Barriers to achieving evidence-based stroke rehabilitation.

OBJECTIVE: To determine the perceived barriers to evidence-based practice by health professionals working within the field of stroke rehabilitation. DESIGN: Focus groups were carried out to identify the perceived barriers; these were followed by a postal questionnaire that asked stroke rehabilitation professionals to rate their agreement with the perceived barriers. SUBJECTS: One hundred and five stroke rehabilitation professionals participated in the focus groups and were sent the postal questionnaire. Eighty-six responses were returned, from 27 physiotherapists, 26 occupational therapists, 22 nurses, 6 speech and language therapists, and 5 other professionals. MAIN OUTCOME MEASURES: Proportion of subjects rating their level of agreement with statements as 1 'agree', 2, 3, 4 or 5 'disagree'. Scores of 1 or 2 were classified as 'agreement', and scores of 4 or 5 were classified as 'disagreement': the percentages of subjects agreeing or disagreeing with each statement were calculated. RESULTS: Twenty barriers were identified, classified under the headings 'ability', 'opportunity' and 'implementation'. Seventy-nine (92%) of all respondents agreed that keeping up to date with research findings was important to them, but only 7 (8%) were happy with the time that they had to do this. Fifty-eight (67%) perceived a need for further training. Only 4 (5%) agreed that it was easy to transfer research findings into their daily practice. A number of significant differences were found between the perceived barriers of different disciplines.

Inhibition of expression of the type I G protein-coupled receptor for vasoactive intestinal peptide (VPAC1) by hammerhead ribozymes.

Vasoactive intestinal peptide (VIP) is a neuromediator expressed widely in the nervous, gastrointestinal, respiratory, and immune systems. Two G protein-coupled receptors (GPCRs), designated VPAC1 and VPAC2, bind VIP with high affinity and transduce increases in [cyclic AMP](i) and [Ca(2+)](i). As there are no potent VPAC1- or VPAC2-selective antagonists, a hammerhead ribozyme (Rz) strategy capable of in vivo application was adopted to inactivate individual domains of VPAC1. Three Rzs were designed to cleave mRNA encoding the amino terminus, the third intracellular loop, and the cytoplasmic tail of human VPAC1 and were introduced by transfection into HEK-293 cells expressing recombinant human VPAC1. Each Rz specifically degraded VPAC1 mRNA and down-regulated VPAC1 protein and VIP-binding activity, as assessed by ribonuclease protection assays, Western blots, and binding of (125)I-VIP. Rz-mediated down-regulation of VPAC1 was associated with up to 75% suppression of VIP signaling of increases in [cyclic AMP](i) and [IP3](i), and of cyclic AMP response element-luciferase reports. The Rz specific for the amino terminus inhibited VPAC1 expression and signaling to the greatest extent. VIP-evoked cellular responses thus appear to be proportional to the level of VPAC1 expression. Specific Rzs may be powerful tools for manipulating tissue-specific contributions of GPCRs in vitro and in vivo.

What is balance?

Balance is a term frequently used by health professionals working in a wide variety of clinical specialities. There is no universally accepted definition of human balance, or related terms. This article identifies mechanical definitions of balance and introduces clinical definitions of balance and postural control. Postural control is defined as the act of maintaining, achieving or restoring a state of balance during any posture or activity. Postural control strategies may be either predictive or reactive, and may involve either a fixed-support or a change-in-support response. Clinical tests of balance assess different components of balance ability. Health professionals should select clinical assessments based on a sound knowledge and understanding of the classification of balance and postural control strategies.

Pharmacokinetics of fosphenytoin in patients with hepatic or renal disease.

