bluuebunny/crossref_metadata_2025 huggingface dataset:
Pristane induced effects on chromatin of rat lymphoid cells
Authors: Garrett Lenora R. , Cuchens Marvin A.
Published: May 2005

The effects of pristane on the conformation of chromatin in cells isolated from the lymphoid tissues of pristane‐treated Copenhagen rats were examined by flow cytometry, thermal denaturation, sensitivity to enzymatic digestion, and histone protein analyses. Decreases were observed in the fluorescent intensities of propidium iodide (PI) stained nuclei isolated from lymphoid cells of pristane‐treated rats when compared with normal rat lymphoid nuclei. Studies to address the possible basis for the pristane‐induced changes in the DNA staining characteristics of lymphocytes demonstrated that (1) there were no decreases in the amount of DNA present in the nuclei, (2) nuclei isolated from pristane treated rats were less sensitive to thermal denaturation, as well as DNase I enzymatic digestion, and (3) there were apparent increases in the expression of the H1 histone proteins. Collectively, these results suggest that pristane elicits a conformational change in the chromatin which may be mediated by altered expression of nuclear‐associated histone proteins.
The IL‐2 mediated amplification of cellular cytotoxicity
Authors: Grimm Elizabeth Ann , Owen‐Schaub Laurie
Published: May 2005

High dose [> 1 nM or 30 IU] interleukin‐2 (IL‐2) can induce MHC ‐unrestricted killing from various lymphoid populations. Although it is well established that CD16+ NK cells are the major source of blood‐derived LAK precursors, other lymphoid cells, including several CD3+ T subsets can be a source of precursor activity. We hypothesize that most, if not all, lymphocytes with cytolytic potential may eventually express MHC‐unrestricted killing, when provided with adequate IL‐2 to intiate required secondary cytokine production. This perspective article presents our cumulative data supporting the role of secondary cytokines in the IL‐2 initiated activation of MHC‐unrestricted killing, first by our observations of synergy with the exogenously added TNFs or IL‐1s in combination with low dose IL‐2, and then by the evidence of endogenous cytokine production and response in lymphocytes stimulated with high dose IL‐2.

Understanding the amplification mechanism(s) of the various effector arms of the immune system is critical to the eventual regulation of graft rejection, autoimmune phenomena, and potentially to the treatment of cancer. Our studies have focused on the cytotoxic lymphocyte effector system, and have addressed the molecular pathways by which IL‐2 induced cytokines influence the quantity and quality of the cytotoxic lymphocyte response.

This article will review the pivotal role that IL‐2 plays in the development of CTL (MHC‐restricted antigen‐specific cytotoxic lymphocytes), followed by the description of how studies in the CTL system led to the observation that IL‐2 alone can activate a heterogeneous collection of MHC‐unrestricted killer lymphocytes, originally known as “Lymphokine Activated Killers” or LAK. We will then describe experiments performed in out laboratory over the past several years demonstrating the positive regulation of LAK activity by exogenous addition of TNF‐α, TNF‐β, IL‐1α or IL‐1β. Finally, we will summarize our data and propose, in the context of the current literature, the endogenous autocrine/paracrine amplification network of secondary cytokines operative in the generation of LAK.
Long‐term expression of gene introduction into normal human T‐lymphocytes by retroviral‐mediated gene transfer
Authors: Fauser A. A.
Published: May 2005

Human T‐lymphocytes are long lived, easily accessible, mature, and capable of proliferation. They are theoretically a suitable target for retroviral mediated gene transfer. To test this hypothesis, normal human T‐cells obtained from bone marrow and peripheral blood were stimulated with phytohemagglutinin (PHA) and infected 24 h later with the retroviral vector N2 which carries the bacterial neo gene. T‐lymphocytes were propagated in culture for up to 14 weeks with interleukin‐2 (IL‐2). Analysis by whole cell RNA dot/blot using a single stranded RNA probe demonstrated persistent expression of the neo gene. Preliminary functional studies revealed that both helper and suppressor functions were preserved in the infected cells in culture. These results demonstrate that normal T‐cells are capable of long‐term expression of genes introduced by retroviral mediated gene transfer and are potential target cells for somatic gene therapy.
Characterization of Synthetic Peptide Substrates for p34cdc2 Protein Kinase
Authors: Marshak Daniel R. , Vandenberg Mark T. , Bae Young Seuk , Yu Ii Je
Published: May 2005

