bluuebunny/crossref_metadata_2025 huggingface dataset:
Self‐assembling biomolecular materials in mediaine
Authors: Bayley Hagan
Published: May 2005

Materials that mimic or extend the properties of natural molecules are being developed for medical appoications. Recent breakthroughs in genetic engineering, polymer synthesis, molecular self‐assmbly and related areas are greatly expanding the variety of structures available for use in physiological settings.
Identification of the major sites of phosphorylation in IGF binding protein‐3
Authors: Hoeck Wolfgang G. , Mukku Venkat R.
Published: May 2005

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) is the major carrier of insulin‐like growth dactor I and II in the circulation. IGFBP‐3 is secreted by various tissues and cell lines as a glycosylated phosphoprotein. We have identified two major serine phosphorylation sites located at amino acids 111 and 113 of the human protein. These serine residues ngighboring amino acids potentially involved in defining a protrein kinase recognition sequence were mutated to alanine using PCR. Single and double point mutants were stably transfected into CHO‐cells and analyzed for their level of phosphorylation. Mutation of both serines reduced phosphorylation by 80% in the full‐length protein and completely abolished phosphorylation in a 17 kDa IGFBP‐3 fragment, derived from digestion with EndoProteinase Lys‐C. The 17kDa fragment containd serines 111 and 113. S111A/S113A, a double serine‐to‐alanine mutant at positions 111 and 113, showed a strongly reduced glycosylation pattern that appears to be the result of anino acid substitutions rather tha n lack of phosphrylation. Mutant S111A/S113A, despite being non‐phosphorylated and non‐glycosylated, is functionally similar to the wild‐type IGFBP‐3 in terms of IGF‐1 binding. These results enhance our non‐glycosylated, is functionally similar to the wild‐type IGFBP‐3 in terms of IGF‐I binding. These results enhance our understanding on the functional role of glycosylation and phosphorylation of IGFBP‐3.
Vitamin D receptor alleles and bone physiology
Authors: White C. P. , Morrison N. A. , Gardiner E. M. , Eisman J. A.
Published: May 2005

The vitamin D endocrine system is central to the control of bone and calcium homeostasis. The active hormonal aform of vitamin D, 1,25 dihydroxyvitamin D (calcitriol), the circulating level of which is tightly regulated, acts through a specific receptor to mediate its genomic actions on almost every aspect of calcium homeostasis. Because of its transactivation function, it possible that a small difference in vitamin D receptor level could be amplified into a biologically significant alteration in physiological setpoint. The recent finding that polymorphisms in the vitamin D receptor gene are predictive of bone density (morrison et al., Nature 367:284–287, 1994) is the first example of an allelic effect in such a homeostatically controlled system. This raises the possibility that such central operators may exist in other regulatory pathways, and could expllain a large part of the observed “ormal” population distribution that exists for all physiological paraameters.
Pleckstrin homology (PH) domains in signal transducton
Authors: Ingley Evan , Hemmings Brian A.
Published: May 2005

A diverse array of molecules involved in signal transduction have recently been recognised as containing a new homology domain, the pleckstrin homology (PH) domain. These include kinases (both serine/threonine and tyrosine specific), all currently known mammalian phospholipase Cs, GTPases, GTPage‐activatng proteins, GTpace‐exchange factors, “adapter” proteins, cyotskeletal proteins, and kinase substrates. This has sparked a new surge of research into elucidating its sturcture and function. The NMR solution structure of the PH domains of β‐spectrin and pleckstrin (the N‐terminal domain) both display a core consisting of seven anti‐parallel β‐sheet strands. The carboxy terminus is folded into a long α‐helix. The molecule is electrostatically polarised and contains a pocket which may be involved in the inding of a ligand. The PH domain overall topological relatedness to the retinoid inding protein family of molecules would suggest a lipid ligand could bind to this pocket. the prime function of the PH domain still remains to be elucidated. However, it has been shown to be important in signal transduction, most probably by mediating protein‐protein interactions. An extended PH domain of the β‐adrenergic receptor kinase (βARK), as well as that of several other molecules, can bind to βγ subunits of the heterotrimeric G‐proteins. The possibility that the PH domain, which is found in so many signalling molecules, being generally inovolved in βγ binding site appear to be concomitant in βARK, detailed analysis indicates that the PH domain is not generally a βγ binding domain. Thus, the race is on to find the ligands of each PH domain and determine a common nature to their interaction.
Identification of a novel heparin binding domain in RHAMM and evidence that it modifies HA mediated locomotion of ras‐transformed cells
Authors: Yang Baihua , Hall Christine L. , Yang Bing Luo , Savani Rashmin C. , Turley Eva A.
Published: May 2005

