Moraxella bovis, Moraxella ovis and Moraxella bovoculi: biofilm formation and lysozyme activity.

AIMS: This study aimed to verify the formation of biofilms by Moraxella bovis, Moraxella ovis and Moraxella bovoculi isolates from ruminants. In addition, the lysozyme activity against the isolates of M. bovis, M. ovis and M. bovoculi in free form and in biofilms was determined. METHODS AND RESULTS: In this study, 54 isolates of Moraxella sp. obtained from bovine and ovine clinical samples were evaluated in vitro for capacity of biofilm formation and lysozyme susceptibility in planktonic and sessile cells. In addition, biofilms produced by four Moraxella sp. isolates were visualized under scanning electron microscope (SEM). It was possible to demonstrate, for the first time, the ability to form biofilms by M. ovis and M. bovoculi. The isolates of Moraxella sp. have the capacity to form biofilms in different intensities, varying among weak, moderate and strong. It was verified that the lysozyme shows activity on Moraxella sp. in planktonic form. However, on biofilms there was a reduction in the production, but without impairing its formation, and on consolidated biofilms the lysozyme did not have the capacity to eradicate the preformed biofilms. CONCLUSIONS: This work shows the capacity of biofilm formation by Moraxella sp. of veterinary importance. The lysozyme susceptibility of Moraxella sp. in planktonic form shows that this enzyme has bacteriostatic activity on this micro-organism and it reduced the production of biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on the results, it is possible to infer that the biofilm formation capacity by Moraxella sp. and the resistance to lysozyme concentrations equal to or greater than the physiological levels of the ruminant tear may be linked not only to the capacity to colonize the conjunctiva, but also to remain in this place even after healing of the lesions, being a reservoir of Moraxella sp. in a herd.

Cardiac toxoplasmosis after heart transplantation diagnosed by endomyocardial biopsy.

We describe a case of cardiac toxoplasmosis diagnosed by routine endomyocardial biopsy in a patient with trimethoprim-sulfamethoxazole (TMP-SMX) intolerance on atovaquone prophylaxis. Data are not available on the efficacy of atovaquone as Toxoplasma gondii prophylaxis after heart transplantation. In heart transplant patients in whom TMP-SMX is not an option, other strategies may be considered, including the addition of pyrimethamine to atovaquone.

A systematic review of pre-operative anaemia and blood transfusion in patients with fractured hips.

We systematically reviewed the observational associations of anaemia with outcomes and the effects of interventions to increase haemoglobin concentrations following hip fracture in older people. Anaemia on hospital admission was associated with increased mortality, relative risk 1.64 (95% CI 1.47-1.82), p < 0.0001. After adjustment for co-morbidities, the association of anaemia with increased mortality remained in four of eight observational studies. There was no association of postoperative transfusion with mortality after adjusting for covariates. Transfusion at 80 g.l(-1) vs 100 g.l(-1) increased acute myocardial infarction, relative risk 1.67 (95% CI 1.01-2.77), p = 0.05. Transfusion threshold was not associated with differences in other outcomes. There were insufficient high-quality studies to inform pre-operative blood transfusion or the use of peri-operative iron or erythropoietin. Studies for most interventions recruited too few participants to determine effects on infections, mortality or function.

In vitro susceptibility of zoospores and hyphae of Pythium insidiosum to antifungals.

OBJECTIVES: The purpose of this study was to compare the in vitro susceptibilities of 22 Brazilian isolates of Pythium insidiosum to antifungals using a standardized inoculum of zoospores and a proposed novel inoculum prepared from cultured mycelia (hyphae) of P. insidiosum. METHODS: A zoospore suspension of P. insidiosum was obtained by the zoosporogenesis technique. The hyphal inoculum was prepared from a suspension of P. insidiosum mycelium. Susceptibility to each drug was evaluated using the CLSI M38-A2 method. RESULTS: Of the 88 MIC comparisons performed, 36 (41%) showed the same MIC value for the two inocula. The agreement (differences not greater than one dilution) between MICs obtained with both types of inocula was 39.8% (35/88). In other MIC comparisons analysed, 17 (19.3%) showed differences of two or three dilutions. CONCLUSIONS: We conclude that the use of hyphal inocula of P. insidiosum for in vitro susceptibility tests could be a suitable method for evaluating antimicrobial susceptibility, particularly when it is not possible to obtain a standardized zoospore inoculum.

