Altered permeability and modulatory character of connexin channels during mammary gland development

Abrupt developmental changes occur in structural form and function of connexin (Cx) channels in the mouse mammary gland. Microarray study shows that the principal connexin isoform in epithelial cells during pregnancy is Cx26, up-regulated and persisting from the virgin. After parturition, there is rapid induction of Cx32. In epithelial plasma membranes, size exclusion chromatography reveals that Cx32 organizes initially with Cx26 as heteromeric (Cx26-Cx32) hemichannels and later in heteromeric and homomeric Cx32 channels. Dramatic alterations of connexin channel function following these developmental changes in channel composition are characterized using native channels reconstituted into liposomes. Changes to channel stoichiometry increase the allowable physical size limits of permeant after parturition; the new Cx32 channels are wider than channels containing Cx26. Most remarkably, heteromeric Cx26-Cx32 channels are selectively permeability to adenosine 3',5' cyclic phosphate (cAMP), guanosine 3',5' cyclic phosphate (cGMP), and inositol 1,4,5-triphosphate (IP(3)), whereas homomeric channels are not. Homomeric Cx26 and heteromeric channels with high Cx26/Cx32 stoichiometry are also inhibited by taurine, an osmolyte playing a key role in milk protein synthesis. Taurine effect is reduced where heteromeric channels contain Cx32 > Cx26 and eliminated when channels contain only Cx32. Connexin channel stoichiometry, permeability, and chemical gating character change in precisely the desired fashion after parturition to maximize molecular and electrical coupling to support coordinated milk secretion.     

Non-Invasive Monitoring of Streptococcus pyogenes Vaccine Efficacy Using Biophotonic Imaging

Streptococcus pyogenes infection of the nasopharynx represents a key step in the pathogenic cycle of this organism and a major focus for vaccine development, requiring robust models to facilitate the screening of potentially protective antigens. One antigen that may be an important target for vaccination is the chemokine protease, SpyCEP, which is cell surface-associated and plays a role in pathogenesis. Biophotonic imaging (BPI) can non-invasively characterize the spatial location and abundance of bioluminescent bacteria in vivo. We have developed a bioluminescent derivative of a pharyngeal S. pyogenes strain by transformation of an emm75 clinical isolate with the luxABCDE operon. Evaluation of isogenic recombinant strains in vitro and in vivo confirmed that bioluminescence conferred a growth deficit that manifests as a fitness cost during infection. Notwithstanding this, bioluminescence expression permitted non-invasive longitudinal quantitation of S. pyogenes within the murine nasopharynx albeit with a detection limit corresponding to approximately 10⁵ bacterial colony forming units (CFU) in this region. Vaccination of mice with heat killed streptococci, or with SpyCEP led to a specific IgG response in the serum. BPI demonstrated that both vaccine candidates reduced S. pyogenes bioluminescence emission over the course of nasopharyngeal infection. The work suggests the potential for BPI to be used in the non-invasive longitudinal evaluation of potential S. pyogenes vaccines.     

Birth defects and anti-heat shock protein 70 antibodies in early pregnancy

Abstract It has been suggested that induction of the heat shock response in the mammalian embryo during the critical period of organogenesis can result in anatomical malformation. We measured serum heat shock protein 70 (Hsp70), anti-Hsp70, and anti-Hsp60 in samples taken from expectant mothers at 16 weeks gestation. Samples from women whose babies were born with a birth defect (n = 30) were compared with controls who gave birth to healthy babies (n = 46). Anti-Hsp70 levels were significantly elevated in patients who later gave birth to babies with cleft lip or palate or neurological abnormalities (n = 10): 260 (223-406) microg/mL compared to 150 (88-207) microg/mL in controls (P < 0.001). No significant differences were found in serum Hsp70 and anti-Hsp60 levels between cases and controls. This finding of increased maternal anti-Hsp70 in patients who later gave birth to babies with these abnormalities suggests a previous stressful event may have contributed to the pathogenesis. Further work is required to determine whether Hsp70 has a direct or indirect role in this pathogenesis or whether anti-Hsp70 is simply a marker of a prior increase in Hsp70 due to a physiological stress that itself resulted in the damage. This work is consistent with previous studies showing a buffering role for Hsps in evolution.     

