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Nanovesicles present in urine the so-called urinary exosomes have been found to be secreted by every epithelial cell type lining the urinary tract system in human. Urinary exosomes are an appealing source for biomarker discovery as they contain molecular constituents of their cell of origin, including proteins and genetic materials, and they can be isolated in a non-invasive manner. Following the discovery of urinary exosomes in 2004, many studies have been performed using urinary exosomes as a starting material to identify biomarkers in various renal, urogenital, and systemic diseases. Here, we describe the discovery of urinary exosomes and address the issues on the collection, isolation, and normalization of urinary exosomes as well as delineate the systems biology approach to biomarker discovery using urinary exosomes.

Nanovesicles present in urine the so-called urinary exosomes have been found to be secreted by every epithelial cell type lining the urinary tract system in human. Urinary exosomes are an appealing source for biomarker discovery as they contain molecular constituents of their cell of origin, including proteins and genetic materials, and they can be isolated in a non-invasive manner. Following the discovery of urinary exosomes in 2004, many studies have been performed using urinary exosomes as a starting material to identify biomarkers in various renal, urogenital, and systemic diseases. Here, we describe the discovery of urinary exosomes and address the issues on the collection, isolation, and normalization of urinary exosomes as well as delineate the systems biology approach to biomarker discovery using urinary exosomes.

Urea transporter gene knockout mice have been created for the study of the urine-concentrating mechanism. The major findings in studies of the renal phenotype of these mice are as follows: (1) Urea accumulation in the inner medullary interstitium is dependent on intrarenal urea recycling mediated by urea transporters; (2) urea transporters are essential for preventing urea-induced osmotic diuresis and thus for water conservation; (3) NaCl concentration in the inner medullary interstitium is not significantly affected by the absence of IMCD, descending limb of Henle and descending vasa recta urea transporters. Studies in urea transporter knockout mouse models have highlighted the essential role of urea for producing maximally concentrated urine.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

Enteroendocrine (EEC) cells have a pivotal role in intestinal nutrient sensing and release hormones that orchestrate food digestion and regulate appetite. EEC cells are found scattered throughout the intestine and have typically been classified based on the primary hormone they contain. I cells represent a subset of EEC cells that secrete cholecystokinin (CCK) and are mainly localized to the duodenum. Recent studies have shown that I cells express mRNAs encoding several gut hormones. In this study, we investigated the hormonal profile of murine fluorescence-activated cell sorting-sorted duodenal I cells using semiquantitative RT-PCR, liquid chromatography tandem mass spectrometry, and immunostaining methods. We report that I cells are enriched in mRNA transcripts encoding CCK and also other key gut hormones, including neurotensin, glucose-dependent insulinotropic peptide (GIP), secretin, peptide YY, proglucagon, and ghrelin (Ghrl). Furthermore, liquid chromatography tandem mass spectrometry analysis of fluorescence-activated cell sorting-purified I cells and immunostaining confirmed the presence of these gut hormones in duodenal I cells. Immunostaining highlighted that subsets of I cells in both crypts and villi coexpress differential amounts of CCK, Ghrl, GIP, or peptide YY, indicating that a proportion of I cells contain several hormones during maturation and when fully differentiated. Our results reveal that although I cells express several key gut hormones, including GIP or proglucagon, and thus have a considerable overlap with classically defined K and L cells, approximately half express Ghrl, suggesting a potentially important subset of duodenal EEC cells that require further consideration.

Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage.

The post-translational modifications (PTMs) phosphorylation and ubiquitylation regulate plasma membrane protein function. Here, we examine the interplay between phosphorylation and ubiquitylation of the membrane protein aquaporin-2 (AQP2) and demonstrate that phosphorylation can override the previously suggested dominant endocytic signal of K63-linked polyubiquitylation. In polarized epithelial cells, although S256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at S269. Despite their close proximity, AQP2 phosphorylation at S269 and ubiquitylation at K270 can occur in parallel, with increased S269 phosphorylation and decreased AQP2 endocytosis occurring when K270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occurring in parallel to maximal S269 phosphorylation and enhanced AQP2 plasma membrane localization. In conclusion, we demonstrate for the first time that although K63-linked polyubiquitylation marks AQP2 for endocytosis, site-specific phosphorylation can counteract polyubiquitylation to determine its final localization. Similar mechanisms might exist for other plasma membrane proteins.