BRAF, NRAS and HRAS mutations in spitzoid tumours and their possible pathogenetic significance.

BACKGROUND: The relationships between so-called spitzoid tumours have proven difficult to understand. OBJECTIVES: To address three questions: does spitzoid tumour morphological similarity reflect molecular similarity? Does Spitz naevus progress into spitzoid melanoma? Are ambiguous spitzoid tumours genuine entities? METHODS: BRAF, NRAS and HRAS mutations were analysed using single-strand conformational polymorphism analysis and sequencing. RESULTS: Both Spitz naevi and spitzoid melanoma had a lower combined BRAF and NRAS mutation frequency compared with common acquired naevi (P = 0.0001) and common forms of melanoma (P = 0.0072), respectively. To look for evidence of progression from Spitz naevi to spitzoid melanoma, HRAS was analysed in 21 spitzoid melanomas, with no mutations identified. The binomial probability of this was 0.03 based on an assumption of a 15% mutation frequency in Spitz naevi with unbiased progression. Under these assumptions, HRAS mutations must be rare/absent in spitzoid melanoma. Thus, Spitz naevi seem unlikely to progress into spitzoid melanoma, implying that ambiguous spitzoid tumours cannot be intermediate degrees of progression. In addition, the data suggest that HRAS mutation is a potential marker of benign behaviour, in support of which none of three HRAS mutant spitzoid cases metastasized. CONCLUSIONS: First, the morphological similarity of spitzoid tumours reflects an underlying molecular similarity, namely a relative lack of dependence on BRAF/NRAS mutations. Second, Spitz naevi do not appear to progress into spitzoid melanoma, and consequently ambiguous spitzoid tumours are likely to be unclassifiable Spitz naevi or spitzoid melanoma rather than genuine entities. Third, HRAS mutation may be a marker of Spitz naevus, raising the possibility that other molecular markers for discriminating Spitz naevi from spitzoid melanoma can be discovered.

Loss of M2 muscarine receptors in the cerebral cortex in Alzheimer's disease and experimental cholinergic denervation.

Cerebral cortex samples from patients with Alzheimer's disease and from rats after experimental cholinergic denervation of the cerebral cortex exhibited reductions in the presynaptic marker choline acetyltransferase activity and in the number of M2 muscarine receptors, with no change in the number of M1 receptors. These results are in keeping with evidence that M2 receptors function in cholinergic nerve terminals to regulate the release of acetylcholine, whereas M1 receptors are located on postsynaptic cells and facilitate cellular excitation. New M1-selective agonists and M2-selective antagonists directed at post- or presynaptic sites deserve consideration as potential agents for the treatment of the disease.

Phosphorylation of the kinase homology domain is essential for activation of the A-type natriuretic peptide receptor.

Natriuretic peptide receptor A (NPR-A) is the biological receptor for atrial natriuretic peptide (ANP). Activation of the NPR-A guanylyl cyclase requires ANP binding to the extracellular domain and ATP binding to a putative site within its cytoplasmic region. The allosteric interaction of ATP with the intracellular kinase homology domain (KHD) is hypothesized to derepress the carboxyl-terminal guanylyl cyclase catalytic domain, resulting in the synthesis of the second messenger, cyclic GMP. Here, we show that phosphorylation of the KHD is essential for receptor activation. Using a combination of phosphopeptide mapping techniques, we have identified six residues within the ATP-binding domain (S497, T500, S502, S506, S510, and T513) which are phosphorylated when NPR-A is expressed in HEK 293 cells. Mutation of any one of these Ser or Thr residues to Ala caused reductions in the receptor phosphorylation state, the number and pattern of phosphopeptides observed in tryptic maps, and ANP-dependent guanylyl cyclase activity. The reductions were not explained by decreases in NPR-A protein levels, as indicated by immunoblot analysis and determinations of cyclase activity in the presence of detergent. Conversion of Ser-497 to Ala resulted in the most dramatic decrease in cyclase activity (approximately 20% of wild-type activity), but conversion to an acidic residue (Glu), which mimics the charge of the phosphoserine moiety, had no effect. Simultaneous mutation of five of the phosphorylation sites to Ala resulted in a dephosphorylated receptor which was unresponsive to hormone and had potent dominant negative inhibitory activity. We conclude that phosphorylation of the KHD is absolutely required for hormone-dependent activation of NPR-A.

