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     

Effect of precursors on biosynthesis of monensins A and B

Precursors of monensins (acetate, propionate, butyrate, isobutyrate) affect the total production and the relative proportion of monensins A and B. Addition of propionate into the fermentation medium causes a prevalence of monensin B whereas butyrate and isobutyrate stimulate the production of monensin A and suppress the production of monensin B.     

Inhibition of Aspartate Aminotransferase by Glycation In Vitro Under Various Conditions

Incubation of 50 mM d -glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 × g supernatant) at 25°C had no effect on enzyme activity. 50 mM d -fructose or d -ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM dl -glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM d -fructose or d -ribose was marked even at a temperature of 4°C but it was less pronounced than at 25°C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM l -aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by d -ribose or d -fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Putative candidate genes for blood pressure control in the SHR.BN-RNO8 congenic substrains

The SHR-Lx congenic strain carrying a differential segment of chromosome 8 of BN and PD origin was recently shown to exhibit a significant decrease in blood pressure as compared to the SHR strain. There were two positional candidate genes for blood pressure control mapped to the differential segment: the rat kidney epithelial potassium channel gene (Kcnj1) and brain dopamine receptor 2 gene (Drd2). Bot these genes were separated into SHR.BN-RNO8 congenic substrains. In this communication, we are presenting the assignment of two further putative candidate genes, which might be involved in blood pressure control to the BN/PD differential segment of the SHR-Lx congenic strain. These are: the gene coding for smooth muscle cell specific protein 22 (Sm22) defined by the D8Mcw1 marker and neuronal nicotinic acetylcholine receptor gene cluster, defined by the D8Bord1 marker. Moreover, the glutamate receptor gene Grik4 which also maps to the differential segment of the SHR-Lx should be taken into account. The genetic separation of all these putative candidate genes of blood pressure control is being performed by recombinations and subsequent selection using (SHR×SHR-Lx) intercross population.     

Activation of erythrocyte membrane Ca2+-ATPase by calpain

Ca2+-ATPase of erythrocyte membranes, prepared from erythrocytes substantially removed of contaminating leukocytes, was found to be activated by calpain isolated from the same source. Saponin or glycodeoxycholate treatment of membranes was essential for elicitation of the calpain response. Unlike the membrane bound ATPase, solubilized ATPase was inactivated by calpain. Digestion of membranes with the protease did not affect the Km (ATP) of Ca2+-ATPase though stimulation of the membrane ATPase by calmodulin could be partially substituted by calpain treatment. As compared with control, Ca2+-ATPase of calpain-digested membranes attained maximal activity at a lower free Ca2+ concentration.     

Loci controlling lymphocyte production of interferon γ after alloantigen stimulation in vitro and their co-localization with genes controlling lymphocyte infiltration of tumors and tumor susceptibility

Low infiltration of lymphocytes into cancers is associated with poor prognosis, but the reasons why some patients exhibit a low and others a high infiltration of tumors are unknown. Previously we mapped four loci (Lynf1–Lynf4) controlling lymphocyte infiltration of mouse lung tumors. These loci do not encode any of the molecules that are involved in traffic of lymphocytes. Here we report a genetic relationship between these loci and the control of production of IFNγ in allogeneic mixed lymphocyte cultures (MLC). We found that IFNγ production by lymphocytes of O20/A mice is lower than by lymphocytes of OcB-9/Dem mice (both H2 ^pz ) stimulated in MLC by irradiated splenocytes of C57BL/10SnPh (H2 ^b ) or BALB/cHeA (H2 ^d ) mice, or by ConA. IFNγ production in MLCs of individual (O20 × OcB-9)F₂ mice stimulated by irradiated C57BL/10 splenocytes and genotyped for microsatellite markers revealed four IFNγ-controlling loci (Cypr4-Cypr7), each of which is closely linked with one of the four Lynf loci and with a cluster of susceptibility genes for different tumors. This suggests that inherited differences in certain lymphocyte responses may modify their propensity to infiltrate tumors and their capacity to affect tumor growth.     

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.