bluuebunny/crossref_metadata_2025 huggingface dataset:
Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression
Authors: Ku Nam-On , Michie Sara , Resurreccion Evelyn Z. , Broome Rosemary L. , Omary M. Bishr
Published: July 2002

Keratin polypeptides 8 and 18 (K8/18) are the major intermediate filament proteins of simple-type epithelia. K18 Ser-33 phosphorylation regulates its binding to 14-3-3 proteins during mitosis. We studied the significance of keratin binding to 14-3-3 in transgenic mice that overexpress wild-type or Ser-33→Ala (S33A) K18. In S33A but not wild-type K18-overexpressing mice, pancreatic acinar cell keratin filaments retracted from the basal nuclear region and became apically concentrated. In contrast, K18 S33A had a minimal effect on hepatocyte keratin filament organization. Partial hepatectomy of K18-S33A-overexpressing mice did not affect liver regeneration but caused limited mitotic arrest, accumulation of abnormal mitotic figures, dramatic fragmentation of hepatocyte keratin filaments, with retention of a speckled 14-3-3ζ mitotic cell nuclear-staining pattern that usually becomes diffuse during mitosis. Hence, K18 Ser-33 phosphorylation regulates keratin filament organization in simple-type epithelia
in vivo
. Keratin binding to 14-3-3 may partially modulate hepatocyte mitotic progression, in association with nuclear redistribution of 14-3-3 proteins during mitosis.
Resorcinarenes with 2-benzimidazolone bridges: Self-aggregation, self-assembled dimeric capsules, and guest encapsulation
Authors: Ebbing Maurits H. K. , Villa María-Jesús , Valpuesta José-María , Prados Pilar , de Mendoza Javier
Published: July 2002

The synthesis and spectroscopic characterization of self-assembled dimeric resorcinarenes 1a–d containing four 2-benzimidazolone (cyclic urea) bridges are reported. The nanometer-size capsules are held together by a cyclic array of complementary hydrogen bonds. Unlike the related imide-bridged resorcinarenes reported by Rebek and coworkers [Heinz, T., Rudkevich, D. M. & Rebek, J., Jr. (1998)
Nature (London)
394, 764–766], these strongly bound dimers aggregate in chloroform solutions yielding different self-organized structures, depending on the nature and length of the four carbon chains attached at the bottom of each resorcinarene platform, as revealed by transmission electron microscopy. Thus, phenethyl groups (dimer 1c⋅1c) produce long fibers, probably arising from tail to tail contacts and subsequent threading of the resulting linear self-assembled polymers, whereas long alkyl chains (dimers 1a⋅1a and 1b⋅1b) induce formation of large reverse vesicles of 0.8–2.2 μm diameter through side to side extensive stacking. Presumably, the rigidity of the dimer precludes folding of the aggregate into smaller vesicles. On the contrary, dimer 1d⋅1d, containing four nine-carbon chains and a cis-double bond, does not substantially aggregate and gives rise to reasonably resolved
1
H NMR spectra. The compound was shown to be dimeric either by matrix-assisted laser desorption ionization–time-of-flight and vapor pressure osmometry. Encapsulation studies were followed by NMR. Propionic or pivalic acid was included in the capsules, probably as head to head hydrogen-bonded dimers in mesitylene-
d
12
, a solvent too big to be a guest by its own. Longer dimeric carboxylic acids or larger substrates, like 2-adamantyl azide or cyclohexylcarbodiimide, do not encapsulate, but mixtures of a long and a short carboxylic acid (i.e., propionic-adamantyl or propionic-cyclohexyl) yield pairwise complexes.
In vitro
reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex
Authors: Omer Arina D. , Ziesche Sonia , Ebhardt Holger , Dennis Patrick P.
Published: July 2002

