bluuebunny/crossref_metadata_2025 huggingface dataset:
Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes
Authors: Navarro Beatriz , Flores Ricardo
Published: July 2002

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398–399 nucleotides.
In vitro
transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during
in vitro
transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active
in vivo
, because the 5′ terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the
in vitro
structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.
Abnormalities of pancreatic islets by targeted expression of a dominant-negative K
ATP
channel
Authors: Miki Takashi , Tashiro Fumi , Iwanaga Toshihiko , Nagashima Kazuaki , Yoshitomi Hideyuki , Aihara Hiroyuki , Nitta Yoshio , Gonoi Tohru , Inagaki Nobuya , Miyazaki Jun-ichi , Seino Susumu
Published: July 2002

ATP-sensitive K
+
(K
ATP
) channels are known to play important roles in various cellular functions, but the direct consequences of disruption of K
ATP
channel function are largely unknown. We have generated transgenic mice expressing a dominant-negative form of the K
ATP
channel subunit Kir6.2 (Kir6.2G132S, substitution of glycine with serine at position 132) in pancreatic beta cells. Kir6.2G132S transgenic mice develop hypoglycemia with hyperinsulinemia in neonates and hyperglycemia with hypoinsulinemia and decreased beta cell population in adults. K
ATP
channel function is found to be impaired in the beta cells of transgenic mice with hyperglycemia. In addition, both resting membrane potential and basal calcium concentrations are shown to be significantly elevated in the beta cells of transgenic mice. We also found a high frequency of apoptotic beta cells before the appearance of hyperglycemia in the transgenic mice, suggesting that the K
ATP
channel might play a significant role in beta cell survival in addition to its role in the regulation of insulin secretion.
Mouse
Eya
genes are expressed during limb tendon development and encode a transcriptional activation function
Authors: Xu Pin-Xian , Cheng Jane , Epstein Jonathan A. , Maas Richard L.
Published: July 2002

Vertebrate limb tendons are derived from connective cells of the lateral plate mesoderm. Some of the developmental steps leading to the formation of vertebrate limb tendons have been previously identified; however, the molecular mechanisms responsible for tendinous patterning and maintenance during embryogenesis are largely unknown. The
eyes absent
(
eya
) gene of
Drosophila
encodes a novel nuclear protein of unknown molecular function. Here we show that
Eya1
and
Eya2
, two mouse homologues of
Drosophila eya
, are expressed initially during limb development in connective tissue precursor cells. Later in limb development,
Eya1
and
Eya2
expression is associated with cell condensations that form different sets of limb tendons.
Eya1
expression is largely restricted to flexor tendons, while
Eya2
is expressed in the extensor tendons and ligaments of the phalangeal elements of the limb. These data suggest that
Eya
genes participate in the patterning of the distal tendons of the limb. To investigate the molecular functions of the
Eya
gene products, we have analyzed whether the highly divergent PST (proline-serine-threonine)-rich N-terminal regions of Eya1–3 function as transactivation domains. Our results demonstrate that
Eya
gene products can act as transcriptional activators, and they support a role for this molecular function in connective tissue patterning.
A mutant RNA polymerase that forms unusual open promoter complexes
Authors: Severinov Konstantin , Darst Seth A.
Published: July 2002

We describe a mutant
Escherichia coli
RNA polymerase (RNAP) that forms stable open promoter complexes even at −20°C but with a shortened melted region that extends downstream to only position −7. In the presence of initiating transcription substrates, the mutant RNAP undergoes a temperature-dependent isomerization, resulting in a promoter complex that is indistinguishable from the wild-type RNAP–promoter complex, with the melted region extended downstream to position +4. We propose that the open complex formed by the mutant RNAP represents an intermediate on the normal promoter-opening pathway and that our results support earlier findings that initial promoter opening occurs in the upstream region of the −10 promoter consensus element and subsequently extends downstream to encompass the transcription start site.
Suppressor mutations in
Escherichia coli
methionyl-tRNA formyltransferase: Role of a 16-amino acid insertion module in initiator tRNA recognition
Authors: Ramesh Vaidyanathan , Gite Sadanand , Li Yan , RajBhandary Uttam L.
Published: July 2002

The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF; EC
2.1.2.9
) is important for the initiation of protein synthesis in eubacteria and in eukaryotic organelles. The determinants for formylation in the tRNA are clustered mostly in the acceptor stem. As part of studies on the molecular mechanism of recognition of the initiator tRNA by MTF, we report here on the isolation and characterization of suppressor mutations in
Escherichia coli
MTF, which compensate for the formylation defect of a mutant initiator tRNA, lacking a critical determinant in the acceptor stem. We show that the suppressor mutant in MTF has a glycine-41 to arginine change within a 16-amino acid insertion found in MTF from many sources. A mutant with glycine-41 changed to lysine also acts as a suppressor, whereas mutants with changes to aspartic acid, glutamine, and leucine do not. The kinetic parameters of the purified wild-type and mutant Arg-41 and Lys-41 enzymes, determined by using the wild-type and mutant tRNAs as substrates, show that the Arg-41 and Lys-41 mutant enzymes compensate specifically for the strong negative effect of the acceptor stem mutation on formylation. These and other considerations suggest that the 16-amino acid insertion in MTF plays an important role in the specific recognition of the determinants for formylation in the acceptor stem of the initiator tRNA.
Two forms of replication initiator protein: Positive and negative controls
Authors: Wu Jiazhen , Sektas Marian , Chen Dongzhao , Filutowicz Marcin
Published: July 2002

