The inactivation of bovine cathepsin B by novel N-chloro-acetyl-dipeptides. Application of the Houghten 'tea bag' methodology to inhibitor synthesis

In this study, a series of N-chloro-acetylated dipeptides were synthesised by the application of Houghten's methodology of multiple analog peptide syntheses. The peptides, all of which contain a C-terminal free acid, were tested as inactivators of bovine cathepsin B, in an attempt at exploiting the known and, amongst the cysteine proteinases, unique carboxy dipeptidyl peptidase activity of the protease. We have succeeded in obtaining a number of effective inactivators, the most potent of which-chloroacetyl-Leu-Leu-OH, inactivates the enzyme with an apparent second-order rate constant of 3.8 x 10(4) M-1 min(-1). In contrast, the esterified analog, chloroacetyl-Leu-Leu-OMe, inactivates the enzyme some three orders of magnitude less efficiently, lending credence to our thesis that a free carboxylic acid moiety is an important determinant for inhibitor effectiveness. This preliminary study has highlighted a number of interesting features about the specificity requirements of the bovine proteinase and we believe that our approach has great potential for the rapid delineation of the subsite specificities of cathepsin B-like proteases from various species. (c) 2005 Elsevier Inc. All rights reserved.

An orally available compound prevents deficits in memory caused by the Alzheimer amyloid-B oligomers.

Despite progress in defining a pathogenic role for amyloid beta protein (Abeta) in Alzheimer's disease, orally bioavailable compounds that prevent its effects on hippocampal synaptic plasticity and cognitive function have not yet emerged. A particularly attractive therapeutic strategy is to selectively neutralize small, soluble Abeta oligomers that have recently been shown to mediate synaptic dysfunction. METHODS: Using electrophysiological, biochemical, and behavioral assays, we studied how scyllo-inositol (AZD-103; molecular weight, 180) neutralizes the acutely toxic effects of Abeta on synaptic function and memory recall. RESULTS: Scyllo-inositol, but not its stereoisomer, chiro-inositol, dose-dependently rescued long-term potentiation in mouse hippocampus from the inhibitory effects of soluble oligomers of cell-derived human Abeta. Cerebroventricular injection into rats of the soluble Abeta oligomers interfered with learned performance on a complex lever-pressing task, but administration of scyllo-inositol via the drinking water fully prevented oligomer-induced errors. INTERPRETATION: A small, orally available natural product penetrates into the brain in vivo to rescue the memory impairment produced by soluble Abeta oligomers through a mechanism that restores hippocampal synaptic plasticity.

B-type supergiants in the SMC: Chemical compositions and comparison of static and unified models

High-resolution UCLES/AAT spectra are presented for nine B-type supergiants in the SMC, chosen on the basis that they may show varying amounts of nuclear-synthetically processed material mixed to their surface. These spectra have been analysed using a new grid of approximately 12 000 non-LTE line blanketed tlusty model atmospheres to estimate atmospheric parameters and chemical composition. The abundance estimates for O, Mg and Si are in excellent agreement with those deduced from other studies, whilst the low estimate for C may reflect the use of the C II doublet at 4267 Å. The N estimates are approximately an order of magnitude greater than those found in unevolved B-type stars or H II regions but are consistent with the other estimates in AB-type supergiants. These results have been combined with results from a unified model atmosphere analysis of UVES/VLT spectra of B-type supergiants (Trundle et al. 2004, A&A, 417, 217) to discuss the evolutionary status of these objects. For two stars that are in common with those discussed by Trundle et al., we have undertaken a careful comparison in order to try to understand the relative importance of the different uncertainties present in such analyses, including observational errors and the use of static or unified models. We find that even for these relatively luminous supergiants, tlusty models yield atmospheric parameters and chemical compositions similar to those deduced from the unified code fastwind.

The present-day chemical composition of the SMC from UVES spectra of the sharp-lined, B-type dwarf AV 304

High-resolution spectroscopic VLT/UVES observations are presented for the B-type main-sequence star, AV 304, in the Small Magellanic Cloud (SMC). These spectra have been analysed using LTE model-atmosphere techniques, to derive stellar atmospheric parameters and chemical compositions. As AV 304 is located within the hydrogen burning main-sequence band, its chemical composition should reflect that of the SMC interstellar medium (ISM). A detailed line-by-line differential analysis has been undertaken relative to a Galactic comparison star. A general metal deficiency for the a-process elements O, Si & S of -0.43 +/- 0.05 dex is found for AV 304, with iron having a similar underabundance. Oxygen may be relatively over- abundant by similar to0.1 dex and carbon and aluminium underabundant by similar to0.2 dex. A large nitrogen underabundance (of -1.2 dex relative to hydrogen and -0.7 dex relative to iron) is found. This is interpreted in terms of the CNO bi-cycle having been suppressed in the SMC. Furthermore, the large nitrogen deficiency is in excellent agreement with that found for SMC H II regions. Indeed, this represents a first for stellar astrophysics - confirming the low base-line nitrogen composition of the SMC ISM (viz. 12+log(N/H) similar to 6.66 +/- 0.10 dex).

