Some observations on the fine structure of the myotendinous junction in myotomal muscles of the tadpole tail

Summary Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble microfibrils (Hanak and Böck, 1971), spine-like or thread-like profiles (Korneliussen, 1973) and intermediary layer (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles.Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those of the triad, such as SR feet , scalloped SR membranes and granular content of the SR sac, suggesting that they are analogous and functionally similar to the triad and other equivalent structures.     

A general strategy for the isolation of carbohydrate chains fromN-,O-glycoproteins and its application to human chorionic gonadotrophin

For the structural analysis of the carbohydrate chains ofN-,O-glycoproteins a straightforward strategy was developed based on the cleavage of theN-linked chains with immobilized peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase-F (PN-Gase-F) fromFlavobacterium meningosepticum, followed by alkaline borohydride treatment of the remainingO-glycoprotein material. This methodology was applied to the isolation of the Asn- and Ser-linked carbohydrate chains of human chorionic gonadotrophin. The structures of the isolated oligosaccharides were verified by 500-MHz¹H-NMR spectroscopy. The Asn-linked sugar chains were shown to be: NeuAc2-3Gal1-4GlcNAc1-2Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man1-3]Man 1-4GlcNAc1-4[Fuc1-6]_0-1GlcNAc and Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man 1-3]Man1-4GlcNAc1-4GlcNAc. Also some minor constituents occurred. The structures of the Ser-linked oligosaccharides were established in the form of their oligosaccharide-alditols as: NeuAc2-3Gal1-3[NeuAc2-6]GalNAc, NeuAc2-3Gal 1-3GalNAc and NeuAc2-3Gal1-3[NeuAc2-3Gal1-4GlcNAc1-6]GalNAc.Abbreviations hCG human chorionic gonadotrophin - hCG- -subunit - hCG- -subunit - ElA enzyme immunoassay - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (EC 3.5.1.52) - SDS sodium dodecyl sulphate - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

The association of the human ε-globin gene with the nuclear matrix: a reconsideration

The association of the human -globin gene with the nuclear matrix was studied in erythroid and non-erythroid cell lines. Using a high salt method to prepare histone depleted nuclei we studied the association of variety of fragments covering a 7.8 kb region which contains the human -globin gene. We furthermore studied the association of a set of DNA fragments covering the 13 kb human G/A-globin gene domain, the 16 kb /-globin gene domain and the 10 kb -globin gene domain with the nuclear matrix of K562 and Raji cells. The results show that all fragments studied are easily released from the nuclear matrix, indicating no specific association.Summarizing our results we could say that a region starting 5.7 kb 5 to the human -globin gene and ending 4 kb 3 to the human -globin gene seems to contain no attachment sites with the nuclear matrix of both erythroid and non-erythroid cells.     

TNF-α induced altered signaling mechanism in human neutrophil

In the present study we investigated the TNF- induced signal transduction mechanism in human neutrophil. Exogenously added TNF- affects both PKC activity and its translocation from cytosol to the membrane. Endogenous protein phosphorylation pattern is inhibited in TNF- induced neutrophil in Ca-dependent and Ca-independent manner, including a major 47 and 66 kDa cytosolic proteins, which may be implicated in superoxide anion generation. However TNF- dose dependently enhances the expression of -PKC isotype but not the -PKC. Morphology and cell cytotoxicity are studied in TNF- treated neutrophil to understand the TNF- induced cell death or apoptosis and these experiment is further confirmed by DNA fragmentation analysis. These results clearly demonstrate that TNF- induces cellular death of human neutrophil at least in part by enhanced expression of Ca-independent -PKC. These observations provide an insight towards understanding the function of -PKC in apoptotic pathway.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.     

Wall morphogenesis in diatoms: Deposition of silica by cytoplasmic vesicles

Summary Several TEM and SEM techniques were applied to examine developing structures in valves of the centric diatomThalassiosira eccentrica (Ehrenb.) Cleve after cytokinesis. It was possible to confirm that in each stage of the silicification process there is a distinction between a growing zone with a loose assemblage of silica spheres and a compacting zone in an older phase of development. The spherical structure of the silica in the growing zone results from the addition of silica by small cytoplasmic vesicles of about 300 to 400 Å in diameter. The vesicle membrane fuses with the silicalemma and the vesicle content is released into the silica-deposition vesicle. The origin of these vesicles, named STV, is still unknown.     