PURPOSE: The pharmacokinetic behavior of fosphenytoin (FOS), the water-soluble prodrug of phenytoin (PHT), has been characterized in normal subjects. This is the first study of the effect of hepatic or renal disease on the rate and extent of conversion of FOS to PHT. METHODS: A single dose of fosphenytoin (250 mg over a period of 30 min) was administered to subjects with hepatic cirrhosis (n = 4), renal disease requiring maintenance hemodialysis (n = 4), and healthy controls (n = 4). Serial plasma concentrations were measured, and pharmacokinetic parameters were calculated. RESULTS: The mean time to reach the peak plasma FOS concentration was similar for each of the three groups. However, the mean time to achieve peak plasma concentrations of PHT tended to occur earlier in the hepatic or renal disease groups than in healthy subjects. The half-life of FOS was 4.5, 9.2, and 9.5 min for the three groups, respectively. There was a trend toward increased FOS clearance and earlier peak PHT concentration in subjects with hepatic or renal disease. This finding is consistent with decreased binding of FOS to plasma proteins and increased fraction of unbound FOS resulting from decreased plasma protein concentrations associated with these disease states. The conversion of FOS to PHT was equally efficient in subjects with hepatic or renal disease and healthy subjects. CONCLUSIONS: Although the differences in pharmacokinetic parameters between the three groups were not statistically significant, these data suggest the need for close clinical monitoring during FOS administration to patients with hepatic or renal disease. To minimize the incidence of adverse effects in this patient population, FOS may need to be administered at lower doses or infused more slowly.

Regulated expression of matrix metalloproteinases and TIMP in nephrogenesis.

The roles of the matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMP), in embryologic development in general, and in nephrogenesis in particular, have not been fully elucidated. The activities of these enzymes and their inhibitors may be critical in the extensive extracellular matrix remodeling that accompanies the formation of the full complement of mature nephrons in the developing kidney. The temporal and spatial expression of two critical basal lamina modifying enzymes, the 72 kDa gelatinase A (MMP-2) and the 92 kDa gelatinase B (MMP-9), as well as TIMP-1, -2, and -3 molecules were evaluated in the developing rat kidney. Additionally, transcripts for the recently described membrane-associated matrix metalloproteinase, MT1-MMP (MMP-14), which can act as an activating receptor for MMP-2/TIMP-2 complexes (Strongin et al.[1995] J. Biol. Chem. 270:5331-5338) were localized by in situ hybridization. Our immunohistochemical data demonstrate distinct localization of MMP-2 within immature nephron structures undergoing epithelial differentiation, while MMP-9 localizes only to the invading vascular structures within immature glomeruli. In contrast, by in situ hybridization, MMP-2 transcripts localize to the background undifferentiated mesenchyme and not to those structures undergoing epithelial differentiation. In a pattern similar to the MMP-2 protein, MT1-MMP transcripts were found within developing epithelial structures. Neither MMP-2, MMP-9 nor MT1-MMP were detected in mature nephrons. TIMP-2 and -3 follow a pattern of expression similar to the MMP-2 protein. We conclude that MMP-2 and TIMP play important roles in the remodeling of basal laminae associated with the epithelial structures of the developing kidney, that these enzymes are temporally and spatially regulated, and that the co-localization of MT1-MMP to sites of basement membrane remodeling suggests a potential role for this molecule as a receptor for and/or modulator of MMP-2/TIMP complexes.

Glomerular mesangial cell-specific transactivation of matrix metalloproteinase 2 transcription is mediated by YB-1.

Mesangial cell (MC) activation plays a pivotal role in the development of the end stage sclerotic lesion characteristic of most forms of chronic glomerular disease. We have previously demonstrated that MC activation is directly linked to high level expression of the matrix metalloproteinase-2 (MMP-2) enzyme (Turck, J., Pollock, A. S., Lee, L., Marti, H.-P., and Lovett, D. H. (1996) J. Biol. Chem. 25, 15074-15083), the transcription of which is regulated in a tissue-specific fashion. Recent studies (Harendza, S., Pollock, A., Mertens, P. R., and Lovett, D. H. (1995) J. Biol. Chem. 270, 18786-18796) delineated a strong cis-acting enhancer element, designated MMP-2 RE1, within the 5'-flanking region of the rat MMP-2 gene. Gel shift, DNA footprint, and transcriptional analyses mapped the enhancer element to a unique 40-base pair (bp) sequence located at -1322 to -1282 bp relative to the translational start site. Bromodeoxyuridine-substituted UV cross-linking of the 40-bp enhancer element with MC nuclear extracts yielded a single protein of 52 kDa, while Southwestern blot analysis with MMP-2 RE1 demonstrated three hybridizing nuclear proteins of 52, 62, and 86 kDa size. Screening of a human MC cDNA expression library with MMP-2 RE1 exclusively yielded clones with the identical sequence of the transcription factor YB-1. Western blot and supershift gel analysis of MC nuclear extracts with an anti-YB-1 antibody confirmed the presence of YB-1 within the shifted complex. Examination of the MMP-2 RE1 sequence revealed an incomplete Y-box sequence (CTGCTGGGCAAG), which specifically interacted with recombinant YB-1 on DMS protection footprinting analysis. YB-1 protein preferentially bound the single-stranded components of the 40-bp MMP-2 RE1 and, with increasing concentrations, formed multimeric complexes. Co-transfection of YB-1 in MC increased the enhancer activity within the context of the native MMP-2 promoter, while transfection of non-MMP-2-synthesizing glomerular epithelial cells with YB-1 led to transcriptional suppression. This study indicates that YB-1 is a major, cell type-specific transactivator of MMP-2 transcription by glomerular mesangial cells.

Gelatinase A is a glomerular mesangial cell growth and differentiation factor.

The members of the matrix metalloproteinase gene family play critical roles in numerous physiologic events, including cellular migration, tissue remodeling in wound healing and development, as well as in the evolution of the inflammatory process. The 72 kDa gelatinase A (formerly denoted 72 kDa Type IV collagenase) is centrally involved in the inflammatory and sclerotic events common to most forms of chronic glomerular disease. In this article recent studies are summarized which demonstrate that this particular enzyme can directly affect the proliferative and differentiation properties of the intrinsic glomerular mesangial cell, both in vitro and in vivo.

Transfection and overexpression of the calcium binding protein calbindin-D28k results in a stimulatory effect on insulin synthesis in a rat beta cell line (RIN 1046-38).

Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.

Matrix metalloproteinase 2 (gelatinase A) regulates glomerular mesangial cell proliferation and differentiation.

A biologic role for the 72-kDa gelatinase A (matrix metalloproteinase 2; MMP-2), beyond simple extracellular matrix turnover, was evaluated in glomerular mesangial cells. To determine the significance of MMP-2 secretion for the acquisition of the inflammatory phenotype, we reduced the constitutive secretion of MMP-2 by cultured mesangial cells with antisense RNA expressed by an episomally replicating vector or with specific anti-MMP-2 ribozymes expressed by a retroviral transducing vector. The phenotype of the transfected, or retrovirally infected, cells was profoundly altered from the activated state and closely approximated that of quiescent cells in vivo. The prominent differences included a change in the synthesis and organization of the extracellular matrix, loss of activation markers, and a virtually total exit from the cell cycle. Reconstitution with exogenous active, but not latent MMP-2, induced a rapid return to the inflammatory phenotype in vitro. This effect was specific to MMP-2, because the closely related MMP-9 did not reproduce these changes. Furthermore, this pro-inflammatory effect of MMP-2 is dependent upon the active form of the enzyme, which can be produced by an autocatalytic activation process on the mesangial cell plasma membrane. It is concluded that MMP-2 acts directly upon mesangial cells to permit the development of an inflammatory phenotype. Specific inhibition of MMP-2 activity in vivo may represent an alternate means of ameliorating complex inflammatory processes by affecting the phenotype of the synthesizing cells, per se.

Characterization of a glomerular epithelial cell metalloproteinase as matrix metalloproteinase-9 with enhanced expression in a model of membranous nephropathy.

