Increased short-term food satiation and sensitivity to cholecystokinin in neurotrophin-4 knock-in mice

Neurotrophin-4 (NT-4) knockout mice exhibited decreased innervation of the small intestine by vagal intraganglionic laminar endings (IGLEs) and reduced food satiation. Recent findings suggested this innervation was increased in NT-4 knock-in (NT-4KI) mice. Therefore, to further investigate the relationship between intestinal IGLEs and satiation, meal patterns were characterized using solid and liquid diets, and cholecystokinin (CCK) effects on 30-min solid diet intake were examined in NT-4KI and wild-type mice. NT-4KI mice consuming the solid diet exhibited reduced meal size, suggesting increased satiation. However, compensation occurred through increased meal frequency, maintaining daily food intake and body weight gain similar to controls. Mutants fed the liquid diet displayed a decrease in intake rate, again implying increased satiation, but meal duration increased, which led to an increase in meal size. This was compensated for by decreased meal frequency, resulting in similar daily food intake and weight gain as controls. Importantly, these alterations in NT-4KI mice were opposite, or different, from those of NT-4 knockout mice, further supporting the hypothesis that they are specific to vagal afferent signaling. CCK suppressed short-term intake in mutants and controls, but the mutants exhibited larger suppressions at lower doses, implying they were more sensitive to CCK. Moreover, devazepide prevented this suppression, indicating this increased sensitivity was mediated by CCK-1 receptors. These results suggest that the NT-4 gene knock-in, probably involving increased intestinal IGLE innervation, altered short-term feeding, in particular by enhancing satiation and sensitivity to CCK, whereas long-term control of daily intake and body weight was unaffected.     

Lysophospholipids increase ICAM-1 expression in HUVEC through a Gi- and NF-kappaB-dependent mechanism

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of G_i, ammonium pyrrolidinedithiocarbamate and BAY 11–7082, inhibitors of the nuclear factor (NF)-B pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a G_i-, NF-B-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes. lysophosphatidic acid; sphingosine 1-phosphate; inflammation; intercellular adhesion molecule-1; nuclear factor-B; human umbilical cord vein endothelial cells     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Inhibition of yeast growth by molybdenum-hydroxylamido complexes correlates with their presence in media at differing pH values

The effects of Mo-hydroxylamido complexes on cell growth were determined in Saccharomyces cerevisiae to investigate the biological effects of four different Mo complexes as a function of pH. Studies with yeast, an eukaryotic cell, are particularly suited to examine growth at different pH values because this organism grows well from pH 3 to 6.5. Studies can therefore be performed both in the presence of intact complexes and when the complexes have hydrolyzed to ligand and free metal ion. One of the complexes we examined was structurally characterized by X-ray crystallography. Yeast growth was inhibited in media solutions containing added Mo-dialkylhydroxylamido complexes at pH 3-7. When combining the yeast growth studies with a systematic study of the Mo-hydroxylamido complexes' stability as a function of pH and an examination of their speciation in yeast media, the effects of intact complexes can be distinguished from that of ligand and metal. This is possible because different effects are observed with complex present than when ligand or metal alone is present. At pH 3, the growth inhibition is attributed to the forms of molybdate ion that exist in solution because most of the complexes have hydrolyzed to oxomolybdate and ligand. The monoalkylhydroxylamine ligand inhibited yeast growth at pH 5, 6 and 7, while the dialkylhydroxylamine ligands had little effect on yeast growth. Growth inhibition of the Mo-dialkylhydroxylamido complexes is observed when a complex exists in the media. A complex that is inert to ligand exchange is not effective even at pH 3 where other Mo-hydroxylamido complexes show growth inhibition as molybdate. These results show that the formation of some Mo complexes can protect yeast from the growth inhibition observed when either the ligand or Mo salt alone are present.     

The development of potent non-peptidic PTP-1B inhibitors

The SAR from our peptide libraries was exploited to design a series of potent deoxybenzoin PTP-1B inhibitors. The introduction of an ortho bromo substituent next to the difluoromethylphosphonate warhead gave up to 20-fold increase in potency compared to the desbromo analogues. In addition, these compounds were orally bioavailable and active in the animal models of non-insulin dependent diabetes mellitus (NIDDM).     

