Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) Seedlings : Intermediary Metabolism and Ethanol Tolerance

Tolerance to ethanol and the ability to metabolize key intermediary substrates under anaerobiosis were studied in Echinochloa crus-galli (L.) Beauv. var oryzicola seeds to further characterize the mechanisms which enable it to germinate and grow without O₂.

Our results indicate that E. crus-galli var oryzicola possesses an inherently high tolerance to ethanol and is able to metabolize low levels of ethanol in the absence of O₂. Concentrations of ethanol 45-fold greater than endogenous levels did not prove toxic to germinating seeds.

Five-day anaerobically grown seedlings of E. crus-galli var oryzicola metabolized added [¹⁴C]sucrose primarily to CO₂ and ethanol. Of the soluble compounds labeled, the phosphorylated intermediates of glycolysis and the oxidative pentose phosphate pathway predominated more under anaerobiosis than in air. In addition, organic acids and lipids were labeled from [¹⁴C]sucrose, the latter indicating that metabolism of carbohydrate via acetyl-CoA occurred in the absence of O₂. Lipids were also labeled when seeds were supplied with [¹⁴C]ethanol or [¹⁴C]acetate. Labeling experiments using the above compounds plus [¹⁴C]NaHCO₃, showed further labeling of organic acids; succinate and citrate being labeled under nitrogen, while fumarate was formed in air.

The above metabolic characteristics would allow for the maintenance of an active alcoholic fermentation system which, along with high alcohol dehydrogenase activity, would continue to recycle NAD and result in continued energy production without O₂. In addition, Echinochloa's ability to metabolize carbohydrate intermediates and to synthesize lipids indicates that mechanisms exist for providing the carbon intermediates for biosynthesis, particularly membrane synthesis for growth, even in the absence of O₂.

     

Significance of the delta 5 and delta 4 steroidogenic pathways in the hamster preovulatory follicle

Isolated hamster granulosa cells and theca from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroid substrates. The purpose of the experiments was to determine, in theca, the relative activities of the delta 5 and delta 4 pathways under controlled conditions, and to compare the ability of granulosa cells and theca to form progesterone from exogenous pregnenolone. The results of the experiments show that the delta 5 pathway in theca predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from delta 5 to delta 4 as the major metabolic pathway. Progesterone formation from exogenous pregnenolone is 7 to 10 times greater in unstimulated granulosa cells than in theca. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theca. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone.     

The effect of embryo quality at freezing on subsequent development of thawed cow embryos

Day 7 to 9 embryos were frozen by a rapid two-step method to ?38°C before being plunged into liquid nitrogen. Glycerol was used as the cryoprotectant and, following thawing, the embryos were cultured for 12 – 24 hours in PBS + 15% heat-treated steer serum. In Experiment 1, embryos were frozen in 2.0 ml glass ampoules or 0.5 ml Cassou straws. Two levels of glycerol (1.0M and 1.4M) gave comparable $\text{in vitro}$ survival rates ($\text{12}\text{20}$ and $\text{13}\text{25}$, respectively). A greater proportion of embryos developed in culture after freezing in straws. In Experiment 2, embryos were classified morphologically before and after freezing into 5 grades (1 = excellent; 2 = good; 3 = fair; 4 = poor; 5 = degenerate). Only embryos of grade 1, 2 and 3 were frozen. The post-thaw survival rates for embryos graded 1, 2 and 3 before freezing were 100% ($\text{11}\text{11}$), 86% ($\text{24}\text{28}$) and 83% ($\text{20}\text{24}$), respectively. Furthermore, the porportion of surviving embryos estimated to be of poor quality (grade 4) was greater for embryos graded 3 before freezing ($\text{13}\text{20}$) than for embryos graded 2 ($\text{6}\text{24}$) or 1 ($\text{1}\text{11}$). The percentage of embryos which developed normally after $\text{in vitro}$ culture for each of the pre-freezing grades 1, 2 and 3 was 91% ($\text{10}\text{11}$), 50% ($\text{14}\text{28}$) and 29% ($\text{7}\text{24}$), respectively. Of the total number of frozen-thawed embryos which developed in culture, $\text{5}\text{31}$ (16%) were of poor quality. The proportion of poor quality developing embryos was greater inembryos graded 3 before freezing ($\text{3}\text{7}$) than those graded 2 ($\text{2}\text{14}$). All of the embryos graded 1 before freezing and which developed in culture were of good quality. Results indicate that, if high post-thaw survival rates are to be obtained, stringent embryo selection processes will be required.     

