Effects of salinity on antibiotic production in sponge-derived Salinispora actinobacteria.

AIMS: To investigate the effects of growth conditions related to marine habitat on antibiotic production in sponge-derived Salinispora actinobacteria. METHODS AND RESULTS: Media with varying salt concentration were used to investigate the effects of salinity in relation to Salinispora growth and rifamycin production. The chemotypic profiles of the model strain Salinispora arenicola M413 was then assessed using metabolomic fingerprints from high-pressure liquid chromatography with diode array detection (HPLC-DAD) and multivariate data analysis, before extending this approach to two other strains of S. arenicola. Fingerprint data were generated from extracts of S. arenicola broth cultures grown in media of varying salt (NaCl) concentrations. These fingerprints were then compared using multivariate analysis methods such as principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). From the analysis, a low-sodium growth condition (1% NaCl) was found to delay the onset of growth of the model S. arenicola M413 strain when compared to growth in media with either 3% artificial sea salt or 3% NaCl. However, low-sodium growth conditions also increased cell mass yield and contributed to at least a significant twofold increase in rifamycin yield when compared to growth in 3% artificial sea salt and 3% NaCl. CONCLUSIONS: The integration of HPLC-DAD and multivariate analysis proved to be an effective method of assessing chemotypic variations in Salinispora grown in different salt conditions, with clear differences between strain-related chemotypes apparent due to varying salt concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The observed variation in S. arenicola chemotypic profiles further suggests diversity in secondary metabolites in this actinomycete in response to changes in the salinity of its environment.

Effects of l-arginine and N(G)-nitro-l-arginine methyl ester treatments on expression of neuronal nitric oxide synthase in the guinea-pig bladder after partial bladder outlet obstruction.

This study was aimed to examine the effects of pharmacological intervention on partial bladder outlet obstruction (PBOO) on expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production and NO-related free radical damage using nitrotyrosine as a marker in the guinea-pig bladder. Partial urethral ligation was performed in young male guinea pigs which were then intraperitoneally administered l-arginine, N(G)-nitro-l-arginine methyl ester (l-NAME) or vehicle (saline) for 2 or 4 weeks. At the respective time points, the bladder was removed for nNOS immunohistochemistry, Western blot analysis, nitrotyrosine enzyme-linked immunosorbent assay test and NO colorimetric assay. In l-arginine-treated animals killed at 2 and 4 weeks, the total number of nNOS positive intramural neurons was significantly increased when compared with the corresponding control. Some neurons projected long extending fibers that were closely associated with the blood vessels. Furthermore, at 4 weeks, the nNOS protein content and NO production as reflected by the concentration of nitrite and nitrate were drastically elevated as measured by Western blot analysis and NO colorimetric assay, respectively. In l-NAME-treated group killed at 2 weeks, the number of nNOS positive neurons was markedly reduced when compared with the controls, but the change was not significant at 4 weeks. In the latter, however, the NO production as reflected by the concentration of nitrite and nitrate was markedly reduced; in addition, the nitrotyrosine concentration was significantly lower than the control. The present results support the role of NO in the pathophysiological changes following PBOO. We suggest the potential therapeutic application of l-arginine and l-NAME in PBOO; however, ultimately balancing the bidirectional effects of NO would be essential.

Effect of maternal age, birth weight and infant sex on total nucleated cell (TNC) count and volume of umbilical cord blood (UCB) collected.

This study evaluates the effect of maternal age, birth weight and infant sex on two main UCB parameters for use and long-term cryopreservation: TNC and volume. Data from 1000 UCB units were collected and analyzed in this study. The results indicate that TNC is correlated to infant birth weight and sex but not maternal age at delivery. Volume is only correlated to birth weight but not maternal age and infant sex.

Trophoblast-specific transcription from the mouse placental lactogen-I gene promoter.

