The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway.

The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface.     

Production, Purification, and Characterization of Recombinant Human Hemoglobin Rainier Expressed inSaccharomyces cerevisiae

Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the β145 tyrosine is substituted with cysteine. The α and β^Rainierglobin cDNAs were cloned in a high copy number vector and expressed inSaccharomyces cerevisiaeunder the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual α and β^Rainierglobin chains. Coexpression of both α and β^RainiercDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the α and β chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A₀. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.     

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.     

Genome Analysis and Physiological Comparison of Alicycliphilus denitrificans Strains BC and K601T

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601^T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601^T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601^T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601^T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601^T. Genes involved in cyclohexanol degradation were only found in strain K601^T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far.     

Using dynamic pupillometry as a simple screening tool to detect autonomic neuropathy in patients with diabetes: a pilot study

Background  

Autonomic neuropathy is a common and serious complication of diabetes. Early detection is essential to enable appropriate interventional therapy and management. Dynamic pupillometry has been proposed as a simpler and more sensitive tool to detect subclinical autonomic dysfunction. The aim of this study was to investigate pupil responsiveness in diabetic subjects with and without cardiovascular autonomic neuropathy (CAN) using dynamic pupillometry in two sets of experiments.     

Antiviral activity of an N-allyl acridone against dengue virus

Background

Dengue virus (DENV), a member of the family Flaviviridae, is at present the most widespread causative agent of a human viral disease transmitted by mosquitoes. Despite the increasing incidence of this pathogen, there are no antiviral drugs or vaccines currently available for treatment or prevention. In a previous screening assay, we identified a group of N-allyl acridones as effective virus inhibitors. Here, the antiviral activity and mode of action targeted to viral RNA replication of one of the most active DENV-2 inhibitors was further characterized.

Results

The compound 10-allyl-7-chloro-9(10H)-acridone, designated 3b, was active to inhibit the in vitro infection of Vero cells with the four DENV serotypes, with effective concentration 50% (EC₅₀) values in the range 12.5-27.1 μM, as determined by virus yield inhibition assays. The compound was also effective in human HeLa cells. No cytotoxicity was detected at 3b concentrations up to 1000 μM. Mechanistic studies demonstrated that virus entry into the host cell was not affected, whereas viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR. The addition of exogenous guanosine together with 3b rescued only partially the infectivity of DENV-2.

Conclusions

The acridone derivative 3b selectively inhibits the infection of Vero cells with the four DENV serotypes without a direct interaction with the host cell or the virion but interfering specifically with the intracellular virus multiplication. The mode of antiviral action for this acridone apparently involves the cellular enzyme inosine-monophospahe dehydrogenase together with another still unidentified target related to DENV RNA synthesis.