Human IL6 stimulates bovine satellite cell proliferation through a Signal transducer and activator of transcription 3 (STAT3)-dependent mechanism.

Bovine satellite cell (bSC) myogenesis and skeletal muscle hypertrophy occur through the orchestrated actions of multiple autocrine and paracrine growth factors. Intimate to the bSC niche is IL6, a dual-purpose cytokine with proinflammatory and mitogenic properties. The objective of the experiment was to examine the effects of IL6 on proliferation and differentiation of bSC in vitro. Treatment of primary bSC cultures with recombinant bovine IL6 (bIL6) failed to alter myogenesis owing to the absence of intracellular signal transduction. The cytokine was able to stimulate phosphorylation of signal transducer and activator of transcription 3 tyrosine 705 (STAT3Y705) in Madin-Darby bovine kidney (MDBK) epithelial cells, thus demonstrating bioactivity. Media supplemented with recombinant human IL6 (hIL6) caused phosphorylation of STAT3Y705 in bSC and increased (P < 0.05) proliferation. Inclusion of a STAT3 inhibitor in the media blunted phosphorylation of the STAT3Y705 and suppressed (P < 0.05) hIL6-mediated bSC proliferation. Morphologic and biochemical measures of bSC differentiation remained unchanged (P > 0.05) following treatment for 48 h with hIL6. These results support a role for hIL6 as a bSC mitogen in vitro. The inability of bIL6 to initiate an intracellular signal in bSC requires further investigation.

Dietary selenium and prolonged exercise alter gene expression and activity of antioxidant enzymes in equine skeletal muscle.

Untrained Thoroughbred horses (6 mares and 6 geldings; 11 yr [SE 1] and 565 kg [SE 11]) were used to evaluate antioxidant gene expression and enzyme activity in blood and skeletal muscle in response to prolonged exercise after receiving 2 levels of dietary selenium for 36 d: 0.1 (CON; = 6) or 0.3 mg/kg DM (SEL; = 6). Horses were individually fed 1.6% BW coastal bermudagrass hay, 0.4% BW whole oats, and a mineral/vitamin premix containing no Se. Sodium selenite was added to achieve either 0.1 or 0.3 mg Se/kg DM in the total diet. On d 35, horses underwent 2 h of submaximal exercise in a free-stall exerciser. Blood samples were obtained before (d 0) and after 34 d of Se supplementation and on d 35 to 36 immediately after exercise and at 6 and 24 h after exercise. Biopsies of the middle gluteal muscle were obtained on d 0, before exercise on d 34, and at 6 and 24 h after exercise. Supplementation with Se above the NRC requirement (SEL) increased serum Se ( = 0.011) and muscle thioredoxin reductase (TrxR) activity ( = 0.051) but had no effect on glutathione peroxidase (GPx) activity in plasma, red blood cell (RBC) lysate, or muscle in horses at rest. Serum creatine kinase activity increased ( < 0.0001) in response to prolonged exercise but was not affected by dietary treatment. Serum lipid hydroperoxides were affected by treatment ( = 0.052) and were higher ( = 0.012) in horses receiving CON than SEL immediately following exercise. Muscle expression of was unchanged at 6 h but increased ( = 0.005) 2.8-fold 24 h after exercise, whereas muscle TrxR activity remained unchanged. Glutathione peroxidase activity increased in plasma (P < 0.0001) and decreased in RBC lysate ( = 0.010) after prolonged exercise. A Se treatment × time interaction was observed for RBC GPx activity (P = 0.048). Muscle and expression and GPx activity did not change during the 24-h period after exercise. Level of dietary Se had no overall effect on expression of , , , , , , or in muscle following exercise. The impact of prolonged exercise on the activities of antioxidant enzymes varied. Furthermore, changes in enzyme activity did not necessarily align with enzyme gene expression following exercise. A higher level of Se intake elevated Se status of untrained horses, increased GPx activity, and lessened lipid peroxidation following exercise, suggesting that Se may be beneficial for mitigating oxidative muscle damage and aiding in postexercise recovery.

Effects of recombinant bovine somatotropin administration at breeding on cow, conceptus, and subsequent offspring performance of beef cattle.

