Neurobiology of the sleep-wake cycle: sleep architecture, circadian regulation, and regulatory feedback

This mini-review article presents the remarkable progress that has been made in the past decade in our understanding of the neural circuitry underlying the regulation of sleep-wake states and circadian control of behaviors. Following a brief introduction to sleep architecture and physiology, the authors describe the neural circuitry and neurotransmitters that regulate sleep and cortical arousal (i.e., wakefulness). They next examine how sleep and wakefulness are regulated by mutual inhibition between sleep-and arousal-promoting circuitry and how this interaction functions analogously to an electronic "flip-flop" switch that ensures behavioral state stability. The authors then discuss the role of circadian and homeostatic processes in the consolidation of sleep, including the physiologic basis of homeostatic sleep drive (i.e., wake-dependent increase in sleep propensity) and the role of the SCN in the circadian regulation of sleep-wake cycles. Finally, they describe the hypothalamic circuitry for the integration of photic and nonphotic environmental time cues and how this integration allows organisms to sculpt patterns of rest-activity and sleep-wake cycles that are optimally adaptive.     

The need to feed: homeostatic and hedonic control of eating

Feeding provides substrate for energy metabolism, which is vital to the survival of every living animal and therefore is subject to intense regulation by brain homeostatic and hedonic systems. Over the last decade, our understanding of the circuits and molecules involved in this process has changed dramatically, in large part due to the availability of animal models with genetic lesions. In this review, we examine the role played in homeostatic regulation of feeding by systemic mediators such as leptin and ghrelin, which act on brain systems utilizing neuropeptide Y, agouti-related peptide, melanocortins, orexins, and melanin concentrating hormone, among other mediators. We also examine the mechanisms for taste and reward systems that provide food with its intrinsically reinforcing properties and explore the links between the homeostatic and hedonic systems that ensure intake of adequate nutrition.     

Identification of an Escherichia coli operon required for formation of the O-antigen capsule

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD, ymcC, ymcB, ymcA, yccZ, etp, and etk, which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli, and Shigella species possess an intact G4C operon.     

Structure of Hsp15 reveals a novel RNA-binding motif

We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome. Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif. This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA. The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site. An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif. This may imply a hitherto unrecognized functional similarity between these three protein classes.     

A guide to the perplexed on the specificity of antibodies

Many investigators are unaware of the potential problems with specificity of antibodies and the need to document antibody characterization meticulously for each antibody that is used. In this review, I consider the principles of antibody action and how they define a set of rules for what information should be obtained by the investigator before using an antibody in a serious scientific investigation.     

Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and anterograde tracing, and combination with immunohistochemistry

Tetramethylbenzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry is more sensitive than other chromogens. Its instability in aqueous solutions and ethanol, however, has limited its application. We now report a method for stabilizing TMB by incubation in combinations of diaminobenzidine (DAB)/cobalt (Co2+)/H2O2. The stabilized TMB product was unaffected by long-term exposures to ethanol, neutral buffers, and subsequent immunohistochemical staining procedures. A procedure is recommended for optimal stabilization of TMB that affords a sensitivity for demonstrating retrogradely labeled perikarya comparable to standard TMB histochemistry. The physical characteristics of the reaction product make it suitable for combination with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunohistochemical staining procedure. This was established by staining retrogradely labeled neurons in the basal forebrain with a monoclonal antibody against choline acetyltransferase. Because the stabilized TMB product exhibited a superior sensitivity over cobalt ion intensification of the DAB-based reaction product (DAB-Co), it offers a distinct advantage over previously described combination procedures.     

Effect of capsid confinement on the chromatin organization of the SV40 minichromosome

Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a ‘molten droplet’ state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement.