bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins
Authors: Cheatham Bentley , Volchuk Allen , Kahn C. Ronald , Wang Lois , Rhodes Christopher J. , Klip Amira
Published: July 2002

A major physiological role of insulin is the regulation of glucose
uptake into skeletal and cardiac muscle and adipose tissue, mediated by
an insulin-stimulated translocation of GLUT4 glucose transporters from
an intracellular vesicular pool to the plasma membrane. This process is
similar to the regulated docking and fusion of vesicles in
neuroendocrine cells, a process that involves SNARE-complex proteins.
Recently, several SNARE proteins were found in adipocytes:
vesicle-associated membrane protein (VAMP-2), its related homologue
cellubrevin, and syntaxin-4. In this report we show that treatment of
permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which
selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of
insulin to stimulate translocation of GLUT4 vesicles to the plasma
membrane. Furthermore, treatment of the permeabilized adipocytes with
glutathione S-transferase fusion proteins encoding soluble forms of
VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4
translocation. These results provide evidence of a functional role for
SNARE-complex proteins in insulin-stimulated glucose uptake and suggest
that adipocytes utilize a mechanism of regulating vesicle docking and
fusion analogous to that found in neuroendocrine tissues.
Developmental signal transduction pathways uncovered by genetic suppressors
Authors: Shaulsky Gad , Escalante Ricardo , Loomis William F.
Published: July 2002

We have found conditions for saturation mutagenesis by restriction
enzyme mediated integration that result in plasmid tagging of disrupted
genes. Using this method we selected for mutations in genes that act at
checkpoints downstream of the intercellular signaling system that
controls encapsulation in
Dictyostelium
discoideum
. One of these genes,
mkcA
, is a
member of the mitogen-activating protein kinase cascade family while
the other,
regA
, is a novel bipartite gene homologous to
response regulators in one part and to cyclic nucleotide
phosphodiesterases in the other part. Disruption of either of these
genes results in partial suppression of the block to spore formation
resulting from the loss of the prestalk genes,
tagB
and
tagC
. The products of the
tag
genes have
conserved domains of serine proteases attached to ATP-driven
transporters, suggesting that they process and export peptide signals.
Together, these genes outline an intercellular communication system
that coordinates organismal shape with cellular differentiation during
development.
Recombinant scrapie-like prion protein of 106 amino acids is soluble
Authors: Muramoto Tamaki , Scott Michael , Cohen Fred E. , Prusiner Stanley B.
Published: July 2002

The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPScformation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPScformation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPScformation, and the production of PrPSc-specific antibodies.
A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.
Authors: Chen R , Udenfriend S , Prince G M , Maxwell S E , Ramalingam S , Gerber L D , Knez J , Medof M E
Published: July 2002

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.
Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.
Authors: Schmidt A , Rutledge S J , Endo N , Opas E E , Tanaka H , Wesolowski G , Leu C T , Huang Z , Ramachandaran C , Rodan S B , Rodan G A
Published: July 2002

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.
Long-time quantum simulation of the primary charge separation in bacterial photosynthesis.
Authors: Makri N , Sim E , Makarov D E , Topaler M
Published: July 2002

Accurate quantum mechanical simulations of the primary charge transfer in photosynthetic reaction centers are reported. The process is modeled by three coupled electronic states corresponding to the photoexcited chlorophyll special pair (donor), the reduced bacteriopheophytin (acceptor), and the reduced accessory chlorophyll (bridge) that interact with a dissipative medium of protein and solvent degrees of freedom. The time evolution of the excited special pair is followed over 17 ps by using a fully quantum mechanical path integral scheme. We find that a free energy of the reduced accessory chlorophyll state approximately equal to 400 cm(-1) lower than that of the excited special pair state yields state populations in agreement with experimental results on wild-type and modified reaction centers. For this energetic configuration electron transfer is a two-step process.
Tat protein induces self-perpetuating permissivity for productive HIV-1 infection
Authors: Li Chiang J. , Ueda Yutaka , Shi Bin , Borodyansky Laura , Huang Lili , Li You-Zhi , Pardee Arthur B.
Published: July 2002