The effects of administration of recombinant bovine ST (bST) on plasma hormone concentrations of cows, conceptus development, and postnatal calf performance were examined. Lactating beef cows ( = 190) were exposed to a fixed-time AI (TAI) protocol from d -10 to 0 (TAI on d 0). Cows were blocked by breed and stratified by days postpartum and then randomly assigned to receive, subcutaneously 1) 2 injections of saline (1 mL of 0.9% saline), 1 on d 0 at TAI and a second injection on d 14 (CTRL; = 53); 2) an injection of 325 mg of bST on d 0 and a saline injection on d 14 (bST0; = 48); 3) a saline injection on d 0 and an injection of 325 mg of bST on d 14 (bST14; = 49); or 4) 2 injections of 325 mg of bST, 1 on d 0 and a second injection on d 14 (bST0+14; = 40). Pregnancy status, crown-to-rump length (CRL) on Day 35, and crown-to-nose length (CNL) on Day 65 were determined via transrectal ultrasonography. Blood samples were collected on d 0, 7, 14, 21, 35, and 65, relative to TAI, to determine plasma concentrations of progesterone (P4), IGF-1, and pregnancy-specific protein B (PSPB) and also on d 18 and 21 for isolation of peripheral blood leukocytes for RNA extraction and measurement of interferon-stimulated genes transcript abundance. Individual calf BW was determined at birth and every 30 d until weaning. A subset of 24 calves was randomly selected for liver biopsies at birth to determine mRNA expression of target genes. Administration of bST to cows increased ( < 0.0001) concentrations of plasma IGF-1 for 14 d after injection compared with CTRL but did not affect fetal CRL and CNL ( = 0.23). Cows receiving bST only on d 0 had a greater ( = 0.05) transcript abundance in myxovirus resistance 2 on d 21 compared with 2bST cows (2.0- and 0.8-fold for bST0 and 2bST, respectively), whereas cows receiving bST14 and CTRL were intermediate (1.2- and 0.9-fold, respectively). Calf BW did not differ ( ≥ 0.100) among treatments on d 0, 30, 60, 90, 120, and 150 relative to birth. Injection of bST only on d 0 tended ( = 0.062) to increase calf liver mRNA expression of at birth compared with the calves born to cows in other treatments. Therefore, during a TAI protocol, the administration of 1 or 2 injections of 325 mg of bST to lactating beef cows enhanced their plasma concentrations of IGF-1 but failed to improve fetal size and plasma concentrations of maternal PSPB and P4 and had no effect on postnatal calf growth performance.

Creep-feeding to stimulate metabolic imprinting in nursing beef heifers: impacts on heifer growth, reproductive and physiological variables.

This experiment compared growth, physiological, and reproductive responses of beef heifers with (MI) or without (CON) access to a creep-feeder, as a manner to stimulate metabolic imprinting while nursing their dams. On day 0, 60 Angus × Hereford heifers were ranked by BW and age (140 ± 3 kg and 68±3 days), and assigned to pairs so all ranking criteria were similar between heifers within each pair. On day 1, pairs were randomly assigned to MI (n=15) or CON (n=15). From day 1 to 51, MI pairs and their dams were allocated to 15 drylot pens where heifers had ad libitum access to a corn-based supplement through a creep-feeder. The CON pairs and their dams were maintained in an adjacent single drylot pen. From day 52 to 111, treatments were managed as a single group on a semiarid range pasture. On day 111, heifers were weaned and allocated to two pastures (one pasture/treatment), receiving hay and a corn-based concentrate until day 326. Heifer BW was recorded before and at the end of the creep-feeding period (day 1 to 51), and on days 112 and 326. On days 0, 51, 111, 187, 261, and 325, jugular blood was collected and real-time ultrasonography for longissimus muscle depth and backfat thickness assessment was performed. Blood was also collected every 10 days from days 113 to 323 for puberty evaluation via plasma progesterone. Liver and subcutaneous fat biopsies were performed on days 51, 111, 261 and 325. Average daily gain was greater (P<0.01) for MI than CON from day 1 to 51, tended (P=0.09) to be greater for CON than MI from day 112 to 326, while BW on day 326 was similar between treatments. On day 51, MI had greater (P ⩽ 0.01) plasma IGF-I and glucose concentrations, as well as mRNA expression of hepatic pyruvate carboxylase and adipose fatty acid synthase than CON. On days 261 and 325, plasma insulin concentrations were greater (P ⩽ 0.03) in CON than MI. Mean mRNA expression of hepatic IGF-I and adipose peroxisome proliferator-activated receptor gamma were greater (P ⩽ 0.05) in MI than CON. No treatment effects were detected for puberty attainment rate. In conclusion, supplementing nursing heifers via creep-feeding for 50 days altered physiological and biochemical variables suggestive of a metabolic imprinting effect, but did not hasten their puberty attainment.

