Effects of reduced frequency of milk removal on gene expression in the bovine mammary gland.

Regulation of milk synthesis and secretion is controlled mostly through local (intramammary) mechanisms. To gain insight into the molecular pathways comprising this response, an analysis of mammary gene expression was conducted in 12 lactating cows shifted from twice daily to once daily milking. Tissues were sampled by biopsy from adjacent mammary quarters of these animals during the two milking frequencies, allowing changes in gene expression to be assessed within each animal. Using bovine-specific, oligonucleotide arrays representing 21,495 unique transcripts, a range of differentially expressed genes were found as a result of less frequent milk removal, constituting transcripts and pathways related to apoptotic signaling (NF-kappaB, JUN, ATF3, IGFBP5, TNFSF12A) mechanical stress and epithelial tight junction synthesis (CYR61, CTGF, THBS1, CLDN4, CLDN8), and downregulated milk synthesis (LALBA, B4GALT1, UGP2, CSN2, GPAM, LPL). Quantitative real-time PCR was used to assess the expression of 13 genes in the study, and all 13 of these were correlated (P < 0.05) with values derived from array analysis. It can be concluded that the physiological changes that occur in the bovine mammary gland as a result of reduced milk removal frequency likely comprise the earliest stages of the involution response and that mechano-signal transduction cascades associated with udder distension may play a role in triggering these events.

Mutation in bovine beta-carotene oxygenase 2 affects milk color.

beta-Carotene biochemistry is a fundamental process in mammalian biology. Aberrations either through malnutrition or potentially through genetic variation may lead to vitamin A deficiency, which is a substantial public health burden. In addition, understanding the genetic regulation of this process may enable bovine improvement. While many bovine QTL have been reported, few of the causative genes and mutations have been identified. We discovered a QTL for milk beta-carotene and subsequently identified a premature stop codon in bovine beta-carotene oxygenase 2 (BCO2), which also affects serum beta-carotene content. The BCO2 enzyme is thereby identified as a key regulator of beta-carotene metabolism.

Improved hospital mortality with a low MET dose: the importance of a modified early warning score and communication tool.

Rapid response systems have been mandated for the recognition and management of the deteriorating patient. Increasing medical emergency team (MET) dose may be associated with improved outcomes. Large numbers of MET calls may divert resources from the program providing the service unless additional personnel are provided. To describe the implementation and outcomes of a multifaceted rapid response system (RRS) in a teaching hospital, we conducted an observational study. The RRS consisted of the introduction of a MET together with 1) redesign of the ward observation chart with the vital sign variables colour-coded to identify variation from normal; 2) mandated minimum frequency of vital sign measurement; 3) three formal levels of escalation based on the degree of physiological instability as measured by a modified early warning score (MEWS); 4) COMPASS© education and e-learning package with a two-hour face-to-face small group tutorial; 5) practise in escalation and communication using the ISBAR (Identify, Situation, Background, Assessment, Response/Recommendation) communication tool. The primary outcome measures were all-cause hospital mortality rate and hospital standardised mortality ratio (HSMR) compared to peer hospitals calculated by the Health Round Table. There were 161,153 separations and 1,994 hospital deaths from July 2008 to December 2012. The MET call rate was 11.3 per 1000 separations in 2012. There was a decline in all-cause hospital mortality from 13.8 to 11 deaths/1000 separations. The HSMR decreased from 95.7 in 2008 to 66 in the second half of 2012 (below the three standard deviation control limit). A low MET dose may be associated with improved hospital mortality when combined with a MEWS and an intervention to improve communication.

Depression in the medical setting: biopsychological interactions and treatment considerations.

