Babru Samal

High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.

LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

Isolation and characterization of the gene encoding a novel, thermostable serine proteinase from the mould Tritirachium album Limber

A number of proteinases are induced and secreted into the culture medium of Tritirachium album Limber when the nitrogen source is limited to exogenous proteins. We have constructed a cDNA library using the polyadenylated RNA isolated during the nutritional induction with bovine serum albumin. A full‐length clone of a gene for a new proteinase (named proteinase R) was identified from this library. This clone contained sequences coding for the 108‐amino‐acid prepro‐leader as well as for the 279‐amino‐acid mature preoteinase. Proteinase R apparently belongs to the subtilisin group of serine proteases that contains disulphide bonds. Homology between proteinase R and proteinase K was found to be about 87% at the nucleotide as well as at the amino acid level. The Brookhaven Protein Data Base co‐ordinate file of proteinase K was used as a template to study the proteinase R substitutions in three‐dimensional space. The majority of the substitutions of proteinase R with respect to proteinase K were found to be on the exterior of the protein model.

Cloning and expression of the gene encoding a novel proteinase from Tritirachium album Limber

We have isolated the genomic and cDNA clones encoding a novel proteinase from the fungus Tritirachium album Limber, named proteinase T, synthesis of which is induced in skim milk medium. The coding sequence for this enzyme is interrupted by two introns in the fungal genome. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 53% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of two disulfide bonds, which might explain the thermal stability of the proteinase. We have expressed the proT cDNA in Escherichia coli. The authenticity of the product has been characterized by Western blotting and N-terminal analysis of the recombinant product.

Drying and storage of polyacrylamide slab gels: A simple procedure

A simple procedure for drying and storing of polyacrylamide slab gels is described. A polyacrylamide slab gel is fixed in acetic acid plus glycerol and then sandwiched between a gel bond plastic sheet and a dialysis membrane in the presence of a minute amount of gelatin and dried on the benchtop at room temperature. The fixed gel can be stored indefinitely.

Hematologic effects of stem cell factor (SCF) and leukemia inhibitory factor (LIF) in vivo: LIF-induced thrombocytosis in SCF-primed mice

Abstract: Stem cell factor (SCF) administered as daily bolus injections in dose‐response experiments in mice causes a progressive and dramatic dose‐dependent panleukocytosis characterized by neutrophilia, eosinophilia, monocytosis, and lymphocytosis. SCF causes circulating platelet numbers to be dose‐dependently increased after 2 weeks of daily injections. Leukemia inhibitory factor (LIF) administered as daily bolus injections in mice causes a peripheral leukopenia that is largely due to peripheral lymphopenia. LIF causes thrombocytosis peaking after approximately 1 w. Coinjection of SCF and LIF for 1 to 2 wk in mice does not cause a much greater thrombocytosis than the maximum thrombocytosis achievable with SCF or LIF alone. On the other hand, daily injection of SCF for 5 days followed by daily injection of LIF for 5 to 6 d in mice causes a very substantial increase in platelets that was lineage‐specific in terms of not being accompanied by a generalized leukocytosis. In contrast, only a very modest thrombocytosis was noted in SCF‐primed LIF‐treated rats. LIF causes a large increase in the cytoplasmic volume of splenic megakaryocytes in mice, but not in rats. In conclusion, SCF‐induced priming followed by LIF‐induced maturation of megakaryocytes causes a substantial selective increase in the numbers of circulating platelets in mice.

Crystallization and preliminary X-ray analysis of leukemia inhibitory factor

Leukemia inhibitory factor (LIF) is a polyfunctional molecule with significant and diverse biological activities. LIF is a glycoprotein secreted by a number of different cell types in vitro. It is induced in fibroblasts, lymphocytes, monocytes and astrocytes by various inducers such as serum, TNF, interleukin‐IP and EGF. Due to extensive and variable glycosylation the molecular weight can range from 38 to 67 kDA. The biological functions of LIF are mediated through a receptor and a signal transducer, gp130, which is also used by factors like interleukm‐6 (IL‐6), cilliary neurotropic factor (CNTF), and oncostatin M (OSM). Here, we report the crystallization of the non‐glycosylated human‐like LIF expressed in E. coli. The present crystals diffract to 2.0 Å using synchrotron radiation. They belong to the monoclinic space group C2, and the cell dimensions are a = 61.5 Å, b = 45.3 Å , c =77.7 Å and β = 112.3°.

Identification of interleukin 6 as a synergistic factor for the differentiation-inducing effect of TNF on leukemic ML-1 cells

Recombinant TNF was capable of inducing differentiation of human leukemic ML-1 cells in the monocytic pathway. Recombinant interleukin 6 did not have the activity but it could significantly increase the activity of recombinant TNF. Both of these molecules were found to play a similar role in PWM-induced conditioned medium from human lymphocytes (LCM). The differentiation inducing effect of LCM could be partially neutralized by antibody to interleukin 6. Fractionation of LCM also identified interleukin 6 as the factor that synergizes with TNF.

Comparative analysis of the effects of recombinant cytokines on the growth and differentiation of ML-1, a human myelogenous leukemic cell line.

We have investigated the effect of a number of cytokines on the human acute myelomonocytic leukemic cell line, ML-1. The differentiation inducing effects of interleukins (IL-1 beta, IL-3 and IL-6), colony stimulating factors (GCSF and GMCSF), TNF, LIF and IFNg, were studied either individually or in combination. Criteria for monocytic differentiation were as follows: an increase in the percentage of cells reducing nitroblue tetrazolium (NBT) salt, an increase in the alkaline phosphatase activity as well as the appearance of macrophagic phenotype. Among all the cytokines tested, only TNF was found to induce differentiation and to inhibit growth of ML-1 cells. IL-3, IL-6, interferon gamma, GCSF and to a smaller extent IL-1 beta and GMCSF synergized the differentiation inducing activity of TNF.