bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancer
Authors: Mueller Elisabetta , Smith Matthew , Sarraf Pasha , Kroll Todd , Aiyer Anita , Kaufman Donald S. , Oh William , Demetri George , Figg William D. , Zhou Xiao-Ping , Eng Charis , Spiegelman Bruce M. , Kantoff Philip W.
Published: July 2002

Peroxisome proliferator-activated receptor γ (PPARγ) is a
nuclear hormone receptor that plays a key role in the differentiation
of adipocytes. Activation of this receptor in liposarcomas and breast
and colon cancer cells also induces cell growth inhibition and
differentiation. In the present study, we show that PPARγ is
expressed in human prostate adenocarcinomas and cell lines derived from
these tumors. Activation of this receptor with specific ligands exerts
an inhibitory effect on the growth of prostate cancer cell lines.
Further, we show that prostate cancer and cell lines do not have
intragenic mutations in the PPARγ gene, although 40% of the
informative tumors have hemizygous deletions of this gene. Based on our
preclinical data, we conducted a phase II clinical study in patients
with advanced prostate cancer using troglitazone, a PPARγ ligand used
for the treatment of type 2 diabetes. Forty-one men with histologically
confirmed prostate cancer and no symptomatic metastatic disease were
treated orally with troglitazone. An unexpectedly high incidence of
prolonged stabilization of prostate-specific antigen was seen in
patients treated with troglitazone. In addition, one patient had a
dramatic decrease in serum prostate-specific antigen to nearly
undetectable levels. These data suggest that PPARγ may serve as a
biological modifier in human prostate cancer and its therapeutic
potential in this disease should be further investigated.
Role of the cytoplasmic tyrosines of β1A integrins in transformation by v-src
Authors: Sakai Takao , Jove Richard , Fässler Reinhard , Mosher Deane F.
Published: July 2002

GD25 cells lacking β1 integrins or expressing β1A with
mutations of conserved cytoplasmic tyrosines (Y783, Y795) to
phenylalanine have poor directed migration to platelet-derived growth
factor or lysophosphatidic acid when compared with GD25 cells
expressing wild-type β1A. We studied the effects of v-src on these
cells. Transformation with v-src caused tyrosine and serine
phosphorylation of wild-type β1A but not of Y783/795F doubly
mutated β1A. v-src-transformed cells had rounded and/or fusiform
morphology and poor assembly of fibronectin matrix. Adhesion to
fibronectin or laminin and coupling of focal contacts to
actin-containing cytoskeleton were preserved in transformed Y783/795F
cells but lost on transformation when β1A was wild type. Transformed
Y783/795F cells also retained ability, albeit limited, to migrate
across filters, whereas transformed cells with wild-type β1A were
unable to transverse filters. Studies of single tyrosine mutants showed
that the more important tyrosine for retaining ability to adhere,
assemble focal contacts, and migrate is Y783. These results suggest
that overactive phosphorylation of cytoplasmic residues of β1A,
particularly Y783, accounts in part for the phenotype of
v-src-transformed cells.
Activation of the Mason–Pfizer monkey virus protease within immature capsids
in
vitro
Authors: Parker Scott D. , Hunter Eric
Published: July 2002

For all retroviruses, the completion of the viral budding process
correlates with the activation of the viral protease by an unknown
mechanism, and, as the structural (Gag) polyproteins are cleaved by the
viral protease, maturation of the immature virus-like particle into an
infectious virion. Unlike most retroviruses, the Mason–Pfizer
monkey virus Gag polyproteins assemble into immature capsids within the
cytoplasm of the cell before the viral budding event. The results
reported here describe a unique experimental system in which
Mason–Pfizer monkey virus immature capsids are removed from the cell,
and the protease is activated
in vitro
by the addition
of a reducing agent. The cleavage of the protease from the precursor
form is a primary event, which proceeds with a half time of 14 min, and
is followed by authentic processing of the Gag polyproteins. Activity
of the viral protease
in vitro
depends on pH, with an
increase in catalytic rates at acidic and neutral pH. The initiation of
protease activity within immature capsids
in vitro
demonstrates that viral protease activity is sensitive to
oxidation-reduction conditions, and that the viral protease can be
activated in the absence of viral budding.
Siah-1 binds and regulates the function of Numb
Authors: Susini Laurent , Passer Brent J. , Amzallag-Elbaz Nathalie , Juven-Gershon Tamar , Prieur Sylvie , Privat Nicolas , Tuynder Marcel , Gendron Marie-Claude , Israël Alain , Amson Robert , Oren Moshe , Telerman Adam
Published: July 2002