Effect of Brahman genetic influence on collagen enzymatic crosslinking gene expression and meat tenderness.

The objective of the study was to examine the effect of Brahman genetics on collagen enzymatic crosslinking gene expression and meat tenderness. Steers were randomly selected to represent a high percentage Brahman genetics (n = 13), Half-Blood genetics (n = 13), Brangus genetics (n = 13), and a high percentage Angus genetics (n = 13). Muscle samples from the Longissimus lumborum muscle were collected at weaning and harvest and reverse transcription quantitative PCR (qPCR) analysis was conducted to measure the mRNA expression of lysyl oxidase (LOX), bone morphogenetic protein 1 (BMP1), and cystatin C (CYS). Steaks from subject animals were collected at harvest, aged for 14 d and subjected to collagen analysis, Warner-Bratzler Shear Force (WBS) and trained sensory panel analysis (tenderness, juiciness, and connective tissue). Data indicated that Half-Blood and Brahman steers had greater (P<0.05) WBS values and tended to receive decreased (P < 0.06) panel tenderness scores than Angus and Brangus steers. Panelists tended to detect more connective tissue in Brahman and Half-Blood steaks when compared to Angus and Brangus steaks (P < 0.07). Crosslinking gene expression data revealed that at weaning Half-Blood steers had more (P < 0.05) mRNA expression of CYS and LOX than Angus and Brangus steers. At weaning and harvest, all genetic groups had similar mRNA expression of BMP1 (P > 0.10). At harvest, Brangus and Angus steers had greater LOX mRNA expression than Brahman cattle (P < 0.05). Pearson's correlation coefficients indicated that only weaning CYS mRNA expression was correlated to WBS, panel tenderness and connective tissue scores (P < 0.05). Expression of LOX was only correlated to these measures at harvest, and BMP1 was correlated to these traits at both time periods (P < 0.05). These results indicate that collagen crosslinking enzyme activity, as indicated by mRNA levels, early in an animal's life may account for some of the variation seen in steak tenderness due to Brahman genetic influence.

Effects of zinc-L-carnosine and vitamin E on aspirin-induced gastroduodenal injury in dogs.

BACKGROUND: Nonsteroidal anti-inflammatory drugs frequently cause gastrointestinal (GI) injury. Zinc-L-carnosine has antioxidant, anti-inflammatory, mucosal protective, and healing properties in rodent models and in some human studies of GI injury. HYPOTHESIS: The combination of zinc-L-carnosine and vitamin E attenuates aspirin-induced gastroduodenal mucosal injury. ANIMALS: Eighteen healthy random-source Foxhound dogs. METHODS: In this randomized, double-blinded, placebo-controlled study dogs were treated with placebo (n = 6; 0X group), 30 mg/30 IU (n = 6; 1X group), or 60 mg/60 IU (n = 6; 2X group) zinc-L-carnosine/vitamin E orally every 12 hours for 35 days. Between Day 7 and 35, GI mucosal lesions were induced with aspirin (25 mg/kg p.o. q8h). Mucosal injury lesions (hemorrhage, erosion, and ulcer) were assessed by gastroduodenoscopy on Days 14, 21, and 35 with a 12-point scoring scale. RESULTS: At baseline (Day -1) gastroscopy scores were not significantly different between groups (mean ± SD: 0X, 4.4 ± 0.8; group 1X, 4.4 ± 0.6; group 2X, 4.2 ± 0.3; P= .55). Gastroscopy scores increased significantly in all groups between Day -1 and Days 14, 21, and 35 (P < .0001). On Day 35, gastroscopy scores were 29.2 ± 5.2 (0X), 27.3 ± 3.7 (1X), and 28.6 ± 3.3 (2X). Mean gastroscopy scores were not significantly different among treatment groups on any of the days (P = .61). CONCLUSIONS AND CLINICAL IMPORTANCE: Administration of the combination of zinc-L-carnosine and vitamin E at 1X or 2X dosing did not attenuate aspirin-induced gastroduodenal mucosal injury.

