Albumin: physiologic and clinical effects on lung function.

Fluid resuscitation is one of the most frequent and necessary practices in clinical medicine and is an integral part of the initial stabilization of critically ill, hypovolemic patients. Longstanding debate and conflicting evidence surround the use of both colloid and crystalloid fluid resuscitation in these patients. The basis of this debate is heavily rooted in the physiological understanding of Starling's forces. In this review, we aim to highlight the ongoing debate of albumin versus crystalloid resuscitation both broadly and as it relates to lung function, and will discuss the current state-of-the-art, starting from an historic perspective and progressing through a review of both physiologic and clinical evidence. Despite the biologic and physiologic plausibility of therapeutic benefit, the current evidence base does not support the routine use of albumin administration to improve patient survival or prevent respiratory dysfunction.

The hunting of the Src.

The non-receptor tyrosine kinase Src is important for many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first proto-oncogene to be discovered in the vertebrate genome. Src has been important, not only as an object of study in itself, but also as an entry point into the molecular genetics of cancer.

Acute respiratory distress syndrome: innovative therapies.

Since its description in 1967, the acute respiratory distress syndrome (ARDS) has become one of the most studied pathophysiological processes in intensive care units worldwide. The current state of knowledge about this severe illness and its associated mediators has come from the study of relevant animal models. In the mid-1970s, the development of the sheep model of ARDS and later, the porcine model, led to the discovery of a wide variety of inflammatory lipid mediators, cytokines, and proteases, to name but a few. Recognition of the presence of such highly toxic mediators associated with the development of ARDS has led to numerous potential targets for drug development toward therapeutic intervention. Through implementation of a standardized definition for ARDS and its less severe sibling acute lung injury (ALI), growth in patient-oriented research has been possible. Substantial numbers of new therapies have been brought forth and examined in recent years, many of which remain controversial. This article is a critical appraisal of the potential therapeutic options investigated in recent years for the management of ALI/ARDS.

Career racing performance in Thoroughbreds treated with prosthetic laryngoplasty for laryngeal neuropathy: 52 cases (1981-1989).

OBJECTIVE: To compare racing performance before and after prosthetic laryngoplasty for treatment of laryngeal neuropathy in inexperienced and experienced Thoroughbred racehorses. DESIGN: Retrospective study. ANIMALS: 52 Thoroughbred racehorses treated with prosthetic laryngoplasty for laryngeal neuropathy. PROCEDURE: Lifetime race records were analyzed by use of a verified regression model. Individual race records and hospital records were also reviewed. RESULTS: Experienced horses had a decline in performance, as measured by performance index, earnings percentage, and mean prediction error, during the 6-month period before prosthetic laryngoplasty. Performance improved after surgery, relative to performance in 1 to 4 races immediately before surgery, but did not attain previous baseline values for performance index and earnings percentage, although racing speed was restored to baseline values. Factors associated with failure to attain baseline levels of performance included other racing-related injuries and disorders, major complications of surgery, and age. Individually, however, many horses had long and successful careers after surgery. Performance of inexperienced horses after surgery was at least equal to that of experienced horses. CONCLUSIONS AND CLINICAL RELEVANCE: In addition to warning clients of the complications associated with prosthetic laryngoplasty, it may be prudent to provide a guarded prognosis for full restoration of racing performance in older horses, unless they are especially talented and are free of other racing-related problems.

v-Src generates a p53-independent apoptotic signal.

Evasion of apoptosis appears to be a necessary event in tumor progression. Some oncogenes, such as c-myc and E1A, induce apoptosis in the absence of survival factors. However, others, such as bcl-2 and v-src, activate antiapoptotic pathways. For v-Src, these antiapoptotic pathways are dependent on the function of Ras, phosphatidylinositol (PI) 3-kinase, and Stat3. Here we asked whether v-Src can activate a proapoptotic signal when survival signaling is inhibited. We show that when the functions of Ras and PI 3-kinase are inhibited, v-src-transformed Rat-2 fibroblasts undergo apoptosis, evidenced by loss of adherence, nuclear fragmentation, and chromosomal DNA degradation. The apoptotic response is dependent on activation of caspase 3. Under similar conditions nontransformed Rat-2 cells undergo considerably lower levels of apoptosis. Apoptosis induced by v-Src is accompanied by a loss of mitochondrial membrane potential and release of cytochrome c and is blocked by overexpression of bcl-2, indicating that it is mediated by the mitochondrial pathway. However apoptosis induced by v-Src is not accompanied by an increase in the level of p53 and is not dependent on p53 function. Thus v-Src generates a p53-independent proapoptotic signal.

Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control.

Cdc6/Cdc18 is a conserved and essential component of prereplication complexes. The 2.0 A crystal structure of an archaeal Cdc6 ortholog, in conjunction with a mutational analysis of the homologous Cdc18 protein from Schizosaccharomyces pombe, reveals novel aspects of Cdc6/Cdc18 function. Two domains of Cdc6 form an AAA+-type nucleotide binding fold that is observed bound to Mg.ADP. A third domain adopts a winged-helix fold similar to known DNA binding modules. Sequence comparisons show that the winged-helix domain is conserved in Orc1, and mutagenesis data demonstrate that this region of Cdc6/Cdc18 is required for function in vivo. Additional mutational analyses suggest that nucleotide binding and/or hydrolysis by Cdc6/Cdc18 is required not only for progression through S phase, but also for maintenance of checkpoint control during S phase.

Hypoproteinemia predicts acute respiratory distress syndrome development, weight gain, and death in patients with sepsis. Ibuprofen in Sepsis Study Group.

OBJECTIVE: Starling's equation indicates that reduced oncotic pressure gradients will favor edema formation, and the current consensus definition of acute respiratory distress syndrome (ARDS) excludes only the hydrostatic pressure contribution. We hypothesized that low serum total protein levels might correlate with the likelihood of ARDS in at-risk patients because serum total protein is the chief determinant of oncotic pressure in humans. DESIGN: Regression analysis to compare outcomes in patients with low serum total protein levels with outcomes in patients with normal serum total protein levels with respect to weight change, development of ARDS, and mortality. SETTING: Intensive care units (ICUs) of seven clinical centers in North America. PATIENTS: A total of 455 ICU patients who met consensus criteria for severe sepsis (178 of whom developed ARDS) from a recently completed prospective clinical trial. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: We found that 92% of the patients developing ARDS had low or borderline serum total protein levels (<6 g/dL). Logistic and multiple regression analyses confirmed that of 18 clinical variables, initial serum total protein level and protein change over time were the most statistically significant predictors of weight gain, prolonged mechanical ventilation, ARDS development, and mortality in the study population. This correlation remained significant after adjustment for the other major predictors of outcome present at baseline (ie, Acute Physiology and Chronic Health Evaluation II score). CONCLUSIONS: Hypoproteinemia is significantly correlated with fluid retention and weight gain, development of ARDS and poor respiratory outcome, and mortality in patients with sepsis. Prospective, randomized trials of serum protein manipulation are needed to establish whether there is a cause-effect relationship to this association.

Shp-2 mediates v-Src-induced morphological changes and activation of the anti-apoptotic protein kinase Akt.

The protein-tyrosine phosphatase Shp-2 is a positive modulator of the Ras/mitogen-activated protein kinase pathway and a putative substrate of the transforming non-receptor tyrosine kinase v-Src. To characterize the role of Shp-2 in cellular transformation and signaling by v-Src, we expressed v-Src in normal and Shp-2-deficient mouse embryo fibroblasts. Expression of Shp-2 was found to be necessary for morphological transformation by v-Src: Shp-2+/+ cells became rounded or spindly upon v-Src expression, whereas Shp-2-deficient cells remained relatively flat. v-Src-induced reorganization of the actin cytoskeleton and the formation of podosomes were compromised in Shp-2-deficient cells. Shp-2 deficiency also reduced v-Src-induced activation of the anti-apoptotic protein kinase Akt. The reduced activation of Akt in Shp-2-deficient cells correlated with a reduction in the association of the p85 regulatory subunit of PI3-kinase with the adapter protein Cbl. Activation of PI3-kinase by v-Src may be mediated by the association of the adapter protein Cbl with the p85 subunit. Since activation of Akt is dependent on PI3-kinase, this suggests that the effect of Shp-2 on Akt activation may be mediated, at least in part, by its effects on the interaction between PI3-kinase and Cbl. The defect in activation of the Akt survival pathway also correlated with enhanced sensitivity of Shp-2-deficient cells to an apoptosis-inducing agent. These results implicate Shp-2 in v-Src-induced cytoskeletal reorganization and activation of the Akt cell survival pathway.

Ras-independent oncogenic transformation by an EGF-receptor mutant.

