The activity of ribosome modulation factor during growth of Escherichia coli under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.

Intra-ovarian roles of activins and inhibins

Granulosa cells are the main ovarian source of inhibins, activins and activin-binding protein (follistatin) while germ (oogonia, oocytes) and somatic (theca, granulosa, luteal) cells express activin receptors, signaling components and inhibin co-receptor (betaglycan). Activins are implicated in various intra-ovarian roles including germ cell survival and primordial follicle assembly; follicle growth from preantral to mid-antral stages; suppression of thecal androgen production; promotion of granulosa cell proliferation, FSHR and CYP19A1 expression; enhancement of oocyte developmental competence; retardation of follicle luteinization and/or atresia and involvement in luteolysis. Inhibins (primarily inhibin A) are produced in greatest amounts by preovulatory follicles (and corpus luteum in primates) and suppress FSH secretion through endocrine negative feedback. Together with follistatin, inhibins act locally to oppose auto-/paracrine activin (and BMP) signaling thus modulating many of the above processes. The balance between activin-inhibin shifts during follicle development with activin signalling prevailing at earlier stages but declining as inhibin and betaglycan expression rise.

Carbon monoxide mediates the anti-apoptotic effects of heme oxygenase-1 in medulloblastoma DAOY cells via K+ channel inhibition

Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K(+) channels also play a crucial role in apoptosis, permitting K(+) efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K(+) channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K(+) currents in DAOY cells, and largely reversed the oxidant-induced increase in K(+) channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K(+) channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K(+) channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

Endocannabinoids in neuroendopsychology: multiphasic control of mitochondrial function

The endocannabinoid system (ECS) is a construct based on the discovery of receptors that are modulated by the plant compound tetrahydrocannabinol and the subsequent identification of a family of nascent ligands, the 'endocannabinoids'. The function of the ECS is thus defined by modulation of these receptors-in particular, by two of the best-described ligands (2-arachidonyl glycerol and anandamide), and by their metabolic pathways. Endocannabinoids are released by cell stress, and promote both cell survival and death according to concentration. The ECS appears to shift the immune system towards a type 2 response, while maintaining a positive energy balance and reducing anxiety. It may therefore be important in resolution of injury and inflammation. Data suggest that the ECS could potentially modulate mitochondrial function by several different pathways; this may help explain its actions in the central nervous system. Dose-related control of mitochondrial function could therefore provide an insight into its role in health and disease, and why it might have its own pathology, and possibly, new therapeutic directions.

Transcription factor NF-kappaB is transported to the nucleus via cytoplasmic dynein/dynactin motor complex in hippocampal neurons

Background Long-term changes in synaptic plasticity require gene transcription, indicating that signals generated at the synapse must be transported to the nucleus. Synaptic activation of hippocampal neurons is known to trigger retrograde transport of transcription factor NF-κB. Transcription factors of the NF-κB family are widely expressed in the nervous system and regulate expression of several genes involved in neuroplasticity, cell survival, learning and memory. Principal Findings In this study, we examine the role of the dynein/dynactin motor complex in the cellular mechanism targeting and transporting activated NF-κB to the nucleus in response to synaptic stimulation. We demonstrate that overexpression of dynamitin, which is known to dissociate dynein from microtubules, and treatment with microtubule-disrupting drugs inhibits nuclear accumulation of NF-κB p65 and reduces NF-κB-dependent transcription activity. In this line, we show that p65 is associated with components of the dynein/dynactin complex in vivo and in vitro and that the nuclear localization sequence (NLS) within NF-κB p65 is essential for this binding. Conclusion This study shows the molecular mechanism for the retrograde transport of activated NF-κB from distant synaptic sites towards the nucleus.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2 h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

