Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli

A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18. The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P. funiculosum anticellulases.     

Viscometric characterization of pennisetin from pearl millet

Pennisetin, the alcohol soluble storage protein of pearl millet (Pennisetum americanum), was isolated in a homogeneous state. The intrinsic viscosity [n] of this protein was found to be in the range of 16.5-17.7 ml/g in 70% (v/v) aqueous ethanol. The [eta] changed marginally when temperature was increased from 20 to 70 degrees C and also in the presence of 10 mM NaCl. The data indicated that pennisetin was a rigid, rod shaped asymmetric hydrodynamic particle with molecular dimensions in the range of 301 x 14.4 A - 317.7 x 14.2 A. During denaturation with guanidine hydrochloride (Gdn.HCl), the intrinsic viscosity of pennisetin increased from 16 to 25ml/g with a mid point at 3.6 M of the denaturant. The native protein structure was unfolded in 6 M Gdn.HCl as shown by the exposure of aromatic amino acid residues buried in the native state and this transition was found to be reversible. The intrinsic viscosity of pennisetin in 5.9 M Gdn.HCl corresponded to Mr 25,000 which was comparable to that determined by SDS-PAGE.     

Characterization of the purified multifunctional cellulase component of Penicillium funiculosum

Summary The endoglucanase component (CMCase I) ofPenicillium funiculosum cellulase was purified to apparent homogeneity by ultrafiltration and gel chromatography. It consists of a single polypeptide chain with a molecular weight of 56000 and is a glycoprotein. Viscometric and end-product analysis revealed the randomness of enzyme action. Multifunctional characteristic of CMCase I was studied with various carbohydrate substrates.NCL Communication No.: 4307     

Hydroxylation of benzoate and its chlorinated derivatives in Aspergillus niger

Comparison of the properties of a purified benzoate-4-hydroxylase, using benzoic acid and its chlorinated derivatives as substrate, suggests that the same enzyme operates during the initial hydroxylation of benzoate, 2-chlorobenzoate (2-cba), and 3-chlorobenzoate (3-cba) in A.niger.     

Nature of leukemia-associated antigenicity of dinitrophenylated human lymphocytes and lymphoblasts

Using dinitrophenylated human lymphocytes and phytohaemagglutinin-stimulated human lymphoblasts as antigens, antibodies were produced in rabbits. The immunological reactivities of the antisera so produced were tested against various types of leukemic cells after absorbing the sera with pooled normal leukocytes. Both the sera showed reactivity with all types of leukemic cells and no specific affinity for lymphoid leukemic cells was seen. This may suggest the presence of some common antigens on all types of leukemic cells or that dinitrophenylation brings about similar changes on all types of normal leukocytes.     

Production of Recombinant Human Bile Salt Stimulated Lipase and Its Variant inPichia pastoris

hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.     

A novel small molecule target in human airway smooth muscle for potential treatment of obstructive lung diseases: a staged high-throughput biophysical screening

Background

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.

Methods

Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.

Results

Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.

Conclusions

This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.     

Thidiazuron-induced morphogenesis in tamarind seedlings

Summary Germination of tamarind seeds in medium containing thidiazuron (TDZ) resulted in induction of nodular protrusions in and around the cotyledonary node meristem. The structures developed radially in well-defined circles and subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. The structures developed into shoots on transfer to medium devoid of growth regulators. Histological studies revealed that the protrusions initiated from the nodal meristem and extended to the non-meristematic region between the two meristems and also in the proximal part of the hypocotyl in seedlings germinated in 9.08 μM TDZ. Newly formed cell layers and less-differentiated meristematic protrusions were also seen. With the increase in the distance from the meristem, the buds were less differentiated; in the proximal part of the hypocotyl only the multiple layers of meristematic cells were noted. With extension of the period of incubation, the TDZ-induced meristematic activity extended laterally in circles towards the neighboring region. The radial spread of the meristematic activity from the center of the nodal meristem was also evident at 18.16 μM TDZ. From the pattern of the morphogenic development and the histological studies it may be hypothesized that in tamarind, TDZ influences the existing meristems specifically. Subsequently de novo organogenesis is triggered in the neighboring cells.