Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa.

The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.     

Effect of protoplast formation and regeneration on the production of nebramycin complex in Streptomyces cremeus subsp. tobramycini

Production of the nebramycin complex in Streptomyces cremeus subsp. tobramycini before and after the protoplast formation and regeneration was comparatively studied. The antibiotic production was estimated by the total activity and component composition of the nebramycin complex. It was found that formation and regeneration of the protoplast led to lowering of the activity and changing of the complex component composition. Strains mainly synthesizing each one of the three basic components of the nebramycin complex were isolated. The strains proved to be unstable by the antibiotic production property and after three subcultures lost the differences in the complex component composition.     

Phosphotungstic Acid-Hematoxylin; Spectropho-Tometry of the Lake in Solution and in Stained Tissue

Job's method of continuous variation (Ann. Chim., ser. 10; 9, 113-203, 1928) was applied to tungstate-hematein and molybdate-hematein lakes. For phosphotungstic acid-hematein (PTA-Hn), sodium tungstate-hematein, or molybdic acid-hematein, it demonstrates that each forms a single complex in solution, with ratios of composition (PTA to Hn) 1:2, 1:2 and 1:1 respectively. PTA-Hn was the best stain, as judged by its ability to differentiate collagenous connective tissues from muscle, and to delineate nuclear and cytoplasmic detail. Hematein is used in place of hematoxylin because a reproducible, potent staining solution can be prepared without lengthy aging periods or uncertain oxidants. With this lake, staining is independent of fixation and obviates the necessity of mordanting procedures prior to staining. Microspectrophotometric evidence indicates that the blue component of the well-recognized polychromasia of these lakes is a true example of bathochromic metachromasia. Staining by the lake at various pH values and by acid and basic dyes after PTA treatment of tissues indicate that PTA-Hn is an acid lake dye and therefore different from the iron and aluminum hematoxylin lakes. Observations on the distribution of metachromasia suggest that it is topographically related to tissue components having a high content of basic protein.     

Mycobacterium smegmatis displays the Mycobacterium tuberculosis virulence-related neutral red character when expressing the Rv0577 gene

Neutral red staining is a cytochemical reaction that has been found to be related to Mycobacterium tuberculosis virulence and, therefore, the component involved in it is thought to be a virulence factor. To study the molecular basis of this reaction we constructed an M. tuberculosis cosmid library in Mycobacterium smegmatis and selected recombinant neutral red positive clones. Heterologous complementation identified Rv0577 as the gene responsible for this trait and we have also shown that it is expressed as a single polycistronic unit together with Rv0576 which could also be involved in the neutral red staining.     

Alcohol inhibition of formalin induced depletion of neurosecretory material in the teleost Clarias batrachus (L).

In the normal C. batrachus a fair amount of stainable neurosecretory material (NSM) is always present in all the component parts of the hypothalamo-neurohypophysial (HN) complex. When formalin was injected intraperitoneally 70 to 90% of the NSM was depleted from the HN complex. When ethyl alcohol was administered immediately after formalin treatment, the depletion of NSM was inhibited, and their staining intensity could be compared with those of untreated controls.     

High-performance liquid chromatographic purification of a ribonucleoprotein complex and its protein component

This paper describes the rapid purification by high-performance liquid chromatographic techniques of milligram quantities of the 7 S ribonucleoprotein complex (RNP 7 S) and its protein component from tench (Tinca tinca) oocyte extracts. High-performance gel permeation chromatography (with a TSK 3000SW column) was found to be unacceptable because of multiple contaminants which coelute with RNP 7 S. In contrast, semipreparative high-performance DEAE ion-exchange chromatography was found to give an excellent separation of the 7 S complex which could be directly adapted to a preparative scale providing rapid purification (less than 1 h) of milligram quantities of the complex. Agarose electrophoresis followed by specific staining of protein and nucleic acid was found to be a convenient and rapid means of evaluating the purification. Finally, reverse-phase high-performance liquid chromatography was found suitable for the purification of the protein component of the 7 S complex.     

Up-regulation of MCM3 Relates to Neuronal Apoptosis After Traumatic Brain Injury in Adult Rats

Minichromosome maintenance complex component 3, one of the minichromosome maintenance proteins, functions as a part of pre-replication complex to initiate DNA replication in eukaryotes. Minichromosome maintenance complex component 3 (MCM3) was mainly implied in cell proliferation and tumorigenesis. In addition, MCM3 might play an important role in neuronal apoptosis. However, the functions of MCM3 in central nervous system are still with limited acquaintance. In this study, we performed a traumatic brain injury (TBI) model in adult rats. Western blot and immunohistochemistry staining showed up-regulation of MCM3 in the peritrauma brain cortex. The expression patterns of active caspase-3 and Bax, Bcl-2 were parallel with that of MCM3. Immunofluorescent staining and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling suggested that MCM3 was involved in neuronal apoptosis. In conclusion, our data indicated that MCM3 might play an important role in neuronal apoptosis following TBI. Further understanding of these insights could serve as the basis for broadening the therapeutic scope against TBI.     