We have previously reported that the hyaluronan (HA) receptor RHAMM (Receptor for Mediated Motility) [Turley et al., 1991] and that HA stimulation of the motility of ras‐transformed fibroblasts is mediated via its interaction with RHAMM. Here we show that RHAMM also contains binding sites for heparin (HP) anbd that interaction of HP with these sites can regulates the locomotion of ras‐transformed fibroblasts. At low concentrations (0.01 mg/ml), HP inhibited HA‐induced locomotion of ras‐transformed cells in a manner independent of RHAMM. At higher, but still physioligical concentrations (0.1 mg/ml), HP alone stimulated cell locomotion and this stimulation appeared to be RHAMM‐dependent as it was blocked by anti‐RHAMM antibodies. Other related glycosaminogolycans such as chondroitin sulfate and dermatin sulfate had no effect on cell motility. In ligand blotting assays, GST‐RHAMM fusion protein was shown to bind biotin‐labelled HP and this binding was displaceable with unabelled HP. In similar lignad binding analyses conducted with truncations of RHAMM fusion protein, the binding region was found to be localizeed in the same 35 amino acid segment of RHAMM that contains the two HA binding domains. Synthetic peptides corresponding to these HA binding domains were retained on and bound effectively to an HP‐Sepharose affinity column. Fusion protein generated by linkage of these peptides to the non‐HP binding amino terminus of RHAMM conferred HP binding capacity to the genetically engineered proteins. Conversely, deletion of the HA binding domains of RHAMM resulted in fusion proteins devoid of HP binding activity. The relative affinities of RHAMM for HA and HP, as determined by competition and transblot assays as well as quantification of binding at various salt concentrations, indicated that RHAMM had lower affinity for HP than that for HA. These results demonstrate the existence of new HP binding motif that has biological relevance to locomotion.
IFI 16 gene encodes a nuclear protein whose expression is induced by interferons in human myeloid leukaemia cell lines
Authors: Dawson Michelle J. , Trapani Joseph A.
Published: May 2005

We have characterized the induction of mRNA and protein products of the human IFI 16 gene in response to IFN‐γ, IFN‐α, and IFN‐β2 (IL‐6). We demonstrate that the IFI 16 gene product is a novel nucleoprotein expressed in association with the differentiation of myeloid precursor cell lines. In Northern blots, IFI 16 mRNA was increased ∼25‐fold above barely detectable levels in unstimulated promyelocytic HL‐60 cells, in response to IFN‐γ. Other myeloid cell lines, U937 and K562, also demonstrated a marked IFN‐γ‐inducibility of IFI 16 mRNA. However, all three cell lines were far less responsive to IFN‐α, and there was no response to IL‐6. By comparison, a panel of T and B cell lines demonstrated high constitutive expression of IFI 16 mRNA that was not regulated by these cytokines. Culture of HL‐60 cells in medium containing dimethylsulfoxide, retinoic acid, and 1,25 dihydroxyvitamin D3, agents that stimulate the differentiation of HL‐60 along myeloid pathways, also caused the induction of IFI 16 mRNA. To characterize the protein product of IFI 16, a monoclonal antibody was raised against a recombinant bacterial protein comprising the amino terminal 159 amino acids of IFI 16 fused to glutathione S‐transferase. The antibody, designated 1G7, was used in Western blotting to demonstrate the strong induction of a cluster of proteins of 85–95 kDa in the nuclear extracts of IFN‐γ‐treated HL‐60. The nuclear localization of IFI 16 antigen was confirmed by immunohistochemical staining of HL‐60 cells treated with IFN‐γ, dimethylsulfoxide, and retinoic acid. IFI 16 was also detected in the nuclei of monocytes, neutrophils, and lymphocytes in normal peripheral blood. Database comparisons of the IFI 16 amino acid sequence revealed 51% identity with the recently cloned myeloid cell nuclear differentiation antigen (MNDA), and extensive similarity to protein products of the Gene 200 cluster of IFN‐inducible genes, Ifi 202 and Ifi 204. The amino terminal domain of IFI 16 encodes a putative nuclear localization signal, 124PGAQKRKK, which is strongly conserved in MNDA and 204. Nuclear IFI 16 was able to bind double‐stranded DNA in vitro and exhibited a similar elution profile from DNA‐cellulose as previously observed for MNDA and 204. Therefore, IFI 16 and MNDA are members of a novel family of human DNA‐binding proteins whose expression is associated with myeloid cell differentiation induced by cytokines and chemical agents.
Temporal variation of c‐fos proto‐oncogene expression during osteoblast differentiation and osteogenesis in developing rat bone
Authors: Machwate M. , Jullienne A. , Moukhtar M. , Marie P. J.
Published: May 2005

