Serum protein concentration and oncotic pressure relationship in the rat

Summary A formula relating oncotic pressure torat serum protein concentration was derived and found to be in complete agreement with the Landis-Pappenheimer formula derived from measurements onhuman plasma.This study was supported by the Danish Medical Research Council and Johan and Hanne Weimann's legacy.     

Self-selecting albino rats exhibit differential preferences for pure macronutrient diets: Characterization of three subpopulations

Analyses of natural feeding behavior in albino male Sprague-Dawley rats demonstrate that, when allowed to self-select from pure macronutrient diets (protein, carbohydrate and fat), these rats of the same genetic strain can be categorized into 3 subpopulations according to either their 24-h or their 12-h nocturnal patterns of nutrient intake. A majority of the animals (HC for high carbohydrate, 50% of the total population) consumed a diet rich in carbohydrate relative to protein or fat, while a smaller population of rats (HF, 30%) preferred the fat diet, and an even smaller population (HP, 20%) chose a high-protein diet. These 3 subpopulations, after a few weeks of maintenance on the diets, differed in their body weight, with the HF rats having a higher body weight than the HP animals, who tended to weigh more than the lightest HC rats. Whereas all subgroups exhibited a similar bimodal distribution of feeding during the nocturnal cycle, with peaks during the early and late dark periods, they were distinguishable on the basis of their nutrient consumption during specific phases of the dark cycle. This difference was most apparent in the early dark phase, when the 3 subgroups exhibited exaggerated preferences for the specific nutrient that was generally preferred over the 24-h cycle. This is in contrast to the middle dark phase, when diet preferences were attenuated or lost, and the late dark phase, when most rats were similar in showing an increased preference for protein and fat and a decreased preference for carbohydrate. The HF group was further distinguished by an unusually strong burst of feeding during the first 2 h of the dark period and an extra peak of feeding in the middle dark period (7th h), both of which were relatively high in fat content.     

Novel protein microarray for the detection of avian influenza virus antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes

ABSTRACT

Avian influenza virus (AIV) can cause serious zoonotic disease, thereby threatening the poultry industry and human health. An efficient and rapid detection approach is crucial to prevent and control the spread of avian influenza. In this study, a novel protein microarray was developed. Haemagglutinin proteins of H5 and H7 subtypes and nucleoprotein (NP) were purified and spotted onto the initiator-integrated poly-(dimethylsiloxane) as antigens. Monoclonal antibodies with inhibition effect were screened and utilized for the synchronous detection of three avian influenza antibodies in different species. In the protein microarray, the cut-off values were 40%, 50% and 30% inhibition for H5 antibody detection; 50%, 50% and 20% for NP antibody detection; 40%, 50% and 40% for H7 antibody detection in chicken, peacock and duck sera, respectively. The 95 serum samples were detected by microarray, and results were compared with the findings of AIV antibody test enzyme-linked immunosorbent assay (ELISA) or haemagglutination inhibition (HI) test. NP antibody detection in the microarray showed 100% (55/55) agreement ratio in chicken using ELISA. Compared with HI, H5 antibody detection in the microarray showed 100% (95/95) agreement ratio in chicken, peacock and duck, whilst those of H7 displayed 98.18% (54/55) agreement in chicken, 100% (20/20) in peacock and 90% (18/20) in duck. In conclusion, this novel protein microarray is a high-throughput and specific method for the detection of AIV antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes. It can be applied to the serological diagnosis and epidemiological investigation of AIV.

RESEARCH HIGHLIGHTS

• A novel protein microarray method has been developed.

• The microarray can detect AIV antibodies and distinguish between H5 and H7 subtypes.

• The study lays the foundation for simultaneous identification of multiple pathogens.

     

Desensitization of acetylcholine induced inositol 1,4,5-trisphosphate formation in neuroblastoma SH-SY5Y cells following repetitive acetylcholine stimulations

In this study, the desensitization of acetylcholine-induced inositol 1,4,5-trisphosphate [I(1,4,5)P₃] formation, upon short-time prestimulations, was investigated in cultures of human neuroblastoma SH-SY5Y cells. Four repeated stimulations for 10 seconds with 10 μM acetylcholine were necessary to induce a desensitization of the I(1,4,5)P₃ formation. The desensitization was observed 4 hours after the initiation of repetitive stimulations. The same effect was obtained by a single prestimulation with 1 mM acetylcholine. Preincubation of the cells with phorbol 12-myristate 13-acetate (PMA) markedly down-regulated the acetylcholine-induced I(1,4,5)P₃ formation. However, the protein kinase C (PKC) inhibitors H7 and staurosporine did not influence the desensitization induced by four repeated stimulations with 20 μM acetylcholine. These results indicate that the signal transduction can be desensitized following repeated stimulations with sub-maximal concentrations of receptor agonist and although activation of PKC can induce the same down-regulation, PKC is most likely not involved in the desensitization induced by repetitive acetylcholine-stimulations.     

