In vitro translocation of periplasmic and outer membrane proteins into inverted plasma membrane vesicles from Escherichia coli was completely prevented by the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD). DCCD was inhibitory to both co- and post-translational translocations, suggesting an involvement of the H+-translocating F1F0-ATPase in either mode of transport. This was verified by (i) the dependence of efficient co-translational translocation upon a low salt, i.e. F1-containing extract from membrane vesicles; (ii) the co-purification of the translocation activity present in this extract and F1-ATPase; (iii) the inability of either vesicles or their low-salt extract, derived from F1F0-ATPase-lacking mutant strains, to support translocation; and (iv) the greatly diminished extent of ATP-dependent, post-translational translocation into F1-deprived vesicles. Membranes devoid of F1 did show, however, residual translocation activity that was also found to be inhibitable by DCCD. These results suggest a dual target for DCCD in bacterial protein export, one being the H+-ATPase and the other an as yet unidentified translocation factor.

Bacterial Outer Membrane Proteins↗

PubMed1987

Anti-tumor effects of tumor necrosis factor in combination with chemotherapeutic agents.

The anti-tumor activity of partially purified tumor necrosis factor (TNF) was analysed in combination with chemotherapeutic drugs against intradermally transplanted Meth A sarcoma. Tumors were allowed to grow until they reached an average diameter of about 8 mm. TNF was given once i.v. (16,000 units as determined on L-M cells) and the chemotherapeutic agents adriamycin (1-10 mg/kg), 5-FU (3-100 mg/kg), cyclophosphamide (50-200 mg/kg) were applied once i.p. 4 hr after TNF injection. The strongest anti-tumor effects in normal BALB/c mice were observed when TNF was combined with the following doses of chemotherapeutic agents: cyclophosphamide 100 mg/kg, adriamycin 5 mg/kg, 5-FU 30-100 mg/kg. The most effective combinations induced complete regressions. When TNF was combined with varying doses of adriamycin and cyclophosphamide, bell-shaped dose-response curves were obtained. Experiments were repeated in Meth A sarcoma-bearing BALB/c nu/nu mice. In this case TNF combinations with cyclophosphamide and 5-FU did not induce tumor regressions. The highest dose of the chemotherapeutic agent was the most effective in drug combinations. Histological analysis revealed a potentiation of the TNF-induced necrosis by cyclophosphamide. Increased hyperemia and extravasation of erythrocytes could be found in tumors of animals treated with the drug combination.

Animals↗

PubMed1987

[Prevention of kinetosis in children. Transdermal therapy with scopolamine].

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Administration, Cutaneous↗

PubMed1987

Intracellular neutral carrier-based Ca2+ microelectrode with subnanomolar detection limit.

In intracellular electrolyte solutions a Ca2+-selective microelectrode based on the synthetic electrically neutral carrier N,N,N',N'-tetracyclohexyl-3-oxapentanediamide (ETH 129) shows an improved detection limit when compared with the so far widely used Ca2+ microelectrodes based on the neutral carrier ETH 1001. Detection limits are found at pCa = 9.2 in Ca2+ buffers containing an intracellular background of K+ (125 mM). Selectivity studies in mixed solutions show a preference of Ca2+ over Na+ of 6 X 10(5), over K+ of 1.6 X 10(6), and over Mg2+ of 5 X 10(6). The microelectrode does not suffer from significant interference by inorganic and organic inhibitors and by lipophilic cations and anions. The low detection limit is unchanged at least during the first eight hours of continuous contact with Ca2+ solutions. The EMF drift during the first hour of use is between 5 and 10 mV and is then reduced to about 1 mV/h. The changes in EMF induced between solution of pCa = 7 and pCa = 8 are reproducible within 24.7 +/- 0.4 mV (SD, n = 8, about 3 h). These electrode characteristics were found for single-barrelled microelectrodes of one micrometer diameter front-filled with a PVC-containing membrane phase. In the absence of poly(vinyl chloride) in the membrane phase irregular EMF response curves were obtained throughout. Preliminary punctures of ferret ventricular muscle cells indicate that the Ca2+ electrode response is not disturbed by the contact of a cytosolic milieu.

Animals↗

PubMed1987

Drug monitoring of quinine by HPLC in cerebral malaria with acute renal failure treated by haemofiltration.