Almost half of patients receiving lithium salts have nephrogenic diabetes insipidus. Chronic lithium exposure induces AQP2 downregulation and changes in the cellular composition of the collecting duct. In order to understand these pathophysiological events, we determined the earliest lithium targets in rat inner medullary collecting duct (IMCD) by examining changes in the IMCD phosphoproteome after acute lithium administration. IMCDs were isolated 9 h after lithium exposure, a time when urinary concentrating impairment was evident. We found 1093 unique phosphopeptides corresponding to 492 phosphoproteins identified and quantified by mass spectrometry. Label-free quantification identified 152 upregulated and 56 downregulated phosphopeptides in response to lithium. Bioinformatic analysis highlighted several signaling proteins including MAP kinases and cell-junction proteins. The majority of the upregulated phosphopeptides contained a proline-directed motif, a known target of MAPK. Four hours after lithium exposure, phosphorylation sites in the activation loops of ERK1/2 and p38 were upregulated. Increased expression of phospho-Ser261-AQP2 (proline-directed motif) was concomitant with the increase in urine output. Pretreatment with MAPK inhibitors reversed the increased Ser261-AQP2 phosphorylation. Thus, in IMCD, ERK1/2 and p38 are early targets of lithium and may play a role in the onset of lithium-induced polyuria.Kidney International advance online publication, 30 April 2014; doi:10.1038/ki.2014.107.

The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and blood pressure (BP). Gain-of-function in NCC-regulating kinases or loss-of-f...

The renal distal nephron [distal convoluted tubule 2 (DCT2) and connecting tubule (CNT)] is crucial for sodium and blood pressure regulation, and is tightly controlled by aldosterone. Due to diffic...

The thiazide-sensitive NaCl cotransporter (NCC) plays an important role in sodium reabsorption in the kidney distal convoluted tubules (DCT). Both angiotensin II (ANGII) and vasopressin (AVP) are h...

Our recent work suggests that AQP2 endocytosis rates can be regulated via phosphorylation. As NCC is phosphorylated at conserved residues in its intracellular NH2 terminus, we hypothesized that a s...

Eleven fatty acid analogues incorporating four-membered carbocycles (cyclobutenes, cyclobutanes, cyclobutanones, and cyclobutanols) were investigated for the ability to inhibit the growth of Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb). A number of the analogues displayed inhibitory activity against both mycobacterial species in minimal media. Several of the molecules displayed potent levels of inhibition against Mtb, with MIC values equal to or below those observed with the anti-tuberculosis drugs D-cycloserine and isoniazid. In contrast, two of the analogues that display the greatest activity against Mtb failed to inhibit E. coli growth under either set of conditions. Thus, the active molecules identified herein may provide the basis for the development of anti-mycobacterial agents against Mtb.

Urea transporter gene knockout mice have been created for the study of the urine-concentrating mechanism. The major findings in studies of the renal phenotype of these mice are as follows: (1) Urea accumulation in the inner medullary interstitium is dependent on intrarenal urea recycling mediated by urea transporters; (2) urea transporters are essential for preventing urea-induced osmotic diuresis and thus for water conservation; (3) NaCl concentration in the inner medullary interstitium is not significantly affected by the absence of IMCD, descending limb of Henle and descending vasa recta urea transporters. Studies in urea transporter knockout mouse models have highlighted the essential role of urea for producing maximally concentrated urine.

The post-translational modifications (PTMs) phosphorylation and ubiquitylation regulate plasma membrane protein function. Here we examine interplay between phosphorylation and ubiquitylation of the membrane protein aquaporin-2 (AQP2) and demonstrate that phosphorylation can override the previously suggested dominant endocytic signal of K63-linked polyubiquitylation. In polarized epithelial cells, although Ser-256 is an important phosphorylation site for AQP2 membrane localization, the rate of AQP2 endocytosis was reduced by prolonging phosphorylation specifically at Ser-269. Despite close association, AQP2 phosphorylation at Ser-269 and ubiquitylation at Lys-270 can occur in parallel, with increased Ser-269 phosphorylation and decreased AQP2 endocytosis occurring when Lys-270 polyubiquitylation levels are maximal. In vivo studies support this data, with maximal levels of AQP2 ubiquitylation occuring in parallel to maximal Ser-269 phosphorylation and enhanced AQP2 plasma membrane localization. In conclusion, we demonstrate for the first time that although K63-linked polyubiquitylation marks AQP2 for endocytosis, site-specific phosphorylation can counteract polyubiquitylation to determine its final localization. Similar mechanisms may exist for other plasma membrane proteins.

