Genetic inversion in the formation of an Hfr strain from a temperature-sensitive F'' gal strain.

An Hfr strain (PB15) that carries a duplicated copy of the galactose operon genes flanking the integrated sex factor is unusually stable since it does not show excision of the repeated deoxyribonucleic acid segment. The right-hand galactose operon is in the normal orientation. Deletion mutations that eliminate the right-hand galactose genes, the sex factor, and some of the left-hand operon have been isolated. Mutants believed to have their left-hand galactose operon inverted were able to be induced for galactose epimerase synthesis by D-fucose but did not show escape synthesis on induction of bacteriophage lambda. Ribonucleic acid specific for the galactose operon was isolated after induction of lysogenic strains presumed to carry the galactose operon in the normal and inverted orientation. Hybridization to the isolated left and right strands of lambdapgal showed that the noninformational strand of the left-hand galactose operon of the deletion mutant of PB15 was transcribed on escape induction. These results show that inversion has occurred.     

L1CAM from human melanoma carries a novel type of N-glycan with Galβ1-4Galβ1- motif. Involvement of N-linked glycans in migratory and invasive behaviour of melanoma cells

Dramatic changes in glycan biosynthesis during oncogenic transformation result in the emergence of marker glycans on the cell surface. We analysed the N-linked glycans of L1CAM from different stages of melanoma progression, using high-performance liquid chromatography combined with exoglycosidase sequencing, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and lectin probes. L1CAM oligosaccharides are heavily sialylated, mainly digalactosylated, biantennary complex-type structures with galactose β1-4/3-linked to GlcNAc and with or without fucose α1-3/6-linked to GlcNAc. Hybrid, bisected hybrid, bisected triantennary and tetraantennary complex oligosaccharides, and β1-6-branched complex-type glycans with or without lactosamine extensions are expresses at lower abundance. We found that metastatic L1CAM possesses only α2-6-linked sialic acid and the loss of α2-3-linked sialic acid in L1CAM is a phenomenon observed during the transition of melanoma cells from VGP to a metastatic stage. Unexpectedly, we found a novel monoantennary complex-type oligosaccharide with a Galβ1-4Galβ1- epitope capped with sialic acid residues A1[3]G(4)2S_2-3. To our knowledge this is the first report documenting the presence of this oligosaccharide in human cancer. The novel and unique N-glycan should be recognised as a new class of human melanoma marker. In functional tests we demonstrated that the presence of cell surface α2-3-linked sialic acid facilitates the migratory behaviour and increases the invasiveness of primary melanoma cells, and it enhances the motility of metastatic cells. The presence of cell surface α2-6-linked sialic acid enhances the invasive potential of both primary and metastatic melanoma cells. Complex-type oligosaccharides in L1CAM enhance the invasiveness of metastatic melanoma cells.     

Evaluation of shotgun sequencing for proteomic analysis of human plasma using HPLC coupled with either ion trap or Fourier transform mass spectrometry

This paper reports on studies directed to the characterization of the proteome of human plasma by the shotgun sequencing approach, namely the use of HPLC coupled to mass spectrometry (MS). The report will present data from two laboratories that allows the comparison of peptide and protein identifications by either accurate mass measurement on a Fourier transform mass spectrometry or MS/MS fragmentation on an ion trap mass spectrometer. Because the dynamic range of the protein components of plasma is one of the largest for a biological sample, the analysis of such a challenging sample was aided by the use of these two MS approaches. The major classes of proteins observed were transport proteins, enzymes, and enzyme inhibitors, blood-clotting factors, membrane-associated proteins including soluble forms of receptors, hormones, immunoglobulins, and other glycoproteins. The protein identifications were also highly consistent with results obtained from 2D gel studies, although a larger number of additional proteins were observed with the shotgun sequencing approach. The quantitation of low to medium level proteins was explored in the ion trap with an add-back of a known amount of human growth hormone (hGH) at a clinically relevant level (5 ug/L). The isotope coded affinity tag (ICAT) approach was used to quantitate successfully different levels of hGH in replicate analysis via the disulfide linked tryptic peptide (T6-T16). These studies suggest that the shotgun sequencing approach can be used to characterize part of the plasma proteome and serve as a starting point for the use of multidimensional analytical approaches for the analysis of complex biological samples.     

Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli.

The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.     

The phylogeny of Mycoplasma bovis as determined by sequence analysis of the 16S rRNA gene

Abstract The nucleotide sequence of the 16S rRNA gene of Mycoplasma bovis has been determined. Comparisons with other 16S rRNA sequences of mycoplasmas showed that Mycoplasma agalactiae is phylogenetically the closet relative. In total, only eight nucleotides differed between the M. bovis and M. agalactiae 16S rRNA sequences. The phylogenetic position of M. bovis with respect to other mycoplasmas was determined by sequence comparisons and from features in the secondary structure of 16S rRNA.     

Nucleotide sequence analysis of RepFIC, a basic replicon present in IncFI plasmids P307 and F, and its relation to the RepA replicon of IncFII plasmids.

RepFIC is a basic replicon of IncFI plasmid P307 which is located within a 3.09-kilobase SmaI fragment. The nucleotide sequence of this region has been determined and shown to be homologous with the RepFIIA replicon of IncFII plasmids. The two replicons share three homologous regions, HRI, HRII, and HRIII, which are flanked by two nonhomologous regions, NHRI and NHRII. A comparison of coding regions reveals that the two replicons have several features in common. RepFIC, like RepFIIA, codes for a repA2 protein with its amino-terminal codons in HRI and its carboxy-terminal codons in NHRI. Although the codons for the repA1 proteins are located in NHRII, the DNA region containing a putative promoter, ribosomal binding site, and initiation codons is located in HRII. This region also codes for an inc RNA. There are nine base-pair differences between the inc RNA of RepFIIA and that of RepFIC, and as a result, RepFIC and RepFIIA replicons are compatible. An EcoRI fragment from the F plasmid which shows homology with RepFIC of P307 has also been sequenced. This fragment contains only a portion of RepFIC, including the genes for the putative repA2 protein and inc RNA. The region coding for a putative repA1 protein is interrupted by the transposon Tn1000 and shows no homology with the repA1 region of RepFIIA and RepFIC of P307. Our comparative and structural analyses suggest that RepFIC and RepFIIA, although different, have a similar replication mechanism and thus can be assigned to the same replicon family, which we designate the RepFIIA family.     

Temperature-sensitive mutations affecting the replication of F-prime factors in Escherichia coli K 12

Summary More temperature-sensitive mutants affecting the replication of the F-gal⁺ episome of Escherichia coli K12 have been isolated. Eight of the mutations were located on F itself and three were located on the chromosome.The temperature sensitive F-gal⁺'s have been integrated into the chromosome to produce Hfr strains. These Hfr strains have transfer origins similar to Hfr Cavalli, and all show aberrant excision and transfer of elongated segments of the chromosome including the integrated F-gal to generate long merodiploids.The chromosomal mutations that govern the replication of F have been termed seg (for segregation). Wild-type F-gal⁺ can be integrated into seg cells at 42° C to give Hfrs, in a process analogous to integrative suppression in the formation of Hfrs from cells carrying mutations that are temperature-sensitive for chromosomal DNA replication (dnaA). A curious feature of an Hfr derived from a seg strain is that it also shows F-genote enlargement as well as normal transfer of chromosomal genetic marker. Preliminary transductional mapping data show that the mutation seg-2 is linked to the threonine locus (minute 0).     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

Determination of Serotonin and Dopamine Metabolites in Human Brain Microdialysis and Cerebrospinal Fluid Samples by UPLC-MS/MS: Discovery of Intact Glucuronide and Sulfate Conjugates

An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.