The
Drosophila
Seven in absentia
(
Sina
) gene product originally was described as a
protein that controls cell fate decisions during eye development. Its
mammalian homolog, Siah-1, recently was found to be involved in
p53-dependent and -independent pathways of apoptosis and
G
1
arrest. We report that Siah-1 interacts directly with
and promotes the degradation of the cell fate regulator Numb.
Siah-1-mediated Numb degradation leads to redistribution of
endogenous cell-surface Notch to the cytoplasm and nucleus
and to augmented Notch-regulated transcriptional activity. These data
imply that through its ability to target Numb for degradation, Siah-1
can act as a key regulator of Numb-related activities, including Notch
signaling.
Functional implications of protein-protein interactions in icosahedral viruses.
Authors: Johnson J E
Published: July 2002

Biological processes often require that a single gene product participate in multiple types of molecular interactions. Viruses with quasiequivalent capsids provide an excellent paradigm for studying such phenomena because identical protein subunits are found in different structural environments. Differences in subunit joints may be controlled by protein segments, duplex or single-stranded RNA, metal ions, or some combination of these. Each of the virus groups examined display a distinctive mechanism for switching interface interactions, illustrating the magnitude of options that are likely to be found in other biological systems. In addition to determining capsid morphology, assembly controls the timing of autocatalytic maturation cleavage of the viral subunits that is required for infectivity in picorna-, noda-, and tetraviruses. The mechanism of assembly-dependent cleavage is conserved in noda- and tetraviruses, although the quaternary structures of the capsids are different as are the molecular switches that control subunit interfaces. The function of the cleavage in picorna-, noda-, and tetraviruses is probably to release polypeptides that participate in membrane translocation of RNA.
In vitro activation of the interferon-induced, double-stranded RNA-dependent protein kinase PKR by RNA from the 3' untranslated regions of human alpha-tropomyosin.
Authors: Davis S , Watson J C
Published: July 2002

The cellular kinase known as PKR (protein kinase RNA-activated) is induced by interferon and activated by RNA. PKR is known to have antiviral properties due to its role in translational control. Active PKR phosphorylates eukaryotic initiation factor 2 alpha and leads to inhibition of translation, including viral translation. PKR is also known to function as a tumor suppressor, presumably by limiting the rate of tumor-cell translation and growth. Recent research has shown that RNA from the 3' untranslated region (3'UTR) of human alpha-tropomyosin has tumor-suppressor properties in vivo [Rastinejad, F., Conboy, M. J., Rando, T. A. & Blau, H. M. (1993) Cell 75, 1107-1117]. Here we report that purified RNA from the 3'UTR of human alpha-tropomyosin can inhibit in vitro translation in a manner consistent with activation of PKR. Inhibition of translation by tropomyosin 3'UTR RNA was observed in a rabbit reticulocyte lysate system, which is known to contain endogenous PKR but was not seen in wheat germ lysate, which is not responsive to a known activator of PKR. A control RNA purified in the same manner as the 3'UTR RNA did not inhibit translation in either system. The inhibition of translation observed in reticulocyte lysates was prevented by the addition of adenovirus virus-associated RNA1 (VA RNAI), an inhibitor of PKR activation. Tropomyosin 3'UTR RNA was bound by immunoprecipitated PKR and activated the enzyme in an in vitro kinase assay. These data suggest that activation of PKR could be the mechanism by which tropomyosin 3'UTR RNA exerts its tumor-suppression activity in vivo.
Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest.
Authors: Gu W , Wind M , Reines D
Published: July 2002

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.
Ceramide-binding and activation defines protein kinase c-Raf as a ceramide-activated protein kinase.
Authors: Huwiler A , Brunner J , Hummel R , Vervoordeldonk M , Stabel S , van den Bosch H , Pfeilschifter J
Published: July 2002

Interleukin 1 is the prototype of an inflammatory cytokine, and evidence suggests that it uses the sphingomyelin pathway and ceramide production to trigger mitogen-activated protein kinase (MAPK) activation and subsequent gene expression required for acute inflammatory processes. To identify downstream signaling targets of ceramide, a radioiodinated photoaffinity labeling analog of ceramide ([125I] 3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramide) was employed. It is observed that ceramide specifically binds to and activates protein kinase c-Raf, leading to a subsequent activation of the MAPK cascade. Ceramide does not bind to any other member of the MAPK module nor does it bind to protein kinase C-zeta. These data identify protein kinase c-Raf as a specific molecular target for interleukin 1 beta-stimulated ceramide formation and demonstrate that ceramide is a lipid cofactor participating in regulation of c-Raf activity.
Independence of metastatic ability and extravasation: metastatic ras-transformed and control fibroblasts extravasate equally well.
Authors: Koop S , Schmidt E E , MacDonald I C , Morris V L , Khokha R , Grattan M , Leone J , Chambers A F , Groom A C
Published: July 2002

