Developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta

Cytomegalovirus (CMV), the major viral cause of congenital disease, infects the uterus and developing placenta and spreads to the fetus throughout gestation. Virus replicates in invasive cytotrophoblasts in the decidua, and maternal immunoglobulin G (IgG)-CMV virion complexes, which are transcytosed by the neonatal Fc receptor across syncytiotrophoblasts, infect underlying cytotrophoblasts in chorionic villi. Immunity is central to protection of the placenta-fetal unit: infection can occur when IgG has a low neutralizing titer. Here we used immunohistochemical and function-blocking methods to correlate infection in the placenta with expression of potential CMV receptors in situ and in vitro. In placental villi, syncytiotrophoblasts express the virion receptor epidermal growth factor receptor (EGFR) but lack integrin coreceptors, and virion uptake occurs without replication. Focal infection can occur when transcytosed virions reach EGFR-expressing cytotrophoblasts that selectively initiate expression of alphaV integrin. In cell columns, proximal cytotrophoblasts lack receptors and distal cells express integrins alpha1beta1 and alphaVbeta3, enabling virion attachment. In the decidua, invasive cytotrophoblasts expressing coreceptors upregulate EGFR, thereby dramatically increasing susceptibility to infection. Our findings indicate that virion interactions with cytotrophoblasts expressing receptors in the placenta (i) change as the cells differentiate and (ii) correlate with spatially distinct sites of CMV replication in maternal and fetal compartments.     

Domain I of ribosomal protein L1 is sufficient for specific RNA binding

Ribosomal protein L1 has a dual function as a ribosomal protein binding 23S rRNA and as a translational repressor binding its mRNA. L1 is a two-domain protein with N- and C-termini located in domain I. Earlier it was shown that L1 interacts with the same targets on both rRNA and mRNA mainly through domain I. We have suggested that domain I is necessary and sufficient for specific RNA-binding by L1. To test this hypothesis, a truncation mutant of L1 from Thermus thermophilus, representing domain I, was constructed by deletion of the central part of the L1 sequence, which corresponds to domain II. It was shown that the isolated domain I forms stable complexes with specific fragments of both rRNA and mRNA. The crystal structure of the isolated domain I was determined and compared with the structure of this domain within the intact protein L1. This comparison revealed a close similarity of both structures. Our results confirm our suggestion that in protein L1 its domain I alone is sufficient for specific RNA binding, whereas domain II stabilizes the L1-rRNA complex.     

Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2

Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.     

Inhibition of S. aureus alpha-hemolysin and B. anthracis lethal toxin by beta-cyclodextrin derivatives

Many pathogens utilize the formation of transmembrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increase if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of beta-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the transmembrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought beta-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcus aureus alpha-hemolysin (alpha-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of beta-cyclodextrin that inhibited the activity of alpha-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by alpha-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.     

Ultrastructure of ependyma in brain third ventricle of the rats exposed to repeated tail-suspension. Scanning electron microscopical study

By means of scanning electron microscopy the ultrastructure of ependyma was studied in the brain third ventricle of the rats repeatedly exposed to 14-day tail-suspension (TS). Animals were subjected to TS for 30 days, then readapted to horizontal position during 30 days and again, repeatedly subjected to TS for 14 days simultaneously with the rats which were in TS for the first time during 14 days. Repeated TS of rats, inspite of repeated redistribution of body liquid mediums in cranial direction, results in considerably less expressed destructive changes in ultrastructure of ependymocyte cilia, then after primary 14- and 30-day TS, showing much greater cerebrospinal fluid (CSF) outflow from brain ventricles into sagittal venous sinus at postponed for a long time, repeated simulation of weightlessness effects in comparison with CSF outflow at primery one.     

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.     