To delineate the implication of c‐fos protooncogenic in the osteogenie process, we have investigated the temporal pattern of c‐fos mRNA expression in fetal and neonatal rat bone during intramembranous and endochondral bone formation. Northern blot analysis of mRNA extracted from calvaria and femur showed that expression of c‐fos, Histone H4, and osteocalcin mRNAs followed a temporal sequence during bone development. The levels of histone H4 mRNA, a marker of cell proliferation, were high at early stages of fetal development of calvaria and femur, and decreased until birth. In both the postnatal calvaria and femur, c‐fos mRNA levels increased transiently at birth and preceded a rise in osteocalcin transcripts, a marker of the mature osteoblast phenotype. The immunohistochemical analysis showed that c‐fos protein was expressed in osteoprogenitor cells in the perichondrium and periosteum, and not in mature osteoblasts which expressed markers of differentiated osteoblasts such as type‐I collagen, bone sialoprotein, and osteocalcin. Thus, the transient c‐fos proto‐oncogene expression during the postnatal life that precedes the osteocalcin expression may be involved in the transition from the precursor state to mature osteoblasts. These results suggest that c‐fos proto‐oncogene may play an important role in osteogenesis during rat postnatal life.
Proximal promoter binding protein contributes to developmental, tissue‐restricted expression of the rat osteocalcin gene
Authors: Heinrichs Arianne A. J. , Bortell Rita , Bourke Michael , Lian Jane B. , Stein Gary S. , Stein Janet L.
Published: May 2005

Osteocalcin is a 6 kD tissue‐specific calcium binding protein associated with the bone extracellular matrix. The osteocalcin gene is developmentally expressed in postproliferative rat osteoblasts with regulation at least in part at the transcriptional level. Multiple, basal promoter and enhancer elements which control transcriptional activity in response to physiological mediators, including steroid hormones, have been identified in the modularly organized osteocalcin gene promoter. The osteocalcin box (OC box) is a highly conserved basal regulatory element residing between nucleotides −99 and −76 of the proximal promoter. We recently established by in vivo competition analysis that protein interactions at the CCAAT motif, which is the central core of the rat OC box, are required for support of basal transcription [Heinrichs et al. J Cell Biochem 53:240–250, 1993]. In this study, by the combined utilization of electrophoretic mobility shift analysis, UV cross linking, and DNA affinity chromatography, we have identified a protein that binds to the rat OC box. Results are presented that support involvement of the OC box‐binding protein in regulating selective expression of the osteocalcin gene during differentiation of the rat osteoblast phenotype and suggest that this protein is tissue restricted.
Humoral hypercalcemia of malignancy: Some enigmas on the clinical features
Authors: Ikeda Kyoji , Ogata Etsuro
Published: May 2005

Humoral hypercalcemia of malignancy (HHM) is a common paraneoplastic syndrome mediated by tumor‐derived parathyroid hormone‐related peptide (PTHRP), which bears structural and functional similarities to PTH. Thus the clinical features of HHM are very similar to those of primary hyperparathyroidism (1° HPT), a prototype of humoral hypercalcemia caused by PTH. On the other hand, HHM syndrome differs from 1° HPT in several aspects, including serum 1,25(OH)2D levels, acid‐base balance, and bone remodeling process, the reason of which remains largely unknown. We approached these questions using a unique animal model of HHM, nude rats implanted with PTHRP‐overproducing human carcinomas. In this review we will summarize the results and discuss the implications in understanding the disease mechanism.
Tyrosine phosphorylation of phosphatase inhibitor 2
Authors: Williams John P. , Jo Hanjoong , Hunnicutt Ruthann E. , Brautigan David L. , McDonald Jay M.
Published: May 2005

Inhibitor 2 is a heat‐stable protein that complexes with the catalytic subunit of type‐1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosphorylated on tyrosine residues. Inhibitor 2 was 32P‐labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type‐1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type‐1 phosphatase activity.
Noncoordinate changes in the steady‐state mrna expressed from aldolase A and aldolase C genes during differentiation of chicken myoblasts
Authors: Meighan‐Mantha Rachel L. , Tolan Dean R.
Published: May 2005

