Colony-forming hematopoietic cells in the mouse embryonic lung

The colony-forming activity of embryo lung cells CBA mice was determined according to the Till and McCulloch technique (1961). After intravenous injection to jung cells (1 x 10(6)) from 14-day embryos the total number of colonies on the area of 1 mm2 of spleen sections from irradiated recipient mice averaged 2.31 +/- 0.39 whereas after transplantation of lung cells from 15-day embryos it averaged 2.34 +/- 0.53. The percent of macrocolonies equalled 52.5% in the former case and only 2.5% in the latter case. Macrocolonies contained cells of the myeloid, erythroid and megakaryocyte lines. Microcolonies predominantly consisted of granulocytes at various stages of differentiation. Thus, polypotent stem hematopoietic cells migrate into the fetal lung in vivo. The colony-forming ability of the lung polypotent stem cells decreases as the period of embryogenesis increases.     

Variability of key biochemical parameters in senescent leaves of sugar beet at nitrate deficiency and surplus accumulation of glucose

Parameters of sugar beet (Beta vulgaris L.) leaf senescence were investigated in the stage of vegetative growth in plants grown at normal nitrate level (N) or under its deficiency (DefN). Accelerated senescence was initiated by the 41-h-long exposure of leaf discs on the surface of water with alternating darkness and light. In plants grown at DefN, the number of leaves and their average area decreased; after the incubation of the discs from such leaves on water, the content of soluble carbohydrates (sCarb) and especially of glucose sharply increased as compared with normal level of nitrate (N), whereas the content of soluble protein (sProt) and Rubisco activity considerably decreased, which is characteristic of the negative hexokinase (HXK1) effect of glucose. The rate of a decrease in the content of sProt in the course of leaf senescence calculated for the leaf of each strorey was lower than the rate of a decrease in RuBisCO activity. A decrease in the content of sProt and RuBisCO activity in all the storeys of leaves grown at joint action of nitrate deficit (DefN) and incubation on water was on the average greater than in each of these treatments separately but less than the sum of these effects. The imperfection of the putative mechanism of signal transduction at DefN and excess glucose and their interaction in senescent sugar beet leaves is discussed as well as the opportunity to use the ratio between sCarb and sProt for the evaluation of the manifestation of the negative hexokinase effect of glucose.     

Antiapoptotic Activity of the Cardiovirus Leader Protein,a Viral “Security” Protein