The
pir
gene of plasmid R6K encodes the protein, π, a replication and transcription factor. Two translational options for the
pir
gene give rise to two forms of π protein: a 35.0-kDa form (π
35.0
) and a shortened 30.5-kDa form (π
30.5
). Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only π
35.0
can bind to a site in the (A⋅T)-rich segment of its γ
ori
and activate the γ
ori in vivo
and
in vitro
. However, unlike π
35.0
, π
30.5
can inhibit
in vivo
and
in vitro
replication (activated by π
35.0
). We propose that the two forms of π might have distinct functions in replication. We show that although both forms of π produce dimers, the nature of these dimers is not identical. The N-terminal 37 amino acid residues appear to control the formation of the more stable π
35.0
dimers, whereas another, apparently weaker interface holds together dimers of π
30.5
. We speculate that the leucine zipper-like motif, absent in π
30.5
, controls very specific functions of π protein.
WIP, a protein associated with Wiskott–Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells
Authors: Ramesh Narayanaswamy , Antón Inés M. , Hartwig John H. , Geha Raif S.
Published: July 2002

Wiskott–Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.
Nef proteins encoded by human and simian immunodeficiency viruses induce the accumulation of endosomes and lysosomes in human T cells
Authors: Sanfridson Annika , Hester Susan , Doyle Carolyn
Published: July 2002

The
nef
gene of human and simian immunodeficiency viruses encodes a 27–32-kDa myristoylated protein that is expressed at high levels early after infection. Many functions have been ascribed to the Nef protein, including the down-regulation of cell surface CD4 and a role in viral infectivity. This report describes a novel effect of the Nef protein on human T cells. Electron microscopy was used to examine human T cell lines stably expressing functionally active simian or human immunodeficiency virus type 1 Nef proteins. These studies revealed that the subcellular morphology of Nef-expressing cells was dramatically altered as compared with control cells. The Nef-expressing cells contained numerous membrane-bound vesicles prominently displayed throughout the cytoplasm. The vesicles were analyzed by immunoelectron microscopy (IEM) and by the accumulation of internalized nonspecific membrane tracer, and thus identified as late endosomes and lysosomes. The accumulation of endosomes and lysosomes in response to the expression of Nef was a consistent finding, observed with several different viral isolates and human T cell lines.
Cell–cell contact changes the dynamics of lamellar activity in nontransformed epitheliocytes but not in their
ras
-transformed descendants
Authors: Gloushankova N. A. , Alieva N. A. , Krendel M. F. , Bonder E. M. , Feder H. H. , Vasiliev J. M. , Gelfand I. M.
Published: July 2002

We investigated the structural and functional alterations of active lamellae during initial cell–cell collision and establishment of cell–cell contacts in wounded cultures of nontransformed rat epitheliocytes (IAR-2 line) and their
ras
-transformed descendants (C4 line). Typically, the leading edges of nontransformed cells formed multiple transient contacts followed by establishment of small, stable contacts that would undergo lateral expansion. Formation and expansion of the contact area was accompanied by accumulation of the cell–cell adhesion molecules E-cadherin, β-catenin, and plakoglobin. During lateral expansion, the circumferential bundles of actin filaments, characteristic of IAR-2 cells, disassembled at the site of stable contact forming a concave arc-like actin bundle between adjacent cells at the expanding edge. Pseudopodial activity was completely inhibited in the contact zone and partially inhibited at the free lamellar edges adjacent to the zone of contact. Con A-coated beads on the plasma membrane at the zone of contact stopped undergoing centripetal transport but now moved along the cell–cell boundary. On the other hand,
ras
-transformed cells developed overlapping lamellae and exhibited no detectable change in activity of lamellae, localization of adhesion molecules, and organization of the actin cytoskeleton. We propose that contact-induced reorganization of cell surface adhesion molecules and the underlying cortical cytoskeleton leads to development of lateral traction that may be an essential element in inducing expansion of the contact and in inhibiting local pseudopodial activity.
Pluripotent hemopoietic stem cells are c-kit
<low
Authors: Doi Hiroshi , Inaba Muneo , Yamamoto Yoshihisa , Taketani Shigeru , Mori Shin-Ichiro , Sugihara Akira , Ogata Hajime , Toki Junko , Hisha Hiroko , Inaba Kayo , Sogo Shinji , Adachi Masakazu , Matsuda Tadashi , Good Robert A. , Ikehara Susumu
Published: July 2002