RNAi-based strategies provide a great therapeutic potential for treatment of various human diseases including kidney disorders, but face the challenge of in vivo delivery and specific targeting. The chitosan delivery system has previously been shown to target siRNA specifically to the kidneys in mice when administered intravenously. Here we confirm by 2D and 3D bioimaging that chitosan formulated siRNA is retained in the kidney for more than 48 hours where it accumulates in proximal tubule epithelial cells (PTECs), a process that was strongly dependent on the molecular weight of chitosan. Chitosan/siRNA nanoparticles, administered to chimeric mice with conditional knockout of the megalin gene, distributed almost exclusively in cells that expressed megalin, implying that the chitosan/siRNA particle uptake was mediated by a megalin-dependent endocytotic pathway. Knockdown of the water channel aquaporin 1 (AQP1) by up to 50% in PTECs was achieved utilizing the systemic i.v. delivery of chitosan/AQP1 siRNA in mice. In conclusion, specific targeting PTECs with the chitosan nanoparticle system may prove to be a useful strategy for knockdown of specific genes in PTECs, and provides a potential therapeutic strategy for treating various kidney diseases.

ABSTRACT PBK/TOPK is a recently cloned serine/threonine kinase which is phosphorylated during mitosis. Earlier work indicated that this kinase is upregulated in a Burkitt's lymphoma cell line (GA-10). To determine whether PBK/TOPK is upregulated in other mitotically active neoplastic cell lines and tissues, Northern analysis was performed on a panel of malignant cell lines and on clinical samples from patients with leukemia or lymphoma. While PBK/TOPK mRNA was not detectable in normal peripheral blood cells and was weakly expressed in hyperplastic tonsillar B-cells, significantly higher levels of mRNA were detected in 8 Burkitt's lymphoma cell lines, 10 other neoplastic cell lines, and 2 clinical samples—one derived from a patient with ALL and a second derived from a patient with relapsed myeloma. In addition, Northern analysis of fetal tissues showed upregulated expression of PBK/TOPK in fetal kidney, lung, spleen, brain, and testis. These data suggest that PBK/TOPK expression is increased in highly proliferative malignant cells and during normal fetal development.

The IFN-γ-inducible proteins monokine induced by IFN-γ (Mig) and chemokine responsive to γ-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-γ-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-γ. In addition to depending on IFN-γ, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-γ R α and β-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-γ contain increased chemotactic activity for enriched human and mouse CD3+ T cells, as well as mouse DX5+ NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.

Information obtained through the analysis of dynamic manipulability can be useful in the design and control of robotic manipulators. In this paper, we investigate the influences of the joint drive systems on the dynamic manipulability, with special attention to the effects of gear reduction ratios. In order to reflect this influence, a new form of the dynamic manipulability ellipsoid is developed, which includes the gear reduction ratios, and a modified formula is derived for the dynamic manipulability measure. This formula indicates that joint drive dynamics can significantly influence the dynamic manipulability of a robot. The most important effect is that changing the gear reduction ratios modifies the nature of the dynamic manipulability distribution throughout the workspace of the manipulator. Examples of a two-link planar arm and a PUMA 560 robot are presented to illustrate the results.

This article describes the emerging roles of the systems engineering (SE) function in supporting enterprise management in information-intensive organizations. “Enterprise Systems Engineering” (ESE) comprises three major roles or “levels” of systems engineering for successful and efficient development or procurement of large complex systems of systems (SoS). While the authors' experience focuses predominantly on government organizations acting as their own SoS integrators, the SoS ESE concept has generic applicability for any organization, public or private, seeking to attain competitive advantage through leveraging of information technology resources and systems. The processes and tools described here have been developed and successfully employed to facilitate government project management and investment decisions and control. [Carlock and Decker, 1998] This paper describes a formal three-level SoS ESE process that, at the top level, organizes and maintains all of the details of the enterprise-wide SoS architecture and strategic development plan in a flexible framework that accommodates the changes expected over a long SoS evolution. The information maintained in this framework allows the organization to know where it is going, how and when it is going to get there, the required capabilities and interfaces of each SoS component, and the impact of changes to system requirements, budgets, schedules, etc., on the overall SoS. The middle level processes allow the organization to perform trade studies among alternative solutions to implement required capabilities based on what is best for the enterprise-wide SoS rather than just local considerations. The end result of the middle level processes is a selected and approved solution and its associated cost, schedule, benefits, and technical baselines. The third level processes implement the approved solutions in accordance with the approved baselines. © 2001 John Wiley & Sons, Inc. Syst Eng 4: 242–261, 2001