Escape of cancer cells from the circulation (extravasation) is thought to be a major rate-limiting step in metastasis, with few cells being able to extravasate. Furthermore, highly metastatic cells are believed to extravasate more readily than poorly metastatic cells. We assessed in vivo the extravasation ability of highly metastatic ras-transformed NIH 3T3 cells (PAP2) versus control nontumorigenic nontransformed NIH 3T3 cells and primary mouse embryo fibroblasts. Fluorescently labeled cells were injected intravenously into chicken embryo chorioallantoic membrane and analyzed by intravital videomicroscopy. The chorioallantoic membrane is an appropriate model for studying extravasation, since, at the embryonic stage used, the microvasculature exhibits a continuous basement membrane and adult permeability properties. The kinetics of extravasation were assessed by determining whether individual cells (n = 1481) were intravascular, extravascular, or in the process of extravasation, at 3, 6, and 24 h after injection. Contrary to expectations, our results showed that all three cell types extravasated with the same kinetics. By 24 h after injection > 89% of observed cells had completed extravasation from the capillary plexus. After extravasation, individual fibroblasts of all cell types demonstrated preferential migration within the mesenchymal layer toward arterioles, not to venules or lymphatics. Thus in this model and for these cells, extravasation is independent of metastatic ability. This suggests that the ability to extravasate in vivo is not necessarily predictive of subsequent metastasis formation, and that postextravasation events may be key determinants in metastasis.
A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.
Authors: Dropulić B , Hĕrmánková M , Pitha P M
Published: July 2002

Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo.
Occurrence of two somatostatin variants in the frog brain: characterization of the cDNAs, distribution of the mRNAs, and receptor-binding affinities of the peptides.
Authors: Tostivint H , Lihrmann I , Bucharles C , Vieau D , Coulouarn Y , Fournier A , Conlon J M , Vaudry H
Published: July 2002

In tetrapods, only one gene encoding a somatostatin precursor has been identified so far. The present study reports the characterization of the cDNA clones that encode two distinct somatostatin precursors in the brain of the frog Rana ridibunda. The cDNAs were isolated by using degenerate oligonucleotides based on the sequence of the central region of somatostatin to screen a frog brain cDNA library. One of the cDNAs encodes a 115-amino acid protein (prepro-somatostatin-14; PSS1) that exhibits a high degree of structural similarity with the mammalian somatostatin precursor. The other cDNA encodes a 103-amino acid protein (prepro-[Pro2, Met13]somatostatin-14; PSS2) that contains the sequence of the somatostatin analog (peptide SS2) at its C terminus, but does not exhibit appreciable sequence similarity with PSS1 in the remaining region. In situ hybridization studies indicate differential expression of the PSS1 and PSS2 genes in the septum, the lateral part of the pallium, the amygdaloid complex, the posterior nuclei of the thalamus, the ventral hypothalamic nucleus, the torus semicircularis and the optic tectum. The somatostatin variant SS2 was significantly more potent (4-6 fold) than somatostatin itself in displacing [125I-Tyr0, D-Trp8] somatostatin-14 from its specific binding sites. The present study indicates that the two somatostatin variants could exert different functions in the frog brain and pituitary. These data also suggest that distinct genes encoding somatostatin variants may be expressed in the brain of other tetrapods.
Characterization of small nontranslated polyadenylylated RNAs in vaccinia virus-infected cells.
Authors: Lu C , Bablanian R
Published: July 2002

Host protein synthesis is selectively inhibited in vaccinia virus-infected cells. This inhibition has been associated with the production of a group of small, nontranslated, polyadenylylated RNAs (POLADS) produced during the early part of virus infection. The inhibitory function of POLADS is associated with the poly(A) tail of these small RNAs. To determine the origin of the 5'-ends of POLADS, reverse transcription was performed with POLADS isolated from VV-infected cells at 1 hr and 3.5 hr post infection. The cDNAs of these POLADS were cloned into plasmids (pBS or pBluescript II KS +/-), and their nucleotide composition was determined by DNA sequencing. The results of this investigation show the following: There is no specific gene encoding for POLADS. The 5' ends of POLADS may be derived from either viral or cellular RNAs. Any RNA sequence including tRNAs, small nuclear RNAs and 5'ends of mRNAs can become POLADS if they acquire a poly(A) tail at their 3' ends during infection. This nonspecific polyadenylylation found in vaccinia virus-infected cells is probably conducted by vaccinia virus poly(A)+ polymerase. No consensus sequence is found on the 5' ends of POLADS for polyadenylylation. The 5' ends of POLADS have no direct role in their inhibitory activity of protein synthesis.
Signals and signs in the nervous system: The dynamic anatomy of electrical activity is probably information-rich
Authors: Bullock Theodore Holmes
Published: July 2002

