Environmental biosensors for organochlorines, cyanobacterial toxins and endocrine disrupting chemicals

Environmental biosensors and related techniques for monitoring organochlorines, endocrine disrupting chemicals and cyanobacterial toxins are described. The practical requirements for an ideal environmental biosensor are analyzed. Specific case studies for environmental applications are reported for triazines chlorinated phenols, PCBs, microcystins, and endocrine disrupting chemicals. A new promising approach is reported for microcystins and alkylphenols that utilize electrooptical detection.     

Induction of resistance against Fusarium wilt of tomato by combination of chitosan with an endophytic bacterial strain: ultrastructure and cytochemistry of the host response

The potential of Bacillus pumilus (PGPR strain SE 34), either alone or in combination with chitosan, for inducing defense reactions in tomato (Lycopersicon esculentum Mill.) plants inoculated with the vascular fungus, Fusarium oxysporum f. sp. radicis-lycopersici, was studied by light and transmission electron microscopy and further investigated by gold cytochemistry. The key importance of fungal challenge in the elaboration of defense mechanisms is discussed in relation to the possibility that an alarm signal provided by the pathogen itself is required for the expression of resistance in plants previously sensitized by biotic agents. Ultrastructural investigations of the infected root tissues from water-treated (control) plants showed a rapid colonization of all tissues including the vascular stele. In root tissues from bacterized tomato plants grown in the absence of chitosan, the limited fungal development coincided with marked changes in the host physiology. The main facets of the altered host metabolism concerned the induction of a structural response at sites of fungal entry and the abnormal accumulation of electron-dense substances in the colonized areas. A substantial increase in the extent and magnitude of the cellular changes induced by B. pumilus was observed when chitosan was supplied to bacterized tomato plants. These changes were characterized by a considerable enlargement of the callose-enriched wall appositions deposited onto the inner cell wall surface in the epidermis and the outer cortex. The use of the wheat germ agglutinin-ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium cells colonizing bacterized tomato roots was not substantially altered. One of the most-typical fungal cell reactions, observed only when bacterized tomato plants were grown in the presence of chitosan, was the formation of abnormal chitin-enriched deposits between the retracted plasma membrane and the cell wall. Results of the present study provide the first evidence that combination of biocontrol approaches is a promising step towards elaborating integrated pest management programmes. Received: 6 June 1997 / Accepted: 8 July 1997     

Distribution of Activator of G-Protein Signaling 3 within the Aggresomal Pathway: Role of Specific Residues in the Tetratricopeptide Repeat Domain and Differential Regulation by the AGS3 Binding Partners Giα and Mammalian Inscuteable

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Giα-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Giα and mammalian Inscuteable (mInsc). Giα rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.The discovery of AGS3 (GPSM1) and related accessory proteins revealed unexpected functional diversity for G-protein signaling systems (8, 36). AGS3 is involved in a number of different cellular activities, including asymmetric cell division during neuronal development (30), neuronal plasticity and addiction (9, 10, 12, 38, 39), autophagy (27), membrane protein trafficking (17), cardiovascular function (7), and metabolism (7). AGS3 is a multidomain protein consisting of seven tetratricopeptide repeats (TPR) in the amino-terminal portion of the protein and four G-protein regulatory (GPR) motifs in the carboxyl region of the protein. Each of the GPR motifs binds and stabilizes the GDP-bound conformation of Gα (Giα, Gtα, and Gi/oα), essentially behaving as a guanine nucleotide dissociation inhibitor. As such, AGS3 may be complexed with up to four Gα and function as an alternative binding partner for Gα independently of the classical heterotrimeric Gαβγ. Despite the clearly demonstrated function of AGS3 and the related protein LGN (GPSM2 or AGS5) in various model organisms and a fairly solid, basic biochemical understanding of the interaction of a GPR motif with Gα, the signals that operate “upstream” and/or “downstream” of AGS3 or an AGS3-Gi/oα complex are not well defined.AGS3 and other GPR proteins may regulate G-protein signaling directly by influencing the interaction of Gα with Gβγ or another Gα binding partner. In addition, a portion of Gα in the cell is complexed with GPR proteins to various degrees, and this interaction is regulated. Ric-8A interacts with an AGS3-Giα complex in a manner somewhat analogous to the interaction of a G-protein-coupled receptor with heterotrimeric Gαβγ, promoting nucleotide exchange and the apparent dissociation of AGS3 and Giα-GDP (37). The specific impact of AGS3 and other GPR proteins on signaling events is likely dependent upon where the individual protein is positioned within the cell and the nature of intra- and intermolecular interactions that influence the interaction of the GPR motif with Gi/oα.The TPR domain of AGS3 is an important determinant of its positioning within the cell through its interaction with specific binding partners (1, 8, 28, 36). As part of a broader effort to address the fundamental questions of AGS3 “positioning” and control of G-protein interaction, we focused upon the roles of individual TPR domains. Endogenous and ectopically expressed wild-type AGS3 is nonhomogeneously distributed in the cytoplasm, with obvious punctate structures, and it may be present at the cell periphery. Disruption of the TPR organizational structure by targeted amino acid substitutions or introduction of a nonsynonymous single-nucleotide polymorphism redistributes AGS3 to punctate structures throughout the cytoplasm that are similar in appearance to the preaggresomal assemblies or aggregates observed in neurodegenerative diseases. Upon cellular stress, both wild-type and TPR-modified AGS3 migrate, in a microtubule-dependent manner, to a perinuclear aggresome. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is differentially regulated by Giα and mammalian Inscuteable (mInsc), which bind to the GPR and TPR domains, respectively, of AGS3. These data present AGS3 and G-proteins as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies and suggest that this involvement can be manipulated to therapeutic advantage.     

