Identical Electrophoretic Mobility Research Articles

Nucleotide and protein sequences for dog masticatory tropomyosin identify a novel Tpm4 gene product.

Jaw-closing muscles of several vertebrate species, including members of Carnivora, express a unique, "masticatory", isoform of myosin heavy chain, along with isoforms of other myofibrillar proteins that are not expressed in most other muscles. It is generally believed that the complement of myofibrillar isoforms in these muscles serves high force generation for capturing live prey, breaking down tough plant material and defensive biting. A unique isoform of tropomyosin (Tpm) was reported to be expressed in cat jaw-closing muscle, based upon two-dimensional gel mobility, peptide mapping, and immunohistochemistry. The objective of this study was to obtain protein and gene sequence information for this unique Tpm isoform. Samples of masseter (a jaw-closing muscle), tibialis (predominantly fast-twitch fibers), and the deep lateral gastrocnemius (predominantly slow-twitch fibers) were obtained from adult dogs. Expressed Tpm isoforms were cloned and sequencing yielded cDNAs that were identical to genomic predicted striated muscle Tpm1.1St(a,b,b,a) (historically referred to as αTpm), Tpm2.2St(a,b,b,a) (βTpm) and Tpm3.12St(a,b,b,a) (γTpm) isoforms (nomenclature reflects predominant tissue expression ("St"-striated muscle) and exon splicing pattern), as well as a novel 284 amino acid isoform observed in jaw-closing muscle that is identical to a genomic predicted product of the Tpm4 gene (δTpm) family. The novel isoform is designated as Tpm4.3St(a,b,b,a). The myofibrillar Tpm isoform expressed in dog masseter exhibits a unique electrophoretic mobility on gels containing 6 M urea, compared to other skeletal Tpm isoforms. To validate that the cloned Tpm4.3 isoform is the Tpm expressed in dog masseter, E. coli-expressed Tpm4.3 was electrophoresed in the presence of urea. Results demonstrate that Tpm4.3 has identical electrophoretic mobility to the unique dog masseter Tpm isoform and is of different mobility from that of muscle Tpm1.1, Tpm2.2 and Tpm3.12 isoforms. We conclude that the unique Tpm isoform in dog masseter is a product of the Tpm4 gene and that the 284 amino acid protein product of this gene represents a novel myofibrillar Tpm isoform never before observed to be expressed in striated muscle.

Screening of neuraminidase inhibitors from traditional Chinese medicine by transverse diffusion mediated capillary microanalysis.

A transverse diffusion mediated capillary microanalysis method has been developed for screening of neuraminidase inhibitors from traditional Chinese medicine. The enzyme, substrate and inhibitors were sequentially injected, mixed efficiently by transverse diffusion of laminar flow profiles, then incubated and separated in the same capillary. To enhance the mixing efficiency of reactants, running buffer was injected by alternately applying +5 kPa and -5 kPa at the capillary inlet and the procedure was repeated three times. The capillary electrophoresis (CE) separation conditions and reactants mixing conditions were optimized. Dual-wavelength detection was employed to eliminate the interference with natural compounds. The method has been applied to determine the kinetics constant of neuraminidase and screen 12 compounds from traditional Chinese medicine. Four compounds have been found to be positive for enzyme inhibition. The results are in good agreement with those reported in the literature. The method realized the mixing of substrate and enzyme with identical electrophoretic mobility. This novel CE method was simple, rapid, economic, and fully automated. Therefore, it was appropriate for neuraminidase inhibitors screening and could be extended to other high-throughput screening of active components from traditional Chinese medicine.

Amphidiploids between tetraploid wheat and Aegilops sharonensis Eig exhibit variations in high-molecular-weight glutenin subunits

Wide crossing is a useful method for transferring traits of interest from wild Triticeae species to wheat. In this study, Aegilopssharonensis was successfully hybridized to three tetraploid wheat species with crossability varying from 0.86 to 1.76 %. F1 hybrids were verified by their display of intermediate characteristics in morphology, cytology, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles. The exogenous x- and y-type subunits of high-molecular-weight glutenin subunits of Ae.sharonensis were expressed in F1 hybrid seeds and showed identical electrophoretic mobilities to those originally present in Ae.sharonensis. In F2 hybrids, the x-type subunit migrated faster, indicating that a decrease in molecular mass had occurred. Cloning of the open reading frames of subunit variants revealed that the x-type subunit had protein sequence variations, including single residue mutations, and insertions and deletions involving one or more repeat motifs. The two deletions causing the size reduction of the exogenous x-subunits of Ae.sharonensis in the F2 seeds were most likely the result of slip-mismatching.

