Pharmacological evaluation of a selective bradykinin B1 antagonist in a canine model of arthritis.

The effects of a selective bradykinin 1 receptor antagonist, compound A, were evaluated in a canine model of acute inflammatory model of arthritis. Despite detection of the B1 receptor in canine type B synoviocytes using a fluorescent ligand, oral administration of compound A (9 and 27 mg/kg) did not improve weight bearing of dogs injected intra-articularly with IL-1ß in a force plate analysis. Analysis of the synovial fluid of IL-1ß-treated dogs indicated high levels of bradykinin postchallenge. Excellent exposure, coupled with evidence of the presence of the B1 receptor during an acute inflammatory model of pain, indicates an inability of the receptor to mediate inflammatory pain in canines.

Fast Curing with High-power Curing Lights Affects Depth of Cure and Post-gel Shrinkage and Increases Temperature in Bulk-fill Composites.

OBJECTIVES: High-power LED curing lights and bulk-fill resin composites are intended to reduce chair time. This study investigated depth of cure, post-gel shrinkage (responsible for shrinkage stress), and heat generation in bulk-fill composites when cured according to minimum curing times recommended by manufacturers of curing lights and composites. METHODS: A regular LED curing light (Demi Ultra, 1350 mW/cm2, Kerr Dental) and two LED curing lights with high-power modes (VALO Grand, 3117 mW/cm2 Xtra Power, Ultradent; and Bluephase PowerCure, 2435 mW/cm2 Turbo and 3344 mW/cm2 3sCure, Ivoclar Vivadent) were tested on three bulk-fill composites (Filtek One Bulk Fill, 3M Oral Care Solutions; Tetric EvoCeram Bulk Fill, Ivoclar Vivadent; Tetric Powerfill, Ivoclar Vivadent). Using minimum times recommended by manufacturers (3, 5, 6, 10, or 20 seconds), depth of cure was determined by Vickers hardness of specimens cured in a slot (n=10). Post-gel polymerization shrinkage was measured using a strain gauge (n=10) and temperature with a thermocouple (n=5). Results were analyzed using two- and one-way analysis of variance (ANOVA) followed by pairwise comparisons or Student-Newman-Keuls post hoc tests (α=0.05). RESULTS: Curing lights and curing protocols significantly affected depth of cure, post-gel shrinkage, and temperature rise (p<0.001). Cure decreased with depth whereby best overall curing performance was achieved by the 20 second exposure at lowest irradiance (Demi Ultra). Fast curing (3-5 seconds) at high irradiance resulted in lesser depth-of-cure performance, except for the BluePhase-Tetric PowerFill combination. Post-gel shrinkage was higher in all composites when cured at high irradiance (p<0.001), while heat generated also tended to be higher. CONCLUSIONS: Although the high-power LED curing lights advertise time savings, not all manufacturer recommended minimum curing times cured bulk-fill materials to the same extent. Moreover, these time savings came at a cost of higher post-gel shrinkage and generated more heat in the bulk-fill composites than the lower irradiance curing protocol.

Intrafetal insulin-like growth factor-I infusion stimulates adrenal growth but not steroidogenesis in the sheep fetus during late gestation.

We investigated the effects of an intrafetal infusion of IGF-I on adrenal growth and expression of the adrenal steroidogenic and catecholamine-synthetic enzyme mRNAs in the sheep fetus during late gestation. Fetal sheep were infused for 10 d with either IGF-I (26 microg/kg.h; n = 14) or saline (n = 10) between 120 and 130 d gestation, and adrenal glands were collected for morphological analysis and determination of the mRNA expression of steroidogenic and catecholamine-synthetic enzymes. Fetal body weight was not altered by IGF-I infusion; however, adrenal weight was significantly increased by 145% after IGF-I infusion. The density of cell nuclei within the fetal adrenal cortex (the zona glomerulosa and zona fasciculata), and within the adrenaline synthesizing zone of the adrenal medulla, was significantly less in the IGF-I-infused fetuses compared with the saline-infused group. Thus, based on cell-density measurements, there was a significant increase in cell size in the zona glomerulosa and zona fasciculata of the adrenal cortex and in the adrenaline-synthesizing zone of the adrenal medulla. There was no effect of IGF-I infusion on the adrenal mRNA expression of the steroidogenic or catecholamine-synthetic enzymes or on fetal plasma cortisol concentrations. In summary, infusion of IGF-I in late gestation resulted in a marked hypertrophy of the steroidogenic and adrenaline-containing cells of the fetal adrenal in the absence of changes in the mRNA levels of adrenal steroidogenic or catecholamine-synthetic enzymes or in fetal plasma cortisol concentrations. Thus, IGF-I infusion results in a dissociation of adrenal growth and function during late gestation.