We report that human immunodeficiency virus type 1 (HIV-1) has evolved a self-perpetuating mechanism to actively generate cells permissive for productive and cytopathic infection. Only activated T cells can be productively infected, which leads to their rapid depletion (2 × 109/day in an infected individual). Establishment of productive HIV-1 infection therefore requires continual activations from the large pool of quiescent T cells. Tat protein, which is secreted by infected cells, activated uninfected quiescent T cellsin vitroandin vivo. These Tat-activated uninfected cells became highly permissive for productive HIV-1 infection. Activation of primary T cells by Tat protein involved integrin receptors and was associated with activation of mitogen-activated protein kinases, including ERK1 and JNK kinase. Accordingly, these primary T cells progressed from G0to the late G1phase of the cell cycle.
Induction of epithelial chloride secretion by channel-forming cryptdins 2 and 3
Authors: Lencer W. I. , Cheung G. , Strohmeier G. R. , Currie M. G. , Ouellette A. J. , Selsted M. E. , Madara J. L.
Published: July 2002

Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl
−
secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl
−
secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl
−
secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl
−
secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 μM), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyfluorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl
−
secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that
in vivo
may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.
Regulation of the human ether-a-gogo related gene (HERG) K+channels by reactive oxygen species
Authors: Taglialatela Maurizio , Castaldo Pasqualina , Iossa Silvana , Pannaccione Anna , Fresi Angela , Ficker Eckhard , Annunziato Lucio
Published: July 2002

Human ether-a-gogo related gene (HERG) K+channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+channels expressed inXenopusoocytes has been explored in the present study. Exposure ofXenopusoocytes to an extracellular solution containing FeSO4(25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+currents carried by other K+channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+currents carried by the HERG channels.
Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein α gene
Authors: Tang Qi-Qun , Jiang Man-Shiow , Lane M. Daniel
Published: July 2002

During adipocyte differentiation, the expression of C/EBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPα gene led to the identification of a potential repressive element within the proximal 5′ flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (
C
/EBPα
u
ndifferentiated
p
rotein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoter-luciferase constructs containing both the proximal 5′ flanking and the entire 5′ untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5′ untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5′ untranslated region. Although mutation of either CUP element in constructs containing both the 5′ flanking and 5′ untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPα gene prior to induction of the adipocyte differentiation program.
Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: A critical role for nuclear targeting
Authors: Massfelder Thierry , Dann Pamela , Wu Terence L. , Vasavada Rupangi , Helwig Jean-Jacques , Stewart Andrew F.
Published: July 2002

Parathyroid hormone-related protein (PTHrP) is a prohormone that is posttranslationally processed to a family of mature secretory forms, each of which has its own cognate receptor(s) on the cell surface that mediate the actions of PTHrP. In addition to being secreted via the classical secretory pathway and interacting with cell surface receptors in a paracrine/autocrine fashion, PTHrP appears to be able to enter the nucleus directly following translation and influence cellular events in an “intracrine” fashion. In this report, we demonstrate that PTHrP can be targeted to the nucleus in vascular smooth muscle cells, that this nuclear targeting is associated with a striking increase in mitogenesis, that this nuclear effect on proliferation is the diametric opposite of the effects of PTHrP resulting from interaction with cell surface receptors on vascular smooth muscle cells, and that the regions of the PTHrP sequence responsible for this nuclear targeting represent a classical bipartite nuclear localization signal. This report describes the activation of the cell cycle in association with nuclear localization of PTHrP in any cell type. These findings have important implications for the normal physiology of PTHrP in the many tissues which produce it, and suggest that gene delivery of PTHrP or modified variants may be useful in the management of atherosclerotic vascular disease.
Receptors induce chemotaxis by releasing the βγ subunit of G
i
, not by activating G
q
or G
s
Authors: Neptune Enid R. , Bourne Henry R.
Published: July 2002

