Bioactive scaffolds with enhanced supramolecular motion promote recovery from spinal cord injury.

The signaling of cells by scaffolds of synthetic molecules that mimic proteins is known to be effective in the regeneration of tissues. Here, we describe peptide amphiphile supramolecular polymers containing two distinct signals and test them in a mouse model of severe spinal cord injury. One signal activates the transmembrane receptor ß1-integrin and a second one activates the basic fibroblast growth factor 2 receptor. By mutating the peptide sequence of the amphiphilic monomers in nonbioactive domains, we intensified the motions of molecules within scaffold fibrils. This resulted in notable differences in vascular growth, axonal regeneration, myelination, survival of motor neurons, reduced gliosis, and functional recovery. We hypothesize that the signaling of cells by ensembles of molecules could be optimized by tuning their internal motions.

Latex allergy in dentistry. Review and report of case presenting as a serious reaction to latex dental dam.

Latex allergy may have severe consequences including development of anaphylaxis. This report describes a patient who underwent a reaction to latex dental dam manifesting as erythema, facial swelling and mild airway compromise. Restorative procedures under latex dental dam were performed under local anaesthesia on two occasions resulting in reactions of increasing severity. Following the first event the cause of the reaction was undetermined, but attributed to a possible allergy to local anaesthetic, and managed with corticosteroids and antihistamines. On a subsequent occasion the swelling was more severe, associated with difficulty in swallowing and mild airway compromise, and was managed as previously with adrenaline also being required. Latex allergy was subsequently confirmed.

The relationship between the position of the retinal area centralis and feeding behaviour in juvenile black bream Acanthopagrus butcheri (Sparidae: Teleostei).

The topography of the neurons in the retinal ganglion cell layer of juvenile black bream Acanthopagrus butcheri changes during development. The region of high cell density the area centralis (AC), relocates from a temporal (central) to a dorsal (peripheral) position within the dorso-temporal retinal quadrant. To ascertain whether the differences in the position of the AC during development are related to feeding behaviour, we monitored fishes that were given a choice of food. A range of feeding behaviour patterns was recorded in individual fishes. The smallest fishes (8-15 mm standard length (SL)) took live food from the water column. Following weaning onto pellets, fishes exhibited a preference for taking food from either the substrate or the surface (but not both). When greater than 20 mm SL, a number of individuals then divided their time between surface and substrate feeding before all fishes became exclusive benthic feeders at a stage between 50 and 80 mm SL. Three individual fishes, for which behaviour patterns were categorized, were killed and the topography of the retinal ganglion cell layer analysed. A range of positions for the AC was found with the smallest fish (12 mm SL) possessing a region of high cell density in the temporal retina. In a larger fish (70 mm SL), feeding from both the substrate and the surface, the AC was found in an intermediate dorso-temporal position. The AC of a fish (51 mm SL) preferentially taking food from the substrate was located in a dorsal position.

Variability in the location of the retinal ganglion cell area centralis is correlated with ontogenetic changes in feeding behavior in the black bream, Acanthopagrus butcheri (Sparidae, teleostei).

The development of neural cell topography in the retinal ganglion cell layer was examined in a teleost, the black bream (Acanthopagrus butcheri). From Nissl-stained wholemounts, it was established that fish between 10 and 15 mm standard body length (SL) possess high cell densities throughout the dorso-temporal retinal quadrant, with peak cell densities located in temporal regions of the retina. However, in fish between 15 and 80 mm SL, a wide variation in the position of the peak cell density is revealed with the locations of the areae centrales (AC) ranging from exclusively temporal to periphero-dorsal retina. Fish larger than 80 mm SL always possess an AC located in the dorsal region of the dorso-temporal retinal quadrant. The topography of ganglion cells within the ganglion cell layer was determined by comparing the numbers of ganglion cells retrogradely-labeled from the optic nerve with the total population of Nissl-stained neurons (ganglion plus displaced amacrine cells) in a range of different-sized individuals. Ganglion cell topography was the same as that recorded for all Nissl-stained neurons. The feeding behavior of juveniles from metamorphosis to 80 mm SL was observed, where fish were given the choice of feeding on live food in mid-water (until 15 mm SL) or obtaining pellets from the surface or the bottom. A range of feeding patterns was recorded, with the smallest fish taking food from mid-water but individuals between 15 and 80 mm SL taking food either from the surface or the bottom or both. A correlation between the preferred mode of feeding and the position of the AC was found, such that those individuals feeding in mid-water or at the surface possess a temporal or intermediate (dorso- temporal) AC, whereas those predominantly feeding from the bottom possess a dorsal AC.