R-ISSR – for Fingerprinting, Mapping and Identification of New Genomic Loci in Rye (Secale cerealeL.)

The results of the research confirming the possibility of applying various combinations of RAPD and ISSR primers in one multiplex PCR and the generation of a new type of R-ISSR products for the rye genome were presented in this work. The following was applied in the research: five rye genotypes including two inbred lines (153/79-1 and Otl-3), hybrid F1 and two bulks (tolerant and susceptible) formed from recombinant inbred lines - RILs (F9) varying in the response to abiotic stress caused by nutrient deficiencies at the seedling stage. While evaluating the possibility of applying R-ISSR to the assessment of the rye variability, five of its genotypes were amplified separately with the RAPD and ISSR primers in each PCR reaction. These primers were combined in R-ISSR amplifications. The products of RAPD, ISSR and R-ISSR amplification were separated in 1.5% agarose gel. 32 R-ISSR combinations were examined, combining 20 and 8 selected RAPD and ISSR primers, respectively. 658 loci were amplified, including 230 RAPD, 180 ISSR and 271 R-ISSR, including 157 new loci. Over 91 loci were found, with an identical electrophoretic mobility for three methods. It was shown that R-ISSR products with electrophoretic mobility on agarose gels, identical to the co-migrating RAPD or ISSR, are not products of RAPD or ISSR, but they possess sequences of heteroamplicons - R-ISSR. The occurrence of sequences of primers used to R-ISSR was demonstrated while sequencing seven selected products of the above type. The ISSR primers with a low Tm were proven to generate repeatable fingerprints in the thermal profile of the reaction specific for RAPD and combined with the RAPD primer - repeatable R-ISSR profiles. A similar range of variability as described in RAPD or ISSR was observed in the R-ISSR profiles. The correlation coefficient between genetic similarity matrices for five rye genotypes, calculated with the Mantel test, amounted to rABC = 0.870.

R-ISSR - Tool for Generation of a New Type of Products, Applied for the Identification of Putative Molecular Marker Linked to QTL Determined Tolerance to Nutrient Deprivation Stress in Rye (Secale cereale L.)

This study presents results confirming the possibility of the application of various combinations of RAPD and ISSR primers in one multiplex PCR for the rye genome and generating a new type of R-ISSR products. The plant material consisted of two inbred lines (Ot0-6 and Ot1-3), F1 and two bulks (tolerant and susceptible), selected from a population of RILs (F9) with different responses to stress caused by nutrient deprivation at the seedling stage. In one PCR reaction, the DNA of five genotypes was amplified separately, with RAPD, ISSR, and RAPD+ISSR primers. In total, 28 R-ISSR combinations were tested by combining 20 and 8 appropriately selected RAPD and ISSR primers. 567 loci were amplified, including 230 RAPD, 136 ISSR and 207 amplified R-ISSR loci, of which 114 were new. It was shown that only one ISSR product was identified amongst sequenced R-ISSR products with identical electrophoretic mobility as co-migrating RAPDs and ISSRs. The remaining ones had heteroamplicons (R-ISSR) sequences. A similar range of variability was observed both in R-ISSR profiles and in RAPD, as well as in ISSR. The correlation coefficient between the matrices of genetic similarity for five rye genotypes calculated by means of the Mantel test was highly significant rAB.C = 0.964.

Electric current during electrophoretic deposition of conjugated polymer: A test with various electrode distances

The transient current during electrophoretic deposition of conjugated polymer is a promising tool for exploring the basic process of the deposition. In this paper, it has been shown that the transit times obtained from various electrode distances give almost identical electrophoretic mobility. The result shown here seems to support our interpretation of the transient current profile during electrophoretic deposition that it reflects the drift motion of colloidal particles in suspension driven by the electric field.

