Date Palm DNA Mini-Preparation without Liquid Nitrogen

We have developed a new mini-procedure for isolation of total cellular DNA from date palm (Phoenix dactylifera L.). The procedure, which does not use liquid nitrogen, has proved useful due to temporary disruptions in supplies of liquid nitrogen that occur in countries where date palm trees are cultivated. DNA suitable for RFLP and PCR analyses is obtained.     

Haem ligands of the ferricytochrome c of Ustilago sphaerogena

The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH-cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14-21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N(2)-acetylhistidine and monocarboxymethyl derivatives of N(2)-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N(2)-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6-8.4.     

Stimulation of gastrin secretion from the perfused rat stomach by somatostatin antiserum

The effect of a high capacity somatostatin antiserum on antral gastrin secretion was examined in an isolated vascularly perfused rat stomach preparation. Infusion of somatostatin antiserum diluted 1:1 and 1:9 with Krebs buffer solution produced significant increases in gastrin secretion throughout the period of infusion. Neither infusion of somatostatin antiserum diluted 1:99 nor infusion of control rabbit serum had any effect on gastrin secretion. The data indicate that antral somatostatin excercises a continous restraint on gastrin secretion in the basal state.     

An active center tryptophan residue in dihydrofolate reductase: chemical modification, sequence surrounding the critical residue, and structural homology considerations.

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei contains three tryptophan residues and the amino acid sequence surrounding each tryptophan has been determined. Oxidation of one of these residues by N-bromosuccinimide at pH 6.5 can be correlated with the complete loss of enzymatic activity. Following denaturation in urea, the oxidized enzyme was alkylated with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Based on amino acid analyses and absorbance measurements at 410 nm, 2.2 mol of hydroxynitrobenzyl groups was incorporated per mol of protein. Presumably, hydroxynitrobenzyl adducts are formed with the two nonessential tryptophans. From the amino acid compositions of the two major thermolytic peptides containing the hydroxynitrobenzyl label and the partial sequences of two cyanogen bromide peptides containing the tryptophans, it was deduced that tryptophan-5 and tryptophan-129 were modified and, therefore, by difference, tryptophan-21 is the functional residue which becomes oxidized. The amino acid sequence surrounding tryptophan-21 is -Leu-

-Trp-His-Leu-Pro-. In reductases from four other species, this region of the sequence is highly homologous; such a conservation in this vicinity of the primary structure may indicate a functional involvement. The proline residues at positions 20 and 24 may serve to position tryptophan-21 into the appropriate configuration for optimum substrate-binding interactions.     

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.     

Effect of elevated blood pressure, renin and aldosterone on renal sodium handling in two-kidney Goldblatt hypertensive rats

The effect of elevated blood pressure, renin and aldosterone on renal Na+ retention in two-kidney Goldblatt hypertensive rats were investigated. The technique involved retrograde perfusion from the renal veins via the kidneys, and then through the renal arteries and dorsal aorta. Sodium retention in the stenosed kidney of 7 and 30-60 days post-stenosis hypertensive rats was 82 and 70% higher than in normotensive sham-operated rats respectively. Sodium rention in the clipped kidney, 1 day post-stenosis, was insignificant. However, the contralateral kidney of the 1 day post-stenosis rats retained 27% more Na+. The 1 and 7 days post-stenosis rats had higher plasma aldosterone concentrations than controls, while the 30-60 days post-stenosis rats showed lower levels. The plasma renin activity of the 1 day post-stenosis rats showed 65% higher activity than the sham controls with no significant change in the 30-60 days post-stenosis. Therefore Na+ retention may be mediated by aldosterone in the 7 days post-stenosis rats. Natriuresis in the non-stenosed kidneys of both the 7 and 30-60 days post-stenosis rats may be modulated by an increase in filtration rate due to hypertrophy.     

MicroRNA profiles in Sorghum exposed to individual drought or heat or their combination

Sorghum is largely grown for food, fodder and for biofuel production in semi-arid regions where the drought or high temperature or their combination co-occur. Plant microRNAs (miRNAs) are integral to the gene regulatory networks that control almost all biological processes including adaptation to stress conditions. Thus far, plant miRNA profiles under separate drought or heat stresses have been reported but not under combined drought and heat. In this study, we report miRNA profiles in leaves of sorghum exposed to individual drought or heat or their combination. Approximately 29 conserved miRNA families represented by 80 individual miRNAs, 26 families represented by 47 members of less conserved or sorghum-specific miRNA families as well as 8 novel miRNA families have been identified. Of these, 25 miRNAs were found to be differentially regulated in response to stress treatments. The comparative profiling revealed that the miRNA regulation was stronger under heat or combination of heat and drought compared to the drought alone. Furthermore, using degradome sequencing, 48 genes were confirmed as targets for the miRNAs in sorghum. Overall, this study provides a framework for understanding of the miRNA-guided gene regulations under combined stresses.

     

Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.