bluuebunny/crossref_metadata_2025_split huggingface dataset:
The prospects for domesticating milk protein genes
Authors: Hennighausen Lothar 
Published: May 2005

AbstractIt is possible to convert milk glands of transgenic animals into bioreactors producing heterologous proteins such as scarce human pharmaceuticals. To predictably and successfully engineer the milk gland, we will need a thorough understanding of its physiology. Expression studies in transgenic animals have located mammary specific and hormone inducible transcription elements in the promoter/upstream regions of milk protein genes, and transfection studies in cell lines or primary cells have identified constitutive and hormone inducible elements. Most importantly, it appears that in addition to individual promoter based transcription elements structural features of milk protein chromosomal loci may contribute to the tight developmental and hormonal regulation.I will discuss milk protein gene regulation with emphasis on regulatory differences between genes and species, and the possibility that transcription elements function only properly within genetically defined chromatin domains. Novel strategies to build mammary expression vectors and to test their functionality without pursuing the standard transgenic route will be presented. Finally, I will discuss homologous recombination with the goal to target milk protein genes. Only through the domestication of milk protein genes will we be able to use their full potential in the mammary bioreactor.
Bistratene A: A novel compound causing changes in protein phosphorylation patterns in human leukemia cells
Authors: Watters Diane J. , Michael Julie , Hemphill Julia E. , Hamilton Susan E. , Lavin Martin F. , Pettit George R. 
Published: May 2005

AbstractBistratene A, a polyether toxin isolated from the colonial ascidian Lissoclinum bistratum, causes incomplete differentiation of human leukemia (HL‐60) cells apparently through a mechanism not involving protein kinase C. In view of the importance of phosphorylation/dephosphorylation in cellular growth and differentiation we have investigated protein phosphorylation in these cells following exposure to bistratene A, using two‐dimensional polyacrylamide gel electrophoresis. Marked increases in the phosphorylation of a protein of 20 kDa, pl 6.7, and a basic protein of 25 kDa were observed after incubation with bistratene A. A comparison was made with cells treated with 12‐O‐tetradecanoylphorbol 13‐acetate and bryostatin 5. While changes in phosphorylation patterns were observed with these two compounds, the 20 kDa and 25 kDa proteins did not undergo phosphorylation changes. The 20 kDa protein was induced rapidly by very low concentrations of bistratene A reaching near maximal levels with 10 nM at 15 min exposure. This protein was found to be localised to the cytoplasm. Phosphoaminoacid analysis demonstrated that the majority of 32P was present in serine and tyrosine residues. The increased phosphorylation of the 20 kDa protein appeared to be due to hyperphosphorylation of existing protein although there was some increase in the amount of the protein. These results suggest that bistratene A will be a useful tool with which to investigate cellular differentiation mechanisms.
Cancer‐Related urinary proteinase inhibitor, EDC1: A new method for its isolation and evidence for multiple forms
Authors: Chawla Rajender K. , Lawson David H. , Ahmad Mushtaq , Travis James 
Published: May 2005

AbstractDuring the past several years, numerous laboratories have reported isolation and purification of proteinase inhibitors from human urine. Many of these molecules were incompletely characterized and some of them may have been artifacts in part because of harsh procedures used for their isolation. Consequently, there is disagreement and confusion regarding the biochemical characteristics of these inhibitors. We previously reported the isolation of a proteinase inhibitor, EDC1, from the urine of a leukemic patient. This molecule, Mr 30 kDa, was antigenically related to plasma inter‐α‐trypsin inhibitor (IATI) and inhibited the growth of a virally transformed B cell line. Immunoreactive EDC1 was also the major component of low molecular weight proteinuria observed in cancer patients. We now report a new method for the isolation EDC1 from urine of patients with adenocarcinomas of colon and lung and melanoma and compare its partial amino acid sequence with that of HI 30, a proteinase inhibitor previously isolated from pooled normal urine by Hochstrasser et al. [Hoppe‐Seyler's Z physiol Chem 357:153–162, 1976]. Our method involves (i) a batchwise cation exchange, (ii) gel filtration chromatography, (iii) anion exchange chromatography on FPLC, and (iv) reverse phase C18 chromatography on HPLC. This method is mild and results in an over all yield of 0.4 to 1.2 mg of EDC1/liter urine. On the basis of the partial N‐terminal amino acid sequence of its N terminal (38 residues) and middle regions (29 residues), EDC1 appears to be identical with H130. Surprisingly, during this isolation procedure, another proteinase inhibitor, Mr 22 kDa, which cross‐reacted with antisera to EDC1 and IATI, was also isolated. The 22 kDa molecule was a major component of the IATI related urinary molecules and was identical with the 30 kDa EDC1 in which first the 15 N terminal residues were clipped. The lower Mr inhibitor was not an artifact formed during storage or isolation procedure because the Western blot analysis of fresh cancer and normal urine revealed the 30 and 22 kDa molecules. Thus, both the 30 kDa EDC1 (HI30) and its clipped variant, the 22 kDa molecule, are physiologic components of IATI related urinary proteinase inhibitors and excretion of both forms may be increased in patients with advanced cancer. © 1992 Wiley‐Liss, Inc.
HMG‐Like chromosomal proteins in Trypanosoma cruzi
Authors: Morales Mónica , Galanti Norbel , Oñate Eddie , Imschenetzky Maria 
Published: May 2005