We have isolated the gene encoding mouse placental lactogen-I and characterized the promoter region of this gene by transient and stable transfection. Promoter sequences extending 274 basepairs (bp) up-stream from the start site of transcription contain all of the elements necessary for maximal expression upon transient transfection into the rat choriocarcinoma Rcho-1 cell line; these Rcho-1 cultures contain both proliferative trophoblast stem cells and terminally differentiated trophoblast giant cells. In stably transfected cell lines, expression from this promoter increases as the percentage of differentiated cells in the culture increases. In contrast to these results in trophoblast cells, the 274-bp promoter as well as a promoter region extending 2700 bp up-stream of the transcriptional start site are unable to drive transcription in a variety of other cell types. Mutational and protein binding analyses indicate that two AP-1 sites are required for maximal expression in Rcho-1 cells, and that the composition of the AP-1 transcription factor may vary as differentiation in the cell culture increases. In addition to these two AP-1 sites, at least one other element appears to be critical for promoter activity in trophoblast cells.

Factors associated with psychosis among patients with severe acute respiratory syndrome: a case-control study.

We observed that a number of patients with severe acute respiratory syndrome (SARS) developed affective psychosis during the acute phase of their illness. We reviewed all SARS-related psychiatric consultations in Hong Kong and investigated the risk factors for psychosis among patients with SARS in a matched case-control study. Patients with SARS-related psychosis received higher total doses of steroids and had higher rates of family history of psychiatric illness. The findings of the present study suggest that steroid toxicity, personal vulnerability, and, probably, psychosocial stressors jointly contributed to the development of psychosis in patients with SARS.

Permanent occlusion of the middle cerebral artery upregulates expression of cytokines and neuronal nitric oxide synthase in the spinal cord and urinary bladder in the adult rat.

The expression pattern of proinflammatory cytokines, neuronal nitric oxide synthase (nNOS), substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord and the bladder in response to permanent middle cerebral artery occlusion (MCAO) was investigated. In this connection, the gene expression of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and interleukin-6 in the lumbosacral spinal cord and the bladder as determined by real-time polymerase chain reaction was upregulated. In the spinal cord, the immunoreactivity of TNF-alpha and IL-1beta was mainly localized in the ventral horn motoneurons contralateral to MCAO. In the bladder, TNF-alpha was mainly expressed in the inflammatory cells. The expression of nNOS immunoreactivity as well as nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining in the spinal cord and bladder was also markedly increased in response to MCAO. Furthermore, the temporal and spatial expression of nNOS paralleled that of TNF-alpha and IL-1beta in the spinal cord. On the other hand, there was no noticeable change in gene expression and immunoreactivity of SP and CGRP. The present results have shown that cytokines and nNOS expression are elevated in areas far removed from the primary site of ischemic infarct, namely, the lumbosacral spinal cord and bladder. This together with some neuronal deaths maybe linked to the dysfunction of the latter in a clinical stroke. On the other hand, the apparent lack of SP and CGRP changes following MCAO suggests that the two neurotransmitters are not directly involved.

Cytokine changes in the horizontal diagonal band of Broca in the septum after running and stroke: a correlation to glial activation.

The relationship between running, glial cell activation and pro-inflammatory cytokines was studied in the context of neuroprotection against ischemic stroke induced by middle cerebral artery occlusion (MCAO). This was investigated in four groups of rats, namely, (1) nonrunner, (2) runner after 12 weeks of treadmill running, (3) nonrunner with MCAO and (4) runner with MCAO. The horizontal diagonal band of Broca (HDB) in the septum was scrutinized for qualitative cum quantitative changes in the microglia and astrocytes. Reverse transcription-polymerase chain reaction and immunoblot work were carried out in the forebrain homogenate to determine, respectively, the gene and protein expression of several pro-inflammatory cytokines. Our results indicated that the runner exhibited less immunoreactivity and reduced numbers of glial cells within the HDB compared with the nonrunner. Interestingly, the mRNA and protein levels of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and interferon-gamma, were significantly downregulated in the runner. Our data also suggest albeit with some inconsistency that the runner/MCAO rats had benefited from running. These observations suggest that running can result in changes to the microenvironment, in which the microglia and astrocytes exist in a state of quiescence concomitant with a reduced expression of pro-inflammatory cytokines, that may lead to beneficial effects seen in ischemic stroke induced by MCAO.

Cortical expression of endothelin receptor subtypes A and B following middle cerebral artery occlusion in rats.