The dichotomy between two groups of workers on neuroelectrical activity is retarding progress. To study the interrelations between neuronal unit spike activity and compound field potentials of cell populations is both unfashionable and technically challenging. Neither of the mutual disparagements is justified: that spikes are to higher functions as the alphabet is to Shakespeare and that slow field potentials are irrelevant epiphenomena. Spikes are not the basis of
the
neural code but of multiple codes that coexist with nonspike codes. Field potentials are mainly information-rich
signs
of underlying processes, but sometimes they are also
signals
for neighboring cells, that is, they exert influence. This paper concerns opportunities for new research with many channels of wide-band (spike and slow wave) recording. A wealth of structure in time and three-dimensional space is different at each scale—micro-, meso-, and macroactivity. The depth of our ignorance is emphasized to underline the opportunities for uncovering new principles. We cannot currently estimate the relative importance of spikes and synaptic communication vs. extrasynaptic graded signals. In spite of a preponderance of literature on the former, we must consider the latter as probably important. We are in a primitive stage of looking at the time series of wide-band voltages in the compound, local field, potentials and of choosing descriptors that discriminate appropriately among brain loci, states (functions), stages (ontogeny, senescence), and taxa (evolution). This is not surprising, since the brains in higher species are surely the most complex systems known. They must be the greatest reservoir of new discoveries in nature. The complexity should not deter us, but a dose of humility can stimulate the flow of imaginative juices.
Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants
Authors: Pang Sheng-Zhi , Jan Fuh-Jyh , Gonsalves Dennis
Published: July 2002

RNA-mediated virus resistance has recently been shown to be the result of post-transcriptional transgene silencing in transgenic plants. This study was undertaken to characterize the effect of transgene length and nontarget DNA sequences on RNA-mediated tospovirus resistance in transgenic plants. Transgenic
Nicotiana benthamiana
plants were generated to express different regions of the nucleocapsid (N) protein of tomato spotted wilt (TSWV) tospovirus. Transgenic plants expressing half-gene segments (387–453 bp) of the
N
gene displayed resistance through post-transcriptional gene silencing. Although smaller
N
gene segments (92–235 bp) were ineffective in conferring resistance when expressed alone in transgenic plants, these segments conferred resistance when fused to the nontarget green fluorescent protein gene DNA. These results demonstrate that (
i
) a critical length of
N
transgene (236–387 bp) is required for a high level of transgene expression and consequent gene silencing, and (
ii
) the post-transcriptional gene silencing mechanism can trans-inactivate the incoming tospovirus genome with homologous transgene segments that are as short as 110 bp. Therefore, the activation of post-transcriptional transgene silencing requires a significantly larger transgene than is required for the trans-inactivation of the incoming viral genome. These results raise the possibility of developing a simple new strategy for engineering multiple virus resistance in transgenic plants.
Platelet signal transduction defect with Gα subunit dysfunction and diminished Gα
q
in a patient with abnormal platelet responses
Authors: Gabbeta Jagadeesh , Yang Xu , Kowalska M. Anna , Sun Ling , Dhanasekaran N. , Rao A. Koneti
Published: July 2002

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca
2+
mobilization. Present studies demonstrated that platelet phospholipase A
2
(cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca
2+
release induced by guanosine 5′-[γ-thio]triphosphate (GTP[γS]) was diminished in the patient’s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of α-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A
2
analog U46619. Binding of
35
S-labeled GTP[γS] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I
2
analog) -induced rise in cAMP (mediated by Gα
s
) and its inhibition (mediated by Gα
i
) by thrombin in the patient’s platelet membranes were normal. Immunoblot analysis of Gα subunits in the patient’s platelet membranes showed a decrease in Gα
q
(<50%) but not Gα
i
, Gα
z
, Gα
12
, and Gα
13
. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein α-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of Gα
q
in thrombin-induced responses.
Correlates of latent and productive HIV type-1 infection in tonsillar CD4
+
T cells
Authors: Røsok Bård , Brinchmann Jan E. , Voltersvik Pål , Olofsson Jan , Bostad Leif , Åsjö Birgitta
Published: July 2002

