Decavanadate possesses α-adrenergic agonist activity and a structural motif common with trans-β form of noradrenaline

Decavanadate, an inorganic polymer of vanadate, produced contraction of rat aortic rings at a relatively high concentration compared to phenylephrine, an agonist of -adrenergic receptor. This effect was blocked by two known a-adrenergic receptor antagonists, prazosin and phenoxybenzamine. Decavanadate, formed by possible dimerization of V5 under acid conditions, possessed a structural feature of two pairs of unshared oxygen atoms at a distance of 3.12 Å, not found in its constituents of V4 or V5. A structural motif of O..O..O using such oxygen atoms is recognized in decavanadate. This matches with a similar motif of N..O..O that uses the essential amino and hydroxyl groups of the side-chain and the m-hydroxyl group in trans-b form of noradrenaline. The interaction of such a structural motif with the membrane receptor is likely to be the basis of the unusual noradrenaline-mimic action of decavanadate.     

Semi-automated imaging system to quantitate Her-2/neu membrane receptor immunoreactivity in human breast cancer.

BACKGROUND: Immunocytochemical methods for quantitating Her-2/neu immunoreactivity rest on subjective semi-quantitative interpretations with resulting interobserver, intraobserver, and fatigue variability. METHODS: To standardize and quantitate measurements of Her-2/neu immunoreactivity, we created epithelial-recognition and specific membrane-recognition algorithms, which could image breast cancer cells against a background of stroma, compartmentalize the cancer cell into nucleus, cytoplasm and membrane, and quantitate the degree of Her-2/neu membrane immunoreactivity based on both gray scale intensity and RGB colorimetric determinations. Image acquisition utilized either scanner or microscope with attached camera with a resolution of 20 pixels/10 microm. Areas of 150 whole slides were screened and the regions of interest manually selected for image processing. Three hundred TMA cores were directly processed. Images were acquired by jpg conversion of svs virtual slides or direct jpg photomicrograph capture. Images were then assessed for quality and processed. RESULTS: The digital algorithms successfully created a semi-automated imaging system whose algorithm-based ordinal values for Her-2/neu both strongly correlated with the subjective measurements (intraclass correlation: 0.84; 95% confidence interval: 0.79-0.89) yet exhibited no run variability. In addition, the algorithms generated immunocytochemical measurements of Her-2/neu on an expanded continuous scale, which more reliably distinguished true Her-2/neu positivity from true Her-2/neu negativity (determined by FISH) than subjective or algorithmic ordinal scale measurements. Furthermore, the continuous scale measurements could better resolve different levels of Her-2/neu overexpression than either subjective or algorithmic ordinal interpretation. Other semi-automated analysis systems have been used to measure Her-2/neu and other cellular immunoreactivities, but these either have required proprietary hardware or have been based on luminosity differences alone. In contrast, our algorithms are independent of proprietary hardware and are based on not just luminosity but also many other imaging properties including epithelial recognition and membrane morphology. CONCLUSION: These features provide a more accurate, versatile, and robust imaging analysis platform.     

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.     

Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila

Bacteria of genus Aeromonas, responsible for a variety of pathological conditions in humans and fish, are ubiquitous waterborne bacteria. Aeromonas produces several virulent factors including a complex of lipopolysaccharide and surface array protein, involved in colonization. UDP-galactose 4-epimerase (GalE) catalyzes the production of UDP-galactose, a precursor for lipopolysaccharide biosynthesis, and thus is an important drug target. GalE exhibits interspecies variation and heterogeneity at its structural and functional level and therefore, the differences between the GalE of the host and the pathogen can be exploited for drug designing. In the present study, we report biochemical and functional characterization of the recombinant GalE of Aeromonas hydrophila. Unlike GalE reported from all other species, the purified recombinant GalE of A. hydrophila was found to exist as a monomer. This is the first report of UDP-galactose 4-epimerase from any species being a monomer. The molecular mass of the 6xHis-rGalE was determined to be 38271.477 (m/z). The 6xHis-rGalE with a K(m) of 0.5 mM for UDP-galactose exhibited optimum activity at 37 degrees C and pH 8-9. Spectrofluorimetric and CD analysis confirmed that the thermal inactivation was due to structural changes and not due to the NAD-dissociation. A relatively more ordered structure of the enzyme at pH 8 and 9 as compared to that at pH 6 or 7 suggests a key role of the electrostatic interactions in maintaining its native tertiary structure.     

