Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma

A cell line was established from an in situ squamous cell carcinoma of the skin (Bowen's disease), and its in vitro karyotypic evolution was cytogenetically analyzed. Initially, considerable genetic heterogeneity was evident. Nine cytogenetically abnormal clones, eight of which were apparently unrelated, were found among the 83 metaphases analyzed from the primary culture and the first passage. With increasing time in culture this complexity was reduced, so that a single clone dominated passages 7-11. The clone that emerged from this genetic convergence had a t(12;17)(p13;q21) as the sole abnormality. Our findings indicate that the cytogenetic multiclonality that has been repeatedly detected in short-term cultures of squamous cell carcinomas is not caused by the in vitro conditions. Instead, the principles of Darwinian selection apply: the altered, but stable, selection pressure facing a newly established and initially multiclonal cell line will lead to a reduction of genetic heterogeneity until the one clone that now has the proliferative advantage outgrows the other subpopulations.     

Microcalorimetric measurements of tissue cells in vitro

A culture flask was designed for the microcalorimetric measurements of tissue cells by an MS 80 standard calvet microcalorimeter. Tissue cells cultured in this flask behaved in the same manner as in the common culture flask used in cytobiological studies. The thermograms of human adenocarcinoma gastric cells (SGc 7901) and HeLa cells were obtained. The heat output power of SGc 7901 cells continuously increased for 70 h with an initial cell number of 3.0 X 10(5). The thermogram was reproducible under strictly controlled conditions. The relationship between the heat output power and the number of SGc 7901 cells within 48 h was obtained. The heat output power was 40 pW/cell to 49 pW/cell when the cell number was in the range 4.5 X 10(5) to 10.4 X 10(5). It was 62.3 +/- 2.9 pW/cell for HeLa cells when the cell number was 6 X 10(5).     

Calcitonin gene-related peptide (CGRP) in normotensive and spontaneously hypertensive rats

With the techniques of specific radioimmunoassay and gel filtration it was found that CGRP was distributed in various tissues of normotensive (WKY) and spontaneously hypertensive rats (SHR) with the highest concentration in the lumbar spinal cord (1197 +/- 94.8 pg/mg tissue) and the lowest in the auricle (15.0 +/- 2.1 pg/mg tissue). In comparison with WKY, CGRP concentration in the plasma was decreased and in the abdominal aorta and hypothalamus was increased in SHR. Gel filtration revealed only one major CGRP molecular form in the tissues. In addition, CGRP reduced the mean arterial pressure (MAP) in SHR in a dose-dependent manner. These data suggest that CGRP may play an important role in the pathogenesis of hypertension and its possible therapy.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti.

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Three residues involved in binding and catalysis in the carbamyl phosphate binding site of Escherichia coli aspartate transcarbamylase

Site-directed mutagenesis was used to create four mutant versions of Escherichia coli aspartate transcarbamylase at three positions in the catalytic chain of the enzyme. The location of all the amino acid substitutions was near the carbamyl phosphate binding site as previously determined by X-ray crystallography. Arg-54, which interacts with both the anhydride oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme was approximately 17,000-fold less active than the wild type, although the binding of substrates and substrate analogues was not altered substantially. Arg-105, which interacts with both the carbonyl oxygen and a phosphate oxygen of carbamyl phosphate, was replaced by alanine. This mutant enzyme exhibited an approximate 1000-fold loss of activity, while the activity of catalytic subunit isolated from this mutant enzyme was reduced by 170-fold compared to the wild-type catalytic subunit. The KD of carbamyl phosphate and the inhibition constants for acetyl phosphate and N-(phosphono-acetyl)-L-aspartate (PALA) were increased substantially by this amino acid substitution. Furthermore, this loss in substrate and substrate analogue binding can be correlated with the large increases in the aspartate and carbamyl phosphate concentrations at half of the maximum observed specific activity, [S]0.5. Gln-137, which interacts with the amino group of carbamyl phosphate, was replaced by both asparagine and alanine. The asparagine mutant exhibited only a small reduction in activity while the alanine mutant was approximately 50-fold less active than the wild type. The catalytic subunits of both these mutant enzymes were substantially more active than the corresponding holoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

How does l-glutamate transport relate to selection of mixed nitrogen sources in Rhizobium leguminosarum biovar trifolii MNF1000 and cowpea Rhizobium MNF2030?

When growing on a mixture of ammonia and l-glutamate as nitrogen sources, Rhizobium leguminosarum biovar trifolii MNF1000 utilizes ammonia exclusively, while cowpea Rhizobium MNF2030 utilizes both compounds at similar rates. l-Glutamate transport in both strain MNF1000 and MNF2030 is active, giving rise to a 60-fold concentration gradient across the membrane of cells of strain MNF2030. Both strains produce two kinetically distinguishable glutamate transport systems under all conditions of growth — a high affinity system with an apparent K _m of 0.06–0.17 M but of relatively low V _max, and a low affinity system with a K _m of 1.2–6.7\ M, but of higher overall capacity. l-Glutamate transport activity in cells of MNF2030 was relatively insensitive to the presence of ammonia in the growth medium. By contrast, ammonia in the growth medium resulted in low activities of glutamate transport in cells of MNF1000 which were provided with a carbon source, offering one explanation for the failure of this strain to use glutamate in the presence of ammonia. However, in cells of MNF1000 growing on glutamate as sole source of carbon and nitrogen, the glutamate transport system is synthesized, even in the presence of accumulated or added ammonia. This suggests that the regulation of the glutamate permease also depends on availability of carbon source.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - HEPES N-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Molecular cloning and expression of a proteinase gene from Lactococcus lactis subsp. cremoris H2 and construction of a new lactococcal vector pFX1

The 6.5 kb HindIII DNA fragment of the Lactococcus lactis subsp. cremoris H2 plasmid pDI21 was cloned into Escherichia coli POP13 with NM1149, and also directly into Lactococcus lactis subsp. lactis 4125 using a newly-constructed broad host-range vector pFX1. Proteinase was experessed in both transformed organisms. The proteinase resembles a PI type since it preferentially degraded -casein. The restriction map of the 6.5 kb proteinase gene fragment has minor differences from those of published plamid proteinase genes. High-efficiency electroporation with pFX1 provides a direct approach for gene cloning in lactococci.Abbreviations cfu colony forming units - HEPES N-[2-hydroxyethyl]piperazine-N-[2-ethanesulphonic acid] Dedicated to Prof. Dr. G. Drews on the occasion of his 65th birthday