Zonadhesin D3-Polypeptides Vary among Species but Are Similar in Equus Species Capable of Interbreeding1

Zonadhesin is a rapidly evolving protein in the sperm acrosome that confers species specificity to sperm-zona pellucida adhesion. Though structural variation in zonadhesin likely contributes to its species-specific function, the protein has not previously been characterized in organisms capable of interbreeding. Here we compared properties of zonadhesin in several animals, including the horse (Equus caballus), donkey (E. asinus), and Grevy's zebra (E. grevyi) to determine if variation in zonadhesin correlates with ability of gametes to cross-fertilize. Zonadhesin localized to the apical acrosomes of spermatozoa from all three Equus species, similar to its localization in other animals. Likewise, in horse and donkey testis, zonadhesin was detected only in germ cells, first in the acrosomal granule of round spermatids and then in the developing acrosomes of elongating spermatids. Among non-Equus species, D3-domain polypeptides of mature, processed zonadhesin varied markedly in size and detergent solubility. However, zonadhesin D3-domain polypeptides in horse, donkey, and zebra spermatozoa exhibited identical electrophoretic mobility and detergent solubility. Equus zonadhesin D3-polypeptides (p110/p80 doublet) were most similar in size to porcine and bovine zonadhesin D3-polypeptides (p105). Sequence comparisons revealed that the horse zonadhesin precursor's domain content and arrangement are similar to those of zonadhesin from other large animals. Partial sequences of horse and donkey zonadhesin were much more similar to each other (>99% identity) than they were to orthologous sequences of human, pig, rabbit, and mouse zonadhesin (52%-72% identity). We conclude that conservation of zonadhesin D3-polypeptide properties correlates with ability of Equus species to interbreed.

Cataracts Are Caused by Alterations of a Critical N-Terminal Positive Charge in Connexin50

To elucidate the basis of the autosomal dominant congenital nuclear cataracts caused by the connexin50 mutant, CX50R23T, by determining its cellular distribution and functional behavior and the consequences of substituting other amino acids for arginine-23. Connexin50 (CX50) mutants were generated by PCR and transfected into HeLa or N2a cells. Expressed CX50 protein was detected by immunoblot analysis and localized by immunofluorescence. Intercellular communication was assessed by microinjection of neurobiotin or by double whole-cell patch-clamp recording. HeLa cells stably transfected with CX50R23T or wild-type CX50 produced immunoreactive CX50 bands of identical electrophoretic mobility. Whereas HeLa cells stably expressing CX50 contained abundant gap junction plaques, CX50R23T localized predominantly in the cytoplasm. HeLa cells expressing wild-type CX50 showed large gap junctional conductances and extensive transfer of neurobiotin, but those expressing CX50R23T did not show significant intercellular communication by either assay. Moreover, CX50R23T inhibited the function of coexpressed wild-type CX50. Three CX50R23 substitution mutants (CX50R23K, CX50R23L, and CX50R23W) formed gap junction plaques, whereas two mutant substitutions with negatively charged residues (CX50R23D, CX50R23E) did not form detectable plaques. Only the mutant with a positive charge substitution (CX50R23K) allowed neurobiotin transfer at levels similar to those of wild-type CX50; none of the other mutants induced transfer. These results suggest that replacement of amino acid 23 in CX50 by any residue that is not positively charged would lead to cataract formation.

Entity evidence for differentiation between Tia and Tib types of soybean Kunitz trypsin inhibitor: detection of a novel transitional variant type between Tia and Tib in wild soybean (Glycine soja Sieb. & Zucc.)

Soybean Kunit trypsin inhibitor (SKTI) has several polymorphic types. Of these SKTI, there are large differences of nine amino acid substitutions between Tia and Tib. So far no transitional type between them has been found. A novel transitional intermediate variant between Tia and Tib was detected in 11 lines from 720 Japanese wild soybeans (Glycine soja Sieb. & Zucc.). This variant showed identical electrophoretic mobility to Tib in the Davis system polyacrylamide gel electrophoresis (PAGE), but higher electric points than other SKTI proteins (Tia, Tib, Tic) in isoelectric focusing PAGE. The genetic analysis of SKTI in F(2) seeds from a cross between the novel variant type and Tib showed that this variant type is inherited as codominant alleles in a multiple allelic system at an SKTI locus. This variant also showed inhibitory activity to trypsin. We propose the genetic symbol Ti b ( i5) for this novel variant. The sequence analysis of Tib ( i5) revealed that six nucleotides were different between Tib ( i5) and Tia, and the nucleotides of these mutated positions were identical to Tib. This causes substitution of five amino acids at the residue position 62 (Tyr-->Phe), 74 (Ser-->Arg), 114 (Met-->Val), 120 (Leu-->Ile) and 137 (Pro-->Thr). These substitutive amino acids are completely in accord with the amino acids of Tib, showing that Tib ( i5) is an intermediate between Tia and Tib types. Tib ( i5) type is widely distributed throughout seven separate areas from northeast to southwest Japan with a 1.5% frequency of total materials examined. This indicated that Tib ( i5) type did not originate from a recent mutation event, but had spread in wild soybean from ancient times.

