Development and validation of a bioanalytical assay for the measurement of total and unbound teicoplanin in human serum.

BACKGROUND: The glycopeptide teicoplanin is considered first-line treatment for severe infections caused by Gram-positive bacteria. Individualized treatment of teicoplanin is gaining interest. As only protein-unbound drug is pharmacologically active, a sensitive assay measuring unbound and total teicoplanin is indispensable for pharmacological research and dose optimization. OBJECTIVES: To develop and validate a UPLC-MS/MS method to quantify unbound and total teicoplanin in human serum. METHODS: The developed assay was validated according to the ICH guideline M10 on Bioanalytical Method Validation and study sample analysis. Unbound teicoplanin was obtained by ultrafiltration. The assay was cross-validated with a quantitative microsphere (QMS) immunoassay in a side-by-side comparison using 40 patient samples. RESULTS: With the developed and validated method, all main teicoplanin components (A2-1, A2-2/A2-3, A2-4/A2-5 and A3-1) can be quantified. Total run time was 5.5 min. Concentration range was 2.5-150 mg/L for total and 0.1-25 mg/L for unbound teicoplanin. Precision (coefficient of variation) and accuracy (bias) of total teicoplanin were 5.97% and 107%, respectively, and 7.17% and 108%, respectively, for unbound teicoplanin.Bland-Altman analysis showed total concentrations measured with the UPLC-MS/MS method were equivalent to the results of the QMS immunoassay. A total of 188 samples from 30 patients admitted to the ICU and haematology department were measured; total concentrations ranged between 2.92 and 98.5 mg/L, and unbound concentrations ranged between 0.37 and 30.7 mg/L. CONCLUSIONS: The developed method provided rapid, precise and accurate measurement of unbound and total teicoplanin. The developed method is now routinely applied in pharmacological research and clinical practice.

Integrated statistical analysis of cDNA microarray and NIR spectroscopic data applied to a hemp dataset.

Both cDNA microarray and spectroscopic data provide indirect information about the chemical compounds present in the biological tissue under consideration. In this paper simple univariate and bivariate measures are used to investigate correlations between both types of high dimensional analyses. A large dataset of 42 hemp samples on which 3456 cDNA clones and 351 NIR wavelengths have been measured, was analyzed using graphical representations. For this purpose we propose clustered correlation and clustered discrimination images. Large, tissue-related differences are seen to dominate the cDNA-NIR correlation structure but smaller, more difficult to detect, variety-related differences can be found at specific cDNA clone/NIR wavelength combinations.

Cloning, expression, and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis.

Proline iminopeptidase produced by Propionibacterium shermanii plays an essential role in the flavor development of Swiss-type cheeses. The enzyme (Pip) was purified and characterized, and the gene (pip) was cloned and expressed in Escherichia coli and Lactococcus lactis, the latter species being an extensively studied, primary cheese starter culture that is less fastidious in its growth condition requirements than P. shermanii. The levels of expression of the pip gene could be enhanced with a factor 3 to 5 by using a strong constitutive promoter in L. lactis or the inducible tac promoter in E. coli. Stable replication of the rolling-circle replicating (rcr) plasmid, used to express pip in L. lactis, could only be obtained by providing the repA gene in trans. Upon the integration of pip, clear gene dosage effects were observed and stable multicopy integrants could be maintained upon growth under the selective pressure of sucrose. The multicopy integrants demonstrated a high degree of stability in the presence of glucose. This study examines the possibilities to overexpress genes that play an important role in food fermentation processes and shows a variety of options to obtain stable food-grade expression of such genes in L. lactis.

Light microscopic and ultrastructural investigation of the dopaminergic innervation of the ventrolateral outgrowth of the rat inferior olive.