Assessment of cardiovascular fibrosis using novel fluorescent probes

Cardiovascular fibrosis resulted from pressure overload or ischemia could alter myocardial stiffness and lead to ventricular dysfunction. Fluorescently labeled collagen-binding protein CNA 35, derived from the surface component of Staphylococcus aureus, and a novel synthetic biphenylalanine containing peptide are applied to stain fibrosis associated collagen and myocytes, respectively. Detailed pathological characteristics of cardiovascular fibrosis could be identified clearly in 2 hours. This staining pair requires only simple staining and brief washing, generating less than 10 ml of waste. The image information collected by this novel fluorescent staining pair is compatible with it collected by the traditional Masson's Trichrome and Picrosirius Red staining which are widely used to stain cardiovascular fibrosis and isolated cells.     

Subunit Composition and Glycosidic Activities of the Cellulase Complex from Clostridium thermocellum JW20

The subunit composition of the extracellular complex from Clostridium thermocellum was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Twenty-six bands, representing proteins with apparent molecular sizes ranging from 37,500 to 185,000 Da, could be detected by silver staining. Cultivation of the bacteria with the substrate Avicel, Sigma cellulose, Solka floc, or cellobiose as the carbon source had no influence on the number of detectable protein bands. By activity staining with the substrate carboxymethyl cellulose or xylan added to the SDS-polyacrylamide gels, 15 of the 26 bands exhibited endoglucanase activity and 13 showed xylanase activity. In 8 of the 26 bands, both activities could be found. As minor activities, β-glucosidase, β-xylosidase, β-galactosidase, and β-mannosidase activities could be demonstrated in the cellulase complex. Upon measuring the release of para-nitrophenol (PNP) from PNP-cellobioside and determining the amount of glucose formed, the presence of exoglucanase activity was indicated. Upon glycoprotein staining of SDS-polyacrylamide gels, 14 of the 26 bands reacted positive, indicating the glycoprotein nature of the respective proteins. Four proteins (apparent molecular sizes, 58,000, 72,500, 94,000, and 110,000 Da) could be enriched from the originally bound cellulase complex by preparative SDS-PAGE. The two smaller proteins exhibited xylanase activity, whereas the 94,000-Da protein had endo- and exoglucanase activity, and the 110,000-Da protein degraded PNP-pyranosides.     

Bovine complex I is a complex of 45 different subunits

Mammalian mitochondrial complex I is a multisubunit membrane-bound assembly with a molecular mass approaching 1 MDa. By comprehensive analyses of the bovine complex and its constituent subcomplexes, 45 different subunits have been characterized previously. The presence of a 46th subunit was suspected from the consistent detection of a molecular mass of 10,566 by electrospray ionization mass spectrometry of subunits fractionated by reverse-phase high pressure liquid chromatography. The component was found associated with both the intact complex and subcomplex Ibeta, which represents most of the membrane arm of the complex, and it could not be resolved chromatographically from subunit SGDH (the subunit of bovine complex I with the N-terminal sequence Ser-Gly-Asp-His). It has now been characterized by tandem mass spectrometry of intact protein ions and shown to be a C-terminal fragment of subunit SGDH arising from a specific peptide bond cleavage between Ile-55 and Pro-56 during the electrospray ionization process. Thus, the subunit composition of bovine complex I has been established. It is a complex of 45 different proteins plus non-covalently bound FMN and eight iron-sulfur clusters.     

A histochemical comparison of methylene-blue/acid fuchsin with hematoxylin and eosin for differentiating calcification of stromal tissue

Benign and malignant connective tissue tumors consist of a fibrous component that contains varying amounts of one or more types of bone or other calcified tissue. Diagnosis of these connective tissue tumors often poses challenges for pathologists, because it is difficult to differentiate the organic matrix of osteoid from hyalinized stroma. To establish a definitive diagnosis, it sometimes is advantageous to demonstrate histologically by special staining either the type of calcification or the presence or absence of calcification. We compared the efficacy of methylene blue-acid fuchsin (MB-AF) to hematoxylin and eosin (H-E) for connective tissue tumors suspected to contain calcifications and to devise an optimal staining technique for calcification that would be specific, simple, and cost- and time-effective. We examined 50 benign and 45 malignant connective tissue tumors that were suspected to contain calcifications. Sections were stained with H-E and MB-AF and evaluated. MB-AF stained bone pink, which contrasted with blue soft tissue. After MB-AF staining, osteoid was faint pink in a blue stromal background. Osteoid was not visualized in H-E stained sections; it was stained the same shade of pink as stromal tissue. Dystrophic calcification and cementum could be identified equally well using either staining technique, but contrast was better after H-E staining. MB-AF staining of bone was comparable to H-E staining and could be used effectively to stain bone and osteoid. MB-AF is a simple, single step procedure. It also stains cementum blue with faint blue rimming and dystrophic calcification bluish-pink, but it cannot be used as a specific stain for types of calcification other than bone and osteoid.     

