Measuring the microelastic properties of biological material.

We have used the atomic force microscope (AFM) to measure the local rigidity modulus at points on the surface of a section of hydrated cow tibia. These data are obtained either from contrast changes that occur as the contact force is altered, or from force versus distance curves obtained at fixed points. These two methods yield the same values for rigidity modulus (at a given point). At low resolution, the elastic morphology and topography mirror the features seen in optical and electron micrographs. At high resolution we see dramatic variations in elastic properties across distances as small as 50 nm.     

Fractionation and quantitation of egg antigens from Schistosoma japonicum by the single-tube kinetic-dependent enzyme-linked immunosorbent assay (k-ELISA): higher antigenic activity in urea-soluble than in aqueous-soluble fractions

To identify sources of high potency antigens for use in serodiagnosis, aqueous-soluble egg antigens from Schistosoma japonicum were extracted with Dulbecco's phosphate-buffered saline. Residual particulates were solubilized with Tris-buffered 8 M urea, yielding a urea-soluble egg antigen fraction. The urea-soluble fraction was further fractionated with Bio Gel A50m and QAE-Sephadex. All fractions were quantitatively assayed for their specific antigenic activities against serum specimens from infected rabbits by the single-tube enzyme-linked immunosorbent assay (k-ELISA). In antigen rate-limiting conditions, the urea-soluble particulate fractions were more antigenically active than the aqueous-soluble fraction. In antigen-excess and antibody-limiting assay conditions, the ideal conditions for serologic assays, the urea-derived antigens also showed superior activities against sera from infected humans. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on gradient gels revealed numerous low molecular weight protein bands in the aqueous-soluble fraction, whereas the urea-soluble fractions appeared to be much simpler with the majority of their proteins concentrated in one or two high molecular weight bands (greater than or equal to 200 kdaltons). Electro-transfer blots of the SDS-PAGE onto nitrocellulose papers and subsequent visualization of antigens by enzyme-linked immunoabsorbence confirmed these findings. The above data suggest that the urea-soluble fraction of S. japonicum eggs is antigenically active and has potential use in the development of a diagnostic reagent.     

Effects of 2,3-diphosphoglyceric acid on the human erythrocyte membrane phosphorylation system

The effect of 2,3-diphosphoglycerate (2,3-P2-glycerate) on the phosphorylation of spectrin in solution by purified membrane cyclic AMP-independent protein kinase and in membrane preparations by the endogenous kinase was investigated. 2,3-P2-Glycerate inhibited spectrin phosphorylation both in solution and in the intact membrane. Kinetic analyses showed that 2,3-P2-glycerate had no effect on the Km for ATP but appeared to lower the Vmax of the reaction. When the effect of 2,3-P2-glycerate was examined in the presence of varying concentrations of spectrin, competitive inhibition kinetics were obtained. Interestingly, low concentrations of 2,3-P2-glycerate were found to effect the release of the membrane kinase from erythrocyte membranes. This release reaction may be related to the ability of 2,3-P2-glycerate to interfere with the interaction between the kinase and spectrin. The data suggest the possibility that the kinase may be bound to spectrin in the erythrocyte membrane. 2,3-P2-glycerate also caused the solubilization of 3-phosphoglyceraldehyde dehydrogenase, but not of cyclic AMP-dependent protein kinase. Taken together, our data indicate that 2,3-P2-glycerate may have a regulatory role in membrane protein phosphorylation and also may regulate the extent of association of the kinase with the membrane.     

Inhibition of protein phosphorylation and induction of protein cross-linking in erythrocyte membranes by diamide

This report presents studies on the effect of diamide on protein phosphorylation in erythrocyte membranes. Diamide, a thiol-oxidizing reagent, nonspecifically inhibits cyclic Amp-dependent and -independent autophosphorylation of red cell memvranes, but not the activity of the solubilized membrane cycle AMP-independent protein kinases. Analysis of diamide-treated membranes by gel electrophoresis indicates that diamide is capable of inducing cross-linking of membrane proteins. The action of diamide, both in the inhibition of membrane autophosphorylation and in the cross-linking of membrane proteins, is very similar to that of Cu2+. o-phenanthroline complex. Our data indicate that diamide inhibits erythrocyte membrane autophosphorylation by perturbing the protein substrates.     

Human erythrocyte proteins associated with adenosine 3',5'-cyclic monophosphate action.

Two adenosine 3',5'-cyclic monophosphate (cyclic-AMP)-binding protein factors (molecular weight 230,000) have been partially purified from human erythrocytes. One of these proteins seems to be different from the cyclic-AMP-binding component of the cyclic-AMP-dependent protein kinases. These protein factors are also capable of binding adenosine. We present data also on two forms of cyclic-AMP-dependent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) partially purified from the cytosol of normal human erythrocytes. Kinase I has been classified as type I enzyme on the basis of its activation when preincubated with protamine, histone or NaCl. The substrate specificities of the two kinases and many of their kinetic parameters are rather similar. Their subunit structure is reminiscent of that of kinases obtained from other sources. The catalytic subunit of both enzymes reversibly cross-react with the regulatory subunit of kinase I from the rabbit red blood cell.     

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

The N¹-methyl-Adenosine (m¹A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m¹A58 modification is essential for viability because it is required for the stability of the initiator-tRNA^Met. However, m¹A58 modification is not required for the stability of several other tRNAs in yeast. This differential m¹A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m¹A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m¹A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA^Met is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m¹A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m¹A58 hypomodified tRNAs. Most m¹A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m¹A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.     

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.     

Focal adhesion kinase maintains,but not increases the adhesion of dental pulp cells

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.