Nature and properties of humic acid prepared from different sources and its effect on nutrient availability

Summary Effect of sources, moisture levels and extractants on extraction, analytical properties, IR-spectra, macromolecular characteristics, interaction with cations and physiological properties of the extracted material (humic acid) were studied. Among the three variables, extractant influenced the degree of humification, IR-spectra, nature and properties of humic acids to a greater extent though the other two had significant contribution. Sodium pyrophosphate found to be an ideal extractant for humic acid. Reduced viscosity of humic acids increased with dilution particularly below 0.1 g/dl and decreased with increasing H⁺ concentration between pH 6.5 and 8.5 irrespective of source, moisture level and extractant. The amount of proton released and drop in initial pH of humic acids on addition of different metal ions indicated formation of mono-, di-hydroxy metal-HA complexes which broke down at pH 8.0 and above. HA-Fe²⁺ and HA-Cu²⁺ complexes were found to be most stable. Humic acids from different sources varied significantly in their effect on available nutrient status of soils though, the latter is main determining factor particularly for calcium and magnesium.     

A cholinergic receptor site on murine lymphocytes with novel binding characteristics

To further analyze functionally important cholinergic receptors on lymphocytes, we studied the binding of the muscarinic antagonist Quinuclidinyl benzilate (QNB) to murine splenic lymphocytes. Studies of displacement of [³H]QNB by unlabelled QNB on lymphocytes revealed at least two binding sites. Scatchard analysis of equilibrium binding isotherms also distinguished two sites with apparent Kds of 480 nM and 16 μM. There was greater specific QNB binding to B cell-enriched lymphocyte fractions than to T cell fractions. Lymphocyte binding demonstrated temperature-dependent dissociability, and specific binding occurred on isolated lymphocyte membranes as well. Both muscarinic and nicotinic ligands competed for QNB binding to lymphocytes with low and nearly equal affinity. Therefore, QNB binding sites on lymphocytes appear to be of low affinity and of mixed muscarinic and nicotinic character.     

Molecular asymmetry in alkaline phosphatase of Escherichia coli

Thermal inactivation of alkaline phosphatase of Escherichia coli has been studied at different temperatures (45 to 70 degrees C) and pHs (7.5, 9.0, and 10.0) for the commercial, buffer-dialyzed (pH 9.0) and EDTA-dialyzed (pH 9.0) enzymes. In each case, the inactivation exhibits biphasic kinetics consistent with the rate equation, (formula; see text) where A0 and A are activities at time zero and t, and k1 and k2 are first-order rate constants for the fast and slow phase, respectively. Values of k1 and k2 change independently with temperature, pH, and pretreatment (dialysis) of the enzyme. Time course of inactivation of the enzyme with excess EDTA and effect of Zn2+ ion concentration on the activity of EDTA-dialyzed enzyme have been investigated. The data suggest that the dimeric enzyme protein has two types of catalytic sites which have equal catalytic efficiency (or specific activity) but differ in several other properties. Structural implications of these results have been discussed.     

Effect of adaptation to high altitude on components of blood and urine of lowlanders compared with high altitude natives

Some of the blood and urinary constituents, oral glucose tolerance and urea clearance were determined in lowlanders at sea level (200 m) and at an altitude of 4, 000 m after their stay of two years. These data were compared with those of natives of high altitude area. The concentration of proteins, cholesterol, creatinephosphokinase and aspartate aminotransferase in blood among lowlanders after 2 year acclimatization were similar to that observed among highlanders. The urinary excretion of creatine and creatinine was of similar magnitude in highlanders and in acclimatized lowlanders but that of 17-keto and 17-hydroxysteroids was higher among highlanders. High altitude acclimatization among lowlanders facilitated appearance of a sharp peak in oral glucose tolerance curves and a decreased fasting blood glucose values. It also induced a restriction in renal filtration as indicated by a depressed urea clearance among lowlanders.     