Antlers as biomonitors of environmental pollution by lead and fluoride: A review

Antlers are periodically cast and re-grown cranial appendages of deer. Both endochondral and intramembranous ossification are involved in the formation of antler bone. After velvet shedding, antlers are completely bony structures which are referred to as hard antlers. Growing antlers accumulate substances with an affinity to mineralized tissues. Among these substances are lead and fluoride. Due to the seasonally limited life span of antlers, the concentrations of lead and fluoride in hard antlers reflect uptake by the forming bone during a defined, species-specific period of some months. Antlers can thus be viewed as naturally standardized environmental samples that are well suited as biomonitors of environmental pollution by bone-seeking contaminants. Because hard antlers are collected by hunters as trophies and kept in private or public collections, material for study can be obtained rather easily. So far, lead concentrations in hard antlers have been reported only from Europe, whereas data for fluoride are available from both Europe and North America. Some studies compared contaminant concentrations in antlers from different regions, while others analyzed time-trends in contaminant levels in antlers from a single region. Using the latter approach, a pronounced drop of lead concentrations during recent decades has been reported for antlers from various European countries. This indicates a marked decline of environmental lead levels that can be attributed mainly to the phase-out of leaded gasoline and, in addition, to a reduction of lead emissions also from stationary sources. In Germany, a corresponding drop has also been recorded for fluoride concentrations in antlers, which is attributed to a decline of fluoride emissions from stationary sources due to improved emission-control measures. There is some evidence that exposure to higher levels of fluoride may lead to an impaired antler mineralization. Using antlers as biomonitors has been shown to be an efficient method for assessing environmental pollution by lead and fluoride at low cost. Further studies using this now well established approach are therefore encouraged.This revised version was published online in May 2005. The following errors were eliminated:1. In the chapter Antler growth and mineralization in the last sentence of the first paragraph a slash was missing between the words reindeer and caribou; 2. In the second paragraph of the same chapter the word reiudees was corrected to reindeer; 3. In the chapter Accumulation of lead and fluoride in growing antlers the word lightest was corrected to highest.     

A cDNA encoding vacuolar type β-d-fructofuranosidase (Osβfruct3) of rice and its expression in Pichia pastoris

A vacuolar type -d-fructofuranosidase (Osfruct3) was cloned from etiolated rice seedlings cDNA library. It encodes an open reading frame of 688 residues. The deduced amino acid sequence had 58% identity to the vacuolar type -d-fructofuranosidase of maize (Ivr1). Osfruct3 exists as a single copy per genome. Northern analyses showed that Osfruct3 undergoes organ-specific expression and is involved in the adjustment of plant responses to environmental signals and metabolizable sugars. Osfruct3 was also heterologously expressed in Pichia pastoris. The recombinant proteins were confirmed to be a vacuolar type -d-fructofuranosidase.     

Molecular and Functional Studies of the Gamma Subunit of the Sodium Pump

This article reviews our studies of the subunit of the sodium pump. is a member of the FXYD family of small, single transmembrane proteins and is expressed predominantly in the kidney tubule. There are two major variants of which function similarly to bring about two distinct effects, one on K_ATP and the other, on K _K, the affinity of the pump for K ⁺ acting as a competitor of cytoplasmic Na⁺. In this way, is believed to provide a self-regulatory mechanism for maintaining the steady-state activity of the pump in the kidney. Our studies also suggest that K⁺ antagonism of cytoplasmic Na⁺ activation of the pump is relevant not only to the presence of in the kidney, but probably some hitherto undefined factor(s) in other tissues, most notably heart. The interesting possibility that not only but other members of the FXYD family regulate ion transport in a tissue-specific manner is discussed.     

