High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation.

Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation.

Molecular analysis of the regulation of nisin immunity.

The genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue. The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism. This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis.

Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5.

A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W.

Menkes disease: recent advances and new insights into copper metabolism.

Copper is a trace element necessary for the normal function of several important enzymes but copper homeostasis is still poorly understood. In recent years remarkable progress has been made in this field following the isolation of the gene defective in Menkes disease. Menkes disease and occipital horn syndrome are X-linked recessive disorders, demonstrating the vital importance of copper, which is also highly toxic in excessive amounts.

Appraisal system for community child health doctors.

An appraisal system for Community Child Health Doctors in Nottingham is described and evaluated. All staff are invited to an annual meeting with their consultant, preparation documents having been filled in by both appraisee and appraiser beforehand. These form the basis of the session, which is predominantly a self-assessment.

Protein engineering of lantibiotics.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply.

A gene replacement strategy for engineering nisin.

A lactococcal expression system was developed which allows the exclusive production of novel nisins encoded by mutated pre-nisin (nisA) genes. This system is based on a combination of a specifically constructed host strain and vectors which facilitate the genetic manipulation of the nisA gene. The wild-type chromosomal gene is effectively replaced with a variant nisA gene, by the technique of gene replacement.

The effect of histidine and cysteine on zinc influx into rat and human erythrocytes.

1. The effect of histidine and cysteine on the initial rate of zinc influx into rat and human erythrocytes in the presence of bovine serum albumin has been investigated. 2.

A cassette vector for protein engineering the lantibiotic nisin.

Expression vectors are described that make use of a plasmid-encoded nisA cassette encoding the peptide component of nisin. Specific mutations can readily be incorporated throughout the coding region of nisA. Using this vector in a nisA mutant host, three variant nisins, with dehydroalanine (Dha) residues changed to Ala residues, were generated.

Metallothionein expression in placental tissue in Menkes' disease. An immunohistochemical study.

Menkes' disease is a recessive X-linked disturbance of copper metabolism, resulting in accumulation of copper in several extra-hepatic tissues including the placenta. Metallothionein (MT) is a low-molecular weight protein with a high affinity for group II metal ions, such as copper. Its synthesis is induced by the presence of the ions.

Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials.

A production method has been developed for the purification of pharmaceutical-grade plasmid DNA for in vivo gene therapy. This method has been applied to the purification of VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the HLA-B7 protein. Purified VCL-1005 is formulated with a cationic lipid and injected directly into established tumors of HLA-B7-negative patients with advanced cancers to heighten the patient's immune response against the cancer.

Characterization of the exon structure of the Menkes disease gene using vectorette PCR.

The gene defective in Menkes disease, an X-linked recessive disturbance of copper metabolism, has been isolated and predicted to encode a copper-binding P-type ATPase. We determined the complete exon-intron structure of the Menkes disease gene, which spans about 150 kb of genomic DNA. The gene contains 23 exons, and the ATG start codon is in the second exon.

High level expression and refolding of mouse interleukin 4 synthesized in Escherichia coli.

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide.

Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages.

A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production. Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria. BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics.

First trimester prenatal diagnosis of Menkes disease by DNA analysis.

Menkes disease is an X linked recessive disorder of copper metabolism characterised by neurological symptoms and connective tissue manifestations. The defective gene in Menkes disease has recently been isolated and the gene product is predicted to be a copper transporting ATPase. The diagnosis of Menkes disease has hitherto been performed by biochemical analysis, based on intracellular accumulation of copper.

Analysis of Mnk, the murine homologue of the locus for Menkes disease, in normal and mottled (Mo) mice.

Menkes disease (MNK) lies immediately proximal to pphosphoglycerate kinase (PGK1) in Xq13 in human. Phenotypic similarities between MNK patients and murine mottled (Mo) mutants strongly suggest that both defects are caused by mutations at the same locus. Human MNK cDNA clones and a genomic subclone derived from a 40-kb YAC clone that includes Pgk1 have been used to position the murine homologue of Menkes disease (MNK, Mnk) immediately proximal to, and within 150-200 kb of, phosphoglycerate kinase (Pgk1) on the mouse X chromosome using interspecific backcross analysis and pulsed-field gel electrophoresis.

Characterization of IS905, a new multicopy insertion sequence identified in lactococci.

IS905 is a multicopy insertion sequence identified in Lactococcus lactis. It is 1313 bp long, bounded by 28-bp imperfect inverted repeats, and encodes a putative transposase of 391 amino acids. One end of IS905 contains sequences that are potentially promoter active.

Hypertensive adolescents detected by a school surveillance programme: a problem of obesity.

BP has been measured in secondary school children in Nottingham as part of a routine health appraisal since 1988. School nurses who have received appropriate training refer children with a BP greater than the 95th centile for age and sex (Task Force USA 1987) initially to a community paediatrician or general practitioner. Of 14,570 children checked, only 23 patients (14 female) with a mean age of 13.

Clinical and biochemical consequences of copper-histidine therapy in Menkes disease.

Menkes disease (MD) is an X-linked recessively inherited neurodegenerative disorder of copper (Cu) metabolism leading to death in early childhood. Symptoms are attributed to deficient activity of Cu-dependent enzymes. Limited experience has been reported concerning clinical and biochemical consequences of parenteral treatment with copper-(histidine)2-complex (Cu-His) in MD.

Insulin-like growth factors (IGF) I and II and IGF binding proteins 1, 2 and 3 during low-dose growth hormone (GH) infusion and sequential euglycemic and hypoglycemic glucose clamps: studies in GH-deficient patients.

To evaluate the short-term effects of growth hormone (GH), insulin and different levels of glycemia on insulin-like growth factors (IGF) I and II and IGF binding proteins (IGFBP) 1, 2 and 3, we studied six GH-deficient adolescents during a night and the following day in the postabsorptive (basal) state followed by sequential euglycemic (5 mmol/l) and hypoglycemic (3 mmol/l) glucose clamps concomitant with an intravenous infusion (starting at 24.00 h) of GH (35 micrograms/h) or saline. Current GH therapy was withdrawn 24 h prior to each study.

Clinical studies of IGFBP-2 by radioimmunoassay.

A specific radioimmunoassay for human IGFBP-2 was developed using a polyclonal antiserum directed against a partial sequence (hIGFBP-2(176-190)). The tracer was prepared by radioiodination of a [Tyr]o-hIGFBP-2(176-190) derivative. The assay was used to study IGFBP-2 levels in numerous clinical and experimental situations.