Multiple forms of an inositol polyphosphate 5-phosphatase form signaling complexes with Shc and Grb2.

BACKGROUND: Shc and Grb2 form a complex in cells in response to growth factor stimulation and link tyrosine kinases to Ras during the resulting signaling process. Shc and Grb2 each contain domains that mediate interactions with other unidentified intracellular proteins. For example, the Shc PTB domain binds to 130 kDa and 145 kDa tyrosine-phosphorylated proteins in response to stimulation of cells by growth factors, cytokines and crosslinking of antigen receptors. The Grb2 SH3 domains bind to an unidentified 116 kDa protein in T cells. We have identified three proteins, of 110 kDa, 130 kDa and 145 kDa, as a new family of molecules encoded by the same gene. In vivo studies show that these proteins form signal transduction complexes with Shc and with Grb2. RESULTS: The 130 kDa and 145 kDa tyrosine-phosphorylated proteins that associate with the Shc PTB domain were purified by conventional chromatographic methods. Partial peptide and cDNA sequences corresponding to these proteins, termed SIP-145 and SIP-130 (SIP for signaling inositol polyphosphate 5-phosphatase), identified them as SH2 domain-containing products of a single gene and as members of the inositol polyphosphate 5-phosphatase family. The SIP-130 and SIP-145 proteins and inositol polyphosphate 5-phosphatase activity associated with Shc in vivo in response to B-cell activation. By using an independent approach, expression cloning, we found that the Grb2 SH3 domains bind specifically to SIP-110, a 110 kDa splice variant of SIP-145 and SIP-130, which lacks the SH2 domain. The SIP proteins hydrolyzed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5)-P3) and Ins (1,3,4,5)-P4, but not PtdIns (4,5)-P2 or Ins (1,4,5)-P3. CONCLUSIONS: These findings strongly implicate the inositol polyphosphate 5-phosphatases in Shc- and Grb2-mediated signal transduction. Furthermore, SIP-110, SIP-130 and SIP-145 prefer 3-phosphorylated substrates, suggesting a link to the phosphatidylinositol 3-kinase signaling pathway.

Molecular diagnosis of Duchenne muscular dystrophy in Singapore.

Duchenne muscular dystrophy (DMD) is a common lethal sex-linked recessive disorder. Seventy percent of the cases are inherited and 30% are due to mutations. The mainstay of prevention is detection of female carriers and antenatal diagnosis of affected foetuses. Before the era of molecular diagnosis, DMD has been clinically defined. Serum creatine kinase (CK) has also been used to screen women at risk for carrier status. With the isolation and sequencing of the DMD gene at Xp21 and the identification of the DMD gene-product dystrophin, DNA technology can be applied for the diagnosis of the affected, for the detection of carriers and in antenatal diagnosis. The multiplex polymerase chain reaction (PCR) technique offers a rapid and simple screening method for deletions of the gene. We were able to detect partial deletions which account for 58.3% of gene defects in our patients. This direct demonstration of the gene defect that causes DMD gives a 100% assurance of accuracy and specificity of the diagnosis. Linkage analysis is especially useful for prenatal diagnosis and carrier detection in the remaining 41.7% of families without detectable deletions or duplications. This approach however is indirect and is dependent on information on genotypes from affected males and key family members. With the availability of increasingly more restriction fragment length polymorphisms (RFLPs), it has become practical to use the haplotype method for accurate carrier detection and prenatal diagnosis.

