bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase
Authors: Huang Chuanshu , Ma Wei-Ya , Ryan Clarence A. , Dong Zigang
Published: July 2002

Solar UV irradiation is the causal factor for the increasing incidence of human skin carcinomas. The activation of the transcription factor activator protein-1 (AP-1) has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. We demonstrate that proteinase inhibitor I (Inh I) and II (Inh II) from potato tubers, when applied to mouse epidermal JB6 cells, block UV-induced AP-1 activation. The inhibition appears to be specific for UV-induced signal transduction for AP-1 activation, because these inhibitors did not block UV-induced p53 activation nor did they exhibit any significant influence on epidermal growth factor-induced AP-1 transactivation. Furthermore, the inhibition of UV-induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-Jun N-terminal kinases as well as P38 kinases. Considering the important role of AP-1 in tumor promotion, it is possible that blocking UV-induced AP-1 activity by Inh I or Inh II may be functionally linked to irradiation-induced cell transformation.
Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells
Authors: Le Maout Sophie , Brejon Manuel , Olsen Olav , Merot Jean , Welling Paul A.
Published: July 2002

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in
Xenopus
oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming
bona fide
IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.
Multi-responsiveness of single anterior pituitary cells to hypothalamic-releasing hormones: A cellular basis for paradoxical secretion
Authors: Villalobos Carlos , Núñez Lucía , Frawley L. Stephen , García-Sancho Javier , Sánchez Ana
Published: July 2002

The classic view for hypothalamic regulation of anterior pituitary (AP) hormone secretion holds that release of each AP hormone is controlled specifically by a corresponding hypothalamic-releasing hormone (HRH). In this scenario, binding of a given HRH (thyrotropin-, growth hormone-, corticotropin-, and luteinizing hormone-releasing hormones) to specific receptors in its target cell increases the concentration of cytosolic Ca
2+
([Ca
2+
]
i
), thereby selectively stimulating the release of the appropriate hormone. However, “paradoxical” responses of AP cells to the four well-established HRHs have been observed repeatedly with both
in vivo
and
in vitro
systems, raising the possibility of functional overlap between the different AP cell types. To explore this possibility, we evaluated the effects of HRHs on [Ca
2+
]
i
in single AP cells identified immunocytochemically by the hormone they stored. We found that each of the five major AP cell types contained discrete subpopulations that were able to respond to several HRHs. The relative abundance of these multi-responsive cells was 59% for lactotropes, 33% for thyrotropes, and in the range of 47–55% for gonadotropes, corticotropes, and somatotropes. Analysis of prolactin release from single living cells revealed that each of the four HRHs tested were able to induce hormone release from a discrete lactotrope subpopulation, the size of which corresponded closely to that in which [Ca
2+
]
i
changes were induced by the same secretagogues. When viewed as a whole, our diverse functional measurements of multi-responsiveness suggest that hypothalamic control of pituitary function is more complicated than previously envisioned. Moreover, they provide a cellular basis for the so-called “paradoxical” behavior of pituitary cells to hypothalamic hypophysiotropic agents.
Activation of calcium-dependent potassium channels in rat brain neurons by neurotrophin-3 and nerve growth factor
Authors: Holm Ninna R. , Christophersen Palle , Olesen Søren P. , Gammeltoft Steen
Published: July 2002

The neurotrophins are signaling factors that are essential for survival and differentiation of distinct neuronal populations during the development and regeneration of the nervous system. The long-term effects of neurotrophins have been studied in detail, but little is known about their acute effects on neuronal activity. Here we use permeabilized whole-cell patch clamp to demonstrate that neurotrophin-3 (NT-3) and nerve growth factor activate calcium-dependent, paxilline-sensitive potassium channels (BK channels) in cortical neurons. Application of NT-3 or nerve growth factor produced a rapid and gradual rise in BK current that was sustained for 30–50 min; brain-derived neurotrophic factor, ciliary neurotrophic factor, and insulin-like growth factor-1 had no significant effect. The response to NT-3 was blocked by inhibitors of protein kinases, phospholipase C, and serine/threonine protein phosphatase 1 and 2a. Omission of Ca
2+
from the extracellular medium prevented the NT-3 effect. Our results indicate that NT-3 stimulates BK channel activity in cortical neurons through a signaling pathway that involves Trk tyrosine kinase, phospholipase C, and protein dephosphorylation and is calcium-dependent. Activation of BK channels may be a major mechanism by which neurotrophins acutely regulate neuronal activity.
Apical sorting of hepatitis B surface antigen (HBsAg) is independent of N-glycosylation and glycosylphosphatidylinositol-anchored protein segregation
Authors: Marzolo María Paz , Bull Paulina , González Alfonso
Published: July 2002