G6f-like is an ITAM-containing collagen receptor in thrombocytes

Collagen activates mammalian platelets through a complex of the immunoglobulin (Ig) receptor GPVI and the Fc receptor γ-chain, which has an immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking of GPVI mediates activation through the sequential activation of Src and Syk family kinases and activation of PLCγ2. Nucleated thrombocytes in fish are activated by collagen but lack an ortholog of GPVI. In this study we show that collagen activates trout thrombocytes in whole blood and under flow conditions through a Src kinase driven pathway. We identify the Ig receptor G6f-like as a collagen receptor and demonstrate in a cell line assay that it signals through its cytoplasmic ITAM. Using a morpholino for in vivo knock-down of G6f-like levels in zebrafish, we observed a marked delay or absence of occlusion of the venous and arterial systems in response to laser injury. Thus, G6f-like is a physiologically relevant collagen receptor in fish thrombocytes which signals through the same ITAM-based signalling pathway as mammalian GPVI, providing a novel example of convergent evolution.

Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists.

Cardiac myocyte hypertrophy involves changes in cell structure and alterations in protein expression regulated at both the transcriptional and translational levels. Hypertrophic G protein-coupled receptor (GPCR) agonists such as endothelin-(ET-1) and phenylephrine stimulate a number of protein kinase cascades in the heart. Mitogen-activated protein kinase (MAPK) cascades stimulated include the extracellularly regulated kinase cascade, the stress-activated protein kinase/c-Jun N-terminal kinase cascade, and the p38 MAPK cascade. All 3 pathways have been implicated in hypertrophy, but recent ex vivo evidence also suggests that there may be additional effects on cell survival. ET-1 and phenylephrine also stimulate the protein kinase B pathway, and this may be involved in the regulation of protein synthesis by these agonists. Thus, protein kinase-mediated signaling may be important in the regulation of the development of myocyte hypertrophy.

Estrogens, brain and behavior: studies in fundamental neurobiology and observations related to women's health

Mechanisms and consequences of the effects of estrogen on the brain have been studied both at the fundamental level and with therapeutic applications in mind. Estrogenic hormones binding in particular neurons in a limbic-hypothalamic system and their effects on the electrophysiology and molecular biology of medial hypothalamic neurons were central in establishing the first circuit for a mammalian behavior, the female-typical mating behavior, lordosis. Notably, the ability of estradiol to facilitate transcription from six genes whose products are important for lordosis behavior proved that hormones can turn on genes in specific neurons at specific times, with sensible behavioral consequences. The use of a gene knockout for estrogen receptor alpha (ERalpha) revealed that homozygous mutant females simply would not do lordosis behavior and instead were extremely aggressive, thus identifying a specific gene as essential for a mammalian social behavior. In dramatic contrast, ERbeta knockout females can exhibit normal lordosis behavior. With the understanding, in considerable mechanistic detail, of how the behavior is produced, now we are also studying brain mechanisms for the biologically adaptive influences which constrain reproductive behavior. With respect to cold temperatures and other environmental or metabolic circumstances which are not consistent with successful reproduction, we are interested in thyroid hormone effects in the brain. Competitive relations between two types of transcription factors - thyroid hormone receptors and estrogen receptors have the potential of subserving the blocking effects of inappropriate environmental circumstances on female reproductive behaviors. TRs can compete with ERalpha both for DNA binding to consensus and physiological EREs and for nuclear coactivators. In the presence of both TRs and ERs, in transfection studies, thyroid hormone coadministration can reduce estrogen-stimulated transcription. These competitive relations apparently have behavioral consequences, as thyroid hormones will reduce lordosis, and a TRbeta gene knockout will increase it. In sum, we not only know several genes that participate in the selective control of this sex behavior, but also, for two genes, we know the causal routes. Estrogenic hormones are also the foci of widespread attention for their potential therapeutic effects improving, for example, certain aspects of mood and cognition. The former has an efficient animal analog, demonstrated by the positive effects of estrogen in the Porsolt forced swim test. The latter almost certainly depends upon trophic actions of estrogen on several fundamental features of nerve cell survival and growth. The hypothesis is raised that the synaptic effects of estrogens are secondary to the trophic actions of this type of hormone in the nucleus and nerve cell body.