Evaluation of capillary and myofiber density in the pectoralis major muscles of rapidly growing, high-yield broiler chickens during increased heat stress.

Skeletal muscle development proceeds from early embryogenesis through marketing age in broiler chickens. While myofiber formation is essentially complete at hatching, myofiber hypertrophy can increase after hatch by assimilation of satellite cell nuclei into myofibers. As the diameter of the myofibers increases, capillary density peripheral to the myofiber is marginalized, limiting oxygen supply and subsequent diffusion into the myofiber, inducing microischemia. The superficial and deep pectoralis muscles constitute 25% of the total body weight in a market-age bird; thus compromise of those muscle groups can have profound economic impact on broiler production. We hypothesized that marginal capillary support relative to the hypertrophic myofibers increases the incidence of microischemia, especially in contemporary high-yield broilers under stressing conditions such as high environmental temperatures. We evaluated the following parameters in four different broiler strains at 39 and 53 days of age when reared under thermoneutral (20 to 25 C) versus hot (30 to 35 C) environmental conditions: capillary density, myofiber density and diameter, and degree of myodegeneration. Our data demonstrate that myofiber diameter significantly increased with age (P > or = 0.0001), while the absolute numbers of capillaries, blood vessels, and myofibers visible in five 400 x microscopic fields decreased (P > or = 0.0001). This is concomitant with marginalization of vascular support in rapidly growing myofibers. The myofiber diameter was significantly lower with hot environmental temperatures (P > or = 0.001); therefore, the absolute number of myofibers visible in five 400X microscopic fields was significantly higher. The incidence and subjective degree of myodegeneration characterized by loss of cross-striations, myocyte hyperrefractility, sarcoplasmic vacuolation, and nuclear pyknosis or loss also increased in hot conditions. Differences among strains were not observed.

Excess cortisol interferes with a principal mechanism of resistance to dehydration in Bos indicus steers.

This study investigated the effects of excess cortisol on physiological mechanisms that resist dehydration in Bos indicus steers (n = 31, 2 yr of age, 193 +/- 21.47 kg mean BW) during a 90-h period. Steers were assigned randomly to one of four groups: 1) no water/no cortisol (n = 8), 2) water/no cortisol (n = 8), 3) no water/cortisol (n = 8), and 4) water/cortisol (n = 7). Animals allocated to cortisol treatment groups were given 0.1 mg x kg BW(-1) x h(-1) of hydrocortisone suspended in isotonic saline for the duration of the study. Total body water, osmolality, hematocrit, urine output, feed and water intake, and plasma concentrations of arginine vasopressin (AVP), angiotensin II (AII), electrolytes, total protein, and albumin were determined at 24-h intervals for 90 h. In the presence of excess plasma cortisol, total body water was maintained in the presence of a water deprivation insult for 90 h, whereas hydration indices, such as total plasma protein and albumin, did not change, supporting the body water data. However, plasma osmolality increased for the water-deprived groups from 24 h (P = 0.008). Hematocrit did not reflect dehydration in any group. Water deprivation induced an increase in endogenous plasma cortisol concentrations after 60 h of the study (P = 0.028). Plasma concentrations of AVP increased with water deprivation (P = 0.006). Excess cortisol decreased the plasma concentration of AVP at 72 h only (P = 0.027) and suppressed plasma concentrations of AII at 24 and 72 h (P < 0.001 and P = 0.036, respectively). Animals treated with excess cortisol maintained urinary output for 48 h before decreasing at 72 h (P = 0.057), although there was no effect on water or feed intake. Water deprivation increased plasma sodium concentrations (P < 0.05) until 72 h, whereas potassium decreased under the influence of excess plasma cortisol (P = 0.001) at 24 h. Water deprivation increased plasma chloride concentration at 72 and 90 h (P = 0.051 and P = 0.026, respectively). Plasma phosphorus decreased at 24 h (P = 0.001) and remained at lesser concentrations for the duration of the study (P = 0.05). These results highlight the complexity of endocrine interactions associated with water balance in Bos indicus steers. We accept our hypothesis that, in the presence of excess cortisol, the renin-angiotensin-aldosterone axis is suppressed; however, homeostasis is achieved through other physiological systems.

