Thermal behavior and elastic properties of phospholipid bilayers under the effect of a synthetic flavonoid derivative, LEW-10.

We have investigated the effect on phospholipidic bilayers of LEW-10, a synthetic flavonoid, derivative of diosmin. Two optical techniques, Quasi-elastic Light Scattering (QLS) and Fourier Transform Infrared Spectroscopy (FT-IR) were used. The results show that in the presence of LEW-10, the phase transition of the bilayers is lowered and that the elastic modulus is decreased. The FT-IR results indicate interactions in the aqueous interface regions of the bilayers. We also discuss LEW-10 comparatively with another derivative, LEW-7/S1, whose effect has been previously studied.     

Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli.

Glycogenin is a self-glucosylating protein involved in the initiation reactions of glycogen synthesis. Initiation occurs in two stages, requiring first the covalent attachment of a glucose residue to Tyr-194 of glycogenin and then elongation to form an oligosaccharide chain. The latter reaction is known to be catalyzed by glycogenin itself. The glycogenin sequence determined from the protein by Campbell and Cohen (Campbell, D. G., and Cohen, P. (1989) Eur. J. Biochem. 185, 119-125) was used to design oligonucleotide probes to screen a rabbit muscle lambda gt11 library. A cDNA was isolated that predicted an amino acid sequence identical to that of Campbell and Cohen, except that Cys residues replaced Ser-88 and Leu-97. Northern analysis indicated a strongly hybridizing message of 1.8 kilobases, present in most tissues including skeletal muscle, but much weaker in kidney and scarcely detectable in liver. A much weaker 3-kilobase message was also detected in muscle. Polymerase chain reaction was used to isolate DNA fragments encoding a portion of glycogenin from rat and cow. The sequence of this segment was > 90% identical at the amino acid level across the three species, indicating that glycogenin is a highly conserved protein. Using the pET-8c vector, the glycogenin protein was expressed in Escherichia coli. Incubation of the recombinant glycogenin with UDP-[14C]glucose and Mn2+ resulted in labeling of the glycogenin protein, indicating that the recombinant glycogenin was enzymatically active and capable of self-glucosylation. Furthermore, after incubation with UDP-glucose, the recombinant glycogenin could serve as a substrate for glycogen synthase, leading to the production of high M(r) polysaccharide. Therefore, production of functional glycogenin did not require the intervention of any other mammalian protein.     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. The coxII/coxIII operon codes for structural and assembly proteins homologous to those in yeast.

The coxII/coxIII operon of Rhodobacter sphaeroides cytochrome c oxidase has been sequenced and characterized by insertional inactivation/complementation analysis. The organization of the genes in this locus (coxII.orf1.orf3.coxIII) is the same as that of the equivalent operon of Paracoccus denitrificans (ctaC.ctaB.ctaG.ctaE), but unlike that of other bacteria whose cytochrome oxidase genes have been characterized so far. The predicted amino acid sequence homology with eukaryotic oxidases is also higher for Rb. sphaeroides (and P. denitrificans) than for other bacterial versions of the enzyme. The inactivation of coxII results in loss of the characteristic cytochrome oxidase spectrum from membranes of the mutant strain. Full recovery requires introduction into the bacterium of the complete operon containing coxII.orf1.orf3.coxIII; partial complementation yielding a spectrally altered enzyme is achieved with a plasmid containing coxII or coxII.orf1.orf3. These results indicate that the peptides ORF1, ORF3, and COXIII are all required for assembly of native cytochrome c oxidase, suggesting an oxidase-specific assembly or chaperonin function for the ORFs in Rb. sphaeroides similar to that observed for the homologous gene products in yeast, COX10 and COX11.     

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.     

Complex expression of the genes coding for plasminogen activators and their inhibitors in HeLa-smooth muscle cell hybrids.