Recombinant human interferon γ exerts an anti-proliferative effect and modulates the expression of human leukocyte antigens A,B,C and DR in human urothelial cell lines

Summary In this study we have treated three malignant (TGrIII) and two pre-malignant (TGrII) urothelial cell lines with recombinant human interferon (rHu-INF). The malignant cells (HCV29-T112C1, Hu1703He and T24) were inhibited in growth by more than 50% after treatment with 100–1000 units of rHu-INF/ml for 4 days as compared to untreated controls. The growth of the pre-malignant cell lines (HCV29 and Hu609) was not influenced to the same extent in the presence of rHu-INF in the culture medium. Treatment with rHu-INF increased the expression of monomorphic human leukocyte antigens (HLA) A,B,C as well as ₂-microglobulin in all the cell lines tested, as demonstrated using a quantitative immunofluorescence assay. The tumourigenic cell lines increased their expression of HLA in a dose-dependent way, whereas treatment of the non-tumourigenic cells with higher concentrations of rHu-INF than 10 units/ml, did not increase the HLA-A,B,C expression further. None of the cell lines expressed HLA-DR unless treated with rHu-INF. No correlation between tumourigenicity and the dose of rHu-INF required for de novo induction of HLA-DR could be demonstrated. After removal of rHu-INF from the medium, the expression of HLA-DR gradually decreased in less than 14 days, indicating that the expression of HLA-DR is not constitutive but dependent upon the presence of rHu-INF. We conclude that human urothelial cells grown in vitro are sensitive to the anti-proliferative and major-histocompatibility-complex-modulating effects of rHu-INF, and that malignant urothelial cells are more sensitive than pre-malignant cells. Finally, our data indicate a possible role for rHu-INF in the management of human bladder cancer.     

Synergistic effect of BMP-2 and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells

Of TGF- superfamily proteins, BMP-2 enhanced alkaline phosphatase (ALP) activity in cultured osteoblastic cells, MC3T3-E1, to the same level promoted by ascorbate, whereas TGF-s (1, 2, 3) reduced ALP activity and altered cell morphology under the same conditions. Activin appeared to have no distinct effect on ALP synthesis. Ascorbate dependent increase in ALP synthesis was markedly stimulated in the presence of BMP-2. The synergistic effect of ascorbate on ALP synthesis was replaced by type I collagen coated on the culture dish. However, BMP-2 appeared not to bind to type I collagen. These findings indicate that BMP-2 acts directly on osteoblastic cells through its receptors and collagenous matrix can neither recruit BMP-2 nor modulate directly the action of BMP-2 in the pericellular area. Treatment of cells grown in ascorbate media with TGF-s decreased rapidly the cellular ALP activity indicating that TGF-s direct cells to the differentiated stage.     

Texturerkennung und Texturreproduktion

The recognition of textures involves multiple local filtering and subsequent averaging of the image to produce a feature space in which textures are represented as cluster centers. Results are presented for a two variate algorithm involving the features brightness and roughness. Both regular and random textures generated by program are used. The final result of classification is expressed in terms of the original textures: the computer paints what it has seen — and makes rather understandable errors at the borders between different textures.     

Histochemical evidence for granulosa steroids in follicle maturation in the African catfish,Clarias lazera

Summary The effects of carp pituitary suspension (CPS) and 11-desoxycorticosterone-acetate (DOCA) on 3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) activity in the ovary of Clarias lazera are described. Strong 3-HSD and G6PD activities are localized in the stroma, of both control and treated fish. A single CPS injection stimulates 3-HSD activity in the granulosa of postvitellogenic, maturing and postovulatory follicles, but DOCA has no such effect on the postvitellogenic and maturing follicles, and only stimulates a weak response in the postovulatory ones.     

Subcellular localisation of some acid hydrolases Triturus cristatus spermatozoa

Summary The histochemical localisation of acid -glycerophosphatase has been studied, by optical and electron microscopy, in the sperm cells of the newt. The enzymatic activity is restricted to some of the acrosomal structures, other parts of the cell being unreactive.The subcellular localisation of this enzyme, and of other four acid hydrolases (Proteinase, DNase, RNase and -glucuronidase) has been studied using biochemical techniques. The sedimentability of the before mentioned hydrolases with the acrosomes detached from the entire cells and their solubilisation by Triton X-100, have been examined.The evidence obtained was discussed in relation to the hypothesis of a lysosomal-like nature of the acrosome.     

