Micropatterning polyvinyl alcohol as a biomimetic material through soft lithography with cell culture

Integrating inorganic materials with specific biological systems requires a complex set of properties that can mimic physiological function to create microchip devices. Several factors in the material including the spatial location of the cellular attachment heavily influence the control of this functionality and must be dictated to parallel in vivo characteristics. In this work, we demonstrate polyvinyl alcohol (PVA) for use in emulating these properties to produce a highly robust and functional biomaterial as a microchip. We show the utility of PVA for cellular patterning using soft lithography. Based on these results, PVA can be applied in a diversity of areas including tissue engineering, biomimetics, and cellular micropatterning.     

On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids

25 aromatic carboxylic acids which are analogs of benzoic acid were tested in the rat diaphragm preparation for effects on chloride conductance (G(Cl)). Of the 25, 19 were shown to reduce membrane G(Cl) with little effect on other membrane parameters, although their apparent K(i) varied widely. This inhibition was reversible if exposure times were not prolonged. The most effective analog studied was anthracene-9-COOH (9-AC; K(i) = 1.1 x 10(-5) M). Active analogs produced concentration-dependent inhibition of a type consistent with interaction at a single site or group of sites having similar binding affinities, although a correlation could also be shown between lipophilicity and K(i). Structure-activity analysis indicated that hydrophobic ring substitution usually increased inhibitory activity while para polar substitutions reduced effectiveness.

These compounds do not appear to inhibit G(Cl) by altering membrane surface charge and the inhibition produced is not voltage dependent. Qualitative characteristics of the I-V relationship for Cl(-) current are not altered. Conductance to all anions is not uniformly altered by these acids as would be expected from steric occlusion of a common channel. Concentrations of 9-AC reducing G(Cl) by more than 90 percent resulted in slight augmentation of G(I). The complete conductance sequence obtained at high levels of 9-AC was the reverse of that obtained under control conditions. Permeability sequences underwent progressive changes with increasing 9-AC concentration and ultimately inverted at high levels of the analog. Aromatic carboxylic acids appear to inhibit G(Cl) by binding to a specific intramembrane site and altering the selectivity sequence of the membrane anion channel.

     

Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.     

GENETIC ANALYSIS OF KILLER WHALE (ORCINUS ORCA) HISTORICAL BONE AND TOOTH SAMPLES TO IDENTIFY WESTERN U.S. ECOTYPES

Little is known about the historical range of killer whale ecotypes in the eastern North Pacific (ENP). It is possible that ranges have shifted in the last few decades because of changes in availability of food. In particular, the southern resident ecotype, currently found primarily in the inland waters of Washington State, is known to prey extensively on salmon, which have declined in recent decades along the outer coasts of Washington, Oregon, and California. To investigate historical distributions of this and the other ENP ecotypes, samples of teeth and bones were obtained from NMFS and museum collections. We amplified a short section of the mitochondrial DNA control region that contains four diagnostic sites that differentiate between haplotypes found in ecotypes of ENP killer whales. Results did not show any southern resident haplotypes in samples from south of the Washington State inland waterways. One whale genetically identified as a northern resident extends the known southernmost distribution of the population from Oregon to California. Items of diet identified from stomach contents of six of the whales genetically identified to ecotype conformed with what is known of the feeding habits of the various ecotypes.     

Genomic heterogeneity among human and nonhuman strains of hepatitis A virus.

Cloned cDNA probes derived from the P1 and P2 regions of the genome of HM175 virus, a reference strain of human hepatitis A virus (HAV), failed to hybridize under standard stringency criteria with RNA from PA21 and PA33 viruses, two epizootiologically related HAV strains recovered from naturally infected New World owl monkeys. Hybridization of these probes to PA21 RNA was only evident under reduced stringency conditions. However, cDNA representing the 5' nontranslated region of the HM175 genome hybridized equally to HM175 and PA21 RNA under standard stringency conditions, while a probe derived from the 3' 1,400 bases of the genome yielded a reduced hybridization signal with PA21 RNA. In contrast, no differences could be discerned between HM175 virus and three other HAV strains of human origin (GR8, LV374, and MS1) in any region of the genome, unless increased stringency conditions were used. These results suggest that PA21 and PA33 are unique among HAV isolates and may represent a virus native to the owl monkey. Despite extremely poor homology within the P1 region, which encodes capsid polypeptides, monoclonal antibody analysis confirmed that the immunodominant neutralization epitopes of HAV were highly conserved between HM175 and PA21 viruses. Furthermore, experimental challenge of the owl monkey with successive PA33 and HM175 inocula confirmed a high but incomplete degree of cross-protection. Only one of six monkeys previously infected with PA33 developed recurrent hepatitis 28 days after intravenous HM175 challenge, while none of six monkeys previously infected with HM175 had demonstrable hepatitis following PA33 challenge. These data provide molecular evidence for the existence of HAV strains unique to nonhuman primate species and indicate that strict conservation of antigenic function may accompany substantial genetic divergence in HAV.