Microdetermination of unbound tryptophan in plasma by a combination of ultrafiltration and high-performance liquid chromatography

Previous methods for measuring unbound plasma tryptophan are not completely satisfactory, and therefore we have developed an improved method for this purpose. Unbound tryptophan is separated from bound tryptophan by centrifugation through Amicon ultrafiltration membrane cones in a short period (within 2 min). The first fraction of filtrate is obtained in 30 s by centrifugation at 3000g at controlled pH and is assayed with a high-performance liquid chromatography system. The first filtrate has a higher tryptophan concentration than that obtained by prolonged centrifugation. We propose that the tryptophan concentration in the first filtrate is the value nearest that of the plasma, since a change of equilibrium between bound and unbound tryptophan during the separation procedure is quite small. The method is also simple and convenient for clinical application.     

Tumor-Targeting Salmonella typhimurium A1-R Arrests a Chemo-Resistant Patient Soft-Tissue Sarcoma in Nude Mice

A patient-derived nude-mouse model of soft-tissue sarcoma has been established and treated in the following groups: (1) untreated controls; (2) gemcitabine (GEM) (80 mg/kg, ip, weekly, 3 weeks); (3) Pazopanib (100 mg/kg, orally, daily, 3 weeks) and (4) Salmonella typhimurium A1-R (5 × 10⁷ CFU/body, ip, weekly, 3 weeks). The sarcoma was resistant to GEM (p = 0.879). Pazopanib tended to reduce the tumor volume compared to the untreated mice, but there was no significant difference (p = 0.115). S. typhimurium A1-R significantly inhibited tumor growth compared to the untreated mice (p = 0.001). S. typhimurium A1-R was the only effective treatment for the soft-tissue sarcoma nude mouse model among all treatments including a newly approved multiple tyrosine kinase inhibitor; Pazopanib. These results suggest tumor-targeting S. typhimurium A1-R is a promising treatment for chemo-resistant soft-tissue sarcoma.     

Catalytic Intermediates of Inducible Nitric-oxide Synthase Stabilized by the W188H Mutation

