Demonstration of adenosine triphosphate hydrolysis in the enamel organ of the mouse incisor.

The hydrolysis of adenosine triphosphate in the mandibular enamel organ demonstrated that the Mg++-activated ATPase was destroyed by pre-treatment with either heat or alcohol, substrate specific for ATP, stimulated by the addition of glutathione or dinitrophenol, and inhibited by oligomycin. The distribution of reaction product was the same with Mg++, Mn++ or Zn++ as the activating cation. Omission of Mg++ from the incubation medium, or replacement with Ca++ or Sr++ resulted in marked hydrolysis of ATP in the cells associated with enamel matrix formation, with loss of enzyme activity in the cells of the zone of enamel matrix maturation. Hydrolysis of ATP by the cells of the stratum intermedium, stellate reticulum and papillary layer was dependent upon Mg++, Mn++, or Zn++.     

Conditions for detecting the mutagenicity of divalent metals in Salmonella typhimurium.

The mutagenesis of metals in bacteria, as reported in the literature, can best be described as inconsistent. We report that cobalt chloride (Co++), ferrous sulfate (Fe++), manganese sulfate (Mn++), cadmium chloride (Cd++), and zinc chloride (Zn++) could be reproducibly detected as mutagens in Salmonella strain TA97 when preincubation exposures were made in sterile, distilled, deionized water, or in Hepes buffer in NaCl2/KCl2, rather than the standard sodium phosphate buffer. Co++ was also mutagenic under standard preincubation conditions. The individual components of Vogel-Bonner medium, i.e., potassium and ammonium phosphate, citrate, and magnesium sulfate, inhibit mutagenesis by these metals. The phosphates and the citrate probably inhibit by chelating the metals, while data are presented to suggest that Mg++ inhibition of metal mutagenesis is due to competitive inhibition for active transport via the magnesium active transport system in Salmonella. The chelator, diethyldithiocarbamate, inhibited the mutagenicity of Co++, Fe++, Zn++, and Mn++, but enhanced the mutagenicity of Cd++. The results presented show that divalent metals can be detected as mutagens in Salmonella, and that their lack of detection as mutagens is not due to an inherent insensitivity of Salmonella but to their interaction with media components and/or passive and active transport processes.     

Isolation and characterization of hyaluronidase from Streptococcus uberis

All tested cultures of Streptococcus uberis produced free hyaluronidase. Hyaluronidase could be isolated by ammonium sulfate precipitation and was further purified by chromatography on DEAE-cellulose, gelfiltration on ultragel ACA44 and isoelectric focusing. The purification factor was estimated to be 1689. The purified hyaluronidase had an isoelectric point at pH 4.9 and a molecular weight of approximately 54000 D. It showed maximal enzyme activity at pH 6.0 and 45 degrees C. The Michaelis constant was estimated to be 7.0 X 10(-2) mg/ml. Hyaluronidase activity was stimulated by Ca++, Mg++, Mn++, Co++, Li+, and K+ and inhibited by Zn++ and Cd++ at final concentrations of 10 mmol/l, respectively.     

Measurement of peritoneal fluid pH in patients with non-serosal invasive gastric cancer

The accurate pH range of peritoneal fluid is clinically valuable for the evaluation of some pathological conditions of the body, however, it is not easy to measure in healthy individuals. The aim of this study was to measure; pH, pCO2, pO2, Na+, K++, Ca++, HCO3-, and O2 saturation of the peritoneal fluid in patients with non-serosal invasive gastric cancer. One hundred and thirty four patients (86 men and 48 women), ranging in age from 24 to 91 years were enrolled in this study. After opening the abdominal wall, the probe of a portable pH meter was placed in the peritoneal fluid in the subhepatic space. In addition, I collected the peritoneal fluid from the subhepatic space to measure, pH, pCO2, pO2, Na+, K++, Ca++, HCO3-, and O2 saturation using an autoanalyzer. The pHs of the peritoneal fluids tested has a mean of 7.73 (range 7.46 - 8.10), and the other parameters were pCO2, 22.81 mmHg; pO2, 136.49 mmHg; Na+, 146.57 mmol/L; K++, 4.80 mmol/L; Ca++, 0.89 mmol/L; HCO3-, 30.54 mmol/L, and O2 saturation, 99.74%. This study describes a practical method of measuring the pH of peritoneal fluid. The result obtained reflects the normal adult peritoneal pH value, which I propose as a reference value.     