Mutations in the ligand-binding domain of the epidermal growth factor receptor have been identified in several types of human cancers, including malignant gliomas. These mutations render signaling by this receptor to be constitutively ligand-independent. In fibroblasts transformed with ligand-independent epidermal growth factor receptor mutants, there is a correlation between the formation of a unique phosphotyrosine protein complex and oncogenic transformation. This phosphoprotein complex includes Grb2, Shc, Sos, tyrosine-phosphorylated form of caldesmon, and two, as yet, unidentified proteins. The presence of Grb2, Shc, and Sos in this complex implicates Ras in ligand-independent signaling by these oncogenic epidermal growth factor receptor mutants. We, therefore, have used retroviral co-infections of cultured primary fibroblasts to determine if Ras activation is required for phosphoprotein complex formation, stress fiber loss, or transformation. As predicted, expression of a dominant-negative Ras mutant (N17Ras) completely abrogates ligand-stimulated soft agar colony growth of primary fibroblasts. In contrast, N17Ras expression has no effect on v-ErbB mediated stress fiber disassembly, soft agar colony growth, or phosphoprotein complex assembly. In addition, our data suggest that ligand-dependent Ras activation may be suppressed by oncogenic v-ErbB expression. Together these observations suggest that oncogenic signaling by v-ErbB does not require Ras activation, and implicate an alternative signal transduction pathway in ligand-independent epidermal growth factor receptor oncogenic signaling.

Factors associated with racing performance of Thoroughbreds undergoing lag screw repair of condylar fractures of the third metacarpal or metatarsal bone.

OBJECTIVE: To evaluate effects of sex, fracture configuration, affected limb, and screw placement on outcome of Thoroughbreds with condylar fractures involving the third metacarpal or metatarsal bone. DESIGN: Cohort study. Animals-56 horses. PROCEDURE: Age, sex, affected limb, fracture configuration, fracture length, fracture fragment width, and distance of the most distal screw from the articular surface were analyzed in logistic regression models. RESULTS: Females were more likely to have displaced fractures and race in fewer races after surgery than males. Sex and fracture configuration were associated with number of postoperative races. Among horses that returned to racing, those with thicker fracture fragments were 11 times as likely as horses with thinner fracture fragments to win a race after surgery. Horses with longer fractures and older horses had fewer postoperative races. Horses in which the most distal screw had been placed further from the joint surface had more races. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that female horses with displaced condylar fractures and male horses with nondisplaced condylar fractures are more likely to be referred for treatment. The effect of sex on outcome for these horses cannot be clearly separated from the effect of fracture configuration. When adjusted for fracture configuration, males were 6 times as likely as females to race after surgery. When adjusted for sex, horses with nondisplaced fractures were 17 times as likely as horses with displaced fractures to race after surgery. Results suggest that the most distal screw should be placed above the epicondylar fossa.

Fluid balance and colloid osmotic pressure in acute respiratory failure: emerging clinical evidence.

Available evidence suggests that both hydrostatic and osmotic forces are important in the development of acute respiratory distress syndrome (ARDS) or, more broadly, acute lung injury (ALI). More than 80% of ARDS patients in a large-scale randomized controlled trial (RCT) exhibited, at least intermittently, pulmonary artery wedge pressures (PAWP) above 18 mmHg. Retrospective analyses have shown that PAWP elevation is associated with increased mortality. Reduction in serum total protein (STP) has been shown, in a recent retrospective analysis of data from a sepsis patient population with a high frequency of ARDS, to be highly predictive of positive fluid balance, weight gain, development of ARDS, prolonged mechanical ventilation, and mortality. These findings suggest that therapy with diuretics and colloids might be of benefit in the prevention or treatment of ALI. A prospective RCT was designed and conducted to evaluate combination therapy with furosemide and albumin over a 5-day period in 37 ALI patients. Both mean serum albumin and mean STP increased promptly and substantially in furosemide + albumin recipients. The furosemide + albumin group also achieved a mean weight loss of 10 kg by the end of the treatment phase, and their weight loss exceeded that of placebo patients throughout. Hemodynamics improved in the treatment group during the 5-day protocol. Oxygenation, as assessed by the ratio between the fraction of inspired oxygen and the partial pressure of oxygen in arterial blood (PaO2/FiO2), was significantly higher within 24 h after commencement of treatment in the furosemide + albumin than the placebo group. No clinically important adverse effects of furosemide + albumin therapy were encountered. These results provide evidence that combined therapy with furosemide and albumin is effective in augmenting serum albumin and STP levels, promoting weight loss, and improving oxygenation and longer-term hemodynamic stability. Although mortality did not differ between groups, the RCT showed a trend toward reduced duration of mechanical ventilation and length of stay in the intensive care unit in patients receiving furosemide + albumin. The findings of the RCT further highlight the importance of both hydrostatic and osmotic forces in hypoxemic respiratory failure, a subject that requires further investigation.