Correlates of virus load and characteristics of virus-producing cells in tonsillar tissue were investigated. Our results suggest that when less than 1:100 tonsillar CD4
+
T cells from individuals infected with HIV type-1 (HIV-1) contain replication competent provirus, the level of CD4
+
T cells in tonsils is comparable to that observed in uninfected individuals. Virus load at or above this level was associated with low CD4 cell numbers in tonsillar tissue. Only a few percent of all infected T cells in tonsillar tissue were active virus producers, with minor differences observed between individuals. Plasma viremia was found to correlate with infectious virus load in tonsillar tissue. With less than 1:1,000 of CD4 cells in lymphoid tissues being involved in active virus production, direct cytopathic effect by HIV-1 on infected CD4 cells is unlikely to fully explain the immunodeficiency seen in AIDS.
Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in
Escherichia coli
enhances thermotolerance
Authors: Yeh Ching-Hui , Chang Pi-Fang Linda , Yeh Kai-Wun , Lin Wan-Chi , Chen Yih-Ming , Lin Chu-Yung
Published: July 2002

A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into
Escherichia coli
using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that
E. coli
does not normally produce class I LMM HSPs. We compared the survivability of
E. coli
XL1-Blue cells transformed with a recombinant plasmid containing a glutathione
S
-transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control
E. coli
cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β-
d
-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two
E. coli
transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5°C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.
Genetically targeted cell disruption in
Caenorhabditis elegans
Authors: Harbinder S. , Tavernarakis Nektarios , Herndon Laura A. , Kinnell Maud , Xu Si Qun , Fire Andrew , Driscoll Monica
Published: July 2002

The elimination of identified cells is a powerful tool for investigating development and system function. Here we report on genetically mediated cell disruption effected by the toxic
Caenorhabditis elegans mec-4
(
d
) allele. We found that ectopic expression of
mec-4
(
d
) in the nematode causes dysfunction of a wide range of nerve, muscle, and hypodermal cells.
mec-4
(
d
)-mediated toxicity is dependent on the activity of a second gene,
mec-6
, rendering cell disruption conditionally dependent on genetic background. We describe a set of
mec-4
(
d
) vectors that facilitate construction of cell-specific disruption reagents and note that genetic cell disruption can be used for functional analyses of specific neurons or neuronal classes, for confirmation of neuronal circuitry, for generation of nematode populations lacking defined classes of functional cells, and for genetic screens. We suggest that
mec-4
(
d
) and/or related genes may be effective general tools for cell inactivation that could be used toward similar purposes in higher organisms.
Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity
Authors: Zhou Yong-Jie , Hanson Eric P. , Chen Yi-Qing , Magnuson Kelly , Chen Min , Swann Patrick G. , Wange Ronald L. , Changelian Paul S. , O’Shea John J.
Published: July 2002

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, γc and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.
Protein kinase A activity is required for the budding of constitutive transport vesicles from the trans-Golgi network
Authors: Muñiz Manuel , Martín María Esther , Hidalgo Josefina , Velasco Angel
Published: July 2002

We have examined the role played by protein kinase A (PKA) in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface.
In vivo
this transport step was inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurred at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlated with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding from the TGN was studied
in vitro
using vesicular stomatitis virus-infected, permeabilized cells. Addition to this assay of C-PKA stimulated vesicle release while it was suppressed by PKA inhibitory peptide, H89, and antibody against C-PKA. Furthermore, vesicle release was decreased when PKA-depleted cytosol was used and restored by addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN.
Hair cell-specific splicing of mRNA for the α
1D
subunit of voltage-gated Ca
2+
channels in the chicken’s cochlea
Authors: Kollmar Richard , Fak John , Montgomery Lisa G. , Hudspeth A. J.
Published: July 2002

The L-type voltage-gated Ca
2+
channels that control tonic release of neurotransmitter from hair cells exhibit unusual electrophysiological properties: a low activation threshold, rapid activation and deactivation, and a lack of Ca
2+
-dependent inactivation. We have inquired whether these characteristics result from cell-specific splicing of the mRNA for the L-type α
1D
subunit that predominates in hair cells of the chicken’s cochlea. The α
1D
subunit in hair cells contains three uncommon exons: one encoding a 26-aa insert in the cytoplasmic loop between repeats I and II, an alternative exon for transmembrane segment IIIS2, and a heretofore undescribed exon specifying a 10-aa insert in the cytoplasmic loop between segments IVS2 and IVS3. We propose that the alternative splicing of the α
1D
mRNA contributes to the unusual behavior of the hair cell’s voltage-gated Ca
2+
channels.
Redox chains in chloroplast envelope membranes: Spectroscopic evidence for the presence of electron carriers, including iron–sulfur centers
Authors: Jäger-Vottero Pascale , Dorne Albert-Jean , Jordanov Jeanne , Douce Roland , Joyard Jacques
Published: July 2002

