Comparative studies with cyclic and linear somatostatin on active avoidance behaviour and open-field activity in rats

The action of cyclic and linear somatostatin on active avoidance and open-field behaviour was investigated in rats. Both peptides inhibited the extinction of the avoidance behaviour and slightly but not significantly increased the intertrial activity (ITR). Both peptides increased ambulation and rearing activity in a dose related fashion, while the grooming and defecation rates were not changed. Since both peptides are capable of increasing the locomotor performance this action might contribute to the effect exerted on the extinction of active avoidance behaviour.     

Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum

Zusammenfassung Es wird die 25–30 fache Anreicherung einer löslichen NADH-Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.) aus R. rubrum durch Gelfiltration an Sephadex G 200 und Chromatographie an DEAE-Cellulose beschrieben. Das Enzym ist bei DEAE-Cellulosechromatographie nur in Gegenwart von FMN oder NADH stabil. Menadion, Ferricyanid, DCPIP, p-Benzochinon und Cytochrom c sind als Elektronenacceptoren wirksam. Cytochrom c ist ein schlechter Acceptor. Das pH-Optimum der Reaktion liegt bei 8,4. Km für NADH ist 3,0 · 10^-5 m. NADPH wird nur mit etwa 3–5% des Wertes von NADH umgesetzt. Die prosthetische Gruppe des Enzyms ist FMN, Km für FMN ist 0,3 · 10^-7 m. Das Enzymprotein wird bei Verdünnung in 0,05 m Puffer inaktiviert; FMN und FAD sowie NADH und NADPH haben einen stabilisierenden Effekt. Durch höhere Pufferkonzentrationen wird das Enzym zunehmend inaktiviert. Die Inaktivierung besteht in einer Labilisierung oder Abspaltung der prosthetischen Gruppe vom Enzymmolekül. Verschiedene Metalle inaktivieren das Enzym ebenfalls.

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Oxidation of reduced nicotinamide adenine dinucleotide in Rhodospirillum rubrum I. Characterization of a soluble NADH dehydrogenase

Summary A soluble NADH^* Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.] has been purified 25–30 fold from R. rubrum by gelfiltration with Sephadex G 200 and ionexchange chromatography on DEAE-Cellulose. During the second purification step the enzyme is stable only in the presence of either FMN or NADH. Electronacceptors which were found to be effective include menadione, ferricyanide, DCPIP, p-benzoquinone and cytochrome c, the latter substance being a poor acceptor. The optimum pH of the reaction is at about 8.4. Km for NADH is 3.0×10^-5 m. NADPH is oxidized at only about 3–5% the rate of NADH. The active prosthetic group of the enzyme is FMN with an appearant Km of 0.3×10^-7 m. When diluted in 0.05 m buffer the enzyme becomes rapidly inactivated. FMN, FAD and also NADH and NADPH exhibit a stabilizing protective effect. Higher concentrations of buffer cause increasing inactivation. The mechanism of the inactivation is thought to be a labilization or detachment of the prosthetic group from the enzyme molecule. Several metals also inactivate the enzyme.

     

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.     

The effect of beta-(Tyr9)melanotropin-(9-18) on active avoidance behavior, electroconvulsive shock-induced amnesia and T-discrimination learning of rats

The effect of beta-(Tyr9)melanotropin-(9-18) was investigated on active avoidance behavior, electroconvulsive shock (ECS)-induced amnesia and T-discrimination learning in rats. The decapeptide inhibited the extinction of active avoidance behavior. It was also able to block ECS-induced amnesia if the treatment was performed immediately, or 4 hr or 20 hr after the ECS. In the T-discrimination paradigm the peptide facilitated spatial discrimination learning and reversal learning. These results suggest that beta-(Tyr9)melanotropin-(9-18) can influence learning and memory processes in different behavioral tests.     

Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum

Summary An active form of ApoNADH Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3] from R. rubrum can be obtained by incubating the apoenzyme at 20° C without flavin. Subsequent lowering the temperature to 0° C decreases the activity to the original level. The whole process can be repeated.The addition of flavin stabilizes the active form, so that no decrease is observed after the temperature has been dropped to 0° C. Here FMN is 30–40 times more effective than FAD. The activated apoenzyme, containing FMN, is again the normal functional form of NADH Dehydrogenase, showing its properties.It is assumed, that during the preparation of the apoenzyme the prosthetic group is not removed from the proteinmolecule but is altered in its binding to or in its position at the molecule. This leads to an equilibrium between a form of low and of higher activity of the apoenzyme, which, by changing the temperature, can be shifted to either side.It is possible to remove the prosthetic group still present at the apoenzyme by dialysis or by gelfiltration with Sephadex. The resulting protein can now be activated only in the presence of flavin.

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Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum II. Über eine reversible, temperaturabhängige Aktivierung der ApoNADH-Dehydrogenase

Zusammenfassung Das Apoenzym der NADH-Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.] aus R. rubrum kann durch Inkubation bei 20° C ohne Flavin in eine aktive Form übergeführt werden. Durch Absenken der Temperatur auf 0° C wird die wenig aktive Form wiederhergestellt. Der gesamte Vorgang kann beliebig wiederholt werden.Flavin stabilisiert die aktive Form, so daß bei Wechsel der Temperatur auf 0° C keine Abnahme der Aktivität mehr eintritt. Dabei hat FMN etwa 30–40fach größere Wirksamkeit als FAD. Das aktivierte, FMN enthaltende Apoenzym ist wieder die normale funktionsfähige Form der NADH-Dehydrogenase und zeigt deren Eigenschaften.Es wird vermutet, daß bei der Herstellung des Apoenzyms die prosthetische Gruppe in ihrer Bindung oder Lage am Enzymmolekül verändert, jedoch nicht abgespalten wird. Daraus resultiert der Zustand eines durch Temperaturwechsel verschiebbaren Gleichgewichtes zwischen einer wenig aktiven und einer aktiven Form des Apoenzyms.Die noch am Apoenzym befindliche prosthetische Gruppe kann durch Dialyse oder Gelfiltration mit Sephadex entfernt werden. Das Apoenzym ist jetzt nur noch bei Zusatz von Flavin aktivierbar.

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Verwendete Abkürzungen NADH reduziertes Nicotinamid-Adenin-Dinucleotid - FMN Flavinmononucleotid - FAD Flavin-adenin-dinucleotid     

Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens strain PIV-1

Quaternary carbon-containing compounds exist in natural and fossil oil-derived products and are used in chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment. Here, we investigated the unknown degradation of the quaternary carbon-containing model compound pivalate (2,2-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens strain PIV-1 (formerly Thauera pivalivorans). We provide multiple evidence for a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed similar to the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA and the cleavage of 3-methyl-3-hydroxyglutaryl-CoA to acetyl-CoA and acetoacetate. The completed genome of Thauera humireducens strain PIV-1 together with proteomic data was used to identify pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. A pivalate-induced gene encoding a putative carboxylic acid CoA ligase was heterologously expressed, and its highly enriched product exhibited pivalate CoA ligase activity. The results provide the first experimental insights into the biodegradation pathway of a quaternary carbon-containing model compound that serves as a blueprint for the degradation of related quaternary carbon-containing compounds.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.     

A cluster of proton/amino acid transporter genes in the human and mouse genomes

We recently cloned and functionally characterized two novel proton/amino acid transporters (PAT1 and PAT2) from mouse. Here we report the isolation of the corresponding cDNAs of the human orthologues and one additional mouse and human PAT-like transporter cDNA, designated PAT3. The PAT proteins comprise 470 to 483 amino acids. The mouse PAT3 mRNA is expressed in testis of adult mice. In the human and mouse genomes the genes of the PAT transporters (designated SLC36A1-3 and Slc36a1-3, respectively) are clustered on human chromosome 5q33.1 and in the syntenic region of mouse chromosome 11B1.3. PAT-like transporter genes are present as well in the genomes of other eukaryotic organisms such as Drosophila melanogaster and Caenorhabditis elegans. For the PAT3 subtype transporter, we could not yet identify its function. The human PAT1 and PAT2 transporters when functionally expressed in Xenopus laevis oocytes show characteristics similar to those of their mouse counterparts.