Two novel navigation-guidance-based methods are presented for on-line-robotic-interception of fast-maneuvering objects. For the first method, an ideal proportional navigation-guidance (IPNG) technique is used during phase I of the two-phase robot trajectory planning and control strategy. For the second method, an augmented form of the IPNG (AIPNG) technique is suggested for phase I, when a reliable estimation of the acceleration of the target can be provided to the interceptor. Both techniques are modified to reflect the greater mobility of a robotic manipulator over an airborne interceptor. The convergence analyses of both methods are detailed and compared to that of a conventional moving-object tracking method. Computer simulations clearly illustrate the effectiveness of the IPNG-based interception methods over pure tracking-based techniques proposed in the cited literature. © 2000 John Wiley & Sons, Inc.

Presents an approach to online, robot-motion planning for moving-object interception. The proposed approach utilizes a navigation-guidance-based technique, that is robust and computationally efficient for the interception of fast-maneuvering objects. Navigation-based techniques were originally developed for the control of missiles tracking free-flying targets. Unlike a missile, however, the end-effector of a robotic arm is connected to the ground, via a number of links and joints, subject to kinematic and dynamic constraints. Also, unlike a missile, the velocity of the robot and the moving object must be matched for a smooth grasp, thus, a hybrid interception scheme, which combines a navigation-based interception technique with a conventional trajectory tracking method is proposed herein for intercepting fast-maneuvering objects. The implementation of the proposed technique is illustrated via numerous simulation examples.

In this paper, a dynamic system consisting of a robot manipulator and a target is analyzed. The target is considered in a general way as a dynamic subsystem having finite mass and moments of inertia (e.g., a rigid body or a second robot). The situation investigated is when the robot establishes interaction with the target in such a way that it intercepts and captures a reference element of the target. The analysis of target capture is divided into three phases in terms of time: the precapture, “free” motion (finite motion); the transition from free to constrained motion in the vicinity of interception and capture (impulsive motion); and the postcapture, constrained motion (finite motion). The greatest attention is paid to the analysis of the phase of transition, the impulsive motion, and dynamics of the system. Based on the use of impulsive constraints and the Jourdainian formulation of analytical dynamics, a novel approach is proposed for the dynamic modeling of target capture by a robot manipulator. The proposed approach is suitable to handle both finite and impulsive motions in a common analytical framework. Based on the dynamic model developed and using a geometric representation of the system’s dynamics, a detailed analysis and a performance evaluation framework are presented for the phase of transition. Both rigid and structurally flexible models of robots are considered. For the performance evaluation analyses, two main concepts are proposed and corresponding performance measures are derived. These tools may be used in the analysis, design, and control of time-varying robotic systems. The dynamic system of a three-link robot arm capturing a rigid body is used to illustrate the material presented.

Zanamivir is a highly selective neuraminidase (NA) inhibitor with demonstrated clinical efficacy against influenza A and B virus infections. In phase II clinical efficacy trials (NAIB2005 and NAIB2008), virological substudies showed mean reductions in virus shedding after 24 h of treatment of 1.5 to 2.0 log 10 50% tissue culture infective doses compared to a placebo, with no reemergence of virus after the completion of therapy. Paired isolates ( n = 41) obtained before and during therapy with zanamivir demonstrated no shifts in susceptibility to zanamivir when measured by NA assays, although for a few isolates NA activity was too low to evaluate. In plaque reduction assays in MDCK cells, the susceptibility of isolates to zanamivir was extremely variable even at baseline and did not correlate with the speed of resolution of virus shedding. Isolates with apparent limited susceptibility to zanamivir by plaque reduction proved highly susceptible in vivo in the ferret model. Further sequence analysis of paired isolates revealed no changes in the hemagglutinin and NA genes in the majority of isolates. The few changes observed were all natural variants. No amino acid changes that had previously been identified in vitro as being involved with reduced susceptibility to zanamivir were observed. These studies highlighted problems associated with monitoring susceptibility to NA inhibitors in the clinic, in that no reliable cell-based assay is available. At present the NA assay is the best available predictor of susceptibility to NA inhibitors in vivo, as measured in the validated ferret model of infection.

