Pyridinedicarboxylates, the first mechanism-derived inhibitors for prolyl 4-hydroxylase, selectively suppress cellular hydroxyprolyl biosynthesis. Decrease in interstitial collagen and Clq secretion in cell culture.

Peptidyl-dipeptidase A (angiotensin converting enzyme; ACE, EC 3.4.15.1), has been purified from pig kidney and striatum by affinity chromatography employing the selective inhibitor lisinopril as ligand. The inclusion of a 2.8 nm spacer arm improved the yield of the enzyme compared with the 1.4 nm spacer arm described in previous work. Two forms of striatal ACE (Mr 180,000 and 170,000), but only a single form of kidney ACE (Mr 180,000), were isolated by this procedure. Both forms of striatal ACE were recognized by a polyclonal antibody to kidney ACE. No significant differences in substrate specificity or inhibitor sensitivity between kidney and striatal ACE could be detected. In particular, the amidated neuropeptide, substance P, was hydrolysed identically by both preparations and no significant hydrolysis of the related tachykinin peptides neurokinin A and neurokinin B could be detected. After chemical or enzymic deglycosylation, kidney and both forms of striatal ACE migrated identically on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 150,000. We suggest that the two detectable forms of ACE in pig brain are not isoenzymes but are the result of differential glycosylation in different cell types in the brain. It appears that ACE, unlike endopeptidase-24.11, does not have the general capacity to hydrolyse and inactivate the tachykinin peptides at a significant rate in brain.     

Heptatriacontan-4-one,tetratriacontanyl octacosanoate and other constituents from Pedalium murex

Two new compounds isolated from the fruits of Pedalium murex are characterized as heptatriacontan-4-one and tetratriacontanyl octacosanoate by spectral studies. Pentatriacontane, sitosterol, hexatriacontanoic acid, hentriacontanoic acid, ursolic acid and vanillin have also been isolated and identified.     

A compound-heterozygous Marfan patient: two defective fibrillin alleles result in a lethal phenotype.

We describe here the identification of defined mutations in both alleles of the fibrillin gene (FBN1) in a compound-heterozygote Marfan syndrome (MFS) child who had a very severe form of MFS resulting in death from cardiac failure at the age of 4 mo. The nonconsanguineous parents were both affected with MFS. The father's heterozygous point mutation has earlier been reported to result in W217G substitution, the mother was here shown to carry a heterozygous point mutation resulting in G2627R substitution, and the child had inherited both these mutations. The mutant FBN1 alleles were demonstrated to be transcribed with equal efficiency compared with the normal alleles, but metabolic labeling of fibroblast cultures from the child and both parents showed reduced biosynthesis and secretion of profibrillin. Also, the respective amounts of fibrillin in cell-culture media and extracellular-matrix extracts were markedly diminished, particularly in the cell cultures from father and child. In addition, immunofluorescence analysis of the cell cultures of all three family members revealed a drastically reduced amount of microfibrils, and virtually no visible fibrils could be seen in the case of the compound-heterozygote child. These findings demonstrate incomplete dominance of fibrillin mutations and underline the fatal consequences of the complete absence of normal fibrillin molecules in the microfibrils.     

Costusoside-I and costusoside-J,two new furostanol saponins from the seeds of Costus speciosus

The structure of costusoside I and costusoside J have been established as 3-O-{β-d-glucopyranosyl (1 → 2)-α-l-rhamnopyranosyl (1 → 2) [α-l-rhamnopyranosyl (1 → 4)]-β-d-glucopyranosyl}-26-O-(β-d-glucopyranosyl)-22α-methoxy 25 R)-furost-5-en-3β, 26-diol and its 22-hydroxy compound respectively, isolated fron the seeds of Costus speciosus.     