Synthetic peptide substrates for the cell division cycle regulated protein kinase, p34cdc2, have been developed and characterized. These peptides are based on the sequences of two known substrates of the enzyme, Simian Virus 40 Large T antigen and the human cellular recessive oncogene product, p53. The peptide sequences are H‐A‐D‐A‐Q‐H‐A‐T‐P‐P‐K‐K‐K‐R‐K‐V‐E‐D‐P‐K‐D‐F‐OH (T antigen) and H‐K‐R‐A‐L‐P‐N‐N‐T‐S‐S‐S‐P‐Q‐P‐K‐K‐K‐P‐L‐D‐G‐E‐Y‐NH2 (p53), and they have been employed in a rapid assay of phosphorylation in vitro. Both peptides show linear kinetics and an apparent Km of 74 and 120 μM, respectively, for the purified human enzyme. The T antigen peptide is specifically phosphorylated by p34cdc2 and not by seven other protein serine/threonine kinases, chosen because they represent major classes of such enzymes. The peptides have been used in whole cell lysates to detect protein kinase activity, and the cell cycle variation of this activity is comparable to that measured with specific immune and affinity complexs of p34cdc2. In addition, the peptide phosphorylation detected in mitotic cells is depleted by affinity adsorption of p34cdc2 using either antibodies to p34cdc2 or by immobilized p13, a p34cdc2‐binding protein. Purification of peptide kinase activity from mitotic HeLa cells yields an enzyme indistinguishable from p34cdc2. These peptides should be useful in the investigation of p34cdc2 protein kinases and their regulation throughout the cell division cycle.
A combination of posttranslational modifications is responsible for the production of neuronal α‐tubulin heterogeneity
Authors: Eddé Bernard , Rossier Jean , Caer Jean‐Pierre Le , Berwald‐Netter Yoheved , Koulakoff Annette , Gros François , Denoulet Philippe
Published: May 2005

We describe the presence of α‐tubulin and MAP2 acetyltransferase activities in mouse brain. The enzyme(s) copurified with microtubules through two cycles of assembly‐disassembly. Incubation of microtubule proteins with [3H]acetyl CoA resulted in a strong labeling of both α‐tubulin and MAP2. To determine the site of the modification, tubulin was purified and digested with Glu‐C endoproteinase. A unique radioactive peptide was detected and purified by HPLC. Edman degradation sequencing showed that this peptide contained €N‐acetyllysine at position 40 of the α‐tubulin molecule. This result demonstrates that mouse brain α‐tubulin was acetylated at the same site as in Chlamydomonas. Isoelectric focusing analysis showed that acetylated α‐tublin was resolved into five isoelectric variants, denoted α3 and α5 to α8. This heterogeneity is not due to acetylation of other sites but results from a single acetylation of Lys40 of an heterogeneous population of α‐tubulin isoforms. These isoforms are produced by posttranslational addition of one to five glutamyl units. Thus, neuronal α‐tubulin is extensively modified by a combination of modifications including acetylation, glutamylation, tyrosylation, and other yet unknown modifications.
Fibroblast heterogeneity in collagenolytic response to cyclosporine
Authors: Tipton David A. , Stricklin George P. , Dabbous Mustafa Kh.
Published: May 2005

To investigate the mechanism of cyclosporine (CS)‐induced fibrotic gingival enlargement, the effect of CS on the collagenolytic activities of 14 different human gingival fibroblast strains derived from healthy individuals with non‐inflammed gingiva was examined in vitro. There was marked heterogeneity among individuals in basal levels of collagenase activity, and there was also variation among the subpopulations derived from one strain. Fibroblasts from different individuals also varied markedly in their collagenolytic response to CS (0.1 to 0.75 μg/ml). In most strains, CS decreased collagenase activity, but in some, the drug caused no change or significantly increased activities. In most of the subpopulations CS significantly decreased collagenolytic activity.

Two of the fibroblast strains and the subpopulations described above were examined for the production of immunoreactive collagenase and tissue inhibitor of metalloproteinase (TIMP). The two strains made similar amounts of collagenase, but differed markedly in TIMP levels; CS affected their collagenase production differently but had similar effects on TIMP. Among the subpopulations there was variation in the production of collagenase, although none made detectable levels of TIMP; they also varied in the production of both proteins in response to CS. In two of the subpopulations and in both strains at some concentrations, the effect of CS on the relative levels of collagenase and TIMP could account for the decreased collagenase activity; i.e., the level of collagenase was unchanged or decreased, and TIMP production was unchanged or increased.