Chemical composition of B-type supergiants in the OB 8, OB 10, OB 48, OB 78 associations of M 31

Absolute and differential chemical abundances are presented for the largest group of massive stars in M31 studied to date. These results were derived from intermediate resolution spectra of seven B-type supergiants, lying within four OB associations covering a galactocentric distance of 5-12 kpc. The results are mainly based on an LTE analysis, and we additionally present a full non-LTE, unified model atmosphere analysis of one star (OB 78-277) to demonstrate the reliability of the differential LTE technique. A comparison of the stellar oxygen abundance with that of previous nebular results shows that there is an off set of between similar to0.15-0.4 dex between the two methods which is critically dependent on the empirical calibration adopted for the R 23 parameter with [O/H]. However within the typical errors of the stellar and nebular analyses (and given the strength of dependence of the nebular results on the calibration used) the oxygen abundances determined in each method are fairly consistent. We determine the radial oxygen abundance gradient from these stars, and do not detect any systematic gradient across this galactocentric range. We find that the inner regions of M31 are not, as previously thought, very "metal rich". Our abundances of C, N, O, Mg, Si, Al, S and Fe in the M31 supergiants are very similar to those of massive stars in the solar neighbourhood.

B-type supergiants in the Small Magellanic Cloud: rotational velocities and implications for evolutionary models

High-resolution spectra for 24 SMC and Galactic B-type supergiants have been analysed to estimate the contributions of both macroturbulence and rotation to the broadening of their metal lines. Two different methodologies are considered, viz. goodness-of-fit comparisons between observed and theoretical line profiles and identifying zeros in the Fourier transforms of the observed profiles. The advantages and limitations of the two methods are briefly discussed with the latter techniques being adopted for estimating projected rotational velocities ( v sin i) but the former being used to estimate macroturbulent velocities. The projected rotational velocity estimates range from approximately 20 to 60 kms(-1), apart from one SMC supergiant, Sk 191, with a v sin i similar or equal to 90 km s(-1). Apart from Sk 191, the distribution of projected rotational velocities as a function of spectral type are similar in both our Galactic and SMC samples with larger values being found at earlier spectral types. There is marginal evidence for the projected rotational velocities in the SMC being higher than those in the Galactic targets but any differences are only of the order of 5 - 10 km s(-1), whilst evolutionary models predict differences in this effective temperature range of typically 20 to 70 km s(-1). The combined sample is consistent with a linear variation of projected rotational velocity with effective temperature, which would imply rotational velocities for supergiants of 70 kms(-1) at an effective temperature of 28 000 K ( approximately B0 spectral type) decreasing to 32 km s(-1) at 12 000 K (B8 spectral type). For all targets, the macroturbulent broadening would appear to be consistent with a Gaussian distribution ( although other distributions cannot be discounted) with an 1/e half-width varying from approximately 20 km s(-1) at B8 to 60 km s(-1) at B0 spectral types.

The use of Raman microscopy to determine and localize vitamin E in biological samples

Alpha-tocopherol (aT), the predominant form of vitamin E in mammals, is thought to prevent oxidation of polyunsaturated fatty acids. In the lung, aT is perceived to be accumulated in alveolar type II cells and secreted together with surfactant into the epithelial lining fluid. Conventionally, determination of aT and related compounds requires extraction with organic solvents. This study describes a new method to determine and image the distribution of aT and related compounds within cells and tissue sections using the light-scattering technique of Raman microscopy to enable high spatial as well as spectral resolution. This study compared the nondestructive analysis by Raman microscopy of vitamin E, in particular aT, in biological samples with data obtained using conventional HPLC analysis. Raman spectra were acquired at spatial resolutions of 2-0.8 microm. Multivariate analysis techniques were used for analyses and construction of corresponding maps showing the distribution of aT, alpha-tocopherol quinone (aTQ), and other constituents (hemes, proteins, DNA, and surfactant lipids). A combination of images enabled identification of colocalized constituents (heme/aTQ and aT/surfactant lipids). Our data demonstrate the ability of Raman microscopy to discriminate between different tocopherols and oxidation products in biological specimens without sample destruction. By enabling the visualization of lipid-protein interactions, Raman microscopy offers a novel method of investigating biological characterization of lipid-soluble compounds, including those that may be embedded in biological membranes such as aT.

