Inspiratory airway obstruction does not affect lung fluid balance in lambs

The purpose of this study was to examine the effects of inspiratory airway obstruction on lung fluid balance in newborn lambs. We studied seven 2- to 4-wk-old lambs that were sedated with chloral hydrate and allowed to breathe 30-40% O2 spontaneously through an endotracheal tube. We measured lung lymph flow, lymph and plasma protein concentrations, pulmonary arterial and left atrial pressures, mean and phasic pleural pressures and airway pressures, and cardiac output during a 2-h base-line period and then during a 2- to 3-h period of inspiratory airway obstruction produced by partially occluding the inspiratory limb of a nonrebreathing valve attached to the endotracheal tube. During inspiratory airway obstruction, both pleural and airway pressures decreased 5 Torr, whereas pulmonary arterial and left atrial pressures each decreased 4 Torr. As a result, calculated filtration pressure remained unchanged. Inspiratory airway obstruction had no effect on steady-state lung lymph flow or the lymph protein concentration relative to that of plasma. We conclude that in the spontaneously breathing lamb, any decrease in interstitial pressure resulting from inspiratory airway obstruction is offset by a decrease in microvascular hydrostatic pressure so that net fluid filtration remains unchanged.     

Muscle fluid shift does not alter EMG global variables during sustained isometric actions

Body fluid redistribution occurs in astronauts traveling in space, potentially altering interstitial water content and hence impedance. This in turn may impact the features of electromyographic (EMG) signals measured to compare in-flight muscle function with pre- and post-flight conditions. Thus, the current study aimed at investigating the influence of similar fluid shifts on EMG spectral variables during muscle contractile activity. Ten men performed sustained isometric actions (120 s) at 20% and 60% of maximum voluntary contraction (MVC) following 1-h rest in the vertical or supine position.From single differential EMG signals, recorded from the soleus (SOL), the medial (MG) and lateral (LG) gastrocnemius muscles, initial value and rate of change over time (slope) of mean power frequency (MNF) and average rectified value (ARV) were assessed. MNF initial value showed dependence on muscle (P < 0.01), but was unaffected by body tilt. MNF rate of change increased (P < 0.001) with increased force and differed across muscles (P < 0.05), but was not influenced (P = 0.85) by altered body position. Thus, fluid shift resulting from vertical to supine tilt had no impact on myoelectrical manifestations of muscle fatigue. Furthermore, since such alteration of body fluid distribution resembles that occurring in microgravity, our findings suggest this may not be a methodological limitation, when comparing EMG fatigue indices on Earth versus in space.     

Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet

We have shown that first-pass intestinal metabolism is necessary for approximately 50% of whole body arginine synthesis from its major precursor proline in neonatal piglets. Furthermore, the intestine is not the site of increased arginine synthesis observed during dietary arginine deficiency. Primed constant intravenous (iv) and intraportal (ip) infusions of L-[U-14C]proline, and iv infusion of either L-[guanido-14C]arginine or L-[4,5-3H]arginine were used to measure first-pass hepatic arginine synthesis in piglets enterally fed either deficient (0.20 g.kg(-1).day(-1)) or generous (1.80 g.kg(-1).day(-1)) quantities of arginine for 5 days. Conversion of arginine to other urea cycle intermediates and arginine recycling were also calculated for both dietary treatments. Arginine synthesis (g.kg(-1).day(-1)) from proline was greater in piglets (P < 0.05) fed the deficient arginine diet in both the presence (generous: 0.07; deficient: 0.17; pooled SE = 0.01) and absence (generous: 0.06; deficient: 0.20; pooled SE = 0.01) of first-pass hepatic metabolism. There was no net arginine synthesis from proline during first-pass hepatic metabolism regardless of arginine intake. Arginine conversion to urea, citrulline, and ornithine was significantly greater (P < 0.05) in piglets fed the generous arginine diet. Calculated arginine fluxes were significantly lower (P = 0.01) for [4,5-3H]arginine than for [guanido-14C]arginine, and the discrepancy between the values was greater in piglets fed the deficient arginine diet (35% vs. 20%). Collectively, these findings show that first-pass hepatic metabolism is not a site of net arginine synthesis and that piglets conserve dietary arginine in times of deficiency by decreasing hydrolysis and increasing recycling.     