The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C/D box small RNAs that guide 2′-O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the
Sulfolobus
homologues of eukaryotic proteins that are known to be present in C/D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an
in vitro
transcript of the
Sulfolobus
sR1 small RNA were reconstituted
in vitro
, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting
S-
adenosyl methionine (SAM)-dependent, site-specific 2′-O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve
in vitro
RNA guide-directed 2′-O-methylation of ribose in a target RNA.
Negative regulation of cell growth and differentiation by TSG101 through association with p21
Cip1/WAF1
Authors: Oh Hyesun , Mammucari Cristina , Nenci Arianna , Cabodi Sara , Cohen Stanley N. , Dotto G. Paolo
Published: July 2002

TSG101 was discovered in a screen for tumor susceptibility genes and has since been shown to have a multiplicity of biological effects. However, the basis for TSG101's ability to regulate cell growth has not been elucidated. We report here that the TSG101 protein binds to the cyclin/cyclin-dependent kinase (CDK) inhibitor (CKI) p21
Cip1/WAF1
and increases stability of the p21 protein in HEK293F cells and differentiating primary keratinocytes, suppressing differentiation in a p21-dependent manner. In proliferating keratinocytes where the p21 protein is relatively stable, TSG101 does not affect the stability or expression of p21 but shows p21-dependent recruitment to cyclin/CDK complexes, inhibits cyclin/CDK activity, and causes strong growth suppression to a much greater extent in p21+/+ than in p21−/− cells. Conversely, suppression of endogenous TSG101 expression by an antisense TSG101 cDNA causes doubling of the fraction of keratinocytes in the S phase of the cell cycle as occurs during p21 deficiency. Our results indicate that TSG101 has a direct role in the control of growth and differentiation in primary epithelial cells, and that p21 is an important mediator of these TSG101 functions.
Evolutionary recruitment of a flavin-dependent monooxygenase for the detoxification of host plant-acquired pyrrolizidine alkaloids in the alkaloid-defended arctiid mothTyriajacobaeae
Authors: Naumann Claudia , Hartmann Thomas , Ober Dietrich
Published: July 2002

Larvae ofTyria jacobaeaefeed solely upon the pyrrolizidine alkaloid-containing plantSenecio jacobaea. Ingested pyrrolizidine alkaloids (PAs), which are toxic to unspecialized insects and vertebrates, are efficientlyN-oxidized in the hemolymph ofT. jacobaeaeby senecionineN-oxygenase (SNO), a flavin-dependent monooxygenase (FMO) with a high substrate specificity for PAs. Peptide microsequences obtained from purifiedT. jacobaeaeSNO were used to clone the corresponding cDNA, which was expressed in active form inEscherichia coli.T. jacobaeaeSNO possesses a signal peptide characteristic of extracellular proteins, and it belongs to a large family of mainly FMO-like sequences of mostly unknown function, including two predictedDrosophila melanogastergene products. The data indicate that the gene forT. jacobaeaeSNO, highly specific for toxic pyrrolizidine alkaloids, was recruited from a preexisting insect-specific FMO gene family of hitherto unknown function. The enzyme allows the larvae to feed on PA-containing plants and to accumulate predation-deterrent PAs in the hemolymph.
Substrates regulate γ-aminobutyric acid transporters in a syntaxin 1A-dependent manner
Authors: Quick Michael W.
Published: July 2002

Several ion channels and pumps are regulated by syntaxin 1A, a component of the synaptic vesicle docking and fusion apparatus. One such regulated protein is the rat brain γ-aminobutyric acid (GABA) transporter GAT1. The N-terminal cytoplasmic domain of GAT1 directly interacts with syntaxin 1A; this interaction induces a decrease in the rate at which GABA and associated ions are transported. GAT1 function also is regulated by transporter substrates, raising the possibility that substrates mediate at least some of their effects by regulating the interaction between GAT1 and syntaxin 1A. In oocytes expressing GAT1 and syntaxin 1A, superfusion of transporter substrates increases the GAT1 transport rate. The substrate-induced rate change (
i
) is prevented by coapplication of GAT1 antagonists, (
ii
) does not occur in oocytes expressing GAT1 alone, and (
iii
) does not occur in oocytes expressing interaction-deficient syntaxin 1A mutants. In oocytes, and in hippocampal neurons that endogenously express both GAT1 and syntaxin 1A, substrate application results in a decrease in the fraction of syntaxin 1A that is bound to GAT1 on a time-scale comparable to the substrate-induced change in transport rates. These data suggest that substrate translocation regulates GAT1–syntaxin 1A interactions and provide a mechanism by which GABA transport can be increased during times of rising synaptic GABA concentrations.
Naturally occurring
rhodopsin
mutation in the dog causes retinal dysfunction and degeneration mimicking human dominant retinitis pigmentosa
Authors: Kijas James W. , Cideciyan Artur V. , Aleman Tomas S. , Pianta Michael J. , Pearce-Kelling Susan E. , Miller Brian J. , Jacobson Samuel G. , Aguirre Gustavo D. , Acland Gregory M.
Published: July 2002