This article examines depression in 6 medical conditions: coronary artery disease (CAD), cancer, human immunodeficiency virus (HIV) infection, Parkinson's disease, pain, and the sex hormone changes of aging. Research is beginning to define specific biological and psychological mechanisms underlying the adverse interactions between depression and these medical conditions. Antidepressant medications, psychosocial therapies, and hormonal manipulations are effective in reducing depressive symptoms. Specific psychosocial interventions may increase longevity in CAD and cancer and may enhance quality of life in HIV infection. Newer antidepressants appear to be safer and better tolerated than older agents for medically ill patients, but do not appear to be as effective for neuropathic pain. Dopamine agonists may benefit depression associated with Parkinson's disease. Hormone replacement therapy may improve subsyndromal depressive symptoms in postmenopausal women and may enhance antidepressant response for older women with major depression.

Reversible ventilation and perfusion abnormalities in unilateral obstructed lung.

An intraluminal carcinoid tumor obstructing the left mainstem bronchus produced hypoxemia through alteration in ventilation/perfusion matching. Studies of regional lung function using 133-xenon (133Xe) and a multiprobe computerized instrumentation system documented a reduction of perfusion to 22 percent and ventilation to 6 percent of the total. There was negligible washout of intravenously injected 133Xe from the left lung consistent with air trapping. Four days after left mainstem bronchial sleeve resection, perfusion, ventilation and washout of injected xenon had significantly improved and by four months postresection, all measurements were virtually normal, although complete restoration of perfusion in relation to ventilation was delayed. Regional lung function studied with a multiprobe system in this patient provided a clinical model for the study of ventilation and perfusion inter-relationships in large airway obstruction and demonstrated that a prolonged time may be required for return of perfusion to normal.

Hyaluronan in radiation-induced lung disease in the rat.

We have used a previously described model of bilateral radiation-induced lung disease in the rat (Ward et al., Radiat. Res., 136, 15-21, 1993) to study the role of hyaluronan in this process. Hyaluronan was measured in the bronchoalveolar lavage fluid, serum and lung tissue of rats after gamma irradiation or sham irradiation. Four weeks after irradiation, during peak alveolitis (12-fold increase in protein in the lavage, 7-fold increase in lavaged cells) hyaluronan was elevated 5.5-fold in serum and 1.5-fold in the bronchoalveolar lavage fluid. Histochemical staining demonstrated hyaluronan was in the intra-alveolar edema fluid but was not increased in the alveolar walls; hyaluronan, measured by high-performance liquid chromatography, also was not elevated in lavaged lung tissue. Hyaluronan was not increased in bron-choalveolar lavage fluid, serum or lung tissue during pulmonary edema (2 weeks) or fibrosis (6 to 20 weeks). The administration of methylprednisolone significantly decreased the alveolitis, including the increase in hyaluronan in the alveolar space and serum, but did not suppress fibrosis. It appears that hyaluronan is a marker of inflammation and cannot be used as a serum marker to predict the onset of radiation pneumonitis. Furthermore, an increase in interstitial hyaluronan does not appear to be a necessary precursor in the evolution of radiation fibrosis.

The pulmonary response to sublethal thoracic irradiation in the rat.

An animal model of radiation-induced lung disease was established using male Wistar rats given sublethal bilateral thoracic irradiation (15 Gy). The rats were studied for up to 20 weeks and compared to sham-irradiated controls. Three distinct syndromes were identified. Two weeks after irradiation there was an increase in wet lung weight without an increase in dry lung weight. Interstitial edema was confirmed ultrastructurally, but aside from minor abnormalities of endothelial cells, both capillary and alveolar basement membranes were intact and there was no alveolar protein leak. At 4 weeks after irradiation, there was an abrupt increase in both wet and dry lung weights, as well as intra-alveolar macrophages, lymphocytes, polymorphs, and protein. These changes persisted for periods of up to 8 weeks. Electron microscopy at 4 weeks revealed prominent interstitial edema and severe endothelial cell damage. There was patchy thickening of the cytoplasm of type I cells as well as some cells which appeared to be transforming from type II to type I cells, suggesting previous epithelial denudation. Mast cell density increased in perivascular and peribronchial areas from 4 weeks, and this and parenchymal mast cell density peaked at 7 weeks. The total collagen content of the lungs (determined biochemically) rose by up to 50% above control values from 5 weeks after irradiation, the bulk of the increase having occurred by 12 weeks. Further increases up to 20 weeks were similar to that seen in growing control animals. Collagen deposition (as defined by electron microscopy and Picrosirius polarization) was prominent in peribronchial and perivascular areas in all animals, but in alveolar walls it was increased severalfold above controls by 20 weeks after irradiation. In summary, this model provides sequential changes of interstitial edema, alveolitis, and interstitial fibrosis which can be studied independently. The temporal relationship between the appearance of mast cells and increased collagen deposition supports the hypothesis that mast cells are intimately related to the development of fibrosis.