Homocysteine and B-group vitamins in renal transplant patients

Increased plasma homocysteine is an independent risk factor for cardiovascular disease. We have investigated homocysteine and B-group vitamin levels in renal transplant patients. Fasting blood was collected from 55 renal transplant recipients with good renal function and 32 age/sex matched control subjects. Total homocysteine was increased in transplant recipients in comparison to controls (10.9+/-1.5 vs. 6.7+/-1.3 micromol/l, P < 0.001). There was no difference in homocysteine between patients receiving cyclosporin (n = 39, homocysteine 11.0+/-1.5 micromol/l) and patients receiving prednisolone + azathioprine (n = 16, 10.8+/-1.6 micromol/l, mean+/-S.D.), although there was a significant correlation between homocysteine and serum cyclosporin concentration in the sub-group of patients receiving that immunosuppressive regimen (r = 0.42, P < 0.05). Levels of B-group vitamins were similar in patients and controls. Plasma homocysteine is increased in renal transplant recipients even in the presence of minor degrees of renal impairment and normal levels of B-group vitamins.

Cyclin G associated kinase interacts with interleukin 12 receptor beta2 and suppresses interleukin 12 induced IFN-gamma production.

BACKGROUND: Although severe encephalopathy has been proposed as a possible contraindication to the use of noninvasive positive-pressure ventilation (NPPV), increasing clinical reports showed it was effective in patients with impaired consciousness and even coma secondary to acute respiratory failure, especially hypercapnic acute respiratory failure (HARF). To further evaluate the effectiveness and safety of NPPV for severe hypercapnic encephalopathy, a prospective case-control study was conducted at a university respiratory intensive care unit (RICU) in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) during the past 3 years. METHODS: Forty-three of 68 consecutive AECOPD patients requiring ventilatory support for HARF were divided into 2 groups, which were carefully matched for age, sex, COPD course, tobacco use and previous hospitalization history, according to the severity of encephalopathy, 22 patients with Glasgow coma scale (GCS) <10 served as group A and 21 with GCS = 10 as group B. RESULTS: Compared with group B, group A had a higher level of baseline arterial partial CO2 pressure ((102 +/- 27) mmHg vs (74 +/- 17) mmHg, P <0.01), lower levels of GCS (7.5 +/- 1.9 vs 12.2 +/- 1.8, P <0.01), arterial pH value (7.18 +/- 0.06 vs 7.28 +/- 0.07, P <0.01) and partial O(2) pressure/fraction of inspired O(2) ratio (168 +/- 39 vs 189 +/- 33, P <0.05). The NPPV success rate and hospital mortality were 73% (16/22) and 14% (3/22) respectively in group A, which were comparable to those in group B (68% (15/21) and 14% (3/21) respectively, all P > 0.05), but group A needed an average of 7 cm H2O higher of maximal pressure support during NPPV, and 4, 4 and 7 days longer of NPPV time, RICU stay and hospital stay respectively than group B (P <0.05 or P <0.01). NPPV therapy failed in 12 patients (6 in each group) because of excessive airway secretions (7 patients), hemodynamic instability (2), worsening of dyspnea and deterioration of gas exchange (2), and gastric content aspiration (1). CONCLUSIONS: Selected patients with severe hypercapnic encephalopathy secondary to HARF can be treated as effectively and safely with NPPV as awake patients with HARF due to AECOPD; a trial of NPPV should be instituted to reduce the need of endotracheal intubation in patients with severe hypercapnic encephalopathy who are otherwise good candidates for NPPV due to AECOPD.

Severe Vitamin E deficiency modulates airway allergic inflammatory responses in the murine asthma model

Allergic asthma is a complex immunologically mediated disease associated with increased oxidative stress and altered antioxidant defenses. It was hypothesized that a-tocopherol (a-T) decreases oxidative stress and therefore its absence may influence allergic inflammatory process, a pathobiology known to be accompanied by oxidative stress. Therefore, selected parameters of allergic asthma sensitization and inflammation were evaluated following ovalbumin sensitization and re-challenge of a-T transfer protein (TTP) knock-out mice (TTP-/-) that have greatly reduced lung a-T levels (e.g.