A constitutively "phosphorylated" guanylyl cyclase-linked atrial natriuretic peptide receptor mutant is resistant to desensitization.

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

Nitrate reduction in the periplasm of gram-negative bacteria.

In contrast to the bacterial assimilatory and membrane-associated, respiratory nitrate reductases that have been studied for many years, it is only recently that periplasmic nitrate reductases have attracted growing interest. Recent research has shown that these soluble proteins are widely distributed, but vary greatly between species. All of those so far studied include four essential components: the periplasmic molybdoprotein, NapA, which is associated with a small, di-haem cytochrome, NapB; a putative quinol oxidase, NapC; and a possible pathway-specific chaperone, NapD. At least five other components have been found in different species. Other variations between species include the location of the nap genes on chromosomal or extrachromosomal DNA, and the environmental factors that regulate their expression. Despite the relatively small number of bacteria so far screened, striking correlations are beginning to emerge between the organization of the nap genes, the physiology of the host, the conditions under which the nap genes are expressed, and even the fate of nitrite, the product of Nap activity. Evidence is emerging that Nap fulfills a novel role in nitrate scavenging by some pathogenic bacteria.

Desipramine and 2-hydroxydesipramine plasma levels in endogenous depressed patients. Lack of correlation with therapeutic response.

Studies of the relationship between plasma concentrations of desipramine hydrochloride and clinical response have shown contradictory results, and only one prior study examined 2-hydroxydesipramine and its relationship to treatment. We therefore performed a study in a large, carefully diagnosed group of depressed patients taking fixed maintenance doses of desipramine to elucidate a potential relationship between clinical response and plasma concentrations of desipramine and 2-hydroxydesipramine. There was no significant correlation between clinical response and steady-state plasma levels of desipramine, 2-hydroxydesipramine, or the sum of desipramine plus 2-hydroxydesipramine. Although some commercial laboratories suggest a specific therapeutic plasma level "range" for desipramine, our data provide no support for such a range, nor for the routine measurement of plasma desipramine and 2-hydroxydesipramine concentrations in depressed patients.

Serum sickness after treatment with rabbit antithymocyte globulin in a heart transplant recipient with previous rabbit exposure.

Antithymocyte globulin (ATG) is a preparation of polyclonal antibodies frequently used to treat acute cellular rejection in organ transplant recipients. Use of rabbit ATG has been associated with serum sickness in liver and kidney transplantation patients previously exposed to rabbits. Here, we report the case of a heart transplantation patient with a history of significant rabbit exposure who had developed migratory diffuse arthralgias 13 days after receiving ATG for acute cellular rejection. Laboratory findings included C-reactive protein elevation, depressed levels of C3 and C4 complement, and strongly positive titers against rabbit immunoglobulin G, all strongly suggestive of serum sickness. To our knowledge, this is the first report of delayed serum sickness related to rabbit ATG after prior rabbit exposure in an adult heart transplantation patient. Early recognition of the symptoms of serum sickness can lead to prompt and appropriate management.

Micafungin alone and in combination therapy with deferasirox against Pythium insidiosum.