Construction of banana bunchy top nanovirus-DNA-3 encoding the coat protein gene and its introducing into banana plants cv. Williams

Banana (Musa sp.) is considered as one of the most important fruit crops worldwide as well as in Egypt. The main goal of this study was to construct the open reading frame (ORF) of banana bunchy top nanovirus (BBTV)-DNA-3 that encodes the viral coat protein (cp) gene for banana transformation. The previously sequenced BBTV-G-DNA-3-ORF that cloned into plasmid pH1 was used as a template for PCR amplification using two specific primers containing Bam H1 site. A new plasmid called pRHA1 containing the amplified ORF under the control of maize polyubiquitin (ubi) promoter was created. The bar gene (herbicide-resistance gene as a selectable marker) cassette (bar gene, Cauliflower mosaic caulimovirus (CaMV) 35S promoter and nos terminator) was released from plasmid pAB6 using Hind III-digestion and subcloned into the Hind III-digested plasmid pRHA1 to create the plasmid pRHA2 via the microprojectile bombardment transformation system. The plasmid pRHA2 was successfully introduced in the applied banana cultivar. Leaf painting test was conducted to confirm the expression of the bar gene in the putative transformed banana lines. The presence and expression of BBTV-G-cp gene were also detected using some molecular (polymerase chain reaction and dot blot using a cold DNA probe) and serological (ELISA and western blot) techniques, respectively, in the obtained transgenic banana lines.     

Impact on Clinical and Cost Outcomes of a Centralized Approach to Acute Stroke Care in London: A Comparative Effectiveness Before and After Model

Background

In July 2010 a new multiple hub-and-spoke model for acute stroke care was implemented across the whole of London, UK, with continuous specialist care during the first 72 hours provided at 8 hyper-acute stroke units (HASUs) compared to the previous model of 30 local hospitals receiving acute stroke patients. We investigated differences in clinical outcomes and costs between the new and old models.

Methods

We compared outcomes and costs ‘before’ (July 2007–July 2008) vs. ‘after’ (July 2010–June 2011) the introduction of the new model, adjusted for patient characteristics and national time trends in mortality and length of stay. We constructed 90-day and 10-year decision analytic models using data from population based stroke registers, audits and published sources. Mortality and length of stay were modelled using survival analysis.

Findings

In a pooled sample of 307 patients ‘before’ and 3156 patients ‘after’, survival improved in the ‘after’ period (age adjusted hazard ratio 0.54; 95% CI 0.41–0.72). The predicted survival rates at 90 days in the deterministic model adjusted for national trends were 87.2% ‘before’ % (95% CI 86.7%–87.7%) and 88.7% ‘after’ (95% CI 88.6%–88.8%); a relative reduction in deaths of 12% (95% CI 8%–16%). Based on a cohort of 6,438 stroke patients, the model produces a total cost saving of £5.2 million per year at 90 days (95% CI £4.9-£5.5 million; £811 per patient).