Many chemoattractants cause chemotaxis of leukocytes by stimulating a structurally distinct class of G protein-coupled receptors. To identify receptor functions required for chemotaxis, we studied chemotaxis in HEK293 cells transfected with receptors for nonchemokine ligands or for interleukin 8 (IL-8), a classical chemokine. In gradients of the appropriate agonist, three nonchemokine G
i
-coupled receptors (the D
2
dopamine receptor and opioid μ and δ receptors) mediated chemotaxis; the β
2
-adrenoreceptor and the M3-muscarinic receptor, which couple respectively to G
s
and G
q
, did not mediate chemotaxis. A mutation deleting 31 C-terminal amino acids from the IL-8 receptor type B quantitatively impaired chemotaxis and agonist-induced receptor internalization, but not inhibition of adenylyl cyclase or stimulation of mitogen-activated protein kinase. To probe the possible relation between receptor internalization and chemotaxis, we used two agonists of the μ-opioid receptor. Morphine and etorphine elicited quantitatively similar chemotaxis, but only etorphine induced receptor internalization. Overexpression of two βγ sequestering proteins (βARK-ct and α
t
) prevented IL-8 receptor type B-mediated chemotaxis but did not affect inhibition of adenylyl cyclase by IL-8. We conclude that: (
i
) Nonchemokine G
i
-coupled receptors can mediate chemotaxis. (
ii
) G
i
activation is necessary but probably not sufficient for chemotaxis. (
iii
) Chemotaxis does not require receptor internalization. (
iv
) Chemotaxis requires the release of free βγ subunits.
Co-expression of the neuronal α
7
and L247T α
7
mutant subunits yields hybrid nicotinic receptors with properties of both wild-type α
7
and α
7
mutant homomeric receptors
Authors: Palma E. , Eusebi F. , Miledi R.
Published: July 2002

Injection of cDNA encoding the neuronal α
7
subunit into
Xenopus
oocytes yields
homomeric
receptors showing responses to AcCho that have low affinity, fast desensitization, nonlinear current–voltage (
I–V
) relation, and sensitivity to α-bungarotoxin (α-BTX) and 5-hydroxytryptamine (5HT), both substances acting as antagonists. Mutation of the Leu-247, located in the channel domain, changes 5HT from an antagonist to an agonist, slows the rate of desensitization, renders the
I–V
relation linear, and increases the affinity for acetylcholine (AcCho). A study was made of receptors expressed after injecting
Xenopus
oocytes with mixtures of cDNAs encoding the wild-type α
7
(WT α
7
) and the L247T α
7
mutated nicotinic AcCho receptors (nAcChoRs). The receptors expressed were again blocked by α-bungarotoxin (100 nM) but exhibited both WT α
7
and α
7
mutant functional characteristics. Out of eight different types of hybrid receptors identified, most were inhibited by 5HT (1 mM) and showed low sensitivity to AcCho, like the WT α
7
receptors, but exhibited a slow rate of desensitization and an
I–V
relation similar to those of α
7
mutant receptors. Together, these findings indicate that the increased nAcChoR affinity and the decreased nAcChoR desensitization after Leu-247 mutation are uncoupled events. We propose that receptor diversity is predicted by permutations of WT α
7
and L247T α
7
subunits in a pentameric symmetrical model and that even partial replacement of Leu-247 with a polar residue within the leucine ring in the channel domain considerably influences the properties of neuronal α
7
nAcChoRs.
“Peptabody”: A new type of high avidity binding protein
Authors: Terskikh Alexey V. , Le Doussal Jean-Marc , Crameri Reto , Fisch Igor , Mach Jean-Pierre , Kajava Andrey V.
Published: July 2002

A new type of high avidity binding molecule, termed “peptabody” was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed inEscherichia coliin a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1surface idiotype with an avidity of about 1 nM, which corresponds to a 2 × 105-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.
In vivo
activation of the cystic fibrosis transmembrane conductance regulator mutant ΔF508 in murine nasal epithelium
Authors: Kelley Thomas J. , Thomas Kirk , Milgram Laura J. H. , Drumm Mitchell L.
Published: July 2002

The gene causing cystic fibrosis (CF) encodes the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. Mutations in this gene result in reduced transepithelial chloride permeability across tissues affected in CF. Consequently, restoring chloride permeability to these tissues may prove therapeutic. Here we report that a combination of forskolin, an adenylate cyclase activator, and milrinone, an inhibitor of class III phosphodiesterases, increases the magnitude of the potential difference across nasal epithelium of mice homozygous for the most common CF mutation, ΔF508, while neither drug alone has a significant effect on potential difference. Transgenic mice lacking CFTR do not respond to the milrinone/forskolin combination, indicating that the effect in ΔF508 mice requires CFTR. These results suggest that, by pharmacological means, at least partial CFTR-mediated electrolyte transport can be restored
in vivo
to CF tissues expressing ΔF508.
Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells
Authors: Zhou Bin-Bing , Li Honglin , Yuan Junying , Kirschner Marc W.
Published: July 2002

