The influence of proteases on the activity of glucoamylase from Aspergillus niger C

Summary Two proteases from Aspergillus niger C post-culture medium were isolated by fractionation on a DEAE-sepharose column and ultrafiltration. The four fractions of glucoamylase activity (GA1, GA2, GA3 and GA4) present in the medium showed different susceptibility to the influence of proteases. The effects of proteases on the different glucoamylase fractions during the growth of the fungus are demonstrated. The activity was found to decrease at the beginning of the culture, but by its end there was a stimulation of GA4 glucoamylase. After treating GA2 and GA3 with protease II, a new additional fraction of glucoamylase was detected.     

Composition of Caulobacter crescentus murein synthesized in the presence of glycine

Abstract Glycine added to the growth medium of Caulobacter crescentus was found to substitute Cterminal alanine in the peptide side chains of the murein of this species. Murein synthesized in vivo and in vitro in the presence of glycerine was poorly crosslinked as was new murein formed in the presence of the amino acid. The reduced cross-linkage seems to be due to the effect of glycine on the formation of trimeric muropeptides as revealed by high-performance liquid chromatography (HPLC) muropeptide analysis of murein formed in the presence and absence of the amino acid.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.     

The Feeding of Chaoborus flavicans Meigen (Diptera,Chaoboridae). and its Predation on Lake Zooplankton

In a study of Chaoborus feeding in a eutrophic lake, selectivity was found to be positive with Crustacea (especially copepodit stages). and negative with Rotatoria. Daily food rations were about 20% for most of the feeding period, but higher (106%). during the month of intensive growth after hatching. Feeding intensity correlated positively with amount of food an temperature, and negatively with Chaoborus concentration. Elimination of Crustacea (in the epilimnion of the central zone of the lake). equalled about 30–40% of Crustacea production in June and September and slightly exeeded the August production (it was almost zero in the remaining months because Chaoborus larvae stayed at the bottom). This applies, however, only in the central zone – about 50% of the lake volume. Chaoborus probably influences both the density of zooplankton and the quantitative relations between zooplankton species.     

The influence of intracellular idarubicin and daunorubicin levels on drug cytotoxicity in childhood acute leukemia

Uptake and efflux of two anthracyclines, idarubicin (IDA) and daunorubicin (DNR), was studied in childhood acute leukemia samples. A comparison of IDA and DNR transport phenomena in relation to drug cytotoxicity and expression of P-glycoprotein (PGP) was made. Intracellular content of IDA/DNR was determined by flow cytometry using the fluorescent properties of the drugs. In vitro drug cytotoxicity was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. PGP expression was analysed by flow cytometry. The uptake and efflux rates were non-significantly higher for IDA than DNR. There were no differences between three types of leukemia with respect to drug content during accumulation and retention. After correction for the cell volume, intracellular concentration of both drugs in each moment of uptake and efflux was significantly lower in relapsed ALL and AML samples in comparison with initial ALL cells. Efflux, but not uptake, of both drugs was inversely correlated with PGP expression and IDA, but not DNR, cytotoxicity. The cytotoxicity was correlated with drug accumulation for both drugs and with drug retention for IDA. In conclusion, it seems that (1) intracellular content was related to the lipophilic properties of the drugs rather than to the type of leukemia, (2) decreased intracellular concentration of both drugs might have an impact on compromised therapy results in AML and relapsed ALL children, (3) IDA presents higher cytotoxicity, which possibly might be decreased by the presence of PGP. These results might have a practical impact on the rational design of new chemotherapy protocols.     

Penicillin-binding proteins of listeria monocytogenes--a re-evaluation

Intact Listeria monocytogenes cells or membranes isolated from them were treated with [3H]penicillin to allow identification of the penicillin binding proteins (PBPs) located in the cytoplasmic membrane. In the former case the PBPs were released from the cells following disruption of the cell wall murein with Listeria monocytogenes bacteriophage lysin. The procedure described by Dougherty et al. (1996) for Escherichia coli, with some modifications, was used to evaluate the M(r)s of the individual PBPs and allowed direct quantitation of their copy number.     