Isolation and characterization of hyaluronidases from Streptococcus dysgalactiae, S. zooepidemicus and S. equi

10 out of 10 cultures each of Streptococcus dysgalactiae and S. zooepidemicus and 6 out of 10 cultures of S. equi tested for hyaluronidase produced this enzyme. Hyaluronidase could be precipitated from the cell-free culture supernatant with ammonium sulphate and purified by chromatography on DEAE-cellulose, isoelectric focussing and preparative polyacrylamide gel electrophoresis. The isoelectric points of the hyaluronidases from S. dysgalactiae and S. equi were near pH 5, of that from S. zooepidemicus near pH 6. The hyaluronidases from S. dysgalactiae, S. zooepidemicus and S. equi had molecular weights of about 55,000 D. Maximal enzyme activities developed between 40 degrees C and 45 degrees C and pH 5.6 and 5.8. The Michaelis constants ranged from 7.5 x 10(-2) to 8.8 x 10(-2) mg/ml. Hyaluronidase activities were stimulated by Ca++, Mg++, Mn++, Co++, K+, and Li+ and inhibited by Zn++ and Cd++.     

Inhibition of the terminal stage of complement-mediated lysis (reactive lysis) by zinc and copper ions.

The effect of various metal ions, Fe++, Fe+++, Cu++, Zn++, Co++, on the terminal stage of reactive lysis (a form of complement-mediated hemolysis in which only late-acting components of complement are required) was studied. Only Cu++ and Zn++ exhibited an inhibitory effect on the lysis of EC5678 (sheep erythrocytes reacted with C56, C7 and C8) induced by C9. The mode of action of these metal ions was further explored. Both Cu++ and Zn++ inhibited the formation of hemolytically active EC56789 from EC5678 and C9. Their effect appeared to be primarily due to the inhibition of C9 binding to EC5678 through their reversible interaction with C9. Furthermore, Cu++ is shown to inactivate irreversibly the hemolytic activity of EC5678. EC5678 pretreated with Cu++ was capable of binding C9, but the resulting EC56789 was hemolytically inactive. Besides their effect on complement, both metal ions were shown to affect directly the erythrocyte membrane, since the mechanical lysis of hemolytic intermediate cells (E, EC567, EC5678) was suppressed by Cu++ and Zn++. The lysis of EC56789, a process of internal activation, was also inhibited by both Cu++ and Zn++.     

Isochromosome consisting of terminal short arm and proximal long arm X in a girl with short stature

A 16-year-old girl with short stature, short neck, shield chest, and cubitus valgus was studied. FISH analyses of her structurally altered X chromosome showed a der(X)- (wcpX+,TelXp/Yp++,SHOX++,STS++,KAL-, 37A12-,DXZ1+,XIST++,97L7++,300O13-,404F- 18-,417G15-,404F18-,140A-,TelXq/Yq-). These results, together with the high-resolution banding analysis, indicated her karyotype to be 46,X,der(X)(Xpter-->Xp22.3::Xq22.3--> cen-->Xq22.3::Xp22.3-->Xpter). The der(X) was an isochromosome, consisting of duplicated terminal short arms and duplicated proximal long arms. This in turn suggested that the chromosome was formed through pericentric inversion of an X chromosome, followed by isochromosome formation through sister chromatid exchange at Xp, close to the centromere. Replication R-banding analysis showed that the abnormal X chromosome was late replicating. Analysis of digestion patterns with a methylation-sensitive restriction endonuclease of the phosphoglycerate kinase 1 gene, located in Xq13.3, indicated that its inactivation patterns were completely skewed.     

Ehrlich ascites cells activate the alternative pathway of the human complement system

Incubation of Ehrlich ascites cells with normal or C1q or C2 deficient human sera results in killing of the cells. Killing occurred also in the absence of free Ca++, which supported by the fact that factor B and C3 were cleaved, leads to the conclusion that the alternative pathway of the complement system is activated on the surface of the Ehrlich ascites cells.     

The effect of phosphatidyl serine on the activation stage of histamine release induced by neutrophil cationic protein.