This work aimed to define the spatial expression of endothelin A (ET(A)) and B (ET(B)) receptors in the cerebral cortex after permanent middle cerebral artery occlusion (MCAO) and to identify the phenotype of cells expressing ET(A) and ET(B) receptors. Cortical expression of ET(A) and ET(B) receptors was determined at the mRNA level by semi-quantitative reverse transcription-polymerase chain reaction and at the protein level by immunofluorescence staining, 12, 24 and 72 h after MCAO. Cells expressing endothelin receptors were phenotyped by double labelling with antibodies, anti-protein gene product (PGP9.5) and anti-ED1, towards neurons and activated microglia/macrophages, respectively. Both ET(A) and ET(B) receptor mRNA expressions increased significantly in the ipsilateral cortex in a time-dependent manner after MCAO. Robust expression of ET(A) receptors was noted in most neurons of the ischemic core and in several neurons in laminae 3 and 4 of the peri-infarct region 24 and 72 h after MCAO. ET(B) receptor immunoreactivity was observed in activated microglia/macrophages, beginning 24 h after MCAO. These results provide the first evidence that the action of endothelin during ischemia may be mediated by neuronal ET(A) receptors and activated microglia/macrophage ET(B) receptors. This differential localization of ET(A) and ET(B) receptors suggests that endothelin is involved in some complex neuron-glial interactions in addition to its vascular modulatory activity during ischemia.

Neuroprotection associated with running: is it a result of increased endogenous neurotrophic factors?

The possible neuroprotective effect of physical exercise was investigated in rats after middle cerebral artery occlusion (MCAO), a focal stroke model. It was found that physical exercise in the form of a 12-week treadmill running programme reduced the volume of infarction caused by MCAO. At the molecular level, reverse transcription polymerase chain reaction revealed that the runner had increased gene expression for nerve growth factor (NGF) over the nonrunner with or without MCAO. Expression of the NGF receptors, p75, was increased only in the absence of MCAO. In addition, runners showed a significantly higher number of cholinergic neurons, which constitutively expressed p75, in the horizontal diagonal band of Broca. The present findings suggest that neuroprotection after physical exercise may be a result of an increase in an endogenous neurotrophic factor nerve growth factor and the proliferation of its receptive cholinergic neurons.

Light and retinopathy of prematurity: does retinal location offer a clue?

Nursery illumination has been implicated in the pathogenesis of retinopathy of prematurity (ROP), although the results of recent studies are conflicting. The data base for this article is a prospective ROP study on 607 infants of birth weight less than or equal to 1700 g including 35 larger siblings from multiple births when 1 infant fulfilled the birth weight criteria. Retinopathy commences preferentially in the nasal retina of the most immature neonate and is less likely to develop, or its onset is delayed, in the superior and inferior regions. These findings cannot be fully accounted for by regional vascular and neuroanatomical variations. Radiometric and physiological evidence suggests that the very immature neonate, most at risk of developing severe ROP, receives the greatest retinal irradiance. Furthermore, ROP commences in the areas of the retina receiving the highest light dose, and its onset is either retarded or inhibited in the darker retinal regions. Further studies are required to determine whether early exposure to light is a factor in the development of ROP. If a causal relationship is proven, here at least is one modality that can easily and immediately be controlled.

Acute inhibition of DNA synthesis in hamster buccal pouch epithelium exposed to indirect acting carcinogens.

Inhibition of DNA synthesis was observed and quantitated in hamster buccal pouch epithelium exposed in vivo and in vitro to indirect acting carcinogens. Topical application of a 0.5% solution of the potent hamster buccal pouch carcinogen 7,12-dimethylbenz[a]-anthracene (DMBA) acutely inhibited epithelial DNA synthesis by 40-65%, as indicated by a decrease in [3H]thymidine incorporation over a period of 24 h. When applied twice at a concentration of 2%, N-methyl-N-benzylnitrosamine (MBN), another potent buccal pouch carcinogen, inhibited epithelial DNA synthesis by 76% within a period of 4 h. A similar acute inhibitory effect on DNA synthesis was observed when explants of buccal pouch mucosa, exhibiting continuous cell replication, were exposed in vitro in the presence of MBN or DMBA for periods up to 12 and 24 h, respectively. The inhibitory effect of DMBA was greater than that of other polycyclic aromatic hydrocarbons of lesser carcinogenic potency in this tissue. This study demonstrates that the metabolic activation of indirect acting carcinogens leading to acute cytotoxicity and inhibition of DNA synthesis occurs rapidly in hamster buccal pouch mucosa exposed to these agents in vitro as well as in vivo. The experimental imposition of an acute inhibitory pressure, applied as demonstrated in this report, may enable the detection of precancerous cells which have acquired the property of resistance to this cytotoxic effect in the course of carcinogenesis. In principle, the in vitro approach, coupled with autoradiography, may be useful in identifying microscopic foci of resistant preneoplastic cells in samples of human oral mucosa. 24R01 34160

Microglial reaction in focal cerebral ischaemia induced by intra-carotid homologous clot injection.