Date Palm Agrobiodiversity (<Emphasis Type="Italic">Phoenix dactylifera</Emphasis> L.) in Siwa Oasis,Egypt: Combining Ethnography,Morphometry, and Genetics

We evaluate date palm (Phoenix dactylifera L.) agrobiodiversity of Siwa oasis, Egypt, located at the crossroads of ancient Trans-Saharan routes, focusing on diversity both as expressed and maintained by the folk categorization system of Siwa inhabitants (through an ethnographic analysis) and as described by genetic sciences and a morphometric tool based on size and geometry of seeds. We verified that some named types are true cultivars, sharing not only a formal identity, important for Isiwan people, but also a genetic identity. However, we also confirm the existence of “ethnovarieties,” i.e., voluntary collections of multiple clones sharing phenotypic characteristics with the same local name, suggesting the genetic richness is higher than the apparent agrobiodiversity estimated by a superficial ethnobotanical approach. Finally, our research offers new insights on the relative importance of feral and cultivated date palms.     

Discovery of a potent CDK2 inhibitor with a novel binding mode, using virtual screening and initial, structure-guided lead scoping

Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.     

Selectivity Studies of the Benzene cis -Dihydrodiol Dehydrogenase Enzyme from Pseudomonas putida ML2 with Vicinal Diol Substrates

The enantiopure (1 S, 2 S )- cis -dihydrodiol metabolites 2B - 5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A - 5A, using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis -dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis -dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis -dihydrodiol enantiomers 2B - 5B. The BCD and NCD enzymes were found to accept cis -dihydrodiol enantiomers of monosubstituted benzene cis -dihydrodiol substrates 2B - 5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10 - 17 were also found to be acceptable substrates for BCD.     

Novel alkoxybenzamide inhibitors of poly(ADP-ribose) polymerase

We have previously described poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors based on a substituted benzyl-phthalazinone scaffold. As an alternative chemical template, a novel series of alkoxybenzamides were developed with restricted conformation through intramolecular hydrogen bond formation; the compounds exhibit low nM enzyme and cellular activity as PARP-1 inhibitors.     

Sun-Compass Orientation in Mediterranean Fish Larvae

Mortality is very high during the pelagic larval phase of fishes but the factors that determine recruitment success remain unclear and hard to predict. Because of their bipartite life history, larvae of coastal species have to head back to the shore at the end of their pelagic episode, to settle. These settlement-stage larvae are known to display strong sensory and motile abilities, but most work has been focused on tropical, insular environments and on the influence of coast-related cues on orientation. In this study we quantified the in situ orientation behavior of settlement-stage larvae in a temperate region, with a continuous coast and a dominant along-shore current, and inspected both coast-dependent and independent cues. We tested six species: one Pomacentridae, Chromis chromis, and five Sparidae, Boops boops, Diplodus annularis, Oblada melanura, Spicara smaris and Spondyliosoma cantharus. Over 85% of larvae were highly capable of keeping a bearing, which is comparable to the orientation abilities of tropical species. Sun-related cues influenced the precision of bearing-keeping at individual level. Three species, out of the four tested in sufficient numbers, oriented significantly relative to the sun position. These are the first in situ observations demonstrating the use of a sun compass for orientation by wild-caught settlement-stage larvae. This mechanism has potential for large-scale orientation of fish larvae globally.     

DTNBP1, a schizophrenia susceptibility gene, affects kinetics of transmitter release

Schizophrenia is one of the most debilitating neuropsychiatric disorders, affecting 0.5-1.0% of the population worldwide. Its pathology, attributed to defects in synaptic transmission, remains elusive. The dystrobrevin-binding protein 1 (DTNBP1) gene, which encodes a coiled-coil protein, dysbindin, is a major susceptibility gene for schizophrenia. Our previous results have demonstrated that the sandy (sdy) mouse harbors a spontaneously occurring deletion in the DTNBP1 gene and expresses no dysbindin protein (Li, W., Q. Zhang, N. Oiso, E.K. Novak, R. Gautam, E.P. O'Brien, C.L. Tinsley, D.J. Blake, R.A. Spritz, N.G. Copeland, et al. 2003. Nat. Genet. 35:84-89). Here, using amperometry, whole-cell patch clamping, and electron microscopy techniques, we discovered specific defects in neurosecretion and vesicular morphology in neuroendocrine cells and hippocampal synapses at the single vesicle level in sdy mice. These defects include larger vesicle size, slower quantal vesicle release, lower release probability, and smaller total population of the readily releasable vesicle pool. These findings suggest that dysbindin functions to regulate exocytosis and vesicle biogenesis in endocrine cells and neurons. Our work also suggests a possible mechanism in the pathogenesis of schizophrenia at the synaptic level.