Ubiquitin-dependent degradation of active Src.

Signaling by members of the Src family of protein tyrosine kinases, such as Src and Fyn, is important in many biological responses, including gene transcription, cell-cycle progression, and cell adhesion and spreading [1] [2]. Unregulated Src kinase activity has been implicated in the progression of colon cancer and transformation of cultured cells [3] [4] [5] [6]. Thus, precise regulation of Src activity is critical for normal cell growth. Src kinase activity is downregulated by the carboxy-terminal Src kinase (Csk), a tyrosine kinase that phosphorylates a conserved tyrosine residue in the carboxy-terminal tail of Src [7] [8]. When phosphorylated, this tyrosine residue mediates an intramolecular interaction that results in a 'closed' or inactive conformation [1] [2] [9] [10]. Here, we report that loss of csk resulted in a reduction in the abundance of the Src and Fyn proteins, which could be restored by reintroducing catalytically active Csk. The effect of Csk on Src expression was not due to an increase in Src message, but to stabilization of the Src protein. Inhibition of proteasome activity also increased the level of Src protein in csk-deficient cells. Src was found to be ubiquitinated, and activation of Src increased the extent of polyubiquitination. Thus, ubiquitin-proteasome-dependent degradation represents an additional mechanism by which active Src can be downregulated.

Cas mediates transcriptional activation of the serum response element by Src.

The Src substrate p130(Cas) is a docking protein containing an SH3 domain, a substrate domain that contains multiple consensus SH2 binding sites, and a Src binding region. We have examined the possibility that Cas plays a role in the transcriptional activation of immediate early genes (IEGs) by v-Src. Transcriptional activation of IEGs by v-Src occurs through distinct transcriptional control elements such as the serum response element (SRE). An SRE transcriptional reporter was used to study the ability of Cas to mediate Src-induced SRE activation. Coexpression of v-Src and Cas led to a threefold increase in SRE-dependent transcription over the level induced by v-Src alone. Cas-dependent activation of the SRE was dependent on the kinase activity of v-Src and the Src binding region of Cas. Signaling to the SRE is promoted by a serine-rich region within Cas and inhibited by the Cas SH3 domain. Cas-dependent SRE activation was accompanied by an increase in the level of active Ras and in the activity of the mitogen-activated protein kinase (MAPK) Erk2; these changes were blocked by coexpression of dominant-negative mutants of the adapter protein Grb2. SRE activation was abrogated by coexpression of dominant-negative mutants of Ras, MAPK kinase (Mek1), and Grb2. Coexpression of Cas with v-Src enhanced the association of Grb2 with the adapter protein Shc and the protein tyrosine phosphatase Shp-2; coexpression of Shc or Shp-2 mutants significantly reduced SRE activation by Cas and v-Src. Cas-induced Grb2 association with Shp-2 and Shc may account for the Cas-dependent activation of the Ras/Mek/Erk pathway and SRE-dependent transcription. 14-3-3 proteins may also play a role in Cas-mediated signaling to the SRE. Overexpression of Cas was found to modestly enhance epidermal growth factor (EGF)-induced activation of the SRE. A Cas mutant lacking the Src binding region did not potentiate the EGF response, suggesting that Cas enhances EGF signaling by binding to endogenous cellular Src or another Src family member. These observations implicate Cas as a mediator of Src-induced transcriptional activation.

Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways.

An increase in the level of active, GTP-bound Ras is not necessary for transformation of chicken embryo fibroblasts (CEF) by v-Src. This suggests that other Ras-independent pathways contribute to transformation by v-Src. To address the possibility that activation of phosphatidylinositol-3-kinase (PI3K) and the mammalian target of rapamycin (mTOR/FRAP), represents one of these pathways, we have examined the effect of simultaneous inhibition of the Ras-MAPK and PI3K-mTOR pathways on transformation of CEF by v-Src. Transformation was assessed by the standard parameters of morphological alteration, increased hexose uptake, loss of density inhibition, and anchorage-independent growth. Inhibition of the Ras-MAPK pathway by expression of the dominant-negative Ras mutant HRasN17 or by addition of the MAPK kinase (MEK) inhibitor PD98059 reduced several of these parameters but failed to block transformation. Similarly, inhibition of the PI3K-mTOR pathway by addition of the PI3K inhibitor 2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one (LY294002) or the mTOR inhibitor rapamycin, although reducing several parameters of transformation, also failed to block transformation. However, simultaneous inhibition of signaling by the Ras-MAPK pathway and the PI3K-mTOR pathway essentially blocked transformation. These data indicate that transformation of CEF by v-Src is mediated by two parallel pathways, the Ras-MAPK pathway and the PI-3K-mTOR pathway, which both contribute to transformation. The possibility that simultaneous activation of other pathways is also required is not excluded.

