Differences in the surface composition of seemingly similar F75 cobalt-chromium micron-sized particulates can affect synovial fibroblast viability.

We present data and analyses concerning the cytotoxicity and bioreactivity associated with the surface composition of fine metal particulates that are similar to those commonly released in the body by prostheses used in total joint replacement surgery. Here we study the bulk and surface compositions of three separately procured cobalt-chromium-molybdenum (CoCrMo) micron-sized particulate powders, each identified by their corresponding vendor as being ASTM F75 grade material. We use energy dispersive spectroscopy (EDS) to verify the bulk metallic composition and X-ray photoelectron spectroscopy (XPS) to examine the surface metallic composition of each CoCrMo powder. Cultured synovial fibroblasts were then exposed to the particulate powders to see how the metallic surfaces might affect cellular viability. Results indicate that while the bulk metallic composition of each CoCrMo powder was similar, the surface metallic compositions were found to be dramatically different and yielded equally dramatic differences in terms of cytotoxicity and bioreactivity of synovial fibroblast in culture.

Analysis of disputed single-parent/child and sibling relationships using 16 STR loci.

This study describes the validation of short tandem repeat (STR) systems for the resolution of cases of disputed parentage where only a single parent is available for testing or where the claimed relationship of both parents is in doubt and also cases where sibship must be tested. Three separate multiplex systems the Second Generation Multiplex, Powerplex 1.2 and FFFL have been employed, giving a total of 16 STR loci. Both empirical and theoretical approaches to the validation have been adopted. Appropriate equations have been derived to calculate likelihood ratios for different relationships, incorporating a correction for subpopulation effects. An F(ST) point estimate of 1% has been applied throughout. Empirically, 101 cases of alleged father, alleged mother and child where analysed using six SLP systems and also using the three multiplex STR systems. Of the 202 relationships tested, 197 were independently resolved by both systems, providing either clear evidence of non-parentage or strong support for the relationship.

Sequence characterization of ENU-induced mutants of glucose phosphate isomerase in mouse.

Four of five mutations producing GPI1 null lethal phenotypes in the homozygous state, which were previously identified from the offspring of male mice, spermatogonially treated with N-ethyl N-nitrosourea (ENU), have been characterized at the nucleotide level by reverse transcription of RNA from heterozygotes for mutant and wild-type alleles and cycle sequencing with cDNA-derived primers. In three of the mutations studied, a single nucleotide substitution, altering the predicted amino acid on translation, was observed in the mutant allele. In Gpi1-sam1H amino acid residue 277, TCA Ser (wild type), is altered to CCA Pro, and in Gpi1-sbm3H and Gpi1-sbm4H amino acid residue 510 Asp GAC (wild type) is altered to GGC Gly. These ENU-induced mutations occur at A-T base pairs in agreement with the current view of the mechanism of action for this mutagen. These changes also occur at residues implicated as being important in the catalytic functioning of the enzyme, from crystallographic studies, and may explain the loss of enzyme function. The fourth identified mutation, Gpi1-sbm2H, is a deletion of amino acid residues Arg134 to Leu162 inclusive, which may arise from incorrect splicing of mRNA; a fifth mutation has remained undetermined.

Structure and organization of the human glucose phosphate isomerase gene (GPI).

Two overlapping yeast artificial chromosome clones containing the human glucose 6-phosphate isomerase gene (GPI) have been isolated. PCR and direct sequencing were used to determine the exon/intron structure of the gene. The gene spans in excess of 40 kb and consists of 18 exons ranging in size from 44 to 153 bp. All splice sites conform to the GT/AG rule.

Donor families experience of organ donation: a New Zealand study.

AIM: To investigate the experiences of New Zealand families who had given approval for organ donation. METHOD: A postal questionnaire was sent to all families in New Zealand over a 5 year period who had agreed to donate organs on the death of their family member. RESULTS: Of the 102 questionnaires sent, 49 were returned completed. No respondents said it was the wrong decision in agreeing to donate organs. 31/49 respondents said that being asked caused no additional stress. 31/49 had previously discussed organ donation within their family. 16/49 knew the information on the donor's driving license. 38/49 said that they understood brain death at the time. 31/49 said the care, understanding and support given by the staff in the intensive care unit helped them most at the time. 23/49 would have liked further support from the transplant coordinators. 32/49 would have liked written information at some stage. Even though they were not asked 25/49 volunteered that they would have liked information about recipient outcome. CONCLUSION: Our results reinforce and support the findings of previous research from New Zealand and other countries. These have been used to confirm and revise procedures in the planning of care for future donor families.

Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man.

Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. We have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage lambda recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composed of 30 repeat units of a novel 50-bp tandemly repeated units. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster.

Measurement of androgen receptor expression in adult liver, fetal liver, and Hep-G2 cells by the polymerase chain reaction.