Pluripotent hemopoietic stem cells (P-HSCs) were thought to be c-kit
+
, but recent reports indicate that they are c-kit
low
. In the present report, we provide evidence using Ly5 congenic mice that P-HSCs are c-kit
<low
. Lineage-negative (Lin
−
)/CD71
−
cells among bone marrow cells (BMCs) from C57BL/6 Ly5.1 mice were separated into major histocompatibility complex class I
high
(class I
high
)/c-kit
low
and class I
high
/c-kit
<low
populations. Each population (500 cells) was transplanted into lethally (9.0 Gy) irradiated C57BL/6 Ly5.2 congenic mice along with Ly5.2 (2 × 10
5
) compromised cells. Donor-derived Ly5.1
+
cells were detected 6 months after transplantation in primary recipients reconstituted with either class I
high
/c-kit
low
or class I
high
/c-kit
<low
cells. BMCs (1 × 10
6
) from the primary recipients were further transplanted into secondary recipients (Ly5.2 mice) to assess their long term repopulating activity. Six months after bone marrow transplantation, Ly5.1
+
cells in all lineages were detected only in secondary recipients that had been given BMCs from the primary recipients reconstituted with class I
high
/c-kit
<low
cells but not in cells that were class I
high
/c-kit
low
. When the BMCs (1 × 10
6
) of these secondary recipients were further transplanted into tertiary recipients, all tertiary recipients that had been given BMCs from the secondary recipients originally reconstituted with Lin
−
/CD71
−
/class I
high
/c-kit
low
cells died within 10 days whereas all six tertiary recipients originally reconstituted with Lin
−
/CD71
−
/class I
high
/c-kit
<low
cells showed donor (Ly5.1
+
)-derived cells in their peripheral blood. In the single tertiary recipient that was killed, donor-derived T cells, B cells, macrophages, and granulocytes also were detected in several major hematolymphoid organs. The remaining five mice continue to survive more than 6 months after the tertiary bone marrow transplantation.
In vivo
13
C NMR measurements of cerebral glutamine synthesis as evidence for glutamate–glutamine cycling
Authors: Sibson N. R. , Dhankhar A. , Mason G. F. , Behar K. L. , Rothman D. L. , Shulman R. G.
Published: July 2002

The cerebral tricarboxylic acid (TCA) cycle rate and the rate of glutamine synthesis were measured in rats
in vivo
under normal physiological and hyperammonemic conditions using
13
C NMR spectroscopy. In the hyperammonemic animals, blood ammonia levels were raised from control values of ≈0.05 mM to ≈0.35 mM by an intravenous ammonium acetate infusion. Once a steady-state of cerebral metabolites was established, a [1-
13
C]glucose infusion was initiated, and
13
C NMR spectra acquired continuously on a 7- tesla spectrometer to monitor
13
C labeling of cerebral metabolites. The time courses of glutamate and glutamine C-4 labeling were fitted to a mathematical model to yield TCA cycle rate (
V
TCA
) and the flux from glutamate to glutamine through the glutamine synthetase pathway (
V
gln
). Under hyperammonemia the value of
V
TCA
was 0.57 ± 0.16 μmol/min per g (mean ± SD,
n
= 6) and was not significantly different (unpaired
t
test;
P
> 0.10) from that measured in the control animals (0.46 ± 0.12 μmol/min per g,
n
= 5). Therefore, the TCA cycle rate was not significantly altered by hyperammonemia. The measured rate of glutamine synthesis under hyperammonemia was 0.43 ± 0.14 μmol/min per g (mean ± SD,
n
= 6), which was significantly higher (unpaired
t
test;
P
< 0.01) than that measured in the control group (0.21 ± 0.04 μmol/min per g,
n
= 5). We propose that the majority of the glutamine synthetase flux under normal physiological conditions results from neurotransmitter substrate cycling between neurons and glia. Under hyperammonemia the observed increase in glutamine synthesis is comparable to the expected increase in ammonia transport into the brain and reported measurements of glutamine efflux under such conditions. Thus, under conditions of elevated plasma ammonia an increase in the rate of glutamine synthesis occurs as a means of ammonia detoxification, and this is superimposed on the constant rate of neurotransmitter cycling through glutamine synthetase.
Single-strand DNA intermediates in phage λ’s Red recombination pathway
Authors: Hill Stuart A. , Stahl Mary M. , Stahl Franklin W.
Published: July 2002