We have used the hepatitis B surface antigen (HBsAg) as a tool to explore mechanisms by which polarized epithelial cells address specific proteins to their apical domain. It recently has been proposed that N-glycans can serve as apical signals recognized by lectin-like sorting receptors in the trans-Golgi network. We found, however, conclusive evidence that the HBsAg follows an apical pathway not mediated by N-glycan signaling. Neither tunicamycin treatment nor replacement of its single glycosylated residue, Asn-146, altered its predominant (>85%) apical secretion from transfected Madin-Darby canine kidney cells (MDCK). Although HBsAg is known to be secreted as a lipoprotein particle, our results suggest that the exocytic machinery involved in its N-glycan-independent pathway overlaps, at least partially, with that of other apically targeted proteins, including the endogenous gp80, as judged by the effects of brefeldin A. We also tested whether its sorting behavior could be ascribed to association with glycosylphosphatidylinositol (GPI)-anchored proteins, which, together with glycosphingolipids, primarily are targeted to the apical domain of MDCK cells. HBsAg was preferentially secreted from the apices of transfected Fisher rat thyroid cells, which, in contrast to MDCK cells, address GPI-proteins and glycosphingolipids to their basal domain. Moreover, complete inhibition of GPI biogenesis by mannosamine treatment did not impair the HBsAg apical secretion, discarding the possibility that HBsAg could be “hitchhiking” with a newly synthesized GPI-protein. Thus, the HBsAg provides a unique model system to search for yet-unknown apical sorting mechanisms that could depend on proteinaceous targeting signals interacting with cognate trans-Golgi network receptors that are at present unidentified.
The antigen-binding domain of a human IgG-anti-F(ab′)2 autoantibody
Authors: Welschof Martin , Terness Peter , Kipriyanov Sergey M. , Stanescu Diana , Breitling Frank , Dörsam Heinz , Dübel Stefan , Little Melvyn , Opelz Gerhard
Published: July 2002

Recent studies revealed an immunoregulatory role of natural IgG-anti-F(ab′)2antibodies in both healthy individuals and patients with certain diseases. The implication of anti-F(ab′)2antibodies in the pathogenesis of diseases prompted us to study the gene segment structure of their antigen-binding domains and their binding characteristics. cDNA was prepared from the lymphocytes of a patient with a high IgG-anti-F(ab′)2serum titer. Variable heavy and light gene segments were amplified by PCR and inserted into a phagemid surface expression vector. Single-chain antibodies displayed on the phage surface were screened for binding to F(ab′)2fragments. The subsequent analysis of 95 single clones demonstrated that they all bound specifically to F(ab′)2. Sequence analyses of 12 clones showed that 11 were identical and 1 contained a silent point mutation in the heavy chain and three amino acid exchanges in the light chain. The heavy chains belonged to the VH3 and the light chains to the Vκ2 gene family. The 11 identical light-chain genes were completely homologous to a germ-line sequence (DPK-15). Binding assays showed that the single-chain antibodies bind to F(ab′)2, but not to Fab, Fc, or intact IgG. This binding pattern was confirmed by surface plasmon resonance studies, which revealed a relatively high affinity (Ka= 2.8 × 107M−1). The strong binding capacity was further demonstrated by competitive inhibition of the serum anti-IgG antibody’s interaction with antigen. The present study defines for the first time to our knowledge the gene segment structure of the antigen-binding domain of two human IgG-anti-F(ab′)2autoantibody clones and describes the binding kinetics of the purified monomeric fragments.
Poly(ADP-ribose) polymerase enhances activator-dependent transcription
in vitro
Authors: Meisterernst Michael , Stelzer Gertraud , Roeder Robert G.
Published: July 2002