Protein kinase mediators of integrin signal transduction

 Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of ’solid-state’ signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion. Received: 14 May 1996 / Accepted: 2 July 1996     

In the absence of major histocompatibility complex class II molecules,invariant chain is translocated to late endocytic compartments by autophagy

It has been suggested that the cytoplasmic amino-terminal tail of invariant chain (Ii) contains a sorting signal that directs trafficking of the major histocompatibility complex (MHC) class II: Ii oligomeric complex to endocytic compartments. This model is based, in part, on the observation that in the absence of MHC class II molecules, Ii is detectable in lysosomal structures, a phenotype that is dependent on an intact NH₂ terminus. However, the route by which Ii gains access to endosomal compartments in the absence of class II molecules remains uncertain. Here we report a mechanism that localizes Ii in lysosomal compartments independently of class II. We show that murine Ii can be detected by immunofluorescence within late endocytic compartments of stably transfected Ltk^? mouse fibroblasts. Immunochemical studies indicate that degradation of Ii in these cells is sensitive to the lysosomotropic agent ammonium chloride, yet the majority of Ii that undergoes this apparent lysosomal degradation is sensitive to the enzyme endoglycosidase H. This finding suggests that Ii may reach the lysosomal compartment by a route that bypasses the Golgi complex. Consistent with this possibility, we found that in contrast to Ii which is complexed to class II molecules, transport of free Ii to lysosomes is prevented by 3-methyladenine, an inhibitor of the autophagic pathway of protein degradation, a process which involves direct transport from the endoplasmic reticulum to lysosomes. These data suggest the route of transport that leads to endosomal localization of Ii in the absence of class II is distinct from that taken when expressed with class II. This forces a re-evaluation of the concept that the cytosolic tail of Ii contains a dominant Golgi-to-endosomal sorting signal.     

Stage-specific protein synthesis by asexual blood stage parasites of Plasmodium falciparum

Highly synchronised cultures of cloned Plasmodium falciparum (clone T9-94) were metabolically radiolabelled with [35S]methionine during eight consecutive non-overlapping intervals, while parasites developed from young rings to mature schizonts. Analysis of equal amounts of trichloroacetic acid precipitable radioactivity from each interval by sodium dodecyl sulphate-polyacrylamide gel electrophoresis fluorography allowed the stage specificity of protein synthesis to be investigated. More than forty polypeptides with molecular weights of 20 000 to 200 000 can be distinguished. While some proteins are synthesised throughout erythrocytic schizogony many are shown to be stage-specific. Among these are a range of high molecular weight proteins synthesised only during nuclear division. Detailed morphological information permits correlations to be made between synthesis of particular polypeptides and parasite structure.     

Oesophageal squamous cell carcinoma with lymphoid stroma

Summary We treated a 70-year-old Japanese man with squamous cell carcinoma of the oesophagus with evidence of lymphoid stroma. The tumour consisted of a main lesion invading the muscular layer of the oesophagus, in association with wide areas of carcinoma in situ. The tumour stroma of the lesion was nondesmoplastic and was uniformly infiltrated mainly by abundant lymphocytes and plasma cells. Immunohistochemically, the lymphocytes consisted of a large number of T lymphocytes and a small number of B lymphocytes. S-100 protein positive cells were marked in the tumour cell nests and necrotic change of tumour cells was frequent. Abundant infiltration of lymphocytes and plasma cells was also wide-spread beneath the carcinoma in situ, together with the lymphoid follicles. Carcinoma with lymphoid stroma can occur not only in the breast, uterine cervix, nasopharynx and stomach but also in the oesophagus.     

Self-selection of dietary casein and soy-protein by the cat

Growing specific-pathogen-free kittens were fed for two weeks a choice between two complete diets differing only in protein content. When casein diets containing 18, 36 and 54% protein were offered in the three possible combinations, the kittens consistently avoided the higher casein diets and kittens offered the two highest levels of casein significantly reduced their total food intake. In one soy-protein choice study, 16, 31 and 63% protein diets were each offered with a protein-free (PF) diet. When diets were similar in physical consistency, kittens selected similar amounts of both diets with the result that the PF:16% group consumed below their requirement of protein. In another soy-protein experiment the 16, 31 and 63% protein diets were offered in their three possible combinations. Kittens in all three groups selected similar amounts of both diets. Except for their avoidance of casein, the kittens did not regulate in a consistent manner their intake of protein and therefore, behaved very differently from the rat in the self-selection of dietary protein.     

Activation of ion transport by combined effects of ionomycin,forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes

The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca²⁺ pathways, (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl^– conductance by protein kinase A and a basolateral K⁺ conductance by Ca²⁺. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K⁺ conductance. The synergism is caused by PKC-dependent increase of the apical Cl^– conductance and Ca²⁺-dependent increase of the basolateral K⁺ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation, (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl^– channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl^– conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T₈₄ cells in that the latter did not respond with an increase of the short-circuit current to addition of phorbol ester. This may be due to a very low expression of PKC.