Quantitative analysis of acid-base balance in Bos indicus steers subjected to transportation of long duration.

There is a lack of information pertaining to the effects of transport stress on the acid-base physiology of ruminants. The effect of transportation and/or feed and water deprivation on acid-base balance was studied using 19 2-yr-old Bos indicus steers. The steers were allocated to one of three groups: 1) control, offered ad libitum access to feed and water (n = 8); 2) water and feed deprived, offered no feed or water for 60 h (n = 6); and 3) transported, offered no feed or water for 12 h, and then transported for 48 h (n = 5). Blood gases, electrolytes, lactate, total protein, albumin, anion gap, strong ion difference, and total weak acids were determined at the conclusion of transportation. Arterial blood pH did not differ among the experimental groups. Partial pressure of carbon dioxide (pCO2) was lower for the water and feed deprived (P = 0.023) group than for the control group. Plasma total protein, albumin and total weak acid concentrations were higher for the transported (P = 0.001, P = 0.03, P = 0.01) and water- and feed-deprived (P = 0.000, P = 0.003, P = 0.001) groups, respectively, compared with the control group. Transported animals had a lower plasma concentration of potassium (P = 0.026) compared with the control animals. This study demonstrates that although blood pH remains within normal values in transported and fasted steers, the primary challenge to a transported or feed- and water-deprived animal is a mild metabolic acidosis induced by elevated plasma proteins, which may be the result of a loss of body water. The loss of electrolytes had little effect on the acid-base balance of the animals.

Dehydration in stressed ruminants may be the result of a cortisol-induced diuresis.

The effect on water and electrolyte balance of stress, simulated by intravenous infusion of cortisol, was studied using 24 18-mo-old Merino wethers (37.0 +/- 0.94 kg mean body weight [BW]) over 72 h. The sheep were allocated to one of four groups: 1) no water/no cortisol (n = 6); 2) water/no cortisol (n = 4); 3) no water/cortisol (n = 6); and 4) water/cortisol (n = 4). Animals allocated to the two cortisol groups were given 0.1 mg x kg BW(-1) x h(-1) of hydrocortisone suspended in isotonic saline to simulate stress for the duration of the experiment. Total body water, plasma cortisol, osmolality and electrolytes, and urine electrolytes were determined at 24-h intervals for 72 h. In the presence of cortisol, total body water was maintained in the face of a water deprivation insult for 72 h. Water deprivation alone did not induce elevated plasma concentrations of cortisol, in spite of a 13% loss of total body water between 48 and 72 h. Infusion of cortisol was found to increase urine output (P = 0.003) and decrease total urinary sodium output (P = 0.032), but had no effect on plasma electrolyte levels or water intake. Water deprivation was found to increase plasma sodium concentrations (P = 0.037). These results indicate that sheep given cortisol to simulate stress suffer from a loss of body water in excess of that associated with a loss of electrolytes, and support the hypothesis that elevated physiological concentrations of cortisol induce a diuresis in ruminants that contributes to dehydration.

Circular versus longitudinal myometrial contractile response to selective tachykinin receptor agonists in rat.

This study compared the nature and magnitude of the contractile response produced in vitro by selective NK1, NK2 and NK3 tachykinin receptor agonists in circularly and longitudinally oriented strips of myometrium from ovariectomised and ovariectomised oestrogen-treated rats. The nature of the responses produced upon stimulation of the tachykinin receptors varied between the different myometrial preparations and the hormonal environment from which the tissue was taken. Variations included: (i) sustained contraction until washout of agonist; (ii) biphasic contraction until washout of agonist; and (iii) monophasic contraction. The major differences in magnitude of contractions were seen in preparations from oestrogen-treated animals in which responses to stimulation of all tachykinin receptors were reduced in comparison to preparations from non-oestrogen treated animals. Furthermore, the responses in circularly oriented myometrium preparations from oestrogen-treated animals were all markedly reduced compared to responses in longitudinally oriented myometrium preparations. These results suggest that the tachykinin receptors in longitudinally and circularly oriented myometrial layers are differentially regulated, especially in tissue isolated from an oestrogen-dominated environment.

Increased expression of neuronal glucose transporter 3 but not glial glucose transporter 1 following severe diffuse traumatic brain injury in rats.