The ventrolateral outgrowth of the inferior olive is involved in the control of compensatory eye movement responses to optokinetic stimuli about the horizontal axis that is perpendicular to the ipsilateral anterior semicircular canal. Combining immunocytochemistry with retrograde tracing of WGA-BSA-gold, we demonstrated in the present study that this olivary subnucleus receives a substantial dopaminergic input, and that the prerubral parafascicular area and its surrounding regions form the sole source of this input. In addition, we investigated the postsynaptic distribution of the dopaminergic terminals in the inferior olive at the ultrastructural level. About a third (32%) of the dopaminergic terminals was found to make synaptic contacts in the olivary neuropil. The majority (81%) of these boutons terminated on cell bodies or extraglomerular dendrites, while the remaining terminals contacted dendritic spines inside glomeruli. In contrast, GABAergic terminals in the inferior olive formed more frequently (66%) synaptic contacts and they terminated more frequently (38%) in glomeruli. Thus, the ventrolateral outgrowth receives a dopaminergic input from the mesodiencephalic junction, and the postsynaptic distribution of this input reveals a characteristic pattern.

A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos.

The first somatic single cells of carrot hypocotyl explants having the competence to form embryos in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D) were identified using semi-automatic cell tracking. These competent cells are present as a small subpopulation of enlarged and vacuolated cells derived from cytoplasm-rich and rapidly proliferating non-embryogenic cells that originate from the provascular elements of the hypocotyl. A search for marker genes to monitor the transition of somatic into competent and embryogenic cells in established suspension cell cultures resulted in the identification of a gene transiently expressed in a small subpopulation of the same enlarged single cells that are formed during the initiation of the embryogenic cultures from hypocotyl explants. The predicted amino acid sequence and in vitro kinase assays show that this gene encodes a leucine-rich repeat containing receptor-like kinase protein, designated Somatic Embryogenesis Receptor-like Kinase (SERK). Somatic embryos formed from cells expressing a SERK promoter-luciferase reporter gene. During somatic embryogenesis, SERK expression ceased after the globular stage. In plants, SERK mRNA could only be detected transiently in the zygotic embryo up to the early globular stage but not in unpollinated flowers nor in any other plant tissue. These results suggest that somatic cells competent to form embryos and early globular somatic embryos share a highly specific signal transduction chain with the zygotic embryo from shortly after fertilization to the early globular embryo.

AtLTP1 luciferase expression during carrot somatic embryogenesis.

The carrot (Daucus carota L.) EP2 gene encodes a Lipid Transfer Protein (LTP) which is expressed during protoderm formation in developing embryos. To develop a vital reporter system for gene expression during somatic embryo development a 1.1 kB fragment of the Arabidopsis thaliana LTP1 promoter was fused to the firefly luciferase (LUC) coding sequence. The AtLTP1 luciferase expression pattern in transformed carrot suspension cultures was identical to the expression pattern of the endogenous carrot EP2 gene. Cell tracking experiments revealed that all somatic embryos were derived from AtLTP1 luciferase expressing cell clusters. However, not all cell clusters that expressed the AtLTP1 luciferase reporter gene developed into a somatic embryo, suggesting that initiation of an embryogenic pathway in tissue culture does not always lead to development of a somatic embryo.

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme.

The bacteriocin pediocin PA-1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA-1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N-terminal domains of PedD and a subgroup of bacteriocin ABC-transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis.

The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.

Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and nucleotide sequence of the alpha-galactosidase cDNA from Cyamopsis tetragonoloba (guar).

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the alpha-galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH2 terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH2 terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/alpha factor and killer factor. A comparison of the derived amino acid sequence of this alpha-galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the alpha-galactosidase from Escherichia coli.

Synthesis and processing of the plant protein thaumatin in yeast.

Various maturation forms of the plant protein thaumatin were expressed in yeast, using a promoter fragment of the glyceraldehyde- 3P -dehydrogenase (GAPDH) gene. Plasmids encoding preprothaumatin were shown to direct the synthesis of a processed form of the plant protein. The important role of signal sequences in the expression of the plant protein in yeast was indicated by the observation that plasmids encoding processed thaumatin forms were only poorly expressed, if at all. Nucleotide sequence analysis of the 843 nucleotide GAPDH promoter fragment revealed a characteristic structure with two regions of dyad symmetry containing translational starts of GAPDH and a putative 38 amino acid peptide. A promoter fragment from which the upstream region was deleted proved to be less efficient in thaumatin expression.

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli.

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.