Effect of ractopamine-hydrochloride and trenbolone acetate on longissimus muscle fiber area, diameter, and satellite cell numbers in cull beef cows.

The objective of this study was to evaluate the effects of coadministration of ractopamine-HCl (RAC) and trenbolone acetate plus estradiol (TBA) on LM fiber cross-sectional area (CSA), diameter, fiber-associated myonuclei, and satellite cell number. Culled crossbred beef cows (n = 98; 11 +/- 1.8 yr old; BCS 4.3 +/- 0.03) from a single ranch in south Florida were fed a concentrate diet for 92 d in a 2 x 2, randomized block design. Cows were blocked by BW on arrival into light (initial BW = 369.75 +/- 2.68 kg and end BW = 501.96 +/- 6.90 kg) and heavy (initial BW = 418.31 +/- 2.75 kg and end BW = 522.15 +/- 7.09 kg) groups before assignment to treatment. Factors included dietary treatment (0 or 15 ppm) and implant status (0 or 80 mg of trenbolone acetate + 16 mg of estradiol). Ractopamine was provided in the diet to 2 pens or half the treatments during the final 35 d of feeding. Cows were slaughtered on d 92. Forty-eight hours postmortem, the 6th-rib portions of the LM were obtained from 10 randomly selected carcasses from each treatment group (n = 40). Cryosections (12 mum) were immunostained for dystrophin and myosin heavy chain I or II for the measurement of fiber CSA and type, respectively. Fiber-associated nuclei and satellite cell numbers were measured in serial cryosections. There was a RAC x TBA interaction (P < 0.05). Type I fiber CSA and diameter were increased (P < 0.05) by TBA and RAC. Type I CSA and diameter were larger (P < 0.05) in TBA + RAC than RAC only. Type II fiber CSA and diameter were not affected by TBA (P = 0.48), RAC (P = 0.15), or TBA + RAC (P = 0.60). Satellite cell numbers and fiber-associated nuclei were not affected (P > 0.05) by implant status or ractopamine supplementation. These results indicate that TBA and RAC preferentially increase the size of type I fibers in cull cows.

Physical mapping of CSF2RA, ANT3 and STS on the pseudoautosomal region of bovine chromosome X.

The pseudoautosomal region (PAR) of bovine chromosome X (BTA X) has a particularly low representation of genes and markers, making comparative gene mapping in this region difficult. We describe the localization of three genes, colony-stimulating factor 2 receptor alpha (CSF2RA), ADP/ATP translocase 3 (ANT3) and steroid sulphatase (STS) on PAR of BTA X using a 5000 rad whole-genome radiation hybrid panel. The relationship of these genes to a number of previously mapped simple sequence repeat (microsatellite) markers is determined by physical and radiation hybrid mapping methods. The resulting radiation hybrid map resolves a discrepancy between the two major bovine linkage maps in the PAR of BTA X.

Idiopathic noncirrhotic portal hypertension in dogs: 33 cases (1982-1998).

OBJECTIVE: To describe clinical signs, diagnostic findings, and outcome in dogs with idiopathic intrahepatic portal hypertension. DESIGN: Retrospective study. ANIMALS: 33 dogs. PROCEDURE: Medical records of dogs with portal hypertension of intra-abdominal origin were reviewed. Dogs with intra-abdominal portal hypertension of vascular causes or with hepatic histopathologic changes consistent with severe diffuse hepatobiliary disease were excluded. History and results of physical examination, clinicopathologic tests, diagnostic imaging studies, histologic examination, and treatment were summarized. Outcome was determined in 26 dogs. RESULTS: Dogs were referred most often because of ascites, intermittent vomiting or diarrhea, and polydipsia of several months' duration. Microcytosis, high serum alkaline phosphatase and alanine transaminase activities, hepatic dysfunction, urine specific gravity < or = 1.021, and abdominal transudate were the predominant clinicopathologic features. Microhepatia, abdominal effusion, and multiple anomalous venous anastomoses were the major findings of diagnostic imaging. Hepatic histopathologic changes were consistent with idiopathic noncirrhotic portal hypertension and were indistinguishable from those of dogs with surgically created portocaval anastomosis. Outcome was determined for 19 dogs released from hospital; 13 dogs remained healthy with mostly palliative treatment for periods of 5 months to 9 years. CONCLUSIONS AND CLINICAL RELEVANCE: The clinical signs, clinicopathologic test results, portal pressure, and gross appearance of the liver of dogs with idiopathic noncirrhotic portal hypertension may be identical to those of dogs with cirrhosis; therefore liver biopsy is crucial. Because the prognosis for idiopathic noncirrhotic portal hypertension is generally favorable, owners of affected dogs should be discouraged from choosing euthanasia.

