Enhanced expression of c-myc and epidermal growth factor receptor (C-erbB-1) genes in primary human renal cancer.

We have analyzed the expression of 12 protooncogenes, c-Ha-ras, c-Ki-ras, N-ras, c-myc, N-myc, c-fos, c-abl, c-fes, c-fms, c-raf, c-erbB-1, and c-erbB-2, in tissues of human renal cell carcinomas and in adjacent normal kidneys. Comparative densitometry of Northern blot analyses demonstrated enhanced level of c-myc gene expression, i.e., greater than threefold increase over normal kidney tissues, in 11 of 15 (73%) of the tumors examined. Increased levels of c-erbB-1 mRNA were likewise observed in seven of 15 (47%). Interestingly, many of the tumors exhibiting elevated levels of c-erbB-1 revealed increases in c-myc mRNA levels. However, Southern blot analysis failed to detect gene amplification or rearrangement in the tumors with elevated levels of c-myc and/or c-erbB-1. Although N-ras, c-fos, and c-raf gene transcripts were detected in both malignant and normal tissues, differences in these protooncogene expressions were not found between the carcinomas and normal kidneys. Significant elevations of expression were found in one of 16 cases of each for c-Ha-ras and c-fms, whereas expression of c-Ki-ras, N-myc, c-fes, c-abl, or c-erbB-2 could not be detected in any of the tissues surveyed. These results suggest that activation of c-myc and c-erbB-1 genes may be involved in the development of human renal cell carcinomas.

Cloning of a thermostable ascorbate oxidase gene from Acremonium sp. HI-25 and modification of the azide sensitivity of the enzyme by site-directed mutagenesis.

A gene encoding a thermostable ascorbate oxidase (ASOM) was cloned from Acremonium sp. HI-25 and sequenced. The gene comprised 1709 bp and was interrupted by a single intron of 57 bp. ASOM consisted of 551 amino acids including a signal peptide with a molecular mass of 61200, and contained four histidine-rich regions with high sequence homology to the corresponding regions of other multicopper oxidases. The ASOM gene was expressed in Aspergillus nidulans under the Aspergillus oryzae Taka-amylase A gene promoter. The recombinant enzyme (An-ASOM) exhibited almost the same enzymatic properties as ASOM. The ASOM gene was mutated by site-directed mutagenesis with reference to the amino acid sequences of plant enzymes to generate enzymes with altered azide sensitivity. Site-directed mutagenesis at the trinuclear active copper site resulted in an increase in azide resistance; the Ala465Leu and Phe463Trp/Ala465Leu mutants exhibited approximately 10 and 20% increases in azide resistance, respectively.

L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies.

Crystals of flavoenzyme L-lactate oxidase from Aerococcus viridans (LOX) have been obtained that diffract to 3.0 A resolution (P2(1)2(1)2(1), a = 118.4 A, b = 138.4 A, c = 194.6 A). Crystallographic studies suggest that the enzyme may exist as an octameric form with non-crystallographic two- and four-fold axes in the center of the octamer. The four-fold axis makes the tetramer tight, and the tetramers lie upon one another by the two-fold axis.

L-lactate oxidase and L-lactate monooxygenase: mechanistic variations on a common structural theme.

Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-alpha-hydroxy acids, including flavocytochrome b2, lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydroxy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many properties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable and is the form that reacts with O2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O2, to give the observed products, pyruvate and H2O2.

An ELISA to determine the biodistribution of human monoclonal antibody in tumor-xenografted SCID mice.

An ELISA technique was developed to assay the distribution of native human monoclonal antibodies (mAbs) in tumor-xenografted SCID mice. This was used in an investigation of its potential as an alternative to the conventional radioisotopic technique for mAb biodistribution assays which would be simpler to implement and might yield results in closer accord with actual mAb activity because it is based on the use and detection of the native mAbs rather than their radioisotope-coupled immunoconjugates. SCID mice bearing xenografted tumors of the human lung adenocarcinoma cell line A549 received injections via the tail vein of four human mAbs that had been obtained from human-mouse heterohybridomas and were known to be reactive with A549. The biodistribution of the mAbs was assayed by the ELISA technique seven days after the mAb injection. The assay yielded tumor/serum ratios for the four reactive mAbs which were in the range of three to six and tumor mAb levels which were in the range of 0.28 to 0.92 %ID/g (percent of injected mAb dose per gram of tumor). The tumor mAb levels were thus lower than the levels commonly found by radioisotopic assay, and further investigation is desirable to determine the cause of the difference. The results indicate that ELISA can provide a simple, practical means of investigating the biodistribution of human mAbs in mice bearing xenografted carcinomas. The application of this procedure would obviate the need for the complex facilities and procedures associated with radioisotopic labelling and assay.