Adult sterol metabolism is not affected by a positive sterol balance in the neonatal Golden Syrian hamster

Dietary components impact metabolism early in life. Some of the diet-induced effects are long lasting and can lead to various adult-based diseases. In the current studies, we examined the short-term effects of dietary cholesterol on neonatal hepatic sterol metabolism and the long-term effects that those early-life diets had on sterol metabolism in adulthood. Neonatal hamsters began consuming solid food as a supplement to milk by 5 days of age; diets contained 0 or 2% added cholesterol (wt/wt). By 10 days of age, plasma and liver cholesterol concentrations were 3.2- and 2.5-fold greater, respectively, in the neonates fed cholesterol. Hepatic sterol synthesis rates were suppressed 65% in cholesterol-fed neonates compared with control neonates. By 20 days of age, plasma and liver cholesterol concentrations were still greater and sterol synthesis rates were now suppressed maximally in neonates fed cholesterol compared with control neonates. The expression level of an apolipoprotein B-containing lipoprotein receptor (low-density lipoprotein receptor-related protein) was greater and the mature form of the sterol regulatory element-binding protein-2 was similar in livers of 20-day-old control neonates compared with control neonates at 10 days of age. To test whether the change in sterol balance in the neonatal period had a lasting effect on hepatic sterol metabolism, all animals were weaned on a low-cholesterol diet. At 70 days of age, hepatic sterol synthesis rates, plasma lipoprotein and liver cholesterol concentrations, and bile acid pool sizes and compositions were measured. Sterol balance in the adults was similar between animals fed either diet early in life, as demonstrated by a lack of difference in any parameter measured. Thus, even though dietary cholesterol suppressed hepatic sterol synthesis rates dramatically in the neonatal hamster, the change has little impact on sterol balance later in life.     

Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving

Tissue culture media or aqueous sucrose solutions containing activated charcoal buffered to pH 5.5 and autoclaved did not undergo appreciable sucrose hydrolysis as reported. Rather, the extent of sucrose hydrolysis in media containing activated charcoal was found to be directly proportional to the hydrogen ion concentration (pH). This finding is consistent with the known mechanism of acid-catalyzed hydrolysis of acetals such as sucrose. Several types of charcoal were identified that acidified culture media to the extent that considerable acid-catalyzed sucrose hydrolysis occurred under autoclave conditions, making it appear as though activated charcoal was responsible for catalyzing sucrose hydrolysis. A simple mathematical expression was empirically derived that can be used to predict the extent of sucrose hydrolysis based on the post-autoclave pH of the media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Luciferase does not alter metabolism in cancer cells

Luciferase transfected cell lines are used extensively for cancer models, revealing valuable biological information about disease mechanisms. However, these genetically encoded reporters, while useful for monitoring tumor response in cancer models, can impact cell metabolism. Indeed firefly luciferase and fatty acyl-CoA synthetases differ by a single amino acid, raising the possibility that luciferase activity might alter metabolism and introduce experimental artifacts. Therefore knowledge of the metabolic response to luciferase transfection is of significant importance, especially given the thousands of research studies using luciferase as an in vivo bioluminescence imaging reporter. Untargeted metabolomics experiments were performed to examine three different types of lymphoblastic leukemia cell lines (Ramos, Raji and SUP-T1) commonly used in cancer research, each were analyzed with and without vector transduction. The Raji model was also tested under perturbed starvation conditions to examine potential luciferase-mediated stress responses. The results showed that no significant metabolic differences were observed between parental and luciferase transduced cells for each cell line, and that luciferase overexpression does not alter cell metabolism under basal or perturbed conditions.     