Nitric-oxide synthase (NOS) catalyzes nitric oxide (NO) synthesis via a two-step process: l-arginine (l-Arg) →N-hydroxy-l-arginine →citrulline + NO. In the active site the heme is coordinated by a thiolate ligand, which accepts a H-bond from a nearby tryptophan residue, Trp-188. Mutation of Trp-188 to histidine in murine inducible NOS was shown to retard NO synthesis and allow for transient accumulation of a new intermediate with a Soret maximum at 420 nm during the l-Arg hydroxylation reaction (Tejero, J., Biswas, A., Wang, Z. Q., Page, R. C., Haque, M. M., Hemann, C., Zweier, J. L., Misra, S., and Stuehr, D. J. (2008) J. Biol. Chem. 283, 33498–33507). However, crystallographic data showed that the mutation did not perturb the overall structure of the enzyme. To understand how the proximal mutation affects the oxygen chemistry, we carried out biophysical studies of the W188H mutant. Our stopped-flow data showed that the 420-nm intermediate was not only populated during the l-Arg reaction but also during the N-hydroxy-l-arginine reaction. Spectroscopic data and structural analysis demonstrated that the 420-nm intermediate is a hydroxide-bound ferric heme species that is stabilized by an out-of-plane distortion of the heme macrocycle and a cation radical centered on the tetrahydrobiopterin cofactor. The current data add important new insights into the previously proposed catalytic mechanism of NOS (Li, D., Kabir, M., Stuehr, D. J., Rousseau, D. L., and Yeh, S. R. (2007) J. Am. Chem. Soc. 129, 6943–6951).Nitric-oxide synthase (NOS) is a heme-containing flavoenzyme that synthesizes nitric oxide (NO) from l-arginine (l-Arg) in a two-step process (Scheme 1). In the first step of the reaction, one molecule of O₂ and two electrons from NADPH are consumed for the conversion of l-Arg to N-hydroxy-l-arginine (NOHA).² In the second step of the reaction, another molecule of O₂ and an additional electron from NADPH are used to convert NOHA to l-citrulline and NO. Previous studies suggest that the two steps of the reaction follow distinct mechanisms meditated by a compound I (Cmpd I) type of ferryl intermediate and a peroxyl intermediate, respectively (1–7). These mechanisms, however, remain elusive, as none of the putative intermediates have been experimentally observed under solution conditions, although (hydro)peroxo intermediates have been identified at cryogenic temperatures by radiolytic reduction methods (8, 9); in addition, a Cmpd I intermediate has been observed after peroxyacid treatment (10).Three isoforms of NOS have been identified in mammals: neuronal NOS, endothelial NOS, and inducible NOS (iNOS). Similar to the P450 class of enzymes, the heme prosthetic group in all three isoforms of NOS is coordinated by a thiolate sidechain group of an intrinsic cysteine residue in the proximal heme pocket. In P450s, the thiolate ligand forms a H-bond with a peptide NH group (11), whereas in NOSs the analogous thiolate ligand accepts a H-bond from the side chain of a conserved tryptophan residue (Trp-188 in iNOS). It is believed that the H-bonding interaction with the tryptophan residue reduces the electron donating capability of the thiolate ligand in NOSs, thereby modulating the oxygen chemistry occurring in the distal heme pocket of the enzymes (1, 12–15). The mutation of the conserved tryptophan (Trp-409) in neuronal NOS to Phe or Tyr was shown to increase the rate of NO synthesis during multiple turnover conditions by decreasing the heme reduction rate and the degree of NO autoinhibition (15, 16). Comparable mutants of iNOS, W188F, and W188Y, could not be overexpressed as stable recombinant forms (17); however, the W188H mutant was successfully expressed, purified, and studied (18).It was shown that the W188H mutation slowed down the l-Arg hydroxylation reaction by stabilizing a new intermediate with a Soret maximum at 420 nm, which had never been observed during the wild type reaction, and that the formation of the 420-nm intermediate coincides with the disappearance of the ternary complex of the enzyme and the formation of a H₄B radical, whereas its decay was concurrent with the recovery of the resting ferric enzyme. Tejero et al. (18) postulated that the 420-nm species is a catalytically competent oxygen-containing intermediate, such as a Cmpd I type of ferryl species. Regardless of the identity of the intermediate, the data demonstrated that the mutation modulates the structural properties and biochemical reactivity of the enzyme. However, the crystallographic data of the W188H mutant of the oxygenase domain of iNOS (iNOS_oxy) revealed that its active site structure is strikingly similar to that of the wild type enzyme (18). In particular, the side chain of His-188, like that of Trp-188 in the wild type enzyme, formed a H-bond with the thiolate ligand of the heme.Open in a separate windowTo determine how the W188H mutation modulates the oxygen chemistry of iNOS_oxy without significantly perturbing the active site structure of the enzyme, we carried out a series of studies of the W188H mutant with optical absorption, resonance Raman, and EPR spectroscopic methods under steady-state and single turnover conditions. We discovered that the mutation introduced a unique out-of-plane distortion to the heme macrocycle that stabilizes the 420-nm intermediate populated during both the l-Arg and NOHA reactions and at the same time destabilizes the NO bound to the ferric heme during the NOHA reaction. The results are summarized and discussed in the context of the previously postulated NOS mechanism (1).     

Gene expression in the vascular wall of the aortic arch in spontaneously hypertensive hyperlipidemic model rats using DNA microarray analysis

Aims

In recent years, there has been an increase in patients with arteriosclerosis and the risk of lifestyle-related diseases. However, the pathogenesis and medication of atherosclerosis have not been elucidated. We developed a rat model of lifestyle-related diseases by feeding a high-fat diet and 30% sucrose solution (HFDS) to spontaneously hypertensive hyperlipidemic rats (SHHR) and reported that this model is a useful model of early atherosclerosis. In order to elucidate the pathogenesis of early atherosclerosis, we searched for atherosclerosis-related genes by microarray analysis using the aortic arch rat model of lifestyle-related diseases.