Human antibodies responsible for binding inhibition and polymerization inhibition of human immunodeficiency virus type 1 reverse transcriptase.

Using a solid-phase non-radioisotopic (non-RI) reverse transcriptase (RT) assay, antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) RT activity (RTI antibody) were investigated for their ability to inhibit binding of RT to a template-primer and DNA polymerization. The RTI antibody inhibited the binding of RT to the template-primer (BI antibody), and directly reacted with the RT-template-primer complex and inhibited enzymatic activity (PI antibody). The RTI antibody interfered with formation of the RT-template-primer complex suggesting that it recognized the antigenic site involved in template-primer binding of RT molecules. Since deoxynucleotide triphosphates (dNTPs) blocked inhibition of the RT activity by the PI antibody, the antigenic site recognized by the PI antibody may be closely related to the dNTP binding site. The seropositivities of the BI and PI antibodies were 84.6% and 91.2%, respectively, in HIV-1-infected individuals; healthy individuals, HTLV-I-positive individuals, autoimmune disease patients and leukemia patients were all seronegative. No significant correlation of residual RT activities was observed when BI and PI antibodies were compared (r = 0.688). It is possible that the epitopes recognized by the BI antibody differs from those recognized by the PI antibody. The assays described are able to detect BI and PI antibodies in the sera of HIV-1-infected individuals.

Effect of PSC 833 on the cytotoxicity of idarubicin and idarubicinol in multidrug-resistant K562 cells.

We examined the effect of PSC 833, a non-immunosuppressive cyclosporin analogue, on the cytotoxicity, accumulation and retention of idarubicin (IDA) and its 13-dihydro metabolite, idarubicinol (IDAol). P-glycoprotein (PGP)-overexpressing multidrug-resistant K562/D1-9 cells were used for these studies. PSC 833 had no effect on the cytotoxicity, intracellular accumulation, or retention of IDA and IDAol in the parent K562 cells. However, intracellular accumulation of IDA and IDAol in K562/D1-9 cells after a 60-min incubation was restored by 0.4 microM PSC 833 to 104% and 116%, respectively, of the level in parent K562 cells. The retention of IDA and IDAol in K562/D1-9 cells was also restored by 0.4 microM PSC 833. Consequently, 0.4 microM PSC 833 increased the sensitivity of K562/D1-9 cells to IDA and IDAol. The resistance index (RI) of IDA decreased from 20-fold to 4.0-fold, and the RI of IDAol decreased from 104-fold to 1.5-fold. These results suggest that the combination of IDA and PSC 833 may be effective in reversing PGP-mediated multidrug resistance in leukemia cells.

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.

Purification and characterization of choline oxidase from Arthrobacter globiformis.

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.

Oxidative pathway of choline to betaine in the soluble fraction prepared from Arthrobacter globiformis.

One strain of bacteria which showed high H2O2-generating activity was isolated from soil and characterized as Arthrobacter globiformis based on its morphological, nutritional, and physiological characteristics. The activities of H2O2 generation, NAD reduction and oxygen consumption in the bacterial cells were examined using choline, betaine aldehyde or betaine as substrate. Choline was oxidized to betaine aldehyde under aerobic conditions in a reaction coupled with H2O2 generation and oxygen consumption. On the other hand, betaine aldehyde seemed to be oxidized to betaine through two distinct oxidative reactions, H2O2 generation (oxygen consumption) under aerobic conditions and NAD reduction under either aerobic or anaerobic conditions. These enzyme activities were found in the supernatant fraction of the sonicated cell preparation.

Surveillance of the serum Candida antigen titer for initiation of antifungal therapy after post-remission chemotherapy in patients with acute leukemia.