Conclusion

A centralized model for acute stroke care across an entire metropolitan city appears to have reduced mortality for a reduced cost per patient, predominately as a result of reduced hospital length of stay.     

Evaluation of glycolamide esters of indomethacin as potential cyclooxygenase-2 (COX-2) inhibitors

A number of novel indomethacin glycolamide esters were synthesized and tested for their cyclooxygenase (COX-1 and COX-2) inhibition properties in vitro. Many of these compounds proved to be selective COX-2 inhibitors, and subtle structural changes in the substituents on the glycolamide ester moiety altered the inhibitory properties as well as potencies significantly. Their in vitro data were rationalized through molecular modeling studies. Few of them displayed anti-inflammatory activity in vivo. Compound 32, [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl]acetic acid 2-morpholin-4-yl-2-oxo ethyl ester, was identified as a promising compound in this class and its good anti-inflammatory activity was demonstrated in the in vivo model.     

In vivo ablation of CD11c-positive dendritic cells increases susceptibility to herpes simplex virus type 1 infection and diminishes NK and T-cell responses

The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.     

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

The role of CD4⁺ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4⁺ T cells are needed for the generation of the protective HSV-1-specific CD8⁺-T-cell response. This study examined the contribution of CD4⁺ T cells in the generation of the primary CD8⁺-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8⁺-T-cell response generated in the draining lymph nodes of CD4⁺-T-cell-depleted C57BL/6 mice and B6-MHC-II^−/− mice is quantitatively and qualitatively distinct from the CD8⁺ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8⁺ T cells express comparable levels of the activation marker CD44 in mice lacking CD4⁺ T cells and normal mice. In contrast, CD8⁺ T cells generated in the absence of CD4⁺ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8⁺ T cells in the CD4⁺-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8⁺ T cells is diminished in the absence of CD4⁺-T-cell help. These results suggest that CD4⁺-T-cell help is essential for the generation of fully functional CD8⁺ T cells during the primary response to HSV-1 infection.Infection due to herpes simplex virus type 1 (HSV-1) results in a wide spectrum of clinical presentations depending on the host''s age, the host''s immune status, and the route of inoculation (47). HSV-1 typically causes mild and self-limited lesions on the orofacial areas or genital sites. However, the disease can be life-threatening, as in the case of neonatal and central nervous system infections (18). The host''s immune responses, particularly CD8⁺ T cells, play an important role in determining the outcome of HSV infections in both the natural human host (18, 19, 28) and experimental murine models (11, 43). Immunodepletion and adoptive transfer studies have demonstrated the role of CD8⁺ T cells in reducing viral replication, resolving cutaneous disease, and providing overall protection upon rechallenge (6, 25, 26). CD8⁺ T cells play a particularly important role in preventing infection of the peripheral nervous system (PNS) and the reactivation of latent virus from neurons in the sensory ganglia of infected mice (21, 24, 36). The mechanisms that CD8⁺ T cells employ include gamma interferon (IFN-γ) production and functions associated with cytolytic granule content at the sites of primary infection (23, 31, 38). In the PNS of infected mice, the mechanisms primarily involve IFN-γ secretion (16, 20, 29), particularly against infected neurons expressing surface Qa-1 (41). Histopathological evidence from HSV-1-infected human ganglion sections show a large CD8⁺-T-cell infiltrate and the presence of inflammatory cytokines, suggesting that the presence of activated, effector memory cells within the PNS is important for maintaining HSV-1 latency in the natural human host (10, 42).The generation of a robust CD8⁺-T-cell response is essential for the control of various infectious pathogens. Some studies suggest that a brief interaction with antigen-presenting cells (APCs) is sufficient for CD8⁺-T-cell activation and expansion into functional effectors (44). However, the magnitude and quality of the overall CD8⁺-T-cell response generated may be dependent on additional factors (49). Recent evidence suggests that CD4⁺ T cells facilitate the activation and development of CD8⁺-T-cell responses either directly through the provision of cytokines or indirectly by the conditioning of dendritic cells (DC) (8, 