Cerebrospinal fluid acid-base balance during muscular exercise

Ventilation, metabolism, arterial blood gases, and blood and cerebrospinal fluid (CSF) acid-base status were measured in exercise studies on seven ponies during mild, moderate, and near-maximal treadmill exercise. CSF and arterial blood were sampled via indwelling catheters. Generally measurements were made during the 3rd, 6th, and 9th minute of steady-state exercise, with CSF sampled only during the 9th minute. Alveolar ventilation (VA) and metabolic rate (VO2) increased proportionately during exercise below the anaerobic threshold, but above this threshold, VA increased at a faster rate than VO2. The similarity of these response to those observed in man suggests the pony is a suitable animal model for study of exercise hyperpnea. No change in CSF acid-base balance occurred with light-to-moderate work; however, with near-maximal work a fall in CSF carbon dioxide partial pressure due to hyperventilation caused CSF to become alkaline (pH = 7.380) relative to rest (pH = 7.330). CSF lactate increased slightly with exercise but had no effect on CSF [HCO3-], which remained constant from rest to severe exercise. We conclude that it is unlikely the hyperpnea at any intensity of exercise results from an increased H+ stimulation at the medullary chemoreceptor.     

ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice

Alpha/beta hydrolase domain-containing protein 4 (ABHD4) catalyzes the deacylation of N-acyl phosphatidyl-ethanolamine (NAPE) and lyso-NAPE to produce glycerophospho-N-acyl ethanolamine (GP-NAE). Through a variety of metabolic enzymes, NAPE, lyso-NAPE, and GP-NAE are ultimately converted into NAE, a group of bioactive lipids that control many physiological processes including inflammation, cognition, food intake, and lipolysis (i.e., oleoylethanolamide or OEA). In a diet-induced obese mouse model, adipose tissue Abhd4 gene expression positively correlated with adiposity. However, it is unknown whether Abhd4 is a causal or a reactive gene to obesity. To fill this knowledge gap, we generated an Abhd4 knockout (KO) 3T3-L1 pre-adipocyte. During adipogenic stimulation, Abhd4 KO pre-adipocytes had increased adipogenesis and lipid accumulation, suggesting Abhd4 is responding to (a reactive gene), not contributing to (not a causal gene), adiposity, and may serve as a mechanism for protecting against obesity. However, we did not observe any differences in adiposity and metabolic outcomes between whole-body Abhd4 KO or adipocyte-specific Abhd4 KO mice and their littermate control mice (both male and female) on chow or a high-fat diet. This might be because we found that deletion of Abhd4 did not affect NAE such as OEA production, even though Abhd4 was highly expressed in adipose tissue and correlated with fasting adipose OEA levels and lipolysis. These data suggest that ABHD4 regulates adipocyte differentiation in vitro but does not affect adipose tissue lipid metabolism in mice despite nutrient overload, possibly due to compensation from other NAPE and NAE metabolic enzymes.     

Skeletal muscle glutamine metabolism during sepsis in the rat

1. The effect of sepsis, induced by caecal ligation plus puncture (CLP) or endotoxin injection, on glutamine metabolism was studied in rat skeletal muscle. 2. The concentration of glutamine in muscle was decreased by CLP or after 24 or 48 hr after injection of endotoxin. However, the concentration was increased 3 hr after injection of endotoxin. 3. The plasma glutamine concentration was decreased by CLP, but it was unchanged after injection of endotoxin. 4. The rate of glutamine release from incubated stripped soleus muscles was increased in the muscles removed from animals subjected to CLP or from animals injected with endotoxin. 5. It is concluded that sepsis results in marked changes in skeletal muscle glutamine metabolism, which may be used as an early indicator of the septic state. During sepsis there is likely to be an increased demand for glutamine by the immune system, kidney and intestine. 6. This study provides evidence that during sepsis the rate of release of glutamine from the skeletal muscle per se is increased to a sufficient extent to satisfy this increased requirement.     

