Antibodies against EphB4 were found to inhibit tumor growth and angiogenesis, some of which are being investigated for anticancer therapy in preclinical studies. Eph receptor antibodies that were conjugated to small molecule drugs caused internalization of drugs and inhibition of tumor growth in vivo. Soluble extracellular domain of EphB4 targeting ephrin-B2 has been used in inhibiting angiogenesis and tumor growth in vivo. We have found that mAb EC8 potently antagonized ephrin-B2 binding to EphB4, which would block forward and reverse signaling by EphB4 and ephrin-B2 interaction. Similar to the observation of EphB4 upregulation in some tumors, when human tissue array was probed with mAb EC8, ephrin-B2 was found to be also overexpressed in tumors in lung, breast, ovary, colon, and prostate over respective normal tissue. Consistent with the previous observation of ephrinB2 expression in tumor-associated vasculature, EC8 delineated ephrin-B2 expression in newly formed vessels within the tumor. Upregulation of ephrin-B2 was also found in colorectal cancer cell lines, COLO205 and HT108 both in cell culture and as a tumor xenograft in mice. Notably, due to the cross-reactive nature of antibody with murine ephrin-B2, mAb EC8 also identified tumorassociated vasculature, simultaneously detecting ephrin-B2 in human tumor as well as ephrin-B2 in murine host. Neovasculatures in adults sprouting from arterial vessels and capillaries, whether caused by VEGF-signaling, tissue injury, or tumor growth, were found to express ephrin-B2. It is unknown how ephrin-B2 upregulation in some of the tumors of epithelium origin would perturb the balance BAY 43-9006 between ephrin-B2 and Eph4 expressed in arterial and venous vessels, respectively, and contribute to the tumor growth and metastasis. Given the observation that overexpression of ephrin-B2 in some tumors is correlated with poor prognosis, it will be an interesting question if the role of ephrin-B2 together with EphB receptors in some tumor is associated with the promotion of the vasculature growth and the adenoma-carcinoma transition, facilitating tumor metastasis. We propose that high affinity and antagonist antibodies such as EC8 would provide a valuable tool for examining the role of ephrin-B2 expression on tumor angiogenesis and migration. Body weight is normally maintained within a narrow range by an appropriate balance between energy intake and energy expenditure. An increase of energy intake leads to excess energy storage in white adipose tissue and weight gain. Genetic background, excessive food consumption, sedentary lifestyle, and decreased physical activity are the main predisposing factors for alteration of energy balance. However a multitude of perinatal factors can alter the metabolic fate of offspring. We previously demonstrated, on a widely used animal model of nutritional programming, that low birth weight, as a consequence of an intrauterine growth restriction, leads to metabolic alterations and feeding behaviour abnormalities when followed by a rapid catch-up growth. In complement to that work we demonstrated that rapid catch-up growth of IUGR rats lead to a reduction of leptin sensitivity at postnatal day 5 and 12 in arcuate nucleus. Since leptin is a critical neurotrophic factor and seems essential for the normal axonal outgrowth of NPY/AgRP and POMC neurons from the ARC to the PVH that occur during that period, a reduced action will have consequence on the ontogeny of hypothalamic regulatory.

NLRP12 was originally suggested to form an inflammasome with PYCARD. However, more recently, NLRP12 has been characterized as a negative regulator of both canonical and noncanonical NF-kB signaling. NLRP12 was shown to interact with and inhibit the accumulation of hyperphosphorylated IRAK1, downstream of TLR signaling, to attenuate canonical NF-kB signaling. Similarly, NLRP12 associates with NIK in the non-canonical NF-kB pathway, which results in the rapid proteosomal degradation of the kinase. NF-kB regulates a variety of inflammatory pathways that may directly contribute to asthma pathogenesis. However, because of its central role in innate immunity, NF-kB and modulators of NF-kB signaling are more typically associated with modulating the host immune response to agents associated with asthma exacerbation. Prior to this research, only one additional publication has explored the in vivo role of NLRP12. In this previous work, NLRP12 was found to attenuate the development of contact hypersensitivity. The underlying mechanism was found to be associated with altered dendritic cell and granulocyte migration in response to chemokine signaling. This finding was consistent with results from human association studies that reported identifying an association between specific mutations in NLRP12 and a subgroup of atopic dermatitis patients. Because contact hypersensitivity and asthma share many of the same immunopathological features, we sought to characterize the in vivo contribution of NLRP12 in common mouse models of allergic airway inflammation. Anti-inflammatory biopharmaceuticals are considered to be a key element in the clinical regulation of innate and allergic airway inflammation in several human lung diseases. Thus, proteins that function as negative regulators of inflammation are of immense clinical and scientific value. NLRP12 has been shown to be a robust inhibitor of various inflammatory pathways and influence the development of contact hypersensitivity. Subepithelial collagen deposition is a commonly observed feature of asthma and is typically associated with airway remodeling and hyper-responsiveness. The deposition of fibrin, collagen and collagen precursors is facilitated by a host of genes and many are regulated, either directly or indirectly by NF-kB. However, the airway remodeling that is typically observed in human asthma is not recapitulated in short term acute OVA mouse models. Because NLRP12 has been shown to function as a negative regulator of NF-kB, it is possible that loss of NLRP12 could result in accelerated airway remodeling. To evaluate this possibility, we PF-04217903 utilized Masson’s Trichrome staining of lung sections to assess collagen and collagen precursor deposition in the lungs. Consistent with the characteristics of this model, we did not observe an increase in Trichrome positive staining following the short term acute OVA airway challenge in any of the mice tested. Thus, the deletion of Nlrp12 did not appear to have an effect on collagen deposition in the OVA mediated allergic airway disease model. NLRP12 has been shown to influence both canonical and noncanonical NF-kB signaling and classical and nonclassical MHC class I gene expression in vitro. In vivo, Nlrp12 deficiency significantly influences the development of contact hypersensitivity. Based on these findings we hypothesized that the Nlrp122/2 mouse would provide a suitable model to study the contribution of this gene.

