Analysis of Biochemical Equilibria Relevant to the Immune Response: Finding the Dissociation Constants

This paper analyzes the biochemical equilibria between bivalent receptors, homo-bifunctional ligands, monovalent inhibitors, and their complexes. Such reaction schemes arise in the immune response, where immunoglobulins (bivalent receptors) bind to pathogens or allergens. The equilibria may be described by an infinite system of algebraic equations, which accounts for complexes of arbitrary size n (n being the number of receptors present in the complex). The system can be reduced to just 3 algebraic equations for the concentrations of free (unbound) receptor, free ligand and free inhibitor. Concentrations of all other complexes can be written explicitly in terms of these variables. We analyze how concentrations of key (experimentally-measurable) quantities vary with system parameters. Such measured quantities can furnish important information about dissociation constants in the system, which are difficult to obtain by other means. We provide analytical expressions and suggest specific experiments that could be used to determine the dissociation constants.     

Targeting tuberculosis and malaria through inhibition of Enoyl reductase: compound activity and structural data

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.     

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear β-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyrano-syl residues of which 80–85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N-acetylglucosaminyltransferase activity in the formation     

Phosphorylation of the Actin Binding Protein Drebrin at S647 Is Regulated by Neuronal Activity and PTEN

Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus - serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.     

Effects of varied air velocity on sweating and evaporative rates during exercise.

This study was designed to determine the extent to which changes in the evaporative power of the environment (Emax) affect sweating and evaporative rates. Six male subjects undertook four 60-min bouts of cycle ergometer exercise at 56% maximal O2 uptake (VO2max).Emax was varied by differences in ambient temperature and airflow; two exercise bouts took place at 24 degrees C and two at 35 degrees C, with air velocity at < 0.2 and 3.0 m/s in both. Total sweat production was estimated from body weight loss, whereas whole body evaporative rate was measured continuously from a Potter beam balance. Body core temperature was measured continuously from a thermocouple in the esophagus (T(es)), with mean skin temperature (Tsk) computed each minute from thermocouples at eight sites. Total body sweat loss was significantly greater (P < 0.05) in the 0.2- than in the 3.0-m/s condition at both 24 and 35 degrees C. Tsk was higher (P < 0.05) in the still-air conditions at both temperatures, but final T(es) was significantly higher (P < 0.05) in still air only in the 35 degrees C environment. Thus the reduced Emax in still air caused a greater heat storage, thereby stimulating a greater total sweat loss. However, in part because of reduced skin wettedness, the slope of the sweat rate-to-T(es) relation at 35 degrees C in the 3.0-m/s condition was 118% that at 0.2 m/s (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast differentiation

Placental development initially occurs in a low-oxygen (O2) or hypoxic environment. In this report we show that two hypoxia-inducible factors (HIFs), HIF1alpha and HIF2alpha, are essential for determining murine placental cell fates. HIF is a heterodimer composed of HIFalpha and HIFbeta (ARNT) subunits. Placentas from Arnt-/- and Hif1alpha-/- Hif2alpha-/- embryos exhibit defective placental vascularization and aberrant cell fate adoption. HIF regulation of Mash2 promotes spongiotrophoblast differentiation, a prerequisite for trophoblast giant cell differentiation. In the absence of Arnt or Hifalpha, trophoblast stem cells fail to generate these cell types and become labyrinthine trophoblasts instead. Therefore, HIF mediates placental morphogenesis, angiogenesis, and cell fate decisions, demonstrating that O2 tension is a critical regulator of trophoblast lineage determination. This novel genetic approach provides new insights into the role of O2 tension in the development of life-threatening pregnancy-related diseases such as preeclampsia.     

Cellular responses to disruption of the permeability barrier in a three-dimensional organotypic epidermal model

Repeated injury to the stratum corneum of mammalian skin (caused by friction, soaps, or organic solvents) elicits hyperkeratosis and epidermal thickening. Functionally, these changes serve to restore the cutaneous barrier and protect the organism. To better understand the molecular and cellular basis of this response, we have engineered an in vitro model of acetone-induced injury using organotypic epidermal cultures. Rat epidermal keratinocytes (REKs), grown on a collagen raft in the absence of any feeder fibroblasts, developed all the hallmarks of a true epidermis including a well-formed cornified layer. To induce barrier injury, REK cultures were treated with intermittent 30-s exposures to acetone then were fixed and paraffin-sectioned. After two exposures, increased proliferation (Ki67 and BrdU staining) was observed in basal and suprabasal layers. After three exposures, proliferation became confined to localized buds in the basal layer and increased terminal differentiation was observed (compact hyperkeratosis of the stratum corneum, elevated levels of K10 and filaggrin, and heightened transglutaminase activity). Thus, barrier disruption causes epidermal hyperplasia and/or enhances differentiation, depending upon the extent and duration of injury. Given that no fibroblasts are present in the model, the ability to mount a hyperplastic response to barrier injury is an inherent property of keratinocytes.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Augmented expression of polysaccharide intercellular adhesin in a defined Staphylococcus epidermidis mutant with the small-colony-variant phenotype

While coagulase-negative staphylococci (CoNS), with their ability to form a thick, multilayered biofilm on foreign bodies, have been identified as the major cause of implant-associated infections, no data are available about biofilm formation by staphylococcal small-colony variants (SCVs). In the past years, a number of device-associated infections due to staphylococcal SCVs were described, among them, several pacemaker infections due to SCVs of CoNS auxotrophic to hemin. To test the characteristics of SCVs of CoNS, in particular, to study the ability of SCVs to form a biofilm on foreign bodies, we generated a stable mutant in electron transport by interrupting one of the hemin biosynthetic genes, hemB, in Staphylococcus epidermidis. In fact, this mutant displayed a stable SCV phenotype with tiny colonies showing strong adhesion to the agar surface. When the incubation time was extended to 48 h or a higher inoculum concentration was used, the mutant produced biofilm amounts on polystyrene similar to those produced by the parent strain. When grown under planktonic conditions, the mutant formed markedly larger cell clusters than the parental strain which were completely disintegrated by the specific beta-1,6-hexosaminidase dispersin B but were resistant to trypsin treatment. In a dot blot assay, the mutant expressed larger amounts of polysaccharide intercellular adhesin (PIA) than the parent strain. In conclusion, interrupting a hemin biosynthetic gene in S. epidermidis resulted in an SCV phenotype. Markedly larger cell clusters and the ability of the hemB mutant to form a biofilm are related to the augmented expression of PIA.