Low abundance does not mean less importance in cysteine metabolism

The cysteine molecule plays an essential role in cells because it is part of proteins and because it functions as a reduced sulfur donor molecule. In addition, the cysteine molecule may also play a role in the redox signaling of different stress processes. Even though the synthesis of cysteine by the most abundant of the isoforms of O-acetylserine(thiol) lyase in the chloroplast, the mitochondria and the cytosol is relatively well-understood, the role of the other less common isoforms homologous to O-acetylserine(thiol)lyase is unknown. Several studies on two of these isoforms, one located in the cytosol and the other one in the chloroplast, have shown that while one isoform operates with a desulfhydrase activity and is essential to regulate the homeostasis of cysteine in the cytosol, the other, located in the chloroplast, synthesizes S-sulfocysteine. This metabolite appears to be essential for the redox regulation of the chloroplast under certain lighting conditions.Key words: cysteine, S-sulfocysteine, desulfhydrase, sulfur metabolism, redox regulation, ArabidopsisCysteine occupies a central position in the plant primary and secondary metabolism due to its biochemical functions. Cysteine is the first organic compound with reduced sulfur synthesized by the plant in the photosynthetic primary sulfate assimilation. The importance of cysteine for plants derives from its role as an amino acid in proteins but also because of its functions as a precursor for a huge number of essential bio-molecules, such as many plant defense compounds formed in response to different environmental adverse conditions.^1,2 All of these bio-molecules contain sulfur moieties that act as functional groups and are derived from cysteine, and therefore, are intimately linked via their biosynthetic pathways.In addition to the final destination of the reduced sulfur atom in the primary and secondary metabolism of cells, the thiol residue of the cysteine molecule is a functional group that translates the physico-chemical signal (redox) of ROS and RNS into a functional signal, altering the properties of small molecules such as GSH or proteins whose enzymatic or functional properties depend on the redox state of its cysteine residues.³Sulfate is the major sulfur form available to plants. Sulfate is taken up to plant cells through specific sulfate transporters and is activated to adenosine 5′-phosphosulfate (APS). The reduction of the activated sulfate form, APS, is linked to plastids and the photosynthetic activity; therefore, APS is reduced to sulfite by the APS reductase using two GSH molecules as donors of the two electrons required in this step. Sulfite is further reduced to sulfide by the sulfite reductase that uses photosynthetically reduced ferredoxine (Fd) as an electron donor of the six required electrons. The biosynthesis of cysteine is further accomplished by the sequential reaction of two enzymes: First, the serine acetyltransferase (SAT) synthesizes the intermediary product, O-acetylserine (OAS), from acetyl-CoA and serine; and second, the O-acetylserine(thiol)lyase (OASTL) incorporates the sulfide to OAS producing the cysteine. Recently, much progress has been made toward understanding the action of the O-acetylserine(thiol)lyase (OASTL) enzyme, one of the enzymes responsible for the biosynthesis of cysteine, using as a model system the plant Arabidopsis thaliana. The focus of the research has been mainly placed on the most abundant enzymes based on their involvement in the primary sulfate assimilation pathway. Biochemical and molecular analysis of the major OASTL knockout mutants in Arabidopsis thaliana revealed that part of the produced sulfide is incorporated to O-acetylserine to form cysteine in the chloroplast with the assistance of the OAS-B isoform. However, most of the chloroplastic sulfide overflows and escapes into the cytosol and the mitochondria, where it is also assimilated into cysteine by the OAS-A1 and OAS-C isoforms, respectively.^4–6The three major OASTL isoforms seem to be redundant under normal growth conditions. However, our investigations on the major cytosolic isoform, the OAS-A1, revealed new insights on the function of this enzyme as a determinant of the antioxidative capacity of the cytosol.⁷ The OASTL homolog, CYS-C1, exhibits OASTL activity, but in fact, it is a β-cyanoalanine synthase enzyme that uses cysteine to detoxify cyanide within the mitochondria.