The role of the glomerular visceral epithelial cell in the physiologic turnover and pathologic breakdown of the glomerular extracellular matrix has remained largely unexplored. In this study a 98-kD neutral proteinase secreted by cultured rat visceral glomerular epithelial cells was shown to be a calcium, zinc-dependent enzyme secreted in latent form. In addition, the protein was heavily glycosylated and demonstrated proteolytic activity against Type I gelatin, Type IV collagen gelatin, and fibronectin. The similarity in molecular mass and substrate specificities to the 92-kD human matrix metalloproteinase-9 (MMP-9, or gelatinase B) suggested the identity of this activity, which was confirmed by immunoprecipitation and Northern blot analysis. The differences in molecular mass (98 vs. 92 kD) were not due to species-specific differences in glycosylation patterns, since cultured rat peritoneal macrophages secreted MMP-9 as a 92-kD enzyme. Furthermore, transfection of the human MMP-9 cDNA into rat glomerular epithelial cells yielded the 98-kD product. Using a specific monoclonal anti-MMP-9 antibody and in situ reverse transcription (ISRT) analysis of MMP-9 mRNA, the expression of this enzyme was evaluated in vivo. Normal rat glomeruli expressed little immunohistochemical or ISRT staining for MMP-9, while in rats with passive Heymann nephritis there was a major increase in MMP-9 protein and mRNA staining within the visceral epithelial cells. The temporal patterns of MMP-9 expression correlated with the period of proteinuria associated with this model, suggesting that a causal relationship may exist between GEC MMP-9 expression and changes in glomerular capillary permeability.

Endotoxin, TNF, and IL-1 decrease cholesterol 7 alpha-hydroxylase mRNA levels and activity.

Endotoxin (LPS) and cytokines increase cholesterol synthesis and the secretion of lipoproteins by the liver in rodents resulting in hypercholesterolemia. Cholesterol 7 alpha-hydroxylase (CAH) is the rate-limiting enzyme in the conversion of cholesterol to bile acids in the liver, the major regulated pathway by which cholesterol is eliminated from the body. Decreases in CAH would increase the quantity of cholesterol available for lipoprotein production. In the present study, we demonstrate that LPS, TNF, or IL-1 administration to Syrian hamsters produces a marked decrease in the levels of CAH mRNA in the liver. This marked decrease occurred even when the basal level of CAH expression was increased by feeding the bile acid sequestrant, colestipol. Additionally, a marked decrease was also observed when the animals were fed a cholesterol-enriched diet. Moreover, the decrease in CAH mRNA occurred very rapidly (decreased 66% by 90 min after LPS administration) and required relatively small doses of LPS (100 ng/100 g body weight). Lastly, the decrease in mRNA levels was accompanied by a decrease in CAH activity. This decrease in CAH could contribute to the increase in hepatic lipoprotein production induced by LPS and cytokines. CAH can be added to the growing list of proteins that regulate lipid metabolism and that are altered during the acute phase response.

Tissue-specific enhancer-promoter interactions regulate high level constitutive expression of matrix metalloproteinase 2 by glomerular mesangial cells.

The 72-kDa gelatinase A (MMP-2) is a central mediator of the response of the intrinsic glomerular mesangial cell to inflammatory stimuli and is regulated in a unique, cell-specific manner. We isolated a 6-kilobase pair genomic fragment of the rat MMP-2 gene and sequenced and characterized 1686-base pair of the 5'-flanking region. Using a series of 5' deletion constructs of the proximal 5'-flanking region, a strong MMP-2 enhancer element was identified. Gel shift and mutational analyses suggest tha the enhancer region represents the binding site for complex transcription factor demonstrating separable DNA-binding and transcriptional activating domains. The presence and activity of the enhancer element was evaluated in several cell types with varying capabilities to synthesize MMP-2 including mesangial cells, glomerular epithelial cells, and the monocytic U937 cell. Although binding activity was present in all cell types studied, enhancer activity was demonstrated only in mesangial and glomerular epithelial cells. Additional transcriptional control resided in a tissue-specific promoter, which supported transcription only in mesangial cells. These results indicate that the final control of mesangial cell-specific synthesis of MMP-2 derives from an interaction between the strong enhancer element and the tissue-specific MMP-2 promoter.

Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney.

The development of progressive glomerulosclerosis in the renal ablation model has been ascribed to a number of humoral and hemodynamic events, including the peptide growth factor, transforming growth factor-beta 1 (TGF-beta 1). An important role has also been attributed to angiotensin II (AII), which, in addition to its hemodynamic effects, can stimulate transcription of TGF-beta 1. We postulated that increased glomerular production of AII, resulting from enhanced intrinsic angiotensinogen expression, stimulates local TGF-beta 1 synthesis, activating glomerular matrix protein synthesis, and leads to sclerosis. Using in situ reverse transcription, the glomerular cell sites of alpha-1 (IV) collagen, fibronectin, laminin B1, angiotensinogen, and TGF-beta 1 mRNA synthesis were determined at sequential periods following renal ablation. The early hypertrophic phase was associated with global, but transient, increases in the mRNA for alpha-1 (IV) collagen. No changes were noted for fibronectin, TGF-beta 1, and angiotensinogen mRNAs. At 24 d after ablation, at which time sclerosis is not evident, endothelial cells, particularly in the dilated capillaries at the vascular pole, expressed angiotensinogen and TGF-beta 1 mRNAs, as well as fibronectin and laminin B1 RNA transcripts. By 74 d after ablation angiotensinogen and TGF-beta 1 mRNAs were widely distributed among endothelial and mesangial cells, and were particularly prominent in regions of evolving sclerosis. These same regions were also notable for enhanced expression of matrix protein mRNAs, particularly fibronectin. All receptor blockade inhibited angiotensinogen, TGF-beta 1, fibronectin, and laminin B1 mRNA expression by the endothelium. We conclude that, as a result of hemodynamic changes, injured or activated endothelium synthesizes angiotensinogen, triggering a cascade of TGF-beta 1 and matrix protein gene expression with resultant development of the segmental glomerular sclerotic lesion.

Discordant regulation of proteins of cholesterol metabolism during the acute phase response.

Recent studies have shown that the administration of endotoxin (LPS) and cytokines to Syrian hamsters increases serum cholesterol levels, hepatic cholesterol synthesis, and the activity, protein levels, and mRNA levels of hepatic HMG-CoA reductase. Despite the greater than 10-fold increase in HMG-CoA reductase mRNA levels, LPS had only minimal effects on hepatic LDL receptor mRNA levels. In the present study, we demonstrate that LPS increases the transcription rate in the liver of HMG-CoA reductase mRNA approximately 4- to 5-fold without affecting LDL receptor mRNA transcription. Most stimuli that regulate HMG-CoA reductase and LDL receptor mRNA levels also regulate, in parallel, HMG-CoA synthase and farnesyl pyrophosphate (FPP) synthetase. However, in chow-fed animals, LPS and cytokines (TNF, IL-1, TNF + IL-1) increased hepatic HMG-CoA reductase mRNA levels without increasing LDL receptor, HMG-CoA synthase, or FPP synthetase mRNA levels. The feeding of cholesterol or bile resin binders regulates the mRNA levels of HMG-CoA reductase, LDL receptor, HMG-CoA synthase, and FPP synthetase. In both cholesterol- and colestipol-fed animals, LPS increased HMG-CoA reductase mRNA levels while either decreasing or causing minimal increases in the mRNA levels of the other proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Calbindin expression in renal tubular epithelial cells. Altered sodium phosphate co-transport in association with cytoskeletal rearrangement.