Histidylated Lipid-modified Sendai Viral Envelopes Mediate Enhanced Membrane Fusion and Potentiate Targeted Gene Delivery

Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals.

The effect of dissolved oxygen concentrations on (1 → 3)- and (1 → 6)-β-glucanase production by Acremonium sp. IMI 383068 in batch culture

The influence of dissolved oxygen tension (DOT) on the production of extracellular (1 → 3)- and (1 → 6)-β-glucanases by the fungus Acremonium sp. IMI 383068 was investigated in batch culture. The controlled DOT levels at which cultures were grown affected the measured (1 → 3)-β-glucanase specific activities, but these effects were less marked on the measured (1 → 6)-β-glucanase specific activities. There appeared to be no direct link between the branching frequency of the fungus as reflected by its hyphal growth unit, and changing DOT levels. Whether pustulan or scleroglucan were used as the sole carbon source also affected production of these enzymes and their response to varying DOT. The measured (1 → 3)-β-glucanase specific activities were generally higher with scleroglucan, mainly because of the production of an extra active (1 → 3)-β-glucanase whereas pustulan grown cells produced a corresponding inactive protein with an identical electrophoretic mobility by SDS-PAGE and N-terminal amino acid sequence. With pustulan grown cells, DOT levels had comparatively little influence on the optimal measured specific activities of the (1 → 3)-β-glucanases, while with scleroglucan, they increased as DOT increased.

Highly restricted pattern of connexin36 expression in chick somite development.

The gap junction protein connexin36 (CX36) has been well studied in the mature central nervous system, but there has been little information regarding its possible roles in embryonic development. We report here the isolation of the full-length chick CX36 coding sequence (predicted M(r) 35.1 kDa) and its strikingly restricted pattern of gene expression in the mesoderm of the chick embryo. In situ hybridization experiments demonstrated CX36 expression in somites by embryonic day 2. The transcripts first appeared dorsomedially within the somite and expanded ventrolaterally to form stripes in the middle of each somite. The CX36 stripes fell within somitic territories enriched in MYOD and FGF8 expression and impoverished in PAX3 transcripts, establishing that CX36 mRNA is expressed in the myotome. We compared the somitic expression pattern of CX36 with those of three other connexins, CX42, CX43, and CX45. At embryonic day 4, CX42 transcripts were localized to the myotome in a pattern resembling that of CX36. In contrast, CX43 was enriched in the dermomyotome, and CX45 was detected in both the myotome and the dermomyotome. Immunoblotting using Cx36 antibodies demonstrated bands of identical electrophoretic mobilities in trunk and retinal homogenates, and Cx36 immunostaining detected punctate immunoreactivity in the myotome. These results demonstrate that some connexins in the developing mesoderm are broadly expressed whereas others are highly localized, and suggest that CX36, CX42, and CX45 are involved in intercellular communication among developing muscle cells.

Nuclear factor kappa B activation by NADPH oxidases

Reactive oxygen species (ROS) initiate activation of the transcription factor NF-κB in a variety of cell systems. Perhaps the most potent biological source of ROS is the NADPH oxidase of phagocytic cells, a multi-component system that catalyzes the formation of superoxide anion. Although phagocytes use this oxidase to kill ingested microorganisms, the products also mediate a broad range of biological oxidation reactions and some evidence exists for activation of NF-κB through this mechanism. Moreover, the components of the phagocyte NADPH oxidase are present in certain non-phagocytic cells and recently discovered homologues of the catalytic component gp91 phox are expressed in a number of tissues. We explored the hypothesis that the products of NADPH oxidases cause the activation of NF-κB. K562 human erythrokeukemia cells transfected with constructs for expression of gp91 phox , plus other essential NADPH oxidase components generated substantial amounts of superoxide when activated with phorbol ester, lesser amounts with arachidonic acid exposure, and none with TNFα. Gel shift assays demonstrated induction of NF-κB in K562 cells exposed to TNFα and specificity was shown by oligonucleotide competition. Supershift assays demonstrated the presence in nuclear complexes of the NF-κB components p65/RelA and p50. Nuclear complexes of identical electrophoretic mobility were induced in phorbol ester-stimulated K562 cells that expressed the complete NADPH oxidase system, but not in cells lacking one of the essential oxidase components. K562 cells were relatively resistant to NF-κB induction by exogenous peroxide, but certain other cell types (HEK293 and HeLaS3) demonstrated such induction upon exposure to reagent hydrogen peroxide or glucose oxidase plus glucose and this was blocked by catalase. Finally, we found a biphasic pattern of gp91 phox expression in rat liver during aging. High levels observed in young animals decreased in middle age, but increased again in old age. Collectively, these studies demonstrate the potential for NADPH-dependent induction of NF-κB and raise the possibility of a role for this pathway in the biology of aging.