AbstractHMG‐like chromosomal proteins from Truypansoma cruzi were studies. Four HMG‐like proteins, designated HMG A, HMGA‐B, HMG‐C, and HMG‐E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG‐A and HMG‐B from T. cruzi. The relative amount of HMG‐C and HMG‐E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG‐E protein is increased in proliferating cells when compared to its level in non‐proliferating cells. HMG‐C is increased in the non‐proliferating cells. Probably, the shifts observed in the relative amount of HMG‐like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG‐like proteins isolated are similar but not identical to HMG proteins from vertebrates. © 1992 Wiley‐Liss, Inc.
A human histone H2B.1 Variant gene, located on chromosome 1, utilizes alternative 3′ end processing
Authors: Collart David , Pockwinse Shirwin , Lian Jane B. , Stein Janet L. , Stein Gary S. , Romain Paul L. , Pilapil Suzie , Heubner Kay , Cannizzaro Linda A. , Croce Carlo M. 
Published: May 2005

AbstractAbstract a variant human H2B histone gene (GL 105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNA regulated differentially during the hela S3 cell cycle. The H2B‐GL105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3′ end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non‐translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B‐GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B‐GL105 histone gene was localized to chromosome region 1q21‐1q23 by chromosomal in situ hybridization and by analysis of rodent‐human somatic cell hybrids using an H2B‐GL105 specific probe. The H2B‐GL105 gene is paired with a functional H2A histone gene and this H2B/H2B gene pair is seperated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF‐1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3′ end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication‐dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation. © 1992 Wiley‐Liss, Inc.
Characteristics of DNA replication in isolated nuclei initiated by an aprotinin‐binding protein
Authors: Coffman Frederick D. , Fresa Kerin L. , Hameed Meera , Cohen Stanley 
Published: May 2005

AbstractIsolated cell nuclei were used as the source of template DNA to investigate the role of a cytosolic aprotinin‐binding protein (ADR) in the initiation of eukaryotic DNA replication. Computerized image cytometry demonstrated that the DNA content of individual nuclei increased significantly following incubation with ADR‐containing preparations, and the extent of DNA synthesis is consistent with that allowed by the limiting concentration of dTTP. Thus, dTTP incorporation into isolated nuclei represents DNA synthesis and not parent strand repair. We found that dTTP incorporation into the isolated nuclei is dependent on DNA polymerase α (a principal polymerase in DNA replication) but that DNA polymerase β (a principal polymerase in DNA repair processes) does not play a significant role in this system. Finally, neither aprotinin nor a previously described cytosolic ADR inhibitor can block the replication of nuclease‐treated calf thymus DNA, while both strongly inhibit replication of DNA in isolated nuclei. This result, coupled with the relative ineffectiveness of nuclease‐treated DNA compared with nuclear DNA to serve as a replicative template in this assay, argues against a significant contribution from repair or synthesis which initiates at a site of DNA damage. These data indicate that ADR‐mediated incorporation of 3H‐dTTP into isolated nuclei results from DNA replicative processes that are directly relevant to in vivo S phase events. © 1993 Wiley‐Liss, Inc.
pH Sensitivity of epidermal growth factor receptor complexes
Authors: Nunez Marissa , Mayo Kevin H. , Starbuck Cindy , Lauffenburger Douglas 
Published: May 2005

AbstractThe association/dissociation binding kinetics of 125I‐labeled mouse epidermal growth factor (EGF) to receptors on human fibroblast cells in monolayer culture have been measured at 4°C as a function of extracellular pH from pH 5–9. At pH 8, steady‐state total binding is maximal. As pH is lowered to 6.5, total binding monotonically decreases dramatically. It changes further only slightly between pH 6.5 and 5 to about 20% of the maximum binding value. Scatchard binding plots at pH 7.5 and above show the commonly observed concave‐upward, non‐linear curve; as pH is lowered, this plot becomes much more linear, indicating that the “high affinity” bound receptor population is greatly diminished. Application of our ternary complex binding model [Mayo et al., J Biol Chem 264:17838–17844, 1989], which hypothesizes complexation of the EGF‐bound receptor with a cell surface interaction molecule, indicates that pH may have some direct effects on ternary complex formation, but the major effect is on EGF‐receptor dissociation. © 1993 Wiley‐Liss, Inc.
Unphosphorylated α‐PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro
Authors: Filipuzzi Ireos , Fabbro Doriano , Imber Roland 
Published: May 2005