Rhodopsin is the G protein-coupled receptor that is activated by light and initiates the transduction cascade leading to night (rod) vision. Naturally occurring pathogenic rhodopsin (
RHO
) mutations have been previously identified only in humans and are a common cause of dominantly inherited blindness from retinal degeneration. We identified English Mastiff dogs with a naturally occurring dominant retinal degeneration and determined the cause to be a point mutation in the
RHO
gene (Thr4Arg). Dogs with this mutant allele manifest a retinal phenotype that closely mimics that in humans with
RHO
mutations. The phenotypic features shared by dog and man include a dramatically slowed time course of recovery of rod photoreceptor function after light exposure and a distinctive topographic pattern to the retinal degeneration. The canine disease offers opportunities to explore the basis of prolonged photoreceptor recovery after light in
RHO
mutations and determine whether there are links between the dysfunction and apoptotic retinal cell death. The
RHO
mutant dog also becomes the large animal needed for preclinical trials of therapies for a major subset of human retinopathies.
Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells
Authors: Chen Jian-Kang , Capdevila Jorge , Harris Raymond C.
Published: July 2002

In addition to its important functions in detoxification of foreign chemicals and biosynthesis of steroid hormones, the cytochrome P450 enzyme system metabolizes arachidonate to 14,15-epoxyeicosatrienoic acid (14,15-EET). This study demonstrates that a P450 arachidonate epoxygenase metabolite can activate cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and delineates an essential role for HB-EGF in the mitogenic effects of this lipid mediator. Blockade of HB-EGF processing or EGF receptor (EGFR) inhibited 14,15-EET-stimulated early mitogenic signals and DNA synthesis. 14,15-EET failed to induce mitogenesis in cell lines expressing minimal HB-EGF, whereas 14,15-EET induced soluble HB-EGF release into the conditioned media of cell lines that both express high levels of HB-EGF and display mitogenic response to this lipid mediator. Moreover, transfection of a bacterial 14,15-epoxygenase established intracellular endogenous 14,15-EET biosynthesis in cultured cell systems, which allowed direct confirmation of involvement of EGFR transactivation in the endogenous 14,15-EET-mediated mitogenic signaling pathway. This mechanism involves EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF.
Large conformational changes of the ɛ subunit in the bacterial F
1
F
0
ATP synthase provide a ratchet action to regulate this rotary motor enzyme
Authors: Tsunoda Satoshi P. , Rodgers Andrew J. W. , Aggeler Robert , Wilce Matthew C. J. , Yoshida Masasuke , Capaldi Roderick A.
Published: July 2002