This paper presents a novel approach to on-line, robot-motion planning for moving-object interception. The proposed approach utilizes a navigation-based technique, which is robust and computationally efficient for the interception of fast-maneuvering objects. Navigation-based techniques were originally developed for the control of missiles tracking free-flying targets. Unlike a missile, however, the end-effector of a robotic arm is connected to the ground, via a number of links and joints, subject to kinematic and dynamic constraints. Also, unlike a missile, the velocity of the robot and the moving object must be matched for a smooth grasp , thus, a hybrid interception scheme, which combines a navigation-based interception technique with a conventional trajectory tracking method is proposed herein for intercepting fast-maneuvering objects. The implementation of the proposed technique is discussed via numerous simulation examples.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-γ and IFN-γ-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-γ proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o− plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o− to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o− into BALB/c IL-12 p40−/− mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-γ protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.

“Systems of systems” differ from single systems in the way in which they are implemented and in how their implementation must be controlled. For this reason, the systems engineering processes necessary to ensure successful implementation of systems of systems differ in several important ways from the systems engineering processes typically applied to individual systems. A successful approach to implementing systems of systems requires a multi-tiered set of systems engineering processes that builds upon the traditional single-system approach.

In this paper we investigate the dynamics of robotic interception and capture of a moving object. This problem, i.e., the interception and capture of a moving object by a robot, is called dynamic mass capture. The effects of structural flexibility of the robot is taken into consideration. In terms of time, the analysis is divided into three phases: before capture (finite motion), at the vicinity of interception and capture (impulsive motion), and after capture (finite motion). Special attention is paid to the modeling of the second phase when the robot captures the target and it becomes part of the end effector, thus, the system's degrees of freedom suddenly change. To decribe this event, a novel approach is proposed. This is based on the use of a class of impulsive constraints, the so-called inert constraints. Jourdain's principle is employed to derive the dynamic equations for both finite and impulsive motions. Simulation results are presented for two examples: a single flexible link and a two-link manipulator capturing moving objects. In the example of the single link, the results are compared with the observations of an experiment, and good agreement is found between experimental and simulation results.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-γ or TNF-α, and the combination of IFN-γ + TNF-α synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-γ and TNF-α are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-γ and TNF-α.

IL-12 is a potent immunoregulatory cytokine that has been shown to mediate tumor regression in a variety of tumor models. We describe the construction of AdCMV-IL-12, a recombinant adenovirus that encodes both subunits of IL-12 under transcriptional control of the CMV promoter. This recombinant virus efficiently infects a wide variety of cell types leading to the production of high levels of biologically active IL-12. Because the liver is a primary site of infection after i.v.-administered adenovirus, we tested the therapeutic efficacy of this virus in a murine hepatic metastasis tumor model. Systemic administration of AdCMV-IL-12 dramatically inhibited the formation of 3-day Renca hepatic metastases (mean of 16 metastases per liver) compared with the control virus AdCMV-βgal (mean of 209) or vehicle alone (mean of 272). Histologic analysis indicated that metastatic growth inhibition was accompanied by a dramatic perivascular infiltrate consisting of T cells, macrophages, and neutrophils. Therapeutic efficacy was not diminished in animals depleted of CD4+ or CD8+ T cells, or in SCID mice, even after NK cell ablation. In the latter case, a hepatic perivascular infiltrate composed of macrophages and neutrophils was observed after AdCMV-IL-12-treatment, while numerous activated Kupffer cells were noted in the hepatic parenchyma. Analysis of therapy-induced changes in hepatic gene expression demonstrated increased levels of IP-10 and Mig RNAs, but no increase in iNOS, Fas, or FasL RNA levels was observed. Our data suggest a model of metastatic growth inhibition mediated by nonlymphocyte effector cells including macrophages and neutrophils and that may involve anti-angiogenic chemokines.