This study demonstrates the variation among individuals as well as intrastrain heterogeneity of human gingival fibroblasts with regard to collagenase activity and the production of collagenase and TIMP. The heterogeneity of the collagenolytic response of different gingival fibroblast strains and their subpopulations to CS treatment may partly explain the susceptibility of only some individuals to CS‐induced gingival enlargement.
Transcriptional element H4‐site II of cell cycle regulated human H4 histone genes is a multipartite protein/DNA interaction site for factors HiNF‐D, HiNF‐M, and HiNF‐P: Involvement of phosphorylation
Authors: van Wijnen Andre J. , Ramsey‐Ewing Anna L. , Bortell Rita , Owen Thomas A. , Lian Jane B. , Stein Janet L. , Stein Gary S.
Published: May 2005

Cell cycle regulated gene expression was studied by analyzing protein/DNA interactions occurring at the H4‐Site II transcriptional element of H4 histone genes using several approaches. We show that this key proximal promoter element interacts with at least three distinct sequence‐specific DNA binding activities, designated HiNF‐D, HiNF‐M, and HiNF‐P. HiNF‐D binds to an extended series of nucleotides, whereas HiNF‐M and HiNF‐P recognize sequences internal to the HiNF‐D binding domain. Gel retardation assays show that HiNF‐D and HiNF‐M each are represented by two distinct protein/DNA complexes involving the same DNA binding activity. These results suggest that these factors are subject to post‐translational modifications. Dephosphorylation experiments in vitro suggest that both electrophoretic mobility and DNA binding activities of HiNF‐D and HiNF‐M are sensitive to phosphatase activity. We deduce that these factors may require a basal level of phosphorylation for sequence specific binding to H4‐Site II and may represent phosphoproteins occurring in putative hyper‐ and hypo‐phosphorylated forms. Based on dramatic fluctuations in the ratio of the two distinct HiNF‐D species both during hepatic development and the cell cycle in normal diploid cells, we postulate that this modification of HiNF‐D is related to the cell cycle. However, in several tumor‐derived and transformed cell types the putative hyperphosphorylated form of HiNF‐D is constitutively present. These data suggest that deregulation of a phosphatase‐sensitive post‐translational modification required for HiNF‐D binding is a molecular event that reflects abrogation of a mechanism controlling cell proliferation. Thus, phosphorylation and dephosphosphorylation of histone promoter factors may provide a basis for modulation of protein/DNA interactions and H4 histone gene transcription during the cell cycle and at the onset of quiescence and differentiation.
Bellini, carpaccio, and receptors in the central nervous system
Authors: Merrill Jean E.
Published: May 2005

With the convergence of science from the fields of neurobiology and immunology, many exciting and challenging surprises have emerged regarding cytokines, neuroendocrine hormones, neuropeptides, excitatory amino acids, and their receptors. For some time neurobiologists have known that subsets of neural cells had different receptors for the same ligand. Those subsets of cells could be as different as neurons and astrocytes and as closely related as astrocytes from different lineages or anatomical areas. The neurobiological puzzle has been to determine the functional meaning of these differences. Immunologists in contrast have long understood the clear cut differences between T and B lymphocytes or T helper/inducer and T cytotoxic/suppressor cells and their response to cytokines. However, it is only very recently that they have discovered preferential use by these cells of different receptors for an identical cytokine ligand. Indeed, identical cytokines in the central nervous system and immune response may induce their pleiotropic responses by utilizing different receptors in these two systems. Immunologic paradigms may help neurobiologists predict the existence of subsets of neural cells and their function. Likewise, neurobiology may enable immunologists to predict roles for receptors in gene families as well as the existence of as yet unidentified receptors.
Purification and characterization of an osteoclast membrane glycoprotein with homology to manganese superoxide dismutase
Authors: Oursler Merry Jo , Li Ling , Osdoby Philip
Published: May 2005