Apoptosis is a common antiviral defensive mechanism that potentially limits viral reproduction and spread. Many viruses possess apoptosis-suppressing tools. Here, we show that the productive infection of HeLa cells with encephalomyocarditis virus (a cardiovirus) was not accompanied by full-fledged apoptosis (although the activation of caspases was detected late in infection) but rather elicited a strong antiapoptotic state, as evidenced by the resistance of infected cells to viral and nonviral apoptosis inducers. The development of the antiapoptotic state appeared to depend on a function(s) of the viral leader (L) protein, since its mutational inactivation resulted in the efflux of cytochrome c from mitochondria, the early activation of caspases, and the appearance of morphological and biochemical signs of apoptosis in a significant proportion of infected cells. Infection with both wild-type and L-deficient viruses induced the fragmentation of mitochondria, which in the former case was not accompanied with cytochrome c efflux. Although the exact nature of the antiapoptotic function(s) of cardioviruses remains obscure, our results suggested that it includes previously undescribed mechanisms operating upstream and possibly downstream of the mitochondrial level, and that L is involved in the control of these mechanisms. We propose that cardiovirus L belongs to a class of viral proteins, dubbed here security proteins, whose roles consist solely, or largely, in counteracting host antidefenses. Unrelated L proteins of other picornaviruses as well as their highly variable 2A proteins also may be security proteins. These proteins appear to be independent acquisitions in the evolution of picornaviruses, implying multiple cases of functional (though not structural) convergence.Cells that are infected with a virus recognize the invader''s presence by their innate immunity machinery and switch on a variety of defensive mechanisms. The infecting virus, on the other hand, may possess tools capable of interfering with host antiviral responses. The outcome of the infection, both in terms of the efficiency of virus growth and the extent of host pathology, depends on the trade-off between these defensive and counterdefensive measures.Cellular innate immunity involves multiple pathways, and one powerful defense is apoptosis, or the programmed self-sacrifice of the infected cell, potentially limiting viral reproduction and spread (10). However, many viruses are able to suppress this defensive mechanism (14, 37). Remarkably, virus-elicited pathology may be specific for a given type of cells and a given virus. Unraveling the interplay between pathways leading to the death or survival of the infected cells is an important task that may provide clues to understanding viral pathogenesis and, possibly, may indicate new directions for searching for antiviral drugs.Picornaviruses are a family of small nonenveloped animal viruses that includes important human and animal pathogens such as polioviruses, rhinoviruses, hepatitis A virus, foot-and-mouth disease viruses, and many others (89). Their genome is represented by a single-stranded 7.2- to 8-kb RNA molecule of positive polarity encoding about a dozen mature proteins (generated by the limited proteolysis of a single polyprotein precursor), nearly all of which are directly involved in the replication of the viral RNA and formation of virions (1).The first picornavirus demonstrated to interact with the host cell apoptotic machinery by both triggering and suppressing the apoptotic response was poliovirus (95). Since then, a wealth of data has been accumulated that shows that the activation of apoptotic pathways is a widespread, though not universal, response to picornavirus infection. Thus, apoptosis-inducing capacity was reported for coxsackieviruses B3, B4, and B5 (22, 54, 82), enteroviruses 70 and 71 (25, 27, 60, 88), human rhinoviruses 1B, 9, 14, and 16 (32, 92, 100), foot-and-mouth disease virus (53, 76), avian encephalomyelitis virus (62, 63), and hepatitis A virus (16, 43) and was the subject of several recent reviews (15, 102). The antiapoptotic activity of picornaviruses was studied predominantly by using poliovirus (3, 8, 13, 72) and coxsackievirus B3 (21, 36, 85).The present study is focused on the interaction of cardioviruses, which are representatives of a genus in the picornavirus family, with the apoptotic machinery of infected cells. Our interest in this topic stemmed from the fact that these viruses, e.g., encephalomyocarditis virus (EMCV) and its strain mengovirus (MV), as well as the less-related Theiler''s murine encephalomyelitis virus (TMEV), while sharing major features of genome organization and reproductive strategy with other family members, encode a unique protein that is not found in other picornaviruses. Indeed, the leader (L) protein, a derivative of the N-terminal portion of the viral polyprotein (55), appears to be a major player in controlling the virus-host interaction. On the one hand, it is devoid of any known enzymatic activity, and L-lacking mutants are viable, at least in certain cultured cells (19, 57, 106). On the other hand, the L protein appears to inhibit host translation (35, 106), suppresses interferon production (46, 83, 98), and impairs nucleocytoplasmic traffic (11, 30, 61, 80, 81). It has been hypothesized that cardiovirus L protein also is involved in the interaction with defensive apoptotic machinery.Previous studies have demonstrated that TMEV infection may induce apoptosis, especially in partially restrictive cells (50, 51). EMCV also exerted a similar effect in certain cell lines (87, 103). The reason(s) underlying variability in the apoptosis-inducing effects of cardioviruses remains unexplained. Here, we demonstrate that the productive cardiovirus infection of susceptible HeLa cells resulted in their cytopathic death, which was not accompanied by clear signs of apoptosis. On the contrary, the infected cells acquired an antiapoptotic state, as evidenced by their failure to develop an apoptotic response to viral and nonviral apoptosis inducers. However, the antiapoptotic state failed to develop in cells infected with a mutant virus with inactivated L, and this mutant instead elicited caspase-dependent apoptosis preceded by cytochrome c efflux. These data suggest that the wild-type (wt) L protein is involved, directly or otherwise, in the control of viral antiapoptotic function(s).     