Three chemically distinct types of oxidants formed by iron-mediated Fenton reactions in the presence of DNA.

Exposure of Escherichia coli to H2O2 leads to two kinetically distinguishable modes of killing: mode I killing occurs maximally near 2 mM H2O2, whereas mode II killing is essentially independent of H2O2 concentrations up to 20 mM. A major portion of H2O2 toxicity is attributed to DNA damage caused by the iron-mediated Fenton reaction. By studying DNA damage during Fenton reactions in vitro, the same complex kinetics were observed and three types of oxidants were distinguished based upon their reactivities toward H2O2 and alcohols and upon iron-chelator effects. Type I oxidants are sensitive to H2O2 but moderately resistant to ethanol; type II oxidants are resistant to both H2O2 and ethanol; type III oxidants are sensitive to H2O2, ethanol, and t-butanol. To explain these results, we hypothesize that type I oxidants are generated upon Fe2+ associated with DNA only through electrostatic interactions and cause mode I killing of E. coli; type II oxidants arise upon Fe2+, which is at least partially base-associated, and cause mode II killing; type III oxidants arise on Fe2+ free in solution and probably do not cause killing. Therefore, particular interactions of DNA with transition metals should be considered to be an integral part of the chemistry and toxicity of H2O2.

Expression and characterization of recombinant hepatitis A virus 3C proteinase.

The 3C proteinase from the hepatitis A virus (HAV) was cloned into a multicopy expression vector in Escherichia coli under control of the tac promoter. The resulting plasmid construction produced 3C proteinase as a soluble and active enzyme constituting approximately 10% of total cellular proteins. The enzyme was purified to apparent homogeneity as judged by SDS gel electrophoresis and HPLC reversed-phase and FPLC ion-exchange chromatography. A colorimetric assay was developed, and synthetic peptides derived from the predicted cleavage sites of the HAV polyprotein were tested for proteolysis of the enzyme. The peptide representing the 2B/2C cleavage site was cleaved most efficiently with a Km and kcat of 2.1 +/- 0.5 mM and 1.8 +/- 0.1 s-1, respectively. Site-directed mutagenesis was then used to identify the cysteine at position 172 as the active site nucleophile. Finally, the purified enzyme showed the expected endoproteinase activity on the P1 precursor protein generated by in vitro transcription/translation.

Characterization of group specific antibodies in primates: studies with SIV envelope in macaques.

Sera from SIV-infected macaques were found to contain antibodies that reacted with conformation-dependent, group-specific determinants on the SIV envelope protein gp130. These conformation-dependent antibodies exhibited virus neutralizing activity; their presence was associated with protection in vaccine studies. The properties of these antibodies are quite similar to those that have been identified in sera from HIV-infected human subjects. These data suggest that the SIV envelope gp130 remains a candidate for subunit vaccine studies.

Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro.

Exposure of Escherichia coli to low concentrations of hydrogen peroxide results in DNA damage that causes mutagenesis and kills the bacteria, whereas higher concentrations of peroxide reduce the amount of such damage. Earlier studies indicated that the direct DNA oxidant is a derivative of hydrogen peroxide whose formation is dependent on cell metabolism. The generation of this oxidant depends on the availability of both reducing equivalents and an iron species, which together mediate a Fenton reaction in which ferrous iron reduces hydrogen peroxide to a reactive radical. An in vitro Fenton system was established that generates DNA strand breaks and inactivates bacteriophage and that also reproduces the suppression of DNA damage by high concentrations of peroxide. The direct DNA oxidant both in vivo and in this in vitro system exhibits reactivity unlike that of a free hydroxyl radical and may instead be a ferryl radical.