The F
1
F
0
ATP synthase is the smallest motor
enzyme known. Previous studies had established that the central stalk,
made of the γ and ɛ subunits in the F
1
part and c
subunit ring in the F
0
part, rotates relative to a stator
composed of α
3
β
3
δab
2
during
ATP hydrolysis and synthesis. How this rotation is regulated has been
less clear. Here, we show that the ɛ subunit plays a key role by
acting as a switch of this motor. Two different arrangements of the ɛ
subunit have been visualized recently. The first has been observed in
beef heart mitochondrial F
1
-ATPase where the C-terminal
portion is arranged as a two-α-helix hairpin structure that extends
away from the α
3
β
3
region, and toward the
position of the c subunit ring in the intact
F
1
F
0
. The second arrangement was observed in a
structure determination of a complex of the γ and ɛ subunits of the
Escherichia coli
F
1
-ATPase. In this, the two
C-terminal helices are apart and extend along the γ to interact with
the α and β subunits in the intact complex. We have been able to
trap these two arrangements by cross-linking after introducing
appropriate Cys residues in
E. coli
F
1
F
0
, confirming that both conformations of the
ɛ subunit exist in the enzyme complex. With the C-terminal domain of
ɛ toward the F
0
, ATP hydrolysis is activated, but the
enzyme is fully coupled in both ATP hydrolysis and synthesis. With the
C-terminal domain toward the F
1
part, ATP hydrolysis is
inhibited and yet the enzyme is fully functional in ATP synthesis;
i.e., it works in one direction only. These results help explain the
inhibitory action of the ɛ subunit in the
F
1
F
0
complex and argue for a ratchet function
of this subunit.
An
Arabidopsis
mutation in translation elongation factor 2 causes superinduction of
CBF/DREB1
transcription factor genes but blocks the induction of their downstream targets under low temperatures
Authors: Guo Yan , Xiong Liming , Ishitani Manabu , Zhu Jian-Kang
Published: July 2002

Low temperature regulates gene expression in bacteria, yeast, and animals as well as in plants. However, the signal transduction cascades mediating the low temperature responses are not well understood in any organism. To identify components in low temperature signaling genetically, we isolated
Arabidopsis thaliana
mutants in which cold-responsive genes are no longer induced by low temperatures. One of these mutations,
los1–1
, specifically blocks low temperature-induced transcription of cold-responsive genes. Surprisingly, cold-induced expression of the early response transcriptional activators, C-repeat/dehydration responsive element binding factors (CBF/DREB1s), is enhanced by the
los1–1
mutation. The
los1–1
mutation also reduces the capacity of plants to develop freezing tolerance but does not impair the vernalization response. Genetic analysis indicated that
los1–1
is a recessive mutation in a single nuclear gene. The
LOS1
gene encodes a translation elongation factor 2-like protein. Protein labeling studies show that new protein synthesis is blocked in
los1–1
mutant plants specifically in the cold. These results reveal a critical role of new protein synthesis in the proper transduction of low temperature signals. Our results also suggest that cold-induced transcription of
CBF/DREB1
s is feedback inhibited by their gene products or by products of their downstream target genes.
Proton-motive force stimulates the proteolytic activity of FtsH, a membrane-bound ATP- dependent protease in
Escherichia
coli
Authors: Akiyama Yoshinori
Published: July 2002

FtsH is a membrane-bound, ATP-dependent metalloprotease in
Escherichia coli
that degrades some integral membrane proteins and cytoplasmic proteins. In this study, we show that FtsH-dependent degradation of both membrane-bound and soluble proteins is retarded when cells are treated with carbonyl cyanide-3-chlorophenylhydrazone or 2,4-dinitrophenol uncouplers, which dissipate the proton-motive force.
In vitro
casein degradation by membrane-integrated FtsH was stimulated by succinate, a respiratory substrate; this stimulation was counteracted by cyanide-3-chlorophenylhydrazone. Potassium thiocyanate, which specifically collapses Δψ, partially canceled the effect of succinate, but ammonium sulfate, which collapses ΔpH, showed little effect. These results indicate that the proton-motive force, in particular the Δψ component, plays a role in efficient degradation of substrates by FtsH in its native state. FtsH variants with altered transmembrane regions did not receive proton-motive force stimulation, suggesting that the proton-motive force activates FtsH, directly or indirectly, through the transmembrane region.
In vitro
formation of a
c
-type cytochrome
Authors: Daltrop Oliver , Allen James W. A. , Willis Anthony C. , Ferguson Stuart J.
Published: July 2002