The osteoclast is the specialized multinucleated cell primarily responsible for the degradation of the inorganic and organic components of bone matrix. Isolated avian osteoclasts have been used to immunize mice and generate an osteoclast‐directed monoclonal antibody library (J. Cell Biology, 100:1592). A subset of these monoclonal antibodies recognizes antigens which are expressed on osteoclasts and which are absent or nearly so on multinucleated giant cells formed in vitro from monocyte or marrow mononuclear cells. One of these antibodies, designated 121F, has been used to identify and purify an osteoclast plasma membrane‐associated glycoprotein. Western blot analysis on disulfide bond‐reduced extracts from osteoclasts or multinucleated giant cells formed in vitro demonstrates that the 121F antibody recognizes a 150 kDa protein detectable only in osteoclasts. This high molecular weight protein has been purified by a combination of immunoaffinity and gel filtration chromatography procedures, in conjunction with electroelution of a single band from SDS‐polyacrylamide gels. Silver staining of the purified antigen on SDS‐polyacrylamide gels has revealed a single protein species larger than 200 kDa in its unreduced form and 150 kDa when disulfides are reduced. Isoelectric focusing of the purified antigen reveals a single species, having a neutral pl point of 6.95. Whereas endoglycosidase treatment and lectin affinity chromatographic analyses demonstrate that the antigen recognized by the 121F antibody possesses complex N‐linked sugars, trifluoromethanesulfonic acid treatment indicates there are no additional O‐linked carbohydrate components. Periodate oxidation and monosaccharide hapten inhibition studies provide no evidence for the antigenic epitope bound by the 121F antibody being carbohydrate in nature. Although the native antigen is blocked at its N‐terminus, amino acid analysis of a hydroxylamine generated peptide disclosed a striking relationship between the osteoclast antigen recognized by the 121F monoclonal antibody and manganese and iron superoxide dismutase. Therefore, in addition to serving as a distinguishing cell type–specific marker for osteoclasts, this cell surface glycoprotein may function directly in osteoclast‐mediated bone resorption.
Buffering of intracellular calcium in response to increased extracellular levels in mortal, immortal, and transformed human breast epithelial cells
Authors: Ochieng Josiah , Tahin Quivo S. , Booth Charles C. , Russo Jose
Published: May 2005

Extracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF‐10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF‐10A) and oncogene transformed (MCF‐10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF‐10M, MCF‐10A, and MCF‐10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF‐10M, but not in MCF‐10A and MCF‐10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.
The effect of a phorbol ester on the lipid microviscosity of two endoplasmic reticulum membrane fractions isolated from krebs II ascites cells
Authors: Maltseva E. L. , Palmina N. P. , Pryme I. F.
Published: May 2005

This paper deals with microviscosity parameters and thermoinduced structural transitions in the lipids of smooth and heavy rough endoplasmic reticulum membranes isolated from Krebs II ascites cells incubated with the phorbol ester 12–0‐tetradecanoyl‐phorbol‐13‐acetate. The phorbol ester was found to bring about a threefold increase in the microviscosity of the lipids in heavy rough membranes. Spin probe I (2,2,6,6‐tetrahydro‐4‐capryloyl‐oxypiperidine‐1‐oxyl), localized in the surface layer of the membrane lipids, gave results which indicate an increased number of thermoinduced structural transitions in the smooth membranes in the treated cells due to the transitions occurring at relatively low temperature and a decreased number of such transitions in the heavy rough fraction especially at high temperature. For 5,6‐benzo‐2,2,4,4‐tetramethyl‐1,2,3,4‐tetrahydro‐γ‐carboline‐oxyl, probe II, mainly distributed in the annular lipids, a decrease in the number of low temperature transitions in the smooth fraction was observed, while an increase occurred in the heavy rough one. The results obtained are discussed in terms of the effect of phorbol esters as promoters of tumor progression.
Secretion of mutant leucine‐specific binding proteins with internal deletions in Escherichia coli
Authors: Adams Mark D. , Oxender Dale L.
Published: May 2005

The leucine‐specific binding protein, encoded by the livK gene, is located in the periplasm of E. coli. The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm. C‐terminal deletions or fusions of the livK gene to trpA (encoding the α subunit of tryptophan synthetase) were secreted with little loss of efficiency [1]. A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein. Between 16 and 113 amino acids were deleted in the amino‐terminal one‐third of the protein. A few of these deletions were located within a few amino acids of the signal sequence processing site. Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal. Secondary structure predictions suggested that the α‐helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components. These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion. During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS‐PAGE, was much greater than could be accounted for by processing of the signal sequence. This anomalous mobility on gels, however, could be eliminated by performing SDS‐PAGE in the presence of 6 M urea.
Regulation of expression of the growth‐state‐related genes 2F1 and 2A9 during entry of quiescent smooth muscle cells into the cell cycle
Authors: Kindy Mark S. , Brown Karen E. , Sonenshein Gail E.
Published: May 2005