The effect of steroids on radiation-induced lung disease in the rat.

We have used a model of bilateral radiation-induced lung disease in the rat to study the effects of corticosteroids. This model is characterized by interstitial edema at 2 weeks after radiotherapy followed by florid alveolitis with an alveolar protein leak which peaks at 4 weeks. Mast cell density peaks at 7 weeks, and there is a progressive increase in lung collagen (fibrosis) from 5 to 20 weeks. Intraperitoneal corticosteroids or saline were given at the time of irradiation or sham irradiation (protocol 1), every second day during weeks 3 and 4 (protocol 2), or three times weekly during weeks 3 to 8 (protocol 3). In protocol 1, steroids protected the lung from interstitial edema at 2 weeks, delayed the alveolitis without reducing its intensity, and significantly reduced the alveolar protein leak. However, radiation fibrosis was not reduced at 20 weeks. Longer steroid administration (protocol 2) suppressed the alveolar protein leak and delayed and significantly reduced the severity of the inflammatory cell response. Although the tissue mast cell and fibrotic responses were suppressed during and for at least 3 weeks after steroids, the ultimate fibrotic reaction was the same in both irradiated groups. In protocol 3, steroids suppressed the alveolitis and delayed the rise in tissue mast cell density, but did not affect the fibrotic response at 20 weeks. These studies suggest that steroids can suppress the alveolitis provided they are used throughout the period of alveolitis. Although they also delay the tissue mast cell response to radiation, the ultimate fibrosis is not altered. This provides further evidence for the dissociation of alveolitis and fibrosis after lung irradiation and has potential implications for management of radiation-induced lung disease in humans.

Ventilation by external high-frequency oscillation in cats.

Eight anesthetized tracheostomized cats were placed in an 8.2-liter airtight chamber with the trachea connected to the exterior. Thirty-two combinations of high-frequency oscillations (HFO) (0.5-30 Hz; 25-100 ml) were delivered for 10 min each in random order into the chamber. Arterial blood gas tensions during oscillation were compared with control measurements made after 10 min of spontaneous breathing without oscillation when the mean arterial PCO2 (PaCO2) was 30.1 Torr. Ventilation due to spontaneous breathing (Vs) and oscillation (Vo) were derived from the chamber pressure trace and a pneumotachograph, respectively. As the oscillation frequency increased, oscillated tidal volume (Vo) decreased from a mean of 39 (0.5 Hz) to 3.3 ml (30 Hz) when 100 ml was delivered to the chamber. From 6-25 Hz, apnea occurred with Vo less than estimated respiratory dead space (VD); the minimum effective Vo/VD ratio was 0.37 +/- 0.05. Although Vo was maximal at 10 Hz at each oscillation volume, the lowest PaCO2 occurred at 2-6 Hz, and arterial PO2 rose as expected during hypocapnia. Above 10 Hz, PaCO2 was determined by Vo and was independent of frequency, whereas at lower frequencies, PaCO2 was related to Vo; below 6 Hz, PaCO2 varied inversely with the calculated alveolar ventilation. As oscillations became more effective, both PaCO2 and Vs fell progressively and were highly correlated; apnea occurred when PaCO2 was reduced by a mean of 4.5 Torr. Mean chamber pressure remained near zero up to 15 Hz, indicating functional residual capacity did not change. We conclude that externally applied HFO can readily maintain gas exchange in vivo, with Vo less than VD at frequencies over 2 Hz.

