Aberrant protein expression in plasma and kidney tissue during experimental obstructive nephropathy.

Kidney failure is a major health problem worldwide. Patients with end-stage renal disease require intensive medical support by dialysis or kidney transplantation. Current methods for diagnosis of kidney disease are either invasive or insensitive, and renal function may decline by as much as 50% before it can be detected using current techniques.

Direct and indirect roles of RECQL4 in modulating base excision repair capacity.

RECQL4 is a human RecQ helicase which is mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome (RTS), a disease characterized at the cellular level by chromosomal instability. BLM and WRN are also human RecQ helicases, which are mutated in Bloom and Werner's syndrome, respectively, and associated with chromosomal instability as well as premature aging. Here we show that primary RTS and RECQL4 siRNA knockdown human fibroblasts accumulate more H(2)O(2)-induced DNA strand breaks than control cells, suggesting that RECQL4 may stimulate repair of H(2)O(2)-induced DNA damage.

Evaluation of relaxin's antifibrotic action by SELDI-TOF mass spectrometry-based profiling of relaxin knockout mice, a model of progressive fibrosis.

Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry was employed to identify potential biomarkers for early-onset fibrosis. These biomarkers were then used to evaluate the efficacy of relaxin to reverse or ameliorate the development of the condition.

SELDI-TOF mass spectrometry-based protein profiling of kidney tissue.

Protein profiling has numerous applications in renal research including the detection of protein biomarkers with aberrant expression levels during disease development. Such information is essential for early diagnosis and will aid the improvement of patient management and minimise the progression of disease. Further to this, data generated from these studies will assist the elucidation of the precise mechanisms of disease development and can lead to the discovery of potential drug targets.

Purification and assays of Saccharomyces cerevisiae homologous recombination proteins.

Homologous recombination is an important means of eliminating DNA double strand breaks from chromosomes. The homologous recombination reaction is mediated by the Rad51 recombinase, which requires a number of ancillary factors for maximal efficiency. The development of purification procedures and biochemical assays for yeast Rad51 and other yeast recombination proteins has allowed investigators to begin dissecting the hierarchy of physical and functional interactions among these protein factors that govern the integrity of the homologous recombination machinery.

Multifaceted role of the Saccharomyces cerevisiae Srs2 helicase in homologous recombination regulation.

Homologous recombination (HR) is a major pathway for the elimination of DNA DSBs (double-strand breaks) induced by high-energy radiation and chemicals, or that arise due to endogenous damage and stalled DNA replication forks. If not processed properly, DSBs can lead to cell death, chromosome aberrations and tumorigenesis. Even though HR is important for genome maintenance, it can also interfere with other DNA repair mechanisms and cause gross chromosome rearrangements.

Biochemical characterization of the RECQ4 protein, mutated in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by growth deficiency, skin and skeletal abnormalities, and a predisposition to cancer. Mutations in the RECQ4 gene, one of five human homologs of the E. coli recQ gene, have been identified in a subset of RTS patients.

Role of ATP hydrolysis in the antirecombinase function of Saccharomyces cerevisiae Srs2 protein.

Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes.

Recombinant expression, purification and characterisation of the HMG domain of human SRY.

The HMG domain is a DNA binding and bending 'architectural' motif involved in chromatin re-modelling during transcription. Recombinant SRY HMG domain protein, 88 amino acids in length, has been produced in E. coli.

Transcription dependence of chromosomal gene targeting by triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) recognize and bind to specific DNA sequences and have been used to modify gene function in cells. To study factors that might influence triplex formation at chromosomal sites in mammalian cells, we developed a restriction protection assay to detect triplex-directed psoralen crosslinks in genomic DNA prepared from TFO-transfected cells. Using this assay, we detected binding of a G-rich TFO to a chromosomal site even in the absence of transcription when high concentrations of the TFO were used for transfection.

Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain.

Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene-3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene-3 (R3) relaxin that is highly expressed in a discrete region of the adult brain.

Genetic heterogeneity in the alveolar rhabdomyosarcoma subset without typical gene fusions.

Previous studies of the PAX3-FKHR and PAX7-FKHR gene fusions in alveolar rhabdomyosarcoma (ARMS) indicated that the corresponding fusiontranscripts are not detectable in 20% of ARMS cases. To investigate the genetic features of this ARMS subset, we identified 23 ARMS cases in which PAX3-FKHR and PAX7-FKHR transcripts were not detected by a standard sensitivity reverse transcription-PCR (RT-PCR) assay. Subsequent analysis with a high sensitivity RT-PCR assay identified low-level expression of PAX3-FKHR or PAX7-FKHR in three cases.

Purification and characterization of relaxin from the tammar wallaby (Macropus eugenii): bioactivity and expression in the corpus luteum.

The objective of this study was to isolate and purify prorelaxin or mature relaxin from the tammar wallaby corpus luteum (CL), determine their structure and bioactivity, and test the hypothesis that enzymatic cleavage of prorelaxin occurs in late gestation. Tammar relaxin peptides were extracted from pooled corpora lutea of late pregnant tammars using a combination of HPLC methods, and they were identified using Western blotting with a human (H2) relaxin antisera and matrix-assisted laser desorption ionization time of flight mass spectrometry. Although no prorelaxin was identified, multiple 6-kDa peptides were detected, which corresponded to the predicted mature tammar relaxin amino acid sequence, with an A chain of 24 amino acids, and different B chain lengths of 28, 29, 30, and 32 amino acids.