An assay was developed to assess early intermediates arising in λ’s Red recombination pathway. Double-strand breaks were delivered
in vivo
to nonreplicating λ chromosomes. Analysis by blot hybridization of total DNA extracts revealed the following: (
i
) long (>1.4 kilobases) single-strand DNA (ssDNA) intermediates; (
ii
) resection proceeding bidirectionally from the break site; (
iii
) single-strand overhangs of 3′ polarity; and (
iv
) in the absence of λ’s
ninR
functions, a requirement of the
red
α gene product for the production of ssDNA. Therefore, the physical characteristics exhibited by these ssDNA molecules are consistent with their being an early recombination intermediate in the Red recombination pathway as proposed previously from genetic and
in vitro
biochemical analyses.
Transient immunomodulation with anti-CD40 ligand antibody and CTLA4Ig enhances persistence and secondary adenovirus-mediated gene transfer into mouse liver
Authors: Kay Mark A. , Meuse Leonard , Gown Allen M. , Linsley Peter , Hollenbaugh Diane , Aruffo Alejandro , Ochs Hans D. , Wilson Christopher B.
Published: July 2002

Although recombinant adenovirus vectors offer a very efficient means by which to transfer genetic information into cellsin vivo, antigen-dependent immunity limits the duration of gene expression and prevents retreatment. Recombinant murine CTLA4Ig and anti-CD40 ligand antibody block costimulatory interactions between T cells and antigen presenting cells. We previously reported that murine CTLA4Ig prolongs adenoviral-mediated gene transfer, but does not allow for secondary expression after readministration of the vector. In studies described here, when anti-CD40 ligand and recombinant murine CTLA4Ig were coadministered around the time of primary vector administration (i) prolonged adenovirus-mediated gene expression (length of experiment up to 1 year) from the livers of >90% of treated mice was observed, and (ii) secondary adenovirus-mediated gene transfer was achieved in >50% of the mice even after the immunosuppressive effects of these agents were no longer present. Nearly two-thirds of these mice had persistent secondary gene expression lasting for at least 200–300 days. Neither agent alone allowed transduction after secondary vector administration. Treated mice had decreased immune responses to the vector as shown by markedly decreased production of neutralizing antibodies, diminished spleen proliferation responses and IFN-γ productionin vitro, and reduced T cell infiltrates in the liver. These results suggest that it may be possible to obtain persistence as well as secondary adenoviral-mediated gene transfer with transient immunosuppressive therapies.
Ultraviolet radiation, but not γ radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389
Authors: Lu Hua , Taya Yoichi , Ikeda Masako , Levine Arnold J.
Published: July 2002

Polyclonal antibodies were produced and purified that selectively react with a p53 epitope containing the murine phosphoserine-389 or the human phosphoserine-392 residue, but not the unphosphorylated epitope. These antibodies, termed alpha-392, were employed to demonstrate that the phosphorylation of this serine-389 residue in the p53 protein occurs
in vivo
in response to ultraviolet radiation of cells containing the p53 protein. After ultraviolet radiation of cells in culture, p53 levels increase and concomitantly serine-389 is phosphorylated in these cells. By contrast, the serine-389 phosphorylation of the p53 protein was not detected by these antibodies in the increased levels of p53 protein made in response to γ radiation or the treatment of cells with etoposide. These results demonstrate an ultraviolet responsive and specific phosphorylation site at serine-389 of the mouse or serine-392 of the human p53 protein. Previous studies have demonstrated that this phosphorylation of p53 activates the protein for specific DNA binding. This study demonstrates
in vivo
a unique phosphorylation site in the p53 protein that responds to a specific type of DNA damage.
A human homolog of the
Saccharomyces cerevisiae REV3
gene, which encodes the catalytic subunit of DNA polymerase ζ
Authors: Gibbs Peter E. M. , McGregor W. Glenn , Maher Veronica M. , Nisson Paul , Lawrence Christopher W.
Published: July 2002

To get a better understanding of mutagenic mechanisms in humans, we have cloned and sequenced the human homolog of the
Saccharomyces cerevisiae REV3
gene. The yeast gene encodes the catalytic subunit of DNA polymerase ζ, a nonessential enzyme that is thought to carry out translesion replication and is responsible for virtually all DNA damage-induced mutagenesis and the majority of spontaneous mutagenesis. The human gene encodes an expected protein of 3,130 residues, about twice the size of the yeast protein (1,504 aa). The two proteins are 29% identical in an amino-terminal region of ≈340 residues, 39% identical in a carboxyl-terminal region of ≈850 residues, and 29% identical in a 55-residue region in the middle of the two genes. The sequence of the expected protein strongly predicts that it is the catalytic subunit of a DNA polymerase of the pol ζ type; the carboxyl-terminal domain possesses, in the right order, the six motifs characteristic of eukaryotic DNA polymerases, most closely resembles yeast pol ζ among all polymerases in the GenBank database, and is different from the human α, δ, and ɛ enzymes. Human cells expressing high levels of an
hsREV3
antisense RNA fragment grow normally, but show little or no UV-induced mutagenesis and are slightly more sensitive to killing by UV. The human gene therefore appears to carry out a function similar to that of its yeast counterpart.
Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells
Authors: Gambaryan Stepan , Wagner Charlotte , Smolenski Albert , Walter Ulrich , Poller Wolfgang , Haase Winfried , Kurtz Armin , Lohmann Suzanne M.
Published: July 2002

