Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.     

Thrombopoietic activity induced by blood withdrawal and administration of antithrombocyte serum in nephrectomized rats. Role of kidneys in the production of thrombopoietin

The thrombopoietic serum activity was examined in rats during thrombocytopenia produced by bleeding or after treatment with antithrombocyte serum (ATS). 6 hours after both treatments the thrombopoietic activity of the serum, i.e. its content of thrombopoietin, is increased. After the ATS treatment of nephrectomized animals a similar increase of thrombopoietic activity as in normal animals could be achieved. In contrast to that, no similar increase of thrombopoietic activity was observed in nephrectomized animals after blood loss. According to the results of the authors the increase of thrombopoietic activity produced by different stimuli can be attributed to different mechanisms.     

Changes in the vulnerable period of the rat myocardium during hypoxia, hyperventilation and heart failure

Changes in the duration and size of the vulnerable period of the myocardium in the presence of respiratory changes were studied in acute experiments on rats. The limits of the vulnerable period were determined by directly stimulating the heart during ventilation via the enlarged respiratory dead space, during hyperventilation and during heart failure. In the control group (normal ventilation without enlargement of the dead space), the vulnerable period lasted 5.7 +/- 0.76 ms. During ventilation via the enlarged dead space, hypercapnic hypoxaemia developed and the vulnerable period was markedly prolonged (18.55 +/- 5.29 ms) by a shift of its inner limit to the left. Hyperventilation caused normoxic to hyperoxic hypocapnia and markedly reduced the duration of the vulnerable period (8.17 +/- 2.21 and 9.31 +/- 2.38 ms respectively). The vulnerable period lengthened the most in heart failure (25.46 +/- 3.93), mainly as a result of a shift of its outer limit. In all the experimental groups there was a shift of the vulnerable period to the right, which was fastest in hypercapnic hypoxaemia and slowest in hyperoxic hypocapnia. The administration of Inderal (3 mg/kg i.p.) or Arfonad (50 mg/kg i.p.) markedly shortened the vulnerable period during hypercapnic hypoxaemia (9.87 +/- 2.78 and 9.32 +/- 2.16 ms respectively), but did not block the shift. Lengthening of the vulnerable period during hypercapnic hypoxaemia was probably due to activation of sympathetic nerves via beta-adrenergic receptors.     

Enzymic synthesis ofL-Lysine fromDL-α-Amino-ε-caprolactam by new microbial strains

The production of L-lysine fromDL-α-amino-ε-caprolactam (DL-ACL) by new strains producingL-α-amino-ε-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis ofL-ACL byCryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified asPseudomonas sp. were determined. Synthesis ofL-α-amino-ε-caprolactamase is induced byDL-ACL orL-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10 % aqueousDL-ACL toL-lysine by a mixture of native cells in a mass ratio of 1: 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 °C and pH 8.0     

Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

Prenatal detection of a fetus hemizygous for the fragile X-chromosome

Summary A male fetus of a pregnancy known to be at risk for X-linked mental retardation with fragile site Xq27 was found to be affected by demonstrating the marker X-chromosome in five of 180 (2.8%) of metaphases derived from amniocytes cultured in medium 199. The results were confirmed in fetal lymphocytes (25 of 86 metaphases, i.e. 29%), and fetal fibroblasts (five of 100 metaphases when cultured in medium 199, and 14 of 100 after exposure to methotrexate for 43 h).This work was supported in part by a research grant from the Deutsche Forschungsgemeinschaft     

The transition point for protein synthesis in late S-G2 may be delayed by genome bromosubstitution without affecting the time of entry into mitosis

Bromosubstitution for most of the S period in synchronous populations of Allium cepa L. meristematic cells resulted in a delay in the late S-G2 transition point where protein synthesis is needed for later mitotic entrance to occur. This retardation in the position of the transition point was not accompanied by the expected delay in the entrance into mitosis, suggesting that such protein synthesis is a requisite, but not a timer for prophase triggering.     

Iron metabolism in iron-deficient male quail

1. Male quails submitted 20 and 120 days to a low iron diet (7 ppm) were compared to female laying quails, exposed for 30 days to the same low iron regime, in order to compare the response of the iron metabolic control under a single (erythropoiesis) or a doubled (erythropoiesis and egg formation) iron demand. 2. Iron deposit in storage organs, the classical hematology and the intestinal iron absorption were analyzed in these animals. 3. In males, after 120 days, the iron deposits were reduced 50 and 75%, but hematological values (hematocrit and hemoglobin concentration) were normal, although in laying quails, after 30 days, an anemic condition was evident in both blood parameters and iron deposits, provoking an iron deficient erythropoiesis. 4. The enhancement of the intestinal iron uptake, confirms the anemic character of these birds.