A number of active prediction planning and execution (APPE) systems have recently been proposed for robotic interception of moving objects. The cornerstone of such systems is the selection of a robot-object rendezvous-point on the predicted object trajectory. Unlike tracking-based systems, which minimize the state difference between the object and the robot at each control period, in this methodology the robot is sent directly to the selected rendezvous-point. A fine-motion tracking strategy would then be employed for grasping the moving object. Herein, a novel strategy for selecting the optimal (earliest) rendezvous-point is presented. For objects with predictable trajectories, this is a significant improvement over previous APPE strategies which select the rendezvous-point from a limited number of non-optimally chosen candidates.

Crosslinking of CD28 receptors on resting T lymphocytes by B7 costimulatory molecules expressed by antigen-presenting cells (APCs) plays a critical role in T-cell activation. Human melanomas express major histocompatibility complex (MHC)-restricted tumor-associated antigens that can be recognized by cytotoxic T lymphocytes (CTL), yet they remain poorly immunogenic. One mechanism for the failure of T-cell response is the lack of expression of costimulatory molecules by human melanoma cells. We have transfected the B7-1 gene into three HLA-A2-expressing human melanoma cell lines, and studied their capacity to stimulate primary human T cells. B7-expressing melanoma cells were excellent inducers of T-cell proliferation, cytokine production, and cytolytic activity in allogeneic mixed lymphocyte cultures through a process dependent on the function of the T-cell receptor as well as interactions between B7:CD28, CD2:LFA-3, and LFA-1:ICAM-1. Subset analysis demonstrated that CD4 + T cells or addition of exogenous interleukin-2 was required for the induction of CD8 + CTL. Untransfected parental melanoma cells were inert as APCs in these cultures. Rotating stimulation of T cells with the three B7-expressing cell lines led to the generation of T-cell lines that were cytolytic for HLA-A2 + melanoma cells and other HLA-A2 + targets that were pulsed with HLA-A2-restricted MART-I peptides. These data demonstrate that expression of B7-1 by human melanoma cells converts them into effective APCs for the in vitro induction of MHC-restricted, melanoma-specific CTL.

On-line planning of gross robot motion for moving-object interception is considered in this article within the context of an active prediction planning, and execution (APPE) strategy. The objective is to find an optimal interception point such that the robot end-effector and the object arrive simultaneously at this target pregrasping point. In this approach, the optimality of the selected rendezvous point on the target trajectory is directly dependent on the robot-trajectory planning technique. Thus, for the solution of the general interception problem, three issues must be addressed: (i) optimal rendezvous-point selection, (ii) optimal robot-trajectory planning, and (iii) replanning in response to gross changes in the predicted target trajectory. The effect of uncertainties in the target-trajectory prediction must be considered at each planning stage. Herein, solutions to the first two problems are briefly reviewed as background to the proposed rendezvous-point replanning strategy. This strategy determines when replanning is necessary, modifies the rendezvous point, and iteratively replans robot “patch trajectories” to new interception points. Simulation results using two different on-line robot-motion-generation strategies are also presented. © 1998 John Wiley & Sons, Inc. 15: 97–114, 1998

We describe the clinical and pathologic features of a lymphoepithelioma-like carcinoma (LELC) that originated in the hepatobiliary system. A woman, aged 71 years, was first seen with a noncholangiolar adenocarcinoma with lymphoid stroma, which was discovered by open liver biopsy in 1993. In 1995, retroperitoneal and peripancreatic lymph nodes were involved by LELC. There currently is no evidence of distant metastasis outside the hepatobiliary peripancreatic region. Review of the biopsy material revealed a well-differentiated adenocarcinoma with transition into LELC. Epstein-Barr virus (EBV) transcripts were expressed in all histologic phases of the tumor by in situ hybridization using immunoalkaline phosphatase-labeled oligonucleotide probes for EBV-encoded RNA 1 on formalin-fixed, paraffin-embedded sections. Polymerase chain reaction analysis for EBV nuclear antigen 2 was consistent with EBV strain type A. The LMP-1 gene was found to be wild type by polymerase chain reaction analysis. To our knowledge, this is the first report of a primary hepatobiliary adenocarcinoma associated with EBV infection that transformed into an undifferentiated LELC.

