Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats

The present report shows that System A-mediated 2-aminoisobutyric acid (AIB) uptake is elevated in hepatocytes isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, beta, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.     

Does AMP participate in photosynthetic phosphorylation?

Osmotically disrupted chloroplasts catalyze a rapid, light and AMP and ATP dependent ³²P_i incorporation into ATP. Light does not stimulate [¹⁴C] AMP incorporation into ATP in this system. AMP in the presence of P_i inhibits electron flow in a manner analogous to ADP inhibition in the absence of P_i. The inhibition of AMP + P_i is reversed on addition of ADP.     

The dissections of craniocervical arteries

Dissection of craniocervical arteries internal carotid artery (ICA), or vertebral artery (VA) is an increasingly recognized entity and infrequent cause of stroke. We investigated 8 patients (4 women and 4 men) with dissections of the craniocervical arteries. Diagnostic procedures for detection of craniocervical dissection included: extracranial ultrasound-color Doppler flow imaging (CDFI) of carotid and vertebral arteries, transcranial Doppler sonography (TCD) and radiological computed tomography (CT) and digital subtractive angiography (DSA) examinations. Ultrasound findings (CDFI of carotid and vertebral arteries) were positive for vessel dissection in seven patients (or 87.5 per cent) and negative in one patient. DSA was consistent with dissection in five patients (or 62.5 per cent), negative in one, while in two patients the examination was not performed due to known allergy to contrast media. Five patients (62.5 per cent) were treated with anticoagulants, one with suppressors of platelet aggregation, and two patients were operated. Six patients (75 per cent) after the treatment showed partial recovery of neurological defects, and an improvement of ultrasound finding of dissected arteries. In one patient, following operation, stroke developed with deterioration of motor deficit, and one patient was readmitted three months later due to a newly developed stroke and soon died. The diagnosis should be suspected in any young or middle-age patient with new onset of otherwise unexplained unremitting headache or neck ache, especially in association with transient or permanent focal neurological deficits.     

Induction of murine macrophage TNF-alpha synthesis by Mycobacterium avium is modulated through complement-dependent interaction via complement receptors 3 and 4 in relation to M. avium glycopeptidolipid

We studied whether complement receptor (CR) mediated Mycobacterium avium interaction modulated macrophage TNF-alpha expression. Compared to control conditions, infections performed with C3-depletion yielded significantly higher TNF-alpha levels. Blockage of the CR4 iC3b site yielded increases in TNF-alpha for all morphotypic variants of a virulent serovar-8 strain (smooth transparent (SmT), smooth opaque (SmO), serovar-specific glycopeptidolipid (ssGPL) deficient knockout mutant) whereas CR3 blockage increased TNF-alpha only for SmT and ssGPL-deficient strains. Thus, complement-mediated binding of M. avium to CR3 and CR4 was shown to modulate TNF-alpha expression. The differential activation of morphotypic and isogenic variants of a single strain provides an excellent model system to delineate signaling pathways.     

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated K_d in the 10^?4 M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.     

Quantitative approach using multiple single parameters versus visual assessment in dobutamine stress echocardiography

ABSTRACT: BACKGROUND: A number of myocardial Doppler-derived velocity, strain myocardial imaging parameters (DMI) and speckle tracking imaging (STI) have been proposed for the quantification of myocardial ischemia during stress echocardiography. The purpose of the study was to identify the best single ultrasound quantitative parameter for prediction of significant coronary stenosis and compare it with visual assessment during dobutamine stress echocardiography (DSE). METHODS: Prospective analysis included data of 151 patients (age 61.8 +/- 9.2) who underwent dobutamine stress echocardiography for known (n=35) or suspected CAD (n=36) or symptomatic chest pain (n=80), excluding patients with previous myocardial infarction. Systolic, post-systolic and diastolic velocities, strain and strain rate parameters were obtained at rest and at peak dobutamine challenge. Derivative markers as E'/A' ratio, post-systolic index and changes from rest to stress were calculated (98 parameters overall, predominantly longitudinal). Coronary angiography was chosen as reference method considering at least one stenosis greater than or equal to 70% per patient as significant CAD. The predictive value of quantitative parameters and wall motion score index (WMSI) was obtained using logistic regression and ROC analysis. RESULTS: The value of single parameters discriminated as independent predictors of CAD appeared to be modest (area under the curve [AUC] ranged from 0.63 to 0.72 for 16 PW-DMI, 12 CC-DMI and 12 STI markers), comparing to AUC of WMSI 0.88. Sensitivity, specificity and accuracy of visual DSE evaluation was 82.4% (95%CI 77.4%; 85.2%), 92.6% (95%CI 83.4%; 97.5%) and 86.0% (95%CI 79.5%; 89.6%), respectively, Youden index 0.75. Sensitivity, specificity and accuracy of single predictors ranged from 40.0% to 93.3% (95% CI 22.7%; 99.2%), from 34.2% to 88.7% (95% CI 25.6%; 94.1%) and from 45.8% to 80.0% (95% CI 37.5%; 87.2%) respectively, Youden index ranged from 0.20 to 0.52. CONCLUSIONS: Multiple single quantitative parameters showed limited predictive ability to identify significant coronary artery stenosis. Visual assessment of DSE appears to be more accurate than single velocity and strain/strain rate markers in the diagnosis of CAD.     

Participation of Children in Medical Decision-Making: Challenges and Potential Solutions

Participation in healthcare decision-making is considered to be an important right of minors, and is highlighted in both international legislation and public policies. However, despite the legal recognition of children’s rights to participation, and also the benefits that children experience by their involvement, there is evidence that legislation is not always translated into healthcare practice. There are a number of factors that may impact on the ability of the child to be involved in decisions regarding their medical care. Some of these factors relate to the child, including their capacity to be actively involved in these decisions. Others relate to the family situation, sociocultural context, or the underlying beliefs and practices of the healthcare provider involved. In spite of these challenges to including children in decisions regarding their clinical care, we argue that it is an important factor in their treatment. The extent to which children should participate in this process should be determined on a case-by-case basis, taking all of the potential barriers into account.