In chickens, as in all vertebrates, tissue‐specific expression of aldolase isozymes A, B, and C is developmentally coordinated. These developmental transitions in aldolase expression have been studied most extensively by charting enzyme activity during normal and abnormal development of specific vertebrate tissues. Indeed, aldolase expression has been a key marker for normal differentiation and for retrodifferentiation during carcinogenesis. Aldolase expression during chicken myoblast differentiation offers a model for investigating the regulatory mechanisms of these developmental transitions at the level of gene expression. For these studies, cDNAs encoding the most isozyme‐specific regions of both chicken aldolase A and C were cloned. The chicken aldolase A cDNA represents the first report of this sequence. Aldolase steady‐state mRNA expression was measured during chicken myoblast differentiation in primary cultures using RNase protection assays with cRNA probes generated from these aldolase cDNA clones. Steady‐state mRNA for aldolase C, the predominant embryonic aldolase isozyme in chickens, did not significantly change throughout myoblast differentiation. In contrast, expression of steady‐state mRNA for aldolase A, the only aldolase isozyme found in adult skeletal muscle, was not detected until after myoblast fusion was approximately 50% completed. Aldolase A expression gradually increased throughout myoblast differentiation until approximately 48 h after fusion was completed when there was a dramatic increase. These results are contrasted with those of Turner et al. (1974) [Dev Biol 37:63–89] that showed a coordinated switch in isozyme activities between the embryonic aldolase C and the muscle‐specific aldolase A. This discordant expression indicates that the aldolase A and C genes may employ different regulatory mechanisms during myoblast differentiation.
Bcl‐2 protooncogene expression in cervical carcinoma cell lines containing inactive p53
Authors: Liang Xiao Huan , Mungal Salvatore , Ayscue Andrea , Meissner John D. , Wodnicki Pawel , Hockenbery David , Lockett Stephen , Herman Brian
Published: May 2005

Bcl‐2 protein expression has been found to block apoptosis and its overexpression has been implicated in lymphoid malignancies where the chromosomal translocation t(14;18) is present. In this study we investigated bcl‐2 transcription and protein expression in cultured cervical carcinoma cell lines and keratinocytes. Western blotting and immunofluorescence microscopy demonstrated bcl‐2 expression in the cytoplasm of 4 out of 5 cervical carcinoma cell lines examined (HeLa, CaSki, C‐33A, and HT‐3, but not SiHa). Bcl‐2 protein expression was undetectable in normal keratinocytes. None of the cell lines examined demonstrated chromosomal translocation or rearrangement at the major breakpoint‐cluster region (MBR) of the bcl‐2 gene using either Southern blot or polymerase chain reaction (PCR) analyses. Northern blot analysis demonstrated low levels of bcl‐2 transcription in HeLa, CaSki, and C‐33A cell lines while reverse transcriptase (RT)‐PCR demonstrated bcl‐2 transcription in all cervical carcinoma cell lines which had bcl‐2 protein expression. Thus, these data suggest that bcl‐2 expression occurs in cervical carcinoma cell lines in the absence of chromosomal translocation or rearrangement of the bcl‐2 gene. However, each of these cervical carcinoma cell lines contains inactive p53, either due to mutation (C‐33A and HT‐3) or via complexation and degradation with human papillomavirus (HPV) 16/18 E6 protein (HeLa and CaSki). Thus, functional p53, which can induce apoptosis in certain cells, is not present in these cervical cells which have increased bcl‐2 expression. Increased bcl‐2 expression under conditions of p53 inactivation may provide cells with a selective advantage for survival and consequently play a role in the development of cervical carcinogenesis.
Analysis of cell‐mediated mineralization in culture of bone‐derived embryonic cells with neurofibromatosis
Authors: Klein B. Y. , Rojansky N. , Gal I. , Shlomai Z. , Liebergall M. , Ben‐Bassat H.
Published: May 2005

von Recklinghausen neurofibromatosis (NF1) is an autosomal dominant genetic disorder associated with congenital pseudoarthrosis and with short stature. To examine whether the NF1 phenotype includes functional osteogenic defects, embryonic bone‐derived cells affected with NF1 were tested in culture for specific alkaline phosphatase (ALP) activity and cell‐mediated mineralization and compared with other embryonic bone derived cells. NF1 showed a relatively higher specific ALP activity, which has further increased in response to dexamethasone + β‐glycerophosphate (βGP) (Dex medium) coordinately with a decrease in cell proliferation. In the control group, two samples showed increased ALP activity, one showed decreased activity and the forth one did not show any change in ALP. NF1 cells were distinguished from other cells regarding day 21 mineralization, they did not mineralize when cultured with ascorbate alone in the absence of Dex medium, whereas control cells did mineralize. Adding βGP resulted in mineralization by NF1 cells but less than in other cells. In addition, NF1 cells responded to dexamethasone by increasing the βGP‐induced mineralization, as opposed to cells from other embryonic bones, which either did not respond or have even decreased mineralization under dexamethasone. Upon cis‐hydroxyproline exposure, Dex medium has also distinguished NF1 cell ALP activity from that of other cell origins. Inhibition of respiratory complex II by malonate showed that most embryonic bone‐derived cells of 12 weeks gestation are malonate resistant; thus, malonate selection was ineffective. This is in contrast to rat marrow stromal cells previously shown to undergo mineralizing cell enrichment in response to malonate. Exposure to levamisole, of Dex‐treated cells, at days 0–11 has inhibited day 21 mineralization in all tested cultures in spite of the increase in day 11‐specific ALP activity. Both malonate and levamisole did not distinguish NF1 cells from the osteogenic phenotype of other cells. Essentially embryonic bone‐derived cells from 12 weeks gestation, cultured in the absence of βGP, retained their mineralization capacity, which does not increase under dexamethasone, as distinguished from NF1 cells which require βGP for mineralization and positively respond to dexamethasone. Therefore, bone‐derived NF1 cells may be useful for studying the regulation of the mineralization process.
1α,25‐dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts
Authors: Sorensen A. M. , Baran D. T.
Published: May 2005

