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PubMed1996

Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA.

Amino Acid Sequence↗
PubMed2000

Cyclooxygenases: structural, cellular, and molecular biology.

The prostaglandin endoperoxide H synthases-1 and 2 (PGHS-1 and PGHS-2; also cyclooxygenases-1 and 2, COX-1 and COX-2) catalyze the committed step in prostaglandin synthesis. PGHS-1 and 2 are of particular interest because they are the major targets of nonsteroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, and the new COX-2 inhibitors. Inhibition of the PGHSs with NSAIDs acutely reduces inflammation, pain, and fever, and long-term use of these drugs reduces fatal thrombotic events, as well as the development of colon cancer and Alzheimer's disease. In this review, we examine how the structures of these enzymes relate mechanistically to cyclooxygenase and peroxidase catalysis, and how differences in the structure of PGHS-2 confer on this isozyme differential sensitivity to COX-2 inhibitors. We further examine the evidence for independent signaling by PGHS-1 and PGHS-2, and the complex mechanisms for regulation of PGHS-2 gene expression.

Animals↗
PubMed2005

The relative thermal stability of tissue macromolecules and cellular structure in burn injury.

When tissue is subjected to higher than physiological temperatures, protein and cell organelle structures can be altered resulting in cell death and subsequent tissue necrosis. A burn injury can be stratified into three main zones, coagulation, stasis and edema, which correlate with the extent of heat exposure and thermal properties of the tissue. While there has been considerable effort to characterize the time-temperature dependence of the injury, relatively little attention has been paid to the other important variable, the thermal susceptibility of the tissue. In the present study, we employ a standard physical chemistry approach to predict the level of denaturation at supraphysiological temperatures of 12 vital proteins as well as RNA, DNA and cell membrane components. Melting temperatures and unfolding enthalpies of the cellular components are used as input experimental parameters. This approach allows us to establish a relation between the level of denaturation of critical cellular components and clinical manifestations of the burn through the characteristic zones of the injury. Specifically, we evaluate the degree of molecular alteration for characteristic temperature profiles at two different depths (Mid-Dermis and Dermis-Fat interface) of 80 degrees C; 20s contact burn. The results of this investigation suggest that the thermal alteration of the plasma membrane is likely the most significant cause of the tissue necrosis. The lipid bilayer and membrane-bound ATPases show a high probability of thermal damage (almost 100% for the former and 85% for the latter) for short heat exposure times. These results suggest that strategies to minimize the damage in a burn injury might focus on the stabilization of the cellular membrane and membrane-bound ATPases. Further work will be required to validate these predictions in an in vivo model.

Burns↗
PubMed2001

Biochemical characterization of a cellular structure retaining vegetally localized RNAs in Xenopus late stage oocytes.

Two pathways operate during Xenopus oogenesis to localize a small number of RNAs to the vegetal cortex. Correct localization of these RNAs is essential to normal development as the proteins they encode are involved in specifying cell type and in patterning the early embryo. Binding these RNAs to the vegetal cortex and thus preserving their localized condition is a critical step, although little is known about how this is achieved. In this study, we have used a biochemical approach to examine the anchoring step. Xlsirts, an abundant localized RNA (locRNA), was selectively enriched in a detergent-insoluble fraction (DIF) prepared from oocytes that had completed the RNA localization process. These putative RNA-anchoring complexes were analyzed by density gradient centrifugation and in RNA-protein binding assays. Cortical Xlsirts and other localized RNAs are specifically found in the heavy region of sucrose gradients and in the pellet, quite different from other cellular RNPs. Four proteins were identified by UV-crosslinking that bound the Xlsirts localization signal in the cortex, but not in the soluble fraction. These are likely members of the anchoring complex and appear to include vera, a characterized Vg1 RNA binding protein. Vera was found to co-sediment with other locRNAs found in the vegetal cortex, suggesting that it is a common component of locRNPs. Finally, we found that locRNPs extracted into the soluble fraction had the same buoyant density as typical ooplasmic RNPs. We propose that locRNAs are organized and anchored in the cortex as typical RNPs.

Animals↗
PubMed2003

A novel Xenopus SWS2, P434 visual pigment: structure, cellular location, and spectral analyses.