Interactions between Viral and Prokaryotic Pathogens in a Mixed Infection with Cardiovirus and Mycoplasma

In the natural environment, animal and plant viruses often share ecological niches with microorganisms, but the interactions between these pathogens, although potentially having important implications, are poorly investigated. The present report demonstrates, in a model system, profound mutual effects of mycoplasma and cardioviruses in animal cell cultures. In contrast to mycoplasma-free cells, cultures contaminated with Mycoplasma hyorhinis responded to infection with encephalomyocarditis virus (EMCV), a picornavirus, but not with poliovirus (also a picornavirus), with a strong activation of a DNase(s), as evidenced by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) immunofluorescence assay and electrophoretic analysis of host DNA. This degradation was reminiscent of that observed upon apoptosis but was caspase independent, judging by the failure of the specific pan-caspase inhibitor Q-VD-OPh to prevent it. The electrophoretic mobility of the enzyme responsible for DNA degradation and dependence of its activity on ionic conditions strongly suggested that it was represented by a DNase(s) of mycoplasma origin. In cells not infected with EMCV, the relevant DNase was dormant. The possibility is discussed that activation of the mycoplasma DNase might be linked to a relatively early increase in permeability of plasma membrane of the infected cells caused by EMCV. This type of unanticipated virus-mycoplasma “cooperation” may exemplify the complexity of pathogen-host interactions under conditions when viruses and microorganisms are infecting the same host. In the course of the present study, it was also demonstrated that pan-caspase inhibitor zVAD(OMe).fmk strongly suppressed cardiovirus polyprotein processing, illustrating an additional pitfall in investigations of viral effects on the apoptotic system of host cells.Traditionally, virologists are predominantly studying properties of individual viruses and much rarely interactions between different viruses. Even less attention is paid to interactions between viruses and microbes, although animal and plant viruses quite often share, in the natural environment, their ecological niches with microorganisms. Interactions of microbes with viruses other than phages are essentially a virgin soil of either virology or microbiology. The scarcity of relevant information may be illustrated by a recent review focused on the interaction between enteric viruses and enteric bacteria (23). There is ground to believe, however, that the impact of microbial flora on viral growth and pathogenicity (and vice versa) is much broader and deeper than is currently thought. One of the reasons for such a belief is the well-known effect of bacteria (and of their metabolic products) on diverse signaling systems of animal cells, in particular those involved in innate immunity (14, 41, 85, 89). A recent relevant example is demonstration that a prokaryotic infection may protect drosophila from a variety of RNA viruses (39). On the other hand, viral infection may significantly alter the outcome of concomitant bacterial infections. For example, it was reported that herpesvirus latency in mice may confer significant resistance to Listeria and Yersinia pathogens (8).The aim of the present report is to demonstrate, in a model system, profound mutual effects of mycoplasma and cardioviruses during their interaction with animal cells. Cardioviruses, a genus in the picornavirus family, are represented by such widely investigated viruses as encephalomyocarditis virus (EMCV) and its strain mengovirus (MV), as well as Theiler''s murine encephalomyelitis virus (TMEV). Cardioviruses are pathogens naturally affecting many animal species, in particular, rodents, pigs, and some primates (49). Recently, TMEV-related viruses have been implicated in human pathology (1, 12, 21, 29, 45, 46, 51, 96). Picornaviruses, which also include such viruses as poliovirus, hepatitis A virus, foot-and-mouth disease virus, and many others, possess a 7.2-8 kb-long single-stranded RNA genome of positive polarity and share general features of their reproduction mechanism (2). The genomic RNA contains a large single reading frame (certain strains of TMEV, but not EMCV, encode also a protein, L*, in an alternative frame; see reference 47) translated into a polyprotein, which is processed into a dozen “mature” proteins by a series of proteolytic events.Interaction of picornaviruses with susceptible cells may have different outcomes: it may result in cytopathic (necrotic) effect (CPE) or apoptotic death, or it may lead to persistent infection. The character of these outcomes has obvious implications for the pathogenesis of the viral disease. Necrotic death is a strong trigger of inflammatory reactions. Apoptosis, which leads to degradation of host DNA and fragmentation of cells into membrane-coated so-called apoptotic bodies and their eventual “consumption” by macrophages and other scavenger cells, could limit not only the spread of the viral progeny but also prevent potential damages to the neighboring cells caused by inflammation. Therefore, apoptosis is generally considered to represent a defensive host reaction. The fate of the picornavirus-infected cell depends on the genetic properties of the both partners of virus-host interaction, as well as on the status of cell differentiation and environmental conditions (3, 13, 69, 70, 79, 80, 86). In particular, cardioviruses may evoke apoptotic reaction in certain, especially partially restrictive, cells (42, 43, 75, 76, 92).Mycoplasmas are the smallest prokaryotic obligate parasites devoid of cell wall and of many synthetic capabilities and able to trigger a variety of diseases, such as pneumonia, uretritis and many others; they also frequently contaminate cultured cells. Mycoplasmas could be attached to plasma membrane of eukaryotic cells but in some cases they are able to invade the cells as well. They may exert a variety of effects on signaling systems of animal cells affecting, in particular, their innate immunity (67, 71).We will demonstrate here that, in several mycoplasma-contaminated cell cultures, infection with EMCV (but not with poliovirus) results in activation of the microorganism-derived DNase(s), which accomplishes degradation of host cell DNA resembling that occurring during apoptosis but which was caspase independent. The same but mycoplasma-free cells respond to EMCV infection with a canonical CPE (70). This type of unanticipated virus-mycoplasma “cooperation” may illustrate the complexity of pathogen-host interactions under conditions when both viruses and microorganisms are present.     