1α,25‐Dihydroxyvitamin D3 (1α, 25‐(OH)2D3) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)2D3‐induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)2D3, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25‐(OH)2D3, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25‐(OH)2D3, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25‐(OH)2D3, suggesting that these two agents increase nuclear calcium in these osteoblast‐like cells by similar mechanisms. In summary, 1α,25‐(OH)2D3 amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25‐(OH)2D3 and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley‐Liss, Inc.
Zeolite a inhibits osteoclast‐mediated bone resorption in vitro
Authors: Schütze N. , Oursler M. J. , Nolan J. , Riggs B. L. , Spelsberg T. C.
Published: May 2005

The effects of Zeolite A on Bone resorbing activity of highly purified avian osteoclasts were analyzed. The present study demonstrates that when 100 pg/ml of acid‐treated Zeolite A is added to the media the number of pits per osteoclast is reduced 3‐fold at 24h after treatment. Secreted cathepsin B enzyme activity was also reduced 3‐fold. A similar reduction in pit number per osteoclast was measured following 48 h of treatment with Zeolite A but there appeared to be less reduction of cathepsin B enzyme activity. The effects on pit number and cathepsin B Protein activity were Zeolit dose dependent. The structure of the compound seemed to be responsible for the effects measured since compounds used to represent constituents of Zeolite A (silicon dioxide and aluminum cholride) failed to inhibit bone resorption or reduce the level of secreted cathepsin B enzyme activity. Thus the molicular architecture of Zeolite A or a derivative there of appears to be inportant. In conclusion, the data indicate that Zeolite A can inhibit bone resorption. Together with previous data on osteoblasts, this might suggest a potential positive activity of intact Zeolite A or a partial substructure of Zeolite A on bone turnover. © Wiley‐Liss, Inc.
Molecular anatomy of an endodermal gland: Investigations on mucus glycoproteins and cell turnover in Brunner's glands of didelphis verginiana using lectins and PCNA immunoreactivity
Authors: Schumacher U. , Krause W. J.
Published: May 2005

Brunner's glandsl are located in the submucosa of the proximal duodenum and are unique to mammalian species. The North American opossum (Didelphis virginiana) is generally regarded as a prototype marsupial that closely resembles fossil didelphids which can be placed at the beginning of mammalian evolution. The current investigation provided an opportunity for the analysis of secretory products from these glands in a species thought to be more closely related to earlier evolutionary forms. Extracts of Brunner's glands were subjected to SDS‐PAGE and Western Blotting. The results indicate the presence of two high molecular weight PAS‐positive glycoprotein bands. In addition to these two PAS‐positive bands, several other glycoprotein bands were detected in the high molecular weight range that bind several lectins which typically recognize O‐linked carbohydrates indicative of mucus type glycoproteins. The same lectins bind to glandular structures in tissue sections. Comparison of lectin binding sites with the pyloric glands of the stomach and duodenal goblet cells indicates that brunner's glands carbohydrate residues resemble those of the pyloric glands more closely than those of the duodenal goblet cells. The low cell turnover rate in brunner's glands is in contrast to the rapid turnover rate of goble cell precursors in the duodenal crypts. The mucus composition and the cell turnover rate correlate well with embryological data and suggest that Brunner's glands of Didelphis evolved from an epithelium more closely associated closely associated with the stomach than that of the duodenum as the topography of the gland would suggest. © Wiley‐Liss, Inc.
Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat
Authors: Gruppuso Philip A. , Boylan Joan M.
Published: May 2005

Abstract The activity and cellular localization of hepatic casein kinase II(CKII) was examined during late fetal development in the rat. Cultured fetal hepatocytes displayed constitutive CKII activity which was not further activated by growth factor exposure. Similarly, fetal liver CKII showed approximately fivefold greater activity than adult liver. The fetal hepatic activity was, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver showed similar CKII activity. In all cases, FPLC ion exchange chromatography followed by Western immunoblotting showed that immunoreactive CKII coincided with kinase activity. However, Parallel determinations of CKII activity and immunoreactive CKII levels showed a higher(five‐to sixfold) CKII specific activity in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. We hypothesize that the regulation of hepatic CKII is relevant to the mitogen‐independent proliferation displayed by fetal rat hepatocytes. © Wiley‐Liss, Inc.
Apoptosis in anititumor strategies: Modulation of cell cycle or differentiation
Authors: Darzynkiewicz Zbigniew
Published: May 2005

