Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken cardiac muscle

We studied the localization of desmin (skeletin), the major protein subunit of muscle-type intermediate filaments, in adult chicken cardiac muscle by high resolution immunoelectron microscopic labeling of ultrathin frozen sections of the intact fixed tissues. We carried out single labeling for desmin and double labeling for both desmin and either vinculin or alpha-actinin. In areas removed from the intercalated disk membranes, we observed desmin labeling between adjacent Z-bands in every interfibrillar space. Where these spaces were wide and contained mitochondria, convoluted strands of desmin labeling bridged between the periphery of neighboring Z-bands and the mitochondria. The intermediate filaments appeared to be organized in a more three-dimensional manner within the interfibrillar spaces of cardiac as compared to skeletal muscle. Near the intercalated disks, desmin labeling was intense within the interfibrillar spaces, but was completely segregated from the microfilament attachment sites (fascia adherens) where vinculin and alpha-actinin were localized. Desmin therefore appears to play no role in the attachment of microfilaments to the intercalated disk membrane. We discuss the role of intermediate filaments in the organization of cardiac and skeletal striated muscle in the light of these and other results.     

Fine Structure of Bacillus subtilis : I. Fixation

The fine structure of Bacillus subtilis has been studied by observing sections fixed in KMnO₄, OsO₄, or a combination of both. The majority of examinations were made in samples fixed in 2.0 per cent KMnO₄ in tap water. Samples were embedded in butyl methacrylate for sectioning. In general, KMnO₄ fixation appeared to provide much better definition of the boundaries of various structures than did OsO₄. With either type of fixation, however, the surface structure of the cell appeared to consist of two components: cell wall and cytoplasmic membrane. Each of these, in turn, was observed to have a double aspect. The cell wall appeared to be composed of an outer part, broad and light, and an inner part, thin and dense. The cytoplasmic membrane appeared (at times, under KMnO₄ fixation) as two thin lines. In cells fixed first with OsO₄ solution, and then refixed with a mixture of KMnO₄ and OsO₄ solutions, the features revealed were more or less a mixture of those revealed by each fixation alone. A homogeneous, smooth structure, lacking a vacuole-like space, was identified as the nuclear structure in a form relatively free of artifacts. Two unidentified structures were observed in the cytoplasm when B. subtilis was fixed with KMnO₄. One a tortuous, fine filamentous element associated with a narrow light space, was often found near the ends of cells, or attached to one end of the pre-spore. The other showed a special inner structure somewhat similar to cristae mitochondriales.     

Localization of phytochrome in oats by electron microscopy

Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction.     

Lentinula edodes produces a multicomponent protein complex containing manganese (II)-dependent peroxidase, laccase and beta-glucosidase

Haemozoin, the malaria pigment, regulates the synthesis of several host cytokines and has been found to be associated with the disease severity. Here we describe that malarial patients produce a significant amount of anti-haemozoin IgM antibodies. Levels of these antibodies were higher among the complicated Plasmodium falciparum cases compared to the non-complicated P. falciparum group and Plasmodium vivax patients. The P. falciparum haemozoin also induced the synthesis of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) by the monocytes of the healthy individuals, but the production of these cytokines by the monocytes was inhibited in the presence of the anti-haemozoin IgM antibodies. Therefore, it seems that the host produces these antibodies (mainly IgM types) during malarial infection that can influence the progression of the disease by inhibiting the production of cytokines.     

Kinetic analysis of the reaction catalyzed by chitinase A1 from Bacillus circulans WL-12 toward the novel substrates, partially N-deacetylated 4-methylumbelliferyl chitobiosides

The kinetic behavior of chitinase A1 from Bacillus circulans WL-12 was investigated using the novel fluorogenic substrates, N-deacetylated 4-methylumbelliferyl chitobiosides [GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)(2)-UMB (4)], and the results were compared with those obtained using 4-methylumbelliferyl N, N'-diacetylchitobiose [(GlcNAc)(2)-UMB (1)] as the substrate. The chitinase did not release the UMB moiety from compound 4, but successfully released UMB from the other substrates. k(cat)/K(m) values determined from the releasing rate of the UMB moiety were: 145.3 for 1, 8.3 for 2, and 0.1 s(-1) M(-1) for 3. The lack of an N-acetyl group at subsite (-1) reduced the activity to a level 0.1% of that obtained with compound 1, while the absence of the N-acetyl group at subsite (-2) reduced the relative activity to 5.7%. These observations strongly support the theory that chitinase A1 catalysis occurs via a 'substrate-assisted' mechanism. Using these novel fluorogenic substrates, we were able to quantitatively evaluate the recognition specificity of subsite (-2) toward the N-acetyl group of the substrate sugar residue. The (-2) subsite of chitinase A1 was found to specifically recognize an N-acetylated sugar residue, but this specificity was not as strict as that found in subsite (-1).     

Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high‐protein feeding to regulate glucose homeostasis in vivo

High‐protein feeding acutely lowers postprandial glucose concentration compared to low‐protein feeding, despite a dichotomous rise of circulating glucagon levels. The physiological role of this glucagon rise has been largely overlooked. We here first report that glucagon signalling in the dorsal vagal complex (DVC) of the brain is sufficient to lower glucose production by activating a Gcgr–PKA–ERK–K_ATP channel signalling cascade in the DVC of rats in vivo. We further demonstrate that direct blockade of DVC Gcgr signalling negates the acute ability of high‐ vs. low‐protein feeding to reduce plasma glucose concentration, indicating that the elevated circulating glucagon during high‐protein feeding acts in the brain to lower plasma glucose levels. These data revise the physiological role of glucagon and argue that brain glucagon signalling contributes to glucose homeostasis during dietary protein intake.     

RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature

We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.