Imaging Brain Activity With Voltage- and Calcium-Sensitive Dyes

This paper presents three examples of imaging brain activity with voltage- or calcium-sensitive dyes and then discusses the methodological aspects of the measurements that are needed to achieve an optimal signal-to-noise ratio.Internally injected voltage-sensitive dye can be used to monitor membrane potential in the dendrites of invertebrate and vertebrate neurons in in vitro preparations.Both invertebrate and vertebrate ganglia can be bathed in voltage-sensitive dyes to stain all of the cell bodies in the preparation. These dyes can then be used to follow the spike activity of many neurons simultaneously while the preparations are generating behaviors.Calcium-sensitive dyes that are internalized into olfactory receptor neurons in the nose will, after several days, be transported to the nerve terminals of these cells in the olfactory bulb. There they can be used to measure the input from the nose to the bulb.Three kinds of noise are discussed. a. Shot noise from the random emission of photons from the preparation. b. Vibrational noise from external sources. c. Noise that occurs in the absence of light, the dark noise.Three different parts of the light measuring apparatus are discussed: the light sources, the optics, and the cameras.The major effort presently underway to improve the usefulness of optical recordings of brain activity are to find methods for staining individual cell types in the brain. Most of these efforts center around fluorescent protein sensors of activity.     

Expression of nitric oxide synthase isoforms in the mouse kidney: cellular localization and influence by lipopolysaccharide and Toll-like receptor 4

We determined the cellular mRNA expression of all intrarenal nitric oxide (NO)-producing NO synthase (NOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS) and inducible NOS (iNOS) in kidneys from wild-type mice (WT) and immune deficient Toll-like receptor 4 (TLR4) mutant mice, during normal physiological conditions and during a short-term (6–16 h) endotoxic condition caused by systemically administered lipopolysaccaride (LPS). Investigations were performed by means of in situ hybridization and polymerase chain reaction amplification techniques. In WT, LPS altered the expression rate of all intrarenal NOS isoforms in a differentiated but NOS-isoform coupled expression pattern, with iNOS induction, and up- and down-regulation of the otherwise constitutively expressed NOS isoforms, e.g. eNOS and nNOS and an iNOS isotype. In TLR4 mutants, LPS caused none or a lowered iNOS induction, but altered the expression rate of the constitutive NOS isoforms. It is concluded that the intrarenal spatial relation of individual NOS-isoforms and their alteration in expression provide the basis for versatile NO-mediated renal actions that may include local interactions between NOS isoforms and their individual NO-target sites, and that the NOS-isoform dependent events are regulated by TLR4 during endotoxic processes. These regulatory mechanisms are likely to participate in different pathophysiological conditions affecting NO-mediated renal functions.     

Upregulation of major histocompatibility complex class I on liver cells by hepatitis C virus core protein via p53 and TAP1 impairs natural killer cell cytotoxicity

The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis C virus (HCV) are still unclear. Here, we present evidence for a cascade of molecular events that the virus initiates to subvert the innate immune attack. The HCV core protein induced p53-dependent gene expression of TAP1 (transporter associated with antigen processing 1) and consecutive major histocompatibility complex (MHC) class I upregulation. Moreover, in p53-deficient liver cell lines, only reconstitution with wild-type p53, but not mutated p53 lacking DNA binding capacity, showed this effect. As a consequence of increased MHC class I expression, a significantly downregulated cytotoxic activity of natural killer (NK) cells against HCV core-transfected liver cells was observed, whereas lysis by HCV-specific cytotoxic T cells was not affected. These results demonstrate a way in which HCV avoids recognition by NK cells that may contribute to the establishment of a chronic infection.     

Identification of essential regions in the cytoplasmic tail of interleukin-1 receptor accessory protein critical for interleukin-1 signaling

Interleukin (IL)-1 plays an important role in inflammation and regulation of immune responses. The activated IL-1 receptor complex, which consists of the IL-1 receptor type I and the IL-1 receptor accessory protein (IL-1RAcP), generates multiple cellular responses including NF-kappaB activation, IL-2 secretion, and IL-2 promoter activation. Reconstitution experiments in EL4D6/76 cells lacking IL-1RAcP expression and IL-1 responsiveness were used to analyze structure-function relationships of the IL-1RAcP cytoplasmic tail. Mutating a potential tyrosine kinase phosphorylation motif and various conserved amino acid (aa) residues had no effect on IL-1 responsiveness. Truncation analyses revealed that box 3 of the TIR domain was required for NF-kappaB activation, IL-2 production, and c-Jun N-terminal kinase (JNK) activation, whereas IL-2 promoter activation was only partially inhibited. Surprisingly, deletion of aa 527-534 resulted in almost complete loss of all IL-1 responsiveness. Replacement of these aa with alanyl residues did not reconstitute NF-kappaB activation, IL-2 production, or JNK activation but partly restored IL-2 promoter activation. Immunoprecipitation data revealed a strong correlation between MyD88 binding with NF-kappaB activation and IL-2 production but not with IL-2 promoter activation. Taken together, our data indicate that box 3 of IL-1RAcP is critical for IL-1-dependent NF-kappaB activation and stabilization of IL-2 mRNA via JNK, whereas aa 527-534 largely contribute to IL-2 promoter activation.     