Structural and functional studies of the adrenal zona glomerulosa in sodium-depleted and sodium-loaded sheep

Cell and Tissue Research - The effects of alterations in sodium status upon the morphology of the adrenal zona glomerulosa in sheep have been examined qualitatively and quantitatively, using...     

Serum and Urine Fibrinogen-Fibrin-related Antigen (F.R.-Antigen) Levels in Renal Disease

The concentration of serum fibrinogen-fibrin-related antigen (F.R.-antigen) was measured in a group of 142 patients with various renal disorders, in 38 of whom urine F.R.-antigen was also estimated. Raised serum F.R.-antigen levels were present in 48% of the patients, with no particular preponderance in any diagnostic category apart from acute reversible intrinsic renal failure in which high levels were invariably present. Significantly-raised serum levels were also present in the patients with microangiopathic haemolytic anaemia and in those with the more severe degrees of renal impairment. Urine F.R.-antigen was increased in 34 of the 38 patients. The amount of F.R.-antigen in the urine correlated with the degree of proteinuria but not with the serum F.R.-antigen levels. The evidence relating to intravascular coagulation in renal disease is reviewed, and it is suggested that there is a high incidence of localized fibrinogen or fibrin degradation in the kidney, which is related more to factors such as the presence of uraemia and microangiopathic haemolytic anaemia rather than to the diagnostic category.     

High Dosage Metolazone in Chronic Renal Failure

Metolazone is a modified quinazolinesulphonamide and in a dose of between 4 and 7·5 mg is an effective diuretic in man with normal renal function. Fourteen patients with non-oedematous stable chronic renal failure (creatinine clearance ranging from 1·2 to 12 ml/min) were given metolazone in doses ranging from 20-150 mg. A noticeable increase in urine flow and sodium excretion occurred, free water clearance increased, and there was a small but significant increase in potassium excretion. No side effects were noted.     

FREE-FLOW ELECTROPHORETIC SEPARATION AND ELECTRICAL SURFACE PROPERTIES OF SUBCELLULAR PARTICLES FROM GUINEA PIG BRAIN

Continuous free-flow electrophoretic separation has been used to obtain relatively pure preparations of synaptosomes and synaptic vesicles from crude fractions of guinea pig brain homogenates. Measurements of the contents of protein, neuraminic acid, and bound acetylcholine; the activities of succinic dehydrogenase, adenosine triphosphatase, choline acetylase, and 5'-nucleotidase; and the uptake of ¹⁴C-labeled choline arid acetylcholine in the presence and absence of hemicholinium, all confirm the electron microscope evidence that the electrophoretic preparations are at least as pure as those obtained by ultracentrifugal methods. The electrophoretic mobility measurements have been used to calculate zeta potentials and surface charge densities for these particles.     

Enzymatic aromatization of steroids. I. Effects of oxygen and carbon monoxide on the intermediate steps of estrogen biosynthesis

At Case Western Reserve University in Cleveland, the known intermediate steps in the aromatization, catalyxed by human placental microsomes, of 4-androstene-3, 17-dione to estrone were evaluated. Oxygen and carbon monoxide atmospheres were used to expose the properties of the participating oxidases. The results were judged to be evidence for the existence of 3 mixed function oxidases active in steroid aromatization. One of the oxidases may be a form of cytochrome P-450. The proposed microsomal components must be physically fractionated for validation of their independent existences.     

Hydrolysis of bradykinin and its higher homologues by angiotensin-converting enzyme (Short Communication)

The hydrolysis of bradykinin and its higher homologues by angiotensin-converting enzyme has been investigated by using an automated ninhydrin technique. The results show an inverse relationship of hydrolysis rate with size and charge of the peptide, which parallels the inactivation in the pulmonary circulation and offers an explanation for the selectivity of metabolism of these kinins by the lungs.