There is a strong evidence that administration of antitumor drugs triggers apoptotic death of target cells. A characteristic feature of appotosis is active participation of the affected cell in its demise. Attempts have been made, therefore, to potentiate the cytotoxicity of a variety of agents by modulating the propensity of cells to respond by apoptosis. Several strategies to enhance apoptosis that involve modulation of the cell cycle or differentiation are discussed. Loss of control of the G1 checkpoint in tumor cells allows one to design treatments that arrest normal cells at the checkpoint and attempt to selectively kill tumor cells with S phase specific drugs. The possibility of a restoration of the apoptosis triggering function of the tumor suppressor gene p53 when the G1 checkpoint function is abolished is expected to increase tumor cells' sensitivity to S phase poisons. Because induction of apoptosis by many antitumor drugs is cell cycle phase specific, drug combinations that preferentially trigger apoptosis at different phases of the cycle, or recruitment of cells to the sensitive phase, offer another antitumor strategy. There is also evidence that apoptosis is potentiated when cell differentiation is triggered follwing DNA damage. This observation suggests that strategies which combine DNA damaging and differentiating drugs, under conditions where the latter are administered following DNA damage caused by the former, may be successful.
Ceramide: A stress signal and mediator of growth suppression and apoptosis
Authors: Obeid Lina M. , Hannun Yusuf A.
Published: May 2005

A novel pathway termed the sphingomyelin cycle has been identified whereby membrane sphingomyelin is hydrolyzed in response to multiple extracellular stimuli (such as tumor necrosis factor α) which cause activation of regulated sphingomyelinases. The product, ceramide, has emerged as a second messenger that mediates many of the cellular effects of these extracellular stimuli. An intriguing relation exists between activation of the sphingomyelin cycle and the action of multiple stress stimuli that induce growth arrest and programmed cell death. Exogenously administered ceramide mimics these growth‐suppressing effects, including the induction of apoptosis. This review will highlight the role of the sphingomyelin cycle in signal transduction and will focus on the role and function of ceramide in the regulation of cell growth in general and apoptosis specifically.
Concept and organization of a clinical gene therapy lab
Authors: Kittler Ellen L. W. , Quesenberry Peter J.
Published: May 2005

Designing a dedicated clinical facility to meet the needs of existing and developing Gene Therapy Protocols presents a unique challenge. Here, we review some of the issues we faced and share some of our design concepts. An optimal Clinical Gene Therapy Lab must meet relevant regulatory guidelines, interface with other hospital labs as well as the clinic and patient care areas, be efficient and flexible in utilization of space, and have the potential to meet future needs without continual renovation. As clinical science expands to include more gene transfer approaches, specific laboratory areas for this type of work will become increasingly necessary.
Possible involvement of focal adhesion kinase, p125FAK, in osteoclastic bone resorption
Authors: Tanaka Sakae , Takahashi Naoyuki , Udagawa Nobuyuki , Murakami Hiroshi , Nakamura Ichiro , Kurokawa Takahide , Suda Tatsuo
Published: May 2005

Involvement of tyrosine phosphorylation in osteoclastic bone resorption was examined using osteoclast‐like multinucleated cells prepared from co‐cultures of mouse osteoblastic cells and bone marrow cells in the presence of 1α,25‐dihydroxyvitamin D3. When osteoclast‐like cells were plated on culture dishes in the presence of 10% fetal bovine serum, they were sharply stained in their peripheral region by anti‐phosphotyrosine antibody. Western blot analysis revealed that 115‐to 130‐kD proteins were tyrosine‐phosphorylated in osteoclast‐like cells. Using immunoprecipitation and immunoblotting, one of the proteins with 115–130 kD was identified as focal adhesion kinase (p125FAK), a tyrosine kinase, which is localized in focal adhesions. Immunostaining with anti‐p 125FAK antibody revealed that p125FAK was mainly localized at the periphery of osteoclast‐like cells. Herbimycin A, a tyrosine kinase inhibitor, not only suppressed tyrosine phosphorylation of p125FAK but also changed the intracellular localization of p125FAK and disrupted a ringed structure of F‐actin‐containing podosomes in osteoclast‐like cells. Antisense oligodeoxynucleotides to p125FAK inhibited dentine resorption by osteoclast‐like cells, whereas sense oligodeoxynucleotides did not. These results suggest that p125FAK is involved in osteoclastic bone resorption and that tyrosine phosphorylation of p125FAK is critical for regulating osteoclast function.
Differential regulation of p34cdc2 and p33cdk2 by transforming growth factor‐β1 in murine mammary epithelial cells
Authors: Fautsch Michael P. , Eblen Scott T. , Anders Robert A. , Burnette Rebekah J. , Leof Edward B.
Published: May 2005