Vascular smooth muscle cells (SMCs) play a key role in the development of major arteries. Furthermore, abnormal growth of vascular smooth muscle cells has been implicated in the progression of major diseases of the cardiovascular system. Here, we report detection in primary cultures of bovine vascular smooth muscle cells of mRNA for two growth‐state‐related genes, 2F1 and 2A9, which code for a mitochondrial ADP/ATP carrier and calcyclin, respectively, and on the characterization of their cell cycle expression. Cultures of exponentially growing smooth muscle cells were made quiescent by serum deprivation. Upon readdition of serum, cells entered the cell cycle synchronously; DNA synthesis began 12 h post‐serum addition. Levels of 2F1 and 2A9 RNA were low in quiescent cells and increased between 2 and 4 h post‐serum addition. No changes in the rates of transcription of the 2F1 or 2A9 genes were detected by nuclear run‐off assays during the time course. Thus the regulation of changes in expression of 2F1 and 2A9 in early G1 is mediated post‐transcriptionally.
Glucocorticoid regulation of rat liver urate oxidase
Authors: Raab Linda Symons , Decker Glenn L. , Jonas Adam J. , Kaetzel Marcia A. , Dedman John R.
Published: May 2005

Urate oxidase, an enzyme involved in purine catabolism, comprises the crystalline core of rat liver peroxisomes. An affinity‐purified monospecific antibody was developed to study the expression of urate oxidase protein levels. Immunoreactive urate oxidase was not detectable in prenatal liver; however, it is present at low levels after birth until approximately day 15 (postnatal age); expression sharply increases just prior to day 20, after which the enzyme is maintained at adult levels. This pattern of expression was similar to that of another peroxisomal enzyme, catalase; these developmental increases reflect the increase in peroxisomal number. Administration of exogenous glucocorticoid hormone to 10‐day‐old rats resulted in a precocious rise (2.5‐fold) in urate oxidase levels. Adrenalectomy at 10 days of age did not cause decreased levels in the fourth week of life. In adult animals, while exogenous glucocorticoid administration did not influence urate oxidase levels, adrenalectomy at 60 days of age decreased urate oxidase levels to 40 percent of control levels. Subsequent administration of exogenous glucocorticoid hormone restored urate oxidase to normal levels. Parallel studies of catalase levels indicate that this glucocorticoid‐sensitive response is not generalized for all peroxisomal proteins. Our results suggest that peroxisomes proliferate during early postnatal development, but after this process is complete, the biogenesis of individual peroxisomal proteins may be independently regulated.
Co‐regulation of heme oxygenase and erythropoietin genes
Authors: Abraham N. G. , Levere R. D. , Lin J. H.‐C. , Beru N. , Hermine O. , Goldwasser E.
Published: May 2005

The mechanism responsible for the accumulation of heme oxygenase and erythropoietin (epo) transcripts due to cobalt chloride (CoCl2) administration was investigated in rat kidney using a rat heme oxygenase and mouse epo probes. We found an increase of heme oxygenase transcripts in kidney in response to CoCl2. Quantitative evaluation of the heme oxygenase mRNA changes, by scanning densitometry, indicated that the levels of mRNA encoding heme oxygenase were increased by about fiftyfold in rat kidney after administration of CoCl2. That the increase in heme oxygenase mRNA levels resulted from enhanced transcription of the heme oxygenase gene was confirmed by nuclear runoff using isolated rat kidney nuclei after CoCl2 administration. Transcription of the heme oxygenase gene is greatly increased in rat kidney within 1 hr of administration of CoCl2 as evidenced from the levels of 32P‐UTP incorporation into the specific transcript. Time course studies showed that stimulation of transcription was increased about fortyfold 3 hr after CoCl2 administration. This stimulation is the most rapid transcriptional response to heavy metals yet described. In addition, Northern blot analysis demonstrated that epo mRNA was first detected 4 hr following CoCl2 administration and reached a maximum at 5 hr. On the other hand, PCR analysis indicated that epo mRNA was increased as early as 1 hr following CoCl2 administration. The fact that CoCl2 caused increased transcription of both the epo and heme oxygenase genes suggests that a common mechanism may be involved in the regulation of these two genes by the heavy metal ion.
Induction of interleukin‐1β production in human dermal fibroblasts by interleukin‐1α and tumor necrosis factor‐α. Involvement of protein kinase‐dependent and adenylate cyclase‐dependent regulatory pathways
Authors: Mauviel Alain , Temime Nathalie , Charron Dominique , Loyau Gérard , Pujol Jean‐Pierre
Published: May 2005

It has previously been demonstrated that interleukin‐1 (IL‐1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL‐1β production by cultured human dermal fibroblasts. We have shown that IL‐1β is constitutively expressed as a cell‐associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL‐1α and tumor necrosis factor‐α (TNF‐α) exerted a dose‐depdent stimulation on the production of the cell‐associated IL‐1β, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL‐1β production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 μg/ml) of PGE2. In contrast, higher concentrations (0.1 and 1. μg/ml) of PGE2, as well as exogenous dibutyryl‐cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL‐1α and TNF‐α. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL‐1β expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2‐induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL‐1α and TNF‐α, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL‐1β induction by the two cytokines studied.
Release of basic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: A trivial phenomenon or a novel secretion mechanism?
Authors: Mignatti Paolo , Rifkin Daniel B.
Published: May 2005