Mammalian cells contain activities that amplify the effects of activators on class II gene transcription
in vitro
. The molecular identity of several of these cofactor activities is still unknown. Here we identify poly(ADP-ribose) polymerase (PARP) as one functional component of the positive cofactor 1 activity. PARP enhances transcription by acting during preinitiation complex formation, but at a step after binding of transcription factor IID. This transcriptional activation requires the amino-terminal DNA-binding domain, but not the carboxyl-terminal catalytic region. In purified systems, coactivator function requires a large molar excess of PARP over the number of templates, as reported for other DNA-binding cofactors such as topoisomerase I. PARP effects on supercoiled templates are DNA concentration-dependent and do not depend on damaged DNA. The PARP coactivator function is suppressed by NAD
+
, probably as a result of auto-ADP-ribosylation. These observations provide another example of the potentiation of trancription by certain DNA-binding cofactors and may point to interactions of PARP with RNA polymerase II-associated factors in special situations.
The pentamer channel stiffening model for drug action on human rhinovirus HRV-1A
Authors: Vaidehi Nagarajan , Goddard William A.
Published: July 2002

Development of effective drugs against the rhinovirus (HRV) responsible for the common cold remains a challenge because there are over 100 serotypes. This process could be significantly aided by an understanding of the atomistic mechanism by which such drugs work. We suggest that the most effective drugs against HRV-1A act by stiffening the pentamer channel of the viral coat through which the RNA is released, preventing the steps leading to uncoating. Using molecular dynamics methods we tested this Pentamer Channel Stiffening Model (PCSM) by examining the changes in strain energy associated with opening the pentamer channel through which the RNA is released. We find that the PCSM strain correlates well with the effectiveness of the WIN (Sterling–Winthrop) drugs for HRV-1A. To illustrate the use of the PCSM to predict new drugs and to prioritize experimental tests, we tested three modifications of the WIN drugs that are predicted to be nearly as effective (for HRV-1A) as the best current drug.
Folding of the β-propeller domain of the integrin αLsubunit is independent of the I domain and dependent on the β2 subunit
Authors: Huang Chichi , Springer Timothy A.
Published: July 2002

We have studied the folding during biosynthesis of the lymphocyte function-associated antigen 1 (LFA-1) αLsubunit using mAb to epitopes that map to seven different regions within the amino acid sequence. The N-terminal portion of αLis predicted to contain a β-propeller domain, consisting of seven β-sheets, and an I domain that is predicted to be inserted between β-sheet 2 and β-sheet 3 of the β-propeller. The I domain of αLfolds before association with the β2subunit, as shown by immunoprecipitation of the unassociated αLsubunit by mAbs specific for four different sequence elements within the I domain. By contrast, the β-propeller domain is not folded in unassociated αLafter a chase of as long as 12 h after synthesis, but does fold upon association with β2. This is shown with mAbs to regions of αL, that precede and follow the I domain in the primary structure. A mAb that maps near the junction of the C terminus of the I domain with the β-propeller domain suggests that this region is partially folded before subunit association. The results show that the I domain and β-propeller domains fold independently of one another, and suggest that the β-propeller domain bears an interface for association with the β subunit.
Biochemical characterization and crystalization of human Zn-α
2
-glycoprotein, a soluble class I major histocompatibility complex homolog
Authors: Sánchez Luis M. , López-Otín Carlos , Bjorkman Pamela J.
Published: July 2002

Zn-α
2
-glycoprotein (ZAG) is a 41-kDa soluble protein that is present in most bodily fluids. In addition, ZAG accumulates in fluids from breast cysts and in 40% of breast carcinomas, which suggests that ZAG plays a role in the development of breast diseases. However, the function of ZAG under physiological and cancerous conditions remains unknown. Because ZAG shares 30–40% sequence identity with the heavy chains of class I major histocompatibility complex (MHC) proteins, we compared the biochemical properties of ZAG with those of classical class I MHC molecules. We purified human ZAG from breast cyst fluid and serum and produced a panel of anti-ZAG monoclonal antibodies. Binding assays and acid elution experiments revealed that, in contrast to class I MHC proteins, ZAG does not bind peptides or the class I light chain, β
2
-microglobulin (β
2
m). Nevertheless, CD studies indicated that ZAG is thermally stable in the absence of bound peptide or associated β2m, as opposed to class I MHC molecules, which require the presence of both β2m and peptides for stability. These data indicate that the function of ZAG has diverged from the peptide presentation and T-cell interaction functions of class I molecules. To gain insight into the function of ZAG and to compare the three-dimensional structures of ZAG and class I MHC molecules, we produced ZAG crystals that diffract beyond 2.7 Å and have initiated an x-ray structure determination.
The distal ectodomain of angiotensin-converting enzyme regulates its cleavage-secretion from the cell surface
Authors: Sadhukhan Ramkrishna , Sen Ganes C. , Ramchandran Ramaswamy , Sen Indira
Published: July 2002