C
-type cytochromes are essential for almost all organisms; they are characterized by the covalent attachment of heme to protein through two thioether bonds to a Cys-Xaa-Xaa-Cys-His peptide motif. Here we show, contrary to opinion of 30 years standing, that a
c
-type cytochrome can form from heme and apoprotein
in vitro
under mild conditions and in the absence of any biosynthesis apparatus. This reaction occurs provided formation of a disulfide bond within the Cys-Xaa-Xaa-Cys-His motif is avoided. There are important implications for understanding
in vivo
cytochrome
c
assembly.
Reversible encapsulation by self-assembling resorcinarene subunits
Authors: Shivanyuk Alexander , Rebek Julius
Published: July 2002

Encapsulation complexes are assemblies in which a reversibly formed host more or less completely surrounds guest molecules. Host structures held together by hydrogen bonds have lifetimes in organic solvents of milliseconds to hours, long enough to directly observe the encapsulated guest by NMR spectroscopy. We describe here the action of alkyl ammonium compounds as guests that gather up to six molecules of the host module to form encapsulation complexes. The stoichiometry of the complexes—the largest hydrogen-bonded host capsules to date—is determined by the size and concentration of the guest.
Trypanosoma cruzitrans-sialidase: A potent and specific survival factor for human Schwann cells by means of phosphatidylinositol 3-kinase/Akt signaling
Authors: Chuenkova Marina V. , Furnari Frank B. , Cavenee Webster K. , Pereira Miercio A.
Published: July 2002

Patients infected withTrypanosoma cruzimay remain asymptomatic for decades and show signs of neuroregeneration in the peripheral nervous system (PNS). In the absence of such neuroregeneration, patients may die in part by extensive neuronal destruction in the gastrointestinal tract. Thus,T. cruzimay,despite their invasion of the PNS, directly prevent cell death to keep nerve destruction in check. Indeed,T. cruziinvasion of Schwann cells, their prime target in PNS, suppressed host-cell apoptosis caused by growth-factor deprivation. The trans-sialidase (TS) ofT. cruziand the Cys-rich domain of TS reproduced the antiapoptotic activity of the parasites at doses (≥3.0 nM) comparable or lower than those ofbona fidemammalian growth factors. This effect was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K). TS also activated Akt, a downstream effector of PI3K. Ectopic expression of TS in an unrelated parasite,Leishmania major,turned those parasites into activators of Akt in Schwann cells. In contrast, the Cys-rich domain of TS did not block apoptosis in Schwann cells overexpressing dominant-negative Akt or constitutively active PTEN, a negative regulator of PI3K/Akt signaling. The results demonstrate thatT. cruzi, through its TS, triggers the survival of host Schwann cells via the PI3K/Akt pathway, suggesting a role for PI3K/Akt in the pathogenesis of Chagas' disease.
Mechanism of transfer of NO from extracellular
S
-nitrosothiols into the cytosol by cell-surface protein disulfide isomerase
Authors: Ramachandran Niroshan , Root Paul , Jiang Xing-Mai , Hogg Phillip J. , Mutus Bulent
Published: July 2002

N
-dansylhomocysteine (DnsHCys) is quenched on
S-nitrosation. The product of this reaction,
N
-dansyl-
S
-nitrosohomocysteine, is a
sensitive, direct fluorogenic substrate for the denitrosation activity
of protein disulfide isomerase (PDI) with an apparent
K
M
of 2 μM.
S
-nitroso-BSA
(BSA-NO) competitively inhibited this reaction with an apparent
K
I
of 1 μM. The oxidized form of DnsHCys,
N,N
-didansylhomocystine, rapidly accumulated in cells
and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human
umbilical endothelial cells and hamster lung fibroblasts were monitored
as a function of extracellular BSA-NO concentration via dynamic
fluorescence microscopy. The observed quenching of the DnsHCys
fluorescence was an indirect measure of cell surface PDI (csPDI)
catalyzed denitrosation of extracellular
S
-nitrosothiols
as decrease or increase in the csPDI levels in HT1080 fibrosarcoma
cells correlated with the rate of quenching and the PDI inhibitors,
5,5′-dithio-
bis
-3-nitrobenzoate and
4-(
N
-(
S
-glutathionylacetyl)
amino)phenylarsenoxide inhibited quenching. The apparent
K
M
values for denitrosation of BSA-NO by
csPDI ranged from 12 μM to 30 μM. Depletion of membrane
N
2
O
3
with the lipophylic antioxidant, vitamin
E, inhibited csPDI-mediated quenching rates of DnsHCys fluorescence
by ≈70%. The
K
M
for BSA-NO increased by
≈3-fold and
V
max
decreased by ≈4-fold.
These findings suggest that csPDI catalyzed NO released from
extracellular
S
-nitrosothiols accumulates in the
membrane where it reacts with O
2
to produce
N
2
O
3
. Intracellular thiols may then be
nitrosated by N
2
O
3
at the membrane-cytosol
interface.
Unraveling the mechanism of the lactose permease of
Escherichia coli
Authors: Sahin-Tóth Miklós , Karlin Arthur , Kaback H. Ronald
Published: July 2002