Histamine release from rat mast cells induced by cationic protein (band 2) from rabbit neutrophil lysosomes occurs in Ca++-deficient medium. At higher concentrations of Ca++ the release is inhibited. Strontium not only supports, but also enhances the release of histamine in the absence of Ca++. Progressive enhancement of release occurs between 1.8 and 14.4 mM Sr++. The release of histamine from mast cells, activated at low temperature (0-4 degrees C) in the presence of 14.4 mM Ca++ and then washed prior to incubation at 37 degrees C, is inhibited. However, if phosphatidyl serine (PS) (10 microgram) is present with 14.4 mM Ca++, the inhibition is reversed. There is also inhibition of release when cells, activated in the presence of 1.8 mM Ca++, are incubated in the second stage with 14.4 mM Ca++, but this inhibition is less pronounced than when the 14.4 mM Ca++ is in the activation stage. PS enhances the release in the presence of both Ca++ and Sr++. The presence of PS in the activation stage enhances the release, but there is no significant enhancement when cells activated in the absence of PS are washed and incubated in the presence of PS. This suggests that PS enhancement of histamine release occurs at the activation stage, probably through the efficient delivery of calcium to the membrane sites, thereby increasing the efficacy of the membrane perturbation by band a protein.     

Biliary neoplasia with extensive intraductal spread associated with liver cirrhosis: a hitherto unreported variant of biliary intraepithelial neoplasia

We describe the histopathologic features of 2 cases of biliary neoplasia with extensive intraductal spread arising in liver cirrhosis. The prevalence of this type of biliary neoplasia may be 0.4% from the review of 468 cases of cirrhotic liver. Histologic analysis revealed that the micropapillary proliferation of the atypical biliary epithelium composed of columnar cells with enlarged nuclei diffusely extended superficially from the septal intrahepatic bile duct to the reactive ductules associated with liver cirrhosis. Both cases exhibited prominent fibrous or sclerotic stroma near the biliary lesion. Immunohistochemical analysis revealed a characteristic cytokeratin and mucin expression pattern (CK7++, CK19++, CK20+, MUC1+/-, MUC2-, MUC5AC+, MUC6-). The tumor cytoplasm was focally positive for laminin gamma2 together with linear staining of the basement membrane. Proliferative activity confirmed by Ki67 staining was relatively high. Both patients were disease-free for 3 years after the operation. We believe that the possibility of biliary neoplasia with extensive intraductal spread should be considered to be a variant of biliary intraepithelial neoplasia.     

Finger-loops, oncogenes, and metals. Claude Passmore Brown memorial lecture

Certain DNA-binding proteins that regulate gene expression contain single or multiple copies of short polypeptide sequences, approximately 30 residues long, consisting of combinations of four Cys or His residues at defined spacing, so that Zn++ is complexed in tetrahedral coordination with the respective thiol-sulfur and/or imidazole-nitrogen atoms. The Zn++ ion evidently serves as a strut that stabilizes folding of the domain into a 'finger-loop', which is capable of site-specific binding to double-stranded DNA. This article reviews the evidence (a) that finger-loop domains have been highly conserved during evolution, (b) that they furnish one of the fundamental mechanisms for regulating gene expression, and (c) that a metal ion (e.g., Zn++) is required for binding of finger-loops to DNA and for their biological functions. The authors' search of amino acid sequences of 38 transforming proteins identified possible finger-loop domains in the myc, fms, fps, raf-1, rfp, src, syn, yes, erbA, int-1, and TGF-alpha gene-products. The search incidentally revealed possible finger-loop domains in human insulin receptor, which may provide a mechanistic explanation for recent observations that insulin, after binding to its cell surface receptor, is translocated to hepatocyte nuclei and becomes bound to chromatin. Zn++-coordination sites in finger-loop domains are proposed as potential targets for metal toxicity; substitution of Ni++, Co++, or Cd++ for Zn++ in finger-loops of transforming proteins is suggested as an hypothetical mechanism for metal carcinogenesis.     

Ca-dependent and Ca-independent histamine release from mast cells induced by active components of compound 48/80

Compound 48/80 and 14C-labelled compound 48/80 were synthesized, and fractionated by thin-layer chromatography into 14 components with various histamine-releasing activities and different Ca++ requirements for their actions. The histamine release induced from rat mast cells in vitro by the most active component, fraction D (molecular weight = 2280, a tridecamer composed of 13 monomer units), was greatly elevated by extracellular Ca++, and was partially reduced by pretreatment of the cells with dinitrophenylated Ascaris antiserum, an IgE. In contrast, the histamine release induced by fraction H (molecular weight = 1580, a nonamer composed of 9 monomer units), was higher in Ca-free medium than in Ca-containing medium, and partially suppressed by pretreatment of mast cells with neurotensin or substance P, both Ca-independent releasers. The binding potencies of the 14C-labelled components estimated at 4 degrees C in the presence of Ca++, where no degranulation of the cells occurs, generally paralleled their histamine-releasing activities. However, Ca++ was inhibitory for the binding of 14C-fraction H. The binding of fraction D to [3H]arachidonic-acid-preloaded mast cells induced a rapid accumulation of the labelled arachidonic acid into phosphatidic acid, phosphatidylinositol and phosphatidylcholine, with concomitant decrease of the labelled arachidonic acid from phosphatidylethanolamine prior to the detectable histamine release.     

