Kinetics and thermodynamics of a novel endoglucanase (CMCase) from <Emphasis Type="Italic">Gymnoascella </Emphasis><Emphasis Type="Italic">citrina</Emphasis> produced under solid-state condition

Gymnoascella citrina produced two isoforms of endoglucanases (CMCase-I and -capital I, Ukrainiancapital I, Ukrainian) under solid-state condition. Purified CMCase-I was novel because it was apparently holoenzyme in nature. The enzyme was monomeric as its native and subunit mass were almost the same, i.e., 43 and 42 kDa, respectively. Ea for carboxymethylcellulose (CMC) hydrolysis was 36.2 kJ mol(-1). The enzyme was stable over a pH range of 3.5-6.5, while temperature optimum was 55 degrees C. Vmax, Km and k (cat )for CMC hydrolysis were 39 U mg(-1) protein, 6.25 mg CMC mL(-1) and 27.5 s(-1), respectively. The pKa1 and pKa2 of ionizable groups of active site were 2.8 and 7.4, respectively. Thermodynamic parameters for CMC hydrolysis were as follows: DeltaH*=33.5 kJ mol(-1), DeltaG*=70.42 kJ mol(-1) and DeltaS*=-114.37 J mol(-1) K(-1). The removal of metals resulted into complete loss of enzymatic activity and was completely recovered in the presence of 1 mM Mn2+, whereas inhibition initiated at 5 mM. The other metals like Ca2+, Zn2+ and K1+ showed no inhibition up to 7 mM, Co2+ completely inhibited the activity, while Mg2+ could not recover the initial activity up to 7 mM. So we are reporting for the first time, kinetics and thermodynamics of CMCase-Iota from G. citrina.     

Bisleuconothine A,an eburnane–aspidosperma bisindole alkaloid from Leuconotis griffithii

A new bisindole alkaloid, bisleuconothine A (1) consisting of an eburnane–aspidosperma type skeleton, was isolated from the bark of Leuconotis griffithii. The structure including absolute stereochemistry was elucidated on the basis of 2D NMR data and X-ray analysis. Bisleuconothine A (1) showed cell growth inhibitory activity against various human cancer cell lines.     

Oligo-riboprobes. Tools for in situ hybridisation

In situ hybridisation detection of mRNAs using riboprobes has become a widely used technique. However, the identification of cells producing closely-related yet distinct mRNAs is difficult with the usual size probes. Moreover, it is not always easy to obtain the required cDNA essential for cRNA probe synthesis. To avoid these problems, we have used synthetic oligodeoxynucleotides to generate short, single stranded RNA probes ("oligo-riboprobes"). These probes can be labelled to very high (10(9) cpm/micrograms) specific activity and can be prepared for any published nucleotide sequence. We have used these probes to localise beta (preprotachykinin) PPT mRNA producing neurons in rat hypothalamus and bowel. The results were compared to that obtained with cRNA probes generated from beta preprotachykinin cDNA.     

Generalized Baum-Welch Algorithm Based on the Similarity between Sequences

The profile hidden Markov model (PHMM) is widely used to assign the protein sequences to their respective families. A major limitation of a PHMM is the assumption that given states the observations (amino acids) are independent. To overcome this limitation, the dependency between amino acids in a multiple sequence alignment (MSA) which is the representative of a PHMM can be appended to the PHMM. Due to the fact that with a MSA, the sequences of amino acids are biologically related, the one-by-one dependency between two amino acids can be considered. In other words, based on the MSA, the dependency between an amino acid and its corresponding amino acid located above can be combined with the PHMM. For this purpose, the new emission probability matrix which considers the one-by-one dependencies between amino acids is constructed. The parameters of a PHMM are of two types; transition and emission probabilities which are usually estimated using an EM algorithm called the Baum-Welch algorithm. We have generalized the Baum-Welch algorithm using similarity emission matrix constructed by integrating the new emission probability matrix with the common emission probability matrix. Then, the performance of similarity emission is discussed by applying it to the top twenty protein families in the Pfam database. We show that using the similarity emission in the Baum-Welch algorithm significantly outperforms the common Baum-Welch algorithm in the task of assigning protein sequences to protein families.     