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. We find that the Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. Our results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities.
Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities
Authors: Wilson Gisela F. , Magoski Neil S. , Kaczmarek Leonard K.
Published: July 2002

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of
Aplysia
, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the “afterdischarge.” In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10.7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5′-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide
(19–36)
. Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.
Cloning and characterization of hSRP1γ, a tissue-specific nuclear transport factor
Authors: Nachury Maxence V. , Ryder Ursula W. , Lamond Angus I. , Weis Karsten
Published: July 2002

Nuclear import of proteins containing a nuclear localization signal (NLS) is dependent on the presence of a cytoplasmic NLS receptor, the GTPase Ran, and p10/NTF2. The NLS receptor is a heterodimeric protein consisting of subunits of approximately 60 and 97 kDa, which have been termed importin α/β, karyopherin α/β, or PTAC 58/97. Members of the 60-kDa/importin α subunit family directly bind to the NLS motif and have been shown to function as adaptors that tether NLS-containing proteins to the p97/importin β subunit and to the downstream transport machinery. Herein we report the identification and characterization of hSRP1γ, a human importin α homologue. The hSRP1γ protein is around 45% identical to the two previously identified human importin α homologues hSRP1α/Rch1 and NPI/hSRP1. hSRP1γ can form a complex with importin β and is able to mediate import of a BSA-NLS substrate in an
in vitro
nuclear import system. Interestingly, hSRP1γ shows a very selective expression pattern and is most abundantly expressed in skeletal muscle, representing more than 1% of the total protein in this tissue. A potential role for hSRP1γ in tissue-specific transport events is discussed.
Identification and characterization of amelogenin genes in monotremes, reptiles, and amphibians
Authors: Toyosawa Satoru , O’hUigin Colm , Figueroa Felipe , Tichy Herbert , Klein Jan
Published: July 2002

Two features make the tooth an excellent model in the study of evolutionary innovations: the relative simplicity of its structure and the fact that the major tooth-forming genes have been identified in eutherian mammals. To understand the nature of the innovation at the molecular level, it is necessary to identify the homologs of tooth-forming genes in other vertebrates. As a first step toward this goal, homologs of the eutherian amelogenin gene have been cloned and characterized in selected species of monotremes (platypus and echidna), reptiles (caiman), and amphibians (African clawed toad). Comparisons of the homologs reveal that the amelogenin gene evolves quickly in the repeat region, in which numerous insertions and deletions have obliterated any similarity among the genes, and slowly in other regions. The gene organization, the distribution of hydrophobic and hydrophilic segments in the encoded protein, and several other features have been conserved throughout the evolution of the tetrapod amelogenin gene. Clones corresponding to one locus only were found in caiman, whereas the clawed toad possesses at least two amelogenin-encoding loci.
Developmental patterns in the cytoarchitecture of the human cerebral cortex from birth to 6 years examined by correspondence analysis
Authors: Shankle William R. , Romney A. Kimball , Landing Benjamin H. , Hara Junko
Published: July 2002