AbstractExpression of the α‐isoform of protein kinase C (α‐PKC) in E. coli yielded the unphosphorylated 74 kD precursor molecule. This precursor form exhibited phospholipid‐ and calcium‐dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity. In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of α‐PKC is not required for binding of substrates, cofactors, or activators. These results, therefore, suggest that posttranslational phosphorylation of α‐PKC is not needed for the formation of a functional enzyme‐substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates.
High resolution distribution of mRNA within the cytoskeleton
Authors: Bassell Gary J. 
Published: May 2005

AbstractIt has been well documented that mRNA is associated with the cytoskeleton, and that this relationship is involved in translation and mRNA sorting. The molecular components involved in the attachment of mRNA to the cytoskeleton are only poorly understood. The objective of this research was to directly visualize the interaction of mRNA with the cytoskeleton, with sufficient resolution to identify the filament systems involved. This work required the development of novel in situ hybridization methods for use with electron microscopy.
ABP50: An actin‐binding elongation factor 1α from Dictyostelium discoideum
Authors: Edmonds Brian T. 
Published: May 2005

AbstractABP50 is a polypeptide elongation factor 1α from Dictyostelium that is associated with the actin cytoskeleton. Upon chemotactic stimulation, ABP50 undergoes a dramatic cytoplasmic redistribution into newly formed surface projections and in vitro binds to and bundles actin filaments. Many questions are raised by this interaction pertaining to the spatiotemporal regulation of protein synthesis and cytoskeletal organization by extracellular signals.
Effect of cytoarchitecture on the transport and localization of protein synthetic machinery
Authors: Luby‐Phelps Katherine 
Published: May 2005

AbstractThe emerging picture of cytoarchitecture imposes constraints on the transport and localization of several components of the protein synthetic machinery. The range in which “free” polysomes can diffuse through the cytoplasm may be restricted to about 50 nm due to obstruction by cytoskeletal barriers. Individual ribosomes and large transcripts will diffuse at least 4–10 times slower in cytoplasm than in dilute aqueous solution and may be sterically excluded from some cytoplasmic domains. The transport of these components from the nucleus to the cell periphery may be restricted to microtubule‐containing channels that traverse the excluding domains. In the peripheral cytoplasm, mitochondria, endoplasmic reticulum, and other membrane‐bound organelles are found only in nonexcluding channels, while actin, nonmuscle filamin (ABP280), and fodrin are concentrated in excluding domains. This suggests that the cytoplasmic volume may be functionally compartmentalized by local differentiations of cytoarchitecture. Excluding compartments may play a structural role, while nonexcluding compartments are the site of vesicle traffic and protein synthesis.
1α,25‐Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells
Authors: Sorensen Ann Marie , Bowman Douglas , Baran Daniel T. 
Published: May 2005

Abstract1α,25‐Dihydroxyvitamin D3 increases intracellular calcium in rat osteoblast‐like cells that possess the classic receptor (ROS 17/2.8) as well as those that lack the classic receptor (ROS 24/1), indicating that a separate signalling system mediates this rapid nongenomic action. To determine the intracellular sites of this calcium increase, cytosolic and nuclear fluorescence (340 nm/380 nm ratio) were measured in Fura 2AM loaded ROS 17/2.8 cells using digital microscopy. Within 5 min, cytosolic fluorescence increased by 29% (P < 0.05) and nuclear fluorescence by 30% (P < 0.01) after exposure to 1α,25‐dihydroxyvitamin D3 (20 nM). This effect was blocked by the inactive epimer 1β,25‐dihydroxyvitamin D3. In an individual cell, cytosolic and nuclear fluorescence increased gradually after 1, 3, and 5 min exposure to vitamin D. Nuclei were then isolated from ROS 17/2.8 cells to directly measure the hormone's effect on nuclear calcium. The calcium content of Fura 2AM loaded nuclei was not affected by increasing the calcium concentration in the incubation buffer from 50 nM to 200 nM. After 5 min, 1α,25‐dihydroxyvitamin D3, 20 nM, increased the calcium of isolated nuclei in medium containing 50 nM calcium and 200 nM calcium. 1β,25‐dihydroxyvitamin D3, 20 nM, had no effect on nuclear calcium but blocked the 1α,25‐dihydroxyvitamin D3 induced rise in the isolated nuclei. The results indicate that the nuclear membrane of the ROS 17/2.8 cells contain calcium permeability barriers and transport systems that are sensitive to and specific for 1α,25‐dihydroxyvitamin D3.1α,25‐Dihydroxyvitamin D3 rapidly increases nuclear calcium levels in both intact cells and isolated nuclei suggesting that rapid nongenomic activation of nuclear calcium may play a functional role in osteoblastic activity.
Evidence for calcium mediated conformational changes in calbindin‐D28K (the vitamin D‐induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques
Authors: Leathers Valerie L. , Norman Anthony W. 
Published: May 2005