PURPOSE: The purpose of this study was to clone and characterize the green rod pigment in Xenopus laevis. METHODS: The cDNA for the Xenopus "green rod" pigment was cloned and sequenced from Xenopus retina mRNA by reverse transcription polymerase chain reaction and the 5' end cloned by rapid amplification of the cDNA ends. The cellular localization of the Xenopus opsin was determined by immunolabeling of flat-mounted retinas using a specific antibody against this opsin. Spectral properties of the expressed protein were determined by absorption spectroscopy using recombinant pigment. RESULTS: A novel Xenopus opsin cDNA containing a full-length coding region has been cloned and sequenced. The deduced amino acid sequence predicts a protein of 362 amino acids, forming 7 hydrophobic helices. Sequence analysis indicates that it belongs firmly to the SWS2 class of visual pigments and has 89%, 80%, and 75% amino acid sequence identity with bullfrog, tiger salamander, and newt SWS2 pigments, respectively. Staining of Xenopus retina with a Xenopus SWS2 opsin-specific polyclonal antibody demonstrated that the SWS2 pigment is expressed in green rods. After expression in COS cells, reconstitution with 11-cis retinal, and purification, the SWS2 pigment exhibits an absolute absorption maximum of 434 nm Thus, the name "SWS2, P434" was assigned for this opsin. The pigment decays rapidly in hydroxylamine in the dark, unlike the red rod pigment, rhodopsin. CONCLUSIONS: A novel green rod opsin cDNA has been cloned and sequenced from the retina of adult Xenopus laevis, which encodes a protein belonging to the SWS2 group of opsins. The expressed opsin possesses cone-opsin-like properties although it was identified only in the Xenopus green rod cells.

Amino Acid Sequence↗
PubMed1986

A microspectrofluorometric study of the effect of anthralin, an antipsoriatic drug, on cellular structures and metabolism.

The microspectrofluorometric approach has been used to investigate in single living cells in culture fundamental questions raised by the use of anthralin, a potent antipsoriatic drug. This method allows fluorescence determinations on the intracellular fate of the drug as well as the recognition of structural and metabolic alterations induced by the drug. In the absence of demonstrable adduct formation with DNA, the antipsoriatic, i.e. antiproliferative effect of anthralin, has been attributed to its action at the level of mitochondria or at the level of glucose-6-phosphate dehydrogenase which initiates the pentose phosphate shunt (cf. its prominent role in nucleic acid synthesis). Upon addition of 2.3 to 23 microM anthralin to the L cell culture, the characteristic structure of the anthralin anion fluorescence spectrum is recognized almost immediately in the cytoplasm (much weaker in the nucleus) but disappears within minutes. The vital mitochondrial fluorescence probe dimethylaminostyryl-pyridinium-methyl-iodine reveals striking structural alterations of the mitochondria within 15 min after addition of the drug. At the same time, there is a stimulation of the transient NAD(P)+ reduction observed upon microinjection into the L cell of the Krebs' cycle substrate malate, or the pentose cycle substrate 6-phosphogluconate. Specially, the injection of the latter to anthralin-treated cells suggests that upon release of the mitochondrial control, there is a tremendous disruption of metabolic activity which could have profound consequences on the proliferative activity of the cell. These findings, while they open new possibilities for the intracellular evaluation of therapeutic agents, create also a challenge in understanding the complex and dynamic interrelationships between intracellular organelles and bioenergetic or biosynthetic pathways.

Animals↗
PubMed1995

Cytoplasmic cellular structures control permeability of outer mitochondrial membrane for ADP and oxidative phosphorylation in rat liver cells.

The kinetics of regulation mitochondrial respiration by external ADP in permeabilized hepatocytes was studied further. In digitonin-permeabilized hepatocytes, the apparent Km for ADP in regulation of respiration was decreased from 275 +/- 35 microM in control to 48 +/- 8 microM by a treatment with trypsin (15 min, 0.125 mg/ml). In liver tissue homogenates, trypsin treatment similarly decreased the Km value for ADP. These results show that ADP diffusion in hepatocytes may be retarded due to some unknown cytoplasmic trypsin-sensitive protein factor(s) which may be lost during isolation of mitochondria. Since we have previously reported a limited permeability of the outer mitochondrial membrane in isolated hepatocytes (Saks et al. 1995, Biochem. Biophys. Res. Commun., 208, 919-926), we conclude that an important site of control of respiration in liver cells in vivo is located at the porin channels of the outer mitochondrial membrane.

Adenosine Diphosphate↗
PubMed1983

Binding of cationized and native ferritin to cellular structures of the electric organ of Torpedo marmorata.