Traumatic brain injury results in an increased brain energy demand that is associated with profound changes in brain glycolysis and energy metabolism. Increased glycolysis must be met by increasing glucose supply that, in brain, is primarily mediated by two members of the facilitative glucose transporter family, Glut1 and Glut3. Glut1 is expressed in endothelial cells of the blood-brain barrier (BBB) and also in glia, while Glut3 is the primary glucose transporter expressed in neurons. However, few studies have investigated the changes in glucose transporter expression following traumatic brain injury, and in particular, the neuronal and glial glucose transporter responses to injury. This study has therefore focussed on investigating the expression of the glial specific 45-kDa isoform of Glut1 and neuronal specific Glut3 following severe diffuse traumatic brain injury in rats. Following impact-acceleration injury, Glut3 expression was found to increase by at least 300% as early as 4 h after induction of injury and remained elevated for at least 48 h postinjury. The increase in Glut3 expression was clearly evident in both the cerebral cortex and cerebellum. In contrast, expression of the glial specific 45-kDa isoform of Glut1 did not significantly change in either the cerebral cortex or cerebellum following traumatic injury. We conclude that increased glucose uptake after traumatic brain injury is primarily accounted for by increased neuronal Glut 3 glucose transporter expression and that this increased expression after trauma is part of a neuronal stress response that may be involved in increasing neuronal glycolysis and associated energy metabolism to fuel repair processes.

Krüppel-like factor 4 (gut-enriched Krüppel-like factor) inhibits cell proliferation by blocking G1/S progression of the cell cycle.

Krüppel-like factor 4 (KLF4) is an epithelial cell-enriched, zinc finger-containing transcription factor, the expression of which is associated with growth arrest. Previous studies show that constitutive expression of KLF4 inhibits DNA synthesis but the manner by which KLF4 exerts this effect is unclear. In the present study, we developed a system in which expression of KLF4 is controlled by a promoter that is induced upon treatment of cells containing the receptors for the insect hormone, ecdysone, with ponasterone A, an ecdysone analogue. The rate of proliferation of a stably transfected colon cancer cell line, RKO, was significantly decreased following addition of ponasterone A when compared with untreated cells. Flow cytometric analyses indicated that the inducible expression of KLF4 caused a block in the G(1)/S phase of the cell cycle. A similar block was observed when ecdysone receptor-containing RKO cells were infected with a replication-defective recombinant adenovirus containing an inducible KLF4 and treated with ponasterone A. Results of these studies provide evidence that the inhibitory effect of KLF4 on cell proliferation is mainly exerted at the G(1)/S boundary of the cell cycle.

Hormonal variation of rat uterine contractile responsiveness to selective neurokinin receptor agonists.

Regulated uterine contractions are important in many reproductive functions such as sperm transport and embryo positioning during implantation. The role of classical neurotransmitters including acetylcholine and norepinephrine in regulating myometrial contractility has been well studied; however, the peripheral role of sensory neurotransmitters such as the neurokinins is less clear. The major neurokinins are substance P, neurokinin A, and neurokinin B, which predominantly activate neurokinin receptors (NK-Rs) 1, 2, and 3, respectively. This study utilized selective receptor agonists to examine the role of NK-Rs in uterine contractility. Uterine tissues, obtained from the major stages of the rat estrous cycle, were stimulated with selective NK-R agonists. Addition of each agonist resulted in a significant contractile response. However, the magnitude and nature of the response were dependent upon the stage of the estrous cycle, with responses to all agonists being significantly decreased in tissue from proestrus and estrus. Furthermore, the nature of NK3-R-mediated contraction was different in tissue from proestrus and estrus compared to metestrus and diestrus. The hormonal dependence of NK-R-mediated contractility was then examined in the ovariectomized estrogen-supplemented rat model. These studies confirmed that the magnitude and nature of uterine contractility in response to NK-R activation depend upon the hormonal environment.

Detection of viral ribonucleic acid and histologic analysis of inflamed synovium in Ross River virus infection.