Changes in matrix metalloproteinase (MMP)-2 and MMP-9 in the fetal amnion and chorion during gestation and at term and preterm labor.

Increased matrix metalloproteinase (MMP)-9 proteolytic activity is associated with term birth, preterm birth and premature rupture of membranes. However, most studies show no changes with MMP-2, which binds tightly to cell and matrix proteins. We hypothesized better protein extraction would reveal new MMP patterns. Human amnion and chorion were collected from 25 patients at preterm or term, extracted with 2% SDS (a high concentration), and the MMP protein levels and pro-enzyme activities were determined by Western immunoblotting and zymography. MMP-2 protein and MMP-2 and -9 pro-enzyme activities in the amnion increased significantly (p<0.05) with labor at term, and were higher than at preterm labor (p<0.05), when extracted with high SDS concentration. There were no changes in chorion MMPs under any condition. These associations suggest MMP-2 may be another regulator of membrane rupture and other labor-associated mechanisms at term parturition, and its role(s) should be examined further.

Infusion of N-proopiomelanocortin-(1-77) increases adrenal weight and messenger ribonucleic acid levels of cytochrome P450 17alpha-hydroxylase in the sheep fetus during late gestation.

In the sheep there is a rapid increase in fetal adrenal growth and steroidogenesis during the last 10-15 days gestation (term = 147+/-3 days gestation). In the rat, peptides derived from the N-terminal region of POMC play a role in compensatory adrenal growth and in potentiation of ACTH-induced steroidogenesis. We therefore investigated the effects of infusion of bovine N-POMC-(1-77) and its biosynthetic derivative, N-POMC-(1-49) on adrenal growth and on the expression of adrenal steroidogenic enzymes in the late gestation sheep fetus. Twenty-seven pregnant ewes were used in this study. Fetal vascular catheters were inserted between 116-125 days gestation, and purified bovine N-POMC-(1-77) (2 microg/ml x h), N-POMC-(1-49) (2 microg/ml x h) and saline were each infused for 48 h between 136 and 138 days gestation. Intrafetal infusion of N-POMC-(1-77) resulted in an increased adrenal/fetal body weight ratio (94.6+/-5.7 mg/kg) compared with that in saline-infused (75.6+/-1.8 mg/kg), but not N-POMC-(1-49)-infused (82.7+/-6.1 mg/kg), fetal sheep. The ratio of CYP17 messenger RNA (mRNA) to 18S ribosomal RNA was also significantly higher in fetal adrenals ofthe N-POMC-(1-77)-infused group (49.1+/-4.7) compared with that in either the N-POMC-(1-49)-infused (20.4+/-6.4) or saline-infused (15.2+/-4.4) group. There was no difference, however, in the ratios of adrenal CYP11A1 mRNA/3beta-hydroxysteroid dehydrogenase/delta5,delta4-isomerase mRNA and CYP21A1 mRNA/18S ribosomal RNA among the N-POMC-(1-77)-, N-POMC-(1-49)-, and saline-infused groups. There was also no significant change in either plasma cortisol or ACTH concentrations in response to the infusion of either N-POMC-(1-77) or N-POMC-(1-49). In summary, intrafetal infusion of N-POMC-(1-77) stimulated fetal adrenal growth and resulted in a specific increase in adrenal CYP17 gene expression in late gestation. N-POMC-(1-77) may therefore play a modulatory role in the increase in fetal adrenal growth and steroidogenesis that occurs before birth.

