Ca(2+) signalling and PKCalpha activate increased endothelial permeability by disassembly of VE-cadherin junctions.

1. The role of intracellular Ca(2+) mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) in both cases was due to release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+). 2. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial (125)I-albumin permeability. Thapsigargin induced activation of PKCalpha and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCalpha activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC(20)), whereas thrombin induced MLC(20) phosphorylation consistent with formation of actin stress fibres. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca(2+)](i) elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca(2+) signalling and activation of PKCalpha in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis.

Although activated Ras proteins are usually associated with driving growth and transformation, they may also induce senescence, apoptosis, and terminal differentiation. The subversion of these anti-neoplastic effects during Ras-dependent tumor development may be as important as the acquisition of the pro-neoplastic effects. None of the currently identified potential Ras effector proteins can satisfactorily explain the apoptotic action of Ras. Consequently, we have sought to identify novel Ras effectors that may be responsible for apoptosis induction. By examining the EST data base, we identified a potential Ras association domain in the tumor suppressor RASSF1. We now show that RASSF1 binds Ras in a GTP-dependent manner, both in vivo and directly in vitro. Moreover, activated Ras enhances and dominant negative Ras inhibits the cell death induced by transient transfection of RASSF1 into 293-T cells. This cell death appears to be apoptotic in nature, as RASSF1-transfected 293-T cells exhibit membrane blebbing and can be rescued by the addition of a caspase inhibitor. Thus, the RASSF1 tumor suppressor may serve as a novel Ras effector that mediates the apoptotic effects of oncogenic Ras.

The importance of being K-Ras.

The ras genes give rise to a family of related proteins that have strong transforming potential. Typical in vitro studies fail to discriminate between the transforming activity of the Ras proteins. Although activating mutations in ras genes are commonly found in human disease, they are not evenly distributed between the different ras members. Instead, they are concentrated in k-ras. With the absence of evidence to suggest that k-ras DNA is more prone to mutation than h-ras DNA, this imbalance in mutational frequency suggests a special biological role for the K-Ras protein in vivo.

Life expectancy estimation with breast cancer: bias of the declining exponential function and an alternative to its use.

BACKGROUND: Life expectancy gain (LEG) is an outcome measure commonly estimated with a declining exponential function in a Markov model. The accuracy of such estimates has not been objectively evaluated. PURPOSE: To compare LEGs from declining exponential function estimates with those calculated from population data, using published screening mammography studies as examples. METHOD: SEER-based population data are used to compare LEG calculation with declining exponential function estimation and empiric population data in a new model, the "nested" Markov. RESULTS: Analyses of the LEG of mammographic screening based on the declining exponential function significantly overestimate LEGs for younger women and underestimate them for older women. Because of offsetting errors, all-age analyses paradoxically appear accurate. CONCLUSION: Declining exponential function estimates of LEGs for chronic diseases with low mortality rates and long time horizons are liable to significant bias, especially with limited age cohorts.

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.

Early infection in bone marrow transplantation: quantitative study of clinical factors that affect risk.

Infections remain common life-threatening complications of bone marrow transplantation. To examine clinical factors that affect infection risk, we retrospectively studied patients who received bone marrow transplants (53 autologous and 51 allogeneic). Over a median of 27 hospital days, 44 patients developed documented infections. Both autologous transplantation and hematopoietic growth factor use were associated with less prolonged neutropenia and decreased occurrence of infection (P < or = .05). In a survival regression model, variables independently associated with infection risk were the log10 of the neutrophil count (hazard ratio [HR], 0.49; 95% confidence interval [CI], 0.32-0.75), ciprofloxacin prophylaxis (HR, 0.42; 95% CI, 0.19-0.95), empirical intravenous antibiotic use (HR, 0.09; 95% CI, 0.03-0.32), and an interaction between neutrophil count and intravenous antibiotic use (HR, 1.86; 95% CI, 1.06-3.29). In this model, infection risk increases steeply at low neutrophil counts for patients receiving no antibiotic therapy. Ciprofloxacin prophylaxis and particularly intravenous antibiotic therapy provide substantial protection at low neutrophil counts. These results can be used to model management strategies for transplant recipients.

Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression.

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.

