[Validation of telemedical fundus images from patients with retinopathy]. / Validierung von telemedizinischen Fundusaufnahmen bei Patienten mit Retinopathie.

BACKGROUND: The degree of retinal microangiopathy is important foran estimation of the vascular risk and for the optimisation of therapy in hypertensive and diabetic patients. Retinal microangiopathy may be determined by examination of the retinal fundus. We examined the reliability of the ophthalmoscopic diagnosis and of the telemedical judgement of fundus images in relation to the presence and the degree of retinopathy. PATIENTS AND METHODS: This comparative observational study included 47 inpatient hypertensive and/or diabetic subjects. The fundus was judged ophthalmoscopically and subsequently, a fundus image was generated by use of a KOWA camera. The images were sent to the Interdisciplinary Center of Ophthalmologic Preventive Medicine. The reliability of the two diagnostic methods was calculated for one of the two eyes, which was selected by a random generator. RESULTS: The largest concordance of the two diagnosis methods was achieved, in descending order, for retinal bleeding, stage of diabetic retinopathy and the papilla findings. Additionally, there were no differences for the stage of hypertensive retinopathy und tortuosity. The reliability of arterio-venous nicking related to the right and the left eye was low or absent, respectively. CONCLUSIONS: The teleophthalmologic diagnosis achieves good results as compared to the ophthalmoscopic judgement in relation to retinopathy assessment criteria.

Selective modulation of endogenous nitric oxide formation in ischemia/reperfusion injury in isolated rat hearts--effects on regional myocardial flow and enzyme release.

The role of nitric oxide (NO) in ischemia/reperfusion injury is controversial. We tested the role of inducible NOS (iNOS) in the ischemia/reperfusion injury in isolated rat hearts using the selective iNOS inhibitor S-methylisothiourea sulfate (SMT) and the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). After 15 min of stabilization in Langendorff mode, hearts were perfused either with normal Krebs-Henseleit buffer, buffer containing 100 microM L-NAME, 0.5 microM SMT or 50 microM SMT for 5 min and were subjected to 25 min of ischemia followed by 30 min of reperfusion. Left ventricular developed pressure (LVDP) and total coronary flow (CF) were recorded continuously. After ischemia/reperfusion, a marked expression of iNOS protein was demonstrated by Western blotting, while virtually no iNOS protein was present in hearts without ischemia/reperfusion. Regional myocardial blood flow (RMBF) was measured with colored microspheres. Coronary vasoactive concentration of L-NAME and SMT depressed myocardial function as shown by decreased LVDP, dP/dt(max) and coronary.ow before ischemia. After ischemia the recovery of the total CF was impaired in L-NAME and 50 microM SMT pretreated hearts which was related to homogenous RMBF decrease in the right and left ventricle compared to that in control group. Low concentration SMT (0.5 microM) showed no coronary vasoactive effects before ischemia and attenuated ischemia/reperfusion injury indicated by lower ischemic contracture at 25 min of ischemia and reduced CK and LDH release during reperfusion. Thus, NOS inhibition did not affect blood flow distribution in rat hearts either in the pre-ischemic or reperfusion period. Selective iNOS inhibition reduced ischemic injury by reducing ischemic contracture and CK as well as LDH release during reperfusion.

A comparison of liquid hot water and steam pretreatments of sugar cane bagasse for bioconversion to ethanol.

Sugar cane bagasse was pretreated with either liquid hot water (LHW) or steam using the same 25 l reactor. Solids concentration ranged from 1% to 8% for LHW pretreatment and was > or = 50% for steam pretreatment. Reaction temperature and time ranged from 170 to 230 degrees C and 1 to 46 min, respectively. Key performance metrics included fiber reactivity, xylan recovery, and the extent to which pretreatment hydrolyzate inhibited glucose fermentation. In four cases, LHW pretreatment achieved > or = 80% conversion by simultaneous saccharification and fermentation (SSF). > or = 80% xylan recovery, and no hydrolyzate inhibition of glucose fermentation yield. Combined effectiveness was not as good for steam pretreatment due to low xylan recovery. SSF conversion increased and xylan recovery decreased as xylan dissolution increased for both modes. SSF conversion, xylan dissolution. hydrolyzate furfural concentration, and hydrolyzate inhibition increased, while xylan recovery and hydrolyzate pH decreased, as a function of increasing LHW pretreatment solids concentration (1-8%). These results are consistent with the notion that autohydrolysis plays an important. if not exclusive, role in batch hydrothermal pretreatment. Achieving concurrently high (greater than 90%) SSF conversion and xylan recovery will likely require a modified reactor configuration (e.g. continuous percolation or base addition) that better preserves dissolved xylan.

