Improved analysis of hemagglutination assays for quantitation of lectin activity

A method to quantitate lectin activity based on hemagglutination assay in microtiter plates is described. In addition to the normal method of visual titer evaluation an electronic particle counter is used for counting of nonagglutinated cells in the microtiter wells; this allows a rapid, quantitative determination of the amount of lectin required to agglutinate 50% of the countable single cells. It is also recommended that counting results should be related to a standard curve of concanavalin A to improve the reproducibility of the assay.     

Cynoglossopsis somaliensis,sp. nov., und die GattungCynoglossopsis (Boraginaceae)

Cynoglossopsis somaliensis H. Riedl, sp. nov., is described as a new species of the hitherto monotypic genusCynoglossopsis Brand which is closest related toCynoglossum L. but has to be included inBoraginoideae-Eritrichieae from the way the nutlets are attached to the gynobasis.

     

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol

Summary Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HC1 (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour for 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

1H NMR studies at 360 Mhz of the methyl groups in native and chemically modified basic pancreatic trypsin inhibitor (BPTI)

In the ¹H NMR spectra obtained at 360 MHz after digital resolution enhancement, the multiplet resonances of the methyl groups in the basic pancreatic trypsin inhibitor (BPTI) were resolved. With suitable double irradiation techniques the individual methyl resonances were assigned to the different types of aliphatic amino acid residues. Furthermore, from pH titration and comparison of the native protein with chemically modified BPTI, the resonance lines of Ala 16 in the active site and Ala 58 at the C-terminus were identified. Potential applications of the resolved methyl resonances as natural NMR probes for studies of the molecular conformations are discussed.     

Verwertung von Trimethylammoniumverbindungen durch Acinetobacter calcoaceticus

Zusammenfassung Die Verwertung von Carnitin und Carnitinderivaten (O-Acylcarnitine, Carnitincarboxyl-derivate) und strukturverwandten Trimethylammoniumverbindungen (Betaine und Stickstoffbasen) durch Acinetobacter calcoaceticus wurde anhand des Wachstums und des quantitativen Nachweises der Metabolite untersucht. Der Stamm wuchs auf l-Carnitin, l-O-Acylcarnitinen und -Butyrobetain als jeweils einziger C-Quelle. Der Verbrauch dieser Verbindungen und das Wachstum korrelierten mit der Spaltung der C-N-Bindung und mit dem gebildeten Trimethylamin. d-Carnitin wurde metabolisiert, wenn als zusätzliche C-Quelle l-Carnitin im Nährmedium vorhanden war, oder wenn die Bakterien mit l-oder dl-Carnitin vorinkubiert worden waren. Mit d-Carnitin als einziger C-Quelle wuchsen die Bakterien jedoch nicht. Die Bakterien oxidierten Cholin zu Glycinbetain in Gegenwart einer zusätzlichen C-Quelle, Glycinbetain selbst wurde nicht assimiliert. In Hinsicht auf den Abbau quaternärer Stickstoffverbindungen besitzt Acinetobacter calcoaceticus im Vergleich zu anderen Carnitin-verwertenden Bakterienarten einen für ihn charakteristischen Stoffwechselweg.

---

Utilization of trimethylammonium-compounds by Acinetobacter calcoaceticus

The utilization of carnitine and carnitine derivatives (O-acylcarnitines, carnitine carboxylderivatives) and structure-related trimethylammonium-compounds (betaines and nitrogen-bases) by Acinetobacter calcoaceticus was studied by means of the control of growth and the quantitative detection of metabolites. The strain grew only on l-carnitine, l-O-acylcarnitines, and -butyrobetaine as the sole carbon sources. The utilization of these compounds and the growth correlated with the cleavage of the C-N bond and thereby with the formation of trimethylamine. d-Carnitine was metabolized, if an additional carbon source, like l-carnitine, was present in the incubation mixture, or if the bacteria were preincubated with l-or dl-carnitine, but no growth was observed on d-carnitine as the sole carbon source. The bacteria oxidized choline to glycinebetaine in the presence of additional carbon sources, glycinebetaine itself was not assimilated. With regard to the catabolism of quaternary nitrogen compounds Acinetobacter calcoaceticus shows a different pathway in comparison with other bacterial species metabolizing carnitine.

     

Progesterone in the uterus. VIII. Uptake of corticosterone and incorporation of progesterone under concurrent injection of corticosterone into rat uterus

A study of the subcellular distribution of radioactivity in rat uterus after injection of labelled corticosterone showed that the radioactivity was observed in all fractions from 5 min. to 120 min. A maximum uptake was observed 10 min. after application of the labelled steroid. Competitive uptake of radioactive progesterone and unlabelled corticosterone was assayed 10 min. after injection of the hormone mixture. The ratio between radioactive progesterone and unlabelled corticosterone was 1 : 1 and 1 : 2 (moles:moles), respectively. Compared with control experiments with rats which had received radioactive progesterone alone, the results gave evidence that progesterone found in all subcellular fractions and in the total homogenate was not depressed by unlabelled corticosterone. However, unlabelled progesterone reduced the tritiated progesterone in uterine tissue. This observation demonstrates that the uptake of progesterone by rat uterus is specific.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Determination of clozapine and its major metabolites in human serum using automated solid-phase extraction and subsequent isocratic high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatographic (HPLC) method with ultraviolet detection is described for the quantification of the atypical neuroleptic clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum or plasma. The method included automated solid-phase extraction on C₁₈ reversed-phase material. Clozapine and its metabolites were separated by HPLC on a C₁₈ ODS Hypersil analytical column (5 μm particle size; 250 mm × 4.6 mm I.D.) using an acetonitrile—water (40:60, v/v) eluent buffered with 0.4% (v/v) N,N,N′,N′-tetramethylethylenediamine and acetic acid to pH 6.5. Imipramine served as internal standard. After extraction of 1 ml of serum or plasma, as little as 5 ng/ml of clozapine and 10 or 20 ng/ml of the metabolites were detectable. Linearity was found for drug concentrations between 5 and 2000 ng/ml as indicated by correlation coefficients of 0.998 to 0.985. The intra- and inter-assay coefficients of variation ranged between 1 and 20%. Interferences with other psychotropic drugs such as benzodiazepines, antidepressants or neuroleptics were negligible. In all samples, collected from schizophrenic patients who had been treated with daily oral doses of 75–400 mg of clozapine, the drug and its major metabolite, N-desmethylclozapine, could be detected, while the concentrations of clozapine N-oxide were below 20 ng/ml in three of sixteen patients. Using the method described here, data regarding relations between therapeutic or toxic effects and drug blood levels or metabolism may be collected in clinical practice to improve the therapeutic efficacy of clozapine drug treatment.