Characterization of the recombinant IKK1/IKK2 heterodimer. Mechanisms regulating kinase activity.

Nuclear factor kappa B (NF-kappaB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB, initiated by an IkappaB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. To better understand the role of IKKs in NF-kappaB activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K(m) values for ATP and IkappaBalpha peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 micrometer, respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency (k(cat)/K(m)) of 47.50 h(-1) micrometer(-1) using an IkappaBalpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) micrometer(-1)), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) micrometer(-1)), or rhIKK1 (0.02 h(-1) micrometer(-1)). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IkappaBalpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K(i) of 0.77 micrometer may play a physiological role in regulation of the enzyme activity.

Evolving role of CNSs in developing risk-anchored preventive interventions.

Emerging emphases on systems of care, cost containment, and preventive interventions require the CNS to recognize risk factors and change health behaviors before complications develop. To lower substantially the rate of nontraumatic lower extremity amputation, high-risk populations must be screened and must receive appropriate management, including education and self-care interventions. In this article, two studies that examined foot risk factors in ambulatory elderly with intact feet are compared. American Diabetes Association and Gillis W. Long Hansen's Disease Center risk criteria were applied to both datasets. Recommendations for education, management, and referrals based on the calculated level of risk are presented.

Inhibition of neutrophil and monocyte defensive functions by nicotine.

To learn more about the effects of smokeless tobacco on the defensive functions of neutrophils, we studied the influence of nicotine on these cells in vitro, looking at their bactericidal activity against oral pathogens, and at their ability to produce microbicidal reactive oxygen species (oxygen radicals). Exposure of human blood neutrophils to nicotine (0.01% to 0.1%) inhibited their ability to kill Actinomyces naeslundii, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum. Although these concentrations of nicotine are high, such concentrations are relevant to phagocytes in the gingival sulcus, because smokeless tobacco contains 0.5% to 3.5% nicotine by dry weight. Nicotine had no such inhibitory effect when the killing assay was performed in an anaerobic environment, implying that nicotine preferentially affected oxygen-dependent killing mechanisms. To further investigate the effects of nicotine on production of oxygen radicals, neutrophils were primed with lipopolysaccharide and triggered with f-met-leu-phe or phorbol ester in the presence of nicotine. Nicotine inhibited production of superoxide anion (measured by reduction of cytochrome c) and hydrogen peroxide (measured by oxidation of phenol red). Nicotine inhibition of superoxide production was reversible by washing away the nicotine. By observing that nicotine inhibited the reduction of cytochrome c by reagent potassium superoxide, we determined that nicotine directly absorbed superoxide. In addition, by examining nicotine inhibition of the uptake of oxygen by neutrophils, we determined that nicotine also interfered with the production of oxygen radicals by these cells. Nicotine also inhibited production of superoxide and interleukin-1 beta by monocytes. Nicotine did not affect the viability of neutrophils and monocytes, as determined by their ability to exclude trypan blue dye. Inhibition of the aerobic antimicrobial functions of neutrophils and monocytes by nicotine may alter the microbial ecology of the oral cavity, and this might be one mechanism by which nicotine compromises the oral health of users of tobacco products.

Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E. coli.

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli.

Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to the aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 leader from bacteriophage T7, methionine-alanine-TFPI coding sequence, and the p22 transcriptional terminator. In this system, TFPI accounted for about 5-10% of the total cell protein. The inclusion bodies containing. TFPI were sulfitolyzed, purified by anion-exchange chromatography, refolded through a disulfide interchange reaction, and further fractionated by Mono S cation exchange chromatography. The Mono S resin resolved a peak of highly active TFPI from relatively inactive and possibly misfolded molecules. The E. coli TFPI was shown to be about two-fold more active, on a molar basis, than full-length human SK hepatoma TFPI in a tissue factor-induced clotting assay in human plasma.

Family-focused nursing care of hospitalized elderly.

The purposes of this research were to gain an understanding of how nurses in acute care settings involve families of elderly patients in planning and giving care, and to determine the extent of that involvement in nursing documentation. Data were obtained from patient records and interviews with nurses. Evaluated were characteristics of elderly patients and families, documentation of family involvement in care, and nurses' descriptions of family care. A comparison of data sources indicated that nurses verbalized far more inclusion of families in care than the written records documented. Results of this research support the importance of clarifying and promoting acute care-nurses' involvement of family members who are central to the care of increasing numbers of elderly persons.

Attitudes to follow-up after uncomplicated surgery--hospital out-patients or general practitioner?

The attitude of 216 patients, 10 hospital doctors and 80 general practitioners (GPs) to hospital follow-up after uncomplicated surgery for non-malignant disease was assessed. Hospital doctors felt that most patients (86%) could have been satisfactorily followed-up by their GP and in most cases (89%), the GP was willing to provide the service. However, 183 patients (85%) found their visit to hospital out-patients worthwhile and only 41 patients (19%) would have preferred to have visited their GP instead. In fact, 157 patients (73%) had already seen their GP before their return to surgical out-patients. In most cases, hospital follow-up appears to be unnecessary. In this series, if suitable post-operative patients were followed-up by their GP, there would be a reduction of 20% in the number of old patients returning to out-patients.

The reliability of the microscopic diagnosis of malaria in the field and in the laboratory.

Four hundred and seventy thick and thin blood films were prepared from 129 villagers in the Solomon Islands. After staining with Giemsa, Leishman's, and Field's stains, they were randomized and examined in the field, using a miniature McArthur microscope. The specimens were then examined in the local central laboratory and by a microbiologist at a hospital in England. Films over which there was disagreement were examined by an expert at the Liverpool School of Tropical Medicine. The rate of false negative diagnoses (for thick films) was 3% for the field worker, 9% for the malaria laboratory, and 27% for the English hospital. Field diagnosis was no less reliable than laboratory diagnosis (P less than 0.001). Field's stain was the most reliable stain for both thick films (P less than 0.001) and thin films (P less than 0.05), for which a new staining technique is described.