However, as well as increasing catastrophe frequency, Klp5/6 in vivo is also reported to increase the rescue frequency and growth rate. Overall, the effects of kinesin-8 on interphase microtubules in budding yeast, fission yeast and human cells suggest that different members of the kinesins-8 family can increase not only the catastrophe frequency but also the rescue frequency, growth rate and duration of pauses of dynamic microtubules in vivo. Furthermore, these phenomena can arise through either direct or indirect effects of the kinesins-8. Resolving the mechanisms responsible for the diverse in vivo properties reported for the kinesins-8 requires in vitro reconstitution experiments to distinguish direct from indirect effects. Perhaps influenced by Kip3 work, most reconstitution experiments so far have focused on depolymerisation of brain microtubules stabilized with taxol or GMPCPP. Kip3 depolymerizes GMPCPP brain microtubules but any effect on dynamic microtubules remains to be demonstrated in vitro. For Kif18a the ability to depolymerise GMPCPP stabilised microtubules in vitro is itself controversial. Kif18a also fails to depolymerise Taxol stabilised microtubules, although in the presence of a nonhydrolysable ATP analogue it does sequester tubulin into ring structures similar to those formed by MCAK, suggesting it shares some of MCAKs functional as well as structural features. In assays on dynamic microtubules Kif18a has no effect on the stability of existing microtubules, but does block polymerisation of microtubules by Evofosfamide acting as a capping protein. In vitro experiments on full-length Klp5 and 6, expressed as a complex in baculovirus, revealed plus-end-directed sliding of brain microtubules at 39 nm s21, whilst Klp6 motor domain alone drives sliding at 56 nm s21. Klp5/6 is reported to have no in vitro depolymerase activity, either on GMPCPP or Taxol stabilised brain microtubules or on shrinking GDP brain microtubules. Since the biochemical behaviour of kinesin can be different for microtubules from different species we have examined the effect of Klp5/6 on dynamic S. pombe microtubules, both in vitro and in vivo. Our data suggest a new working model for Klp5/6 in vivo. We propose Klp5/6 tubulin complexes initially promote the birth of new microtubules, and thereafter continuously land on the growing microtubule tip, attempting to keep pace with the tip as it grows, but only succeeding at cell ends, where the microtubule tips lodge and their growth slows down in compression. Only then, within the context of the cell end, does Klp5/6 promote microtubule catastrophe. This mechanism, which allows Klp5/6 both to promote nucleation and to amplify an intrinsic tendency for microtubule tips to catastrophise under compression, may have relevance to other kinesins-8. Klp6440His was gel filtered and an aliquot taken from the peak of monodisperse protein. This repurified protein produced both a dose-dependent increase in the number of microtubules formed from S. pombe tubulin and a dosedependent reduction in mean microtubule length, consistent with it promoting microtubule nucleation. We considered two possible classes of nucleation mechanism. First, Klp5 and Klp6 binding to tubulin heterodimers might alter their conformation to favour assembly. Second, dimeric Klp5 and Klp6 might link tubulin heterodimers together to stabilise nascent microtubule nuclei.