Lobucavir-induced proliferative changes in mice

Nucleoside analogues are used in the treatment of viral infections, including those caused by human immunodeficiency virus, cytomegalovirus, and herpes virus. These drugs are beneficial in the treatment of human disease, but are associated with toxicities that often limit their intended therapeutic use, including anemia, neutropenia, peripheral neuropathy, and myopathy. Some of these compounds have been reported to be carcinogenic in rodents. To investigate the carcinogenic potential of lobucavir, a nucleoside analogue, three groups of 60 male and female mice were orally administered lobucavir at daily doses of 10, 50, and 250 mg/kg (males) or 30, 150, and 750 mg/kg (females) over a period of 104 weeks. Two identical groups of 60 male and female mice each served as controls. The morphology and the incidence of neoplasms is described and compared with the tumor spectrum of other nucleoside analogues. Light microscopically, lobucavir-induced neoplastic lesions consisted of upper digestive tract squamous cell neoplasia in males and females; cervical, vaginal, and cutaneous squamous cell neoplasia in females; and Hardarian gland adenomas and adenocarcinomas in male mice. These results suggest that long-term administration of lobucavir causes neoplasia in mice, the spectrum of which resembles that observed after long-term administration of zidovudine or ganciclovir.     

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The K_m values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [γ-³²P]ATP or thiophosphorylated with [³⁵S]GTPγS. The incubation of the ³²P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of ³²P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed.     

A unique pancreatic ductal adenocarcinoma with carcinosarcomatous histology, immunohistochemical distribution of hCG-beta, and the elevation of serum alpha-feto-protein

Pancreatic undifferentiated carcinomas with a neoplastic mesenchymal component (carcinosarcoma) have not been well described to date. The author experienced an autopsy case of a unique pancreatic ductal adenocarcinoma with carcinosarcomatous histology. The patient was a 90 year old Japanese male who died of cahexia with generalized tumor extension. Post-mortem examinations revealed some distinctive or representative components discerned in the tumor tissue. One was the well differentiated ductal adenocarcinoma. The second and the major finding was undifferentiated short spindle shaped or small round sarcomatous cells, which lacked an epithelial nature but showed positivity for CD10+, CD56+, Ki67++, p53++, and were focally positive for Desmin and vimentin. These two components were mixed and constituted the histology of the carcinosarcoma. In another area, anaplastic, large, pleomorphic tumor cells showed the focal immunohistochemical distribution of alpha-feto-protein and human chorionic gonadotropin. An ultrastructural study revealed adenocarcinoma cells with apical mucin secreting granules and well developed ductal differentiation, whereas undifferentiated sarcomatous cells showed primitive fibroblastic or mesenchymal characters without specific differentiation. Conclusively these findings suggested that this well differentiated adenocarcinoma gradually enlarged, accumulated genetic alternations, and then transformed into large and undifferentiated tumor cells, rapidly growing small sarcomatous cells, and a histology of carcinosarcoma.     

Small oncocytic papillary renal cell carcinoma in diabetic glomerulosclerosis

Histologic and immunohistochemical features of oncocytic papillary renal cell carcinoma (RCC) have not been fully elucidated. The author herein report a case of oncocytic papillary RCC (OPRCC). A 71-year-old man with diabetes mellitus and diabetic nephropathy was found to have a small right renal tumor by CT. He had been treated with hemodialysis for chronic renal failure for 10 years. A nephrectomy was performed. Grossly, a small (1.5cm) encapsulated yellow tumor was found in the kidney. Histologically, the tumor was completely encapsulated, and consisted entirely of atypical oncocytes arranged in a diffuse papillary structure with fibrovascular cores. The oncocytes showed grade 3 atypia and pseudostratification. A few mitotic figures were seen, and psammoma bodies, foamy macrophages, and hemosiderin were scattered. Histochemically, the tumor cells were positive for colloidal iron, and negative for mucins (Alcian blue/PAS). Immunohistochemical results of the tumor were as follows: α-methylacyl-coenzyme A rasemase (AMACR) +++, vimentin +++, cytokeratin (CK) 18 +++, CD10 +++, S-100 protein +, MUC1 ++, MUC2 ++, MUC5AC ++, MUC6 ++, panCK Cam5.2 +, CK7 +, CK8 +, CK14 +, CK19 +, CK20 +, p53 +, HepPar1 +, CD68 +, platelet-derived growth factor-α (PDGFRA) +, PanCK AE1/3 -, PanCK WSS -, PanCK MNF115 -, CK 35BE12 -, CK5/6 -, EMA -, desmin -, smooth muscle antigen -, α-fetoprotein -, CEA -, estrogen receptor -, progesterone receptor -, HER2 -, p63 -, and KIT -. Ki67 labeling was 6%. These results suggest that OPRCC can express colloidal iron, low molecular weight CKs, S100 protein, MUC1, MUC2, MUC5AC, MUC6, p53, PDGFRA, and HepPar1.     