Protection by Desferrioxamine Against Histopathological Changes of the Liver in the Post-Oligaemic Phase of Clinical Haemorrhagic Shock in Dogs: Correlation with Improved Survival Rate and Recovery

Haemorrhagic shock was produced in anaesthetized dogs, by rapid arterial bleeding to mean arterial blood pressure 35 mmHg, and maintained oligaemic for 4 h followed by return of withdrawn blood(R0WB). Dogs were observed for 72 h after ROWB for survival and recovery, and, for histopathological (HP) studies on liver, dogs were sacrificed 2 h after ROWB in non-survival experiments. Desferrioxamine mesylate (25mg/kg) was administered intra-muscularly at 2,3 and 4h after blood loss in survival experiments and for HP studies the drug was given at 4 h in one group and at 2 h plus 4 h after blood loss in the second group. With the drug given at 3 or 4h, survival was 70% and 100% while in the 2h and the untreated groups it was 50%. Recovery was rapid in all the drug treated survivors, few became conscious within 30min. showed slight activity by 4-6 h, all were almost normally active by 24 and fully so by 72 h after ROWB. All the 5 control survivors remained unconscious/drowsy upto 24 h; 3 were sluggish at 72 h. By group analysis, serum iron elevation during the oligaemic and at the end of the post-oligaemic phase was less in the drug-treated animals. HP changes of shock in the liver studied by light microscopy, were markedly reduced in severity and were less prevalent in the drug-treated dogs. The salutory effects of desferrioxamine may be due to inhibition of iron catalyzed free-radical production and tissue damage, through its strong iron chelating action. It may have a therapeutic advantage in this emergency condition without the disadvantages of toxicity inherent in prolonged use.     

Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone.

An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.     

Characterization and molecular cloning of thermostable alpha-amylase from Streptomyces sp.To1

Summary A new thermophilic Streptomyces sp. TO1, isolated from Tunisian soil, produced a thermostable alpha-amylase and pullulanase. The gene encoding for the alpha-amylase activity was cloned into the multicopy cloning plasmid pLM1 using S. lividans ZX1 as host strain. The ZX1 / pLM1 strain has the same activity than the initial TO1 strain and about 25 fold higher activity than the ZX1 strain. This alpha-amylase has an optimum of pH and temperature at 6 and 70 °C respectively.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

Effects of tRNALeu1 overproduction in Escherichia coli

Strains of Escherichia coli have been produced which express very high levels of the tRNA^leu₁ isoacceptor. This was accomplished by transforming cells with plasmids containing the leuV operon which encodes three copies of the tRNA^Leu₁ gene. Most transformants grew very slowly and exhibited a 15-fold increase in cellular concentrations of tRNA^Leu₁ As a result, total cellular tRNA concentration was approximately doubled and 56% of the total was tRNA^Leu₁. We examined a number of parameters which might be expected to be affected by imbalances in tRNA concentration: in vivo tRNA charging levels, misreading, ribosome step time, and tRNA modification. Surprisingly, no increase in intracellular ppGpp levels was detected even though only about 40% of total leucyl tRNA was found to be charged in vivo. Gross ribosomal misreading was not detected, and it was shown that ribosomal step times were reduced between two- and threefold. Analyses of leucyl tRNA isolated from these slow-growing strains showed that at least 90% of the detectable tRNA^Leu₁ was hypomodified as judged by altered mobility on RPC-5 reverse-phase columns, and by specific modification assays using tRNA(m¹G)-methyltransferase and pseudo-uridylate synthetase. Analysis of fast-growing revertants demonstrated that tRNA concentration per se may not explain growth inhibition because selected revertants which grew at wild-type growth rates displayed levels of tRNA comparable to that of control strains bearing the leuV operon. A synthetic tRNA^Leu₁ operon under the control of the T7 promoter was prepared which, when induced, produced six- to sevenfold increases in tRNA^Leu₁ levels. This level of tRNA^Leu₁ titrated the modification system as judged by RPC-5 column chromatography. Overall, our results suggest that hypomodified tRNA may explain, in part, the observed effects on growth, and that the protein-synthesizing system can tolerate an enormous increase in the concentration of a single tRNA.