstrictor substances.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal kallikrein-kinin system and the depressor effect of angiotensin converting enzyme inhibitors MK 421, SA 446, and captopril in rats.

Responses in urinary kallikrein and kinin excretion and systolic blood pressure to MK 421, SA 446 or captopril were studied in normotensive rats fed on a regular or a low sodium diet to assess the role of renal kallikrein-kinin system in their hypotensive effect. MK 421, SA 446 or captopril were infused at a rate of 6 mg/kg/day by osmotic minipump implanted intraperitoneally for up to 6 days. The magnitude of fall in systolic blood pressure was greater on a low sodium diet when compared to on a regular diet, whereas the pattern of the fall was similar on both diets. The magnitude of falls in plasma angiotensin II and aldosterone concentration induced by MK 421, SA 446 and captopril was not significantly different between both regular and low sodium diets. Urinary kallikrein and kinin excretion and sodium excretion were increased during infusion of MK 421, SA 446 or captopril on a low sodium diet, however any significant changes were not found in each of them on a regular diet. The present results suggest that on a low sodium diet the augmented hypotensive response to angiotensin converting enzyme inhibitors in the rats might be due to the enhanced renal kallikrein-kinin system in addition to suppressed renin-angiotensin system.

Effects of nifedipine on renal vascular responses to vasoactive agents in rabbits.

The present study was designed to investigate the effects of the Ca-antagonist, nifedipine, on receptor-mediated renal vascular responses to vasoconstrictors (angiotensin II, norepinephrine and vasopressin) and vasodilators (bradykinin and prostaglandin E2). Renal blood flow was estimated by noncannulating electromagnetic flowmetry in anesthetized rabbits. Vasoactive substances were infused directly into the renal artery. After the intravenous administration of nifedipine (50 micrograms/kg), decreases in renal blood flow in response to angiotensin II infused at rates of 2.5, 5 and 10 ng/kg/min were attenuated by 64% (p less than 0.01), 45% (p less than 0.05) and 42% (p less than 0.05), respectively. Decreases in renal blood flow in response to vasopressin infused at rates of 10, 20 and 50 mU/kg/min were also attenuated by nifedipine, 50% (p less than 0.05), 57% (p less than 0.05) and 38% (p less than 0.05), respectively. However, renal vasoconstrictor responses to norepinephrine (25, 50 and 100 ng/kg/min) did not change significantly after the administration of nifedipine. Increases in renal blood flow induced by the intrarenal infusion of bradykinin (2.5, 5 and 10 ng/kg/min) and prostaglandin E2 (20, 50 and 100 ng/kg/min) were not affected by nifedipine. These results suggest that receptor-mediated vasoconstrictor responses of the renal vascular bed to angiotensin II and vasopressin are produced mainly by Ca++ influx but that of norepinephrine is not. Furthermore, it is confirmed that the renal vasodilator effect of bradykinin and prostaglandin E2 is not altered by nifedipine in anesthetized rabbits.

Chronic effects of norepinephrine and vasopressin on urinary prostaglandin E and kallikrein excretions in conscious rats.

To assess in vivo functional interactions of vasopressor substances, norepinephrine and vasopressin, with renal prostaglandins and kallikrein-kinin system which are responsible for the vasodepressor mechanism in the kidney, we evaluated chronic effects of norepinephrine (1.8 mg/kg/day ip) and vasopressin (7.2 U/kg/day ip) on urinary prostaglandin E excretion and urinary kallikrein excretion in conscious rats. Both norepinephrine and vasopressin induced a sustained increase in systolic blood pressure. Norepinephrine induced slight but significant increases in urinary prostaglandin E excretion and urinary kallikrein excretion which were sustained for up to 6 days. Vasopressin induced a marked increase in urinary prostaglandin E excretion which was sustained for up to 6 days, whereas it induced a sustained decrease in urinary kallikrein excretion. Circulating angiotensin II levels was not changed by norepinephrine, but was decreased by vasopressin. These results indicate that renal prostaglandin E may not correlate with renal kallikrein-kinin and renin-angiotensin system in the responses to norepinephrine and vasopressin, and that vasopressin may be a more potent stimulator of the synthesis or release of renal prostaglandin E.