A novel active prediction, planning, and execution (APPE) system is presented for the robotic interception of moving objects. An APPE system's objective is simply to move the robot to the earliest pregrasping location. A fine-motion tracking algorithm can take over the motion control at that point, utilizing proximity sensors mounted on the robot's end-effector. This approach eliminates the necessity of tracking the motion of the object, as required by conventional tracking-based techniques, where the distance between the robot's end-effector and the object is reduced continuously. In this paper, the proposed APPE system is first briefly introduced, and its individual modules are then discussed in detail. Simulation and experimental results are presented in support of the developed optimal-interception strategy.

The levels of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-1α released from human peripheral blood leucocytes (PBL) following interaction with influenza virus clone 7a (virulent, produces high fever in ferrets) and A/Fiji (attenuated, produces relatively low fever in ferrets) were low and similar for the two viruses. Neither strain induced interferon (IFN)-γ, and release of IL-8 (which occurs on incubation of PBLs alone) was reduced after interaction with the two viruses. The levels of IL-1 and IL-6 detected in the plasma of infected ferrets were low and did not correlate with the onset, duration or magnitude of the fevers produced by clone 7a and A/Fiji. Relatively large amounts (100,000 pg/kg) of IL-1 and TNF-α were needed to produce appreciable fever in rabbits, and such quantities of IL-6 were not pyrogenic. Hence, as for previous observations, no evidence could be obtained that induction of known pyrogenic cytokines is responsible for the febrile response in influenza. The possibility that some other mediator(s) may be involved cannot be ruled out. J. Med. Virol. 52:336–340, 1997. © 1997 Wiley-Liss, Inc.

The thiazide-sensitive NaCl cotransporter (NCC) of the renal distal convoluted tubule (DCT) controls ion homeostasis and arterial BP. Loss-of-function mutations of NCC cause renal salt wasting with arterial hypotension (Gitelman syndrome). Conversely, mutations in the NCC-regulating WNK kinases or kelch-like 3 protein cause familial hyperkalemic hypertension. Here, we performed automated sorting of mouse DCTs and microarray analysis for comprehensive identification of novel DCT-enriched gene products, which may potentially regulate DCT and NCC function. This approach identified protein phosphatase 1 inhibitor-1 (I-1) as a DCT-enriched transcript, and immunohistochemistry revealed I-1 expression in mouse and human DCTs and thick ascending limbs. In heterologous expression systems, coexpression of NCC with I-1 increased thiazide-dependent Na(+) uptake, whereas RNAi-mediated knockdown of endogenous I-1 reduced NCC phosphorylation. Likewise, levels of phosphorylated NCC decreased by approximately 50% in I-1 (I-1(-/-)) knockout mice without changes in total NCC expression. The abundance and phosphorylation of other renal sodium-transporting proteins, including NaPi-IIa, NKCC2, and ENaC, did not change, although the abundance of pendrin increased in these mice. The abundance, phosphorylation, and subcellular localization of SPAK were similar in wild-type (WT) and I-1(-/-) mice. Compared with WT mice, I-1(-/-) mice exhibited significantly lower arterial BP but did not display other metabolic features of NCC dysregulation. Thus, I-1 is a DCT-enriched gene product that controls arterial BP, possibly through regulation of NCC activity.

During hypovolemia and hyperkalemia, the kidneys defend homeostasis by Na+retention and K+secretion, respectively. Aldosterone mediates both effects, but it is unclear how the same hormone can evoke such different responses. To address this, we mimicked hypovolemia and hyperkalemia in four groups of rats with a control diet, low-Na+diet, high-K+diet, or combined diet. The low-Na+and combined diets increased plasma and kidney ANG II. The low-Na+and high-K+diets increased plasma aldosterone to a similar degree (3-fold), whereas the combined diet increased aldosterone to a greater extent (10-fold). Despite similar Na+intake and higher aldosterone, the high-K+and combined diets caused a greater natriuresis than the control and low-Na+diets, respectively ( P < 0.001 for both). This K+-induced natriuresis was accompanied by a decreased abundance but not phosphorylation of the Na+-Cl−cotransporter (NCC). In contrast, the epithelial Na+channel (ENaC) increased in parallel with aldosterone, showing the highest expression with the combined diet. The high-K+and combined diets also increased WNK4 but decreased Nedd4-2 in the kidney. Total and phosphorylated Ste-20-related kinase were also increased but were retained in the cytoplasm of distal convoluted tubule cells. In summary, high dietary K+overrides the effects of ANG II and aldosterone on NCC to deliver sufficient Na+to ENaC for K+secretion. K+may inhibit NCC through WNK4 and help activate ENaC through Nedd4-2.