Basic fibroblast growth factor (bFGF), a potent angiogenesis inducer, lacks a signal sequence. Therefore, it has been proposed that bFGF is primarily released from dead or damaged cells. Other proteins devoid of secretion signals, interleukin 1β (IL‐1β) and the muscle lectin L‐14, have been shown to be released via exocytosis, a novel secretion pathway independent of the “classic” endoplasmic reticulum‐Golgi route. In the light of these findings and of our own recent results, we discuss evidence that bFGF can be released from single, uninjured cells and mediate functions is an autocrine manner. As is the case for IL‐1β and L‐14, externalization of bFGF may occur via exocytosis, a pathway utilized during development and differentiation.
A metalloproteinase inhibitor as an inhibitor of neovascularization
Authors: Moses Marsha A. , Langer Robert
Published: May 2005

Metalloproteinases and their endogenous inhibitors are key components of an enzyme system which is important in a number of fundamental biochemical and cellular processes. Our recent work has focused on the role of a particular metalloproteinase, collagenase, and the role of an endogenous inhibitor of this enzyme in the control of neovascularization. The proteolytic degradation of extracellular matrix components by capillary endothelial cells (EC) has been shown to be one of the key prerequisites of the angiogenic process. As part of a study of the effect(s) of the inhibition of collagenase on neovascularization, we have recently reported the purification, characterization and partial NH2‐terminal sequence of a cartilage‐derived inhibitor (CDI) of angiogenesis in vivo and in vitro. Evidence is presented which suggests that one means of controlling deregualted vascular growth characteristic of a number of “angiogenic diseases” may be at the level of the control of metalloproteinase activity.
Interleukin 4 inhibits murine osteoclast formation in vitro
Authors: Shioi Atsushi , Teitelbaum Steven L. , Ross F. Patrick , Welgus Howard G. , Suzuki Hidenori , Ohara Junichi , Lacey David L.
Published: May 2005

Interleukin 4 (IL‐4) is a product of activated T cells and mast cells with effects on immunologic and hematopoietic processes. We now report that IL‐4 inhibits the formation of osteoclasts from murine bone marrow cells cocultured with stromal cells. Numerous (3,000–4,000 cells/2 cm2) tartrate‐resistant acid‐phosphatase‐positive multinucleated cells with the capacity to generate cAMP in response to salmon calcitonin (ED50 = 10−10 M) developed within 10–12 days of culture. IL‐4 (ID50 = 10 U/ml) inhibited osteoclast generation in doses similar to those that induce proliferation of IL‐4‐responsive T cells. Additionally, the rat antimurine IL‐4 monoclonal antibody 11B11 antagonizes the IL‐4‐inhibitory effect on osteoclast formation. These results suggest that IL‐4 impedes agonist‐induced in vitro bone resorption by inhibiting osteoclastogenesis.
Protein/DNA interactions involving ATF/AP1‐, CCAAT‐, and HiNF‐D‐related factors in the human H3‐ST519 histone promoter: Cross‐competition with transcription regulatory sites in cell cycle controlled H4 and H1 histone genes
Authors: van Wijnen André J. , Lian Jane B. , Stein Janet L. , Stein Gary S.
Published: May 2005

Protein/DNA interactions of the H3‐ST519 histone gene promoter were analyzed in vitro. Using several assays for sequence specificity, we established binding sites for ATF/AP1‐, CCAAT‐, and HiNF‐D related DNA binding proteins. These binding sites correlate with two genomic protein/DNA interaction domains previously established for this gene. We show that each of these protein/DNA interactions has a counterpart in other histone genes: H3‐ST519 and H4‐F0108 histone genes interact with ATF‐ and HiNF‐D related binding activities, whereas H3‐ST519 and H1‐FNC16 histone genes interact with the same CCAAT‐box binding activity. These factors may function in regulatory coupling of the expression of different histone gene classes. We discuss these results within the context of established and putative protein/DNA interaction sites in mammalian histone genes. This model suggests that heterogeneous permutations of protein/DNA interaction elements, which involve both general and cell cycle regulated DNA binding proteins, may govern the cellular competency to express and coordinately control multiple distinct histone genes.
The endocytic hyaluronan receptor in rat liver sinusoidal endothelial cells is Ca+2‐independent and distinct from a Ca+2‐dependent hyaluronan binding activity
Authors: Yannariello‐Brown Judith , McGary Carl T. , Weigel Paul H.
Published: May 2005

