N-Acetylcysteine amide (NACA) and diNACA inhibit H2O2-induced cataract formation ex vivo in pig and rat lenses.

Oxidative stress plays a central role in cataract formation suggesting that antioxidants might slow cataract progression. The anticataract activity of N-acetylcysteine amide (NACA) and (2 R, 2 R')-3,3'-disulfanediyl bis(2-acetamidopropanamide) (diNACA) and/or N-acetylcysteine (NAC), were evaluated in porcine and rat lens models. Cataractogenesis via oxidation was induced with H2O2 and/or glucose oxidase (GO). Porcine lenses were incubated in 0.1 mM, 1 mM, or 10 mM NAC, NACA or diNACA for 24 h. Lenses were then transferred to media containing 0.75 mM H2O2 and 4.63U of GO in order to maintain a constant H2O2 level for an additional 8 h. At the end of incubation, lenses were imaged under darkfield microscopy. Separately, rat lenses were extracted from 3-week-old Wistar rats and incubated with either 10 mM NACA or 10 mM diNACA for 24 h prior to treatment with 0.2U GO to generate a steady source of ∼0.6 mM H2O2. Rat lenses were analyzed by LC-MS/MS to quantify changes in cysteine, cystine, glutathione (GSH) or oxidised glutathione (GSSG) levels in the lens epithelium, cortex or core. Pre-treatment with NACA or diNACA followed by oxidation with H2O2 and/or GO to stimulate cataract formation afforded rapid assessment in ex vivo porcine (32 h) and rat (48 h) lens models. Pre-treatment of isolated porcine lenses with 0.1 mM, 1 mM or 10 mM of either NAC, NACA or diNACA followed by H2O2/GO treatment resulted in reduced lens opacity relative to the lenses exposed to H2O2/GO, with NACA and diNACA reducing opacities to a greater extent than NAC. Rat lenses incubated with 10 mM NACA or 10 mM diNACA without exposure to H2O2 showed no signs of opacities. Pre-treatment of rat lenses with 10 mM NACA or 10 mM diNACA, followed by GO cataract induction resulted in reduced opacities compared to control (GO alone). LC-MS/MS analyses revealed that NACA, but not diNACA, increased cysteine, cystine and GSH levels in rat lens epithelium and cortex regions. Taken together, both NACA and diNACA inhibited cataract formation to a greater extent than NAC (all at 1-10 mM) in an ex vivo porcine lens model. Both NACA and diNACA (both at 10 mM) reduced cataract formation in rat lenses. Based on LC-MS/MS analyses, NACA-induced reduction in opacity observed in rat lenses was attributed to enhanced cysteine and GSH levels while the diNACA-induced reduction in opacity induced did not consistently increase cysteine, cystine and GSH levels and, therefore, appears to involve a different antioxidant mechanism. These screening studies warrant further testing of NACA and diNACA as anticataract agents.

Automated annotation and visualisation of high-resolution spatial proteomic mass spectrometry imaging data using HIT-MAP.

Spatial proteomics has the potential to significantly advance our understanding of biology, physiology and medicine. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a powerful tool in the spatial proteomics field, enabling direct detection and registration of protein abundance and distribution across tissues. MALDI-MSI preserves spatial distribution and histology allowing unbiased analysis of complex, heterogeneous tissues. However, MALDI-MSI faces the challenge of simultaneous peptide quantification and identification. To overcome this, we develop and validate HIT-MAP (High-resolution Informatics Toolbox in MALDI-MSI Proteomics), an open-source bioinformatics workflow using peptide mass fingerprint analysis and a dual scoring system to computationally assign peptide and protein annotations to high mass resolution MSI datasets and generate customisable spatial distribution maps. HIT-MAP will be a valuable resource for the spatial proteomics community for analysing newly generated and retrospective datasets, enabling robust peptide and protein annotation and visualisation in a wide array of normal and disease contexts.

Determinants of hypoxic contracture in isolated heart muscle preparations.

Further studies on hypoxic contracture are described, using resting and contracting isolated muscle preparations from several species. The effect of temperature, pH, substrate, calcium concentration, osmolality and inotropic interventions was explored. The 'protective' effect of acidosis was not contingent on its negative inotropic influence. No evidence was adduced that hypoxic contracture can be modulated other than by altering energy supply or demand. The study does not discriminate between rigor and rised cytosolic calcium as mechanisms causing hypoxic contracture, but demonstrates that hypoxic contracture is not directly dependent on the availability of intracellular calcium.

MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3.

BACKGROUND: Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. RESULTS: MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. CONCLUSIONS: MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.

Effects of delta1-trans-tetrahydrocannabinol on mechanical performance of isolated heart muscle preparations.

1 The effects of four concentrations of delta1-trans-tetrahydrocannabinol (delta1-THC) (0.1, 1, 5 and 20 microgram/ml) were compared to that of the vehicle (ethanol, 0.5 microliter/ml) on the mechanical performance of isolated cardiac muscle of the cat and rat. 2 In rat isolated papillary muscles, delta1-THC (20 microgram/ml) caused a decline in mechanical performance (-8% in developed tension and -11% in Vmax) in contrast to the apparent lack of effect of ethanol. 3 All other parameters of mechanical performance studied in both rat and cat papillary muscles were unaffected by delta1-THC when compared with ethanol. 4 It was concluded that delta1-THC in concentrations up to 20 microgram/ml had negligible effect on contractile performance, the time course of contraction or muscle elasticity in rat and cat isolated papillary muscle preparations under the conditions studied.