Angiotensin-converting enzyme (ACE) is a type I ectoprotein that is cleaved off the cell surface by a plasma membrane-bound metalloprotease. However, CD4, another type I ectoprotein does not undergo such cleavage-secretion. In this study, we investigated the structural determinants of the ACE protein that regulate the cleavage-secretion process. Substitution and deletion mutations revealed that the cytoplasmic domain, the transmembrane domain, and the juxtamembrane region encompassing the major and the minor cleavage sites of ACE do not regulate its cleavage. Moreover, a chimeric protein containing the distal extracellular domain of CD4 and the juxtamembrane, transmembrane, and the cytoplasmic domains of ACE, although transported to the cell surface, was not cleavage-secreted. In contrast, the distal extracellular domain of ACE was shown to be the important determinant: a protein containing the distal extracellular domain of ACE and the juxtamembrane, transmembrane, and cytoplasmic domain of CD4 was efficiently cleaved off the cell surface. The chimeric protein was cleaved within the CD4 sequence and the responsible enzymatic activity was inhibited by Compound 3, a relatively specific inhibitor of the ACE secretase activity. These results demonstrate that, in a chimeric protein, the distal extracellular domain of a cleavable protein, such as ACE, can induce a proteolytic cleavage within the juxtamembrane domain of an uncleaved protein such as CD4.
The gamma subunit of the Na,K-ATPase induces cation channel activity
Authors: Minor Nicole T. , Sha Qun , Nichols Colin G. , Mercer Robert W.
Published: July 2002

The γ subunit of the Na,K-ATPase is a hydrophobic protein of approximately 10 kDa. The γ subunit was expressed in Sf-9 insect cells and
Xenopus
oocytes to ascertain its role in Na,K-ATPase function. Immunoblotting has shown that the γ subunit is expressed in Sf-9 cells infected with recombinant baculovirus containing the cDNA for the human γ subunit. Confocal microscopy demonstrates that the γ subunit can be delivered to the plasma membrane of Sf-9 cells independently of the other Na,K-ATPase subunits and that γ colocalizes with α1 when these proteins are coexpressed. When Sf-9 cells were coinfected with α1 and γ, antibodies to the γ subunit were able to coimmunoprecipitate the α1 subunit, suggesting that γ is able to associate with α1. The γ subunit is a member of a family of single-pass transmembrane proteins that induces ion fluxes in
Xenopus
oocytes. Evidence that the γ subunit is a functional component was supported by experiments showing γ-induced cation channel activity when expressed in oocytes and increases in Na
+
and K
+
uptake when expressed in Sf-9 cells.
Retinoic acid has light-adaptive effects on horizontal cells in the retina
Authors: Weiler Reto , Schultz Konrad , Pottek Mark , Tieding Silke , Janssen-Bienhold Ulrike
Published: July 2002

Ambient light conditions affect the morphology of synaptic elements within the cone pedicle and modulate the spatial properties of the horizontal cell receptive field. We describe here that the effects of retinoic acid on these properties are similar to those of light adaptation. Intraorbital injection of retinoic acid into eyes of dark-adapted carp that subsequently were kept in complete darkness results in the formation of numerous spinules at the terminal dendrites of horizontal cells, a typical feature of light-adapted retinae. The formation of these spinules during light adaptation is impaired in the presence of citral, a competitive inhibitor of the dehydrogenase responsible for the generation of retinoic acid
in vivo
. Intracellularly recorded responses of horizontal cells from dark-adapted eyecup preparations superfused with retinoic acid reveal typical light-adapted spatial properties. Retinoic acid thus appears to act as a light-signaling modulator. Its activity appears not to be at the transcriptional level because its action was not blocked by actinomycin.
Transfer of human serum IgG to nonobese diabetic Igμ
null
mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren’s syndrome
Authors: Robinson Christopher P. , Brayer Jason , Yamachika Shigeo , Esch Thomas R. , Peck Ammon B. , Stewart Carol A. , Peen Elisabeth , Jonsson Roland , Humphreys-Beher Michael G.
Published: July 2002