OBJECTIVE: To document the histology of Ross River virus (RRV) arthritis and to examine inflamed synovium for viral RNA. METHODS: Biopsy tissue from the inflamed knees of 12 patients with RRV infection was studied using conventional and immunostaining techniques. Reverse transcriptase-polymerase chain reaction technology was used to probe for the presence of viral RNA in the synovial biopsy samples and in serum. RESULTS: Hyperplasia of the synovial lining layer, vascular proliferation, and mononuclear cell infiltration were the main histologic changes. RRV RNA was found in knee biopsy tissue that was obtained from 2 patients at 5 weeks after the onset of symptoms. CONCLUSION: RRV RNA was identified in inflamed synovium more than a month after symptoms began. Inflammation was apparent in the absence of detectable virus in the majority of patients.

The Zymark BenchMate. A compact, fully-automated solution-phase reaction work-up facility for multiple parallel synthesis.

The rapid growth of multiple parallel synthesis in our laboratories has created a demand for a robust, easily accessed automated system for solution-phase reaction work-up, since the manual work-up of large numbers of small-scale reactions is both time-consuming and tedious, and is a rate limiting step in the generation of large numbers of compounds for test. Work-up in chemical organic synthesis consists of a series of post-reaction operations designed using differential chemical properties to remove excess reagent or starting material, reagent products and, where possible reaction by-products. Careful consideration of post-reaction operations as a clean-up step can obviate the requirement for purification. Generally, work-up can be resolved into four operations: filtration, solvent addition (dilution, trituration), washing and separation (partition) and it is the selection and ordering of these four basic operations that constitutes a chemical work-up. Following the proven success of centralized Zymate robotic systems in the compilation, execution and work-up of complex reaction sequences, a centralized chemical work-up service has been in operation for over 12 months. It now seemed prudent that the needs of multiple parallel synthesis would be better served by the development of a compact, automated system, capable of operating in a standard chemistry laboratory fume-hood. A Zymark BenchMate platform has been configured to perform the four basic operations of chemical solution work-up. A custom-built filtration station, incorporating an integrated tipping facility for the sample tube has also been developed. Compilation of each work-up is through a set of Visual Basic procedure screens, each dedicated to a particular work-up scenario. Methods are compiled at the chemist's own PC and transferred to the BenchMate via a diskette.

Differentiation-dependent expression of gelatinase B/matrix metalloproteinase-9 in trophoblast cells.

The purpose of this study was to evaluate the Rcho-1 trophoblast culture system as a model for studying trophoblast invasion and to examine stage-specific expression of enzyme(s) potentially participating in rat trophoblast giant cell invasive behavior. The invasive behavior of the differentiating Rcho-1 trophoblast cells was demonstrated using Matrigel invasion chambers. Gelatin zymography and Western blot analysis of conditioned medium from differentiating Rcho-1 trophoblast cell cultures and rat ectoplacental cone outgrowths revealed a differentiation-dependent increase in gelatinase B/matrix metalloproteinase (MMP-9). Nothern blot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses of Rcho-1 trophoblast or ectoplacental cone cells also showed increasing expression of MMP-9 accompanying cell differentiation. Rcho-1 trophoblast cells stably transfected with MMP-9 promoter/luciferase reporter constructs exhibited a differentiation-dependent increase in MMP-9 promoter activation. In conclusion, trophoblast giant cell differentiation is characterized by transcriptional activation of the MMP-9 gene and appearance of the invasive phenotype.

Signaling pathways controlling trophoblast cell differentiation: Src family protein tyrosine kinases in the rat.

Trophoblast giant cell differentiation is characterized by endoreduplication and expression of members of the prolactin (PRL) gene family and can be simulated in vitro via manipulations of the Rcho-1 trophoblast cell line. The regulation of trophoblast cell proliferation and differentiation involves tyrosine protein kinase signaling pathways. Treatment of Rcho-1 trophoblast cells with tyrosine kinase inhibitors disrupted differentiation-dependent expression of members of the PRL gene family and cytoskeletal organization. Activated p60c-src, p62c-yes, and p53/56lyn were present in the Rcho-1 rat trophoblast cell line and in differentiated trophoblast cells isolated from the developing rat placenta. p60c-src and p62c-yes were active in proliferating and differentiating trophoblast cells. During proliferation, p62c-yes exhibited distinct associations with other phosphoproteins (34, 66, 76, and 150 kDa). p53/56lyn was activated only in differentiating trophoblast cells. p53/56lyn showed a differentiation-dependent accumulation in cytoskeletal and membrane fractions, whereas p60c-src levels were virtually invariant in both fractions. Expression patterns of csk, a negative regulator of Src family kinase activities, were not consistent with its involvement in the differentiation-dependent activation of p53/56lyn; however, there was some indication of the participation of a tyrosine phosphatase in the regulation of p53/56lyn. In conclusion, p60c-src, p62c-yes, and p53/56lyn patterns of activation in trophoblast cells are consistent with their involvement in the control of trophoblast cell proliferation and differentiation.