Expression and immune recognition of SV40 Tag in transgenic mice that develop metastatic osteosarcomas.

Mature adult mice of the C57BL/6-TgN(Amy1TAg)501Knw transgenic mouse lineage, 501, containing a liver alpha-amylase promoted-SV40 Tag hybrid gene, routinely develop SV40 Tag-induced metastatic osteosarcomas. This form of alpha-amylase was known to be expressed in the liver, salivary glands, pancreas, and fat. Cells in the normal rib adjacent to the periosteum also express alpha-amylase suggesting that transgene expression is correctly targeted to generate osteosarcomas. 501 mice express SV40 Tag in the salivary glands but do not develop abnormalities in these organs by the time of their death from SV40-induced osteosarcomas. Mice of the C57BL/6 strain make a strong and effective anti-tumor immune response to SV40 Tag immunization. However, immunization of 501 mice with SV40 Tag early in life does not alter or prevent SV40 Tag-induced osteosarcomagenesis. 501 mice mount a significantly less effective cytotoxic T-lymphocyte response following SV40 Tag immunization while 501 osteosarcoma-derived cells are fully susceptible to SV40 Tag-specific T-cell lysis. This suggests that partial tolerance, not loss of antigen presentation by tumor cells, characterizes this mouse model of endogenous bone tumor development. To determine whether the immune recognition of endogenous SV40 Tag could influence tumorigenesis, the metastatic potential and time of death from tumor was investigated in CD4-null mutant 501 mice and beta-2 microglobulin-null mutant 501 mice. The size and number of metastases in these strains and longevity of these strains varied. We suggest that components of both the innate and adaptive immune response control tumor appearance and progression.

Activated Raf inhibits myogenesis through a mechanism independent of activator protein 1-mediated myoblast transformation.

Skeletal myogenesis is acutely affected by growth factors and subsequent activation of their respective intracellular signaling cascades. Components of the mitogenic Ras/Raf/mitogen-activated protein kinase (MAPK) signaling module are potent inhibitors of myoblast differentiation. However, the means by which these kinases prevent myocyte formation and activation of the muscle gene program is unknown. Activator protein 1 (AP-1) is a transcriptional regulator the actions of which are up-regulated by signaling events, including elevated MAPK. Because activated Raf inhibits avian myogenesis in a MAPK-dependent fashion, we investigated the role of AP-1 as a mediator of the Raf-imposed block to myogenesis. Avian myoblasts overexpressing activated Raf contain elevated levels of AP-1 DNA binding and transcriptional activity. Introduction of an AP-1 dominant inhibitory protein (AFOS) into Raf-expressing myoblasts prevented acquisition of a transformed morphology. Interestingly, these cells remained differentiation-defective. Myogenic cells cotransduced with RCAS(A)-Raf BXB and RCAS(B)-AFOS remained mononuclear and myosin-negative and did not activate significantly muscle-specific reporter genes. These results argue that Raf inhibits muscle differentiation independent of AP-1-mediated cell transformation. Our results provide evidence for AP-1 as a critical component of the transforming capacity of activated Raf and evidence that AP-1 is not involved in the myogenic inhibitory effects of the kinase.

Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli.

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.

Prediction of neurologic morbidity in extremely low birth weight infants.

OBJECTIVE: (1) Identify major determinants of adverse neurodevelopmental outcome in extremely low birth weight (ELBW) infants. (2) Compare neural networks and regression analysis in the prediction of major handicaps and Bayley scores (MDI and PDI) in individual ELBW neonates followed to 18 months. STUDY DESIGN: Retrospective cohort study of regional tertiary care NICU database. A database with 21 selected variables was divided into training (n = 144) and test sets (n = 74). The training set was used to train a neural network and develop regression equations to predict outcomes in the test set. RESULTS: Determinants (descending order of contribution to variance): Major handicap: intraventricular hemorrhage (IVH) grade, necrotizing enterocolitis > or = stage II, black race, and no chorioamnionitis; low MDI: IVH grade, plurality, bronchopulmonary dysplasia (BPD), lower maternal grade, and no chorioamnionitis; low PDI: IVH grade, BPD, periventricular leukomalacia, lower maternal grade, and no chorioamnionitis. Regression techniques and neural networks were comparable and had relatively low sensitivity and correlation with adverse outcomes. CONCLUSION: Much of the variance in ELBW neurologic outcome cannot be explained by either regression analysis or neural network approaches.

Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism.

Chronic overexpression of the oncogenic form of Ras is a potent inhibitor of skeletal myogenesis. However, the intracellular signaling pathways that mediate the repressive actions of Ras on myogenic differentiation have yet to be identified. We examined the role of Raf-mediated signaling as a modulator of avian myogenesis. Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes. Most notably, the embryonic chick myoblasts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or RCAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures. While residual myofibers were apparent in the RCAS-Raf BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic phenotype. The altered morphology is not a result of reinitiation of the myonuclei cell cycle nor is it due to apoptosis. Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity. Supplementation of the culture media with the MAPK kinase (MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls. Our results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to myogenesis.

Baculovirus expression, purification and evaluation of recombinant pneumococcal surface adhesin A of Streptococcus pneumoniae.

Pneumococcal surface adhesin A (PsaA), with a molecular mass of approximately 37 kD by SDS-PAGE, is a common surface protein expressed by all 90 serotypes of Streptococcus pneumoniae. S. pneumoniae serotype 6B genomic DNA was amplified to generate a DNA fragment carrying the full-length psaA sequence and was cloned into a baculovirus expression system. We expressed either cell-associated or cell-free nonfusion PsaA polypeptides using two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni 5B1-4 (High-Five). Recombinant PsaA (rPsaA) polypeptides were partially purified by partitioning in PBS/Triton X-114 buffers and by weakly basic ion exchange filter chromatography. Membrane-bound 'hydrophobic rPsaA' (hrPsaA) expressed by either Sf9 or High-Five cells had a molecular mass of approximately 38 kD by SDS-PAGE and partitioned in a Triton X-114 phase, it reacted with both rabbit polyclonal and five monoclonal anti-PsaA antibodies by dot blot or Western blot analysis. High-Five-cell-expressed 'soluble rPsaA' (srPsaA) with a molecular mass of approximately 37 kD by SDS-PAGE, was isolated from the serum-free culture medium and did not partition in the Triton X-114 phase; it reacted with anti-PsaA rabbit polyclonal and mouse monoclonal antibodies by ELISA and Western blot analysis. Both rPsaA polypeptide forms were immunogenic in Swiss-Webster adult female mice. In an infant mouse model of bacteremia, survival rates for mice given mouse anti-rPsaA immune serum (from mice immunized with High-Five-expressed srPsaA; 20 microl, 1:50,000 titer) 24 h before bacteremic challenge were greater than for the control group (48 h postchallenge, 20 vs. 90% survival rates) when challenged with S. pneumoniae serotype 6B. These results indicate that rPsaA is immunogenic and elicits protective antibody in mice similar to native protein.

Metallothionein-I/II knockout mice are sensitive to acetaminophen-induced hepatotoxicity.

The purpose of this study was to examine whether intracellular metallothionein (MT) protects against acetaminophen hepatotoxicity. MT-I/II knockout (MT-null) and control mice were given acetaminophen (150-500 mg/kg i.p.), and liver injury was assessed 24 h later. MT-null mice were more susceptible than controls to acetaminophen-induced lethality and hepatotoxicity, as evidenced by elevated serum enzyme activities and histopathology. Zinc pretreatment, a method of MT induction, protected against acetaminophen hepatotoxicity in control mice, but not in MT-null mice. The susceptibility of MT-null mice to acetaminophen hepatotoxicity was not due to the increased acetaminophen bioactivation, as cytochrome P-450 enzymes, and acetaminophen-reactive metabolites in bile and urine were not increased in MT-null mice. Western blots of liver cytosol indicated that acetaminophen covalent binding at 4 h increased with acetaminophen dose, but there was no consistent difference between control and MT-null mice. Acetaminophen injection depleted cellular glutathione similarly in both control and MT-null mice, but produced more lipid peroxidation in MT-null mice, as evidenced by the abundance of thiobarbiturate-reactive substances, and by immunohistochemical localization of 4-hydroxynonenal and malondialdehyde protein adducts. MT-null hepatocytes were more susceptible than control cells to oxidative stress and cytotoxicity produced by N-acetylbenzoquinoneimine, a reactive metabolite of acetaminophen, as determined by oxidation of 2', 7'-dichlorofluorescin diacetate and lactate dehydrogenase leakage. In summary, this study demonstrated that MT deficiency renders animals more vulnerable to acetaminophen-induced hepatotoxicity. The increased sensitivity does not appear to be due to increased acetaminophen activation, glutathione depletion, or covalent binding, but appears to be associated with the antioxidant role of MT.

