Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity

The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.     

Linking implicit and explicit memory: common encoding factors and shared representations

Dissociations between implicit and explicit memory have featured prominently in theories of human memory. However, similarities between the two forms of memory have been less studied. One open question concerns whether implicit and explicit memory share encoding resources. To explore this question, we employed a subsequent memory design in which several novel scenes were repeated once during an fMRI session and explicit memory for the scenes was unexpectedly tested afterward. Subsequently remembered scenes produced more behavioral priming and neural attenuation-two conventional measures of implicit memory-than did subsequently forgotten scenes. Moreover, brain-behavior correlations between these two implicit measures were mediated by subsequent memory. Finally, tonic activity, possibly reflecting the natural time course of attention, was predictive of subsequent memory. These results suggest that implicit and explicit memory are subject to the same encoding factors and can rely on similar perceptual processes and representations.     

DEVELOPMENT AND APPLICATION OF A MASS-ESTIMATION METHOD FOR WEDDELL SEALS

Traditional methods of acquiring mass data limit the ability to collect large samples from across populations of some pinnipeds, or to sample without great disturbance to the animals. In order to collect substantial samples of mass data from the Weddell seal (Leptonychotes weddellii) population in Erebus Bay, Antarctica, we developed the equipment and methods for estimating the mass of Weddell seals using digital photographs. Resulting regression models predict the mass of adult female seals to within ±13.8% of estimated mass, and ±25.9% of estimated mass for pups. We show the protocols developed are repeatable and efficient enough to be applied to a large number of animals in a relatively short period of time and may be useful for studies of other marine mammals. We caution that prediction intervals exist around mass estimates and must be accounted for when estimates are applied to biological questions. In a limited application of the method, differences in mass transfer between experienced and inexperienced maternal females and their pups were detected when prediction error variance around mass estimates was explicitly included. Similar mass‐estimation methods may therefore be useful in consideration of biological questions requiring large samples of mass previously unattainable.     

Role and regulation of nodal/activin receptor-like kinase 7 signaling pathway in the control of ovarian follicular atresia

Although the role of the TGF beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by the following: 1) colocalization of Nodal and its type I receptor Activin receptor-like kinase 7 (ALK7) proteins in the granulosa cells was only observed in atretic antral follicles, whereas they were present in theca cells and granulosa cells of healthy follicles, respectively; 2) addition of recombinant Nodal or overexpression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; 3) constitutively active ALK7 (ALK7-ca) overexpression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, overexpression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased X-linked inhibitor of apoptotic proteins (Xiap) expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2, and cytochrome c in the granulosa cells. Dominant-negative Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, whereas up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.     

Oxidative stress and diabetic cardiovascular complications

Diabetes diagnoses are increasing at an alarming rate worldwide. The majority of diabetes-related deaths arise from cardiovascular complications such as myocardial infarction, stroke, and peripheral vascular disease. Oxidative stress has been demonstrated to be present in animal models as well as in patients with diabetes and has been suggested as a possible contributor to the accelerated atherosclerosis seen in diabetics. The generation of reactive oxygen species in diabetes occurs via several mechanisms and is initiated not only by glucose, but also by other substances that are found at elevated levels in diabetic patients. The resulting oxidative stress leads to a number of proatherogenic events. The elucidation of the mechanisms of oxidative stress in diabetes and their relationship with atherosclerosis could potentially identify molecular targets of therapy for this condition and its cardiovascular consequences.     

Analysis of expansion of myeloid progenitors in mice to identify leukemic susceptibility genes

The myeloid progenitor cell compartment (MPC) exhibits pronounced expansion in human myeloid leukemias. It is becoming more apparent that progression of myelodysplastic syndromes and myeloproliferative diseases to acute myelogenous leukemia is the result of defects in progenitor cell maturation. The MPC of bone marrow was analyzed in mice using a cell culture assay for measuring the relative frequency of proliferative myeloid progenitors. Response to the cytokines SCF, IL-3, and GM-CSF was determined by this assay for the leukemic mouse strain BXH-2 and ten other inbred mouse strains. Significant differences were found to exist among ten inbred mouse strains in the nature of their MPC in bone marrow, indicating the presence of genetic polymorphisms responsible for the divergence. The SWR/J and FVB/J strains show consistently low frequencies of myeloid progenitors, while the DBA/2J and SJL/J inbred strains show consistently high frequencies of myeloid progenitors within the bone marrow compartment. In addition, in silico linkage disequilibrium analysis was conducted to identify possible chromosomal regions responsible for the phenotypic variation. Given the importance of this cell compartment in leukemia progression and the soon to be released genomic sequence of 15 mouse strains, these differences may provide a valuable tool for research into leukemia.     