Kappacin, a novel antibacterial peptide from bovine milk.

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S.

Assembly of synthetic peptide vaccines by chemoselective ligation of epitopes: influence of different chemical linkages and epitope orientations on biological activity.

In this paper, we describe the assembly of synthetic peptide vaccines composed of a T helper cell epitope and a B cell epitope that were synthesized separately and then attached using three different chemoselective ligation methods: oxime bond formation, thioether bond formation and disulfide bond formation. The resulting vaccines were tested in animals to investigate their efficacy. We found that thioether bond formation gave the highest yield of material and that the chemistry involved did not adversely affect immunogenicity and biological activity of the peptide vaccine.

Research and development of an implantable, axial-flow left ventricular assist device: the Jarvik 2000 Heart.

Advances in technology and increased clinical need have led to the development of a new type of blood pump. The Jarvik 2000 Heart is an electrically powered, axial-flow left ventricular assist device that has been developed during the past 13 years. Unlike first-generation left ventricular assist devices, which were developed in the 1970s and were designed to totally capture the cardiac output, the Jarvik 2000 is designed to normalize the cardiac output by augmenting the function of the chronically failed heart for extended periods.

High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells.

To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK(-) cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 x 10(-6) under standard culture conditions. When cells were microinjected with TFOs designed to bind to a 30-bp polypurine site situated between the two TK genes, recombination was observed at frequencies in the range of 1%, 2,500-fold above the background.

Percutaneous Application of Fibrin Sealant to Achieve Hemostasis Following Arterial Catheterization.

OBJECTIVES: The present study sought to investigate the safety and efficacy of a new fibrin sealant using percutaneous delivery techniques. BACKGROUND: The femoral approach has become the preferred means of access for percutaneous diagnostic and interventional procedures. Control of access site bleeding following catheterization, however, remains a significant problem in interventional cardiology.

Foetal fluid balance and hormone status following nephrectomy in the foetal sheep.

1. The role of the kidneys in the maintenance of normal foetal plasma (FP) composition and hormone concentrations was examined in the present study. Five ovine foetuses were chronically cannulated and nephrectomized (nephx) at 100 +/- 1 days of gestation and maintained for 14 days.

Solid-phase synthesis of ovine Leydig cell insulin-like peptide--a putative ovine relaxin?

The primary structure of ovine Leydig cell insulin-like peptide (Ley I-L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B-chain, an Arg-X-X-X-Arg sequence exists within the Ley I-L B-chain although it is located four residues towards the C-terminus from the corresponding position within relaxin.

Synthesis and secondary structural studies of penta(acetyl-Hmb)A beta(1-40).

The Fmoc solid phase synthesis of A beta(1-40), a strongly aggregating peptide found in Alzheimer's disease brain, was performed using 2-hydroxy-4-methoxybenzyl (Hmb) backbone amide protection. Hmb-Gly residues were incorporated using N(alpha)-Fmoc-Hmb-Gly-OH rather than N,O-bisFmoc-Hmb-Gly-OPfp. Amino acid acylation of the sterically hindered Hmb-amino acids was monitored using 'semi-on-line' MALDI-TOF-MS in a novel application of this technique which significantly simplified the successful incorporation of these residues.

A genetically engineered, nonthrombogenic cellular lining for LVADs: in vitro preconditioning before in vivo implantation.

Because of the clinical success of left ventricular assist devices (LVADs) used for short-term "bridge to transplant" and the limited availability of donor organs, heart assist devices are being considered for long-term implantation as an alternative to heart transplantation. In an effort to improve biocompatibility, our laboratory has developed a nonthrombogenic cellular lining from genetically engineered smooth muscle cells (GE-SMC) for the Thermocardiosystems Heartmate LVAD. Smooth muscle cells have been transduced with the gene for endothelial nitric oxide synthase (NOS III) and produce NO at concentrations that reduce platelet deposition and smooth muscle cell proliferation when tested in vitro.

Minimally invasive access of the normal pericardium: initial clinical experience with a novel device.

The pericardial space is being investigated as a reservoir for local drug delivery to the heart and coronary arteries. Intrapericardial drug delivery is currently limited because the pericardial space is normally small and difficult to access by standard pericardiocentesis without invasive surgery or risk of cardiac injury. Clinical trials are being conducted to evaluate a novel, minimally invasive, pericardial access device (PerDUCER, Comedicus Inc.

Studies of the continuing susceptibility of group A streptococcal strains to penicillin during eight decades.

Background: In view of the widespread use of penicillin for >50 years for the treatment of group A streptococcal infections, we examined the question of whether there has been a change in susceptibility to penicillin in group A streptococcal strains collected during a span of 80 years (1917 to 1997).

Methods: One hundred thirty-three group A streptococcal strains collected during 80 years were tested for changes in penicillin susceptibility. Three tests were used: (1) the microtiter broth minimal inhibitory concentration (MIC); (2) the minimal bactericidal concentration (MBC); and (3) the penicillin E strip MIC.

Progress in the development of a transcutaneously powered axial flow blood pump ventricular assist system.

Development of the Jarvik 2000 intraventricular assist system for long-term support is ongoing. The system integrates the Jarvik 2000 axial flow blood pump with a microprocessor based automatic motor controller to provide response to physiologic demands. Nine devices have been evaluated in vivo (six completed, three ongoing) with durations in excess of 26 weeks.