AbstractThe role of the vitamin D‐induced calcium binding protein termed calbindin‐D (CaBP) in the biological response to 1,25‐dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross‐linking reagent methyl‐4‐azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin‐D28K. Calcium‐dependent interactions were evident with purified chick intestinal CaBP‐immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with MR = 60,000 and 130,000, which were photoaffinity cross‐linked with CaBP in a calcium‐dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol‐specific phospholipase C (PI‐PLC)‐treated supernatant fractions from chick intestinal brush borders. PI‐PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G‐150 chromatography of Triton X‐100 solubilized incubations between calbindin‐D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin‐D28K undergoes calcium‐dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium‐binding proteins such as calmodulin or troponin C.
Concomitant changes in mitochondria and intermediate filaments during heat shock and recovery of chicken embryo fibroblasts
Authors: Collier Nancy C. , Sheetz Michael P. , Schlesinger Milton J. 
Published: May 2005

AbstractUtilizing video‐enhanced differential interference contrast microscopy of chicken embryo fibroblasts, we observed dramatic changes in the localization and morphology of mitochondria shortly after cells were subjected to a mild heat shock. At normal temperatures mitochondria were distributed in the cell cytoplasm as elongated, tubular, and dynamic organelles but upon heat shock they moved to the perinuclear region and formed a tight ring of short swollen and—in some cases—fused vesicles. Vital dye staining of mitochondria with rhodamine 123 and indirect immunofluorescence staining with antibodies against the mitochondrial‐matrix protein, HSP 60, confirmed these results. Using cells double labeled with antibodies to vimentin and the HSP 60 protein, we found that the changes in mitochondria were accompanied by perturbations of the intermediate filament network that we and others have reported previously for heat shocked cells. Microtubules remained largely unaltered by our heat shock treatment and the redistribution of intermediate filaments and mitochondria occurred even in the presence of taxol, a microtubule stabilizing drug.The effects of heat shock on mitochondria were reversed when cells were returned to normal temperatures and their recovery to their normal state coincided with return of normal intermediate filament morphology. This recovery was blocked in cells treated with actinomycin D during heat shock, a result indicating that a heat shock protein may be required for recovery. These data are consistent with previously published observations that mitochondria are associated with the intermediate filament network but they extend this interaction to a cell system responding to a physiological stress normally experienced by the intact organism.
Heat‐induced morphological and biochemical changes in the nuclear lamina from Ehrlich ascites tumor cells in vivo
Authors: Krachmarov Chavdar P. , Traub Peter 
Published: May 2005

AbstractMembrane‐depleted nuclei from Ehrlich ascites tumor (EAT) cells isolated at low ionic strength in the presence of EDTA exhibit highly decondensed chromatin fibers and a loss of morphologically identifiable nucleoli. Treatment of these nuclei with nucleases and 2 M NaCl followed by low‐speed centrifugation permitted the facile isolation of the nuclear lamina layer. Under the same conditions, but after heat‐shock treatment of the living cells, the chromatin appears in a more condensed state, the nucleoli are well‐defined, and the nuclear lamina layer was destabilized in concert with the appearance of an internal nuclear matrix and nucleolar skeleton. Furthermore, we also found both an increase in the protein mass as well as the appearance of a relatively large number of new proteins in this fraction, which are phosphorylated. The major proteins of the nuclear lamina, the lamins, and the residual vimentin remained insoluble. These heat‐shock‐induced changes were also accompanied by a dephosphorylation of lamins A and C but not of lamin B.
Identification of a 170 kDa membrane kinase with increased activity in KB‐V1 multidrug resistant cells
Authors: Sampson Kathleen E. , McCroskey Mark C. , Abraham Irene 
Published: May 2005

AbstractUsing an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of 32P incorporation in proteins that were renatured after being subjected to SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB‐V1 cells as compared to the sensitive parental KB‐3‐1 cells. The KB‐V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca++, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P‐glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF‐7/AdrR and murine melanoma B16/AdrR. cells, but the activity was not present in murine leukemia P‐388 sensitive or multidrug resistant cells.