Hepatocellular carcinoma is the most commonly fatal malignant tumour worldwide. The role of androgen receptors, which have been found in hepatocellular carcinoma, is controversial. Sequence specific polymerase chain reaction (PCR) was used to quantify, for the first time, the expression of androgen receptor in four adult liver biopsy specimens (HL-A to HL-D), fetal liver, and Hep-G2 cells. The measurement of androgen receptor is expressed as a ratio (androgen receptor: beta-actin) of the value of androgen receptor to the value of a control gene, beta-actin. The value of the androgen receptor: beta-actin ratios for HL-A, HL-B, HL-C, HL-D, fetal liver, and Hep-G2 were 0.37, 0.86, 0.37, 0.44, 0.87, and 0.66 respectively. To verify sequence specific amplification of the androgen receptor, the PCR androgen receptor fragment was sequenced. The resultant sequence data for both strands of the double stranded PCR androgen receptor fragment had 100% similarity with the published androgen receptor mRNA sequence (complete codons).

Characterization of the 5' end of the gene for human glucose phosphate isomerase (GPI).

We have isolated the gene coding for human glucose phosphate isomerase, and here we report the characterization of its 5' end including the first two exons. The gene is greater than 50 kb in size and contains a minimum of eight exons. We have no evidence that this gene is part of a multigene family or that there are any pseudogenes. The potential transcription start site has been determined by primer extension analysis and is 52 bp upstream from the translation initiation site of the protein. Sequences 5' to the transcription initiation site and within the first intron are extremely GC rich and form part of a CpG island. Five potential Sp1 sites (GGGCGG) have been located at positions -57, -61, -95, +183, and +575. The most 5' of these (GGGGCGGGGG) is likely to be the main Sp1 binding site, as it conforms precisely to a 10-bp consensus sequence. At position -44 there is a putative TATA box (CATAAA). Thus in common with an increasing number of genes, the putative promoter region of glucose phosphate isomerase shows structural similarities to both housekeeping and facultative gene promoters.

Complementation of metabolic defect in phosphoglycerate kinase (PGK)-deficient line of Chinese hamster ovary cells by introduction of human PGK cDNA.

A full-length copy of the coding sequence of the human phosphoglycerate kinase (PGK) gene was introduced into a glycolysis-negative, PGK-deficient line of Chinese hamster ovary (CHO-K1) cells by gene transfer. Transformants were isolated either by cotransfer of the bacterial aminoglycoside phosphotransferase (AGPT) gene, or by direct selection for expression of the PGK gene. Integration of the human PGK gene has been demonstrated by Southern blot analysis and its expression by starch gel electrophoresis. PGK transformants behaved phenotypically as predicted by the properties of their wild type parent: mannose was no longer toxic, but instead was metabolized via glycolysis to lactic acid, and cell growth was no longer dependent on glutamine oxidation. Thus a complex phenotypic change has been mediated by gene transfer. The combination of R1.1.7 cells and the PGK plasmid provides another system to facilitate the study of mammalian gene expression.

Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences.

Neuroleukin is a neurotrophic factor of relative molecular mass (Mr) 56,000 (56K) found in skeletal muscle, brain, heart and kidneys which supports the survival of embryonic spinal neurones, skeletal motor neurones and sensory neurones. Neuroleukin is also a lymphokine product of lectin-stimulated T cells and induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Mouse neuroleukin has been cloned, the complete nucleotide sequence has been determined and its complementary DNA has been transiently expressed in monkey COS-1 cells. The serum-free supernatant of the transfected, but not of control mock-transfected, cells was shown to mimic the properties of neuroleukin isolated from mouse salivary glands. In our work on the molecular genetics of carbohydrate metabolism we have recently isolated a mouse glucose-6-phosphate isomerase (or phosphoglucose isomerase, PGI) cDNA clone using the yeast PGI gene (PGI 1) as a probe. We report here that there is complete sequence identity between the 759 nucleotides at the 3' end of this clone (coding and non-coding) and the sequence of mouse neuroleukin.

Patient compliance with MAO inhibitor therapy.

Exaggerated fears of monoamine oxidase inhibitors (MAOIs) and of their interactions with foods often restrict their use. A review of the literature reveals seven food items most likely to produce a hypertensive crisis in combination with MAOI administration: aged cheeses, smoked or pickled fish, beef or chicken liver, dry fermented sausage, pods of broad beans, brewer's yeast products, and certain alcoholic beverages. Improved understanding of the dietary restrictions, benefits, and mechanism of action of the MAOIs can enhance cooperation with the prescribed treatment program.

Practical aspects of MAO inhibitor therapy.

Instructions for patients regarding the observation of dietary and medication restrictions during MAOI therapy are presented. Principles of physician management of MAOI therapy, including avoidance and management of hypertensive crises and the potential for drug-drug interactions, are briefly reviewed.