The early diagnostic efficacy of serial Candida antigen detection by the assay kit CAND-TEC (a latex particle agglutination test) was evaluated in 12 episodes in 10 patients with acute leukemia after post-remission chemotherapy. To determine the timing to initiate antifungal chemotherapy, we performed the CAND-TEC assay serially in each patient. When the patients revealed febrile neutropenia after antileukemic chemotherapy and the Candida antigen titer was increased compared to that measured before the antileukemic chemotherapy (even if the increased titer was at a lower level, e.g., from negative to 1:1 positive or from 1:1 to 1:2), azole antifungal agents (fluconazole or miconazole) were administered intravenously. In 9 (81.8%) of the 11 evaluable cases, the antifungal chemotherapy was effective and the titers decreased to less than or equal to the previous titers in all cases. In 2 cases, the antifungal chemotherapy was not effective, and the titers did not decrease. These results suggest that serial Candida antigen detection provides a useful method in the early diagnosis of invasive candidiasis in febrile neutropenia and in determining the timing of the initiation of early antifungal chemotherapy. This method might also be useful in preventing the excess use of antifungal agents; thus preventing the proliferation of azole-resistant Candida infection.

Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials.

An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described. Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases. Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed. Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured. The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT. The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively. To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay. The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance.

Behavioral significance of monkey hypothalamic glucose-sensitive neurons.

Feeding-related neuronal activity of lateral hypothalamic glucose-sensitive and glucose-insensitive neurons was investigated in behaving monkeys. The behavioral paradigm was a high fixed ratio of bar pressing for food reward signaled by light and tone cues. Twenty-seven percent of the neurons tested were glucose-sensitive. The population of neurons which changed in firing rate during the feeding task was higher among glucose-sensitive cells than among glucose-insensitive cells. The activity of many glucose-sensitive neurons decreased during the bar pressing and reward periods. A small population of glucose-sensitive neurons responded to cue stimuli. The results suggest that glucose-sensitive neurons are mainly involved in the drive and/or reward mechanism of feeding behavior, and that these cells may have specific roles in neural control of hunger-motivated food acquisition.

Enzyme reactor for urinary acylcarnitines assay by reversed-phase high-performance liquid chromatography.

An immobilized enzyme reactor, made up acylcarnitine hydrolase, carnitine dehydrogenase and diaphorase in sequence, was developed for the sensitive and selective determination of urinary free and individual acylcarnitines by a reversed-phase high-performance liquid chromatography. A 100-microliter urine sample was directly injected onto the TSKgel ODS 80Ts column and eluted by a step-gradient procedure. The eluent was mixed with the substrate solution of beta-NAD+ (1.0 mmol/l), resazurin (25 mumol/l) and Tris acetate (0.2 mol/l, pH 9.0). The mixture was passed through the immobilized enzyme reactor at 40 degrees C. Acylcarnitines were hydrolyzed and the converted to rezorufin which was measured by monitoring the fluorescence intensity at lambda EX = 560 nm and lambda EM = 580 nm. Free, acetyl-, glutaryl-, propionyl-, butyryl-, isobutyryl-, valeryl- and isovalerylcarnitine were determined within 55 min with detection limits (< 1 mumol/l) and within-run and day-to-day imprecision (C.V. < 6%). Free, acetyl- and isobutyrylcarnitine were found in normal urine. On the other hand, propionylcarnitine was detected in the urine of children with propionic aciduria and methylmalonic aciduria and multiple acylcarnitines were found in the urine of children with glutaric aciduria (type II).

Pre- and postnatal stimulation of pulmonary surfactant protein D by in vivo dexamethasone treatment of rats.

Fetal (days 18 and 20 of gestation), neonatal (days 0, 2 and 4 of neonate) and adult rats were injected with dexamethasone (1 mg/kg) in vivo and 24 hours later the effect on the contents of surfactant protein D (SP-D) in the rat lungs were examined in comparison with surfactant protein A, disaturated phosphatidylcholine and phosphatidylglycerol. In vivo dexamethasone treatment resulted in significant increases of SP-D content as the other 3 components of surfactant in both fetuses and neonates, but not in adults. Responsiveness to glucocorticoid treatment on SP-D content was maximum on day 1 of neonate (2.7 times control value). The contents of surfactant components examined tend to respond better to steroid in postnatal rats. These data demonstrated that glucocorticoid treatment in vivo for short durations exhibits the stimulatory effect on the contents of SP-D in the fetal and neonatal rat lungs.

[Studies for dynamics of reverse transcriptase inhibiting antibody in sera from HIV-1 infected individuals].

Antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) reverse transcriptase activity (RTI-antibody), Binding inhibition antibody (BI-antibody) and polymerization inhibition antibody (PI-antibody) were investigated for their ability to inhibiting RT activity in 248 HIV-1 infected individuals and 99 healthy individuals. In BI-antibody, high titer samples were determined more in than in RTI- and PI-antibodies. No significance was indicated between AC, ARC and AIDS is any antibody, however, progression from AC to AIDS was poled to high titer and low titer in RTI- and BI-antibodies. Moreover, time course of each antibody levels in the same infected individuals were resulted in no change, going up or down through all the experimental term, though all samples were collected in AC. These results were suggested that the determination factor of each stage in HIV progression would be multiple, and that the various dynamics of RTI-, BI- and PI-antibodies in the same infected individuals might be caused in the term from HIV infection to AIDS progression, prognosis or appearing of the drug resistant strain but stages of the disease.

An improved non-radioisotopic reverse transcriptase assay and its evaluation.

We developed an improved, highly sensitive non-radioisotopic (non-RI) reverse transcriptase (RT) assay (RTA). While the original non-RI method previously reported made use of primer immobilization, our improved method was based on a primer-template immobilization procedure. We tested the template specificity, reproducibility and linearity of the new method in assays of human immunodeficiency virus type-1 (HIV-1) RT. The sensitivities of the method previously reported, the improved method and the sensitive radioisotopic (RI-) RTA were compared in assays of recombinant HIV-1 RT, partially purified HIV-1 particles, and the culture supernatant derived from HIV-1-infected cells. For each of these samples except the culture supernatant the improved method was the most sensitive. It appeared that the fetal bovine serum presented in the culture medium interfered with the assay reaction. The curve describing inhibition of the assay reaction by fetal bovine serum showed that the highest degree of sensitivity in the assay was obtained when the culture supernatant sample was diluted four times. With this degree of dilution, the sensitivity of the new method for assay of culture supernatant sample was still half that of the sensitive RI-RTA. Culture supernatants of five peripheral blood mononuclear cell samples obtained from HIV-1-seropositive carriers were assayed by both the improved method and the sensitive RI-RTA; and with each of the methods, however all virus-positive cultures could be detected. The improved non-RI RTA was considered especially useful for assay of culture supernatants for purposes of virus isolation because of its advantages of excellent sensitivity and lack of requirement for radioisotopes.

[Ask-Upmark kidney: a case report].

A case of Ask-Upmark kidney is presented. An 18-year-old male patient referred to this facility presented with symptoms of hypertension, microscopic hematuria and proteinuria. A hormonal study revealed a high plasma renin activity level. Intravenous pyelography and abdominal computed tomography revealed thinning of the cortex with calyceal dilatation. Arteriography revealed a deep cortical groove in the middle portion of the kidney without renal arterial stenosis. Plasma renin activity of the left renal vein was significantly higher than that of the right renal vein. A left simple nephrectomy was performed under the diagnosis of Ask-Upmark kidney. Postoperatively, plasma renin activity returned to the normal range and a decrease in blood pressure was noted. Recent reports have suggested Ask-Upmark kidney to be a consequence of vesicoureteral reflux rather than a true congenital malformation. Our case indicated no evidence of vesicoureteral reflux and suggests that the lesion was congenital rather than acquired.

[Bellini duct carcinoma of kidney with invasive growth pattern: a case report].

We report a case of Bellini duct carcinoma of the left kidney with invasive growth pattern. A 39-year-old man was admitted to our hospital with the chief complaint of gross hematuria. Ultrasonography showed left renal swelling but normal reniform configuration of the kidney was maintained. Computed tomography demonstrated a low density tumor infiltrating into the renal cortex and with tumor extension into the renal vein. Renal angiography revealed a hypovascular tumor. We suspected a left renal cell carcinoma with tumor extension into the left renal vein, and performed radical nephrectomy. Macroscopically, the resected kidney had a normal outer contour. The tumor with infiltrative growth pattern existed in renal medulla. Histopathologic examination revealed a papillary adenocarcinoma originated in Bellini duct (pT3bN2M0). The patient underwent systemic chemotherapy (M-VAC). This case showed invasive growth pattern, which were different from the usual renal cell carcinoma and Bellini duct carcinoma.

[Treatment of scrotal hydrocele using fibrin adhesive].

For the purpose of adhesion of the tunica vaginalis in scrotal hydrocele, we used fibrin adhesive, Bolheal. Between September 1993 and December 1994, 13 patients with scrotal hydrocele received occlusion therapies using Bolhead. The therapy was effective in 1 of the 6 patients (17%) administered 2.4 mg of fibrinogen and 750 units of thrombin into the vaginal cavity after aspiration of fluids, and in 5 of the 7 patients (71%) administered 240 mg of fibrinogen and 7.5 units of thrombin. Therefore, fibrin adhesive may be useful in the treatment of scrotal hydrocele.