48, 51). Those studies suggested that the latter mechanism is the dominant pathway, wherein CD4⁺ T cells assist CD8⁺-T-cell priming via the engagement of CD40 ligand (CD154) on CD4⁺ T cells and CD40 expressed on DC (4, 30, 33). This interaction results in the activation and maturation of DC, making them competent to stimulate antigen-specific CD8⁺-T-cell responses (35, 37).The requirement for CD4⁺-T-cell help in the generation of primary and secondary CD8⁺-T-cell responses to antigen varies. Primary CD8⁺-T-cell responses to infectious pathogens, such as Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), influenza virus, and vaccinia virus, can be mounted effectively independently of CD4⁺-T-cell help (3, 12, 22, 34). In contrast, primary CD8⁺-T-cell responses to nonmicrobial antigens display an absolute dependence on CD4⁺-T-cell help (4, 5, 30, 33, 46). This observed difference in the requirement for CD4⁺-T-cell help may ultimately be a product of the initial inflammatory stimulus generated following immunization (49). Microbial antigens trigger an inflammatory response that can lead to the direct activation and priming of APCs, such as DC, thereby bypassing the need for CD4⁺-T-cell help. Nonmicrobial antigens, however, trigger an attenuated inflammatory response that does not directly activate and prime DCs. In the absence of this inflammation, CD4⁺ T cells are thought to condition and license DC functions through CD154/CD40 interactions, which leads to the subsequent activation of antigen-specific CD8⁺-T-cell responses (5, 49). Even in the case of pathogens where primary CD8⁺-T-cell responses were independent of CD4⁺-T-cell help, the secondary responses to these pathogens were found to be defective in the absence of CD4⁺-T-cell help (3, 12, 34, 40).The requirement for CD4⁺-T-cell help in priming CD8⁺-T-cell responses against HSV-1 infection is not well defined. Earlier studies with HSV-1 suggested that CD4⁺ T cells play an important role in the generation of primary CD8⁺-T-cell responses, detected in vitro, to acute infection with HSV-1 (14), principally through the provision of interleukin 2 (IL-2) for optimal CD8⁺-T-cell differentiation and proliferation. Subsequent studies, utilizing an in vivo approach, indicated that CD4⁺ T cells were not required for CD8⁺-T-cell-mediated cytolytic function (23). CD4⁺ T cells are thought to provide help by conditioning DC in a cognate, antigen-specific manner, thereby making them competent to stimulate HSV-1-specific CD8⁺-T-cell responses (37). By contrast, findings from other studies show that CD4⁺-T-cell-depleted mice were able to fully recover from acute infection with HSV-1 (38). These studies imply that the absence of CD4⁺ T cells does not prevent priming of CD8⁺ T cells in vivo.Studies from this laboratory have identified two distinct HSV-1-specific CD8⁺-T-cell subpopulations generated during the primary response, based upon the ability to synthesize IFN-γ following antigenic stimulation in vitro (1). To better understand the need for CD4⁺-T-cell help, we examined the functional characteristics and phenotypes of these CD8⁺-T-cell populations generated during a primary response to acute infection with HSV-1 in mice lacking CD4⁺ T cells. Our findings show that primary CD8⁺-T-cell responses to HSV-1 are compromised in the absence of CD4⁺-T-cell help. Specifically, the HSV-1 gB-specific CD8⁺ T cells produced in the absence of CD4⁺ T cells were found to be active with regard to cytolysis in vivo but were functionally impaired in the production of IFN-γ and TNF-α compared with intact C57BL/6 mice. Virus-specific CD8⁺ T cells were also reduced in number in CD4-depleted mice and in B6 mice lacking major histocompatibility complex (MHC) class II expression (B6-MHC-II^−/−) compared to wild-type (WT) mice. In addition, our data showed higher virus burdens in the infectious tissues obtained from mice lacking CD4⁺ T cells than in those from intact mice. Collectively, these findings demonstrate that CD4⁺-T-cell help is essential for the generation of primary CD8⁺-T-cell responses following acute cutaneous infection with HSV-1.     

Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis

Background

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr^−/−Apobec1^−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.

Methods/Principal Findings

In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr^−/−Apobec1^−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10⁹ genome copies/mouse. Whereas Ldlr^−/−Apobec1^−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr^−/−Apobec1^−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.

Conclusions/Significance

Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.