In vitro activity of oxazaphosphorines in childhood acute leukemia: preliminary report

Glufosfamide (beta-D-glucosyl-ifosfamide mustard) is a new agent for cancer chemotherapy. Its pharmacology is similar to commonly used oxazaphosphorines, but it does not require activation by hepatic cytochrome P-450 and preclinically demonstrates lower nephrotoxicity and myelosuppression than ifosfamide. The aim of the study was a comparison of the drug resistance profiles of glufosfamide and other oxazaphosphorines in childhood acute leukemias. Leukemic cells, taken from children with ALL on diagnosis (n = 41), ALL on relapse (n = 12) and AML on diagnosis (n = 13) were analyzed by means of the MTT assay. The following drugs were tested: glufosfamide (GLU), 4-HOO-ifosfamide (IFO), 4-HOO-cyclophosphamide (CYC) and mafosfamide cyclohexylamine salt (MAF). In the group of initial ALL samples median cytotoxicity values for GLU, IFO, CYC and MAF were 15.5, 33.8, 15.7 and 7.8 microM, respectively. In comparison with initial ALL samples, the relative resistance for GLU and IFO in relapsed ALL samples was 1.9 (p = 0.049) and 1.3 (ns), and in initial AML samples 31 (p < 0.001) and 5 (p = 0.001), respectively. All oxazaphosphorines presented highly significant cross-resistance. Glufosfamide presented high activity against lymphoblasts both on diagnosis and on relapse.     

On the systematic position of Ophiosacculus Macy, 1935 (digenea: Lecithodendriidae), with the erection of the Ophiosacculinae n. subfam

The phylogenetic relationships and systematic position of the digenean genus Ophiosacculus Macy, 1935 has been controversial and opinions of different authors on its systematic position and content are contradictory. Molecular analysis based on the partial sequences of the large subunit ribosomal DNA gene of the type and only valid species of the genus, Ophiosacculus mehelyi (Mödlinger, 1930), as well as previously published sequences of members of several families of Plagiorchiata (including the Allassogonoporidae, Lecithodendriidae and Pleurogenidae as potential relatives of Ophiosacculus) has shown that Ophiosacculus forms a clade with the typical representatives of the Lecithodendriidae from bats. Ophiosacculus is basal to the cluster containing Lecithodendrium, Prosthodendrium and Pycnoporus and has quite pronounced differences in the sequenced fragment compared to these genera. Based on the results of the molecular study, morphological characteristics of Ophiosacculus (in particular, possession of a seminal vesicle lying freely in parenchyma) and the fact that the type-specimen of Gyrabascus brevigastrus Macy, 1835 (type-species of the monotypic genus Gyrabascus and type-genus of the subfamily Gyrabascinae) belongs to Allassogonoporus, a new subfamily, the Ophiosacculinae, with Ophiosacculus as the type-genus, is established within the Lecithodendriidae. Molecular study did not support a close phylogenetic relationship between Allassogonoporus and Ophiosacculus, although several authors previously allocated both these genera to the Allassogonoporidae. Morphological study revealed the position of the genital pore in O. mehelyi to be at the posterior margin of the ventral sucker. An amended diagnosis of Ophiosacculus and a diagnosis of Ophiosacculinae n. subfam. are given.     

Covalent incorporation of selenium into oligonucleotides for X-ray crystal structure determination via MAD: proof of principle. Multiwavelength anomalous dispersion

Selenium was incorporated into an oligodeoxynucleotide in the form of 2'-methylseleno-uridine (U(Se)). The X-ray crystal structure of the duplex left open bracket d(GCGTA)U(Se)d(ACGC) right open bracket (2) was determined by the multiwavelength anomalous dispersion (MAD) technique and refined to a resolution of 1.3 A, demonstrating that selenium can selectively substitute oxygen in DNA and that the resulting compounds are chemically stable. Since derivatization at the 2'-alpha-position with selenium does not affect the preference of the sugar for the C3'-endo conformation, this strategy is suitable for incorporating selenium into RNA. The availability of selenium-containing nucleic acids for crystallographic phasing offers an attractive alternative to the commonly used halogenated pyrimidines.     

Does molecular docking reveal alternative chemopreventive mechanism of activation of oxidoreductase by sulforaphane isothiocyanates?

Isothiocyanates (ITC) are well-known chemopreventive agents extracted from vegetables. This activity results from the activation of human oxidoreductase. In this letter, the uncompetitive activatory mechanism of ITC was investigated using docking and molecular dynamics simulations. This indicates that NAD(P)H:quinone oxidoreductase can efficiently improve enzyme-substrate recognition within the catalytic site if the ITC activator supports the interaction in the uncompetitive binding site.