Mitotic chromosome biorientation in fission yeast is enhanced by dynein and a minus-end-directed, kinesin-like protein

Chromosome biorientation, the attachment of sister kinetochores to sister spindle poles, is vitally important for accurate chromosome segregation. We have studied this process by following the congression of pole-proximal kinetochores and their subsequent anaphase segregation in fission yeast cells that carry deletions in any or all of this organism's minus end-directed, microtubule-dependent motors: two related kinesin 14s (Pkl1p and Klp2p) and dynein. None of these deletions abolished biorientation, but fewer chromosomes segregated normally without Pkl1p, and to a lesser degree without dynein, than in wild-type cells. In the absence of Pkl1p, which normally localizes to the spindle and its poles, the checkpoint that monitors chromosome biorientation was defective, leading to frequent precocious anaphase. Ultrastructural analysis of mutant mitotic spindles suggests that Pkl1p contributes to error-free biorientation by promoting normal spindle pole organization, whereas dynein helps to anchor a focused bundle of spindle microtubules at the pole.     

Antimicrobial susceptibility and molecular mechanisms of resistance to beta-lactams of gram-negative microorganisms--causative agents of nosocomial infections

Profiles and mechanisms of resistance to beta-lactam antibiotics of isolates of Gram-negative microorganisms, which are causative agents of infections in Intensive Care Unit of hospital surgery department, were studied. Two hundred and ten clinical isolates were studied: Pseudomonas aeruginosa--86 strains (40.9%), Acinetobacter baummanii--45 strains (21.4%), Klebsiella pneumoniae--52 strains (24.8%), Escherichia coli--23 strains (11%), Enterobacter spp.--4 strains (1.9%). Profiles of antibiotic resistance were studied by the method of serial microdilutions; detection of most widespread and clinically significant genes of beta-lactamases of Gram-negative bacteria was performed by polymerase chain reaction. Carbapenems and cefoperazone/sulbactam were the most active antibiotics. Local features of distribution of beta-lactamase coding genes (TEM, SHV, CTX) in K. pneumoniae and E. coli isolates were revealed. Eleven strains of P. aeruginosa resistant to carbapenems and possessing genetic determinants of VIM-group, which codes metallo-beta-lactamases, were isolated. Obtained data allows to assess the parameters of resistance to beta-lactam antibiotics and to reveal the main mechanisms of such resistance in etiologic agents of nosocomial infections, that, in its turn, allows to choose preparations for etiotropic therapy.     

The influence of ethylene perception on sex expression in melon (<Emphasis Type="Italic">Cucumis melo </Emphasis>L.) as assessed by expression of the mutant ethylene receptor, <Emphasis Type="Italic">At-etr1-1</Emphasis>, under the control of constitutive and floral targeted promoters

Sexual diversity expressed by Curcurbitaceae species is a primary example of developmental plasticity in plants. Ethylene, which promotes femaleness (carpel development), plays a key role in sex determination. We sought to determine the critical location for ethylene perception in developing floral primodia. The dominant negative Arabidopsis ethylene response mutant gene, etr1-1, was introduced into melon (Cucumis melo L.) plants under control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, or floral-targeted Apetela3 (AP3) and Crab’s Claw (CRC) promoters, which in Arabidopsis, promote expression in petal and stamen, and carpel and nectary primordia, respectively. Based on effects of exogenous ethylene, it was predicted that inhibition of ethylene perception by carpel primordia would inhibit carpel development. Constitutive expression of etr1-1 caused several phenotypes associated with ethylene insensitivity, verifying that etr1-1 inhibits ethylene perception in the heterologous melon system. Carpel-bearing bud production was essentially abolished in 35S::etr1-1 melons, providing direct demonstration of the requirement for ethylene perception for carpel development. CRC::etr1-1 plants, however, showed enhanced femaleness as manifested by earlier and increased number of carpel-bearing buds, and production of female (rather than bisexual) buds. Despite increased carpel-bearing bud formation, a greater proportion of the CRC::etr1-1 carpel-bearing buds aborted before anthesis. AP3::etr1-1 plants showed increased maleness by nearly exclusive staminate flower production, and poorly developed carpels in the rare bisexual flowers. These results indicate that ethylene perception by the stamen (or petal) primordia plays a critical role in promoting carpel development at the time of sex determination, while ethylene perception by the carpel is important for maturation of carpel-bearing flowers to anthesis.