Regulation of deoxyribonucleic acid synthesis in proliferating and differentiating trophoblast cells: involvement of transferrin, transforming growth factor-beta, and tyrosine kinases.

This report investigates the regulation of DNA synthesis in trophoblast stem cells and differentiating trophoblast cells. Experiments in this study were performed on the Rcho-1 trophoblast cell line, which was established from a transplantable rat choriocarcinoma. Rcho-1 trophoblast cells can be manipulated to proliferate or differentiate along the trophoblast giant cell pathway. DNA synthesis in quiescent trophoblast stem cells (maintained in serum-free medium) was stimulated by fetal bovine serum and donor horse serum or transferrin to a level approximately 30- and 10-fold above the basal level, respectively. Transferrin and horse serum were ineffective at maintaining trophoblast cell proliferation. In contrast, serum-starved differentiating trophoblast cells synthesize DNA at maximal levels and could not be further stimulated by the addition of exogenous factors. Fetal bovine serum-stimulated proliferation was effectively inhibited by transforming growth factor-beta 1. Experiments with the tyrosine kinase inhibitor genistein implicate tyrosine kinase involvement in the regulation of DNA synthesis and proliferation in trophoblast stem cells and DNA synthesis in differentiating trophoblast cells. Proliferating and differentiating trophoblast cells differ in their levels of tyrosine kinase activities and express unique tyrosine-phosphorylated proteins. In summary, DNA synthesis and proliferation in trophoblast stem cells are under extrinsic control, whereas DNA synthesis in differentiating trophoblast cells is under intrinsic control. Both mechanisms require tyrosine kinase activity, but the nature of the tyrosine kinase pathways in each process appears to be distinct.

Differential responses of phenotypically distinct rat trophoblast cell lines to MHC class I antigen-inducing cytokines.

Phenotypically distinct rat trophoblast cell lines, the Rcho-1 and R8RP.3 cells, were compared for their responses to cytokines known to induce major histocompatibility (MHC) class I antigens, tumour necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma). Cell enzyme immunosorbent assays and flow cytometry experiments showed that only IFN-gamma could induce RT1 class I antigens on the Rcho-1 cells. Non-adherent cells were slightly less responsive than adherent, giant cell-like Rcho-1 cells. By contrast, RT1 class I antigens on the R8RP.3 cells were induced by both TGF-beta 1 and IFN-gamma. The cytokines also had different effects on mitochondrial enzyme activity in the two lines. TNF and TGF-beta 1 mRNAs were demonstrated in both lines by using Northern blot hybridization. Rcho-1 but not R8RP.3 cells contained two TNF messages (approximately 2.2, 1.9 kb). Steady state levels of transcripts from the TNF gene, and, to a lesser extent, the TGF-beta 1 gene, were increased in cultures of Rcho-1 cells that contained high proportions of giant cells. Thus, phenotypically distinct rat trophoblast cell lines do not respond identically to TNF, TGF-beta 1 or IFN-gamma, transcription of cytokine genes does not prevent the cells from responding to paracrine cytokine signals, and the cells contain novel TNF transcripts that might be important in cell maturation or differentiation.

Hypoxaemia during paediatric dental anaesthesia in the community.