Sodium-phosphate transport in the opossum kidney (OK) cell line was studied in an OK clonal cell line that was transfected with an episomal vector expressing high levels of rat calbindin (28 kDa). High level expression of calbindin buffered the influx of calcium induced by ionomycin by 53% and raised the basal intracellular calcium from 100 +/- 6 to 150 +/- 8 nM. The decrement in sodium phosphate uptake induced by parathyroid hormone or forskolin was identical in the two cell lines. However, phorbol esters (10(-10)-10(-7) M), which decreased sodium phosphate uptake in the parental OK line, increased it in the calbindin-expressing line. Similarly, the parental clone did not respond to phosphate deprivation, while the calbindin-expressing clone did increase phosphate uptake in response to phosphate deprivation. In the calbindin-expressing cells, phorbol 12-myristate 13-acetate or low phosphate medium, which increased phosphate transport, produced actin filament aggregation, dissociation of the myristoylated alanine-rich C kinase substrate protein from sub-apical actin, and membrane-associated tyrosine phosphate staining. Agonists that reduced sodium phosphate uptake (cAMP, parathyroid hormone) did not affect these cellular features. The cytoskeletal rearrangement, redistribution of the myristoylated alanine-rich C kinase substrate protein, and membrane tyrosine phosphorylation are suggested to be involved in the events by which phosphate transport is increased in this cell line.

Asymmetric origins of the mature glomerular basement membrane.

Renal plasma filtration is a critical physiologic function that depends upon the precise composition and arrangement of the constituent extracellular matrix proteins within the glomerular basement membrane (GBM). The GBM develops during renal embryogenesis by the fusion of discrete basement membranes produced independently by endothelial and visceral epithelial cells, and, possibly from matrix secreted by the mesangial cells. In the mature animal, however, the epithelial cell has generally been accepted as the sole source of all GBM constituent proteins. Although the final structures and distributions of the component proteins have been defined by histochemical techniques, the individual contributions of the three resident glomerular cell types to the maintenance and turnover of the mature GBM remain uncertain. We report the application of a new technique, in situ reverse transcription (ISRT), for the localization of RNA transcripts of nine major GBM protein components within the closely apposed cells of the glomerulus. Using this technique, we demonstrate that in normal adult rat glomeruli the RNA transcripts for heparan sulfate proteoglycan and the laminin-S chain are primarily expressed by visceral epithelial cells, while Type IV alpha-1 and alpha-2 collagen transcripts were restricted to the endothelial cells in a heterogeneous pattern. RNA transcripts for entactin and the laminin-A and -B2 chains were expressed by all three glomerular cell types, while laminin-B1 and fibronectin transcripts were limited to the mesangium. These findings demonstrate that GBM synthesis in the mature animal is not restricted to the epithelial cell and that all intrinsic glomerular cells contribute to the production of GBM protein components. The ISRT technique also provided the additional, and unexpected, finding that appreciable synthetic heterogeneity exists within individual glomerular cell types.

Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors.

We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.

Role for monokines in the metabolic effects of endotoxin. Interferon-gamma restores responsiveness of C3H/HeJ mice in vivo.

To examine the role of cytokines in mediating the lipogenic effects of endotoxin (LPS), we studied the effects of LPS and cytokines on hepatic fatty acid synthesis in LPS-sensitive C3H/OuJ mice and in LPS-resistant C3H/HeJ mice, whose macrophages are defective in the ability to produce tumor necrosis factor (TNF) and IL-1 in response to LPS. HeJ mice were 16-fold less sensitive than OuJ mice to the lipogenic effect of LPS. In OuJ mice, 10 micrograms of LPS caused a maximal increase in hepatic lipogenesis (3.86 +/- 0.41-fold), whereas in HeJ mice the maximal increase was only 1.79 +/- 0.32-fold after 100 micrograms of LPS. This lipogenic response paralleled the decreased ability of LPS to increase hepatic and splenic levels of mRNAs for TNF and IL-1 and serum levels of TNF in HeJ mice. In contrast, the maximal effect of TNF on lipogenesis was greater and the sensitivity to TNF was increased 2.4-fold in HeJ mice compared to OuJ mice. Administration of IFN-gamma before LPS in HeJ mice had no effect on IL-1 mRNA, but partially restored the LPS-induced increase in hepatic and splenic mRNA for TNF and serum TNF levels, which may account for the partial restoration of sensitivity to the lipogenic effect of LPS after IFN-gamma treatment. These results indicate that cytokines produced by mononuclear leukocytes mediate the lipogenic effects of LPS.