The monitoring of quinine by HPLC in 3 patients suffering from cerebral malaria with acute renal failure and treated by haemofiltration is reported. The recommended dose of quinine in this situation is reduced to 10 to 15 mg.kg-1.day-1. However, in the first patient, when given quinine 10 mg kg-1.day-1 the plasma concentration was mainly below the recommended therapeutic range of 5 to 15 mg/l. In consequence, the dose of quinine in the second patient was elevated to quinine dihydrochloride 15.1 mg.kg-1.day-1 which produced plasma concentrations in the low therapeutic range. In the third patient, an unreduced dose of quinine dihydrochloride 25.7 mg.kg-1.day-1 was employed, resulting in plasma concentrations above 15 mg/l, which is generally assumed to be toxic, although, no sign of acute quinine toxicity was seen. The antimalarial effect in all three patients was satisfactory. Quinine was estimated in the haemofiltrate in two patients and was found to be below the limit of sensitivity (0.25 mg/l). Plasma quinine did not change during or shortly after haemofiltration. It is concluded that in case of acute renal failure in cerebral malaria the dose of quinine should be reduced, but that the common recommendation of 10 to 15 mg.kg-1.day-1 may be too low, and that haemofiltration has no marked influence on the total body clearance of quinine.

Acute Kidney Injury↗

PubMed1987

Cytoplasmic microvesicles in chromophobe cell renal carcinoma demonstrated by freeze fracture.

In the chromophobe cell type of renal carcinoma, cytoplasmic microvesicles (frequently with "inner vesicles") demonstrable by transmission electron microscopy are one of the most important diagnostic features. The present paper reports on these microvesicles in freeze fracture replicas. Their diameter is mainly between 140 and 300 micron, but smaller and very much larger vesicles may also occur. The vesicle membrane is devoid of, or contains only scanty intramembranous particles. Cytoplasmic invaginations, probably the precursors of "inner vesicles" can also be detected. Connections with the agranular endoplasmic reticulum, mitochondria or other cell components could not be documented. Larger vacuoles limited by a membrane and containing large numbers of microvesicles have been interpreted as autophagic vacuoles. In freeze fracture replicas, there are marked differences between the chromophobe cell and the clear cell type of renal carcinoma, providing further evidence that the chromophobe cell type is a distinct entity.

Carcinoma, Renal Cell↗

PubMed1987

Isolation and partial characterization of the cytochrome c oxidase of Micrococcus luteus (lysodeikticus).

The cell membrane of Micrococcus luteus (lysodeikticus) contains a respiratory chain composed of hemes a, b, and c, which contain 171, 457, and 407 pmol/mg protein, respectively. Cytochrome c oxidase, the heme a containing component, has been purified after solubilization in Triton X-100, by gel filtration on Sepharose 4B-CL ammonium sulfate precipitation and ion-exchange and affinity chromatographies on a yeast cytochrome c-Sepharose 4B column. The purified complex, which contains three polypeptides of apparent Mr 47,000, 31,000, and 19,000, has CN-sensitive ferrocytochrome c oxidase activity (Ki = 0.35 microM) and a characteristic absorption spectrum with maxima in the oxidized form at 595 and 426 nm and in the reduced form at 601 and 444 nm. The purified enzyme contains 17.4 nmol/mg protein and its copper content is 23.2 nmol/mg protein. The enzyme was purified about 100-fold with respect to its content in crude membranes. The total heme a yield, also with respect to crude membranes content, was 6.8%.

Chromatography, Ion Exchange↗

PubMed1987

Hydrogenosomal ATP:AMP phosphotransferase of Trichomonas vaginalis.

1. ATP:AMP phosphotransferase (adenylate kinase) is present in Trichomonas vaginalis, primarily with hydrogenosomal localization. 2. Adenylate kinase has been purified from hydrogenosome-enriched fractions by solubilization with Triton X-100 and KCl followed by affinity chromatography and gel filtration. 3. The enzyme has a Mr = 28,000, a broad pH optimum of pH 7-9, requirement for Mg2+ and specificity for adenine and deoxyadenine nucleotides. 4. The activity is competetively inhibited by P1,P5-di(adenosine-5') pentaphosphate (Ki 200 nM) and reversibly inactivated by 5,5'-dithiobis-(2-nitrobenzoate). 5. Catalytic properties of this enzyme are similar to those of enzymes from other organisms. Other properties indicate its uniqueness, however, since its molecular mass and Ki for P1,P5-di(adenosine-5'-)-pentaphosphate bring it closer to the mitochrondrial isoenzyme, while it shares a requirement for reduced thiol groups with the cytosolic isoenzyme.

Adenylate Kinase↗

PubMed1987

High level expression of the EcoP1 modification methylase gene and characterisation of the gene product.

We have cloned the gene coding for the EcoP1 modification methylase in an expression system based on the phage lambda pL promoter and the cI857-coded thermoinducible repressor. We have used this system to purify the enzyme on the 20-30-mg scale and have examined some of its enzymatic properties. The enzyme is a tetramer of Mr 72,000 subunits and is approx. 40% alpha-helical. Experiments with the methyl donor, S-adenosyl methionine, radioactively labelled in different positions indicate that a methyl group is transferred to the enzyme during the reaction in what is most likely a covalent bond.