An overactive renin-angiotensin-aldosterone system (RAAS) has a central role in the pathogenesis of hypertension and cardiac hypertrophy, precursors of cardiac failure. Natriuretic peptides and NO acting through their second messenger, cGMP, increase natriuresis and diuresis, and inhibit renin release; however the mechanism by which this inhibition of the RAAS system functions is obscure. We recently reported cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), elucidated its first known function of inhibiting the cystic fibrosis transmembrane conductance regulator in rat intestine, and initially described its location in rat kidney juxtaglomerular (JG) cells, the ascending thin limb, and the brush border of proximal tubules. Here, we demonstrate inhibition of isoproterenol- or forskolin-stimulated renin release by 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP), a selective activator of cGK, and prevention of this inhibition by a selective inhibitor of cGK, Rp-8-pCPT-cGMPS. In systems of differing complexity, inhibition by 8-pCPT-cGMP was nearly complete in isolated perfused kidney and microdissected afferent arterioles but only ≈25% in isolated JG cells. Expression of either cGK II or cGK I in JG cells by using adenoviral vectors enhanced the inhibition of forskolin-stimulated renin release by 8-pCPT-cGMP to 50%. Our results indicate that cGK II, and possibly cGK I, can mediate cGMP inhibitory effects on renin release and are physiological components of the cGMP signal transduction system which opposes the RAAS.
Molecular remedy of complex I defects: Rotenone-insensitive internal NADH-quinone oxidoreductase of
Saccharomyces cerevisiae
mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells
Authors: Seo Byoung Boo , Kitajima-Ihara Tomomi , Chan Edward K. L. , Scheffler Immo E. , Matsuno-Yagi Akemi , Yagi Takao
Published: July 2002

The
NDI1
gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of
Saccharomyces cerevisiae
mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable
NDI1
-transfected cells were obtained by screening with antibiotic G418. The
NDI1
gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunofluorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate. The activities were inhibited by flavone, antimycin A, and KCN but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes. Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the
NDI1
-transfected cells were able to grow in the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1. These results indicated that the
S. cerevisiae
Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. It is concluded that the
NDI1
gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.
In vitro
loss of heterozygosity targets the
PTEN/MMAC1
gene in melanoma
Authors: Robertson Gavin P. , Furnari Frank B. , Miele Mary E. , Glendening Michael J. , Welch Danny R. , Fountain Jane W. , Lugo Tracy G. , Huang H.-J. Su , Cavenee Webster K.
Published: July 2002

Gross genetic lesions of chromosome 10 occur in 30–50% of sporadic human melanomas. To test the functional significance of this observation, we have developed an
in vitro
loss of heterozygosity approach in which a wild-type chromosome 10 was transferred into melanoma cells, where there was selection for its breakage and regional deletion to relieve its growth suppressive effects. The overlap of these events was at band 10q23, the site of the recently isolated
PTEN/MMAC1
tumor suppressor gene, suggesting it as a potential target. Although the gene was expressed in the parental cells, both of its chromosomal alleles contained truncating mutations.
In vitro
loss of heterozygosity resulted in loss of the chromosomally introduced wild-type
PTEN/MMAC1
, and ectopic expression of the gene caused cell growth suppression. Thus, this approach identified
PTEN/MMAC1
as a target in malignant melanoma and may provide an alternative means to localizing tumor suppressor genes.
A novel signal recognition particle targets light-harvesting proteins to the thylakoid membranes
Authors: Schuenemann Danja , Gupta Shalini , Persello-Cartieaux Fabienne , Klimyuk Victor I. , Jones Jonathan D. G. , Nussaume Laurent , Hoffman Neil E.
Published: July 2002

The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, “transit complex”, and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.
An SmtB-like repressor from
Synechocystis
PCC 6803 regulates a zinc exporter
Authors: Thelwell Craig , Robinson Nigel J. , Turner-Cavet Jennifer S.
Published: July 2002