Integrin-linked kinase regulates phosphorylation of serine 473 of protein kinase B by an indirect mechanism.

The serine threonine kinase protein kinase B regulates cellular activities as diverse as glycogen metabolism and apoptosis. Full activation of protein kinase B requires 3-phosphoinositides and dual phosphorylation on threonine-308 and serine-473. CaM-K kinase and 3-phosphoinositide dependent-kinase-1 phosphorylate threonine-308. Integrin-linked kinase reportedly phophorylates serine-473. Consistent with this, in a model COS cell system we show that expression of wild-type integrin-linked kinase promotes the wortmannin sensitive phosphorylation of serine-473 of protein kinase B and its downstream substrates, and inhibits C2-ceramide induced apoptosis. In contrast, integrin-linked kinase mutated in a lysine residue critical for function in protein kinases is inactive in these experiments, and furthermore, acts dominantly to block serine-473 phosphorylation induced by ErbB4. However, alignment of analogous sequences from different species demonstrates that integrin-linked kinase is not a typical protein kinase and identifies a conserved serine residue which potentially regulates kinase activity in a phosphorylation dependent manner. Mutation of this serine to aspartate or glutamate, but not alanine, in combination with the inactivating lysine mutation restores integrin-linked kinase dependent phosphorylation of serine-473 of protein kinase B. These data strongly suggest that integrin-linked kinase does not possess serine-473 kinase activity but functions as an adaptor to recruit a serine-473 kinase or phosphatase.

Detergent binding to unmyristylated protein kinase A--structural implications for the role of myristate.

Myristylation often governs the targeting of protein kinases to the plasma membrane. It is now known that a key member of the src family of protein tyrosine kinases, pp60v-src, binds to the lipid bilayer of the plasma membrane via a myristylated amino terminal sequence. The mechanism of this interaction is not known; however, myristic acid (Myristic acid may also be referred to as Myristate) and residues 2 through 14 are also absolutely required (Resh and Ling, 1990). This review presents an analysis of crystal structures of detergent-modified recombinant and myristylated mammalian catalytic subunit of protein kinase A. Crystals of unmyristylated recombinant catalytic subunit of protein kinase A are grown in the presence of Mega 8, a glucamide-type of detergent, and only this detergent binds, which results in a resolution extension (Knighton et al., 1991a). Comparisons of these two structures reveal that the detergent association with the recombinant enzyme binds in exactly the same hydrophobic pocket of the protein occupied by myristic acid in the mammalian protein (Karlsson et al., 1993; Zheng et al., 1993a). Removal of the detergent through soaking results in the local unwinding of the first helix, helix A, and disorder of the canonical recognition sequence of the phosphorylation site, Ser 10 (Zheng et al., 1993b). These results suggest that anchoring the myristic acid inside the protein results in formation of a stable structural template, which includes the myristylated amino terminal sequence important for the recognition by protein kinases. This "inside out" motif might provide a structural paradigm for the recognition of myristylated proteins, including pp60v-src.

Mammalian topoisomerase I has base mismatch nicking activity.

The all-type nicking enzyme (ATE) from human HeLa cells or calf thymus can nick DNA at the first phosphodiester bond 5' to all 8 possible mismatched bases. The strand disparity of this nicking is influenced by the neighboring nucleotide sequences. After nicking, the ATE covalently binds to the 3' end of the DNA product to form a cleavable complex, whose formation is insensitive to camptothecin, a specific inhibitor of eukaryotic topoisomerase I (Topo-I). During the purification of ATE from calf thymus, a Mg(2+)-independent relaxation activity, characteristic of eukaryotic Topo-I, copurifies with the mismatch-nicking activity. The ATE from calf thymus may be a breakdown product of Topo-I. N-terminal amino acid analysis indicates that one of the polypeptides with ATE activity contains the C-terminal portion of Topo-I. Moreover, active human Topo-I, expressed as a fusion protein in Escherichia coli, is also capable of nicking all 8 base mispairs in the absence of Mg2+. This mismatch-specific nicking activity may be a novel property of the mammalian Topo-I.

Protein kinase C inhibitor proteins. Purification from sheep brain and sequence similarity to lipocortins and 14-3-3 protein.