Colonization of germ-free transgenic mice with genotyped Helicobacter pylori strains from a case-control study of gastric cancer reveals a correlation between host responses and HsdS components of type I restriction-modification systems

Helicobacter pylori infects the stomachs of half of all humans. It has a relatively benign relationship with most hosts but produces severe pathology, including gastric cancer, in others. Identifying the relative contributions of host, microbial, and environmental factors to the outcome of infection has been challenging. Here we describe one approach for identifying microbial genes that affect the magnitude of host responses to infection. Single colony purified H. pylori isolates were obtained from 25 cases and 71 controls in a Swedish case-control study of gastric cancer. Strains were first phenotyped based on their ability to produce adhesins that recognize two classes of human gastric epithelial receptors. Thirteen binding strains and two non-binding controls were then subjected to whole genome genotyping using H. pylori DNA microarrays. A cohort of "variable" genes was identified based on a microarray-determined call of "absent" in at least one member of the strain panel. Each strain was subsequently introduced into two types of germ-free transgenic mice, each programmed to express a different host factor postulated to pose increased risk for development of severe pathology. Expression of biomarkers of host defense was quantitated 4 weeks after inoculation, and the magnitude of the response correlated with bacterial genotype. The proportion of genes encoding HsdS homologs (specificity subunit of hetero-oligomeric type I restriction-modification systems) was significantly higher in the pool of 18 variable genes whose presence directly correlated with a robust host response than their proportion in the remaining 352 members of the variable gene pool. This suggests that the functions of these HsdS homologs may include control of expression of microbial determinants that affect the extent of gastric responses to this potentially virulent pathogen.     

Cholesterol as stabilizer of the oxytocin receptor

The function of the oxytocin receptor system is strongly dependent on steroids as demonstrated by several physiological studies. One key element of this dependence on steroids may be the interaction of cholesterol and the oxytocin receptor. In this study, we show that cholesterol stabilizes the solubilized human oxytocin receptor against thermal inactivation and proteolytic degradation. In the absence of additional cholesterol, the soluble receptor inactivates within minutes. Maximal stabilization of the oxytocin receptor requires a continuous supply with cholesterol from a cholesterol-rich environment. A structure-activity analysis of various cholesterol analogues and their effect on the thermal stability of the oxytocin receptor showed that the stabilizing function of cholesterol was highly specific. The structural requirements of a potent stabilizing steroid are very similar to those necessary to support the high-affinity state of the receptor. Moreover, in the presence of cholesterol, the oxytocin receptor is significantly more stable against alterations of pH value (pH 4-12). The results show that cholesterol acts as a general stabilizer of the oxytocin receptor.     

Effect of mineralocorticoid deficiency on ion and urea transporters and aquaporin water channels in the rat

Mineralocorticoid deficiency is associated with impaired urinary concentration and dilution. The present investigation was undertaken to determine the effects of selective mineralocorticoid deficiency on renal sodium and urea transporters and aquaporin water channels and whether these perturbations can be reversed by maintenance of extracellular fluid volume. Mineralocorticoid deficiency was induced by bilateral adrenalectomies with glucocorticoid replacement. Mineralocorticoid deficient rats receiving plain drinking water (MDW) were compared with mineralocorticoid deficient rats receiving saline-drinking water (MDS) in order to maintain extracellular fluid volume, and with controls (CTL). In MDW rats, there was a significant decrease in renal outer medulla Na-K-2Cl co-transporter and outer medulla Na-K-ATPase as well as an increase in inner medulla aquaporins 2 and 3. There were no significant changes in aquaporin-1, aquaporin-4, or urea transporters. These alterations were reversed with maintenance of extracellular fluid volume in MDS rats. Our findings indicate that mineralocorticoid deficiency in the rat is associated with alterations in factors involved in the countercurrent concentrating mechanism (Na-K-2Cl, Na-K-ATPase) and osmotic water equilibration in the collecting duct (AQP2, AQP3). Maintenance of sodium balance and extracellular fluid volume is associated with normalization of these perturbations.     

Mechanisms by which a Very-Low-Calorie Diet Reverses Hyperglycemia in a Rat Model of Type 2 Diabetes

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Inhibition of interleukin 2 production by prostaglandin E2 is not absolute but depends on the strength of the stimulating signal

In view of the eminently important role of interleukin-2 (IL-2) in T-cell responses, and in view of reports about immune stimulatory effects of PGE2, we reinvestigated the question whether PGE2 inhibits IL-2 production. It was found that PGE2 does not inhibit IL-2 production in murine spleen cell cultures after optimal stimulation (5 micrograms/ml concanavalin A) but does inhibit at suboptimal stimulation conditions. The failure of PGE2 to inhibit IL-2 production at optimal concanavalin A concentration was demonstrated by two independent IL-2 assays namely by the co-stimulator assay and by the proliferation of IL-2-dependent T-cell clone W-2. Our observations indicated that the inhibitory effect of PGE2 depends on the strength of the stimulating signal. IL-2 production in cultures with 5 micrograms/ml concanavalin A was also not suppressed by PGE1, by prostaglandin D2, thromboxane B2 (T X B2), and prostaglandin F2.