⁸ Furthermore, Arabidopsis cells contain four additional O-acetylserine(thiol)lyase isoforms encoded by the CYS-D1 (At3g04940), CYS-D2 (At5g28020), CS26 (At3g03630) and CS-LIKE (At5g28030) genes with unknown function. Are these four isoforms authentic OASTL and are, therefore, redundant enzymes or do they have different activities and, therefore, different functions?Our recent research on the less-common isoforms, CS-like and CS26, shed light on this issue, and we are decoding two important aspects of the sulfur metabolism in plants.^9,10 The CS-LIKE protein was identified by sequence homology upon the completion of the sequencing of the Arabidopsis genome. Because of its cytosolic localization, it is thought to have an auxiliary function with respect to the major cytosolic isoform, the OAS-A1. The characterization of the purified recombinant protein has shown that the CS-LIKE isoform catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate; thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and it is designated as DES1 (Fig. 1). This enzyme is important for maintaining the homeostasis of cysteine in the cell, and the loss of function of this protein in knockout mutant plants results in higher levels of cysteine and glutathione. This increased level of soluble thiols results also in a higher antioxidant capacity of the plant, which, in turn, becomes more resistant to abiotic stress phenomena such as the presence of heavy metals or hydrogen peroxide. This observation may indicate that the regulation of this enzyme may be a key component of the plant physiological processes that involve redox reactions. Cytosolic cysteine degrading enzymes with desulfhydrase activity has been found in plants, but the protein responsible for this activity remained unisolated until now that it is revealed with our investigation on DES1.¹¹ From the standpoint of biotechnology, plants with this modified enzyme may result in abiotic stress-resistant lines that deserve to be studied.Open in a separate windowFigure 1Biosynthesis of cysteine and S-sulfocysteine in the chloroplast and cytosol of Arabidopsis and subcellular localization of the responsible enzymes. The cytosolic and plastidial O-acetylserine(thiol)lyase, L-cysteine desulfhydrase and S-sulfocysteine synthase are shown in red. A single representative of a grana thylakoid is shown as a grey oval compartment.The other less common enzyme studied, called CS26 and localized in the chloroplast, has proved to be an enzyme with S-sulfocysteine synthase activity.¹⁰ This enzyme synthesizes the incorporation of thiosulfate to O-acetylserine to form S-sulfocysteine (RSSO₃⁻). This activity, discovered for the first time in plants, was previously reported in bacteria where the biosynthesis of cysteine can be accomplished by two enzymes encoded by the cysK and cysM genes.^12,13 This enzyme activity is essential for the chloroplast function under long-day growing conditions but seems to be superfluous under short-day conditions. Morphologic and biochemical phenotype comparisons of the knockout oas-b and cs26 highlight the importance of the metabolite S-sulfocysteine and not the cysteine in the redox control of the chloroplast. Under long-day growth conditions, the cs26 mutants exhibit a reduction in size and show leaf paleness, have reductions in the chlorophyll content and photosynthetic activity, and are not able to properly detoxify reactive oxygen species, which are accumulated to high levels. None of these changes are observed in the oas-b mutant.Although we do not know the function of the S-sulfocysteine molecule in the chloroplast, two aspects are important to note. On the one hand, the enzyme CS26 can be located in the chloroplast''s lumen in opposition to the enzyme OAS-B, which is located in the stroma. The second aspect is the difference in chemical reactivity of S-sulfocysteine and cysteine. The S-sulfocysteine has two sulfur atoms with different degrees of oxidation, −1 and +5; therefore, it may act as an oxidant molecule by reacting with reduced thiols forming a disulfide bridge and releasing sulfite.¹⁴ We have suggested that a putative target of S-sulfocysteine can be the STN7 kinase, which contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved cysteines that are critical for its activity. A disulfide bridge between these two cysteines is required for the kinase activity, but how the redox states of these two cysteines are regulated in the lumen remains an open question.¹⁵ In general, how the thiol oxidation of proteins located in the thylakoid lumen takes place is still unclear because no sulfhydryl oxidases have been identified in this compartment. In fact, this process is highly important because the chaperones and peptidyl-prolyl cis-trans isomerases, such as the AtFKBP13, need to be oxidized in order to be functional in the lumen and to regulate the folding of the Rieske protein.^16–18     

