Force spectroscopy of hepatocytic extracellular matrix components.

We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular "footprints" indicated that the cells secrete large polymers (e.g., 3.5mum contour length and estimated MW 1000kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.

[Lipoma of the larynx and hypopharynx]. / Tluszczak krtani i gardla dolnego.

A case of lipoma of the larynx and hypopharynx is described in 56 year female. Expiration dyspnoea and change of the voice were manifested from due years. The tumor was removed per os and complete cure was obtained. Final diagnosis lipoma was appointmented of histological examination.

Two distinct types of mutations conferring to Escherichia coli K12 capability of D-tryptophan utilization.

We showed that the ability of Escherichia coli K12 tryptophan auxotrophs to utilize D-tryptophan as a substitute for L-tryptophan may result from two types of mutations. The first type consisted in changes in the dadR regulatory site of the dad operon increasing the synthesis of D-amino acid dehydrogenase. The mutations of the second type mapped within the dad A structural gene. They changed the apparent substrate specificity of D-amino acid dehydrogenase. We suppose that the change may be due to an altered enzyme structure which make it more accessible to D-tryptophan.

Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K12.

Evidence is presented that alanine racemase activity in E. coli K12 is due to two distinct gene products. The predominant isozyme is inducible by either alanine stereoisomer and repressible by glucose. The gene dadX coding for its structure is located by the dadA gene determining the structure of D-amino acid dehydrogenase. The regulatory site for the expression of both genes, dadR, is located on the other side of dadA. The orientation of the dad operon established by multiple-point crosses and deletion mapping is as follows: fadR ...dadRAX ...hemA. The dadX alanine racemase activity is unusually refractory to changes of incubation temperature. It differs strikingly from that of the other isozyme, probably the product of the alr gene. The latter isozyme shows a typical dependence upon incubation temperature. The synthesis of alr alanine racemase is constitutive in respect of both alanine and glucose. In dadX mutants, in which alanine racemase activity equals only 15% of that in wild-type cells grown in the absence of an inducer or catabolite repressor, the dad operon cannot be induced by D-alanine. We presume, therefore, that L-alanine is involved more directly than D-alanine in dad operon regulation.

Mutants of Salmonella typhimurium able to utilize D-histidine as a source of L-histidine.

Secondary mutants able to utilize d-histidine, dhu, were isolated in histidine auxotrophs of Salmonella typhimurium. Mutations of one class (dhuA) are closely linked with the hisP locus which codes for a component of histidine permease. The specific activity of l-histidine permeation was estimated as increased two- to seven-fold in dhuA mutants. The dhuB mutants which have not been mapped also had elevated specific activity of l-histidine permeation. The uptake of d-histidine, barely detectable in the parental strains, was prominent in dhuA mutants and showed an apparent Michaelis constant about 1,000-fold higher than that observed with l-histidine. No change was detected in the kinetics of l-histidine permeation. d- and l-histidine competed in the uptake process. Tertiary mutants which lost the ability to grow on d-histidine were isolated by ampicillin counter-selection in dhuA his(-) strains. All of them mapped in the dhuA hisP region. Most of them had all known properties of hisP mutants. It is inferred from these data that the dhuA mutations increase synthesis of components critical to d- and l-histidine permeation.