In the same report, an 11-yearold girl with asymptomatic microscopic hematuria and low serum urate was a compound heterozygote for W258X/R477H. Her mother had a heterozygous W258X mutation associated with renal hypouricaemia. In addition, a 15 year old girl with haematuria, proteinuria and renal hypouricaemia was noted to have a single heterozygous W258X mutation, whilst a 36-year-old female, with recurrent Cycloheximide 66-81-9 episodes of uric acid stones in the right ureter had a serum uric acid of 1.8 mg/dL and an elevated FEurate of 28.1%. She was heterozygote for a R90H variant in URAT1. Va´zquezMellado et al. reported patients heterozygous for C850G in URAT1 with primary gout and low serum uric acid concentrations whilst Ichida, et al. reported 5 individuals with a W258X heterozygous change, one of whom had a history of acute kidney injury and renal stones. Recently, the GLUT9 glucose transporter, encoded by SLC2A9 gene has been shown to have an important functional role in transporting uric acid from the renal tubular cells through the basolateral membrane into interstitium. Two additional reports have confirmed this finding. Matsuo et al. detected two heterozygous mutations in GLUT9 in two hypouricaemic subjects whom were negative for URAT1 mutations and confirmed their reduced transport activity in Xenopus oocyte expression system whilst Dinour et al. recently described two families with recessively inherited hypouricaemia who were negative for URAT1 mutations. Among hypouricaemic subjects in both families, three subjects had nephrolithiasis and three subjects had a history of exercise induced acute kidney injury. In these families, homozygous carriers of SLC2A9 mutations had very low serum levels of uric acid and extremely high values of FEurate. In this report we present combined data from Macedonian and English patients who are heterozygous carriers of SLC22A12 sequence variants. Although the presentations of the patients were varied, a systematic search for hypouricaemia identified patients with possible hereditary renal hypouricaemia and who are suitable for mutational analysis of SLC22A12 and SLC2A9 genes to try and identify abnormalities in the encoded urate transporters URAT1 and GLUT9, respectively. We identified seven patients harboring five SLC22A12 variants. Interestingly, three Macedonian patients carried mutations at amino acid position 434 of URAT1. Some of these variants in SLC22A12 were discovered following significant clinical episodes including nephrolithiasis in patients SK-1, NC-1 and NC-2. There were however, no episodes of exercise induced acute kidney injury that we are aware of. The sequence variant R434H was identified in one of our patients with hypouricaemia. This variant has a reported heterozygosity index of 0.011, although this variant was not detected in our normal control subjects. Additional studies are required to demonstrate whether this variant is a common cause of hypouricaemia. Of note, in our cohort of patients, we found no novel sequence variants in the GLUT9 transporter, leaving a number of patients with unexplained hypouricaemia. However, given the complexity of proximal tubule urate handling, other urate transporter protein variants may account for hypouricaemia in the remaining patients.

Proposed models of urate transport in the proximal tubule suggest an initial uptake of uric acid from the filtrate by URAT1, coupled to organic acid transporters. GLUT9, in two different isoforms, allows for basolateral exit of urate from the proximal tubule and regulation of urate entry/exit at the apical membrane. Finally, in the late proximal tubule there are transporter proteins mediating uric acid secretion. As uric acid excretion is mediated through molecular transporters, certain drugs such as fenofibrate, valproic acid, trimethoprim and losartan may be used to manipulate these processes, thus allowing manipulation of serum uric acid levels. In humans, genetic defects in the activity of xanthine oxidase or an acquired defect in liver enzyme function or renal uric acid handling may result in hypouricaemia. Acquired hypouricaemia may be seen in a number of clinical disorders, including Fanconi syndrome, type 1 and type 2 diabetes mellitus, thyrotoxicosis, pseudohypoparathyroidism type 1b, pseudoaldosteronism due to licorice ingestion, distal renal tubular acidosis, obstructive jaundice and severe acute respiratory syndrome. Idiopathic renal hypouricaemia is an inherited form of hypouricaemia that is characterized by excessive urinary wasting of uric acid leading to an increased clearance of uric acid. The majority of Torin 1 patients are asymptomatic, but some may present with uric acid nephrolithiasis or acute kidney injury following severe exercise. In 2002, Enomoto et al. reported that mutations in gene SLC22A12 encoding the URAT1 transporter were responsible for most cases of idiopathic renal hypouricaemia. Recently Anzai et al. found mutations in SLC2A9, encoding GLUT9, in patients with severe renal hypouricaemia. It is noteworthy that reports of idiopathic renal hypouricaemia secondary to mutations in uric acid transporters URAT1 and GLUT9 were initially reported from Japan, Korea and China. More recently, three Jewish Israeli families of Iraqi origin have been reported to have renal hypouricaemia, with a common mutation in SLC22A12. Inactivating mutations in SLC22A12 have not yet, to our knowledge, been reported in a Caucasian population. The typical presentation of idiopathic renal hypouricaemia is that of exercise induced acute kidney injury with a preceding history of loin pain with nausea and vomiting for several hours after physical exercise. The exact mechanism of renal damage is unclear, but may relate to damage from oxygen free radicals. In contrast to this dramatic presentation, most patients are well with no overt clinical symptoms, although renal stones and hematuria may be presenting symptoms and signs. Here we present data from Skopje and Newcastle upon Tyne where we have investigated the underlying genetic cause of hypouricaemia in patients of European descent. We present mutations in SLC22A12 encoding URAT1 alongside their clinical, biochemical and functional characterization. This data highlights the importance of renal urate transporters in determining serum urate concentrations. Idiopathic renal hypouricaemia is a disorder that has been characterized previously in patients from Far Eastern countries including Japan, Korea and China.