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren’s syndrome. NOD.Igμ
null
mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igμ
null
mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab′)
2
fragments from parental NOD mice or human primary Sjögren’s syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [
3
H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren’s syndrome.
Localization of signal recognition particle RNA in the nucleolus of mammalian cells
Authors: Jacobson Marty R. , Pederson Thoru
Published: July 2002

The signal recognition particle (SRP) of eukaryotic cells is a cytoplasmic ribonucleoprotein machine that arrests the translational elongation of nascent secretory and membrane proteins and facilitates their transport into the endoplasmic reticulum. The spatial pathway of SRP RNA processing and ribonucleoprotein assembly in the cell is not known. In the present investigation, microinjection of fluorescently tagged SRP RNA into the nucleus of mammalian cells was used to examine its intranuclear sites of localization. Microinjection of SRP RNA into the nuclei of normal rat kidney (NRK) epithelial cells maintained at 37°C on the microscope stage resulted in a very rapid initial localization in nucleoli, followed by a progressive decline of nucleolar signal and an increase of fluorescence at discrete sites in the cytoplasm. Nuclear microinjection of a molecule corresponding to a major portion of the
Alu
domain of SRP RNA revealed a pattern of rapid nucleolar localization followed by cytoplasmic appearance of signal that was similar to the results obtained with full-length SRP RNA. In contrast, a molecule corresponding to the S domain of SRP RNA did not display nucleolar localization to the extent observed with full-length SRP RNA. An SRP RNA molecule lacking helix 6 of the S domain displayed normal nucleolar localization, whereas one lacking helix 8 of the S domain did not. These results, obtained by direct, real-time observation of fluorescent RNA molecules inside the nucleus of living mammalian cells, suggest that the processing of SRP RNA or its ribonucleoprotein assembly into the SRP involves a nucleolar phase.
Targeted gene disruption reveals a role for natural secretory IgM in the maturation of the primary immune response
Authors: Ehrenstein Michael R. , O’Keefe Theresa L. , Davies Sarah L. , Neuberger Michael S.
Published: July 2002

Accelerated development of the secondary immune response may be attributable in part to the rapid delivery of antigen to lymphoid follicles by circulating antibody elicited on primary immunization. Here we provide evidence indicating that the nonspecific IgM present in naive mice (natural antibody) plays a role in the acceleration of the primary response. Targeted deletion of the Ig μspolyadenylation site by use of Cre recombinase allowed the creation of mice that, although harboring a normal number of B cells expressing surface IgM, completely lacked serum IgM while retaining the other Ig isotypes. These mice retained a broadly normal B lymphocyte distribution (although containing a somewhat expanded peritoneal B1a subset) but exhibited substantial delays in mounting affinity-matured IgG responses to T cell-dependent antigens. The T cell-independent response, however, was augmented. The data indicate that the IgM present before antigen challenge (as well, possibly, as that elicited immediately after immunization) accelerates maturation of the primary response, presumably by complexing with the antigen and facilitating lymphocyte activation and/or antigen trapping.
The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels
Authors: Ali Shariq , Chen Xun , Lu Ming , Xu Jason Z. , Lerea Kenneth M. , Hebert Steven C. , Wang WenHui
Published: July 2002

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K
+
secretory channels in the kidney. Addition of 1 μM forskolin or 100 μM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in
Xenopus
oocytes. In contrast, application of 1 μM forskolin, within 3 min, significantly increased whole-cell K
+
current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (
i
) Addition of 100 μM 8-bromo-cAMP mimics the effect of forskolin and (
ii
) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 μM) and cAMP (100 μM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.
Guanine nucleotide exchange factor GEF115 specifically mediates activation of Rho and serum response factor by the G protein α subunit Gα13
Authors: Mao Junhao , Yuan Huidong , Xie Wei , Wu Dianqing
Published: July 2002