This study examined the microglial reaction in a simulated thrombo-embolus ischaemia in rats given an intracarotid injection of a suspension of homologous blood clot. All rats including the controls receiving vehicle injection were perfused at 5 hours, and 1, 3 and 7 days post-operation. The brains were removed and processed for immunohistochemistry using a panel of monoclonal antibodies: OX-42, OX-18 and OX-6 for labeling of microglia. In rats given saline injection OX-42 immunoreactive microglial cells were observed to be distributed quite evenly throughout the whole brain. When injection of clot suspension was given, microglial cells responded vigorously, particularly in the ipsilateral hippocampus. Microglial reaction was also detected in the ipsilateral cerebral cortex, caudate as well as septal nuclei. The majority of the detected reactive microglial cells were hypertrophied showing thick or stout processes. Some rod-like and amoeboid microglia were also observed. Rarely did the reactive microglia express OX-6 immunoreactivity. All microglial cells were unreactive for OX-18. The actual mechanisms leading to the microglial activation as well as functions of reactive microglia in focal cerebral ischaemia remain speculative. In the absence of direct evidence, it could only be suggested that they may act as sensor cells for detection of subtle alterations in the microenvironment, probably in response to focal ischaemia and/or leakage of serum-derived factors induced by thrombo-embolus stroke.

An electron microscopic study of neuronal degeneration and glial cell reaction in the retina of glaucomatous rats.

The present investigation was focused on the ultrastructural changes in the neurons and glial cells in the retina of rats with experimentally-induced glaucoma. An experimental glaucoma model was created by limbal-derived vein cauterization. Animals were sacrificed at 1, 3 weeks and 3 months post-operation. Retinae were dissected and processed for electron microscopy. Neuronal degeneration was observed in all the different layers of the retina at both 1 and 3 weeks post-operation. Some degenerating neurons were found in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL). And the dying neurons presented apoptotic-like more than necrotic neurons. Many degenerating axons and axon terminals were observed between neurons in the GCL, inner plexiform layer (IPL), INL, and outer plexiform layer (OPL). Activated astrocytes and microglial cells were present in close association with degenerating neurons and axons. The Müller cells in the INL also presented longer and darker processes with more microfilaments than in normal cells. Degenerating neuronal debris, degenerating axonal profiles and electron-dense bodies were often found in the cytoplasm of macrophages. The results suggest that both microglial cells and astrocytes are activated in the process of neuronal degeneration in the retina of experimentally-induced glaucomatous rats. It is hypothesized that they may play a protective role in removing degenerating neuronal elements in the retina after the onset of glaucoma.

Induction of major histocompatibility class II antigen on microglial cells in postnatal and adult rats following intraperitoneal injections of lipopolysaccharide.

Microglial cells, notably the ramified form, were induced to express major histocompatibility complex (MHC) class II antigen in postnatal and adult rats given intraperitoneal injections of lipopolysaccharide (LPS). The immunoreactive microglia which occurred in cell colonies or clusters were detected immunohistochemically with the monoclonal antibody OX-6. Some of the widely distributed MHC II positive cells were round or amoeboidic located preferentially in the perivascular area. In view of the widespread occurrence of microglial cells showing OX-6 immunoreactivity which is negligible in normal animals, it is suggested that the effect of LPS on microglia in vivo is a widespread phenomenon and is independent of age. It is suggested that the endotoxin not only triggers off the immunological potentiality of these cells but also elicits the entry of some mononuclear cells into the brain parenchyma.

Induction of microglial reaction and expression of nitric oxide synthase I in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection.

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy.

Nitric oxide, microglial activities and neuronal cell death in the lateral geniculate nucleus of glaucomatous rats.