Potent inhibitors of protein kinase C have been isolated from sheep brain by DEAE-cellulose, phenyl-Sepharose CL-4B and Mono Q anion-exchange chromatography. Analysis by one- and two-dimensional SDS/polyacrylamide gel electrophoresis showed the purified preparation to contain three bands ranging over 29-33 kDa in molecular mass, each consisting of several charge isomers with similar pI values (5.4-5.7). Peptide mapping, amino acid analysis and sequencing suggested that the proteins are related, with the possibility that some species are distinct gene products. The concentration of inhibitor proteins required for half-maximal inhibition of protein kinase C activity is 1.7 microM. Inhibitory activity could not be affected by increasing the substrate, cofactor or ATP concentration in the standard protein kinase C assay, but was abolished by heat treatment. The inhibitor preparation did not affect the binding of phorbol dibutyrate to protein kinase C and could inhibit phosphorylation over a wide range of calcium concentrations. Inhibitory activity could be removed by immunoprecipitation of the purified inhibitor proteins with polyclonal antibodies raised against synthetic peptides, the sequences corresponding to those of peptide fragments obtained from protein digests. Amino acid sequence analysis of the inhibitors confirms they are novel proteins although similarities exist with a neuronal specific protein termed 14-3-3 and the carboxy terminus of the calcium-lipid binding series (endonexin/calpactin/lipocortin).

Substitution of phosphatidylserine by lipid A in the activation of purified rabbit brain protein kinase C.

Three lipid A derivatives (hexaacyl monophosphoryl lipid A, hexaacyl diphosphoryl lipid A, and disaccharide precursor IVA) were shown to activate protein kinase C from rabbit brain. These derivatives substituted for phosphatidylserine in a concentration-dependent manner and did not compete for binding of [3H]phorbol dibutyrate to its receptor site. Instead, phorbol dibutyrate binding was increased on raising the concentration of the derivatives in a similar manner to phosphatidylserine. The phorbol ester 12-0-tetra-decanol 13-acetate augmented the activation of protein kinase C by the lipid A derivatives.

Total parenteral nutrition-related infections. Prospective epidemiologic study using semiquantitative methods.

Studies of total parenteral nutrition-related infection have incorrectly relied on positive results on culture of the cannula tip to confirm the source. We undertook a prospective study of total parenteral nutrition-related infections in adult patients by obtaining blood from all total parenteral nutrition lines for pour-plate culture twice weekly and culturing intravascular line segments by the technique of Maki. Twelve of 100 courses of total parenteral nutrition (12 percent) in 69 patients resulted in infections--five (5.0 percent) had sepsis, and seven (7.0 percent) had local infection. In five of these 12, pour-plate culture gave positive results (five of 38 pour plates) with counts of 8 colony-forming units per ml (cfu/ml) for Candida tropicalis, and 25 to more than 1,000 for bacterial isolates. In nine of 12, culture of the intravascular line segment gave positive results with more than 50 cfu/ml. Pathogens isolated from intravascular line segments included Staphylococcus epidermidis (three cases), Candida species (three cases), Staphylococcus aureus (two cases), Serratia marcescens (one case) and mixed bacterial pathogens (one case). In contrast, pour-plate culture gave positive results in only seven of 88 uninfected (control) courses (318 pour plates), and culture of intravascular line segments gave positive results in two of 65 uninfected courses (p less than 0.001). No differences existed among patients with and without infection with regard to age, underlying disease, surgery, systemic antibiotic usage, or the presence of other infections. The duration of total parenteral nutrition was longer in courses without infection than in courses with infection (14.7 +/- 9.4 days versus 11.0 +/- 4.0 days; p less than 0.02). In six of 12 courses with infection, the line had been violated compared with 22 of 88 courses without infection (p less than 0.001). T-connectors for the centra administration of intralipid were associatd with infection (p less than 0.02). The value of routine pour-plate culture was illustrated in three courses in which the positive pour-plate culture results antedated positive blood culture results or line removal.

Nutritionally deficient Streptococcus: investigation of the hidden culprit in culture-negative endocarditis.