Cloning, Molecular↗

PubMed1987

Immunohistochemical localization of L-ornithine decarboxylase in developing rat brain.

L-Ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis and a marker enzyme of tissue proliferation and maturation, was localized immunocytochemically in the developing rat central nervous system. It can be noted that the distribution of the enzyme protein underlies temporal alterations. Conclusions are drawn from the localization of the enzyme and possible functional roles played by ornithine decarboxylase in discrete brain areas.

Animals↗

PubMed1987

The uprighting effect of the Fränkel appliance on the mandibular canines and premolars during eruption.

The effect of the Fränkel (FR) appliance on the eruptive path of the mandibular canines and premolars was evaluated in patients who were monitored from 8 to 13 years of age. The sample treated with the FR appliance consisted of 84 subjects with Class I or Class II malocclusion associated with crowding in the mandibular dental arch. Twenty-seven subjects of approximately the same age and with almost an identical space deficiency in the mandibular arch not subjected to FR treatment served as a comparison group. Permanent teeth were not extracted during treatment in any of the subjects in either group. The primary criterion of patient selection was based on the availability of standardized serial lateral and posteroanterior cephalograms, permitting an exact identification of the canine and premolar contours during eruption. Increases in width between the tips of the mandibular canines and the buccal tips of the mandibular premolars during eruption were significantly greater in the FR group than in the comparison group (P less than 0.001). The expanding effect of the FR appliance was evidenced particularly in the region of the mandibular first premolars. The distance between the buccal tips of these teeth increased by an average of 3.5 mm more in the FR group than in the comparison group. Likewise, the width between the root landmarks of the first mandibular premolars increased during eruption in the FR group 2 1/2 times more than in the untreated sample, a difference that was statistically significant (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Activator Appliances↗

PubMed1987

Spontaneous missense mutations in the rplX gene for ribosomal protein L24 from Escherichia coli.

Temperature-resistant pseudorevertants of the temperature-sensitive Escherichia coli mutant KNS19, harboring a mutation in rplX, the gene for ribosomal protein L24, were isolated, cloned, and sequenced. The codon GAC for the amino acid Asp in the temperature-sensitive mutant corresponding to position 84 in the protein chain mutated either back to the wild type (Gly) or to codons for the amino acids Tyr and Glu. Furthermore, rplX genes from two other mutants with an altered protein L24 were cloned and sequenced. The mutations were localized at position 56 (Gly to Asp) and at position 62 (Glu to Lys) in the rplX gene. The latter two mutants lacked a conditional lethal phenotype. The results suggest that the amino acid Gly at positions 56 and 84 in the protein might be involved in loop formations.

Amino Acid Sequence↗

PubMed1987

Analysis of carcinoembryonic antigen determinants by monoclonal antibodies and lectins.

Two out of five determinants recognized by monoclonal antibodies (MoAbs) on carcinoembryonic antigen (CEA) were analyzed by blocking the CEA-MoAb interaction with 9 different lectins in a solid phase radioimmunoassay. Two batches of CEA were first studied for their binding to lectins at the solid phase. Lotus, soybean and wheat germ lectins were most active, but the reaction pattern was different with the two CEA batches. The corresponding sugars were capable of inhibiting the lectin-CEA binding. MoAbs were fixed to the solid phase, and their reaction with CEA was tried to be blocked by preincubating the antigen with lectins. Lotus lectin and wheat germ agglutinin were clearly blocking the MoAb-CEA interaction, but this was strikingly dependent on the CEA batch used and the particular CEA determinant investigated. These reactions could not be blocked by sugars. The data provide evidence for a role of sugars in the sterical configuration of CEA determinants. In view of the heterogeneity of the findings, however, the conclusion is drawn that the protein moiety plays the more important role in the configuration of the CEA determinants studied.

Antibodies, Monoclonal↗

PubMed1987

Effect of substrate heat-denaturation on ATP- and ubiquitin-dependent proteolysis.

The susceptibility of human serum albumin (HSA) to degradation by the ATP- and ubiquitin-dependent proteolytic system is enhanced by heat-denaturation. The steady-state levels of formed ubiquitin conjugates, however, are equal with denatured and native HSA. In contrast, both the hemin sensitive Pi release and proteolysis with native HSA are less than one fourth of that with denatured HSA. The rate limiting step for the degradation of HSA by the ATP- and ubiquitin-dependent proteolytic system must be beyond the ubiquitin conjugation steps. Differences concerning the effects of heat denaturation of mitochondria-containing stroma and HSA on ATP- and ubiquitin-dependent proteolysis are discussed.

Adenosine Triphosphate↗