Nuclear proteins of the bovine esophageal epithelium. I. Monoclonal antibody W2 specifically reacts with condensed nuclei of differentiated superficial cells

1993 ◽  
Vol 104 (2) ◽  
pp. 237-247 ◽  
Author(s):  
T.K. Tang ◽  
T.M. Hong ◽  
C.Y. Lin ◽  
M.L. Lai ◽  
C.H. Liu ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Metal Ion ◽  
Gradient Centrifugation ◽  
Basal Layer ◽  
Small Cells ◽  
Target Antigen ◽  
Esophageal Epithelium ◽  
Nuclear Condensation ◽  
Calcium Binding Site

Cells from three layers of the bovine esophageal epithelium, representing different stages of differentiation, were dissociated and separated by Percoll gradient centrifugation into fractions of small, medium and large sizes. A majority of the large cells possessed condensed nuclei, a characteristic feature of terminal differentiation of the superficial epithelium. The small cells resembled the proliferate cells of the basal layer. In vitro culture of the esophageal epithelial cells resulted in proliferation of the small cells, colony formation, and, in some cases, differentiation into cells with condensed nuclei. Nuclei, or nuclear subfractions derived from cells of the different layers, were used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bound specifically to different regions of the esophageal tissue. One such antibody, designated W2, labeled the condensed nuclei from the superficial layer of stratified esophageal and corneal epithelia in situ, as well as the large cells from esophageal culture in vitro. Thus, the expression of the W2 antigen may be associated with the process of nuclear condensation during epithelial differentiation. Immunoisolation of the target antigen of W2 from extracts of large cells of the bovine esophagus yielded a band of M(r) approximately 33,000 on nonreducing polyacrylamide gels. This band dissociated into two polypeptides, of M(r) approximately 22,000 and approximately 11,000, upon treatment with dithiothreitol. Amino acid sequence analysis of the larger polypeptide showed extensive homology to a group of small calcium-binding proteins, including two helix-turn-helix motifs designated as the EF-hand, characteristic of the configuration of the metal-ion coordinating ligands of the calcium-binding site. Similarly, the sequence at the amino terminus of the polypeptide of approximately 11,000 indicated that it was the light chain counterpart of the same calcium-binding protein complex.

Nuclear proteins of the bovine esophageal epithelium. II. The NuMA gene gives rise to multiple mRNAs and gene products reactive with monoclonal antibody W1

1993 ◽  
Vol 104 (2) ◽  
pp. 249-260 ◽  
Author(s):  
T.K. Tang ◽  
C.J. Tang ◽  
Y.L. Chen ◽  
C.W. Wu
Keyword(s):  
Alternative Splicing ◽  
Polymerase Chain Reaction Amplification ◽  
Basal Layer ◽  
Small Cells ◽  
Target Antigen ◽  
Cdna Clones ◽  
Esophageal Epithelium ◽  
Mitotic Apparatus ◽  
Cdna Sequences ◽  
Alternative Mrna Splicing

Treatment of small cells derived from the basal layer of bovine esophageal epithelium, with Triton X-100, urea and sonication resulted in a nuclear residue that was used as an immunogen for generation of monoclonal antibodies directed against nuclear components. One such antibody, designated W1, was found to label the nuclei of all cells examined. In interphase cells, the target antigen of antibody W1 was diffusely distributed in the nucleus. During metaphase, however, the W1 antigen formed prominent crescents at the poles of the mitotic spindle, diminished gradually in anaphase, and finally redistributed into the regenerating daughter nuclei. Western blotting with antibody W1 yielded a prominent polypeptide of M(r) approximately 230,000. The amino acid sequence, deduced from the nucleotide sequence of several overlapping cDNA clones that span the entire coding region, revealed that the W1 polypeptide was identical to the Nuclear Mitotic Apparatus (NuMA) protein, with a long alpha-helical central core flanked by two nonhelical domains. Interestingly, most cDNA sequences were identical to each other, except for six sequence blocks which were either inserted or deleted in individual cDNA clones. Analysis of the cDNA sequences of various clones, coupled with polymerase chain reaction amplification of cellular mRNA and genomic Southern blotting with region-specific probes, all indicated that multiple mRNA species were present in U-251 human glioma cells, derived from alternative splicing of the RNA transcript from a single NuMA/W1 gene. Besides the predominant form of the mRNA giving rise to the polypeptide of M(r) approximately 230,000, two other forms of mRNA, which arise as a result of alternative splicing and which use different translation termination codons, may yield lower molecular weight polypeptide products. Consistent with this notion, polypeptides of M(r) approximately 195,000 and approximately 194,000 have been observed in this and other studies on the NuMA/W1 protein. These data suggest that multiple isoforms of the NuMA polypeptides generated by alternative mRNA splicing may play some important functions which remain to be characterized.

scholarly journals Atomic resolution probe for allostery in the regulatory thin filament

2016 ◽  
Vol 113 (12) ◽  
pp. 3257-3262 ◽  
Author(s):  
Michael R. Williams ◽  
Sarah J. Lehman ◽  
Jil C. Tardiff ◽  
Steven D. Schwartz
Keyword(s):  
Binding Site ◽  
Human Disease ◽  
Calcium Binding ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Calcium Binding Site