Axl deficiency does not affect adipogenesis or adipose tissue development

To evaluate a potential role of Axl, the high-affinity receptor of growth arrest-specific protein 6 (GAS6) in adiposity, murine embryonic fibroblasts (MEF) derived from mice with genetic deficiency of Axl (Axl(-/-)) or wild-type littermates (Axl(+/+)) were differentiated into mature adipocytes. In addition, Axl(-/-) and Axl(+/+) mice were kept on standard fat diet (SFD) or on high-fat diet (HFD) for 15 weeks. Deficiency of Axl in MEF did not affect differentiation, as shown by a similar uptake of Oil Red O and expression of the adipogenic markers aP2 and peroxisome proliferator activator receptor γ (PPARγ) at the end of the differentiation. In the first 7 weeks of HFD feeding, Axl(-/-) mice gained less weight than their wild-type littermates. Weight gain for both genotypes on either SFD of HFD over 15 weeks was, however, not significantly different, resulting in comparable body weights, as well as subcutaneous (s.c.) and gonadal (GON) fat mass. Adipocyte size in the fat tissues was not affected by Axl deficiency. Gene expression analysis indicated that the absence of Axl in vivo may be compensated for by the other TAM family members Mer and Tyro3. Glucose and insulin tolerance tests (ITT) in Axl(-/-) and Axl(+/+) mice did not reveal significant differences in glucose homeostasis. Thus, Axl deficiency had no significant effect on adipogenesis in vitro or in vivo.     

Lung fluid balance during acute renal failure in sheep

To determine whether uremia changes lung vascular permeability, we measured the flow of lymph and proteins from the lungs of acutely uremic sheep. Acute renal failure was induced by either bilateral nephrectomy or by reinfusing urine. Both models of renal failure increased the plasma creatinine from 0.8 +/- 0.3 to 11 +/- 1 mg/dl in 3 days but caused no significant change in the flow of lymph from the lungs. To determine whether uremia increased the protein clearance response to elevated pulmonary microvascular pressures, we inflated a balloon in the left atrium for 2 h before and 3 days after inducing acute renal failure. In seven sheep, before removing the kidneys, the 20 cmH2O elevation of left atrial pressure increased the protein clearance 3.9 +/- 3.0 ml/h (from 9.5 +/- 4.9 to 13.4 +/- 5.4 ml/h). Three days after the bilateral nephrectomy the same increase in left atrial pressure increased the protein clearance 6.4 +/- 3.6 ml/h (from 6.1 +/- 2.1 to 12.5 +/- 5.2 ml/h), which was a significantly larger increase than that measured before the nephrectomy (P less than 0.05). Sham nephrectomy in seven sheep caused the protein clearance response to the elevated left atrial pressure to fall from 4.7 +/- 1.9 ml/h before the sham nephrectomy to 2.6 +/- 1.4 ml/h 3 days later (P less than 0.05). Uremia due to reinfusion of urine in five sheep did not affect the protein clearance response to elevations in left atrial pressure. Neither model of acute uremia increased the postmortem extravascular lung water volume.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caffeine does not enhance radiosensitivity of normal liver tissue in vivo

The aim of this study was to determine whether caffeine enhanced radiosensitivity of normal liver tissue in a rat radiation-induced liver disease model. Buffalo rat McA-RH7777 hepatocellular cancer cells and BRL3A normal liver cells were irradiated, and cell cycle distribution and apoptosis rates were analyzed. A rat model of radiation-induced liver disease was established, rats were randomized into four groups: control; caffeine alone; irradiation (IR) alone; and caffeine plus IR (Caff + IR) group. Apoptosis rates in normal rat liver tissue after IR were evaluated by TUNEL staining and caspase-3 Western blot. Transaminase activity was measured and histopathological examination was done after IR. Caffeine abrogated IR-induced G2 phase arrest (Caff + IR vs. IR: 40.9 ± 4.0 vs. 60.7 ± 5.5%, at 12 h after IR) and increased apoptosis rates (Caff + IR vs. IR: 56.1 ± 6.8 vs. 35.5 ± 4.0%, at 72 h after IR) in McA-RH7777 cells, but did not affect IR-induced G2 phase arrest and apoptosis rates at any time point after IR in BRL3A cells. Caffeine did not enhance apoptosis, transaminase activity, or histopathological injury of normal rat liver tissue at any time points after IR. This study suggests that caffeine might not enhance radiosensitivity of normal liver tissue in vivo. In an earlier study, we reported that caffeine enhanced radiosensitivity of human hepatocellular cancer in a nude mice model. Together, these results offer feasibility of clinical application.