We studied the effect of pH on ligand binding in wild-type lactose
permease or mutants in the four residues—Glu-269, Arg-302, His-322,
and Glu-325—that are the key participants in H
+
translocation and coupling between sugar and H
+
translocation. Although wild-type permease or mutants in Glu-325 and
Arg-302 exhibit marked decreases in affinity at alkaline pH, mutants in
either His-322 or Glu-269 do not titrate. The results offer a
mechanistic model for lactose/H
+
symport. In the ground
state, the permease is protonated, the H
+
is shared between
His-322 and Glu-269, Glu-325 is charge-paired with Arg-302, and
substrate is bound with high affinity at the outside surface. Substrate
binding induces a conformational change that leads to transfer of the
H
+
from His-322/Glu-269 to Glu-325 and reorientation of
the binding site to the inner surface with a decrease in affinity.
Glu-325 then is deprotonated on the inside because of rejuxtaposition
with Arg-302. The His-322/Glu-269 complex then is reprotonated from
the outside surface to reinitiate the cycle.
The complex ATP–Fe
2+
serves as a specific affinity cleavage reagent in ATP-Mg
2+
sites of Na,K-ATPase: Altered ligation of Fe
2+
(Mg
2+
) ions accompanies the E
1
P→E
2
P conformational change
Authors: Patchornik Guy , Goldshleger Rivka , Karlish Steven J. D.
Published: July 2002

In the presence of ascorbate/H
2
O
2
,
ATP–Fe
2+
or AMP-PNP–Fe
2+
complexes act as
affinity cleavage reagents, mediating selective cleavage of the alpha
subunit of Na,K-ATPase at high affinity ATP–Mg
2+
sites.
The cleavages reveal contact points of Fe
2+
or
Mg
2+
ions. In E
1
and E
1
Na
conformations, two major cleavages are detected within the conserved
708
TGDGVNDSPALKK sequence (at V712 and nearby), and one
(E
1
Na) or two (E
1
) minor cleavages near V440.
In media containing sodium and ATP, Fe
2+
substitutes for
Mg
2+
in activating phosphorylation and ATP hydrolysis. In
the E
1
P conformation, cleavages are the same as in
E
1
. Fe
2+
is not bound tightly. By contrast, in
the E
2
P conformation, the pattern is different. A major
cleavage occurs near the conserved sequence
212
TGES,
whereas those in TGDGVNDSPALKK are less prominent. Fe
2+
is
bound very tightly. On E
2
P hydrolysis, the Fe
2+
dissociates. The results are consistent with
E
1
↔E
2
conformation-dependent movements of
cytoplasmic domains and sites for P
i
and Mg
2+
ions, inferred from previous Fe-cleavage experiments. Furthermore,
these concepts fit well with the crystal structure of Ca-ATPase
[Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000)
Nature (London)
405, 647–655]. Altered ligation of
Mg
2+
ions in E
2
P may be crucial in facilitating
nucleophilic attack of water on the O—P bond. Mg
2+
ions
may play a similar role in all P-type pumps. As affinity cleavage
reagents, ATP–Fe
2+
or other nucleotide–Fe
2+
complexes could be widely used to investigate nucleotide binding
proteins.
Design of a retroviral-mediated ecdysone-inducible system and its application to the expression profiling of the PTEN tumor suppressor
Authors: Stolarov Javor , Chang Kenneth , Reiner Andrew , Rodgers Linda , Hannon Gregory J. , Wigler Michael H. , Mittal Vivek
Published: July 2002