Cyclin‐dependent kinases (cdks) are a family of proteins whose function plays a critical role in cell cycle traverse. Transforming growth factor‐β1 (TGF‐β1) is a potent growth inhibitor of epithelial cells. Since cdks have been suggested as possible biochemical markers for TGF‐β growth inhibition, we investigated the effect of TGF‐β1 on cdc2 and cdk2 in a normal mouse mammary epithelial cell line (MME) and a TGF‐β‐resistant MME cell line (BG18.2). TGF‐β1 decreases newly synthesized cdc2 protein levels within 6 h after addition. Coincident with this decrease in newly synthesized cdc2 protein was a marked reduction in its ability to phosphorylate histone H1. This decrease in kinase activity is not due to a change in steady‐state levels of cdc2 protein, since mRNA and total protein levels of cdc2 are not reduced until 12 h after TGF‐β1 addition. This suggests that the kinase activity of cdc2 is dependent on newly synthesized cdc2 protien. Moreover, the protein synthesis of another cyclin‐dependent kinase, cdk2, is not effected by TGF‐β1 addition, but its kinase activity is substantially reduced. Thus, it appears that TGF‐β decreases the kinase activity of both cdc2 and cdk2 by distinct mechanisms.
Dysfunction of protein kinase FA/GSK‐3α in lymphocytes of patients with schizophrenic disorder
Authors: Yang Shiaw‐Der , Yu Jau‐Song , Lee Tsong‐Tze , Yang Chuan‐Ching , Ni Mei‐Hui , Yang Yong‐Yi
Published: May 2005

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP. Mg‐dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase‐3α (kinase FA/GSK‐3α) (the activating factor of ATP.Mg‐dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK‐3α immunoblotting and kinase activity analysis in an anti‐kinase FA/GSK‐3α immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK‐3α in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK‐3α activity in the high levels of 14.8 ± 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK‐3α activity in the low levels of 2.8 ± 1.6 units/mg, indicating that the different levels of kinase FA/GSK‐3α activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide intial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK‐3α, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes. © 1995 Wiley‐Liss, Inc.
Changes associated with tyrosine phosphorylation during short‐term hypoxia in retinal microvascular endothelial cells in vitro
Authors: Koroma Barba M. , de Juan Eugene
Published: May 2005

The occlusion of capillary vessels results in low oxygen tension in adjacent tissues which triggers a signaling cascade that culminates in neovascularization. Using bovine retinal capillary endothelial cells (BRCEC), we investigated the effects of short‐term hypoxia on DNA synthesis, phosphotyrosine induction, changes in the expression of basic fibroblast growth factor receptor (bFGFR), protein kinase C (PKCα), heat shock protein 70 (HSP70), and SH2‐containing protein (SHC). The effect of protein tyrosine kinase (PTK) and phosphatase inhibitors on hypoxia‐induced phosphotyrosine was also studied. Capillary endothelial cells cultured in standard normoxic (pO2 = 20%) conditions were quiesced in low serum containing medium and then exposed to low oxygen tension or hypoxia (pO2 = 3%) in humidified, 5% CO2, 37°C, tissue culture chambers, on a time‐course of up to 24 h. DNA synthesis was potentiated by hypoxia in a time‐dependent manner. This response positively correlated with the cumulative induction of phosphotyrosine and the downregulation of bFGFR (Mr ∼ 85 kDa). Protein tyrosine kinase inhibitors, herbimycin‐A, and methyl 2,5‐dihydroxycinnamate, unlike genistein, markedly blocked hypoxia‐induced phosphotyrosine. Prolonged exposure of cells to phosphatase inhibitor, sodium orthovanadate, also blocked hypoxia‐induced phosphotyrosine. The expression of HSP70, PKCα, and SHC were not markedly altered by hypoxia. Taken together, these data suggest that short‐term hypoxia activates endothelial cell proliferation in part via tyrosine phosphorylation of cellular proteins and changes in the expression of the FGF receptor. Thus, endothelial cell mitogenesis and neovascularization associated with low oxygen tension may be controlled by abrogating signaling pathways mediated by protein tyrosine kinase and phosphatases. © 1995 Wiley‐Liss, Inc.
Role of apoptosis in the regulation of virus‐induced T cell responses, immune suppression, and memory
Authors: Welsh Raymond M. , Selin Liisa K. , Razvi Enal S.
Published: May 2005