Calcium binding and dissociation within the cardiac thin filament (CTF) is a fundamental regulator of normal contraction and relaxation. Although the disruption of this complex, allosterically mediated process has long been implicated in human disease, the precise atomic-level mechanisms remain opaque, greatly hampering the development of novel targeted therapies. To address this question, we used a fully atomistic CTF model to test both Ca2+ binding strength and the energy required to remove Ca2+ from the N-lobe binding site in WT and mutant troponin complexes that have been linked to genetic cardiomyopathies. This computational approach is combined with measurements of in vitro Ca2+ dissociation rates in fully reconstituted WT and cardiac troponin T R92L and R92W thin filaments. These human disease mutations represent known substitutions at the same residue, reside at a significant distance from the calcium binding site in cardiac troponin C, and do not affect either the binding pocket affinity or EF-hand structure of the binding domain. Both have been shown to have significantly different effects on cardiac function in vivo. We now show that these mutations independently alter the interaction between the Ca2+ ion and cardiac troponin I subunit. This interaction is a previously unidentified mechanism, in which mutations in one protein of a complex indirectly affect a third via structural and dynamic changes in a second to yield a pathogenic change in thin filament function that results in mutation-specific disease states. We can now provide atom-level insight that is potentially highly actionable in drug design.

scholarly journals In Vitro Analysis of Roles of a Disulfide Bridge and a Calcium Binding Site in Activation of Pseudomonas sp. Strain KWI-56 Lipase

2000 ◽  
Vol 182 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Junhao Yang ◽  
Koei Kobayashi ◽  
Yugo Iwasaki ◽  
Hideo Nakano ◽  
Tsuneo Yamane
Keyword(s):  
Binding Site ◽  
Disulfide Bond ◽  
Calcium Binding ◽  
Burkholderia Cepacia ◽  
Active Form ◽  
Translation System ◽  
Expression Systems ◽  
Lipase Gene ◽  
Calcium Binding Site

ABSTRACT The expression of lipase from Pseudomonas sp. strain KWI-56 (recently reclassified as Burkholderia cepacia) had been found to be dependent on an activator gene (act) downstream of its structural gene (lip). In this work, the mature lipase was synthesized in an enzymatically active form with a cell-free Escherichia coli S30 coupled transcription-translation system by expressing a recombinant lipase gene (rlip) encoding the mature lipase in the presence of its purified activator or by coexpression of rlip andact. The in vitro expression systems were used for studying the folding process of the lipase. The addition of dithiothreitol in the expression systems decreased the activity dramatically without affecting the synthesis level of the lipase, whereas the in vitro-synthesized active lipase was relatively stable even in the presence of dithiothreitol. This phenomenon was further investigated by constructing mutant lipase genes only in vitro by PCR without gene cloning. Replacements of cysteine residues (Cys190 and Cys270) forming a sole putative disulfide bond to serine residues decreased the lipase activity greatly, suggesting that the disulfide bond was essential for the proper folding of the lipase. In addition, replacing Asp242 and Asp288, which were deduced to be part of a Ca2+ binding site, also greatly decreased the activities of the in vitro-synthesized lipases. The role of the Ca2+ binding site in the activation of the lipase is also discussed.

scholarly journals Computational and biochemical analysis of type IV Pilus dynamics and stability

2021 ◽  
Author(s):  
Yasaman Karami ◽  
Aracelys López-Castilla ◽  
Andrea Ori ◽  
Jenny-Lee Thomassin ◽  
Benjamin Bardiaux ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Ex Vivo ◽  
Targeted Mutagenesis ◽  
Human Pathogens ◽  
Calcium Binding Site ◽  
Type Iv ◽  
Biochemical Analyses ◽  
Dynamics Simulations

SUMMARYType IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 μs all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

A novel calcium-binding site of von Willebrand factor A2 domain regulates its cleavage by ADAMTS13

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4623-4631 ◽  
Author(s):  
Minyun Zhou ◽  
Xianchi Dong ◽  
Carsten Baldauf ◽  
Hua Chen ◽  
Yanfeng Zhou ◽  
...  
Keyword(s):  
Binding Site ◽  
Calcium Binding ◽  
Metal Ion ◽  
Wild Type ◽  
Calcium Binding Site ◽  
Dynamic Simulations ◽  
Primary Hemostasis ◽  
Significant Difference ◽  
Thrombogenic Potential ◽  
A2 Domain

Abstract The proteolysis of VWF by ADAMTS13 is an essential step in the regulation of its hemostatic and thrombogenic potential. The cleavage occurs at strand β4 in the structural core of the A2 domain of VWF, so unfolding of the A2 domain is a prerequisite for cleavage. In the present study, we present the crystal structure of an engineered A2 domain that exhibits a significant difference in the α3-β4 loop compared with the previously reported structure of wild-type A2. Intriguingly, a metal ion was detected at a site formed mainly by the C-terminal region of the α3-β4 loop that was later identified as Ca2+ after various biophysical and biochemical studies. Force-probe molecular dynamic simulations of a modeled structure of the wild-type A2 featuring the discovered Ca2+-binding site revealed that an increase in force was needed to unfold strand β4 when Ca2+ was bound. Cleavage assays consistently demonstrated that Ca2+ binding stabilized the A2 domain and impeded its unfolding, and consequently protected it from cleavage by ADAMTS13. We have revealed a novel Ca2+-binding site at the A2 domain of VWF and demonstrated a relationship between Ca2+ and force in the regulation of VWF and primary hemostasis.