Tumor necrosis factor-induced toxic liver injury results from JNK2-dependent activation of caspase-8 and the mitochondrial death pathway

In vitro studies of hepatocytes have implicated over-activation of c-Jun N-terminal kinase (JNK) signaling as a mechanism of tumor necrosis factor-alpha (TNF)-induced apoptosis. However, the functional significance of JNK activation and the role of specific JNK isoforms in TNF-induced hepatic apoptosis in vivo remain unclear. JNK1 and JNK2 function was, therefore, investigated in the TNF-dependent, galactosamine/lipopolysaccharide (GalN/LPS) model of liver injury. The toxin GalN converted LPS-induced JNK signaling from a transient to prolonged activation. Liver injury and mortality from GalN/LPS was equivalent in wild-type and jnk1-/- mice but markedly decreased in jnk2-/- mice. This effect was not secondary to down-regulation of TNF receptor 1 expression or TNF production. In the absence of jnk2, the caspase-dependent, TNF death pathway was blocked, as reflected by the failure of caspase-3 and -7 and poly(ADP-ribose) polymerase cleavage to occur. JNK2 was critical for activation of the mitochondrial death pathway, as in jnk2-/- mice Bid cleavage and mitochondrial translocation and cytochrome c release were markedly decreased. This effect was secondary to the failure of jnk2-/- mice to activate caspase-8. Liver injury and caspase activation were similarly decreased in jnk2 null mice after GalN/TNF treatment. Ablation of jnk2 did not inhibit GalN/LPS-induced c-Jun kinase activity, although activity was completely blocked in jnk1-/- mice. Toxic liver injury is, therefore, associated with JNK over-activation and mediated by JNK2 promotion of caspase-8 activation and the TNF mitochondrial death pathway through a mechanism independent of c-Jun kinase activity.     

Regulatory T cells dampen pulmonary inflammation and lung injury in an animal model of pneumocystis pneumonia

CD4+CD25+FoxP3+ regulatory T cells are decreased in patients infected with HIV and have been shown to be critical in mediating Ag tolerance in the lung. Because a subset of Pneumocystis-infected individuals develop substantial lung injury, which can be modeled in immune reconstituted scid mice, we used mouse models of Pneumocystis carinii to investigate the role of regulatory T cells in opportunistic infection and immune reconstitution. In this study, we show that CD4+CD25+FoxP3+ cells are part of the host response to Pneumocystis in CD4+ T cell-intact mice. Moreover, lung injury and proinflammatory Th1 and Th2 cytokine levels in the bronchoalveolar lavage fluid and lung homogenate were increased following CD4+CD25- immune reconstitution in Pneumocystis-infected SCID mice but not in CD4+CD25+ T cell-reconstituted animals. The ability of CD4+CD25+ T cells to control inflammation and injury during the course of Pneumocystis was confirmed by treatment of wild-type C57BL/6 mice with anti-CD25 mAb. These data show that CD4+CD25+ T cells control pulmonary inflammation and lung injury associated with Pneumocystis infection both in the setting of immune reconstitution as well as new acquisition of infection.     

Interleukin-17 acts independently of TNF-alpha under arthritic conditions

The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-alpha. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-alpha-deficient mice that TNF-alpha is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/BxN serum transfer arthritis to a similar degree in TNF-alpha-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.     

Evidence of a maternal effect that protects against water stress in larvae of the American dog tick, Dermacentor variabilis (Acari: Ixodidae)

We report that the ability to absorb water vapor from the air in larvae of the American dog tick, Dermacentor variabilis, changes depending upon moisture conditions where the eggs develop. When development occurs at lower relative humidities, resultant larvae can replenish water stores, maintain water balance, and survive at relative humidities as low as 75-85% RH, a range that agrees with previously published values for the critical equilibrium humidity or CEH. In contrast, exposure to high relative humidity conditions during development elevates the CEH to 93-97% RH. These larvae can survive only at relative humidities that are close to saturation, as 93% RH is a dehydrating atmosphere. For these larvae, absorption at 97% RH can be prevented by blocking the mouthparts with wax, indicating that an upward shift has occurred in the moisture threshold where the active mechanism for water vapor absorption operates. Based on transfer experiments between low and high relative humidities, the CEH of larvae is determined by the relative humidity experienced by the mother rather than the moisture conditions encountered by eggs after they are laid. The fact that no changes in body water content, dehydration tolerance limit and water loss rate were observed implies that adjustments to the CEH conferred by the mother have the adaptive significance of enabling larvae to maintain water balance by limiting the range of hydrating atmospheres.