Prenatal stress increases corticotropin-releasing factor (CRF) content and release in rat amygdala minces.

Corticotropin-releasing factor (CRF) is a neuropeptide found throughout the central nervous system that has a proposed role in modulating emotional and behavioral states, including stress and anxiety. The amygdala, which is important in the control of emotional and autonomic responses to stress, contains CRF nerve terminals, CRF cell bodies, and CRF receptors. In rats, exposure to prenatal stress results in offspring that display a hyperemotional state and increased anxiety. In this study the effects of prenatal stress on CRF release was measured in amygdala minces (1 mm3) obtained from adult (8-16 weeks of age) male offspring of dams subjected to daily saline injection (0.1 ml, s.c.) from gestational day 14 to 21. CRF release from amygdala was time- and calcium-dependent, and stimulated by KCl-induced depolarization. Depolarization-induced CRF release was significantly increased by 42% from the amygdala of prenatally stressed offspring versus controls. Prenatally stressed offspring also showed a 49% increase in CRF levels in the amygdala. The increased amounts of CRF released in response to depolarization were likely the consequence of increased tissue content of CRF, as fractional release under basal or KCl-stimulated conditions was not different in the prenatal stress group versus control. This suggests that a long-lasting up-regulation of the CRFergic neurotransmission may occur in the amygdala, which may be important in the generation of hyperemotional offspring after exposure to prenatal stress.

Psychiatric morbidity in endocrine disorders.

Psychiatric disturbances are frequently observed during the course of endocrine disorders. This article discusses the history, current knowledge, assessment, and treatment of psychiatric morbidity in endocrine disorders. The primary focus is on biologic links between psychiatric symptoms and endocrine dysfunction. Psychiatric disorders associated with abnormalities of the pituitary, thyroid, parathyroids, adrenals, and gonads are discussed as well as the chronic illness of diabetes mellitus.

Effects of prenatal stress on defensive withdrawal behavior and corticotropin releasing factor systems in rat brain.

Exposure of pregnant rats to stress results in offspring that exhibit abnormally fearful behavior and have elevated neuroendocrine responses to novelty and aversive stimuli. This study examined the effects of prenatal stress on plasma corticosterone, adrenal weight, defensive withdrawal behavior, and the density of receptors for corticotropin releasing factor (CRF) in the amygdala. Pregnant Sprague-Dawley rats were stressed by daily handling and saline injection (s.c., 0.9%, 0.1 mL) during the last week of gestation. Male offspring were studied at adulthood (60-120 days of age). Adrenal hypertrophy and increased plasma corticosterone were observed in the prenatally stressed offspring. Defensive withdrawal, an ethological measure of the conflict between exploratory behavior and retreat, was quantified in naive offspring, and in offspring exposed to restraint stress (2 h). Restraint stress increased defensive withdrawal in both control and prenatally stressed offspring. Both naive and restraint-stressed prenatally stressed offspring exhibited increased defensive withdrawal compared to control offspring. There was a significant interaction between prenatal stress and restraint stress, suggesting increased vulnerability of prenatally stressed offspring. The effects of restraint in the defensive withdrawal test were reduced by intracerebroventricular administration of the CRF antagonists, alpha-helical CRF9-41 (20 microg every hour) or D-phe(12), Nle(21, 38), C(alpha)-MeLeu(37)]-CRF((12-41)) (5 microg every hour) during the restraint period. The difference between control and prenatally stressed offspring was abolished by the CRF antagonists, suggesting that increased activation of CRF receptors may be a factor in the behavioral abnormalities of prenatally stressed rats. Measurement of CRF receptors in amygdala revealed a 2.5-fold increase in binding in prenatally stressed offspring. In light of previous work from this laboratory demonstrating increased content and release of CRF in amygdala from prenatally stressed offspring, the present study suggests that the increased fearfulness of prenatally stressed rats may be a consequence of increased activity of CRFergic systems in the amygdala.

Failure of systemic N-acetyl cysteine to protect the rat lung against bleomycin toxicity.