ORF slr0798, now designated
ziaA
, from
Synechocystis
PCC 6803 encodes a polypeptide with sequence features of heavy metal transporting P-type ATPases. Increased Zn
2+
tolerance and reduced
65
Zn accumulation was observed in
Synechococcus
PCC 7942, strain R2-PIM8(
smt
), containing
ziaA
and upstream regulatory sequences, compared with control cells. Conversely, reduced Zn
2+
tolerance was observed following disruption of
ziaA
in
Synechocystis
PCC 6803, and
ziaA
-mediated restoration of Zn
2+
tolerance has subsequently been used as a selectable marker for transformation. Nucleotide sequences upstream of
ziaA
, fused to a promoterless
lacZ
gene, conferred Zn
2+
-dependent β-galactosidase activity when introduced into R2-PIM8(
smt
). The product of ORF sll0792, designated ZiaR, is a Zn
2+
-responsive repressor of
ziaA
transcription. Reporter gene constructs lacking
ziaR
conferred elevated Zn
2+
-independent expression from the
ziaA
operator–promoter in R2-PIM8(
smt
). Gel retardation assays detected ZiaR-dependent complexes forming with the
zia
operator–promoter and ZiaR–DNA binding was enhanced by treatment with a metal-chelator
in vitro
. Two mutants of ZiaR (C71S/C73S and H116R) bound to, and repressed expression from, the
ziaA
operator–promoter but were unable to sense Zn
2+
. Metal coordination to His-imidazole and Cys-thiolate ligands at these residues of ZiaR is thus implicated in Zn
2+
-perception by
Synechocystis
PCC 6803.
Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence
Authors: Mitchell Deborah M. , Zhou Mingyue , Pariyarath Rajalakshmi , Wang Hongxing , Aitchison John D. , Ginsberg Henry N. , Fisher Edward A.
Published: July 2002

When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated with the microsomes. Furthermore, by crosslinking sec61β to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB100–ubiquitin conjugates could be detected. When lipid synthesis and lipoprotein assembly/secretion were stimulated by adding oleic acid (OA) to the chase medium, apoB100 was deubiquitinated, and its interaction with sec61β was disrupted, signifying completion of translocation concomitant with the formation of lipoprotein particles. MTP participates in apoB100 translocation and lipoprotein assembly. In the presence of OA, when MTP lipid-transfer activity was inhibited at the end of pulse labeling, apoB100 secretion was abolished. In contrast, when the labeled apoB100 was allowed to accumulate in the cell for 60 min before adding OA and the inhibitor, apoB100 lipidation and secretion were no longer impaired. Overall, the data imply that during most of its association with the endoplasmic reticulum, apoB100 is close to or within the translocon and is accessible to both the ubiquitin-proteasome and lipoprotein-assembly pathways. Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events.
Deletion ofPlasmodium berghei-specific CD4+T cells adoptively transferred into recipient mice after challenge with homologous parasite
Authors: Hirunpetcharat Chakrit , Good Michael F.
Published: July 2002

The immune response to malaria parasites includes T cell responses that reduce parasites by effector T cell responses and by providing help for antibody responses. Some parasites are more sensitive to antibody and others are more sensitive to cell-mediated immunity. We demonstrate that cultured CD4+T cells that produce interferon γ and interleukin 2, but not interleukin 4, in response to stimulation with the rodent parasitePlasmodium bergheican reduce but not eliminate parasitesin vivoafter adoptive transfer. Although cells can persistin vivofor up to 9 months in uninfected mice, infection results in elimination of up to 99% of specific T cells in different tissues, as judged by tracking T cells labeled with the fluorescent dye 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester. T cells specific for ovalbumin are unaffected.In vivoactivation and division of transferred T cellsper seare not responsible for deletion because T cells positive for 5-(and -6)-carboxyfluorescein diacetate succinimidyl ester divide up to six times within 7 days in uninfected mice and are not deleted. Understanding the factors responsible for parasite-mediated specific deletion of T cells would enhance our knowledge of parasite immunity.
Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth
Authors: Chun Tae-Yon , Gregg David , Sarkar Dipak K. , Gorski Jack
Published: July 2002

PR1 cells are a prolactin (PRL)-secreting cell line derived from a pituitary lactotroph tumor found in 17β-estradiol-treated Fischer 344 rats. We examined the effect of estrogen on cell proliferation and PRL synthesis under various culture conditions. Estrogen, at extremely low concentrations, induces cell proliferation in this cell line, whereas antiestrogen inhibits proliferation. Interestingly, the proliferation response is much more sensitive than the PRL response because 0.01 pM estradiol or diethylstilbestrol induces half-maximal growth induction [≈0.1% estrogen receptor (ER) occupancy is required], whereas 0.01 nM concentration is required for half-maximal PRL induction (≈50% ER occupancy is required). The proliferation response is not as sensitive to antiestrogen as the PRL response, because 10 nM concentration of the pure antiestrogen ICI 182,780 could not inhibit 1 nM estradiol- or diethylstilbestrol-induced proliferation. The same concentration of ICI 182,780 decreased PRL secretion to 1% of estradiol- or diethylstilbestrol-induced prolactin secretion suggesting a possible dichotomy of ER control of proliferation and PRL synthesis. The
K
d
of ER binding in these cells is about 3 × 10
−11
M. These results with the PR1 cells extend previous studies in other estrogen- regulated systems and suggest that only a small pool of ER is required for cell proliferation in contrast with the regulation of expression of specific genes. They also raise questions as to how a dimeric receptor functions when only one ligand site is occupied or when both an estrogen and an antiestrogen occupy one dimer.
Archaeal translation initiation revisited: The initiation factor 2 and eukaryotic initiation factor 2B α-β-δ subunit families
Authors: Kyrpides Nikos C. , Woese Carl R.
Published: July 2002