Hepatic prolactin receptor gene expression increases in the sheep fetus before birth and after cortisol infusion.

We have investigated the effect of increasing gestational age and cortisol on prolactin receptor (PRLR) gene expression in the fetal sheep liver during late gestation. RNA was extracted from the liver of sheep fetuses between 90 and 144 days (d) gestation (n = 18) and after intrafetal infusion of either cortisol (2-2.5 mg cortisol i.v./24 h; n = 6) or saline (n = 6) between 109 and 116 d gestation. A ribonuclease protection assay for the mRNAs encoding the long (PRLR1) and short (PRLR2) forms of the PRLR was developed using an antisense RNA probe complementary to ovine PRLR2. There was a significant increase (p < 0.05) in the relative levels of liver PRLR1: GAPDH mRNA and PRLR2: GAPDH mRNA levels in fetal sheep between 90 and 144d gestation (PRLR1 mRNA: 90-95 d 0.6 +/- 0.1, 131-133 d 1.2 +/- 0.2, 141-144 d 3.6 +/- 0.5; PRLR2 mRNA: 90-95 d 0.7 +/- 0.1; 131-133 d 1.4 +/- 0.2, 141-144 d 3.0 +/- 0.4). The relative levels of liver PRLR1 and PRLR2: GAPDH mRNA levels were higher (p < 0.05) after cortisol administration (1.7 +/- 0.3 and 0.9 +/- 0.1 respectively) when compared with the saline infused group (0.7 +/- 0.1 and 0.5 +/- 0.1 respectively). We have demonstrated therefore that there is in increase in the levels of the mRNA encoding PRLR1 and PRLR2 in the fetal sheep liver during late gestation and that physiological increases in fetal cortisol stimulate PRLR1 and PRLR2 expression in the liver of the sheep fetus. These data suggest that fetal PRL may play a role in the growth and maturation of the fetal liver which occurs before birth.

Role of interferon-gamma in lung inflammation following cecal ligation and puncture in rats.

Interferon-gamma (IFN-gamma) has been implicated in the mortality of animal models of endotoxemia. On the other hand, the specific role of IFN-gamma in the development of organ inflammation in a model of polymicrobial sepsis has not been elucidated. In this study, we hypothesized that IFN-gamma plays an important role in lung inflammation after cecal ligation and puncture (CLP). To verify this hypothesis, lung tissue was removed 5 h after CLP or from sham controls. The mRNA expression (by RT-PCR) of IFN-gamma was increased in lung homogenates of CLP rats compared to sham controls. Using immunohistochemistry, we show for the first time the increased presence of IFN-gamma staining cells in the lung following CLP. Only very small amounts of positive staining for IFN-gamma was observed in lungs of sham controls. The presence of IFN-gamma in the lung 5 h after CLP correlated with a twofold increases in lung superoxide generation and MPO activity (index of neutrophil sequestration). Plasma and lung nitrite levels (breakdown product of nitric oxide) were also significantly increased in CLP rats. IFN-gamma antibody (1.2 mg/kg, i.v.) administered immediately after CLP significantly decreased lung superoxide levels to levels similar to the sham controls without affecting MPO activity, or lung or plasma nitrite levels. These results provide evidence that IFN-gamma may contribute to lung inflammation 5 h following CLP via increased production of superoxide.

Glucocorticoids decrease phenylethanolamine N-methyltransferase mRNA expression in the immature foetal sheep adrenal.

This study examined the impact of a chronic physiological elevation of plasma cortisol levels on adrenal catecholamine synthetic enzyme and proenkephalin A mRNA expression in foetal sheep. Cortisol (2.5-3. 0 mg.5 ml-1.24 h-1, n=9) or saline (0.9% saline, n=6) was infused into foetal sheep for 7 days between 109 days and 116 days gestation. Foetal plasma cortisol concentrations were higher (P<0.0005) in the cortisol infused foetuses when compared with the saline infused group (43.07+/-4.13 nmol.l-1 vs 1.67+/-0.10 nmol.l-1). There were no differences, however, in the plasma ACTH levels between the two groups. Using Northern blot analysis, adrenal phenylethanolamine N-methyltransferase (PNMT) mRNA expression was found to be reduced (P<0.005) fivefold in the cortisol infused foetuses when compared with the controls, as was the relative area of the adrenal medulla which stained positively with anti-PNMT (28.1+/-2.5% vs 44.8+/-4.8%, P<0.007). No effect of cortisol infusion was observed on adrenal tyrosine hydroxylase mRNA and protein expression or proenkephalin A mRNA expression. We conclude that before birth, adrenaline synthesis may be suppressed by a novel direct, or indirect, inhibitory effect of glucocorticoids on PNMT mRNA expression.