Sirtuin 1 and 7 mediate resveratrol-induced recovery from hyper-anxiety in high-fructose-fed prediabetic rats

Hyperglycaemia in diabetes is either caused by reduced availability of insulin (type 1 diabetes, T1D) or insulin resistance to the cells (type 2 diabetes, T2D). In recent years, the prevalence of T2D has increased to an alarming proportion, encompassing 95% of the total diabetic burden, probably due to economy-driven changes in lifestyle. Recent epidemiological studies show comorbid depression, anxiety and related mental illness. To explore the molecular mechanisms underlying this comorbid conditions, we used Sprague–Dawley rats on high-fructose diet for 8 weeks to induce prediabetic condition. Rats with this metabolic syndrome also showed hyper-anxiety when they were subjected to anxiety-related behavioural assays. Rats were administered with resveratrol, an activator of sirtuins, and metformin, a standard antidiabetic drug, simultaneously with fructose. We observed that resveratrol was more effective in protecting from both the metabolic (prediabetic) and affective (anxiety) disorders than metformin. Molecular studies showed that recovery was associated with the upregulation of few nuclear sirtuins that act epigenetically – Sirt 1 and 7, which were significantly attenuated in the striatum of prediabetic rats. In conclusion, our study showed that hyper-anxiety associated with prediabetic condition is ameliorated by resveratrol through modulation of sirtuins, which is more or less similar to metformin.     

A possible role for p190RhoGAP in PKCepsilon-induced morphological effects

We have previously shown that protein kinase C (PKC) epsilon induces neurite outgrowth via its regulatory domain. This is accompanied by PKC-induced stress fibre loss. Here, we show that the regulatory domain (RD) of PKCepsilon induces processes also in NIH-3T3 fibroblasts, similar to what has been observed with p190RhoGAP. This study also shows that p190RhoGAP induces neurite outgrowth in SK-N-BE(2) neuroblastoma cells. We therefore investigated whether p190RhoGAP may be downstream of PKCepsilon. We could detect a co-localization of p190RhoGAP and PKCepsilon at membrane ruffles and an increased association between the proteins in fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The association is also seen in neuroblastoma cells, and nerve growth factor (NGF) treatment of SH-SYSY/TrkA cells decreases the interaction. However, overexpressed PKCepsilon did not coprecipitate overexpressed p190RhoGAP in CHO cells, indicating that the proteins do not interact directly. This raises the possibility that p190RhoGAP is involved in mediating the morphological effects of PKCepsilon.     

Advancing cartilage tissue engineering: the application of stem cell technology

The treatment of cartilage pathology and trauma face the challenges of poor regenerative potential and inferior repair. Nevertheless, recent advances in tissue engineering indicate that adult stem cells could provide a source of chondrocytes for tissue engineering that the isolation of mature chondrocytes has failed to achieve. Various adjuncts to their propagation and differentiation have been explored, such as biomaterials, bioreactors and growth hormones. To date, all tissue engineered cartilage has been significantly mechanically inferior to its natural counterparts and further problems in vivo relate to poor integration and deterioration of tissue quality over time. However, adult stem cells--with their high rate of proliferation and ease of isolation--are expected to greatly further the development and usefulness of tissue engineered cartilage.     

Effects of vitamin restriction and supplementation on rat intestinal epithelial cell apoptosis

Diet influences intestinal growth and function and vitamins modulate intestinal cell turnover. We have assessed the effects of chronic, moderate (50% of control) vitamin restriction and supplementation on intestinal epithelial cell (IEC) apoptosis and the relevance of this to alterations in tissue oxidative stress and antioxidant status. Feeding a vitamin-restricted diet to male, weanling WNIN rats for 20 weeks significantly increased IEC apoptosis, but only in the villi region, as evident from increased annexin V staining, M30 positivity, histological observations, DNA ladder formation, and reduced expression of Bcl-2. This was associated with elevated levels of lipid peroxides and protein carbonyls in the intestinal mucosa despite the increased activities of superoxide dismutase, catalase, and glutathione peroxidase. Consistent with the increased oxidative stress and apoptosis, structural and functional integrity of the villi were compromised as evident from the lowered ratio of villus height:crypt depth and the decreased activities of the membrane marker enzymes alkaline phosphatase and Lys-Ala dipeptidyl aminopeptidase. These changes were reversed by supplementation with a vitamin mixture or vitamin E alone, whereas riboflavin or folic acid supplementation reduced the apoptotic rates, but only partially. Further, oxidative stress was the least in vitamin E- or vitamin mixture-supplemented rats and correlated well with their IEC apoptotic rates. Increased tissue oxidative stress seems to mediate the vitamin-restriction-induced apoptosis of the IECs in rats.     