This paper uses correspondence analysis to examine the developmental patterns in the cytoarchitecture of the human cerebral cortex from birth to 72 months. The study is based on data collected by the late J. L. Conel, which consist of over 4 million individual measurements of six microscopic neuroanatomic features for each of six cortical layers in 46 cytoarchitecturally distinct regions. We analyze 1,727 profiles of development over eight age-points (term birth, 1, 3, 6, 15, 24, 48, and 72 postnatal months) resulting from the combinations of neuroanatomic feature, cortical layer, and brain cytoarchitectural region in the Conel data. The profiles for any given combination of feature and layer are found to be remarkably similar in all regions of the brain, and therefore the developmental patterns of different cytoarchitectural regions are not distinguishable from one another. Developmental change is most rapid at the earlier stages; of the total change in profile patterns observed, more than one-third occurs between birth and 6 months, about one-third occurs between 6 and 15 months, and less than one-third occurs between 15 and 72 months. The majority of the variance in developmental profiles is accounted for by the six microscopic, neuroanatomic features. Correspondence analysis shows that Conel’s data are highly consistent and reliable.
The stimulatory action of tolbutamide on Ca
2+
-dependent exocytosis in pancreatic β cells is mediated by a 65-kDa mdr-like P-glycoprotein
Authors: Barg Sebastian , Renström Erik , Berggren Per-Olof , Bertorello Alejandro , Bokvist Krister , Braun Matthias , Eliasson Lena , Holmes William E. , Köhler Martin , Rorsman Patrik , Thévenod Frank
Published: July 2002

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca
2+
concentration of 0.17 μM. This effect was not detectable in the complete absence of cytoplasmic Ca
2+
and when exocytosis was elicited by guanosine 5′-
O
-(3-thiotriphosphate) (GTPγS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl
−
-channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl
−
channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (
i
) tolbutamide stimulates Ca
2+
-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (
ii
) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl
−
conductance. We speculate that the activation of granular Cl
−
fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.
Regulation of ROMK1 channel by protein kinase A via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism
Authors: Liou Horng-Huei , Zhou Shi-Sheng , Huang Chou-Long
Published: July 2002

ROMK inward-rectifier K
+
channels control renal K
+
secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Having found that direct interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP
2
) is essential for channel activation, we investigate here the role of PIP
2
in regulation of ROMK1 by PKA. By using adenosine-5′-[γ-thio]triphosphate) (ATP[γS]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP
2
. Rather, phosphorylation by PKA + ATP[γS] lowers the concentration of PIP
2
necessary for activation of the channels. In solution-binding assays, anti-PIP
2
antibodies bind PIP
2
and prevent PIP
2
–channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 for inhibition by the antibodies, indicating an enhanced interaction between PIP
2
and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP
2
interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP
2
–channel interaction.
Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain
Authors: Ye Li-Hong , Kishi Hiroko , Nakamura Akio , Okagaki Tsuyoshi , Tanaka Takeshi , Oiwa Kazuhiro , Kohama Kazuhiro
Published: July 2002

Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain. The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1–Pro-41 sequence of MLCK binds to actin to inhibit this interaction. However, it is not known whether the myosin-binding domain modifies the actin–myosin interaction. We designed MLCK⋅cDNA to overexpress the Asp-777–Glu-972 sequence in
Escherichia coli
. The purified Asp-777–Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (
V
max
= 7.36 ± 0.44-fold,
K
m
= 1.06 ± 0.20 μM,
n
= 4). When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816–Glu-972, lost the stimulatory activity. We synthesized the Ala-777–Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (
V
max
= 3.03 ± 0.22-fold,
K
m
= 6.93 ± 1.61 μM,
n
= 3). When this peptide was further divided into Asp-777–Met-795 and Ala-796–Ser-815 peptides, the stimulatory activity was found in the latter. We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides. Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active.
Two distinct forms of the 64,000
M
r
protein of the cleavage stimulation factor are expressed in mouse male germ cells
Authors: Wallace A. Michelle , Dass Brinda , Ravnik Stuart E. , Tonk Vijay , Jenkins Nancy A. , Gilbert Debra J. , Copeland Neal G. , MacDonald Clinton C.
Published: July 2002

Polyadenylation in male germ cells differs from that in somatic cells. Many germ cell mRNAs do not contain the canonical AAUAAA in their 3′ ends but are efficiently polyadenylated. To determine whether the 64,000
M
r
protein of the cleavage stimulation factor (CstF-64) is altered in male germ cells, we examined its expression in mouse testis. In addition to the 64,000
M
r
form, we found a related ≈70,000
M
r
protein that is abundant in testis, at low levels in brain, and undetectable in all other tissues examined. Expression of the ≈70,000
M
r
CstF-64 was limited to meiotic spermatocytes and postmeiotic spermatids in testis. In contrast, the 64,000
M
r
form was absent from spermatocytes, suggesting that the testis-specific CstF-64 might control expression of meiosis-specific genes. To determine why the 64,000
M
r
CstF-64 is not expressed in spermatocytes, we mapped its chromosomal location to the X chromosome in both mouse and human. CstF-64 may, therefore, be absent in spermatocytes because the X chromosome is inactivated during male meiosis. By extension, the testis-specific CstF-64 may be expressed from an autosomal homolog of the X chromosomal gene.
Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9
Authors: Chakrabarti Subhra Ranjan , Sood Rashmi , Ganguly Surajit , Bohlander Stefan , Shen Zhiyuan , Nucifora Giuseppina
Published: July 2002