We have shown that envelope membranes from spinach chloroplasts contain (
i
) semiquinone and flavosemiquinone radicals, (
ii
) a series of iron-containing electron-transfer centers, and (
iii
) flavins (mostly FAD) loosely associated with proteins. In contrast, we were unable to detect any cytochrome in spinach chloroplast envelope membranes. In addition to a high spin [1Fe]
3+
type protein associated with an EPR signal at
g
= 4.3, we observed two iron–sulfur centers, a [4Fe-4S]
1+
and a [2Fe-2S]
1+
, associated with features, respectively, at
g
= 1.921 and
g
= 1.935, which were detected after reduction by NADPH and NADH, respectively. The [4Fe-4S] center, but not the [2Fe-2S] center, was also reduced by dithionite or 5-deazaflavin/oxalate. An unusual Fe-S center, named X, associated with a signal at
g
= 2.057, was also detected, which was reduced by dithionite but not by NADH or NADPH. Extremely fast spin–relaxation rates of flavin- and quinone-free radicals suggest their close proximity to the [4Fe-4S] cluster or the high-spin [1Fe]
3+
center. Envelope membranes probably contain enzymatic activities involved in the formation and reduction of semiquinone radicals (quinol oxidase, NADPH-quinone, and NADPH-semiquinone reductases). The physiological significance of our results is discussed with respect to (
i
) the presence of desaturase activities in envelope membranes and (
ii
) the mechanisms involved in the export of protons to the cytosol, which partially regulate the stromal pH during photosynthesis. The characterization of such a wide variety of electron carriers in envelope membranes opens new fields of research on the functions of this membrane system within the plant cell.
Target of rapamycin proteins and their kinase activities are required for meiosis
Authors: Zheng Xiao-Feng , Schreiber Stuart L.
Published: July 2002

The phosphatidylinositol kinase-related kinases, including Tor1p, Tor2p, FRAP/RAFT, FRP/ATR, ATM, Mec1p, Rad3, and Tel1p, function in signal transduction pathways involved in cell cycle progression and surveillance. The rapamycin-sensitive kinase activities of Tor1p and Tor2p are required for the nutrient-activated protein translation essential for G
1
cell cycle progression in haploid yeast cells. In addition, Tor2p’s kinase activity is necessary for its unique rapamycin-insensitive function involved in the assembly of the actin cytoskeleton. In the current study using diploid yeast, we found that the kinase activities of the Tor proteins are also required for two discrete steps during yeast meiosis—the switch between the mitotic and meiotic cell cycles and a later step during meiosis involved in the packaging of resultant haploid cells (spores) into asci. Based on what is known of the mitotic functions of Tor and FRAP proteins, these results likely reflect the requirement for signaling pathways leading to regulated protein translation during meiosis. Mec1p, which is required for meiotic recombination, and the Tor proteins are, therefore, homologous kinases with distinct, yet essential, roles in meiosis.
A 60-kDa plant microtubule-associated protein promotes the growth and stabilization of neurotubules
in vitro
Authors: Rutten Twan , Chan Jordi , Lloyd Clive W.
Published: July 2002

The search for microtubule-associated proteins (MAPs) in plants is relatively recent. In particular, the “classical MAPs,” which stimulate the polymerization and stabilization of microtubules, have only been examined in heterogeneous fractions. As a first step in dissecting the role of individual MAPs, we have chromatographically purified a single 60-kDa protein from a carrot MAP fraction and analyzed its effects on tubulin assembly. MAP60 promoted the formation of long, morphologically regular brain microtubules
in vitro
, an effect inhibited by preincubation of the MAP with affinity-purified antibodies against this protein. MAP60 also increased the stability of microtubules to dilution and significantly enhanced cold stability to the normally cold-sensitive neurotubules. These
in vitro
properties are consistent with a role for MAP60 in regulating the turnover/assembly of dynamic plant microtubules
in vivo
.
Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma
Authors: Petersen Joerg , Dandri Maura , Gupta Sanjeev , Rogler Charles E.
Published: July 2002