Live attenuated influenza virus expressing human interleukin-2 reveals increased immunogenic potential in young and aged hosts

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8(+) T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8(+) T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8(+) T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.     

Role of efflux pumps in adaptation and resistance of Listeria monocytogenes to benzalkonium chloride

In this study, potential mechanisms underlying resistance and adaptation to benzalkonium chloride (BC) in Listeria monocytogenes were investigated. Two groups of strains were studied. The first group consisted of strains naturally sensitive to BC which could be adapted to BC. The second group consisted of naturally resistant strains. For all adapted isolates, there was a correlation between the resistance to BC and ethidium bromide, but this was not the case for the naturally resistant isolates. To investigate the role of efflux pumps in adaptation or resistance, reserpine, an efflux pump inhibitor, was added to the strains. Addition of reserpine to the sensitive and adapted strains resulted in a decrease in the MIC for BC, whereas no such decrease was observed for the resistant strains, indicating that efflux pumps played no role in the innate resistance of certain strains of L. monocytogenes to this compound. Two efflux pumps (MdrL and Lde) have been described in L. monocytogenes. Studies showed low and intermediate levels of expression of the genes encoding the efflux pumps for two selected resistant strains, H7764 and H7962, respectively. Adaptation to BC of sensitive isolates of L. monocytogenes resulted in significant increases in expression of mdrl (P < 0.05), but no such increase was observed for lde for two adapted strains of L. monocytogenes, LJH 381 (P = 0.91) and C719 (P = 0.11). This indicates that the efflux pump Mdrl is at least partly responsible for the adaptation to BC.     

Inducible expression of the gene of Zinnia elegans coding for extracellular ribonuclease in Nicotiana tabacum plants

The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2′-promoter of the mannopine synthase (MAS 2′) gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction of MAS 2′ promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus. The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus content in the inoculum and ribonuclease activity.     

The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

Molecular Biology - Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only...     

Different Roles of Eukaryotic MutS and MutL Complexes in Repair of Small Insertion and Deletion Loops in Yeast

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair.     

A preliminary method for estimating the age of brown kiwi (Apteryx mantelli) embryos

Morphological features of a collection of unknown-age wild kiwi (Apteryx mantelli) embryos from early development to point of hatch are described. Using these features, we assign developmental stages to each embryo and compare the progress of development to similar-staged ostrich (Struthio camelus) and chicken (Gallus gallus) embryos. Two ageing schemes for the kiwi embryos are developed by comparing measurements of their hindlimb segments, bills and crown–rump lengths with those of ostrich and chicken embryos at various stages of development. One of the 20 kiwi embryos was of known age. Both the ostrich model and the chicken model gave identical predictions for the marker and four other embryos. Developmental timing of some features differed between all three species, most markedly in the bill, with growth in the kiwi bill being relatively faster to achieve its larger relative and absolute size at hatch.