Apoptosis is an important mechanism enabling the selection of the non‐self‐reactive T cell repertoire and for maintaining homeostasis in the immune system after it has expanded to combat infections. Highly activated, proliferating T cells become susceptible to apoptosis driven by a number of stimuli, and T cells activated during a viral infection become susceptible to “activation induced cell death” after repeated stimulation through the T cell receptor (TcR). This is a major mechanism for the immune deficiencies observed during many viral infections. During infections with a high antigen load this can lead to a selective deletion of virus‐specific cytotoxic T lymphocytes (CTL) and to the establishment of persistent infection. More commonly, the CTL control the infection first, and high levels of apoptosis in the expanded lymphocyte population occur after antigen and growth factors become limiting. This cell death does not seem to depend on TcR specificity, as the residual population contains a remarkably stable population of memory CTL precursors that approximate the frequency per CD8 cell of that seen during the peak of the acute infection. Subsequent infections with heterologous viruses result in an expansion and then an apoptotic elimination of T cells, with the consequence being a reduction in precursor CTL specific for the first virus. Thus, apoptosis shapes the quality and quantity of T cell memory. © 1995 Wiley‐Liss, Inc.
Relationships between translation of pro α1(I) and pro α2(I) mRNAs during synthesis of the type I procollagen heterotrimer
Authors: Hu Geng , Tylzanowski Przemyslaw , Inoue Hiroyuki , Veis Arthur
Published: May 2005

Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro α chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co‐localization of the synthesis of the nascent pro α1(I) and pro α2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro α‐chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro α1 homotrimer or the normal heterotrimer. Recombinant human pro α1(I) and pro α2(I) CDNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell‐free rabbit reticulocyte lysate ± canine pancreatic microsomes. Cycloheximide (100 μg/ml) was added and the polysomes were collected and fractionated on a 15–50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT‐PCR. Polysomes from K16 (heterotrimer‐producing), W8 (pro α1(I) homotrimer), and A2′ (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro α1(I) and pro α2(I) messages proceeded independently in the cell‐free, membrane‐free systems, but were coordinately altered in the presence of membrane. The cell‐free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro α chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding. © 1995 Wiley‐Liss, Inc.
Stable overexpression of human HSF‐1 in murine cells suggests activation rather than expression of HSF‐1 to be the key regulatory step in the heat shock gene expression
Authors: Mivechi Nahid F. , Shi Xaio‐You , Hahn George M.
Published: May 2005

Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF‐1). After heat shock, HSF‐1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF‐1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor‐1 (HSF‐1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF‐1 expression. Human HSF‐1 cDNA was cloned into a retroviral vector (pvhhsf‐1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF‐1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF‐1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross‐linking experiments indicate that the overexpressed human HSF‐1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF‐1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF‐1 does not increase the transcription of murine HSP‐70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47–50 kDa protein after heat shock and an apparent lack of induction of one HSP‐70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF‐1 show a 4‐fold increase in the basal expression of luciferase when the plasmids containing the human HSP‐70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF‐1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF‐1 transfected and untransfected cells. In conclusion, human HSF‐1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF‐1 after heat shock. However, the overexpression of HSF‐1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF‐1. © 1995 Wiley‐Liss, Inc.
Antithetic effects of ryanodine and ruthenium red on osteclast‐mediated bone resorption and intracellular calcium concentrations
Authors: Ritchie Candace K. , Strei Theresa A. , Maercklein Peter B. , Fitzpatrick Lorraine A.
Published: May 2005

In the process of bone remodeling, osteoclasts are responsible for resorption of bone. High levels of intracellular calcium decrease the bone resorbing activity of osteoclasts and increase detachment of osteoclasts from the bone surface. The regulatory role of intracellular calcium in bone resorption is not clearly understood. To understand this phenomenon, we studied the effects of the intracellular calcium modulators ryanodine and ruthenium red on bone resorption using the disaggregated osteoclast pit assay. Changes in intracellular calcium concentrations after treatment with these compounds were detected with the fluoroprobe fura2. With ryanodine, a significant, dose‐dependent decrease in bone resorption was detected. This inhibition of bone resorption was reversible upon the removal of ryanodine. Ryanodine increased intracellular calcium concentrations, suggesting that the mechanism of inhibition by ryanodine was via alterations in intracellular stores of calcium. After treatment with ruthenium red, osteoclasts resorbed significantly more bone compared to vehicle‐treated cells. This increase in bone resorption correlated with a decrease in intracellular calcium concentrations. The addition of parathyroid hormone or ruthenium red to osteoclast cultures containing ryanodine did not attenuate the decrease in bone resorption caused by ryanodine, suggesting that the mechanism of ryanodine inhibition of bone resorption may involve the “locking” of a calcium channel in an open position. © 1995 Wiley‐Liss, Inc.