Structure and Stability Analysis of Cytotoxic Complex of Camel α-Lactalbumin and Unsaturated Fatty Acids Produced at High Temperature

Abstract

α-Lactalbumin (α-La), together with oleic acid can be converted to a complex, which kills tumor cells selectively. Cytotoxic α-La -oleic acid and a-La -linoleic acid complexes were generated by adding fatty acid to camel holo a-La at 60°C (referred to as La-OA-60 and La-LA-60 state, respectively). Structural properties of these complexes were studied and compared to the camel α-La. The experimental results show that linoleic acid induces a-La partial unfolding but oleic acid does not change the protein structure significantly. Also the stability of La-OA-60 and La- LA-60 toward thermal denaturation was measured. The order of temperature at the transition midpoint is as follows: La-LA-60 < La-0A-60 < α-La. La-0A-60 complex inhibited tubulin polymerization in vitro. Although the structures of La-0A-60 and La-LA-60 were different, these two complexes had similar cytotoxic effect to DU145 human prostate cancer cells. Samples of La-0A-60 that have been renatured after denaturation lost the specific biological activity toward tumor cells.     

Distinct Roles for Rho Versus Rac/Cdc42 GTPases Downstream of Vav2 in Regulating Mammary Epithelial Acinar Architecture

Non-malignant mammary epithelial cells (MECs) undergo acinar morphogenesis in three-dimensional Matrigel culture, a trait that is lost upon oncogenic transformation. Rho GTPases are thought to play important roles in regulating epithelial cell-cell junctions, but their contributions to acinar morphogenesis remain unclear. Here we report that the activity of Rho GTPases is down-regulated in non-malignant MECs in three-dimensional culture with particular suppression of Rac1 and Cdc42. Inducible expression of a constitutively active form of Vav2, a Rho GTPase guanine nucleotide exchange factor activated by receptor tyrosine kinases, in three-dimensional MEC culture activated Rac1 and Cdc42; Vav2 induction from early stages of culture impaired acinar morphogenesis, and induction in preformed acini disrupted the pre-established acinar architecture and led to cellular outgrowths. Knockdown studies demonstrated that Rac1 and Cdc42 mediate the constitutively active Vav2 phenotype, whereas in contrast, RhoA knockdown intensified the Vav2-induced disruption of acini, leading to more aggressive cell outgrowth and branching morphogenesis. These results indicate that RhoA plays an antagonistic role to Rac1/Cdc42 in the control of mammary epithelial acinar morphogenesis.     

Electronic, ductile, phase transition and mechanical properties of Lu-monopnictides under high pressures

The structural, elastic and electronic properties of lutatium-pnictides (LuN, LuP, LuAs, LuSb, and LuBi) were analyzed by using full-potential linearized augmented plane wave within generalized gradient approximation in the stable rock-salt structure (B1 phase) with space group Fm-3m and high-pressure CsCl structure (B2 phase) with space group Pm-3m. Hubbard-U and spin-orbit coupling were included to predict correctly the semiconducting band gap of LuN. Under compression, these materials undergo first-order structural transitions from B1 to B2 phases at 241, 98, 56.82, 25.2 and 32.3 GPa, respectively. The computed elastic properties show that LuBi is ductile by nature. The electronic structure calculations show that LuN is semiconductor at ambient conditions with an indirect band gap of 1.55 eV while other Lu-pnictides are metallic. It was observed that LuN shows metallization at high pressures. The structural properties, viz, equilibrium lattice constant, bulk modulus and its pressure derivative, transition pressure, equation of state, volume collapse, band gap and elastic moduli, show good agreement with available data.