A thermostable leucine aminopeptidase from<Emphasis Type="Italic"> Bacillus kaustophilus</Emphasis> CCRC 11223

Two degenerate primers established from the consensus sequences of bacterial leucine aminopeptidases (LAP) were used to amplify a 360-bp gene fragment from the chromosomal DNA of thermophilic Bacillus kaustophilus CCRC 11223 and the amplified fragment was successfully used as a probe to clone a leucine aminopeptidase (lap) gene from a genomic library of the strain. The gene consists of an open reading frame (ORF) of 1,494 bp and encodes a protein of 497 amino acid residues with a calculated molecular mass of 53.7 kDa. The complete amino acid sequence of the cloned enzyme showed greater than 30% identity with prokaryotic and eukaryotic LAPs. Phylogenetic analysis showed that B. kaustophilus LAP is closely related to the enzyme from Bacillus subtilis and is grouped with the M17 family. His₆-tagged LAP was generated in Escherichia coli by cloning the coding region into pQE-30 and the recombinant enzyme was purified by nickel-chelate chromatography. The pH and temperature optima for the purified enzyme were 8 and 65°C, respectively, and 50% of its activity remained after incubation at 60°C for 32 min. The enzyme preferentially hydrolyzed l-leucine-p-nitroanilide (l-Leu-p-NA) followed by Cys derivative.Communicated by G. Antranikian     

Potential and practical adrenomedullary PET radiopharmaceuticals as an alternative to m-iodobenzylguanidine: m-(omega-[18F]fluoroalkyl)benzylguanidines

To investigate adrenomedullary radiopharmaceuticals for positron emission tomography (PET), we have developed no-carrier-added m-(omega-[18F]fluoroalkyl)benzylguanidines. m-(omega-[18F]Fluoroalkyl)benzylguanidines were prepared in two steps starting from N,N'-bis(tert-butyloxycarbonyl)-N' '-(omega-methanesulfonyloxyalkyl)benzylguanidines in 20-30% radiochemical yields (decay corrected for 100 min) and with high radiochemical purity (>97%) and shown to be stable (>90%) in an in vitro metabolic stability assay. The binding of m-(3-[18F]fluoropropyl)benzylguanidine ((18F]3) to SK-N-SH human neuroblastoma cells was temperature dependent, and binding levels at 4 degrees C were reduced to half of that at 37 degrees C, which was similar to the reduction rate observed for [123I]MIBG. Tissue distribution studies in mice showed the highest uptake in the adrenals (%ID/g = 27.2 +/- 5.0%) with relatively high uptake in the myocardium (%ID/g = 9.3 +/- 0.5%). The results suggest that this radiotracer holds promise as a useful adrenomedullary radiopharmaceutical for PET imaging.     

Glutamate causes a loss in human cerebral endothelial barrier integrity through activation of NMDA receptor

l-Glutamate is a major excitatory neurotransmitter that binds ionotropic and metabotropic glutamate receptors. Cerebral endothelial cells from many species have been shown to express several forms of glutamate receptors; however, human cerebral endothelial cells have not been shown to express either the N-methyl-D-aspartate (NMDA) receptor message or protein. This study provides evidence that human cerebral endothelial cells express the message and protein for NMDA receptors. Human cerebral endothelial cell monolayer electrical resistance changes in response to glutamate receptor agonists, antagonists, and second message blockers were tested. RT-PCR and Western blot analysis were used to demonstrate the presence of the NMDA receptor. Glutamate and NMDA (1 mM) caused a significant decrease in electrical resistance compared with sham control at 2 h postexposure; this response could be blocked significantly by MK-801 (an NMDA antagonist), 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethyoxybenzoate (an intracellular Ca2+ antagonist), and N-acetyl-L-cystein (an antioxidant). Trans(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid, a metabotropic receptor agonist (1 mM), did not significantly decrease electrical resistance. Our results are consistent with a model where glutamate, at excitotoxic levels, may lead to a breakdown in the blood brain barrier via activation of NMDA receptors.     

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow During Focal Cerebral Ischemia in Rats

This investigation was performed to evaluate the effects of ACPD [(1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O₂ consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10^–5 M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 ± 11 vs. 110 ± 11 ml/min/100 g, O₂ consumption: 2.9 ± 0.4 vs. 5.4 ± 1.1 ml O₂/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 ± 0.2 vs. 6.9 ± 0.3 ml O₂/100 ml) and oxygen consumption of the IC (4.3 ± 1.5 vs. 2.9 ± 0.4) compared to the control IC. Our data demonstrated that topical application of 10^–25 M ACPD to the ischemic area worsened cerebral O₂ balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.