The distribution of polycationic and polyanionic binding sites in the electric organ of Torpedo marmorata was investigated by incubation of tissue with native (NF) ferritin. 1) Collagen fibrils from the electric organ carry rosettes of polyanionic sites on their surface with a periodicity of 60 nm, corresponding to the pattern of crossbanding in collagen fibrils. The CF-binding sites are abut 30 nm in size and project 20 nm beyond the surface of the fibril. 2) As revealed by incubation of tissue homogenates, CF heavily stains the intraperiod line of the axonal myelin and also tubular structures in the axonal cytoplasm. 3) Neither the extracellular aspects of the pre- nor the postsynaptic membrane became labeled with either NF or CF. After incubation of tissue homogenates. labeling of the electron-dense material of the cytoplasmic aspect of the postsynaptic membrane was observed with NF and, in particular, with CF. The ventral basal lamina of the electroplaque cell revealed uniform labeling with NF. In contrast, CF-binding sites were distributed in the lamina densa of the basal lamina as a lattice of discrete binding sites, approximately 45 nm in diameter. The presence of polyanionic sites in the basal lamina, which also proceeds through the synaptic cleft, suggests the existence of a diffusion barrier for the released neurotransmitter acetylcholine. It is proposed that this facilitates hydrolysis of acetylcholine in the synaptic cleft and recirculation of the products of hydrolysis to the axon terminal.

Animals↗
PubMed1975

The representability of laparoscopic ovarian biopsies for the cellular structure and function of the ovaries.

Model biopsy specimens from 100 surgically removed ovaries were studied. They were matched in respect of size, dimensions and examination technique to 110 ovarian biopsy specimens obtained during laparoscopy from 60 amenorrhoeic patients. It was found that ovarian biopsy specimens with an all-round edge length of 4 to 5 mm permit usable conclusions in respect of the structure and hence also of the function of normal ovaries.

Adolescent↗
PubMed1979

Effects of various media on tissular and cellular structures of the superior cervical ganglion of the rat.

The superior cervical ganglia of the rat have been incubated in vitro for 1 h in basal medium Eagle (BME) with Hanks' salts, BME with Earle's salts, Kreb's solution and NCTC 109 medium. Comparison of the cell areas, established by a semi-automatic quantitative method, shows that the three former induce a 30--35% neuronal retraction, whereas NCTC 109 has no effect. Thus this latter medium seems the best one for studies using incubation of these cells.

Animals↗
PubMed1984

Cellular structure of the conjunctival epithelium of rabbits.

In rabbits, five different cell types were distinguished within the superficial cell layer of the conjunctival epithelium according to their ultrastructural appearance of cell organelles. These cell types also show a characteristic distribution within the various regions of the lids. The second cell type which is not found in the conjunctiva of primates is characterized by large osmiophilic granules. It is assumed that these cells are particularly important for the production of the lipid layer of the tear film preventing the rabbit cornea from evaporation.

Animals↗
PubMedDate not supplied

Metabolic action and cellular structure of the myocardium during coronary angiography.

Experimental perfusion of coronary arteries of guinea pig heart with contrast media causes various changes in electrolyte metabolism, depending on the chemical structures of the administered contrast media. These electrolyte changes are related to the tolerance of the myocardium to the contrast agents. Electron microscopic sections of human myocardium after coronary perfusion with Urografin-76 reveal that no alterations of subcellular structure occur.

Animals↗
PubMed1990

Structure, cellular distribution, and functional characteristics of the guinea pig leucocyte common antigen.

The guinea pig leucocyte common antigen (LCA), expressed on different hemopoietic cells, was examined using the monoclonal antibody (mAb) H 201. Immunohistology and FACS analysis revealed that T and B lymphocytes, macrophages, and thymocytes express the H 201 epitope in comparable density. The level of LCA-expression increased during the course of maturation and activation of T cells. Differences in the molecular weight of LCA were observed, which depended on the nature of various cell populations, indicating that in each case alternative variants of LCA are expressed. The molecular weight of guinea pig LCA ranged from 175 kDa on thymocytes, up to a 230-kDa variant found on B lymphocytes. Antigen- or alloantigen-induced T cell activation in vitro was moderately affected by the continuous presence of mAb 201. In contrast, the PHA-mediated T cell proliferation was strongly and selectively enhanced, supporting the assumption of an LCA involvement in the "alternative pathway" of T cell activation.

Animals↗
PubMed1984

Studies on cellular structure and ice location in frozen organs and tissues: the use of freeze-substitution and related techniques.

Recent studies have led to the conclusion that extracellular ice per se can damage whole organs and tissues. Thus information on the amount and distribution of ice is an important factor in the design of cooling regimens that avoid intracellular ice formation and attempt to localize the ice formed in areas of the tissue where its disruptive effects can be minimized. Furthermore, ultrastructural studies at subfreezing temperatures can enhance the interpretation of information gained from morphological and function studies conducted before cooling and after rewarming. Although many techniques exist for observing and recording structure in the frozen state, not all are applicable to tissues or organs. Freeze-substitution and isothermal freeze-fixation provide two flexible techniques to explore the frozen state. Isothermal freeze-fixation is most suitable for studies close to the melting point, while freeze-substitution can be used at lower temperatures, extending as far as -120 degrees C. A careful choice of technique can provide an accurate assessment of the amount and distribution of the ice phase and the structure of the tissue matrix.

Animals↗