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein α subunits were characterized in transfection systems. Gαq, Gα12, and Gα13, but not Gαi, activate SRF through RhoA. When Gαq, α12, or α13 were coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Gα13, but not Gαq or Gα12, showed synergistic activation of SRF with GEF115. The synergy between Gα13 and GEF115 depends on the N-terminal part of GEF115, and there was no synergistic effect between Gα13 and another Rho-specific exchange factor Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibited Gα13- and Gα12-induced, but not GEF115 itself- or Gαq-induced, SRF activation. The DH-domain-deletion mutant also suppressed thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Gα12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Gα12 and Gα13. Thus, the inhibition of Gα12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Gα13 and GEF115 indicates that GEF115 mediates Gα13-induced activation of Rho and SRF.
Tumor necrosis factor-α induces adhesion molecule expression through the sphingosine kinase pathway
Authors: Xia Pu , Gamble Jennifer R. , Rye Kerry-Anne , Wang Lijun , Hii Charles S. T. , Cockerill Peter , Khew-Goodall Yeesim , Bert Andrew G. , Barter Philip J. , Vadas Mathew A.
Published: July 2002

The signaling pathways that couple tumor necrosis factor-α (TNFα) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFα induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFα resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dose-dependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNFα on endothelial cells leading to extracellular signal-regulated kinases and NF-κB activation, whereas ceramide or sphingosine was not. Furthermore,N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNFα-induced extracellular signal-regulated kinases and NF-κB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNFα-induced endothelial cell activation.
Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors
Authors: Valés-Gómez Mar , Reyburn Hugh T. , Erskine Robert A. , Strominger Jack
Published: July 2002

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.
Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation
in vitro
and
in vivo
Authors: Maruno Kaname , Absood Afaf , Said Sami I.
Published: July 2002

Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.
Opposite roles of apolipoprotein E in normal brains and in Alzheimer’s disease
Authors: Russo Claudio , Angelini Giovanna , Dapino Debora , Piccini Alessandra , Piombo Giuseppe , Schettini Gennaro , Chen Shu , Teller Jan K. , Zaccheo Damiano , Gambetti Pierluigi , Tabaton Massimo
Published: July 2002

We have characterized the interaction between apolipoprotein E (apoE) and amyloid β peptide (Aβ) in the soluble fraction of the cerebral cortex of Alzheimer’s disease (AD) and control subjects. Western blot analysis with specific antibodies identified in both groups a complex composed of the full-length apoE and Aβ peptides ending at residues 40 and 42. The apoE–Aβ soluble aggregate is less stable in AD brains than in controls, when treated with the anionic detergent SDS. The complex is present in significantly higher quantity in control than in AD brains, whereas in the insoluble fraction an inverse correlation has previously been reported. Moreover, in the AD subjects the Aβ bound to apoE is more sensitive to protease digestion than is the unbound Aβ. Taken together, our results indicate that in normal brains apoE efficiently binds and sequesters Aβ, preventing its aggregation. In AD, the impaired apoE–Aβ binding leads to the critical accumulation of Aβ, facilitating plaque formation.
Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis
Authors: Galli Cinzia , Piccini Alessandra , Ciotti Maria Teresa , Castellani Loriana , Calissano Pietro , Zaccheo Damiano , Tabaton Massimo
Published: July 2002

Some clues suggest that neuronal damage induces a secondary change of amyloid β protein (Aβ) metabolism. We investigated this possibility by analyzing the secretion of Aβ and processing of its precursor protein (amyloid precursor protein, APP) in an
in vitro
model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [
35
S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Aβ, full-length APP, α- and β-APP secreted forms. Immunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer. Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa Aβ were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the β-APP
s
/α-APP
s
ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Aβ sequence showed that the Aβ signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from β-secretase cleavage and suggest that an overproduction of Aβ may be the consequence of neuronal damage from various causes.
Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose
in vivo
Authors: Martin William H. , Hoover Dennis J. , Armento Sandra J. , Stock Ingrid A. , McPherson R. Kirk , Danley Dennis E. , Stevenson Ralph W. , Barrett Eugene J. , Treadway Judith L.
Published: July 2002