The object of this paper is to measure the incidence of hypoxaemia during paediatric dental anaesthesia in the community and thus support or refute the recommendations for monitoring in the Poswillo report. Fifty unpremedicated children, ASA grade 1, between the ages of 2 and 16 years undergoing general anaesthesia for teeth extraction were studied. Patients were anaesthetised with a standard anaesthetic which consisted of 30% oxygen in 70% nitrous oxide and supplemented with halothane. A pulse oximeter was used to measure the oxygen saturation continuously. A 12% incidence of hypoxaemia was recorded and a 32% incidence of a 5% fall in the oxygen saturation from the baseline. Three children became hypoxaemic during the operative period and three during the recovery period. Although the incidence of hypoxaemia in this study was significantly lower than in previous studies, the incidence suggests that careful monitoring is essential and the recommendations that have been put forward in the Poswillo report are fully justified.

Recapitulation of the pathway for trophoblast giant cell differentiation in vitro: stage-specific expression of members of the prolactin gene family.

The trophoblast giant cell lineage is characterized by endoreduplication and expression of members of the PRL gene family. This report describes the functional consequences following in vitro manipulation of a rat trophoblast cell line, termed Rcho-1. Rcho-1 cells can be cultured under conditions that promote proliferation or differentiation. Proliferation is maintained by culturing the cells in the presence of fetal bovine serum under subconfluent conditions. Differentiation is induced by growing the cells to confluence and removing the mitogenic source. Differentiation is characterized by continued synthesis of DNA in the absence of proliferation (endoreduplication) and the sequential expression of members of the PRL gene family. Western and Northern blot analyses demonstrated that placental lactogen-I (PL-I) was first expressed, followed sequentially by PL-II, PRL-like protein-A, and PRL-like protein-C. The ontogeny of expression of members of the PRL gene family by the Rcho-1 cells recapitulated the pattern of in situ expression by trophoblast giant cells of the junctional zone of the chorioallantoic placenta. A notable difference between in vivo trophoblast giant cell differentiation and in vitro Rcho-1 cell differentiation is the termination of PL-I expression in normal trophoblast giant cells developing in vivo and the continued expression of PL-I in differentiated Rcho-1 cell cultures. The Rcho-1 cell line provides a unique in vitro model for investigating the initiation and maintenance of the trophoblast giant cell differentiation pathway.

Recurrent sinus arrest in association with neuroleptic malignant syndrome.

We report a case of neuroleptic malignant syndrome where the catatonia clearly followed the administration of neuroleptics and where the neurovegetative disturbance was remarkably severe, including episodes of tracheal spasm, apnoea and episodes of bradycardia, and sinus arrest requiring insertion of a temporary external pacing wire. To our knowledge, such severe disturbance has not previously been reported.

Heterologous expression and characterization of prolactin-like protein-A. Identification of serum binding proteins.

In this report, we describe the heterologous expression of prolactin-like protein-A (PLP-A) in Chinese hamster ovary (CHO) cells, the characterization of recombinant PLP-A, and the identification of serum PLP-A-binding proteins. CHO cell and native placental PLP-A showed similar immunoreactive characteristics and electrophoretic mobilities. N-terminal sequencing verified the identity and purity of the recombinant PLP-A species and the site of cleavage of the signal peptide from the mature secreted PLP-A species. Recombinant PLP-A lacked activity in standardized prolactin and growth hormone in vitro bioassays. Antibodies generated to recombinant PLP-A facilitated the cellular localization of PLP-A and the identification of high molecular weight PLP-A complexes. Cross-linking analyses of radioiodinated PLP-A with serum harvested from late gestation rats indicated the presence of two major cross-linked complexes migrating under reducing conditions at 130 and 250 kDa and two minor cross-linked complexes migrating at 70 and 110 kDa. Binding of PLP-A to serum proteins was specific for PLP-A and not effectively competed by other members of the prolactin/growth hormone family. The PLP-A binding species were also found in serum from non-pregnant female and male rats.

Respiratory deficiency in experimental idiopathic scoliosis.

Clinically significant respiratory failure in a group of animals with an experimentally produced structural thoracic scoliosis was identified. A detailed physiologic investigation of their respiratory function showed that their respiratory compromise was associated with the development of a smaller and stiffer chest. Frank respiratory failure, however, was rare and seen only in animals with the most severe spinal deformities when it was also associated with a general failure to thrive. Animals with less severe deformities had little measurable restriction of pulmonary function, did not develop respiratory failure, and grew normally.