Subacute bacterial endocarditis in a 72-year-old woman was found to be caused by nutritionally deficient ("satelliting") Streptococcus sanguis. The organism, which required pyridoxal compounds or thiol substitutes, was identified after the application of relatively unusual but simple laboratory procedures. Normally, the organism's nutritional requirements would be fulfilled by components of the human blood that are used to inoculate clinical blood cultures, provided that the dilutional effect of the media is less than 10. The difficulty of diagnosis in such cases appears to lie in the laboratory inability to identify the organisms, rather than in failure to grow in properly obtained blood cultures.

The impact of hospitalization on the aerobic fecal microflora.

Quantitative bacteriological analysis of the aerobic fecal microflora of 75 patients indicated that, at the time of admission to hospital, Escherichia coli were the predominant fecal aerotolerant bacteria. Subsequent fecal samples showed a progressive supplantation of E coli by Klebsiella, Enterobacter and Proteus. At the end of 21 days of hospitalization, E coli remained predominant in only 30 patients. None of the patients had received antibiotics, undergone surgery or been subjected to x-ray studies of the gastrointestinal tract. The cause of the change of fecal flora in these patients is unknown, and no change of flora was observed in a control group of nonhospitalized persons, also studied for 21 days. The appearance of Klebsiella, Enterobacter and Proteus as predominant in the fecal flora of hospitalized patients may be an important factor in the natural history of hospital-associated infections.

The relation between aerobic fecal and oropharyngeal microflora in hospitalized patients.

The aerobic fecal and oropharyngeal bacterial flora was examined in 75 patients hospitalized, but not given antibiotics; in 70 patients given antibiotics during hospitalization and in 25 nonhospitalized controls. In all subjects, when first examined, normal throat flora were predominant. At the end of 21 days, however, a gram-negative bacilli became predominant in 17 (22.7%) of the Hospital Group and 33 (47.1%) of the Antibiotic Group. Newly appearing genera of gram-negative bacilli in the pharynx were almost always represented those present in the fecal flora. The Hospital Group all had recognizable components of the normal oropharyngeal flora present at 21 days, but 12 (17.1%) of the Antibiotic Group had no demonstrable normal oropharyngeal flora at 21 days. The findings suggest that hospitalization alone can be associated with the appearance of gram-negative bacilli in the oropharynx, and that the intestinal tract is their most likely point of origin.

Transient bacteremia associated with barium enema.

A group of 175 patients had barium enema. Pour-plate blood cultures were obtained immediately before and after the procedure and 5, 10, 15, and 30 minutes later. Bacteremia was demonstrable in 20 (11.4%) patients. In some, blood cultures were positive for as long as 15 minutes after barium enema; all were negative at 30 minutes. Among the bacteria associated with the 20 episodes of bacteremia were Escherichia coli, Klebsiella, enterococci, Proteus morganii, Bacteroides, and Veillonella. The incidence of bacteremia among patients with ulcerative colitis, regional enteritis, rectal polyps, colonic or rectal carcinoma, nonspecific diarrhea, or other lower intestinal tract disorders was not much different from patients free of rectosigmoid disease. The results of this study suggest that a history of recent barium enema may be important in patients who have endocarditis.

Transient bacteremia associated with percutaneous liver biopsy.

Transient bacteremia associated with percutaneous liver biopsy was studied by pour-plate blood cultures, which were obtained immediately before and after the procedure and 5, 10, 15, and 30 min later in 89 patients. Part of the liver tissue was also cultured in all patients. Histological diagnoses included hepatitis, cirrhosis, cholangitis, fatty liver, granulomata, metastatic liver disease, lymphoma, and miscellaneous disorders. All blood cultures obtained before liver biopsy were sterile. Bacteremia was demonstrable in 12 patients (13.48%). In most of these patients, blood cultures were positive for as long as 15 min after liver biopsy; all cultures were negative at 30 min. Among the bacteria associated with 12 episodes of bacteremia were Escherichia coli, Klebsiella, Bacteroides, enterococci, diphtheroids, Staphylococcus aureus, alpha-hemolytic Streptococcus, and Streptococcus pneumoniae. The patients with positive liver biopsies had a higher incidence of bacteremia (83.3%) than did the patients whose liver biopsies were sterile (8.r%); this difference is stastically significant (P smaller than 0.01). Thus, liver biopsy can be associated with transient bactermia.