To investigate host and viral mechanisms determining hepadnaviral persistence and hepatocarcinogenesis, we developed a mouse model by transplanting woodchuck hepatocytes into the liver of mice that contain the urokinase-type plasminogen activator transgene (uPA) and lack mature B and T lymphocytes due to a recombination activation gene 2 (RAG-2) gene knockout. The woodchuck hepatocytes were transplanted via intrasplenic injection and were found to integrate into the recipient mouse liver cord structure. Normal adult woodchuck hepatocytes proliferated and reconstituted up to 90% of the uPA/RAG-2 mouse liver. uPA/RAG-2 mice containing woodchuck hepatocytes were infectable with woodchuck hepatitis virus (WHV) and showed WHV replication for at least 10 months with titers up to 1 × 10
11
virions per ml in the peripheral blood. WHV-infected hepatocytes from chronic carrier woodchucks also established a persistent infection in uPA/RAG-2 mice after an 8- to 12-week lag period of viremia. Although WHV envelope, core, and X proteins were produced in the uPA/RAG-2 mice, no inflammatory host immune response was observed in the liver of WHV-replicating mice. A first antiviral test demonstrated a greater than four orders of magnitude drop in WHV titer in response to interferon α treatment. WHV replication was up-regulated by dexamethasone treatment. Comparison of precancerous lesions in donor woodchucks versus recipient uPA/RAG-2 mice revealed an enrichment of dysplastic precancerous hepatocytes in transplanted mice. Clonal amplification of hepatocytes from a woodchuck with hepatocellular carcinomas was demonstrated by the detection of unique WHV DNA integration patterns in hepatocellular carcinomas that arose in uPA/RAG-2 mice. In the absence of B or T cell-mediated immune responses, WHV establishes a persistent noncytotoxic infection of woodchuck hepatocytes in uPA/RAG-2 chimeric mouse livers. Further studies of the kinetics of hepadnavirus infection and replication in quiescent and proliferating hepatocytes should increase our understanding of hepadnavirus spread and aid in the design of therapies to block or cure persistent infection.
Genetic induction of immune tolerance to human clotting factor VIII in a mouse model for hemophilia A
Authors: Evans Gregory L. , Morgan Richard A.
Published: July 2002

Patients with severe coagulation factor VIII deficiency require frequent infusions of human factor VIII (hFVIII) concentrates to treat life-threatening hemorrhages. Because these patients are immunologically hFVIII-naive, a significant treatment complication is the development of inhibitors or circulating alloantibodies against hFVIII, which bind the replaced glycoprotein, increase its plasma clearance, and inhibit its activity, preventing subsequent treatments from having a therapeutic effect. A genetic approach toward the induction of immunologic unresponsiveness to hFVIII has the conceptual advantage of a long-term, stable elimination of undesired immune responses against hFVIII. Here, we report that in a factor VIII (FVIII)-deficient mouse model for severe hemophilia A, genetic modification of donor bone marrow cells with a retroviral vector encoding hFVIII, and transplant to hemophiliac mouse recipients, results in the induction of immune tolerance to FVIII in 50% of treated animals after immunization with hFVIII, despite the fact that hFVIII protein or activity is undetectable. In tolerized animals, the titers of anti-hFVIII binding antibodies and of hFVIII inhibitor antibodies were significantly reduced, and there was evidence for hFVIII unresponsiveness in CD4+T cells. Importantly, the plasma clearance of hFVIII was significantly decreased in tolerized animals and was not significantly different from that seen in a FVIII-naive hemophiliac mouse. This model system will prove useful for the evaluation of genetic therapies for hFVIII immunomodulation and bring genetic therapies for hFVIII tolerance closer to clinical application for patients with hemophilia A.
Reaching the global minimum in docking simulations: A Monte Carlo energy minimization approach using Bezier splines
Authors: Trosset Jean-Yves , Scheraga Harold A.
Published: July 2002

The docking problem faces two major challenges: the global optimization of a multivariable function, such as the energy, and the ability to discriminate between true and false positive results, i.e., native from nonnative structures based on the input energy function. Among all energy evaluation tools, only a local energy-minimization method using an accurate enough potential function is able to discriminate between native and nonnative structures. To meet these requirements, a Monte Carlo with energy-minimization method has been incorporated into a new ECEPP/3 docking program. The efficiency of the simulation results from the use of an energy-grid technique based on Bezier splines and from a simplification of the receptor by switching on the energy of only important residues of the active site. Simulations of a thrombin-inhibitor complex show that the global minimum of the energy function was reached in every independent run within less than 3 min of time on an IBM RX 6000 computer. For comparison, 10 standard independent Monte Carlo simulations with 10
6
steps in each were carried out. Only three of them led to a conformation close to the x-ray structure. The latter simulations required an average of 24 min and about 10 hr with and without the grid, respectively. Another important result is that the Bezier spline technique not only speeds up the calculation by reducing the number of operations during the energy evaluation but also helps in reaching the global minimum by smoothing out the potential energy surface.
Structural homology between the Rap30 DNA-binding domain and linker histone H5: Implications for preinitiation complex assembly
Authors: Groft Caroline M. , Uljon Sacha N. , Wang Rong , Werner Milton H.
Published: July 2002

