Variation in vector competence for dengue viruses does not depend on mosquito midgut binding affinity

Background

Dengue virus genotypes of Southeast Asian origin have been associated with higher virulence and transmission compared to other genotypes of serotype 2 (DEN-2). We tested the hypothesis that genetic differences in dengue viruses may result in differential binding to the midgut of the primary vector, Aedes aegypti, resulting in increased transmission or vectorial capacity.

Methodology/Principal Finding

Two strains of each of the four DEN-2 genotypes (Southeast Asian, American, Indian, and West African) were tested to determine their binding affinity for mosquito midguts from two distinct populations (Tapachula, Chiapas, Mexico and McAllen, Texas, USA). Our previous studies demonstrated that Southeast Asian viruses disseminated up to 65-fold more rapidly in Ae. aegypti from Texas and were therefore more likely to be transmitted to humans. Results shown here demonstrate that viruses from all four genotypes bind to midguts at the same rate, in a titer-dependent manner. In addition, we show population differences when comparing binding affinity for DEN-2 between the Tapachula and McAllen mosquito colonies.

Conclusions

If midgut binding potential is the same for all DEN-2 viruses, then viral replication differences in these tissues and throughout the mosquito can thus probably explain the significant differences in dissemination and vector competence. These conclusions differ from the established paradigms to explain mosquito barriers to infection, dissemination, and transmission.     

Inhibition of cells in culture by polyamines does not depend on the presence of ruminant serum

Using T-lymphocyte (T-LC) and granulocyte colony (GC) assays with truly proliferating cells, the inhibitory dose-response relationships of spermine and spermidine in the presence of selected sera have been examined. In contrast to previous studies which used [3H]thymidine uptake as an index of proliferation, in vitro inhibition by polyamines was shown to require neither foetal calf serum (FCS) nor the addition of any exogenous polyamine oxidase. Cells grown in the absence of FCS were between 5-50% as sensitive to polyamines as in its presence. By using specific inhibitors of polyamine oxidase, it was shown that polyamine-elicited mitotic inhibition in the absence of FCS was still dependent on a polyamine oxidase, and evidence is presented to show that the source of the enzyme is the cells themselves.     

Sex difference in thermal preference of adult mice does not depend on presence of the gonads

Background

The thermoneutral zone (TNZ) is a species-specific range of ambient temperature (T _a), at which mammals can maintain a constant body temperature with the lowest metabolic rate. The TNZ for an adult mouse is between 26 and 34 °C. Interestingly, female mice prefer a higher T _a than male mice although the underlying mechanism for this sex difference is unknown. Here, we tested whether gonadal hormones are dominant factors controlling temperature preference in male and female mice.

Methods

We performed a temperature preference test in which 10-week-old gonadectomized and sham-operated male and female C57BL/6J mice were allowed to choose to reside at the thermoneutral cage of 29 °C or an experimental cage of 26, 29, or 32 °C.

Results

All mice preferred a T _a higher than 26 °C, especially in the inactive phase. Choosing between 29 and 32 °C, female mice resided more at 32 °C while male mice had no preference between the temperatures. Hence, the preferred T _a for female mice was significantly higher (0.9?±?0.2 °C) than that for male mice. However, gonadectomy did not influence the T _a preference.

Conclusions

Female mice prefer a warmer environment than male mice, a difference not affected by gonadectomy. This suggests that thermal-sensing mechanisms may be influenced by sex-specific pathways other than gonadal factors or that the thermoregulatory set point has already been determined prior to puberty.

     

Repair of potentially lethal damage does not depend on functional TP53 in human glioblastoma cells

The functionality of G(1)-phase arrest was investigated in relation to repair of potentially lethal damage (PLD) in human glioblastoma Gli-06 cells. Confluent cultures were irradiated and plated for clonogenic survival either immediately or 24 h after gamma irradiation. Bivariate flow cytometry was performed to assess the distribution over the cell cycle. Levels of TP53 and CDKN1A protein were assessed with Western blotting and levels of CDKN1A mRNA with RT-PCR. Confluence significantly reduced the number of proliferating cells. Marked PLD repair was found in the absence of an intact G(1) arrest. No accumulation of TP53 was observed, and the protein was smaller than the wild-type TP53 of RKO cells. No increased expression of CDKN1A at the mRNA or protein levels was found in Gli-06 cells. The TP53 of Gli-06 cells was unable to transactivate the CDKN1A gene. From this study, it is evident that PLD repair may be present without a functional TP53 or G(1) arrest.     

Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals

Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine. The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins. Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system. Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein. To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation. Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro. The release of these lipoproteins was examined in proteoliposomes. We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes. Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.     