An inhibitor of human liver glycogen phosphorylase
a
(HLGPa) has been identified and characterized
in vitro
and
in vivo
. This substance, [R-(R*,S*)]-5-chloro-
N
-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (CP-91149), inhibited HLGPa with an IC
50
of 0.13 μM in the presence of 7.5 mM glucose. CP-91149 resembles caffeine, a known allosteric phosphorylase inhibitor, in that it is 5- to 10-fold less potent in the absence of glucose. Further analysis, however, suggests that CP-91149 and caffeine are kinetically distinct. Functionally, CP-91149 inhibited glucagon-stimulated glycogenolysis in isolated rat hepatocytes (
P
< 0.05 at 10–100 μM) and in primary human hepatocytes (2.1 μM IC
50
).
In vivo
, oral administration of CP-91149 to diabetic
ob/ob
mice at 25–50 mg/kg resulted in rapid (3 h) glucose lowering by 100–120 mg/dl (
P
< 0.001) without producing hypoglycemia. Further, CP-91149 treatment did not lower glucose levels in normoglycemic, nondiabetic mice. In
ob/ob
mice pretreated with
14
C-glucose to label liver glycogen, CP-91149 administration reduced
14
C-glycogen breakdown, confirming that glucose lowering resulted from inhibition of glycogenolysis
in vivo.
These findings support the use of CP-91149 in investigating glycogenolytic versus gluconeogenic flux in hepatic glucose production, and they demonstrate that glycogenolysis inhibitors may be useful in the treatment of type 2 diabetes.
Domain requirements for the Dock adapter protein in growth- cone signaling
Authors: Rao Yong , Zipursky S. Lawrence
Published: July 2002

Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The
Drosophila
adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.
A substrate of the centisome 63 type III protein secretion system of
Salmonella typhimurium
is encoded by a cryptic bacteriophage
Authors: Hardt Wolf-Dietrich , Urlaub Henning , Galán Jorge E.
Published: July 2002

Salmonella enterica
has evolved a type III protein secretion system that allows these enteropathogens to translocate effector molecules directly into the host cell cytoplasm. These effectors mediate a variety of responses, including cytoskeletal rearrangements, cytokine production, and in certain cells, the induction of apoptosis. We report here the characterization of a substrate of this secretion system in
S. enterica
serovar
typhimurium
(
Salmonella typhimurium
) that is homologous to the SopE protein of
Salmonella dublin
implicated in bacterial entry into cultured epithelial cells. The
sopE
locus is located within a cluster of genes that encode tail and tail fiber proteins of a cryptic P2-like prophage, outside of the centisome 63 pathogenicity island that encodes the invasion-associated type III secretion system. Southern hybridization analysis revealed that
sopE
is present in only a subset of
S. enterica
serovars and that the flanking bacteriophage genes are also highly polymorphic. Encoding effector proteins that are delivered through type III secretion systems in highly mobile genetic elements may allow pathogens to adapt rapidly by facilitating the assembly of an appropriate set of effector proteins required for successful replication in a new environment.
The yeast CLC chloride channel functions in cation homeostasis
Authors: Gaxiola Roberto A. , Yuan Daniel S. , Klausner Richard D. , Fink Gerald R.
Published: July 2002

A defect in the yeast
GEF1
gene, a CLC chloride channel homolog leads to an iron requirement and cation sensitivity. The iron requirement is due to a failure to load Cu
2+
onto a component of the iron uptake system, Fet3. This process, which requires both Gef1 and the Menkes disease Cu
2+
-ATPase yeast homolog Ccc2, occurs in late- or post-Golgi vesicles, where Gef1 and Ccc2 are localized. The defects of
gef1
mutants can be suppressed by the introduction of
Torpedo marmorata CLC-0
or
Arabidopsis thaliana CLC-c
and
-d
chloride channel genes. The functions of Gef1 in cation homeostasis provide clues to the understanding of diseases caused by chloride channel mutations in humans and cation toxicity in plants.
Migrating transformed MDCK cells are able to structurally polarize a voltage-activated K
+
channel
Authors: Reinhardt J. , Golenhofen N. , Pongs O. , Oberleithner H. , Schwab A.
Published: July 2002