Postnatal development of stearoyl coenzyme A desaturase gene expression and adiposity in bovine subcutaneous adipose tissue.

This investigation addressed the hypothesis that stearoyl coenzyme A desaturase (SCD) gene expression would serve as a postnatal marker of adipocyte differentiation in bovine s.c. adipose tissue. Samples of tailhead s.c. adipose tissue were obtained by biopsy from preweaning steer calves 2.5 wk, 5 mo, and 7.5 mo of age and from yearling steers 12 mo of age. Samples also were obtained at slaughter when the steers were 18 mo of age. The steers sampled as yearlings were fed native pasture from weaning until 12 mo of age, and the steers sampled at slaughter were fed a high-concentrate diet from 12 to 18 mo of age. Major peak adipocyte volumes for the 2.5-wk-, 5-mo-, and 7.5-mo-old steers were 14, 270, and 700 pL, respectively (P < .001). The steers did not gain weight during pasture feeding, and at 12 mo of age peak adipocyte volume had decreased (P = .009) to 270 pL. At this time, a second, smaller population of adipocytes had appeared with a peak volume of 115 pL. At slaughter, adjusted fat thickness of the steers was 1.60 +/- .13 cm, the USDA yield grade of the carcasses was 3.51 +/- .31, and peak adipocyte volume had increased (P = .01) to over 2,500 pL. The number of adipocytes per 100 mg of adipose tissue doubled (P = .006) between 2.5 wk and 5 mo of age, concurrent with the nearly 20-fold increase in peak adipocyte volume, indicating that this was a period of apparent adipocyte hyperplasia. Uncoupling protein mRNA was undetectable at all stages of postnatal growth, indicating that differentiating tailhead s.c. adipocytes do not acquire brown adipocyte characteristics postnatally. Lipogenesis expressed on a cellular basis was low in all preweaning samples and increased significantly above preweaning values only in the 18-mo-old steers. Stearoyl coenzyme A desaturase mRNA concentration also was low in all preweaning samples, but it peaked (P = .07) at 12 mo of age. Because the peak in SCD mRNA concentration preceded a significant rise in lipogenesis and lipid filling, we conclude that the level SCD gene expression may be indicative of the extent of terminal differentiation in bovine tailhead s.c. adipose tissue.

Metabolism and morphology of brown adipose tissue from Brahman and Angus newborn calves.

The objective of this study was to compare adipocyte morphology and lipogenesis between breed types (Angus vs Brahman) in brown adipose tissue (BAT) and white adipose tissue (WAT) from newborn calves. The Brahman calves (n = 7) were born during the fall season, whereas the Angus calves were born in fall (n = 6) or the following spring (n = 4). At parturition, Brahman cows were lighter than fall Angus cows, but were heavier than spring Angus cows (P < .05). Birth weights and perirenal BAT weights were greater in spring-born, but not in fall-born Angus calves, than in Brahman calves (P < .05). Fall-born Angus BAT contained 63% more (P < .05) adipocytes/100 mg tissue and contained a greater proportion (P < .05) of adipocytes with mean diameters of 40 to 50 microm, and fewer adipocytes with diameters of 60 microm or greater, than Brahman BAT. Brahman BAT contained two-to-three times as many beta-receptors as Angus BAT (P < .05), although the dissociation constant (Kd) was not different between breed types. Mitochondria in Brahman BAT were primarily spherical, whereas Angus BAT mitochondria were elongated, and mitochondrial cross-sectional area tended (P = .08) to be greater in Brahman BAT than in Angus BAT. The mitochondrial uncoupling protein (UCP) mRNA concentration (per 10(6) cells) was greater in Brahman BAT than in BAT from fall-born Angus calves. Lipogenesis from acetate was greater in Angus BAT than in Brahman BAT (P < .05), and glucose and palmitate contributed a greater proportion of carbon to lipogenesis in Brahman BAT than in Angus BAT. These differences in lipogenesis between breed types were not observed in s.c. WAT. The WAT from both breed types contained adipocytes with distinct brown adipocyte morphology, suggesting an involution of BAT to WAT in utero. We conclude that differences in UCP gene expression cannot cause the greater peak thermogenesis of Angus calves; however, differences between breed types in lipid metabolism and(or) mitochondrial morphology may contribute to this phenomenon.

The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product.

The fission yeast cdc18(+) gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18(+) gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C-2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.