Abnormal development of floral meristem triggers defective morphogenesis of generative system in transgenic tomatoes

Parthenocarpy and fruit malformations are common among independent transgenic tomato lines, expressing genes encoding different pathogenesis-related (PR) protein and antimicrobal peptides. Abnormal phenotype developed independently of the expression and type of target genes, but distinctive features during flower and fruit development were detected in each transgenic line. We analyzed the morphology, anatomy, and cytoembryology of abnormal flowers and fruits from these transgenic tomato lines and compared them with flowers and fruits of wild tomatoes, line YaLF used for transformation, and transgenic plants with normal phenotype. We confirmed that the main cause of abnormal flower and fruit development was the alterations of determinate growth of generative meristem. These alterations triggered different types of anomalous growth, affecting the number of growing ectopic shoots and formation of new flowers. Investigation of the ovule ontogenesis did not show anomalies in embryo sac development, but fertilization did not occur and embryo sac degenerated. Nevertheless, the ovule continued to differentiate due to proliferation of endothelium cells. The latter substituted embryo sac and formed pseudoembryonic tissue. This process imitated embryogenesis and stimulated ovary growth, leading to the development of parthenocarpic fruit. We demonstrated that failed fertilization occurred due to defective male gametophyte formation, which was manifested in blocked division of the nucleus in the microspore and arrest of vegetative and generative cell formation. Maturing pollen grains were overgrown microspores, not competent for fertilization but capable to induce proliferation of endothelium and development of parthenocarpic ovary. Thus, our study provided new data on the structural transformations of reproductive organs during development of parthenocarpic fruits in transgenic tomato.     

Three-dimensional structure of α-crystallin domain dimers of human small heat shock proteins HSPB1 and HSPB6

Small heat shock proteins (sHSPs) are a family of evolutionary conserved ATP-independent chaperones. These proteins share a common architecture defined by a signature α-crystallin domain (ACD) flanked by highly variable N- and C-terminal extensions. The ACD, which has an immunoglobulin-like fold, plays an important role in sHSP assembly. This domain mediates dimer formation of individual protomers, which then may assemble into larger oligomers. In vertebrate sHSPs, the dimer interface is formed by the symmetrical antiparallel pairing of two β-strands (β7), generating an extended β-sheet on one face of the ACD dimer. Recent structural studies of isolated ACDs from a number of vertebrate sHSPs suggest a variability in the register of the β7/β7 strand interface, which may, in part, give rise to the polydispersity often associated with the full-length proteins. To further analyze the structure of ACD dimers, we have employed a combination of X-ray crystallography and solution small-angle X-ray scattering (SAXS) to study the ACD-containing fragments of human HSPB1 (HSP27) and HSPB6 (HSP20). Unexpectedly, the obtained crystal structure of the HSPB1 fragment does not reveal the typical β7/β7 dimers but, rather, hexamers formed by an asymmetric contact between the β4 and the β7 strands from adjacent ACDs. Nevertheless, in solution, both ACDs form stable dimers via the symmetric antiparallel interaction of β7 strands. Using SAXS, we show that it is possible to discriminate between different putative registers of the β7/β7 interface, with the results indicating that, under physiological conditions, there is only a single register of the strands for both proteins.     

A point mutation in the coat protein gene affects long distance transport of the tobacco mosaic virus

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic distribution of the mutant virus was delayed by 5-6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.     

Human RFP2 gene promoter: unique structure and unusual strength

Human gene RFP2 is a candidate tumor suppressor located at 13q14.3 and deleted in multiple tumor types. To explore regulation of RFP2, we determined structure of the 5'-untranslated region of RFP2 gene and its promoter. RFP2 promoter area is TATA-less, highly enriched in G and C nucleotides, and contains multiple quadruplex forming GGGGA-repeats. Deletion analysis of 5'-flanking sequences demonstrated that repeat containing fragment possesses activity seven times exceeding that of the combined SV40 promoter/enhancer. Other unusual features of the RFP2 promoter include anomalously high electrostatic fields induced by sequence-dependent dipoles and very low nucleosome forming potential. A "minimized" version of the RFP2 promoter could be used for overexpression of the various transgenes in the mammalian cells.     

Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.     

FABP4 and omentin-1 gene expression in epicardial adipose tissue from coronary artery disease patients

Omentin-1 and fatty acid-binding protein 4 (FABP4) are adipose tissue adipokines linked to obesity-associated cardiovascular complications. The aim of this study was to investigate epicardial adipose tissue (EAT) omentin-1 and FABP4 gene expression in obese and non-obese patients with coronary artery disease (CAD). Omentin-1 and FABP4 mRNA levels in EAT and paired subcutaneous adipose tissue (SAT) as well as adipokine serum concentrations were assessed in 77 individuals (61 with CAD; 16 without CAD (NCAD)). EAT FABP4 mRNA level was decreased in obese CAD patients when compared to obese NCAD individuals (p=0.001). SAT FABP4 mRNA level was decreased in CAD patients compared to NCAD individuals without respect to their obesity status (p=0.001). Omentin-1 mRNA level in EAT and SAT did not differ between the CAD and NCAD groups. These findings suggest that omentin-1 gene expression in adipose tissue is not changed during CAD; downregulated FABP4 gene expression in SAT is associated with CAD while EAT FABP4 gene expression is decreased only in obesity-related CAD.     

Evaluation of diagnostic efficiency of the recombinant protein modeling immunodominant epitope V3 of envelope gp120 for immunoenzyme detection for HIV-1 infection antibodies

The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.     

Localization of a lysine residue near the site of initiating substrate binding of T7 bacteriophage RNA polymerase

A highly selective affinity label was introduced into the T7 phage RNA polymerase by means of GMP ortho-formylphenyl ester and [alpha-32P]UTP nearby the enzyme's active site, which was located using limited cleavage technique. Hydroxylamine, bromine, N-chlorosuccinimide, and cyanogen bromide were employed as the reagents. Analysis of gel-electrophoretic patterns of the cleavage products led to a conclusion that Lys631 is the target of labelling. The region nearby this residue has a high degree of sequence homology with regions of RNA polymerases from T3 and SP6 phages and yeast mitochondria.