Integrin activation and focal complex formation in cardiac hypertrophy.

Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.

Identification of RPE65 in transformed kidney cells.

The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS-1 and COS-7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS-7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full-length encoding region was amplified from HEK293 and COS-7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS-7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.

Beta3-integrin-mediated focal adhesion complex formation: adult cardiocytes embedded in three-dimensional polymer matrices.

In vivo studies show that beta3-integrin-mediated focal adhesion formation (FAF) causes recruitment of nonreceptor tyrosine kinases to the cytoskeleton in pressure-overloaded myocardium. To define the mechanism of beta3-integrin-mediated signaling, we developed a cell culture model (adult feline cardiocytes embedded in a 3-dimensional matrix of native type 1 collagen, fibronectin, and vitronectin) wherein beta3-integrin-mediated focal adhesion kinase occurs. Focal adhesion kinase was analyzed immunocytochemically using confocal microscopy. Initial studies suggested that cardiocytes cultured in a 3-dimensional matrix formed focal adhesions consisting of both beta3-integrin and the muscle-specific isoform, beta1-integrin (beta1D). The focal adhesions were associated with focal adhesion kinase on both costameres and intercalated disks. To determine the cause of beta1D-integrin-mediated focal adhesion kinase in this model, time course studies were done. Beta3-integrin-mediated focal adhesion kinase occurred within 30 minutes after embedding cardiocytes and persisted for >24 hours, whereas beta1D-integrin-mediated focal adhesion kinase was present from the outset. Because confocal microscopy showed that laminin was present on the surface of freshly isolated cardiocytes, we hypothesized that this was causative of beta1D-integrin-mediated focal adhesion kinase. Freshly isolated cardiocytes washed with acidic medium (2 minutes, pH 3.0) to remove laminin and then embedded in a 3-dimensional matrix showed complete absence of beta1D-integrin-mediated focal adhesion kinase, but beta3-integrin-mediated focal adhesion kinase occurred with a time course similar to that seen in cultured, unwashed cardiocytes. Acid washing did not alter the binding ability of beta1D-integrin, because acid-washed cardiocytes in the presence of laminin showed beta1D-integrin-mediated focal adhesion kinase. Thus, cardiocytes embedded in a 3-dimensional matrix show beta3-integrin-mediated focal adhesion kinase and provide an in vitro model to study beta3-integrin-mediated signaling in response to hemodynamic cardiac loading.

Differential activation of p70 and p85 S6 kinase isoforms during cardiac hypertrophy in the adult mammal.

An adult feline right ventricular pressure overload (RVPO) model was used to examine the two S6 kinase (S6K) isoforms, p70(S6K) and p85(S6K), that are involved in translational and transcriptional activation. Biochemical and confocal microscopy analyses at the level of the cardiocyte revealed that p70(S6K) is present predominantly in the cytosol, substantially activated in 1-h RVPO (>12 fold), and phosphorylated in the pseudosubstrate domain at the Ser-411, Thr-421, and Ser-424 sites. p85(S6K), which was localized exclusively in the nucleus, showed activation subsequent to p70(S6K), with a sustained increase in phosphorylation for up to 48 h of RVPO at equivalent sites of p70(S6K), Thr-421 and Ser-424, but not at Ser-411. Neither isoform translocated between the cytosol and the nucleus. Further studies to determine potential upstream elements of S6K activation revealed: (i) similar time course of activation for protein kinase C isoforms (alpha, gamma, and epsilon) and c-Raf, (ii) absence of accompanying phosphatidylinositol 3-kinase activation, (iii) activation of c-Src subsequent to p70(S6K), and (iv) similar changes in adult cardiocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate. Thus, these studies suggest that a protein kinase C-mediated pathway couples pressure overload to growth induction via differential activation of S6K isoforms in cardiac hypertrophy.

Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction.

The purpose of this study was to test the hypothesis that energy metabolism is impaired in residual intact myocardium of chronically infarcted rat heart, contributing to contractile dysfunction. Myocardial infarction (MI) was induced in rats by coronary artery ligation. Hearts were isolated 8 wk later and buffer-perfused isovolumically. MI hearts showed reduced left ventricular developed pressure, but oxygen consumption was unchanged. High-energy phosphate contents were measured chemically and by 31P-NMR spectroscopy. In residual intact left ventricular tissue, ATP was unchanged after MI, while creatine phosphate was reduced by 31%. Total creatine kinase (CK) activity was reduced by 17%, the fetal CK isoenzymes BB and MB increased, while the "adult" mitochondrial CK isoenzyme activity decreased by 44%. Total creatine content decreased by 35%. Phosphoryl exchange between ATP and creatine phosphate, measured by 31P-NMR magnetization transfer, fell by 50% in MI hearts. Thus, energy reserve is substantially impaired in residual intact myocardium of chronically infarcted rats. Because phosphoryl exchange was still five times higher than ATP synthesis rates calculated from oxygen consumption, phosphoryl transfer via CK may not limit baseline contractile performance 2 mo after MI. In contrast, when MI hearts were subjected to acute stress (hypoxia), mechanical recovery during reoxygenation was impaired, suggesting that reduced energy reserve contributes to increased susceptibility of MI hearts to acute metabolic stress.

Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents.

We have studied the spontaneous and nerve-evoked synaptic currents during the initial period of nerve-muscle contact in Xenopus cell cultures. The precise timing of the contact was achieved by physically manipulating embryonic muscle cells into contact with co-cultured spinal neurons. Previous studies have shown that physical contact of the muscle membrane induces pulsatile release of acetylcholine (ACh) from the growth cone of these neurons, resulting in spontaneous synaptic currents (SSCs) in the muscle cell within seconds following the contact. In the present work, we first showed that these SSCs at the manipulated nerve-muscle contacts are similar to those observed at naturally occurring synapses. We then examined the possible cellular mechanisms responsible for the marked variation in SSC amplitude and showed that it most likely results from differences in either the amount of ACh contained in each release event or the extent of close membrane apposition near the release sites. During the first 20 min following the nerve-muscle contact, there was an increase in the frequency and mean amplitude of the SSCs. During a similar period, the evoked synaptic currents (ESCs), which were induced by suprathreshold electrical stimulation of the neuronal soma, also showed an increase in the mean amplitude and a reduction in the delay of onset following the stimulus. These postcontact changes in the efficacy of synaptic transmission may be related to an increase in the total area of close membrane apposition between the nerve and muscle cells. This was suggested by the finding that neurite-muscle adhesion increases over a similar postcontact period. The transition from low- to high-efficacy transmission during the early phase of contact may reflect the process of selective adhesion between the cells, and thus signify the formation of specific synapse. Analysis of the fluctuation in the ESC amplitude at the early nerve-muscle contact suggests that evoked release of ACh occurs as multiples of a quantal unit. However, this unit is apparently related to only a small subpopulation of SSCs of relatively high amplitudes.(ABSTRACT TRUNCATED AT 400 WORDS)

Divorced parents in family therapy in a residential treatment setting.

Divorced parents are required to participate together in the family therapy of their child placed in a residential treatment center. Different sources of resistance and treatment techniques are identified and discussed through a theoretical analysis and case study material. The therapy of these fractured families contributed to an elimination of recidivism and, according to followup reports, to significant and sustained improvement in the children's functioning in school, home, and community activities.