The E-26 transforming specific (ETS)-related gene
TEL
, also known as
ETV6
, is involved in a large number of chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The encoded protein contains two functional domains: a helix–loop–helix (HLH) domain (also known as pointed domain) located at the N terminus and a DNA-binding domain located at the C terminus. The HLH domain is involved in protein–protein interaction with itself and other members of the ETS family of transcription factors such as FLI1. TEL is a transcription factor, and we and others have shown that it is a repressor of gene expression. To understand further the role of TEL in the cell, we have used an
in vivo
interaction system to identify proteins that interact with TEL. We show that a protein, UBC9, interacts specifically with TEL
in vitro
and
in vivo
. UBC9 is a member of the family of ubiquitin-conjugating enzymes. These enzymes usually are involved in proteosome-mediated degradation; however, our data suggest that interaction of TEL with UBC9 does not lead to TEL degradation. Our studies show that UBC9 binds to TEL exclusively through the HLH domain of TEL. We also show that TEL expressed as fusion to the DNA-binding domain of Gal4 completely represses a Gal4-responsive promoter, but that the coexpression of UBC9 in the same system restores the activity of the promoter. Targeted point mutation of conserved amino acids in UBC9 essential for enzymatic ubiquitination of proteins does not affect interaction nor transcriptional activity. Based on our data, we conclude that UBC9 physically interacts with TEL through the HLH domain and that the interaction leads to modulation of the transcription activity of TEL.
Targeted disruption of the β adducin gene (
Add
2
) causes red blood cell spherocytosis in mice
Authors: Gilligan Diana M. , Lozovatsky Larissa , Gwynn Babette , Brugnara Carlo , Mohandas Narla , Peters Luanne L.
Published: July 2002

Adducins are a family of cytoskeleton proteins encoded by three genes (α, β, γ). In a comprehensive assay of gene expression, we show the ubiquitous expression of α- and γ-adducins in contrast to the restricted expression of β-adducin. β-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin’s role
in vivo
, we created β-adducin null mice by gene targeting, deleting exons 9–13. A 55-kDa chimeric polypeptide is produced from the first eight exons of β-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. β-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of β-adducin in RBCs leads to decreased membrane incorporation of α-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in γ-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin’s importance to RBC membrane stability
in vivo
.
The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus
Authors: Krebs Christopher J. , Jarvis Erich D. , Pfaff Donald W.
Published: July 2002

Estrogen (E) and progesterone (P) orchestrate many cellular
responses involved in female reproductive physiology, including
reproductive behaviors. E- and P-binding neurons important for lordosis
behavior have been located within the ventromedial hypothalamus (VMH),
and several hormone-responsive genes have been observed there as well.
In attempts to identify additional E- and P-responsive genes in the VMH
that may contribute to sexual behaviors, we used the differential
display mRNA screening technique. One of the genes identified encodes
the 73-kDa heat shock cognate protein (
Hsc73
). Quantitative
in situ
hybridization analysis of brains from naturally
cycling female rats revealed a significant increase in
Hsc73
mRNA in the VMH and arcuate nucleus of animals during
proestrus compared with those at diestrus-1. To confirm that these
increases were steroid hormone dependent, we compared vehicle-treated
ovariectomized females with ovariectomized females treated with
estradiol benzoate and P. Northern analysis and
in situ
hybridizations showed that the
Hsc73
gene is enhanced by E
and P in the pituitary and subregions of the VMH. Incidentally, by
examining the primary amino acid sequence of rat, human, and chicken
progesterone receptors, we noticed that putative Hsc73 binding sites
are conserved across species with similar sites existing in the
androgen and glucocorticoid receptors. Together these findings suggest
a possible mechanism through which E could influence the activities of
progesterone, androgen, and glucocorticoid receptors, by enhancing the
expression of Hsc73 in cells where these proteins colocalize.
Neurotrophic factors [activity-dependent neurotrophic factor (ADNF) and basic fibroblast growth factor (bFGF)] interrupt excitotoxic neurodegenerative cascades promoted by a PS1 mutation
Authors: Guo Qing , Sebastian Lois , Sopher Bryce L. , Miller Miles W. , Glazner Gordon W. , Ware Carol B. , Martin George M. , Mattson Mark P.
Published: July 2002