As cells progress through the multistep process of neoplastic transformation, they eventually acquire the property of invasive behavior. Although both plasminogen activators (PA) and their inhibitors (PAI) contribute to this process, their regulation in normal and transformed cells remains poorly defined. Because somatic cell hybrids provide useful tools for examining the transformation pathway, tumorigenic and invasive HeLa cells were fused with human normal vascular smooth muscle cells and tested for invasion-related parameters, including the expression of PA and PAI genes, and matrix degradation. Both parental cell lines produced large amounts of PAI activities with no detectable PA in either cellular or secreted form. Opposite findings were obtained with the hybrid cell lines, which demonstrated the presence of receptor-bound and secreted PA but absence of enzymatically measurable PAI activities. Both urokinase-type and tissue-type PA were found in cell-associated and secreted form in the hybrid cells. In addition, expression of the urokinase-type PA receptor gene was found in the three hybrid cells and the vascular smooth muscle cells but not in the HeLa cells. Expression of active, receptor-bound and secreted PA provided the nontumorigenic hybrid cells with the enzymatic tools to degrade extracellular proteins in a plasminogen-dependent manner. Thus, the hybrid cells lost tumorigenicity while retaining the tissue-degrading capability of HeLa cells. These hybrid cell lines should prove to be important reagents for investigating the complex regulatory control of PA and PAI gene expression.     

Genetic exchange of transposon and integrative plasmid markers in Mycoplasma pulmonis.

Matings of genetically marked derivatives of Mycoplasma pulmonis resulted in the exchange of chromosomal DNA and the appearance of doubly marked transconjugants. Transposons Tn916 and Tn4001, and a series of integrative plasmids derived from their cloned antibiotic resistance genes, were used to construct antibiotic-resistant mycoplasmal derivatives to examine this phenomenon at the molecular level. Genetic exchange occurred on agar surfaces at frequencies ranging from 3.3 X 10(-4) to 6.4 X 10(-8) transconjugants per CFU. Examination of chromosomal DNA from transconjugants by hybridization revealed that the transposons or integrated plasmids were in the same chromosomal locations as in the parental strains, indicating that exchange involved the transfer of chromosomal DNA and homologous recombination. Transfer was not affected by DNase, polyethylene glycol, EDTA, or calcium chloride but was affected by treatment of either parent with trypsin. Mixing of mating strains before plating had no effect on mating frequencies, but mating did occur in liquid media. The ability to exchange chromosomal markers was limited to selected strains of M. pulmonis; mating did not occur with Acholeplasma laidlawii or M. gallisepticum. Heat and UV inactivation studies revealed that nonviable cells could act as donors in matings. The evidence presented supports a conjugationlike mechanism involving specific trypsin-sensitive membrane components.     

Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C

The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning of the nucleic acid-binding domain of the rat HnRNP C-type protein

A cDNA encoding the nucleic acid-binding domain of the hnRNP C-type protein has been cloned by DNA-affinity screening of pituitary-derived expression libraries. An analysis revealed sequence identity with the human C-type cDNA and demonstrated the presence of a peptide sequence contained within the single-stranded DNA-binding protein, UP2, which was absent from the human cDNA. Structural analysis of the protein encoded by the rat cDNA demonstrated a net charge of +15 with 14.56% and 6.33% lysines and arginines, respectively, and an amino acid sequence that is consistent with an extensive helix-loop-helix-turn-helix structure.     

DISCOVERY OF LATE CRETACEOUS PALYNOFLORA FROM FILDES PENINSULA, KING GEORGE ISLAND, ANTARCTICA AND ITS SIGNIFICANCE

This paper makes an analysis and study on altogether 8 palyniferous samples from the volcano-sedimentary rock series in the Half Three Point area of the Fildes Peninsula, King George Island, Antarctica, the rock series being grey tuffaceous siltstone in lithological characters, about 5m in thickness. Only after making a number of analyses, could we find the relatively abundant sporopollen fossils from 4 samples (Nos. GWP 4—7). But the fossils are poorly preserved, and most of them can hardly be identifi...