Type I Interferon-Sensitive Recombinant Newcastle Disease Virus for Oncolytic Virotherapy

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic because of its potent ability to induce apoptosis. Several studies have demonstrated that NDV is selectively cytotoxic to tumor cells but not normal cells due to defects in the interferon (IFN) antiviral responses of tumor cells. Many naturally occurring strains of NDV have an intact IFN-antagonistic function and can still replicate in normal human cells. To avoid potential toxicity issues with NDV, especially in cancer patients with immunosuppression, safe NDV-oncolytic vectors are needed. We compared the cell killing abilities of (i) a recombinant NDV (rNDV) strain, Beaudette C, containing an IFN-antagonistic, wild-type V protein (rBC), (ii) an isogenic recombinant virus with a mutant V protein (rBC-Edit virus) that induces increased IFN in infected cells and whose replication is restricted in normal human cells, and (iii) a recombinant LaSota virus with a virulent F protein cleavage site that is as interferon sensitive as rBC-Edit virus (LaSota V.F. virus). Our results indicated that the tumor-selective replication of rNDV is determined by the differential regulation of IFN-α and downstream antiviral genes induced by IFN-α, especially through the IRF-7 pathway. In a nude mouse model of human fibrosarcoma, we show that the IFN-sensitive NDV variants are as effective as IFN-resistant rBC virus in clearing the tumor burden. In addition, mice treated with rNDV exhibited no signs of toxicity to the viruses. These findings indicate that augmentation of innate immune responses by NDV results in selective oncolysis and offer a novel and safe virotherapy platform.Several naturally occurring or engineered oncolytic viruses are emerging as novel tools for selective growth in and killing of a variety of tumor cells (1, 21, 34, 41). It has been consistently reported that during tumor evolution, diminished interferon (IFN) responsiveness coevolves as a frequent genetic defect (4, 31, 32, 41). Any defects in responsiveness to interferon will afford permissiveness of tumors for replication of oncolytic viruses by blunting the antiviral innate immune system. Thus, it was suggested that oncolytic viruses could be engineered to induce strong IFN response and/or to be defective in antagonizing the IFN signaling. This would result in virus replication in tumor cells with IFN defects but in reduced or crippled virus replication in normal cells, with the absence of toxicity (42). A variety of oncolytic viruses have been engineered to exploit tumor-specific genetic defects (3, 12, 24, 42, 46) and shown to be potent oncolytic agents.Newcastle disease virus (NDV), an avian paramyxovirus, is a promising broad-spectrum oncolytic agent (27, 29, 30, 37). Nonengineered, naturally occurring strains of NDV such as 73-T (6), MTH68 (7), PV701 (28, 35), and NDV-HUJ (11) have been successfully employed in several clinical studies for tumor regression. NDV is inherently oncolytic and tumor selective, sparing normal cells (9, 15, 37). The tumor selectivity of NDV is considered to be due to a defective IFN response in tumor cells (10, 23, 37). NDV is a strong inducer of type I IFN in many types of cells (18). In normal cells, a robust IFN-mediated antiviral response limits the replication of NDV (9, 23). This known sensitivity of NDV to cellular antiviral mechanisms affords a wide safety margin for its use in humans.Recent studies have indicated that improved therapeutic vectors of NDV could be engineered through reverse genetics for enhanced oncolytic efficacy from an increased anti-tumor response and interleukin 2 (IL-2) receptor-mediated targeting (5, 9, 44, 46). Therefore, we reasoned that recombinant NDVs (rNDVs) that are susceptible to cellular innate immune responses would be safer and more effective oncolytic agents. Even though NDV is an avian virus and induces a strong IFN response in normal human cells, it still expresses IFN-antagonizing activity. Ablation of the expression of V protein, which is responsible for this anti-IFN activity, may further reduce the ability of NDV to infect and kill normal human cells without affecting tumor cell infection and lysis. Here, we describe the relative oncolytic efficacies of three rNDV strains differing in IFN antagonism. The rNDV variants with an IFN-sensitive phenotype had parallel therapeutic efficacies in xenotransplanted human fibrosarcoma cells in a nude mouse model and offer great potential as recombinant vectors in therapy of human malignancies.