Studies of mutagenesis and neoplastic transformation by bivalent metal ions and ionizing radiation

We examined the influence of nontoxic concentrations of each of two essential (Zn++ and Mn++) and one nonessential (Ni++) bivalent metal ions on spontaneous and radiation-induced neoplastic transformation and specific gene mutations in mammalian cells. All three metals induced low levels of transformation in mouse BALB/3T3 cells but exerted no mutagenic effect in CHO cells (hprt locus) over a broad range of concentrations. Continuous incubation for 8 or 15 days with each of the metal ions did not enhance the frequency of cell killing, transformation, or mutations induced by acute exposure to x-rays. Zn++, however, had a small but consistent protective effect on the induction of all three endpoints by x-irradiation.     

Proliferation of marginal zone cells mimicking malignant lymphoma

An enlarged axillary lymph node from a 58-year-old woman showed a proliferation of marginal zone cells in nodules or large band-forming aggregates within the cortex. The marginal zone cells also infiltrated the adjacent fatty tissue. They showed polytypic surface immunoglobulins (IgM++, IgG+, kappa+, lambda+). Their immunophenotype (IgD-, CD23-, KiB3-) differed from that of the small lymphocytes of the mantle zone. They were also positive for alkaline phosphatase. The lesion is reactive and must be differentiated from low-grade malignant lymphomas, especially centrocytic lymphoma.     

Comparative studies on antibody and lectin-dependent macrophage-mediated tumor lysis

Three characteristics of the mechanisms of antibody-dependent macrophage-mediated cytolysis (ADMC) and lectin-dependent macrophage-mediated cytolysis (LDMC) in a C3H/He mouse-MM46 syngeneic tumor system were compared: the role of divalent cations, the effect of cyclic nucleotides, and the role of protease and oxygen compounds as lytic substances. Mg++, but not Ca++, was required for ADMC but neither was required for LDMC. Ca++ inhibited both LDMC and ADMC. Dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) and its agonists, such as cholera toxin, prostaglandin E1 (PGE1) and E2 (PGE2), inhibited ADMC, but had no effect on LDMC. Dibutyryl cyclic guanosine 3',5'-monophosphate (cGMP) had no effect on either ADMC or LDMC. Isoproterenol and isobutylmethylxanthine had no effect on either ADMC or LDMC. Protease inhibitors, such as pepstatin, bestatin, chymostatin, elastatinal, leupeptin, and bovine pancreatic trypsin inhibitor, did not affect either ADMC or LDMC. Tosylphenylalanyl-chloromethylketone inhibited both ADMC and LDMC, and also inhibited macrophage-tumor cell contact in both systems. Neither catalase nor superoxide dismutase clearly inhibited either ADMC or LDMC. The different sensitivities of ADMC and LDMC to pharmacological agents suggest that these two types of mediator-dependent macrophage-mediated cytolysis have partly different cytolytic processes as well as different recognition sites.     

In vitro studies of the interaction of isolated Lp(a) lipoprotein and other serum lipoproteins with glycosaminoglycans

The interaction of isolated Lp(a) lipoprotein or other lipoprotein classes with different glycosaminoglycans (GAG) bound to activated Sepharose was studied. In contrast to LDL, the Lp(a) lipoprotein did not bind to the GAG tested if sodium was used as a buffer cation. In the presence of Ca++, however, even the Lp(a) lipoprotein was bound to GAG. This type of binding, probably mediated by divalent cation bridges, is apparently not a simple function of the GAG used. Addition of GAG in solution revealed that this binding may be the only one existing under physiological conditions, and it appears possible that the Lp(a) lipoprotein is bound more firmly to GAG than is LDL under such conditions.