Death of CD4(+) T-cell lines caused by human immunodeficiency virus type 1 does not depend on caspases or apoptosis

A critical aspect of AIDS pathogenesis that remains unclear is the mechanism by which human immunodeficiency virus type 1 (HIV-1) induces death in CD4(+) T lymphocytes. A better understanding of the death process occurring in infected cells may provide valuable insight into the viral component responsible for cytopathicity. This would aid the design of preventive treatments against the rapid decline of CD4(+) T cells that results in AIDS. Previously, apoptotic cell death has been reported in HIV-1 infections in cultured T cells, and it has been suggested that this could affect both infected and uninfected cells. To evaluate the mechanism of this effect, we have studied HIV-1-induced cell death extensively by infecting several T-cell lines and assessing the level of apoptosis by using various biochemical and flow cytometric assays. Contrary to the prevailing view that apoptosis plays a prominent role in HIV-1-mediated T-cell death, we found that Jurkat and H9 cells dying from HIV-1 infection fail to exhibit the collective hallmarks of apoptosis. Among the parameters investigated, Annexin V display, caspase activity and cleavage of caspase substrates, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) signal, and APO2.7 display were detected at low to negligible levels. Neither peptide caspase inhibitors nor the antiapoptotic proteins Bcl-x(L) or v-FLIP could prevent cell death in HIV-1-infected cultures. Furthermore, Jurkat cell lines deficient in RIP, caspase-8, or FADD were as susceptible as wild-type Jurkat cells to HIV-1 cytopathicity. These results suggest that the primary mode of cytopathicity by laboratory-adapted molecular clones of HIV-1 in cultured cell lines is not via apoptosis. Rather, cell death occurs most likely via a necrotic or lytic form of death independent of caspase activation in directly infected cells.     

Systemic immune deviation in the brain that does not depend on the integrity of the blood-brain barrier

OVA injected into the brain of normal mice evoked a deviant immune response (brain-associated immune deviation (BRAID)) that was deficient in OVA-specific delayed-type hypersensitivity. This response was not dependent on an intact blood-brain barrier since BRAID was induced even when OVA was injected into a newly created lesion site with extensive BBB leakage. However, newly activated microglia at the injection site 2 days after ablation of the striatum correlated with the loss of BRAID. At day 4 after trauma, when activated microglia were only visible further away from the injection site, BRAID was again able to be induced. In contrast to immune deviation elicited via the eye, an intact spleen was not required for BRAID, nor was BRAID adoptively transferable with spleen cells. In contrast i.v. injection of cervical lymph node cells harvested 8 days after OVA injection into the striatum was able to transfer BRAID into naive animals. Together, these data indicate that immune privilege in the brain is actively maintained and is mediated by an immune deviation mechanism that differs from eye-derived immune deviation and arises even when the BBB is compromised.     

Auxin-induced H+-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA Indole-3-acetic acid - MES 2-[N morpholino-ethane-sulfonic acid - TRIS 2-Amino-2-hydroxymethyl-1,3-propanediol We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft.     

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.     

Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode

The main protease (M^pro) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for trans-cleavage activity. However, the auto-processing mechanism of M^pro, i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of “immature” M^pro. Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature M^pro, are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and trans-cleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the “immature” C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of M^pro appears to require only two “immature” monomers approaching one another to form an “intermediate” dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of M^pro from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV M^pro.     

Induction of immune tolerance during administration of monoclonal antibody to L3T4 does not depend on depletion of L3T4+ cells

Treatment of mice with mAb to L3T4 profoundly depletes T helper cells. This treatment inhibits humoral and cellular immunity, retards autoimmunity, and permits the induction of Ag-specific tolerance. Treatment of BALB/c mice with F(ab')2 anti-L3T4 inhibits humoral immunity without depleting L3T4+ cells, which is evidence that mAb to L3T4 may inhibit T helper cell function in vivo. In this report, we demonstrate that F(ab')2 anti-L3T4 also permits the induction of immune tolerance in a manner that is independent of T cell depletion. C57BL/6 mice were treated with 1 mg of F(ab')2 anti-L3T4 every other day for 18 days and from the onset were challenged weekly with the immunogen 2C7, a rat mAb to chicken ovalbumin. These mice failed to respond to 2C7 not only during the treatment period but also for at least 5 mo thereafter. This immune tolerance was Ag-specific; the mice rapidly produced antibodies to subsequent challenge with another Ag, human gamma-globulin. Unlike intact anti-L3T4, which immediately depletes L3T4+ cells by greater than 90%, F(ab')2 anti-L3T4 did not initially deplete cells and caused only a partial reduction by the end of the 18-day treatment. This partial reduction of L3T4+ cells did not contribute to the induction of tolerance because mice that were first challenged with 2C7 3 days after stopping the F(ab')2 anti-L3T4 treatment, when L3T4+ cells were lowest, had a normal Ir to 2C7. These findings demonstrate that mAb to L3T4 permits induction of Ag-specific immune tolerance by a mechanism independent of its ability to deplete L3T4+ cells. They also show that F(ab')2 anti-L3T4 treatment does not impair humoral immunity when immunization is initiated after treatment is stopped. Because L3T4 is homologous to CD4 in humans, our findings suggest that F(ab')2 anti-CD4 may offer significant advantages over the use of intact anti-CD4 as an immunosuppressive agent in humans.     