As the amount of available sequence data increases, it becomes apparent that our understanding of translation initiation is far from comprehensive and that prior conclusions concerning the origin of the process are wrong. Contrary to earlier conclusions, key elements of translation initiation originated at the Universal Ancestor stage, for homologous counterparts exist in all three primary taxa. Herein, we explore the evolutionary relationships among the components of bacterial initiation factor 2 (IF-2) and eukaryotic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in introducing the initiator tRNA into the translation mechanism and performing the first step in the peptide chain elongation cycle. All Archaea appear to posses a fully functional eIF-2 molecule, but they lack the associated GTP recycling function, eIF-2B (a five-subunit molecule). Yet, the Archaea do posses members of the gene family defined by the (related) eIF-2B subunits α, β, and δ, although these are not specifically related to any of the three eukaryotic subunits. Additional members of this family also occur in some (but by no means all) Bacteria and even in some eukaryotes. The functional significance of the other members of this family is unclear and requires experimental resolution. Similarly, the occurrence of bacterial IF-2-like molecules in all Archaea and in some eukaryotes further complicates the picture of translation initiation. Overall, these data lend further support to the suggestion that the rudiments of translation initiation were present at the Universal Ancestor stage.
Expression of monocarboxylate transporter mRNAs in mouse brain: Support for a distinct role of lactate as an energy substrate for the neonatal vs. adult brain
Authors: Pellerin Luc , Pellegri Giovanni , Martin Jean-Luc , Magistretti Pierre J.
Published: July 2002

Under particular circumstances like lactation and fasting, the blood-borne monocarboxylates acetoacetate, β-hydroxybutyrate, and lactate represent significant energy substrates for the brain. Their utilization is dependent on a transport system present on both endothelial cells forming the blood-brain barrier and on intraparenchymal brain cells. Recently, two monocarboxylate transporters, MCT1 and MCT2, have been cloned. We report here the characterization by Northern blot analysis and by
in situ
hybridization of the expression of MCT1 and MCT2 mRNAs in the mouse brain. In adults, both transporter mRNAs are highly expressed in the cortex, the hippocampus and the cerebellum. During development, a peak in the expression of both transporters occurs around postnatal day 15, declining rapidly by 30 days at levels observed in adults. Double-labeling experiments reveal that the expression of MCT1 mRNA in endothelial cells is highest at postnatal day 15 and is not detectable at adult stages. These results support the notion that monocarboxylates are important energy substrates for the brain at early postnatal stages and are consistent with the sharp decrease in blood-borne monocarboxylate utilization after weaning. In addition, the observation of a sustained intraparenchymal expression of monocarboxylate transporter mRNAs in adults, in face of the seemingly complete disappearance of their expression on endothelial cells, reinforces the view that an intercellular exchange of lactate occurs within the adult brain.
Neuroprotection with noninvasive neurotrophin delivery to the brain
Authors: Wu Dafang , Pardridge William M.
Published: July 2002

Brain-derived neurotrophic factor (BDNF) is neuroprotective in the ischemic hippocampus if the neurotrophin is injected directly into the brain. However, the efficacy of BDNF via peripheral (i.v.) administration is limited by the lack of transport of the neurotrophin through the brain capillary wall, which makes up the blood-brain barrier (BBB)
in vivo
. The present studies describe a molecular reformulation of BDNF that incorporates polyethylene glycol (PEG) moieties at surface carboxyl residues, to optimize plasma pharmacokinetics, and links pegylated BDNF to the OX26 mAb, which undergoes receptor-mediated transport through the BBB via the brain capillary endothelial transferrin receptor. The BDNF-PEG 2000-biotin conjugated to OX26/streptavidin was administered i.v. daily to rats for 1 week after a 12-min period of transient forebrain ischemia. The neuronal density in the CA1 sector of the hippocampus was decreased 68 ± 10% at 1 week after the ischemia. There was no neuroprotective effect of the unconjugated BDNF or unconjugated OX26 mAb. However, the hippocampal CA1 neuronal density was normalized by i.v. administration of the BDNF-PEG 2000-biotin conjugated to OX26/streptavidin. These studies demonstrate that peripherally administered BDNF may have neuroprotective effects in brain, if the neurotrophin is reformulated to (
i
) optimize plasma pharmacokinetics with carboxyl-directed pegylation, and (
ii
) enable transport through the BBB by coupling to brain transport vectors.
Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals
Authors: Buck Jochen , Sinclair Meeghan L. , Schapal Lissy , Cann Martin J. , Levin Lonny R.
Published: July 2002