Inotropic beta-blocking potency (pA2) and partial agonist activity of propranolol, practolol, sotalol and acebutolol.

The beta-blocking potencies (pA2 values) and cumulative dose responses of the direct effects of dl-propranolol, practolol, acebutolol and sotalol have been characterised in respect of the mechanical performance of isolated cat papillary muscle preparations. pA2 values calculated from antagonism of each of the drugs to the inotropic effect of isoprenaline at 29 degrees C were: dl-propranolol 8.3, practolol 5.6, acebutolol 7.5 and sotalol 4.8. The responses in reserpinized muscles were similar to those in non-reserpinized preparations. dl-Propranolol at concentrations of 10(-5) and 10(-4) M depressed peak developed force (DF) and rate of force development (+dF/dt) by 20% and 60% respectively, with similar but smaller changes in maximum unloaded shortening velocity ('Vmax'). Practolol and sotalol increased DF and +dF/dt by less than 10% at 10(-5) M and by 10-20% at 10(-4) M, with similar but smaller changes in Vmax. Sotalol induced prolonged biphasic contractions at 10(-4) M. Acebutolol had no effect on DF, +dF/dt, or Vmax. Time to peak force and half isometric relaxation time were not altered by any drug. Practolol and acebutolol, generally considered to have partial agonist activity, exerted this to a negligible degree in respect of inotropic effect at less than or equal to 10(-5) M.

Engelmann's disease: a 45-year follow-up.

We report a 45-year follow-up of a patient with Engelmann's disease previously described in 1950, showing progression of the disease with unique involvement of the femoral capital epiphyses. The case is compared with others to add some information about the later stages of a disease which is not fully understood.

Fluid-fluid level in an intraosseous ganglion.

A case of an intraosseous ganglion with a fluid-fluid level visualised on MR imaging is presented. We discuss its possible pathogenesis and review other lesions that may exhibit this sign.

Magnetic resonance appearance of normal inguinal nodes.

AIM: To identify adult inguinal lymph node anatomical subgroups using magnetic resonance imaging (MRI), to derive a normal range for nodal number and size and to describe their morphology. MATERIALS AND METHODS: Eighty-three oncology patients with low stage pelvic tumours had inguinal lymph node assessment by MRI. Nodes were divided into proximal superficial (PS), distal superficial (DS) and deep inguinal (DI) subgroups, their number counted in two planes, and their transaxial short axis diameter recorded. Consistency of the largest node was recorded for each anatomical subgroup and two vertical distances measured, between the skin surface and the ipsilateral pubis, and between the skin surface and the deepest node. RESULTS: Transaxial plane maximum nodal number at the three sites was: PS 5, DS 8, DI 3; and in the coronal plane: PS 7, DS 5, DI 3. Nodal size ranges were: PS 3-10 mm (mean 4 mm), DS 3-15 mm (mean 6 mm) and DI3-10 mm (mean 6 mm). There was no correlation between nodal size or number and age or gender. Nodes were usually uniformly solid (PS 44%; DS 37%, DI 45%), or fatty (PS 39%; DS 33%; DI 25%). The range of distances between the skin and deepest lymph node was 2.5-16 cm depending on patient fatness. CONCLUSION: The mean number of nodes counted in the axial plane was six and in the coronal plane five. A maximum short axis diameter of 15 mm was recorded for inguinal lymph nodes.

The 'penumbra sign' on T1-weighted MR imaging in subacute osteomyelitis: frequency, cause and significance.

OBJECTIVE: We studied the frequency and cause of a feature exhibited on T1-weighted (T1-W) magnetic resonance (MR) imaging termed the 'penumbra sign' in a series of patients presenting with osteomyelitis and correlated it with the double-line sign described as a T2-weighted (T2-W) or short tau inversion recovery (STIR) feature of both the Brodie's abscess and avascular necrosis. MATERIALS AND METHODS: The clinical, radiographic, MR imaging, microbiological and histological findings in 32 patients referred to an orthopaedic oncology service, but subsequently proven to have osteomyelitis, were reviewed. The presence or absence of a rim of tissue lining an abscess cavity typified by minor signal hyperintensity relative to the main abscess contents on T1-W MR imaging (the 'penumbra sign') was identified. The sign was correlated with the radiographic and other findings. RESULTS: The penumbra sign was identified in 24 cases (75%) and appears to be a more sensitive sign than the corresponding double-line sign which was evident in only 29% of these on T2-W or fast STIR images. The lesions were unilocular in 11 cases (46%) and multilocular in 13 (54%). The thickness of the penumbra ranged from 2 to 5mm. On histological examination the tissue comprising the penumbra sign was found to be highly vascularized granulation tissue containing thick walled arterioles. CONCLUSION: The penumbra sign is characteristically seen on T1-W MR images in subacute osteomyelitis and is due to a thick layer of highly vascularized granulation tissue which may not be visible as the double-line sign on T2-W or fast STIR sequences. This characteristic, but not pathognomonic, MR finding supports the diagnosis of bone infection and helps to exclude the presence of a tumour.