This study evaluated the in vitro and in vivo activity of micafungin alone and in combination with the iron chelator deferasirox against Pythium insidiosum. Micafungin showed a poor in vitro activity when it was used alone, but synergistic interactions were observed for 88.2% of the strains when the drug was combined with deferasirox. Smaller lesions were observed in infected rabbits receiving the combination therapy, although it favored disease dissemination to the lungs. The present results show that micafungin alone is ineffective against P. insidiosum, and the combination micafungin-deferasirox might have deleterious effects for the host.

Alpha-adrenoreceptors and muscarine receptors in human pial arteries and microvessels: a receptor binding study.

Human pial arteries and intraparenchymal microvessels were isolated for enzyme assays and radioligand binding studies of receptors. Special attention was paid to contamination with brain tissue, which was assessed by luxol staining and cerebroside assays for myelin and by scanning electron microscopy. The amount of contamination was approximately 1% for pial vessels and 14% for microvessel preparations. Significant levels of alpha 1-adrenoreceptors (binding sites for [3H]prazosin) and alpha 2-adrenoreceptors (sites labeled by [3H]azidoclonidine) were found in both types of vessels, suggesting that each receptor can modify contractility in these human vessels. Levels of muscarine receptors (sites labeled with [3H]quinuclidinyl benzilate) and choline acetyltransferase activity were considered significant only in pial vessels.

Effect of chronic cholinergic denervation on the m1 muscarinic receptor mechanism.

The first three parts of the transduction mechanism for m1 muscarinic receptors (m1 receptors, receptor-G protein coupling, and the activation of phospholipase C) were studied in the rat hippocampus following unilateral or bilateral surgical lesions of the fimbria/fornix. One nM 3H-pirenzepine was used to label m1 receptors selectively. No changes in m1 receptor numbers were found between age 1.7 and 29 months old during normal aging or one year after cholinergic denervation. The interaction between m1 receptors and their associated G protein was examined by competition between 1 nM 3H-pirenzepine and oxotremorine-M in the presence and absence of a guanine nucleotide. The percentage of guanine nucleotide-sensitive high affinity binding sites for the agonist was similar in rats 1.7-29 months old and in rats 1 year after denervation. The ability of oxotremorine-M to activate phospholipase C, via m1, m3, and m5 receptors was also unchanged more than a year after cholinergic denervation of the hippocampus. We concluded that the initial steps in the m1 receptor transduction mechanism remain remarkably stable after denervation.

Dopamine D2 receptors in the striatum and frontal cortex following chronic administration of haloperidol.

The effects of chronic treatment with a neuroleptic on D2 dopamine receptors in the striatum and frontal cortex were studied. Exposure to haloperidol for 21 days caused an upregulation in the striatum but not in the cortex of D2 receptors. These results indicate that dopamine-regulating mechanisms in the cortex may differ from those in the striatum and suggest that the anti-psychotic action of neuroleptics may be due in part to blockade of receptors in the cortex.

Autoradiographic localization of M1 and M2 muscarine receptors in the rat brain.

The distribution of M1 and M2 muscarine receptors in the rat brain was investigated by in vitro autoradiography. Muscarine receptors were visualized after complete receptor uncoupling in ethylenediaminetetraacetic acid buffer containing 1 mM N-ethyl maleimide and saturation with the ligand [3H]quinuclidinyl benzilate. Pirenzepine, an M1-selective antagonist, was used in our assays as a counter ligand to occlude M1 sites, allowing the primary ligand, [3H]quinuclidinyl benzilate, to label the remaining M2 muscarine receptors. In adjacent section, M1 muscarine receptors were labelled with [3H]quinuclidinyl benzilate in the presence of sufficient carbachol, and M2-selective agonist, to inhibit the binding to M2 sites. Our results reveal a heterogeneous distribution of M1 and M2 receptors. Increased densities of carbachol-resistant and pirenzepine-sensitive sites (M1 receptor subtype) were apparent over many forebrain structures including the olfactory tubercle, caudate-putamen, nucleus accumbens, hippocampus, amygdala and cerebral cortex. In contrast, pirenzepine-resistant and carbachol-sensitive sites (M2 receptor subtype) were distributed throughout the brain with increased densities apparent over regions known to contain large numbers of cholinergic cell bodies. M2 receptor localization patterns were largely coincident with the regional distribution and intensity of acetylcholinesterase positive sites. Since the M2 receptor pattern appears to parallel regional innervation densities, we conclude that the M2 receptor may serve as a marker for cholinergic pathways. The findings also suggest that M1 muscarine receptors are involved in the presumptive postsynaptic actions of acetylcholine in many forebrain structures.