Binding of free immunoglobulin light chains to VpreB3 inhibits their maturation and secretion in chicken B cells.

The VpreB3 gene product was first characterized as an immunoglobulin (Ig) mu heavy chain-binding protein in mouse precursor B (pre-B) cells. Although its function is unknown, it has been proposed to participate in the assembly and transport of the pre-B cell receptor. We have identified a VpreB3 orthologous gene in chicken that is located close to the immunoglobulin light chain (LC) gene cluster and specifically expressed in the bursa of Fabricius. By overexpressing VpreB3 in the DT40 IgM(+) immature chicken B cell line, we have characterized VpreB3 as an endoplasmic reticulum-resident glycoprotein that binds preferentially to free IgLC. However, binding to IgHC is observed in IgLC-deficient DT40 cells. Interaction of VpreB3 with free IgLC is partly covalent and induces retention of free IgLC in the endoplasmic reticulum, preventing their secretion without affecting IgM surface expression. Our results demonstrate that this evolutionarily conserved molecule may play a role in the regulation of the maturation and secretion of free IgLC in B cells. We discuss possible implications in the regulation of the immune response.

Induction of Grp78/BiP by Translational Block: ACTIVATION OF THE Grp78 PROMOTER BY ATF4 THROUGH AN UPSTREAM ATF/CRE SITE INDEPENDENT OF THE ENDOPLASMIC RETICULUM STRESS ELEMENTS

Mammalian cells respond to endoplasmic reticulum (ER) stress by attenuation of protein translation mediated through the PERK-eIF2alpha pathway and transcriptional activation of genes such as Grp78/BiP encoding ER chaperone proteins. The disruption of PERK function or the blocking of eIF2alpha Ser51 phosphorylation fails to attenuate translation after ER stress and also results in substantial impairment of Grp78/BiP induction by ER stress. While the activation of the Grp78 promoter by the ATF6 pathway through the endoplasmic reticulum stress elements (ERSEs) is well documented, the molecular mechanism linking PERK activation to Grp78 stress induction is unknown. We report here that ATF4, a transcription factor whose translation is up-regulated by the PERK-eIF2alpha pathway, can activate the Grp78 promoter independent of the ERSE. The ATF4-activating site is localized to an ATF/CRE sequence upstream of the ERSEs and is distinct from the C/EBP-ATF composite site previously identified as the ATF4 binding site in the ER stress-inducible chop promoter. In vitro translated ATF4 binding to the ATF/CRE site requires other nuclear co-factors from non-stressed cells, forming a complex that exhibits identical electrophoretic mobility as a thapsigargin-stress induced complex. Here we have identified the closely related ATF1 and CREB1 as nuclear co-factors that form in vivo complexes with endogenous ATF4. ER stress induces CREB1 phosphorylation and ATF1/CREB1 binding to the Grp78 promoter. Through the use of adenoviral vector expression systems, we provide evidence that when ATF4 function is suppressed and its binding partners are not able to compensate for its function, Grp78 induction by Tg and Tu is partially inhibited. Our studies resolve a mechanism responsible for inhibition of Grp78 mRNA induction by ER stress in cells that are functionally null for PERK or devoid of eIF2alpha phosphorylation.

Selection of the dominant follicle and insulin-like growth factor (IGF)-binding proteins: evidence that pregnancy-associated plasma protein A contributes to proteolysis of IGF-binding protein 5 in bovine follicular fluid.

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn(2+) metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band ( approximately 400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.

Polymorphism of Low Mr Glutenin Subunits in Triticum tauschii

A collection of 173 Triticum tauschii accessions was analysed to evaluate the variability of low molecular weight (Mr) glutenin subunits. These proteins were analysed by one-step one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and were divided into B-, C- and D-subunits in accordance with their electrophoretic mobility. Extensive polymorphism, both in the number and electrophoretic mobility, was detected in lowMr glutenin subunits present in T. tauschii. Thirty different patterns for B-subunits and forty-three for C-subunits were identified, some of which were with identical electrophoretic mobility than those observed in hexaploid wheat. Glutenin subunits with the same electrophoretic mobilities of low Mr D-glutenin subunits as well as subunits encoded at the Glu-D4 and Glu-D5 loci, were also detected in accessions of T. tauschii. These results provide new basic knowledge regarding the genetics variability of the low Mr glutenin subunits, as well as their potential to create novel germplasm for the improvement of wheat quality in breeding programs.