Maturation of social reward in adult male Syrian hamsters does not depend on organizational effects of pubertal testosterone

The rewarding value of female sexual stimuli develops across puberty, as sexually-naïve adult, but not prepubertal, male hamsters show a conditioned place preference (CPP) for both vaginal secretions and a receptive female. Similarly, only adults show an endogenous testosterone surge when they encounter vaginal secretions. Testosterone by itself can condition a place preference in male rodents. Therefore, Experiment 1 assessed whether the endogenous testosterone surge elicited by vaginal secretions is necessary to show a CPP. Both gonad-intact and gonadectomized, testosterone-treated adult males showed a CPP for vaginal secretions, indicating that the rewarding value of this social cue is independent of an endogenous testosterone surge. However, organizational effects of pubertal testosterone could be necessary for adolescent development of social reward, as pubertal testosterone organizes adult-typical expression of sexual behavior. To investigate this possibility, in Experiment 2, sexually-naïve prepubertal and adult male hamsters were gonadectomized and received testosterone-filled capsules four weeks later. Testing began after two weeks of testosterone replacement. Adult males showed a CPP for both vaginal secretions and a receptive female, whether or not they experienced pubertal testosterone. Thus, the acquisition of positive valence of sexual stimuli is not organized by pubertal testosterone. Taken together, the ability of female sexual stimuli to serve as an unconditioned reward to adult male hamsters is independent of the chemosensory-induced endogenous testosterone surge and also organizational effects of pubertal testosterone. Instead, sexual reward may be dependent either on activational effects of testosterone or gonadal hormone-independent mechanisms.     

Acetylcholine-dependent potentiation of temporal frequency representation in the barrel cortex does not depend on response magnitude during conditioning

The response properties of neurons of the postero-medial barrel sub-field of the somatosensory cortex (the cortical structure receiving information from the mystacial vibrissae can be modified as a consequence of peripheral manipulations of the afferent activity. This plasticity depends on the integrity of the cortical cholinergic innervation, which originates at the nucleus basalis magnocellularis (NBM). The activity of the NBM is related to the behavioral state of the animal and the putative cholinergic neurons are activated by specific events, such as reward-related signals, during behavioral learning. Experimental studies on acetylcholine (ACh)-dependent cortical plasticity have shown that ACh is needed for both the induction and the expression of plastic modifications induced by sensory-cholinergic pairings. Here we review and discuss ACh-dependent plasticity and activity-dependent plasticity and ask whether these two mechanisms are linked. To address this question, we analyzed our data and tested whether changes mediated by ACh were activity-dependent. We show that ACh-dependent potentiation of response in the barrel cortex of rats observed after sensory-cholinergic pairing was not correlated to the changes in activity induced during pairing. Since these results suggest that the effect of ACh during pairing is not exerted through a direct control of the post-synaptic activity, we propose that ACh might induce its effect either pre- or post-synaptically through activation of second messenger cascades.     

Mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of de novo fatty acid synthesis in Arabidopsis

The photorespiratory Arabidopsis (Arabidopsis thaliana) mutant gld1 (now designated mtkas-1) is deficient in glycine decarboxylase (GDC) activity, but the exact nature of the genetic defect was not known. We have identified the mtkas-1 locus as gene At2g04540, which encodes beta-ketoacyl-[acyl carrier protein (ACP)] synthase (mtKAS), a key enzyme of the mitochondrial fatty acid synthetic system. One of its major products, octanoyl-ACP, is regarded as essential for the intramitochondrial lipoylation of several proteins including the H-protein subunit of GDC and the dihydrolipoamide acyltransferase (E2) subunits of two other essential multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. This view is in conflict with the fact that the mtkas-1 mutant and two allelic T-DNA knockout mutants grow well under nonphotorespiratory conditions. Although on a very low level, the mutants show residual lipoylation of H protein, indicating that the mutation does not lead to a full functional knockout of GDC. Lipoylation of the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase E2 subunits is distinctly less reduced than that of H protein in leaves and remains unaffected from the mtKAS knockout in roots. These data suggest that mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of lipoate biosynthesis in leaves and may occur independently of this pathway in roots.