Immunization of cross-bred cattle against Hyalomma anatolicum anatolicum by purified antigens

Extracts prepared from unfed larvae of Hyalomma anatolicum anatolicum were purified by immunoaffinity chromatography using anti-gut IgG as ligand. Affinity purified antigen (Aff-GHLAg) was used to immunize cross-bred (Bos taurus×B. indicus) calves of 6–7 months of age. Immunized calves rejected 70.6% larvae, 54.5% nymphs and 61.9% adults. No significant changes in the engorged weight of females was observed; however, significant decrease in the engorgement weight of larvae and nymphs was recorded. There was a significant decrease in the emerging nymphs (p<0.05) and adults (p<0.01) of the tick stages fed on immunized animals. SDS-PAGE analysis revealed three antigenic proteins of 100, 59.4 and 37kDa responsible for induction of resistance in the host.     

Transfection of rabbit articular chondrocytes by the calcium phosphate procedure: Improvement of efficiency and reproducibility

The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding -galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (-galactosidase assay).Results revealed that addition of 4 U ml^–1 of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a giant calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.Abbreviations CPRG Chlorophenol red--D-galactopyranoside - EDTA Ethylenediaminetetraacetic acid - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - HBS Hepes buffered saline - RAC Rabbit articular chondrocytes - X-gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside     

Combination of immunological and lectin reactions in affinity histochemistry: Proposition of the term affinitin

Summary Using the series system cell receptormistletoe lectin antiferritin-antibodyferritin, the possibilities for combination of lectin and immunological reactions for histochemistry are discussed.The system cell antigen antibody labelled mistletoe (or other) lectin is recommended for visualization of cell antigens (mistletoe lectin as common immunoglobulin reagent). It is pointed out that lectin reactions do not belong to immunhistochemistry but to affinity histochemistry. For all receptor specific proteins (antibodies, lectins, enzymes, haptoglobin and other) the term affinitin is proposed. In consideration of this new definition a common scheme is formulated: Affinitin reacts with affinitin receptor forming affinity product.     

Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Evolution of phosphagen kinase VII. Isolation of glycocyamine kinase from the polychaete Neanthes diversicolor and the cDNA-derived amino acid sequences of alpha and beta chains

Glycocyamine kinase (GK) was isolated from the marine polychaete Neanthes diversicolor by gel filtration, DEAE-cellulose chromatography, butyl-Toyopearl hydrophobic chromatography, and chromatofocusing. The GK was eluted as a single peak on the latter three chromatographies, and the molecular mass for the native GK was estimated to be about 80 kDa. The SDS–PAGE showed that the isolated GK consists of two distinct subunits in equal proportion, and chains, with molecular masses of 42.2 and 43.8 kDa, respectively. The present results suggest that the Neanthes GK has a heterodimeric structure. The cDNAs for and chains of Neanthes GK were amplified by PCR and their cDNA-derived amino acid sequences were determined. The and chains are composed of 374 and 390 amino acids, and the molecular masses were calculated to be 42,392 and 43,966 Da, respectively, in good agreement with the apparent masses on SDS–PAGE. The chain has a characteristic N-terminal extension of 15 amino acids, and all of the sequence differences between and chains were restricted in the N-terminal region of 50 residues. The overall sequence identity was 92%. The occurrence of heterodimeric nature in Neanthes GK is of great interest from the evolutionary point of view, because the heterodimeric structure is only known for creatine kinase MB-isozyme specific for mammalian heart muscle among phosphagen kinases.     

Photosynthetic potential of phytoplankton in the deep water of Lake Baikal,Russia

We examined the photosynthetic activity of the phytoplankton community collected from the surface to a depth of 1000m in the south basin of Lake Baikal. Experiments were conducted in June (mixing period) and August (stratified period). The carbon fixation rate was measured by the use of a ¹³C tracer after the incubation of samples under light conditions in the upper water column. Photosynthetic fixation of ¹³C was detected for samples collected from a depth of 500m, indicating the viability of phytoplankton in deep water. The community composition was dominated by Bacillariophyceae in deep water. The finding of lower activity at a depth of 200m than that at a depth of 500m in August suggests that the spring diatom bloom could be a significant source of viable populations at a depth of 500m. Photosynthetic activity was not detected in samples collected at a depth of 1000m.