Cortisol differentially regulates pituitary-adrenal function in the sheep fetus after disconnection of the hypothalamus and pituitary.

We have investigated the effects of a 5 day infusion of cortisol into fetal sheep, in which the hypothalamus and pituitary were surgically disconnected (HPD), on fetal pituitary-adrenal function. Fetal HPD and vascular catheterization were carried out at between 104 and 124 days gestation. Cortisol was administered (3.5 mg 24 h-1) for 120 h between 134 and 140 days (HPD + F group; n = 5) and saline was administered during the same gestational age range to HPD (HPD group; n = 12) and intact fetal sheep (Intact group; n = 6). Cortisol infusion into the HPD fetal sheep did not suppress the mRNA levels for Proopiomelanocortin (POMC) in the fetal anterior pituitary at 139/140 days gestation (POMC mRNA: 18S rRNA: Intact 0.40 +/- 0.05; HPD 0.56 +/- 0.07; HPD + F 0.49 +/- 0.07). Similarly, there was no significant effect of either HPD or cortisol infusion on the plasma concentrations of immunoreactive (ir) ACTH or ACTH(1-39). The adrenal: fetal body weight ratio was significantly higher, however, in the HPD + F (88.4 +/- 8.7 mg kg-1) and Intact groups (84.1 +/- 5.6 mg kg-1) when compared with the HPD fetal sheep (63.7 +/- 5.4 mg kg-1). The ratio of total IGF-II mRNA: 18S rRNA was similar in the adrenals of the Intact (0.48 +/- 0.09), HPD (0.78 +/- 0.09) and HPD + F (0.71 +/- 0.11) groups. The ratios of CYPIIA1, 3 beta-HSD and CYP21A1 mRNA: 18S rRNA were significantly lower in adrenals from the HPD group when compared to those in the Intact group and were not restored to normal by cortisol infusion. We have therefore demonstrated that cortisol does not act directly at the fetal pituitary to suppress POMC synthesis or ACTH secretion in late gestation. Cortisol does, however, stimulate fetal adrenal growth after HPD in the absence of any effects on adrenal IGF-II or steroidogenic enzyme mRNA levels. The data provide evidence that an intact hypothalamic-pituitary axis and cortisol each play an important role in the stimulation of adrenal growth and steroidogenesis which occurs during the last 10-15 days of gestation in the sheep.

Differential effects of placental restriction on IGF-II, ACTH receptor and steroidogenic enzyme mRNA levels in the foetal sheep adrenal.

We have investigated the effects of restriction of placental growth on foetal adrenal growth and adrenal expression of mRNAs for Insulin-like Growth Factor II (IGF-II), the IGF binding protein IGFBP-2, Steroidogenic Factor 1 (SF-1) and adrenocorticotrophic hormone (ACTH) receptor (ACTH-R) and the steroidogenic cytochrome P-450 enzymes: cholesterol side chain cleavage (CYP11A1), 17alpha-hydroxylase (CYP17) and 21-hydroxylase (CYP21A1); and 3beta-hydroxysteroid dehydrogenase/Delta5Delta4 isomerase (3betaHSD). Endometrial caruncles were removed from non-pregnant ewes before mating (placental restriction group; PR). The total adrenal: foetal weight ratio was higher in PR (n=6 foetuses) than in control foetuses (n=6 foetuses). There was no difference in plasma ACTH concentrations between the PR and control foetuses between 130 and 140 days gestation. Adrenal IGF-II mRNA levels were lower (P<0.05) in the PR group, however, adrenal IGFBP-2 mRNA levels were not different between the PR and control groups. Adrenal ACTH-R mRNA levels were also lower whilst CYP11A1 mRNA levels were increased (P<0.005) in the PR group. We conclude that foetal adrenal growth and steroidogenesis are stimulated as a consequence of foetal growth restriction and that factors other than ACTH are important in foetal adrenal activation during chronic, sustained hypoxaemia.