Cell migration of transformed renal epithelial cells (MDCK-F) depends–in addition to cytoskeletal mechanisms–on the polarized activity of a Ca
2+
-sensitive K
+
channel in the rear part of the cells. However, because of the lack of specific markers for this channel we are not able to determine whether a polarized distribution of the channel protein underlies its functional polarization. To determine whether the migrating MDCK-F cells have retained the ability to target K
+
channels to distinct membrane areas we stably transfected the cells with the voltage-dependent K
+
channel Kv1.4. Stable expression and insertion into the plasma membrane could be shown by reverse transcription–PCR, genomic PCR, Western blot, and patch-clamp techniques, respectively. The distribution of Kv1.4 was assessed with indirect immunofluorescence by using conventional and confocal microscopy. These experiments revealed that Kv1.4 is expressed only in transfected cells where it elicits the typical voltage-dependent, rapidly inactivating K
+
current. The Kv1.4 protein is clustered at the leading edge of protruding lamellipodia of migrating MDCK-F cells. This characteristic distribution of Kv1.4 provides strong evidence that migrating MDCK-F cells are able to insert ion channels into the plasma membrane in an asymmetric way, which reflects the polarization of migrating cells in the plane of movement. These findings suggest that not only epithelial cells and nerve cells, but also migrating cells, can create functionally distinct plasma membrane areas.
Carbohydrate vaccines in cancer: Immunogenicity of a fully synthetic globo H hexasaccharide conjugate in man
Authors: Slovin S. F. , Ragupathi G. , Adluri S. , Ungers G. , Terry K. , Kim S. , Spassova M. , Bornmann W. G. , Fazzari M. , Dantis L. , Olkiewicz K. , Lloyd K. O. , Livingston P. O. , Danishefsky S. J. , Scher H. I.
Published: July 2002

The complex carbohydrate molecule globo H hexasaccharide has been
synthesized, conjugated to keyhole limpet hemocyanin, and administered
with the immunologic adjuvant QS-21 as a vaccine for patients with
prostate cancer who have relapsed after primary therapies such as
radiation or surgery. Globo H is one of several candidate antigens
present on prostate cancer cells that can serve as targets for immune
recognition and treatment strategies. The vaccine, given as five
subcutaneous vaccinations over 26 weeks, has been shown to be safe and
capable of inducing specific high-titer IgM antibodies against globo H.
Its immunogenicity was confirmed in prostate cancer patients with a
broad range of stages and tumor burdens. Observations of several
patients who had evidence of disease relapse restricted to a rising
biochemical marker, prostate-specific antigen (PSA), indicated that a
treatment effect could occur within 3 months after completion of the
vaccine therapy. This effect was manifested as a decline of the slope
of the log of PSA concentration vs. time plot after treatment compared
with values before treatment. Five patients continue to have stable PSA
slope profiles in the absence of any radiographic evidence of disease
for more than 2 years. The concept of using PSA slope profiles in
assessing early treatment effects in biological therapies such as
vaccines awaits further validation in phase II and III trials. The use
of a variety of lesser known candidate glycoprotein and carbohydrate
antigens in prostate cancer serves as a focus for the development of a
multivalent vaccine of the treatment of relapsed prostate cancer in
patients with minimal tumor burden.
Interaction between RGS7 and polycystin
Authors: Kim Emily , Arnould Thierry , Sellin Lorenz , Benzing Thomas , Comella Natalia , Kocher Olivier , Tsiokas Leonidas , Sukhatme Vikas P. , Walz Gerd
Published: July 2002

Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Gα subunits and thereby modulate a number of G protein-dependent signaling cascades. Currently, little is known about the regulation of RGS proteins themselves. We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway. The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by
PKD1
, a gene involved in autosomal-dominant polycystic kidney disease. Furthermore, membranous expression of C-terminal polycystin relocalized RGS7. Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins.
Agrin in Alzheimer’s disease: Altered solubility and abnormal distribution within microvasculature and brain parenchyma
Authors: Donahue John E. , Berzin Tyler M. , Rafii Michael S. , Glass David J. , Yancopoulos George D. , Fallon Justin R. , Stopa Edward G.
Published: July 2002

Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer’s disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with β-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to β-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.