The three-dimensional structure of the human Rap30 DNA-binding domain has been solved by multinuclear NMR spectroscopy. The structure of the globular domain is strikingly similar to that of linker histone H5 and its fold places Rap30 into the “winged” helix–turn–helix family of eukaryotic transcription factors. Although the domain interacts weakly with DNA, the binding surface was identified and shown to be consistent with the structure of the HNF-3/
fork head
–DNA complex. The architecture of the Rap30 DNA-binding domain has important implications for the function of Rap30 in the assembly of the preinitiation complex. In analogy to the function of linker histones in chromatin formation, the fold of the Rap30 DNA-binding domain suggests that its role in transcription initiation may be that of a condensation factor for preinitiation complex assembly. Functional similarity to linker histones may explain the dependence of Rap30 binding on the bent DNA environment induced by the TATA box-binding protein. Cryptic sequence identity and functional homology between the Rap30 DNA-binding domain and region 4 of
Escherichia coli
σ
70
may indicate that the σ factors also possess a linker histone-like activity in the formation of a prokaryotic closed complex.
A thermodynamic analysis of the sequence-specific binding of RNA by bacteriophage MS2 coat protein
Authors: Johansson Hans E. , Dertinger Dagmar , LeCuyer Karen A. , Behlen Linda S. , Greef Charles H. , Uhlenbeck Olke C.
Published: July 2002

Most mutations in the sequence of the RNA hairpin that specifically binds MS2 coat protein either reduce the binding affinity or have no effect. However, one RNA mutation, a uracil to cytosine change in the loop, has the unusual property of increasing the binding affinity to the protein by nearly 100-fold. Guided by the structure of the protein–RNA complex, we used a series of protein mutations and RNA modifications to evaluate the thermodynamic basis for the improved affinity: The tight binding of the cytosine mutation is due to (
i
) the amino group of the cytosine residue making an intra-RNA hydrogen bond that increases the propensity of the free RNA to adopt the structure seen in the complex and (
ii
) the increased affinity of hydrogen bonds between the protein and a phosphate two bases away from the cytosine residue. The data are in good agreement with a recent comparison of the cocrystal structures of the two complexes, where small differences in the two structures are seen at the thermodynamically important sites.
Light chain-associated amyloid deposits comprised of a novel κ constant domain
Authors: Solomon Alan , Weiss Deborah T. , Murphy Charles L. , Hrncic Rudi , Wall Jonathan S. , Schell Maria
Published: July 2002

Light chain-associated amyloidosis is characterized by the deposition as fibrils of monoclonal light chain-related components consisting predominately of the variable domain (VL) or the VLplus up to ≈60 residues of the constant domain (CL). Here, we describe a patient (designated BIF) with light chain-associated amyloidosis and κ Bence Jones proteinuria in whom, notably, >80% of the amyloid deposits were comprised of CL-related material. The extracted amyloid protein consisted of 99 aa residues identical in sequence to the main portion of the Cκregion (positions 109–207) of the precursor Bence Jones protein. Remarkably, the CLs from both molecules contained a Ser→Asn substitution at position 177. This heretofore undescribed Cκalteration did not result from somatic mutation but rather was germline encoded. When tested in ourin vitrofibrillogenic kinetic assay, Bence Jones protein BIF was highly amyloidogenic. Notably, endopeptidase treatment of amyloid fibrils prepared from the native light chain revealed the VLto be markedly susceptible to enzymatic digestion, whereas the CLwas protease-resistant. Our findings provide evidence that the fragmented light chains typically present in this disease result from proteolytic degradation and suggest that, in this case, conformational differences in VL/CLpacking within the fibrils may account for the unusual composition of the amyloid deposits. Additionally, we posit that the previously unrecognized Asn177substitution represents yet another Cκallotype, provisionally designatedKm4.
Copy number control of a transposable element, the
I
factor, a LINE-like element in
Drosophila
Authors: Chaboissier Marie-Christine , Bucheton Alain , Finnegan David J.
Published: July 2002

The
I
factor is a LINE-like transposable element in
Drosophila
. Most strains of
Drosophila melanogaster
, inducer strains, contain 10–15 copies of the
I
factor per haploid genome located in the euchromatic regions of the chromosome arms. These are not present in a few strains known as reactive strains.
I
factors transpose at low frequency in inducer strains but at high frequency in the female progeny of crosses between reactive and inducer flies. We have found that the activity of the
I
factor promoter is sensitive to the number of copies of the first 186 nucleotides of the
I
factor sequence, which constitutes the 5′-untranslated region. The activity of the
I
factor decreases as the copy number of this sequence increases.