The present study was initiated to investigate neuronal degeneration, microglial reactivity and possible roles of NO in the lateral geniculate nucleus (LGN) of glaucomatous rats. An experimental one-eye glaucoma model was created by cauterization of the limbal-derived veins. Neuronal cell viability was studied by immunostaining with antibody against neuronal nuclei. Changes of expressions of nitric oxide synthase I (NOS I), NOS II, ED 1, OX6 and OX42 in the LGN were studied by immunohistochemistry. NADPH-d histochemistry was also employed. In the experimental glaucomatous rats, the number of NeuN labelled neurons was significantly decreased in both the ipsi- and contra-lateral sides of the ventral LGN (vLGN) but not the dorsal LGN (dLGN) at 1 month post-operation and beyond. Expressions of NOS I and NADPH-d were notably increased from 1 week post-operation in the ipsilateral vLGN. In the contralateral side of the vLGN, however, this change was only observed from 1 month post-operation. No NOS II immunoreaction was observed in LGN of both the normal control and glaucomatous rats. Increased microglial reactivity as indicated by OX-42 immunoreactivity was first observed in both sides of the LGN at 1 week post-operation, and this was most significant especially at 1 and 2 months post-operation. The present results suggest that NO and microglial cells may play some important roles in the pathologic processes of neuronal degeneration in the LGN of glaucomatous rats.

Expression of immunoregulatory cytokines in neurons of the lateral hypothalamic area and amygdaloid nuclear complex of rats immunized against human IgG.

Present results showed that interleukin-1 (IL-1), IL-6 and transforming growth factor-beta (TGF-beta) were constitutively expressed in the supraoptic and paraventricular nuclei of the rat hypothalamus. Immunoreactive cells were also detected, but to a lesser extent, in other parts of hypothalamus as well as in the cerebral cortex. In rats immunized with IgG, there was moderate increase in immunoreactivities of the cytokines. A notable feature, however, was the induction of the cytokine expression in the lateral hypothalamic area and the amygdaloid nuclear complex, suggesting that the neurons in these two areas are involved in possible immune regulation.

Positive and negative modulation by AMPA- and kainate-receptors of striatal kainate injection-induced neuronal loss in rat forebrain.

We investigated the roles of ionotropic glutamate receptor subtypes in mediating striatal kainate injection-induced neuronal loss in rat forebrain, using subtype-specific antagonists and histochemical staining. Our study demonstrates that kainate injected unilaterally into the striatum induces a massive neuronal loss in the rat ipsilateral forebrain through activation of kainate receptors and, to a limited extent, a consequent involvement of M-methyl-D-aspartate (NMDA) receptors, whereas activation of alpha-amino-3-hydroxy-5-methylisoxazol-4-propionate (AMPA) receptors shows a neuroprotective effect. These and previous results suggest that three subtypes of ionotropic glutamate receptors play differential roles in mediating excitatory amino acid (EAA)-induced neurodegeneration.

Emperipolesis of lymphoid cells in vagal efferent neurons following an intraneural injection of ricin into the vagus nerve in rats.

Injection of a minute amount of the toxic lectin, Ricinus communis agglutinin-60 (RCA-60) into the vagus nerve resulted in a selective destruction of the vagal efferent neurons in the ipsilateral dorsal motor nucleus (DMN). This has elicited a massive influx of mononuclear leucocytes, notably macrophages and T-lymphocytes, as detected with ED-1 and OX-19 antibodies, respectively. A small number of B-lymphocytes as identified by OX-33 antibody, were also observed in the neuropil of DMN. The influx of mononuclear leucocytes into the neuropil of DMN was by way of diapedesis, peaking in frequency at 4-6 days after the RCA administration. The infiltrated lymphocytes were closely associated with or penetrated the soma of the vagal neurons, some bearing intact axo-somatic synaptic contacts. The entrapped lymphocytes in neurons underwent degeneration and subsequently disintegrated. Macrophages and plasma cells in the neuropil did not appear to penetrate the neuronal soma. It is concluded that emperipolesis of lymphocytes, presumably cytotoxic T-cells, in RCA-poisoned neurons may represent a form of effector-target cell contact leading to cytotoxicity. In doing so, however, the invading lymphocytes were destroyed by the contents of RCA picked up by the neurons. The absence of macrophages and plasma cells in the RCA-poisoned neurons suggests the cellular specificity of emperipolesis.