Characteristics of homogeneously small keratinocytes from newborn rat skin: possible epidermal stem cells

1991 ◽  
Vol 261 (6) ◽  
pp. C964-C972 ◽  
Author(s):  
J. H. Pavlovitch ◽  
M. Rizk-Rabin ◽  
P. Jaffray ◽  
H. Hoehn ◽  
M. Poot
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Population ◽  
Calcium Binding ◽  
Small Cells ◽  
Proliferative Potential ◽  
Rat Skin ◽  
Newborn Rat ◽  
Basal Keratinocytes

The aims of the present study were to characterize the phenotype, growth kinetics, and proliferative activation in culture of a population of poorly differentiated homogeneously small (HS) keratinocytes. These slow-cycling cells were separated by unit gravity sedimentation from a population of actively proliferating basal keratinocytes in newborn rat skin. This population (approximately 1% of the total basal keratinocytes) consisted of extremely small cells with little cytoplasm or RNA. Their positive KL4 staining demonstrates that they were keratinocytes. HS keratinocytes did not, however, contain epidermal calcium binding protein. Acridine orange, bivariate Hoechst, and ethidium bromide flow cytometry of in vitro bromodeoxyuridine-labeled cells as well as Ki67 staining showed that HS keratinocytes were in the G0 stage of the cell cycle and did not actively proliferate in vivo. [3H]thymidine label-retaining cells were found only in the HS cell population, showing that HS cells may originate from a central position in the epidermal proliferative unit. Growth of HS cells in vitro was characterized by a delayed but progressive increase in RNA before entry into the cell cycle. The clonogenic efficiency of HS cells in primary culture was much less than that of larger cells. Subclones of HS cell colonies exceeded primary colonies in their cloning efficiency and proliferative potential, suggesting that HS cells, although normally prevented from dividing, retain a high self-renewal capacity. They also maintain the ability to differentiate. The results are consistent with the concept that HS cell population may represent the epidermal-specific progenitor cells which act as stem cells in this tissue.

Desmosomal distribution in the epidermis of a non-contracting human skin equivalent

1992 ◽  
Vol 50 (1) ◽  
pp. 896-897
Author(s):  
L.X. Oakford ◽  
S.D. Dimitrijevich ◽  
R. Gracy
Keyword(s):  
Human Skin ◽  
Basal Layer ◽  
Skin Equivalent ◽  
Tissue Component ◽  
Intact Skin ◽  
Similar Sequence ◽  
Sequence Of Events ◽  
Suprabasal Keratinocytes ◽  
Human Skin Equivalent

In intact skin the epidermal layer is a dynamic tissue component which is maintained by a basal layer of mitotically active cells. The protective upper epidermis, the stratum corneum, is generated by differentiation of the suprabasal keratinocytes which eventually desquamate as anuclear comeocytes. A similar sequence of events is observed in vitro in the non-contracting human skin equivalent (HSE) which was developed in this lab (1). As a part of the definition process for this model of living skin we are examining its ultrastructural features. Since desmosomes are important in maintaining cell-cell interactions in stratified epithelia their distribution in HSE was examined.

scholarly journals Role of Jagged1-mediated Notch Signaling Activation in the Differentiation and Stratification of the Human Limbal Epithelium

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1945
Author(s):  
Sheyla González ◽  
Maximilian Halabi ◽  
David Ju ◽  
Matthew Tsai ◽  
Sophie X. Deng
Keyword(s):  
Notch Signaling ◽  
Progenitor Cell ◽  
Basal Layer ◽  
Notch Signaling Pathway ◽  
Basal Cells ◽  
Proliferation Markers ◽  
Limbal Epithelium ◽  
Asymmetric Divisions ◽  
Signaling Activation

The Notch signaling pathway plays a key role in proliferation and differentiation. We investigated the effect of Jagged 1 (Jag1)-mediated Notch signaling activation in the human limbal stem/progenitor cell (LSC) population and the stratification of the limbal epithelium in vitro. After Notch signaling activation, there was a reduction in the amount of the stem/progenitor cell population, epithelial stratification, and expression of proliferation markers. There was also an increase of the corneal epithelial differentiation. In the presence of Jag1, asymmetric divisions were decreased, and the expression pattern of the polarity protein Par3, normally present at the apical-lateral membrane of basal cells, was dispersed in the cells. We propose a mechanism in which Notch activation by Jag1 decreases p63 expression at the basal layer, which in turn reduces stratification by decreasing the number of asymmetric divisions and increases differentiation.

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