Isolated and cultured rat liver sinusoidal endothelial cells (LECs) retain the ability to specifically bind 125I‐hyaluronan (HA) internalize it using a coated pit pathway [Biochem J, 257:875–884, 1989]. Here we have determined the effect of Ca+2 on the binding and endocytosis of HA by LECs. 125I‐HA binding to intact LECs at 4°C occurred both in the absence (10 mM EGTA) or the presence of physiologic concentrations of Ca+2 (1.8 mM). However, the specific binding of 125I‐HA to LECs increased linearly with increasing Ca+2 concentrations. After permeabilization with the nonionic detergent digitonin, the Ca+2‐independent HA binding activity increased ∼743%, while the Ca+2‐dependent binding activity was enhanced only ∼46%. Therefore, the Ca+2‐dependent HA binding activity appears not to be intracellular, whereas the Ca+2‐independent HA receptor is found both inside LECs and on the cell surface. When LECs were allowed to endocytose 125I‐HA at 37°C in 10 mM EGT A or in 1.8 mM Ca+2, no differences were seen in the extent or rate of endocytosis. When LECs were allowed to endocytose 125I‐HA in the presence of 10mM Ca125, the amount of cell‐associated radiocctivity increased appoximately 20–50‐fold. However, this additional cell‐associated 125I‐HA was not sensitive to hyperosmolarity and was removed by washing the cells in 10mM EGTA at 4°C. Therefore, the Ca+2‐dependent cell‐associated 125I‐HA had accumulated on the cell surface and had not been internalized. From these studies we conclude the LECs have at least two trypes of specfic HA binding sites. One, the previously characterized HA receptor, is Ca+2‐indepoendent, localized both extracellularly and intracellular, and medicates he efficient binding and subsequent endocytosis of HA using a coated pit pathway. The other newly recognized HA binding activity is Ca+2‐dependent, localized wxtracellularly, and is not responsible for the endocytosis of HA in rat LECs.
Characterization of the human myeloid cell nuclear differentiation antigen: Relationship to interferon‐inducible proteins
Authors: Burrus G. R. , Briggs J. A. , Briggs R. C.
Published: May 2005

The human myeloid cell nuclear differentiation antigen (MNDA) is expressed specifically in cells of the granulocyte/monocyte lineage. The MNDA has been isolated by using a monoclonal antibody affinity matrix and reversed‐phase high performance liquid chromatography. Its NH2‐terminal sequence has been obtained, as well as additional sequence information derived from peptides produced by cyanogen bromide and SV8 protease cleavages. Meaningful similarities were observed in extended regions between the MNDA and the reported β interferon‐inducible proteins, 202 and 204, from Ehrlich ascites mouse tumor cells. An amphipathic, basic α‐helical region, showing no similarity to the 202 and 204 proteins, exhibited close similarity to a region in the interferon response factor‐2, a protein which binds the interferon stimulated response element. The relatively high number of S(T)PXX motifs present in the partial amino acid sequence of the MNDA, described herein, suggests that the MNDA binds DNA and is a transcription factor.
Defective insulin secretion in niddm: Integral part of a multiplier hypothesis
Authors: Robertson R. Paul
Published: May 2005

Non‐insulin dependent diabetes mellitus (NIDDM) is characterized by a specific defect in glucose recognition by the pancreatic islet beta cell. This is in clear distinction to patients with insulin dependent diabetes mellitus (IDDM) who undergo pancreatic islet beta cell death and no longer have the ability to synthesize, store, and release insulin. Defective glucose‐induced first phase insulin responses in patients with NIDDM can be partially restored by exogenous insulin treatment and by other pharmacologic therapy. These observations provide strength for the theory of glucose desensitization of the pancreatic beta cell as an important secondary defect in the pathogenesis of abnormal insulin secretion in NIDDM. However, even though defective insulin secretion is an essential part of the pathogenesis of NIDDM, in itself it is not sufficient. A multiplicative effect is required involving interaction between tissue resistance to insulin action and defective insulin secretion whose product is the syndrome of NIDDM.
Protein kinase C involved in zymosan‐induced release of arachidonic acid and superoxide but not in calcium ionophore‐elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate
Authors: Duyster Justus , Schulze‐Specking Agnes , Fitzke Edith , Dieter Peter
Published: May 2005