Renal tubules are highly active transporting epithelia and are at risk of protein aggregation due to high protein turnover and/or oxidative stress. We hypothesized that the risk of aggregation was increased upon hormone stimulation and assessed the state of the intracellular protein degradation systems in the kidney from control rats and rats receiving aldosterone or angiotensin II treatment for 7 days. Control rats formed both aggresomes and autophagosomes specifically in the proximal tubules, indicating a need for these structures even under baseline conditions. Fluorescence sorted aggresomes contained various rat keratins known to be expressed in renal tubules as assessed by protein mass spectrometry. Aldosterone administration increased the abundance of the proximal tubular aggresomal protein keratin 5, the ribosomal protein RPL27, ataxin-3, and the chaperone heat shock protein 70-4 with no apparent change in the aggresome-autophagosome markers. Angiotensin II induced aggregation of RPL27 specifically in proximal tubules, again without apparent change in antiaggregating proteins or the aggresome-autophagosome markers. Albumin endocytosis was unaffected by the hormone administration. Taken together, we find that the renal proximal tubules display aggresome formation and autophagy. Despite an increase in aggregation-prone protein load in these tubules during hormone treatment, renal proximal tubules seem to have sufficient capacity for removing protein aggregates from the cells.

Over the past decade, metabolomics has emerged as an important technique for systems biology. Measuring all the metabolites in a biological system provides an invaluable source of information to explore various cellular processes, and to investigate the impact of environmental factors and genetic modifications. Nuclear magnetic resonance (NMR) spectroscopy is an important method routinely employed in metabolomics. NMR provides comprehensive structural and quantitative information useful for metabolomics fingerprinting, chemometric analysis, metabolite identification and metabolic pathway construction. A successful metabolomics study relies on proper experimental protocols for the collection, handling, processing and analysis of metabolomics data. Critically, these protocols should eliminate or avoid biologically-irrelevant changes to the metabolome. We provide a comprehensive description of our NMR-based metabolomics procedures optimized for the analysis of bacterial metabolomes. The technical details described within this manuscript should provide a useful guide to reliably apply our NMR-based metabolomics methodology to systems biology studies.

Aquaporin-2 (AQP2) water channels in principal cells of the kidney collecting duct are essential for urine concentration. Due to application of modern technologies, progress in our understanding of AQP2 has accelerated in recent years. In this article, we highlight some of the new insights into AQP2 function that have developed recently, with particular focus on the cell biological aspects of AQP2 regulation.AQP2 is subjected to a number of regulated modifications, including phosphorylation and ubiquitination, which are important for AQP2 function, cellular localization and degradation. AQP2 is likely internalized via clathrin and non-clathrin-mediated endocytosis. Regulation of AQP2 endocytosis, in addition to exocytosis, is a vital mechanism in determining overall AQP2 membrane abundance. AQP2 is associated with regulated membrane microdomains. Studies using membrane cholesterol depleting reagents, for example statins, have supported the role of membrane rafts in regulation of AQP2 trafficking. Noncanonical roles for AQP2, for example in epithelial cell migration, are emerging.AQP2 function and thus urine concentration is dependent on a variety of cell signalling mechanisms, posttranslational modification and interplay between AQP2 and its lipid environment. This complexity of regulation allows fine-tuning of AQP2 function and thus body water homeostasis.

The mammalian kidney is responsible for a multitude of homeostatic functions, which are mediated by both structural and functional diversity along the renal tubule. In this article, we focus on the major functions of the terminal portion of the renal tubule, the medullary collecting duct system. The role of the medullary collecting ducts in determining the composition of the final urine through controlled water, sodium, chloride, potassium and urea reabsorption, ammonia transport, and acid-base homeostasis is discussed. The molecular identity of the major channels and transporters that contribute to medullary collecting duct function are described in detail, including; aquaporins, urea transporters, the epithelial sodium channel (ENaC), the Na,K-ATPase, H-ATPase, Rh glycoproteins, and sodium bicarbonate transporters. Knowledge gained from studies in knockout mice is also discussed.