14-3-3 proteins fine-tune plant nutrient metabolism

14-3-3 Proteins regulate many cellular processes by binding to phosphorylated proteins. Previous findings suggest a connection between three 14-3-3 isoforms and plant nutrient signaling. To better understand how these 14-3-3s regulate metabolism in response to changes in plant nutrient status, putative new targets involved in nitrogen (N) and sulfur (S) metabolisms have been identified. The interactions between these 14-3-3s and multiple proteins involved in N and S metabolism and altered activity of the target proteins were confirmed in planta. Using a combination of methods, this work elucidates how 14-3-3s function as modulators of plant N and S metabolic pathways.     

An antagonistic monoclonal anti–Plexin-B1 antibody exerts therapeutic effects in mouse models of postmenopausal osteoporosis and multiple sclerosis

Osteoporosis and multiple sclerosis are highly prevalent diseases with limited treatment options. In light of these unmet medical needs, novel therapeutic approaches are urgently sought. Previously, the activation of the transmembrane receptor Plexin-B1 by its ligand semaphorin 4D (Sema4D) has been shown to suppress bone formation and promote neuroinflammation in mice. However, it is unclear whether inhibition of this receptor–ligand interaction by an anti–Plexin-B1 antibody could represent a viable strategy against diseases related to these processes. Here, we raised and systematically characterized a monoclonal antibody directed against the extracellular domain of human Plexin-B1, which specifically blocks the binding of Sema4D to Plexin-B1. In vitro, we show that this antibody inhibits the suppressive effects of Sema4D on human osteoblast differentiation and mineralization. To test the therapeutic potential of the antibody in vivo, we generated a humanized mouse line, which expresses transgenic human Plexin-B1 instead of endogenous murine Plexin-B1. Employing these mice, we demonstrate that the anti–Plexin-B1 antibody exhibits beneficial effects in mouse models of postmenopausal osteoporosis and multiple sclerosis in vivo. In summary, our data identify an anti–Plexin-B1 antibody as a potential therapeutic agent for the treatment of osteoporosis and multiple sclerosis.     

ICAD Deficiency in Human Colon Cancer and Predisposition to Colon Tumorigenesis: Linkage to Apoptosis Resistance and Genomic Instability

We previously showed that DNA fragmentation factor, which comprises a caspase-3-activated DNase (CAD) and its inhibitor (ICAD), may influence the rate of cell death by generating PARP-1-activating DNA breaks. Here we tested the hypothesis that ICAD-deficient colon epithelial cells exhibiting resistance to death stimuli may accumulate additional genetic modifications, leading to a tumorigenic phenotype. We show that ICAD deficiency may be associated with colon malignancy in humans. Indeed, an examination of ICAD expression using immunohistochemistry in an array of both colon cancer and normal tissues revealed that ICAD expression levels were severely compromised in the cancerous tissues. Upon DNA damage caused by a low dose of irradiation, ICAD cells acquire a tumorigenic phenotype. Colon epithelial cells derived from ICAD mice showed a significant resistance to death induced by the colon carcinogen dimethylhydrazine in vitro and in mice. Such resistance was associated with a decrease in PARP-1 activation. In an animal model of dimethylhydrazine-induced colon tumorigenesis, ICAD^−/− mice developed significantly higher numbers of tumors with markedly larger sizes than the wild-type counterparts. Interestingly, the phenotype of the ICAD^−/− mice was not associated with a significant increase in the precancerous aberrant crypt foci suggesting a potential link to tumor progression rather than initiation. More importantly, ICAD deficiency was associated with severe genomic instability as assessed by array comparative genomic hybridization. Such genomic instability consisted most prominently of amplifications but with sizable deletions as compared to the wild-type counterparts affecting several cancer-related genes including RAF-1, GSN, LMO3, and Fzd6 independently of p53. Altogether, our results present a viable case for the involvement of ICAD deficiency in colon carcinogenesis and show that apoptosis and genomic instability may comprise the means by which such deficiency may contribute to the process of increasing susceptibility to carcinogen-induced tumorigenesis.     

Discovery of new S-adenosylmethionine decarboxylase inhibitors for the treatment of Human African Trypanosomiasis (HAT)

Modification of the structure of trypanosomal AdoMetDC inhibitor 1 (MDL73811) resulted in the identification of a new inhibitor 7a, which features a methyl substituent at the 8-position. Compound 7a exhibits improved potencies against both the trypanosomal AdoMetDC enzyme and parasites, and better blood brain barrier penetration than 1.     

