Mechanism and inhibition of delta 24-sterol methyltransferase from Candida albicans and Candida tropicalis

The S-adenosyl-L-methionine: delta 24-sterol methyltransferase from Candida albicans has been solubilized with a mixture of octyl glucoside and sodium taurodeoxycholate. The enzyme has an apparent molecular weight of approximately 150,000 as measured by gel filtration chromatography. Zymosterol is the preferred substrate for the microsomal methyltransferase. Other nuclear double bond isomers support reduced rates of methenylation, while sterols which bear methyl groups at C-4 or C-14 are not substrates. Initial velocity and product inhibition studies are consistent with a rapid equilibrium ordered kinetic mechanism. A series of novel sterol analogues which contain heteroatoms substituted for C-24 or C-25 have been kinetically characterized as dead-end inhibitors of the methyltransferase, revealing three distinct mechanisms of interaction with the enzyme. Sterols which contain positively charged moieties in these positions are particularly potent inhibitors, supporting the proposed intermediacy of C-24 and C-25 carbocations. The methyltransferase is reversibly inhibited by low concentrations of 24-thiasterols, while behavior consistent with mechanism-based enzyme inactivation is apparent at higher concentrations. Possible mechanisms for this novel inactivation reaction are discussed.     

On the clonal origin of tumours--lessons from studies of intestinal epithelium

Clonal studies of adult chimaeric mouse epithelium have demonstrated the monoclonal composition of crypts of Lieberkühn. In neonatal life, however, polyclonal crypts have been found, indicating that crypts are of polyclonal origin. We here relate these findings to studies of mosaic tissues which have addressed the question whether solid tumours are of monoclonal or polyclonal origin. The issues has so far remained unresolved because the expected frequencies of polyclonal tumours, given polyclonal origins, have not previously been estimated. A general approach for the calculation of such expected values is suggested. The consistent reports of tumours with polyclonal components suggest that autocrine or paracrine mechanisms play an important role during tumorigenesis.     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)     

Degradation of 3-chlorobiphenyl by in vivo constructed hybrid pseudomonads

Abstract 3-Chlorobiphenyl-degrading bacteria were obtained from the mating between Pseudomonas putida strain BN10 and Pseudomonas sp. strain B13. Strains such as BN210 resulted from the transfer of the genes coding the enzyme sequence for the degradation of chlorocatechols from B13 into BN10, whereas B13 derivatives such as B131 have acquired the biphenyl degradation sequence from BN10. During growth of the hybrid strains on 3-chlorobiphenyl 90% chloride was released. Activities of phenylcatechol 2,3-dioxygenase, benzoate dioxygenase, catechol 1,2-dioxygenase, chloromuconate cyloisomerase and 4-carboxymethyl-enebut-2-en-4-olide hydrolase were found in 3-chlorobiphenyl-grown cells. The hybrid strains were found to convert some congeners of the Aroclor 1221 mixture such as mono- and dichloro-substituted biphenyls.     

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5'' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.     

Names and naming: Instances from the ORU-IGBO

Conclusion People have many names. But, changing one's name is no small thing. It can signify a major step in life or an embarrassing story, as in the last case.A person has a complex individual identity. His collective identity is clear from the moment he is conceived. He is one with a body of people, his kinsmen, who share one common ancestor and through him access to land and resources. The continuation of this group and its name is perpetuated through paternal descent.Kingroup membership ensures a person's place in life, his right to exist, eat, live and enjoy. But, who is he, apart from being a kinsman? Which kinsman is he? How are people going to interact with him? His individual identity will become clear through time. He is a re-incarnation of ancestor x. The circumstances of his birth were such. He behaves like this. He has these personal traits. This has happened to him. He has achieved that. There is no permanent role attached to an individual. He is neither bound to class nor caste. He is dynamic and changes constantly. His life is one of achievement, constant striving and upward mobility till death — when it all begins again. His name signals his state of being in time and existence.Sabine Jell-Bahlsen is an anthropologist and filmmaker.     

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.     

Fanconi's anaemia: correlation of genetic complementation group with psoralen/UVA response

Summary The correlation found by Moustacchi (1987) between cellular response to a crosslinking challenge and genetic heterogeneity in Fanconi's anaemia is confirmed for an earlier set of complementation groups (Zakrzewski and Sperling 1980). This allows the matching of the two independently established complementation groupings and better characterization of their DNA repair-related biochemical properties.     

Mitochondrial protein import: identification of processing peptidase and of PEP, a processing enhancing protein

Transport of nuclear-encoded precursor proteins into mitochondria includes proteolytic cleavage of amino-terminal targeting sequences in the mitochondrial matrix. We have isolated the processing activity from Neurospora crassa. The final preparation (enriched ca. 10,000-fold over cell extracts) consists of two proteins, the matrix processing peptidase (MPP, 57 kd) and a processing enhancing protein (PEP, 52 kd). The two components were isolated as monomers. PEP is about 15-fold more abundant in mitochondria than MPP. It is partly associated with the inner membrane, while MPP is soluble in the matrix. MPP alone has a low processing activity whereas PEP alone has no apparent activity. Upon recombining both, full processing activity is restored. Our data indicate that MPP contains the catalytic site and that PEP has an enhancing function. The mitochondrial processing enzyme appears to represent a new type of "signal peptidase," different from the bacterial leader peptidase and the signal peptidase of the endoplasmic reticulum.     

Bioconversion of 3-chlorobenzoate to 2-chloromuconate controlled by on line HPLC

Summary Strain RD330 a transposon mutant of Alcaligenes eutrophus JMP134 was considered to be dienelactone hydrolase defective (Don et al. 1985). During a bioconversion experiment with 3CB (3-chlorobenzoate) 2CMA (2-chloro-cis,cis-muconate) was accumulated by RD330 with an overall amount of 31%, but no dienelactone could be detected. Enzyme tests revealed that both enzymes 2CMA-cycloisomerase and dienelactone-hydrolase were induced at low levels in RD330 by 3CB and its metabolites.The control of 3CB addition during the bioconversion experiment was performed by on line HPLC (high pressure liquid chromatography).