Physaloptera infection in 18 dogs with intermittent vomiting.

Physaloptera infections were diagnosed endoscopically in 18 dogs. Each case had vomiting as the primary clinical sign, and four cases had regurgitation as a concurrent sign. Fecal flotations, using magnesium sulfate solution, were performed in 12 of the 18 cases and were negative for Physaloptera eggs. In 12 of the 18 cases, only one worm was seen during endoscopic examination. Fifteen of 18 cases were treated with pyrantel pamoate, and 10 of 12 cases with follow-up had resolution of their vomiting.

Hearing loss associated with neonatal ECMO: a clinical investigation.

To answer the question as to the prevalence of sensori-neural hearing loss (SNHL) among neonates receiving ECMO, a retrospective chart review was conducted on 198 infants having surgery between November 1987 and January 1995. One hundred fifty-seven (79.7%) survived. One hundred thirty infants met our criteria of having a pre-discharge auditory brainstem evoked response (ABR) test and at least one follow-up behavioral audiologic examination. Strict criteria were set for normal hearing on both the ABR and follow-up examinations. Only follow-up results are reported. At the time of the most recent follow-up examination, two children could not be adequately studied, 106 exhibited normal hearing, 21 (16%) exhibited either unilateral or bilateral conductive hearing loss and three (2.3%) exhibited unilateral or bilateral SNHL. Only one child is using amplification. With the largest sample size to date, we found a lower prevalence of SNHL after ECMO than has been previously noted in the literature. Although the prevalence of hearing loss is low, the post-ECMO group of infants must be considered at risk for hearing loss. The prevalence of hearing loss cannot be based solely on a pre-discharge ABR, i.e. ongoing follow-up testing is necessary.

Signaling through mitogen-activated protein kinase and Rac/Rho does not duplicate the effects of activated Ras on skeletal myogenesis.

The ability of basic helix-loop-helix muscle regulatory factors (MRFs), such as MyoD, to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, we tested two transformation-defective H-Ras 12V effector domain variants for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins revealed no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 revealed that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V.

Identification of tissue-specific DNase I hypersensitive sites in the rabbit flavin-containing monooxygenase form 2 gene.

Previous studies demonstrated that the flavin-containing monooxygenases (FMO) are expressed in a tissue-specific manner. To begin an elucidation of mechanisms regulating this expression pattern, genomic clones for rabbit FMO2 were isolated and characterized. Two clones were isolated from a lambda EMBL3 genomic library and shown to span approximately 30 kilobase pairs but to contain only about 800 base pairs of coding information. Primer extension analysis was used to map the transcription start site, extending the previously published cDNA sequence by 17 base pairs. The FMO2 promoter does not utilize a classical TATA box, nor are HTF islands present (DNA domains rich in cleavable sites for 5-methyl-cytidine/guanosine-sensitive restriction enzymes). Rather, homology with promoters controlled by initiator elements is observed. Previous studies demonstrated FMO2 expression in rabbit pulmonary Clara and type II cells [Overby, Nishio, Lawton, Plopper, and Philpot: Exp. Lung Res. 18, 131, (1992)]. In the present study, a highly enriched Clara/type II cell population was prepared, and the FMO2 gene was analyzed for DNase I-hypersensitive and methylated regions. These data were then contrasted with those obtained from a similar analysis of the nonexpressed, hepatocyte FMO2 gene. No difference in the methylation status was observed. However, Clara/type II cell-specific DNase I-hypersensitive sites are located within the promoter region of the FMO2 gene. Thus, tissue-specific transcription factors likely are more prominent than methylation in regulating FMO2 expression. Consistent with this observation, both polyomavirus enhancer activator 3 (E26 transformation specific) and thyroid transcription factor 1 consensus sequences are present within the tissue-specific DNase I-hypersensitive domain.