Although an excitotoxic mechanism of neuronal injury has been proposed to play a role in chronic neurodegenerative disorders such as Alzheimer’s disease, and neurotrophic factors have been put forward as potential therapeutic agents, direct evidence is lacking. Taking advantage of the fact that mutations in the presenilin-1 (PS1) gene are causally linked to many cases of early-onset inherited Alzheimer’s disease, we generated PS1 mutant knock-in mice and directly tested the excitotoxic and neurotrophic hypotheses of Alzheimer’s disease. Primary hippocampal neurons from PS1 mutant knock-in mice exhibited increased production of amyloid β-peptide 42/43 and increased vulnerability to excitotoxicity, which occurred in a gene dosage-dependent manner. Neurons expressing mutant PS1 exhibited enhanced calcium responses to glutamate and increased oxyradical production and mitochondrial dysfunction. Pretreatment with either basic fibroblast growth factor or activity-dependent neurotrophic factor protected neurons expressing mutant PS1 against excitotoxicity. Both basic fibroblast growth factor and activity-dependent neurotrophic factor stabilized intracellular calcium levels and abrogated the increased oxyradical production and mitochondrial dysfunction otherwise caused by the PS1 mutation. Our data indicate that neurotrophic factors can interrupt excitotoxic neurodegenerative cascades promoted by PS1 mutations.
A MHC-encoded ubiquitin-like protein (FAT10) binds noncovalently to the spindle assembly checkpoint protein MAD2
Authors: Liu Yuan-Ching , Pan Julian , Zhang Chunyu , Fan Wufang , Collinge Mark , Bender Jeffrey R. , Weissman Sherman M.
Published: July 2002

Recently a number of nonclass I genes were discovered in the human MHC class I region. One of these, FAT10, encodes a protein consisting of two domains with homology to ubiquitin. FAT10 mRNA is expressed constitutively in some lymphoblastoid lines and dendritic cells and in certain other cells after γ-interferon induction. FAT10 protein expression is controlled at several levels including transcription, translation, and protein stability. Yeast two-hybrid screening of a human lymphocyte library and immunoprecipitation studies revealed that FAT10 noncovalently associated with MAD2, a protein implicated in a cell-cycle checkpoint for spindle assembly during anaphase. Thus, FAT10 may modulate cell growth during B cell or dendritic cell development and activation.
Two mechanisms for termination of individual Ca
2+
sparks in skeletal muscle
Authors: Lacampagne Alain , Klein Michael G. , Ward Christopher W. , Schneider Martin F.
Published: July 2002

Ca
2+
sparks are brief, localized elevations of
myoplasmic [Ca
2+
] caused by release of increments of
Ca
2+
via sarcoplasmic reticulum Ca
2+
release
channels in muscle. The properties of individual sparks provide
information regarding the opening of sarcoplasmic reticulum
Ca
2+
channels within functioning cells. Here we use
high-speed confocal microscopy to show that individual Ca
2+
sparks activated by membrane depolarization in single frog skeletal
muscle fibers can be terminated prematurely by repolarization. Thus,
either voltage sensor deactivation on repolarization or release channel
inactivation during continued depolarization can terminate the
Ca
2+
release channel activity underlying voltage-activated
Ca
2+
sparks in skeletal muscle.
UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley
Authors: Logemann Elke , Tavernaro Annette , Schulz Wolfgang , Somssich Imre E. , Hahlbrock Klaus
Published: July 2002

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (
Petroselinum crispum
) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxy
arabino
heptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding
S
-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding
S
-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.