Porcine troponin I: a thermostable species marker protein

In this study, we confirmed our previous hypothesis that the 24 kD thermostable skeletal muscle protein (TSMP) recognized by a panel of porcine-specific monoclonal antibodies (MAbs) is skeletal troponin I (sTnI). The TSMP and sTnI purified from porcine muscle have identical electrophoretic mobilities, isoelectric characteristics, and specific antigenicities. The heterogeneity of sTnI between porcine and other species, and between porcine sTnI and other troponin subunits or cardiac isoforms can be immunologically differentiated by the MAbs. Heat treatment of sTnI up to 126 °C for 120 min did not diminish its solubility and antigenicity. The antigenic specificity and thermal stability of sTnI indicate its potential as a thermostable species marker for the identification of the origin of meats in severely heated products.

Oligomerization and Cell-Binding Properties of the Avian Reovirus Cell-Attachment Protein ςC

Avian reovirus protein ςC, the viral cell-attachment protein, is a minor component of the outer-capsid shell of the viral particle that is synthesized in small amounts in infected cells. We cloned the ςC-encoding ORF in vector pIL-2f, expressed it in Escherichia coli, and partially purified the resulting recombinant protein from inclusion bodies. Rabbit polyclonal antibodies raised against the recombinant protein specifically recognized the viral polypeptide in ELISA, immunoprecipitation, and Western blotting. To study the oligomerization capacity and cell-binding affinity of protein ςC, the ςC-encoding ORF was also expressed in chicken embryo fibroblasts (CEFs) and in reticulocyte lysates. In all three systems protein ςC is expressed as a multimer with identical electrophoretic mobility to the naturally occurring protein. Cell-binding experiments show that both in vitro and in vivo expressed protein ςC display affinity for CEF receptors, and this property is exclusively associated with the oligomeric form of the protein. The fact that incubation of CEF cells with the recombinant protein expressed in bacterial cells completely blocks the binding of purified reovirions indicates both that binding of this protein to cells is specific and saturable, and that reovirions and protein ςC bind to the same class of cell receptor. Saturation binding experiments, performed with the recombinant protein expressed in E. coli and with purified reovirions, showed that the number of cellular receptor sites (CRSs) for avian reovirus S1133 is 1.8 × 104 per CEF cell, whereas the number of cellular receptor units (CRUs) for ςC is 2.2 × 105 per CEF cell. These results are consistent with previous reports on the binding of mammalian reoviruses.

The GTP-binding protein RhoA localizes to the cortical granules of Strongylocentrotus purpuratus sea urchin egg and is secreted during fertilization

The sea urchin egg has thousands of secretory vesicles known as cortical granules. Upon fertilization, these vesicles undergo a Ca2+-dependent exocytosis. G-protein-linked mechanisms may take place during the egg activation. In somatic cells from mammals, GTP-binding proteins of the Rho family regulate a number of cellular processes, including organization of the actin cytoskeleton. We report here that a crude membrane fraction from homogenates of Strongylocentrotus purpuratus sea urchin eggs, incubated with C3 (which ADP-ribosylates specifically Rho proteins) and [32P]NAD, displayed an [32P]ADP-ribosylated protein of 25 kDa that had the following characteristics: i) identical electrophoretic mobility in SDS-PAGE gels as the [32P]ADP-ribosylated Rho from sea urchin sperm; ii) identical mobility in isoelectro focusing gels as human RhoA; iii) positive cross-reactivity by immunoblotting with an antibody against mammalian RhoA. Thus, unfertilized S. purpuratus eggs contain a mammalian RhoA-like protein. Immunocytochemical analyses indicated that RhoA was localized preferentially to the cortical granules; this was confirmed by experiments of [32P]ADP-ribosylation with C3 in isolated cortical granules. Rho was secreted and retained in the fertilization membrane after insemination or activation with A23187. It was observed that the Rho protein present in the sea urchin sperm acrosome was also secreted during the exocytotic acrosome reaction. Thus, Rho could participate in those processes related to the cortical granules, i.e., in the Ca2+-regulated exocytosis or actin reorganization that accompany the egg activation.