Gallic acid ameliorates COPD-associated exacerbation in mice

Molecular and Cellular Biochemistry - COPD is an inflammatory lung disease, which is often exacerbated with microbial infections resulting in worsening of respiratory symptoms. Gallic acid (GA), a...     

Changes in skeletal muscle SR Ca2+ pump in congestive heart failure due to myocardial infarction are prevented by angiotensin II blockade

In order to understand the mechanisms of exercise intolerance and muscle fatigue, which are commonly observed in congestive heart failure, we studied sarcoplasmic reticulum (SR) Ca(2+)-transport in the hind-leg skeletal muscle of rats subjected to myocardial infarction (MI). Sham-operated animals were used for comparison. On one hand, the maximal velocities (Vmax) for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities in skeletal muscle of rats at 8 weeks of MI were higher than those of controls. On the other hand, the Vmax values for both SR Ca(2+)-uptake and Ca(2+)-stimulated ATPase activities were decreased significantly at 16 weeks of MI when compared with controls. These alterations in Ca(2+)-transport activities were not associated with any change in the affinity (1/Ka) of the SR Ca(2+)-pump for Ca2+. Furthermore, the stimulation of SR Ca(2+)-stimulated ATPase activity by cyclic AMP-dependent protein kinase was not altered at 8 or 16 weeks of MI when compared with the respective control values. Treatment of 3-week infarcted animals with angiotensin-converting enzyme (ACE) inhibitors such as captopril, imidapril, and enalapril or an angiotensin receptor (AT1R) antagonist, losartan, for a period of 13 weeks not only attenuated changes in left ventricular function but also prevented defects in SR Ca(2+)-pump in skeletal muscle. These results indicate that the skeletal muscle SR Ca(2+)-transport is altered in a biphasic manner in heart failure due to MI. It is suggested that the initial increase in SR Ca(2+)-pump activity in skeletal muscle may be compensatory whereas the depression at late stages of MI may play a role in exercise intolerance and muscle fatigue in congestive heart failure. Furthermore, the improvements in the skeletal muscle SR Ca(2+)-transport by ACE inhibitors may be due to the decreased activity of renin-angiotensin system in congestive heart failure.     

Characterization of NADP-Isocitrate Dehydrogenase from Nodules of Pigeonpea (Cajanus cajan)

NADP-isocitrate dehydrogenase from nodules of pigeonpea (Cajanus cajan L. cv UPAS-120) was partially purified to about 57 folds and its properties were studied. The enzyme showed an absolute requirement for a divalent cation which was fulfilled either by Mn⁺² or Mg⁺² and to a smaller extent by Co⁺². The enzyme exhibited a sigmoidal response to increasing concentrations of Mn²⁺ (S_0.5=0.3mM). The apparent Km values for isocitrate, NADP and Mg²⁺ were 21, 23 and 280 μM, respectively. It had an optimum pH of 8.0–8.2. The enzyme activity was not affected by various organic acids, amino acids and amides. NADH inhibited the activity non-competitively with respect to NADP. An apparent inhibition by ATP and ADP was due to chelation of divalent cation. NADPH acted competitively against NADP and non-competitively against isocitrate. Glutamate caused uncompetitive inhibition with respect to NADP and competitive against isocitrate. Kinetic studies suggested the reaction mechanism to be probably random sequential. Possible regulation of the enzyme activity in the nodules via cellular redox state and the levels of reaction products is discussed.     

A procedure for large-scale purification of domoic acid from toxic blue mussels (Mytilus edulis)

Pure domoic acid is required for use in research to investigate the biological effects of this new shellfish toxin. It may also prove to be a useful tool in studies exploring the basis of Alzheimer's disease. In this paper we describe a procedure which is effective in obtaining adequate quantities of pure domoic acid from blue mussel (Mytilus edulis). The procedure involves tissue homogenization, treatment of homogenate with chloroform and methanol, and separation of different phases with the addition of water. The aqueous-methanolic phase (upper layer) contains water soluble components including domoic acid, the chloroform phase (lower layer) contains lipoid moieties, and the interphase contains denatured proteins. The aqueous phase containing domoic acid was removed, rotory evaporated to get rid of methanol, followed by ultrafiltration to remove high molecular weight contaminants. The filtrate was lyophilized, resuspended in 1 N HCl, centrifuged and the resulting clear solution subjected to column chromatography on C18 reversed phase silica gel. Fractions containing domoic acid were pooled, and lyophilized. A brownish dry powder contained pure domoic acid with 60–65% yield from the original tissue homogenate. Another 10–15% of domoic acid was mixed with its isomer, and can be further resolved to obtain an overall recovery of 75–80% of the starting material.