Characterization of a novel third member of the human cytomegalovirus glycoprotein H-glycoprotein L complex.

A prerequisite for understanding the molecular function of the human cytomegalovirus (HCMV) gH (UL75)-gL (UL115) complex is a detailed knowledge of the structure of this complex in its functional form, as it is present in mature virions. The gH protein is known to be a component of a 240-kDa envelope complex designated as gCIII (D. R. Gretch, B. Kari, L. Rasmussen, R. C. Gehrz, and M. F. Stinski, J. Virol. 62:875-881, 1988). However, the exact composition of the gCIII complex remains unknown. In this report, we attempted reconstitution of the gCIII complex by coexpression of gH and gL in the baculovirus expression system. Formation of recombinant gH-gL complexes of approximately 115 kDa was demonstrated; however, no higher-molecular-mass (approximately 240-kDa) recombinant gH-gL complexes were detected, suggesting that the presence of gH and gL alone is not sufficient for reconstitution of the gCIII complex. To identify other mammalian and/or HCMV factors which may be necessary for gCIII formation, immunoprecipitates of gH and gL from HCMV-infected fibroblasts and purified HCMV virions were examined. This analysis did reveal a number of coprecipitating proteins which associate either transiently or integrally with gH and gL. One coprecipitating protein of 145 kDa was shown to be an integral component of gCIII, along with gH and gL. Characterization of the 145-kDa protein demonstrates that it is structurally and antigenically unrelated to gH and gL and that it appears to be virally encoded. Together, these data indicate that the 145-kDa protein is a third novel component of the mature HCMV gH-gL complex.     

The ultrastructural composition of basement membranes in vivo

The ultrastructure of basement membranes has a homogeneous appearance. The enormous cell biological importance of basement membranes and their components for cell proliferation, migration and differentiation implies that their composition is more complex than their structure suggests. To elucidate the molecular composition of basement membranes in vivo, we optimised immunogold histochemistry to allow the determination of the molecular arrangement of matrix molecules. Basically, we apply a mild fixation and embed the tissues in the hydrophilic LR-Gold. This preserves the basement membrane with a quality similar to freeze substitution. The application of two antibodies directed toward the C- and N-terminal ends of a molecule and coupled to gold particles of different sizes allows determination of the orientation of a molecule within the basement membrane. We were able to demonstrate that the molecular orientation of the laminin-1 molecule changes in the basement membrane according to cell biological needs. We also showed that ultrastructurally identical basement membranes like the ones of the proximal and distal tubules of the kidney have a differing molecular arrangement. Integrin alpha7 influences the molecular composition of the basement membranes at the myotendinous junction. With the help of double labelling at the ultrastructural level we could show that nidogen-1 is co-localised with laminin-1 and only found in fully developed, mature basement membranes. In general, laminin-1, nidogen-1 and collagen type IV are localised over the entire width of basement membranes, with laminin-1 and nidogen-1 co-localised, in accordance with the current basement membrane models. Incidentally, our investigations warn us, that not every matrix protein found at the light microscopic level as a linear staining pattern underneath an epithelium (basement membrane zone) is a real basement membrane component when investigated at the ultrastructural level. Instead, one and the same molecule, e.g. endostatin, can be a basement membrane component in one organ and a matrix molecule in another.     

Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.     

Giemsa Stain for the Diagnosis of Bovine Babesiosis. I. Staining Properties of Commercial Samples and Their Component Dyes

SYNOPSIS. Studies on the composition of commercial Giemsa stain and its effect upon staining quality are reported. These studies were supplemented by observations on the preparation of the components of Giemsa stain and their staining properties in aqueous solution, in Nocht's solution, and in laboratory prepared Giemsa stains containing one azure component. Five groups of commercial batches were differentiated on the basis of their staining reactions on thick and thin films of bovine blood containing Babesia bigemina and B. argentina. Spectrophotometric and chromatographic analysis showed that four groups differed in the proportions of the thiazine components present, while the fifth-group did not appear to be Giemsa stain. Comparison of their staining effects with those obtained with each component in laboratory prepared stains indicated that the major effects of commercial batches on both blood cells and parasites were due to the thiazine component or components in highest proportions, with satisfactory staining of protozoa associated with those batches containing high proportions of methylene blue and azure B and low proportions of the remaining thiazine components.
The function of each component of Giemsa stain is defined and the need for the proper balancing of thiazine eosinates with free azure is shown. Close correlation was obtained between analysis by spectrophotometry and chromatography and direct staining tests when samples initially with low MX values were re-examined spectrophotometrically after removal of their methylene violet content. The existence of a leuco form of eosin is reported and its possible significance to the Romanowsky effect is discussed.     