Cardiac beta-adrenoceptors during normal growth of male and female rats.

1 A binding assay involving (-)-[3H]dihydroalprenolol (DHA) and KCl-washed cardiac membranes were used to assess the numbers and affinities of beta-adrenoceptors in hearts from male and female rats varying in age from about 2 weeks to 18 months. 2 Although female rats grow more slowly and attain lower adult weights than male rats, heart weights increased in approximate proportion to body weight with little sex difference. 3 As heart weight increased about three fold, beta-receptors increased three fold. Since the number of myocardial cells is believed to be nearly constant during postnatal growth, the numbers of receptors/cell presumably increases similarly. 4 As heart weight increased, the number of beta-receptors per g of tissue decreased according to the equation: total pmol/g = 4.33 - 1.43 x heart weight, equally in males and females. 5 Dissociation constants for DHA (2 to 4 nM) remained the same, and equal, in male and female rats during their growth. 6 An excellent correlation was found between the decline in beta-receptors/g tissue during growth and the decline in the area of the external sarcolemma/g tissue. The data suggest that the number of receptors per unit area remains constant during growth, and thus that cell surface area is a major factor determining normal numbers of receptors per cardiocyte.

Effect of propranolol on beta-adrenoceptors in rat hearts.

The question of whether the chronic administration of propranolol modifies the numbers or properties of cardiac beta-adrenoceptors was examined because of many reports suggestive of cardiac hypersensitivity following the withdrawal of beta-antagonists. In three studies, rats were given (+/-)-propranolol, 30 mg/day, orally or intraperitoneally, for 1 to 7 weeks. The numbers and affinities of specific binding sites for radioactive dihydroalprenolol in whole hearts from 60 control and 75 test animals were found to be the same. Thus catecholamine deprivation does not exert an important regulatory effect on most beta-receptors in the heart, as it does in the brain.

Distribution and function of beta-adrenoceptors in different chambers of the canine heart.

1 An improved binding assay involving (-)-[(3)H]-dihydroalprenolol (DHA) and KCl-washed cardiac membranes was developed to study beta-adrenoceptors in the canine heart quantitatively.2 Receptor numbers varied from 3.8 to 7.1 pmol/g fresh tissue, showing a steady increase from left atrium --> right atrium --> right ventricle --> interventricular septum --> left ventricle. With one minor exception, the same pattern was found for adenylate cyclase activity and Na(+), K(+)-activated ATPase activity.3 The binding of DHA was inhibited in the expected manner by beta-adrenoceptor agonists and antagonists, and was stereospecific, in confirmation of previous studies. Dissociation constants determined from Scatchard analyses included DHA: 2.5 nM; (-)adrenaline: 230 nM; (-)noradrenaline: 1167 nM. Kinetic analyses of the binding of DHA yielded a K(D) of about 4 nM.4 The distribution of beta-receptors is closely related to that of blood flow and the arrival plus retention of a circulating catecholamine, but is markedly different from that of endogenous noradrenaline, and thus adrenergic nerve terminals. Most receptors thus appear not at synapses but diffusely localized where they can react with circulating adrenaline.5 Evidence is discussed that beta-receptors at synapses respond primarily to neural noradrenaline, less to circulating adrenaline, and hardly at all to circulating noradrenaline; responses mediate increased cardiac output during exercise. In contrast most cardiac beta-receptors appear to respond only to adrenaline, and to be used, except at times of severe circulatory stress, during psychological stress.