In order to see whether systemically administered N-acetyl cysteine (NAC) could protect the lung from bleomycin lung injury, 24 rats were given a constant subcutaneous infusion of NAC (mean 195 mg.kg-1.day-1) for 7 days while 23 rats were given a similar volume of saline. Two days after the start of infusion, half of each group received an endotracheal injection of bleomycin and the other half received a similar volume of saline. All animals were killed 7 days after intratracheal injection and their lungs were prepared for wet-weight to dry-weight ratio (W:D) and morphometric assessment of histological changes. In animals given intratracheal saline, NAC infusion had no effect on weight gain, W:D or morphometry compared to those animals given saline infusions. However, all bleomycin-treated animals had obvious evidence of lung damage. Compared to either group of animals treated with intratracheal saline, the bleomycin-treated groups gained less weight (p less than 0.001), had a higher W:D (p less than 0.001) and an increase in the volume densities of alveolar and duct walls (p less than 0.001), intra-alveolar cells (p less than 0.05) and consolidation (p less than 0.001). There were no significant differences between the two bleomycin-treated groups in any parameter measured. It is concluded that administration of NAC by a constant subcutaneous infusion was not protective against bleomycin lung injury.

Synthesis and preliminary pharmacology of an internal standard for assay of neostigmine.

The synthesis of the diethyl analog of neostigmine, its preliminary pharmacology, and its use as an internal standard for the GLC assay of neostigmine are described. Both the diethyl analog and neostigmine undergo thermal demethylation in the injection port. The column selected produced satisfactory resolution and short retention times for neostigmine and the diethyl analog. The diethyl analog apparently possesses acetylcholinesterase-inhibiting properties, as evidenced by potentiation of the contractile response to acetylcholine in the ileum. In addition, acetylcholine levels in the brain were elevated slightly. Water solutions of the diethyl analog appeared to lose biological activity with time. the diethyl analog appears to be suitable for use as an internal standard for the GLC assay of neostigmine.

Corticotropin-releasing factor and defensive withdrawal: inhibition of monoamine oxidase prevents habituation to chronic stress.

There is growing evidence for a role of extrahypothalamic corticotropin-releasing factor (CRF) in the pathogenesis of anxiety. A modified form of the defensive withdrawal test was used to test the anxiogenic effects of acute administration of intracerebroventricular (1 microg, i.c.v.) CRF in adult male rats. Habituation to the mild stress of daily handling and subcutaneous (s.c.) saline injection over 2-6 weeks abolished the anxiogenic effects of exogenous CRF. At 6 weeks this habituation also resulted in attenuation of baseline withdrawal behavior. CRF receptor binding was significantly decreased in the amygdala of chronically handled animals and may have been responsible for this habituation phenomenon. Comparison of rats treated with the monoamine oxidase (MAO) inhibitor, phenelzine [3 mg/kg, s.c., daily for 2-6 weeks] to the saline-treated groups revealed a failure to habituate to the chronic handling, as the baseline withdrawal (after injection of artificial CSF) by the phenelzine-treated animals was not different from the baseline withdrawal by unhandled rats. In comparison to rats treated chronically with saline, phenelzine treatment enhanced the anxiogenic effect of CRF. In summary, habituation to a mild chronic stress decreased baseline defensive withdrawal. Intraventricular administration of CRF produced an anxiogenic response as measured in the defensive withdrawal test, which was lost through exposure to mild chronic stress. Two or 6 weeks of daily handling and SC saline injection caused a downregulation of CRF receptors in the amygdala, which could account for the behavioral habituation and the loss of CRF-induced defensive withdrawal. Phenelzine treatment concurrent with mild chronic stress prevented habituation and maintained the anxiogenic effect of CRF in spite of the downregulation of CRF receptors in the amygdala.

Mapping a quantitative trait locus for the concentration of beta-lactoglobulin in milk, and the effect of beta-lactoglobulin genetic variants on the composition of milk from Holstein-Friesian x Jersey crossbred cows.