Chronic stress--the key to parturition?

It is clear that the timing of parturition is dependent on a cascade of endocrine signals from an intact fetal hypothalamo-pituitary-adrenal axis. What is not known, however is the nature or source of the central neural stimulation which results in the stimulation of adrenocorticotrophic hormone (ACTH) synthesis and secretion in late gestation. The changes which occur in the synthesis and posttranslational processing of the ACTH precursor, proopiomelanocortin (POMC), in the fetal anterior pituitary before birth and the consequence of these changes for expression of the corticosteroidogenic enzymes in the fetal adrenal are described in this review. Evidence for the functional heterogeneity of corticotrophic cell types in the fetal sheep pituitary and the proposal that there is a maturational change in the populations of corticotrophic cells in late gestation are discussed. Finally, the development of cortisol negative feedback in the late gestation fetal hypothalamo-pituitary axis and the relevance of chronic stress to the timing of parturition are also discussed.

L-asparaginase from Erwinia carotovora. An improved recovery and purification process using affinity chromatography.

A large-scale process was developed to purify L-asparaginase from submerged cultures of Erwinia carotovora. Cells from 880 L of fermentation broth were harvested and washed using a plate and frame type filter press. A cellular acetone powder was prepared from the washed cells by suspending the cells twice in acetone and the residual acetone was removed by washing the acetone powder in the filter press with 10 mM phosphate buffer (pH 7.0). The cellular acetone powder was extracted with 10 mM borate buffer at pH 9.5. The enzyme-rich borate extract was recovered by filtration and clarified by an in-line bag filter. The filtrate was adjusted to pH 7.5 and filtered through a 1-micron bag filter precoated with Celite and then through a 0.22-micron cartridge filter. The cell-free extract, containing 21 x 10(6) IU of enzyme and 448 g of total protein, was applied to an L-asparagine Sepharose 6 Fast Flow affinity column (9 L) using a bag filter loaded with Cell Debris Remover as an in-line prefilter. The affinity gel was prepared by coupling L-Asn at pH 9.0 to epoxy-activated Sepharose 6 Fast Flow beads. A total of 14 x 10(6) IU of enzyme (35 g protein) was eluted at pH 9.0 in 10.5 L. The eluted enzyme was determined to be greater than 90% pure using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The total process time from whole broth to affinity column elution was 68 h and the enzyme yield was 38%. This improved process for the 880 L fermentation broth produced a cell-free extract of high specific activity, shortened the process time, increased the column capacity, and yielded a product with high purity.

Large-scale recovery and purification of L-asparaginase from Erwinia carotovora.

A large-scale process was developed to purify gram quantities of a therapeutic enzyme, L-asparaginase, from submerged cultures of Erwinia carotovora. Cells were harvested from 150 L of fermentation broth and washed. A cellular acetone powder was prepared and extracted with pH 9.5 borate buffer. After continuous centrifugation and filtration to remove cell debris, the acetone powder extract was adjusted to pH 7.7 and adsorbed onto a 16-L CM-Sepharose Fast Flow column, with a precolumn packed with Cell Debris Remover. The enzyme was desorbed from the catin-exchange column at pH 9.0 and further purified with an affinity column of L-asparagine Sepharose CL-4B. After dialysis-concentration to remove buffer salt, the enzyme was depyrogenated, formulated, sterile filled, and lyophilized as a single-dose final product. The final-product evaluation included analysis of the content of protein, sodium chloride, glycine, sodium, glucose hydrate, phosphate, and endotoxin, as well as reconstitution, potency, pH, specific activity, uniformity of fill, and sterility. The product was further subjected to visual examination, sodium dodecyl sulfate polyacrylamide gel electrophoresis, native gel electrophoresis, isoelectric focusing, amino acid analysis, N-terminal sequencing, peptide mapping, and immunological comparison.