Mammals have nine differentially regulated isoforms of G protein-responsive transmembrane-spanning adenylyl cyclases. We now describe the existence of a distinct class of mammalian adenylyl cyclase that is soluble and insensitive to G protein or Forskolin regulation. Northern analysis indicates the gene encoding soluble adenylyl cyclase (sAC) is preferentially expressed in testis. As purified from rat testis cytosol, the active form of sAC appears to be a fragment derived from the full-length protein, suggesting a proteolytic mechanism for sAC activation. The two presumptive catalytic domains of sAC are closely related to cyanobacterial adenylyl cyclases, providing an evolutionary link between bacterial and mammalian signaling molecules.
Comparison of the intracellular signaling responses by three chimeric fibroblast growth factor receptors in PC12 cells
Authors: Raffioni Simona , Thomas Didier , Foehr Erik D. , Thompson Leslie M. , Bradshaw Ralph A.
Published: July 2002

Stably transfected PC12 cell lines expressing similar amounts of chimeric receptors composed of the extracellular domain of the human platelet-derived growth factor (PDGF)β receptor and the transmembrane and intracellular domains of the fibroblast growth factor receptors (FGFRs) 1, 3, and 4 undergo ligand-induced differentiation. The FGFR1 chimera (PFR1) is the most potent of the three, and PFR4 requires more frequent (every 24 hr) addition of ligand to maintain the response. Both PFR1 and -3 also show significant ligand-independent autophosphorylation but PFR4 does not. All of the chimeras activated phospholipase Cγ, Shc, FGFR substrate (FRS)2, and the mitogen-activated protein kinases, ERK1 and 2. PFR4 was moderately weaker in stimulating these effects as well; PFR1 and -3 were comparable. None of the chimeras induced Sos association or were coprecipitated with Shc. Cotransfection of a dominant-negative Shc derivative, with tyrosine at 239, 240, and 317 replaced with phenylalanine, in the PFR-expressing cells was without effect on PDGF-induced neurite outgrowth. The same derivative substantially inhibited the response of these cells to NGF. These results indicate that FGFR1, 3, and 4 (
i
) are capable of signaling in a similar fashion; (
ii
) primarily use FRS2 and, perhaps, PLCγ; and (
iii
) do not utilize Shc. The results also suggest that the principal difference between FGFR1, 3, and 4 is in the strength of the tyrosine kinase activity and that qualitative differences in signaling capacity are likely to be less important.
Electrically driven motor in the outer hair cell: Effect of a mechanical constraint
Authors: Adachi M. , Iwasa K. H.
Published: July 2002

The outer hair cell has a unique voltage-dependent motility associated with charge transfer across the plasma membrane. To examine mechanical changes in the membrane that are coupled with such charge movements, we digested the undercoating of the membrane with trypsin. We inflated the cell into a sphere and constrained the surface area by not allowing volume changes. We found that this constraint on the membrane area sharply reduced motor-associated charge movement across the membrane, demonstrating that charge transfer is directly coupled with membrane area change. This electromechanical coupling in the plasma membrane must be the key element for the motile mechanism of the outer hair cell.
Treatment of noninfectious intermediate and posterior uveitis with the humanized anti-Tac mAb: A phase I/II clinical trial
Authors: Nussenblatt Robert B. , Fortin Eric , Schiffman Rhett , Rizzo Luiz , Smith Janine , Van Veldhuisen Paul , Sran Pushpa , Yaffe Anita , Goldman Carolyn K. , Waldmann Thomas A. , Whitcup Scott M.
Published: July 2002

To evaluate the safety and potential therapeutic activity of humanized anti-IL-2 receptor mAb (Daclizumab) therapy in the treatment of patients with severe, sight-threatening, intermediate and posterior noninfectious uveitis, a nonrandomized, open-label, pilot study was performed. Patients with uveitis were treated with a minimum of 20 mg of prednisone, cyclosporine, antimetabolites, or any combination of these agents were eligible. Patients were weaned off their systemic immunosuppressive agents according to a standardized schedule, while ultimately receiving Daclizumab infusions every 4 weeks. Anti-IL-2 receptor antibody therapy, given intravenously with intervals of up to 4 weeks in lieu of standard immunosuppressive therapy, appeared to prevent the expression of severe sight-threatening intraocular inflammatory disease in 8 of 10 patients treated over a 12-month period, with noted improvements in visual acuity. One patient met a primary endpoint with a loss of vision of 10 letters or more from baseline in one eye and another patient discontinued therapy because of evidence of increased ocular inflammation. All patients were able to tolerate the study medications without the need for dose reduction. We report effective long-term use of anti-IL-2 therapy for an autoimmune indication. These initial findings would suggest that anti-IL-2 receptor therapy may be an effective therapeutic approach for uveitis and, by implication, other disorders with a predominant Th1 profile.