AIM: To identify quantitative trait loci (QTL) affecting the concentration of beta-lactoglobulin in milk, and to evaluate the effect of beta-lactoglobulin genetic variants on the concentration of fat, protein and casein in bovine milk. METHODS: A herd of 850 F2 Holstein-Friesian x Jersey crossbred cows was produced through mating six Holstein-Friesian x Jersey F1 bulls of high genetic merit with F1 cows from the national herd. A total of 1,610 herd-test records from 556 second-parity crossbreds were analysed. The concentration of fat, protein and casein in milk was measured at peak, mid- and late lactation, during the production seasons of 2003-2004 and 2004-2005. Liveweight was measured daily. DNA from the F2 animals, their F1 dams and sires, and selected grandsires was genotyped across the genome, initially with 285 microsatellite markers, and subsequently with 6,634 single nucleotide polymorphisms (SNP). RESULTS: A highly significant QTL for the concentration of beta-lactoglobulin in milk was identified, which coincided with the position of the beta-lactoglobulin gene on bovine Chromosome 11. No other consistently significant QTL for the concentration of beta-lactoglobulin in milk were detected. Cows with the BB beta-lactoglobulin genotype produced milk with a 30% lower concentration of beta-lactoglobulin than cows with the AA genotype. The beta-lactoglobulin polymorphism also explained variation in the proportion of casein in total protein. In addition, the percentage of fat was higher for BB than AA animals, whereas the percentage of total protein, mean daily milk yield and liveweight did not differ between AA and BB animals. CONCLUSIONS: A significant QTL determining the concentration of beta-lactoglobulin in milk was identified. Selection of animals for the beta-lactoglobulin B-allele may enable the production of milk naturally enriched for casein, thus allowing a potential increase in the yield of cheese. There may be additional future value in production of bovine milk more like human milk, where decreasing the concentration of beta-lactoglobulin is desirable.

Alveolar type I and type II cells.

The alveolar epithelium comprises two main cell types: the alveolar type I and alveolar type II cell. The type I cell is a complex branched cell with multiple cytoplasmic plates that are greatly attenuated and relatively devoid of organelles; these plates represent the gas exchange surface in the alveolus. On the other hand, the type II cell acts as the "caretaker" of the alveolar compartment. It responds to damage of the vulnerable type I cell by dividing and acting as a progenitor cell for both type I and type II cells. In addition, it synthesises, stores and releases pulmonary surfactant into the alveolar hypophase, where it acts to optimise conditions for gas exchange.

Effect of artificial ventilation and anaesthesia on surfactant turnover in rats.

We have tested the hypothesis that breathing releases pulmonary surfactant via distortion of the alveolar type II cell. Gas exchange was maintained in the anaesthetized rat by applying high frequency (10 Hz) oscillations (HFO) to the chest wall; this resulted in apnoea within two to three breaths. After instrumentation under anaesthesia for 30 min, rats were infused with [3H]choline and [14C]choline, and we compared the tubular myelin-rich (PLalv-1) and -poor (PLalv-2) alveolar phospholipids and the microsomal and lamellar body phospholipids (PLlb) together with their specific activities after three forms of ventilation for 90 min: HFO (group 1), conventional mechanical ventilation (group 2) and spontaneous breathing (group 3). Group 4 was killed after surgical instrumentation and in group 5 the lungs were removed immediately after induction of anaesthesia. Groups 1-3 did not differ in any measured variable. Groups 1-4, which were anaesthetized for 30-120 min, had a lower PLalv-2 than did group 5. In contrast, PLlb was greater in groups 1-3, which were anaesthetized for 120 min, than in groups 4 and 5. In conclusion, we have successfully maintained normal gas exchange during complete apnoea by applying external HFO in rats for periods up to 90 min. Compared to mechanically ventilated or spontaneously breathing anaesthetized rats, surfactant turnover was unaltered by HFO, despite a markedly reduced tidal volume. However, the barbiturate anaesthetic itself appeared to inhibit surfactant turnover. We suggest that distortion of the type II cell may be the stimulus for surfactant release at tidal volumes above resting values.