Role of pituitary POMC-peptides and insulin-like growth factor II in the developmental biology of the adrenal gland.

During fetal life, it is critical that there is coordinate regulation of the growth, zonation and differentiation of the fetal adrenal cortex to ensure that cells in key tissues and organs are exposed in a programmed temporal sequence to the actions of glucocorticoids. Glucocorticoids are essential for maturation of key target organs before birth, including the lung, brain, liver, gut, kidney and adrenal, and the prepartum increase in glucocorticoid synthesis and secretion by the fetal adrenal gland is critical for the successful transition to postnatal life. It is also evident that premature or abnormal exposure of embryonic or fetal tissues to glucocorticoids during critical windows of development can irreversibly alter the programmed development of organ systems. Premature or abnormal exposure of the fetus to excess glucocorticoids may occur either as a consequence of endogenous stimulation of the fetal hypothalamo-pituitary-adrenal axis (HPAA) or as a consequence of exposure to exogenous glucocorticoids in a therapeutic context. Administration of synthetic glucocorticoids to women at risk of preterm labour, for example, is a routine clinical practice designed to improve respiratory function and neonatal outcome. It is clearly important to understand what endogenous factors regulate the growth and functional maturation of the adrenal cortex during development and the consequent likelihood of exposure of developing tissues to excess corticosteroids. To date, investigations have centred on the role of ACTH 1-39 in the stimulation of adrenal growth and steroidogenesis in long gestation species, such as the primate and sheep, where maturation and differentiation of organ systems occurs predominantly before birth. In this review, we will focus on the evidence that in addition to ACTH 1-39, other pro-opio-melanocortin (POMC) derived peptides, which are synthesized, processed and secreted by the fetal pituitary, play a role in the coordinate regulation of the specific phases of growth and functional development of the fetal adrenal gland in vivo. We will discuss our recent findings on the direct in vivo actions of N-POMC 1-77 and separately, insulin like growth factor II (IGF-II), as adrenal growth factors. These studies provide an understanding of the separate regulatory mechanisms which control activation of adrenal growth and stimulation of adrenal steroidogenesis in the late gestation fetus.

Comparative viability of peninsular and island axial pattern flaps incorporating the cranial superficial epigastric artery in dogs.

Experimental island and peninsular axial pattern flaps that incorporated the cranial superficial epigastric artery and vein were developed in 6 Beagles. Mean percentage of flap area that survived, for both flaps, was 87%, and percentage of surviving flap area was not significantly different for island versus peninsular flaps. In 1 dog, ligation of an aberrant, perforating branch of the cranial epigastric artery resulted in necrosis of 53% of the flap area. The cranial superficial epigastric axial pattern flaps have potential application for closure of skin defects within their arc of rotation and may be particularly useful for closure of large defects on the ventral aspect of the thorax. A peninsular flap was used to close a defect of the ventral portion of the thoracic wall in a clinical case.

Oligodendroglial dysplasia in two bullmastiff dogs.

Leukodystrophies are inherited neurological disorders involving central nervous system white matter. They are uncommon in animals but a few, breed-specific entities have been described. In 2002, two young-adult, purebred Bullmastiff dogs from central New York State presented to their referring veterinarians displaying moderate to severe ataxia of all limbs, spastic tetraparesis that was worse in the pelvic limbs, and a diffuse, action-related, whole-body tremor. Clinical signs were insidious in onset and slowly progressive. Anatomic diagnoses considered were a C1-C5 lesion or, based on the whole-body tremor, a diffuse central nervous system disorder. No gross lesions were apparent in the brain or spinal cord. Histopathologically, numerous, multifocal, sharply demarcated, small, ovoid to angular areas of myelin pallor (plaques) were present throughout the major white matter tracts of the brainstem and spinal cord. These plaques, which often were traversed by axons, did not stain with luxol fast blue for myelin and were associated with minimal astrocytosis. Ultrastructural findings include occasional hypertrophic glia in white matter, rare unmyelinated segments of axons, and focal proliferation of tubule-containing cytoplasmic glial cell processes (oligodendroglial). The described clinical and morphological findings and age of onset are similar to the well-characterized, presumably hereditary, bovine syndrome known as Charolais ataxia or oligodendroglial dysplasia. This article presents the first description of a leukodystrophy in the Bullmastiff breed and the first report of oligodendroglial dysplasia in animals other than Charolais cattle.

