Application of supercritical fluid chromatography to characterize a labile digitalis-like factor.

A sodium pump inhibitor (digitalis-like factor), isolated from the peritoneal dialysate of volume-expanded, hypertensive patients with kidney failure who were treated with this dialysis modality, was further purified and characterized by means of supercritical fluid chromatography, a separation technique whose application to very-low-concentration biomolecules is new. Previous studies suggested that after high-performance liquid chromatography (HPLC) purification, this inhibitor was the only factor correlated with volume status and blood pressure in these patients. When this same HPLC fraction was furthered purified on 2-dimensional supercritical fluid chromatography, a single peak coeluted with [Na,K]ATPase inhibitory activity.

Sodium pump inhibition and regional expression of sodium pump alpha-isoforms in lens.

Both hypertension and cataract formation have been associated with reductions in sodium pump activity, possibly as a result of an endogenous inhibitor. The objective of the present study was to answer 4 closely related questions: (1) Is the lens sodium pump effectively inhibited by a labile, digitalis-like factor we have identified in the peritoneal dialysate from hypertensive patients in end-stage renal failure? (2) How does that inhibition compare to that induced by ouabain? (3) Does sodium pump isoform distribution determine the degree of lens sodium pump inhibition? (This question was precipitated by the unanticipated finding that the labile DLF was more effective in inhibiting lens sodium pump than was anticipated.) (4) Is sodium pump activity altered in lens in response to increased salt intake, a maneuver known to increase endogenous digitalis-like factor? We found that whereas ouabain produced equivalent or significantly less inhibition of lens Na(+), K(+)-ATPase from calf or rabbit, respectively, compared with brain, labile digitalis-like factor preferentially inhibited lens compared with brain.

Reading the future: the increased relevance of laboratory medicine in the next century.

Through intelligent process control and data management, the laboratory may become the most frequently used--and the most important--source of diagnostic information in medicine. The central laboratory of the future is destined to become an esoteric testing center, whereas routine testing--administered at the patient bedside or at home--will become more economical. Point-of-care testing will soon become the most profitable way to provide laboratory services.

Rickettsia serosurvey in Kimberley, Western Australia.

To determine if antibodies to rickettsiae (scrub typhus, spotted fever, and typhus group rickettsiae) occur among persons living in the Kimberley (northern tropical) region of Western Australia, 920 sera collected in a non-random manner in 1996 from patients in Kununurra, Broome, Fitzroy Crossing, Wyndham, Derby, and Halls Creek were tested by micro-immunofluorescence for antibodies to a panel of rickettsial antigens. Of 920 sera examined, 52 (5.6%) were positive for antibodies to one or more of the three groups of rickettsial microorganisms.

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.

Higher cholesterol and insulin levels in pregnancy are associated with increased risk for pregnancy-induced hypertension.

Hyperinsulinemia and dyslipidemia are known to be associated with essential hypertension but their role in pregnancy-induced hypertension remains unclear. We performed a case-control study comparing cholesterol, insulin, and glucose levels in the early third trimester of pregnancy among 31 women who developed pregnancy-induced hypertension (PIH) (either preeclampsia [n = 6] or nonproteinuric gestational hypertension [n = 25]), with 31 women remaining normotensive through pregnancy. As compared with women remaining normotensive, women subsequently developing PIH had higher fasting cholesterol levels (279 v 247 mg/dL; P = .

Robotic automation of coagulation analysis.

Laboratory automation systems (LAS) have been installed in over 22 sites across North America providing automation of many preanalytical and analytical tasks in clinical laboratories. Only a few laboratories have automated the analysis of citrated whole blood for the diagnosis of hemostasis disorders. The analysis of coagulation factors in citrated blood requires a large amount of labor in order to provide rapid turnaround; thus automation of this analytical process is attractive.

Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells.

Human osteoblast-like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual-color fluorescence-activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO-1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO-1 antigen exclusively (STRO-1+/ALP-), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone-related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis.

New Orientia tsutsugamushi strain from scrub typhus in Australia.

In a recent case of scrub typhus in Australia, Orientia tsutsugamushi isolated from the patient's blood was tested by sequence analysis of the 16S rDNA gene. The sequence showed a strain of O. tsutsugamushi that was quite different from the classic Karp, Kato, and Gilliam strains.

Mapping and polyhedrin gene analysis of the Epiphyas postvittana nucleopolyhedrovirus genome.