Feeding damage by Diuraphis noxia results in a nutritionally enhanced phloem diet

The feeding behavior of Diuraphis noxia Mordvilko (Homoptera: Aphididae) on susceptible hosts causes both ultrastructural and tissue level damage which may affect phloem composition. Genetic evidence suggests that endosymbiotic bacteria in most aphids overproduce limiting amino acids to benefit hosts but that D. noxia depends less on endosymbionts for these nutrients, possibly due to an enriched diet. To determine whether D. noxia feeding damage results in higher concentrations of essential amino acids, stylet exudates were analyzed from wheat (Triticum aestivum) damaged to different degrees. Comparison of samples from undamaged and damaged susceptible wheat revealed changes in amino acid composition and an increase in levels of essential amino acids, indicating a nutritionally enhanced ingesta. The changes in stylet exudates paralleled changes in leaf exudates, indicating that the effects are systemic. Feeding damage is not observed on a resistant wheat host, var. Halt, and leaf exudates from infested Halt did not show changes in amino acid composition. Mean relative growth of nymphs was significantly lower on Halt than on susceptible Arapahoe, indicating that Halt is a less suitable host. Both varieties show similar amino acid levels in non-infested samples, suggesting that D. noxia infestation does not enhance the phloem environment in Halt. This study provides evidence that aphid feeding can generate a nutritionally enhanced phloem diet.     

Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.     

Overlapping neuronal circuits in addiction and obesity: evidence of systems pathology

Drugs and food exert their reinforcing effects in part by increasing dopamine (DA) in limbic regions, which has generated interest in understanding how drug abuse/addiction relates to obesity. Here, we integrate findings from positron emission tomography imaging studies on DA's role in drug abuse/addiction and in obesity and propose a common model for these two conditions. Both in abuse/addiction and in obesity, there is an enhanced value of one type of reinforcer (drugs and food, respectively) at the expense of other reinforcers, which is a consequence of conditioned learning and resetting of reward thresholds secondary to repeated stimulation by drugs (abuse/addiction) and by large quantities of palatable food (obesity) in vulnerable individuals (i.e. genetic factors). In this model, during exposure to the reinforcer or to conditioned cues, the expected reward (processed by memory circuits) overactivates the reward and motivation circuits while inhibiting the cognitive control circuit, resulting in an inability to inhibit the drive to consume the drug or food despite attempts to do so. These neuronal circuits, which are modulated by DA, interact with one another so that disruption in one circuit can be buffered by another, which highlights the need of multiprong approaches in the treatment of addiction and obesity.     

Itinerant exosomes: emerging roles in cell and tissue polarity

Cells use secreted signals (e.g. chemokines and growth factors) and sophisticated vehicles such as argosomes, cytonemes, tunneling nanotubes and exosomes to relay important information to other cells, often over large distances. Exosomes, 30-100-nm intraluminal vesicles of multivesicular bodies (MVB) released upon exocytic fusion of the MVB with the plasma membrane, are increasingly recognized as a novel mode of cell-independent communication. Exosomes have been shown to function in antigen presentation and tumor metastasis, and in transmitting infectious agents. However, little is known about the biogenesis and function of exosomes in polarized cells. In this review, we discuss new evidence suggesting that exosomes participate in the transport of morphogens and RNA, and thus influence cell polarity and developmental patterning of tissues.     

Selective inhibition of cyclooxygenase-2 (COX-2) by 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3, a less calcemic vitamin D analog

Inducible cyclooxygenase-2 (COX-2) has been implicated to play a role in inflammation and carcinogenesis and selective COX-2 inhibitors have been considered as anti-inflammatory and cancer chemopreventive agents. 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), the active hormonal form of vitamin D3 also has been considered to be a cancer chemopreventive agent in addition to its important role in maintaining calcium homeostasis. Based on these observations, we studied the direct effect of 1alpha,25(OH)2D3 and one of its less calcemic synthetic analogs, 1alpha,25(OH)2-16-ene-23-yne-D3 on the activity of both COX-1 and COX-2 in an in vitro enzyme assay. Preliminary data indicated that both 1alpha,25(OH)2D3 and 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited selectively the activity of COX-2 with no effect on the activity of COX-1. Out of the two compounds, 1alpha,25(OH)2-16-ene-23-yne-D3 was found to be more effective with an IC50 of 5.8 nM. Therefore, the rest of the experiments were performed using 1alpha,25(OH)2-16-ene-23-yne-D3 only. 1alpha,25(OH)2-16-ene-23-yne-D3 inhibited the proliferation of lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) with a reduction in the expression of COX-2 along with other inflammatory mediators like inducible nitric oxide synthase (iNOS) and interleukin-2 (IL-2). Furthermore, 1alpha,25(OH)2-16-ene-23-yne-D3 also inhibited carrageenan induced inflammation in an air pouch of a rat and effectively reduced the expression of COX-2, iNOS, and IL-2 in the tissues of the same air pouch. In both cases, 1alpha,25(OH)2-16-ene-23-yne-D3 did not show any effect on the expression of COX-1. In summary, our results indicate that 1alpha,25(OH)2-16-ene-23-yne-D3, a less calcemic vitamin D analog, exhibits potent anti-inflammatory effects and is a selective COX-2 inhibitor.