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan‐ and phorbol‐ester‐induced release of arachidonic acid, prostaglandin E2, and superoxide was dose‐dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan‐ and phorbol‐ester‐induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.
1α,25‐Dihydroxyvitamin D3 modulation in lipid metabolism in established bone marrow‐derived stromal cells, MC3T3‐G2/PA6
Authors: Shionome Manabu , Shinki Toshimasa , Takahashi Naoyuki , Hasegawa Kohji , Suda Tatsuo
Published: May 2005

MC3T3‐G2/PA6 (PA6) cells established from newborn mouse calvaria are preadipocytic stromal cells, which differentiate into adipocytes in response to glucocorticoids. We examined the effects of 1α,25‐dihydroxyvitamin D3[1α,25(OH)2D3] on adipogenesis in PA6 cells were cultured with 10−8 M dexamethasone, adipocytes containing oil red O‐positive droplets first appeared on day 7 (3 days after confluence was attained) and the maximal synthesis of neutral lipids occurred on day 12. Simultaneous addition of 1α,25(OH)2D3 at 10−9 M completely blocked this dexamethasone‐induced neutral lipid synthesis throughout the 14‐day culture period. Dose‐response studies of vitamin D3 derivatives showed that 1α,25(OH)2D3 was the most potent in inhibiting neutral lipid synthesis in PA6 cells, followed by 1α‐hydroxyvitamin D3, 25‐hydroxyvitamin D3 and 24R,25‐dihydroxyvitamin D3, in that order. Dexamethasone greatly enhanced incorporation of [14C]‐acetic acid into triacylglycerol in PA6 cells. The incorporation was markedly inhibited by the addition of 10−9 M 1α,25(OH)2D3 Instead, 1α,25(QH)2D3 greatly increased incorporation of [14C]‐acetic acid into phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, irrespective of the presence or absence of dexamethasone. These results suggest that 1α,25(OH)2D3 modulation of lipid metabolism in bone marrow stromal cells is receptor mediated.
Modulation of cultured chicken growth plate chondrocytes by transforming growth factor‐β1 and basic fibroblast growth factor
Authors: Wu Licia N. Y. , Genge Brian R. , Ishikawa Yoshinori , Wuthier Roy E.
Published: May 2005

Expression of several cellular and matrix proteins which increase significantly during the maturation of growth plate cartilage has been shown to be affected by various endocrine and autocrine factors. In the studies reported here, transforming growth factor‐β (TGF‐β1) and basic fibroblast growth factor (bFGF) were administered to primary cultures of avian growth plate chondrocytes at pre‐ or post‐confluent stages to study the interplay that occurs between these factors in modulating chondrocytic phenotype. Added continuously to pre‐confluent chondrocytes, TGF‐β1 stimulated the cells to produce abundant extracellular matrix and multilayered cell growth; cell morphology was altered to a more spherical configuration. These effects were generally mimicked by bFGF, but cell shape was not affected. Administered together with TGF‐β1, bFGF caused additive stimulation of protein synthesis, and alkaline phosphatase (AP) activity was markedly, but transiently enhanced. During this pre‐confluent stage, TGF‐β1 also increased fibronectin secretion into the culture medium. Added to post‐confluent cells, TGF‐β1 alone caused a dosage‐dependent suppression of AP activity, but bFGF alone did not. Under these conditions, TGF‐β1 and bFGF had little effect on general protein synthesis, but TGF‐β1 alone caused large, dosage‐dependent increases in synthesis of fibronectin, and to some extent type II and X collagens. Given together with bFGF, TGF‐β1 synergistically increased secretion of fibronectin. These findings reveal that regulation of phenotypic expression in maturing growth plate chondrocytes involves complex interactions between growth factors that are determined by timing, level, continuity, and length of exposure.
Teratocarcinoma F9 cells induced to differentiate with sodium butyrate produce both tissue‐type and urokinase‐type plasminogen activators
Authors: Takeda Masashi , Kosaka Mitsuko , Nishina Yukio , Sawada Ken , Matsumoto Keishi , Nishimune Yoshitake
Published: May 2005

Sodium butyrate (NaB) can induce teratocarcinoma cell differentiation as retinoic acid (RA). However, the function of these two agents seems to be a little different [Kosaka et al., Exp Cell Res, 192 : 46–51, 1991]. F9 cells treated with NaB synthesize both tissue‐type (tPA) and urokinase‐type (uPA) plasminogen activator, though RA induces only tPA production. Urokinase‐type PA is demonstrated to exist in association with membrane and to localize its activity to the close environment of the cell surface. This may cause the specific cell morphology and characteristics of differentiated F9 cells induced with NaB.
The prospects for domesticating milk protein genes
Authors: Hennighausen Lothar
Published: May 2005

It 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

Bistratene 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

During 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.