The light brown apple moth, Epiphyas postvittana, is a major insect pest of a variety of fruit crops grown in New Zealand and we are studying a nucleopolyhedrovirus, EppoNPV, isolated from this insect. Restriction endonuclease analysis of EppoNPV DNA shows that this is a single strain of virus with a genome size of approximately 119 kbp and a complete library of the EppoNPV genome has been cloned. A strategy of single-stranded sequencing of the termini of REN fragment clones was employed to map the virus genome.

Regulation of the sodium pump in pregnancy-related tissues in preeclampsia.

Objective: This study examined the expression of the three alpha-isoforms of the sodium pump in preeclampsia. Reductions in sodium pump number and activity in smooth muscle may underlie hypertension in preeclampsia.

Study Design: Northern and Western analyses were used to determine whether sodium pump alpha-isoform regulation in myometrium, placenta, and umbilical artery of women with preeclampsia differed from those with normotensive pregnancies.

A labile sodium pump inhibitor from the peritoneal dialysate of hypertensive renal failure patients: estimates of potency.

Estimations of concentration of the labile sodium pump inhibitor isolated from human peritoneal dialysate were made using supercritical fluid chromatography coupled to flame ionization detection to determine the quantity of this factor in half of a purified preparation of the factor compared to the bioactivity of the other half in different assays. Ouabain was used for comparison. The labile factor appeared to be 30 times more effective than ouabain against canine renal [Na,K]ATPase.

Expression, purification, and initial kinetic characterization of the large subunit of the human mitochondrial DNA polymerase.

Faulty replication of the human mitochondrial genome is thought to be the cause of many diseases; moreover, the low selectivity of the mitochondrial DNA polymerase has been implicated as the cause of many side effects observed in the treatment of viral infections such as HIV. To better understand how the mitochondrial genome is replicated, we cloned a cDNA encoding the large subunit of human DNA polymerase gamma, the enzyme that replicates the mitochondrial genome. The large subunit was recombinantly expressed and purified to near homogeneity.

The pathogenesis of bone loss following total knee arthroplasty.

Bone loss following total knee arthroplasty (TKA) may be focal or diffuse. It may be caused mechanically, either by unloading of the bone leading to disuse osteoporosis, or by overloading of the bone leading to trabecular fractures and bone destruction. Osteolysis, instigated by an inflammatory reaction to particulate wear debris, is an important and common cause of bone loss after TKA.

0.9% sodium chloride injection with and without heparin for maintaining peripheral indwelling intermittent-infusion devices in infants.

The use of 0.9% sodium chloride injection with and without heparin sodium for maintaining peripheral indwelling intermittent-infusion devices (PIIIDs) in infants was studied. In this double-blind study, children up to one year of age who had a 24-gauge PIIID through which a continuous i.

Specificity of the vascular smooth muscle contractile response to a labile digitalis-like factor in peritoneal dialysate: the influence of potassium.

Although multiple lines of evidence have suggested that a circulating endogenous digitalis-like factor (DLF) might contribute to the pathogenesis of sodium-sensitive hypertension, the subject remains controversial. This study was designed to compare the influence of potassium on vascular responses to an endogenous DLF isolated from peritoneal dialysate (PD) in volume-expanded patients with other sodium pump inhibitors, such as ouabain and bufalin, and the influence of serotonin as an index of specificity. An increase in bath potassium (K+) concentration from 2.

Regulation of bone cells by particle-activated mononuclear phagocytes.

Bone loss around replacement prostheses may be related to the activation of mononuclear phagocytes (MNP) by prosthetic wear particles. We investigated how osteoblast-like cells were regulated by human MNP stimulated by particles of prosthetic material. Particles of titanium-6-aluminium-4-vanadium (TiAIV) stimulated MNP to release interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6 and prostaglandin E2 (PGE2).

Tissue-specific regulation of the sodium pump in DOCA-salt hypertension.

Alterations in sodium pump activity have been associated with volume-sensitive hypertension, but little is known regarding the molecular regulation of the catalytically active alpha-subunit of the sodium pump in these models. We examined changes in the mRNA abundance of the alpha-isoforms in tissues that might participate in sodium and volume regulation in the deoxycorticosterone acetate (DOCA)-high salt rat model. These tissues included kidney, heart, aorta, pituitary, and hypothalamus.

Integrin expression and function on human osteoblast-like cells.

The integrin family of cell adhesion molecules are a series of cell surface glycoproteins that recognize a range of cell surface and extracellular matrix (ECM)-associated ligands. To date, the precise role of individual integrin molecules in bone cell-ECM interactions remains unclear. Cell binding assays were performed to examine the ability of normal human bone cells (NHBCs) to adhere to different ECM proteins in vitro.