Analytical purification of the slow, high affinity NGF receptor: identification of a novel 135 kd polypeptide.

Although nerve growth factor (NGF) action is mediated by the slow, high affinity NGF receptor, little is known regarding its molecular composition or mode of action. We have used reversible chemical cross-linkers and affinity chromatography strategies to purify the slow NGF receptor covalently cross-linked to its NGF ligand. Subsequent uncoupling of the cross-links reveals that the receptor-ligand complex is composed of only a novel 135 kd polypeptide interacting with NGF. The previously characterized 85 kd fast, low affinity NGF receptor is not a component of the cross-linked slow receptor-ligand complex. This newly identified 135 kd polypeptide is either the entire slow NGF receptor, or it might be one component of a larger, multisubunit slow NGF holo-receptor.     

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo

DNA methylation had been implicated in the assembly of multiprotein repressory complexes that affect chromatin architecture thereby rendering genes inactive. Proteins containing methyl binding domains (MBDs) are major components of these complexes. MBD3 is a component of the HDAC associated chromatin remodeling complex Mi2/NuRD. The addition of MBD2 to the Mi2/NuRD complex creates MeCP1, a complex that is known to inactivate methylated promoters. The undermethylated state of the mouse preimplantation embryo prompted us to investigate the known repressory complexes at this developmental stage. We found individual components of Mi2/NuRD: MBD3, Mi2, HDAC1 and HDAC2 to be expressed from a very early stage of embryo development and to localize in close proximity with each other and with constitutive heterochromatin by the blastula stage. Expression of MBD2, a component of MeCP1, starts in the blastula stage. Then it is also found to be in proximity with heterochromatin (based on DAPI staining) and with MBD3, Mi2 and HDAC1. In contrast, expression of MeCP2, an MBD containing component of a third repressory complex (MeCP2/Sin3A), is not seen in the preimplantation embryo. Our results suggest that both Mi2/NuRD and MeCP1 complexes are already present at the very early stages of embryo development, while a MeCP2 complex is added to the arsenal of repressory complexes post-implantation at a stage when DNA methylation takes place.     

Determination of nystatin component composition using HPLC and TLC with densitometry]

The component composition of nystatin produced by an improved strain of Streptomyces noursei was determined by HPLC on Milichrom chromatograph (USSR). It was shown that the antibiotic consisted of nystatins A1, A2, A3 and B and admixture substances. The data appeared to be in good agreement with the results of the complex TLC investigation, by using densitometry. The component composition of the samples was evidenced by SIEAP mass spectrometry. Physiochemical and biological characteristics of separate components are presented.     

Immunolocalization of a subnucleolar nucleoprotein complex containing RNA polymerase 1 in ascites hepatoma cells using monoclonal antibodies

We have isolated a discrete subnucleolar macromolecular nucleoprotein complex by direct treatment of Novikoff ascites hepatoma nucleoli by MspI restriction digestion. Using a monoclonal antibody made against the subnucleolar nucleoprotein complex that was shown to inhibit RNA polymerase (pol) 1 activity in vitro, we localized an Mr approximately 55,000 protein subunit which was demonstrated previously by an enzyme-linked immunosorbent assay and Western blotting to share epitopes with the RNA pol 1 moiety of the subnucleolar complex. By indirect immunofluorescence the distribution of the Mr approximately 55,000 component of the subnucleolar nucleoprotein complex was examined at various phases of the cell cycle. At prophase, it was localized in large (approximately 1.5 microns in diameter) ball-like structures associated with the nuclear periphery and nuclear peripheral chromatin, suggesting that these structures might be related to preribosomal elements. After chromatin condensation and the pairing of daughter chromosomes, the large ball-like spheres increased in size and were associated with propidium iodide staining at one side of the nucleus; whereas throughout and especially at the opposite side of the nucleus, smaller, round, punctate structures of approximately 0.5 micron in diameter were visibly labeled that were not associated with propidium iodide staining. At later stages of the cell cycle, these small round structures were again associated with propidium iodide staining, suggesting that they may be related to prenucleolar and/or preribosomal elements which would likely contain the appropriate nucleic acid in association with RNA pol 1 and cofactors of RNA pol 1.     

Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell

The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."