Molecular cloning of gp42, a cell-surface molecule that is selectively induced on rat natural killer cells by interleukin 2: glycolipid membrane anchoring and capacity for transmembrane signaling.

We have previously shown that in vitro culture of rat natural killer (NK) cells in high concentrations of recombinant interleukin 2 (rIL-2) leads to the expression of a surface glycoprotein with a molecular mass of approximately 42 kD. This glycoprotein, gp42, is not induced on other lymphocytes and thus provides a lineage-specific marker for rIL-2-activated NK cells. We here present the nucleotide sequence for gp42 cDNA. The open reading frame encodes 233 amino acids with three potential sites for N-linked glycosylation. The deduced amino acid sequence lacks an apparent transmembrane domain and instead contains a hydrophobic COOH terminus that is characteristic of glycosylphosphatidylinositol (GPI)-anchored surface proteins. Consistent with this, gp42 is cleaved from the NK-like cell line, RNK-16, by phosphatidylinositol-specific phospholipase C (PI-PLC), as is gp42 expressed on CHO cells that have been transformed with gp42 cDNA. On rIL-2-activated NK cells, gp42 is resistant to PI-PLC, though our studies suggest that gp42 on these cells is still expressed as a GPI-anchored molecule. Antibody to gp42 stimulates in RNK-16 cells an increase in inositol phosphates and in intracellular calciu, signals that are associated with the activation of lymphocytes, including NK cells. rIL-2-activated NK cells, however, lack this response to gp42 as well as to other stimuli. Thus, gp42, the only NK-specific activation antigen, is a GPI-anchored surface molecule with the capacity to stimulate transmembrane signaling.

Transfection-mediated expression of a dominant cAMP-resistant phenotype in the opossum kidney (OK) cell line prevents parathyroid hormone-induced inhibition of Na-phosphate cotransport. A protein kinase-A-mediated event.

Sodium-phosphate cotransport in the PTH-responsive opossum kidney (OK) cell line is inhibited by PTH, cAMP, and activators of protein kinase C. In order to probe the role of cAMP, we stably transfected OK cells with an expression vector for a cAMP-binding mutation of the murine protein kinase A regulatory subunit. Two-dimensional electrophoresis of cAMP-binding proteins from transfected cells indicated a 20-fold overexpression of the mutant regulatory unit. Protein kinase A from these cells had a 20-fold increase in the concentration of cAMP required for half-maximal activation, 2.8 microM vs. 0.15 microM for wild type cells. In the transfected cells, Na-phosphate cotransport was insensitive to up to 1 mM 8-Br-cAMP and 1 microM PTH, while these same agonists caused a significant inhibition of transport in the wild type cells. The effects on Na-phosphate cotransport of the protein kinase C activators oleoyl-acetyl glycerol and tetradecanoyl-phorbol acetate, which were marked in the wild type cells, were still present, although attenuated, in the transfected mutants. With prolonged passage, the cAMP-insensitive phenotype reverted to wild type cAMP sensitivity despite continued selection for the cotransfected neo marker. The revertant cells had a normal cAMP requirement for half-maximal activation of protein kinase A, 0.13 microM, and the PTH and cAMP-sensitive inhibition of Na-phosphate cotransport was restored. We suggest that an intact and normally cAMP-sensitive protein kinase A pathway is an absolute requirement for PTH inhibition of Na-phosphate cotransport in the OK cell.