Main methods

Four-month-old male Sprague–Dawley rats and SHHR were each divided into two normal diet (ND) groups and two HFDS groups. After a four-month treatment, the expression of mRNA in the aortic arch was detected using the oligo DNA microarray one-color method and quantified using real-time PCR.

Key findings

In this study, we detected 39 genes in microarray analysis. Esm1, Retnlb Mkks, and Grem2 showed particularly marked changes in gene expression in the SHHR-HFDS group. Compared with the SD-ND group, the SHHR-HFDS group had an increase in Mkks gene expression of about 26-fold and an approximately 22-fold increase in the expression of Grem2. Similarly, the expression of Esm1 increased by about 12-fold and that of Retnlg by about 10-fold as shown by quantitative real-time PCR.

Significance

This study suggested that these four genes might be important in early atherosclerosis development.     

Optogenetic Probing and Manipulation of the Calyx-Type Presynaptic Terminal in the Embryonic Chick Ciliary Ganglion

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca²⁺ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8–14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca²⁺-sensor, the Ca²⁺ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca²⁺ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca²⁺. It is suggested that the photo-activation of ChRFR facilitated the release of Ca²⁺ from intracellular Ca²⁺ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.     

Collagenase in Stomach of Crown-of-thorns Starfish Acanthaster planci

Acetobacter pasteurianus strains IFO3283, SKU1108, and MSU10 were grown under acetic acid fermentation conditions, and their growth behavior was examined together with their capacity for acetic acid resistance and pellicle formation. In the fermentation process, the cells became aggregated and covered by amorphous materials in the late-log and stationary phases, but dispersed again in the second growth phase (due to overoxidation). The morphological change in the cells was accompanied by changes in sugar contents, which might be related to pellicle polysaccharide formation. To determine the relationship between pellicle formation and acetic acid resistance, a pellicle-forming R strain and a non-forming S strain were isolated, and their fermentation ability and acetic acid diffusion activity were compared. The results suggest that pellicle formation is directly related to acetic acid resistance ability, and thus is important to acetic acid fermentation in these A. pasteurianus strains.     

Conjugation to octa-arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.     

A 3-Mbp fragment on rat chromosome 1 affects susceptibility both to stroke and kidney injury under salt loading in the stroke-prone spontaneously hypertensive rat: a genetic approach using multiple congenic strains

We have previously reported that a major quantitative trait locus (QTL) responsible for susceptibility to salt-induced stroke in the stroke-prone spontaneously hypertensive rat (SHRSP) is located in a 3-Mbp region on chromosome 1 covered by SHRSP.SHR-(D1Rat23-D1Rat213)/Izm (termed Pr1.31), a congenic strain with segments from SHRSP/Izm introduced into the stroke-resistant SHR/Izm. Here, we attempted to narrow down the candidate region on chromosome 1 further through analyses of subcongenic strains constructed for the target region. Simultaneously, salt-induced kidney injury was evaluated through the measurement of urinary albumin and the gene expression of renal tubular injury markers (Kim-1 and Clu) to explore a possible mechanism leading to the onset of stroke. All subcongenic strains examined in this study showed lower susceptibility to salt-induced stroke than SHRSP. Interestingly, Pr1.31 had the lowest stroke susceptibility when compared with newly constructed subcongenic strains harboring fragments of the congenic sequence in Pr1.31. Although Kim-1 and Clu expression after 1 week of salt loading in Pr1.31 did not differ significantly from those in SHRSP, the urinary albumin level of Pr1.31 was significantly lower than those of the other subcongenic strains and that of SHRSP. The present results indicated that, although the congenic fragment in Pr1.31 harbored the gene(s) related to salt-induced organ damages, further genetic dissection of the candidate region was difficult due to multiple QTLs suggested in this region. Further analysis using Pr1.31 will unveil genetic and pathophysiological mechanisms underlying salt-induced end organ damages in SHRSP.