Complications and long-term results after partial laryngectomy for the treatment of idiopathic laryngeal paralysis in 45 dogs.

Forty-five dogs with severe respiratory signs caused by idiopathic, acquired laryngeal paralysis were treated by partial laryngectomy. The predominant postoperative complications were coughing in 28 dogs and pneumonia in 15 dogs. Eight dogs required a second operation to alleviate persistent or recurrent upper airway obstruction. Results of surgery were considered excellent in 11 dogs (25%), good in 18 dogs (40%), fair in 6 dogs (13%), and poor in 10 dogs (22%). Poorer results were obtained by surgical residents than by more experienced surgeons. Death in the immediate postoperative period was related to pneumonia (8 dogs) and laryngeal collapse (1 dog). Nine dogs died later of respiratory disease. Although partial laryngectomy is effective for the treatment of laryngeal paralysis, it is not recommended because of the high incidence of postoperative complications.

A premature increase in circulating cortisol suppresses expression of 11beta hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid in the adrenal of the fetal sheep.

We have investigated the effect of intrafetal cortisol administration, before the normal prepartum cortisol surge, on the expression of 11beta hydroxysteroid dehydrogenase (11betaHSD) type 2 mRNA in the fetal adrenal. We also determined whether increased fetal cortisol concentrations can stimulate growth of the fetal adrenal gland or increase expression of adrenal steroidogenic enzymes. Cortisol (hydrocortisone succinate: 2.0-3.0 mg in 4.4 ml/24 h) was infused into fetal sheep between 109 and 116 days of gestation (cortisol infused; n = 12), and saline was administered to control fetuses (saline infused; n = 13) at the same age. There was no effect of cortisol infusion on the fetal adrenal:body weight ratio (cortisol: 101.7 +/- 5.3 mg/kg; saline: 108.2 +/- 4.3 mg/kg). The ratio of adrenal 11betaHSD-2 mRNA to 18S rRNA expression was significantly lower, however, in the cortisol-infused group (0.75 +/- 0.02) compared with the group receiving saline (1.65 +/- 0.14). There was no significant effect of intrafetal cortisol on the relative abundance of adrenal CYP11A1, CYP17, CYP21A1, and 3betaHSD mRNA. A premature elevation in fetal cortisol therefore resulted in a suppression of adrenal 11betaHSD-2. Increased intra-adrenal exposure to cortisol at this stage of gestation is, however, not sufficient to promote adrenal growth or steroidogenic enzyme gene expression.

Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis.

Legionella pneumophila is a facultative intracellular gram-negative rod that causes pneumonia in humans. Free-living amoebas are thought to serve as a reservoir for Legionella infections. Signature-tagged mutagenesis was employed to identify Legionella pneumophila genes necessary for survival in the amoeba Acanthamoeba castellanii. Six mutant strains were defective in assays of invasion and intracellular growth. Four mutants also exhibited invasion and replication defects in Hartmannella vermiformis, an amoeba linked to hospital outbreaks of Legionella pneumonia. The six mutants also were tested in macrophages derived from peripheral blood mononuclear cells. Two mutants had intracellular replication defects, and two different strains entered cells less efficiently. Two transposon insertions were in known L. pneumophila genes, lspK and aroB. The other four were in novel genes. One gene has similarity to a cytochrome c-type biogenesis protein of Pseudomonas fluorescens. Another has similarity to a transcriptional activator regulating flagellar biosynthesis in Vibrio cholera. The third is similar to traA of Rhizobium sp. strain NGR234, which is involved in conjugal transfer of DNA. The fourth has no homology. By using survival in amoeba as a selection, we have isolated mutant strains with a range of phenotypes; and we have potentially identified new L. pneumophila virulence genes.