We have engineered the ecdysone-inducible mammalian expression system for general retroviral delivery to cultured mammalian cells. We inducibly expressed PTEN in the glioblastoma cell line, U87MG, lacking this gene. Because nearly all cells are recruited on induction, we find both up- and down-regulated genes by cDNA microarray analysis. The changes we see are similar to those observed after treatment with LY294002, an inhibitor of phosphatidylinositol 3-OH kinase, fully consistent with the model that PTEN antagonizes phosphatidylinositol 3-OH kinase. Both treatments result in suppressed expression of the transforming growth factor (TGF)-β gene and the genes of the cholesterol biosynthesis pathway. Our results illustrate the power of using a fully inducible expression system in conjunction with cDNA microarray analysis for exploring gene function.
A major quantitative trait locus on chromosome 3 controls colitis severity in IL-10-deficient mice
Authors: Farmer Mark A. , Sundberg John P. , Bristol Ian J. , Churchill Gary A. , Li Renhua , Elson Charles O. , Leiter Edward H.
Published: July 2002

Colitic lesions are much more severe in C3H/HeJBir (C3H) than C57BL/6J (B6) mice after 10 backcrosses of a disrupted interleukin-10 (Il10) gene. This study identified cytokine deficiency-induced colitis susceptibility (Cdcs) modifiers by using quantitative trait locus (QTL) analysis. A segregating F2population (n= 408) of IL-10-deficient mice was genotyped and necropsied at 6 weeks of age. A major C3H-derived colitogenic QTL (Cdcs1) on chromosome (Chr.) 3 contributed to lesions in both cecum [logarithm of odds ratio (LOD) = 14.6)] and colon (LOD = 26.5) as well as colitis-related phenotypes such as spleen/body weight ratio, mesenteric lymph node/body weight ratio, and secretory IgA levels. Evidence for other C3H QTL on Chr. 1 (Cdcs2) and Chr. 2 (Cdcs3) was obtained.Cdcs1interacted epistatically or contributed additively with loci on other chromosomes. The resistant B6 background also contributed colitogenic QTL:Cdcs4(Chr. 8),Cdcs5(Chr. 17, MHC), andCdcs6(Chr. 18). Epistatic interactions between B6 QTL on Chr. 8 and 18 contributing to cecum hyperplasia were particularly striking. In conclusion, a colitogenic susceptibility QTL on Chr. 3 has been shown to exacerbate colitis in combination with modifiers contributed from both parental genomes. The complex nature of interactions among loci in this mouse model system, coupled with separate deleterious contributions from both parental strains, illustrates why detection of human inflammatory bowel disease linkages has proven to be so difficult. A human ortholog of the Chr. 3 QTL, if one exists, would map to Chr. 4q or 1p.
Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.
Authors: Xie Q W , Leung M , Fuortes M , Sassa S , Nathan C
Published: July 2002

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.
The molecular basis of Sanfilippo syndrome type B.
Authors: Zhao H G , Li H H , Bach G , Schmidtchen A , Neufeld E F
Published: July 2002

The Sanfilippo syndrome type B is a lysosomal storage disorder caused by deficiency of alpha-N-acetylglucosaminidase; it is characterized by profound mental deterioration in childhood and death in the second decade. For understanding the molecular genetics of the disease and for future development of DNA-based therapy, we have cloned the cDNA and gene encoding alpha-N-acetylglucosaminidase. Cloning started with purification of the bovine enzyme and use of a conserved oligonucleotide sequence to probe a human cDNA library. The cDNA sequence was found to encode a protein of 743 amino acids, with a 20- to 23-aa signal peptide immediately preceding the amino terminus of the tissue enzyme and with six potential N-glycosylation sites. The 8.5-kb gene (NAGLU), interrupted by 5 introns, was localized to the 5'-flanking sequence of a known gene, EDH17B, on chromosome 17q21. Five mutations were identified in cells of patients with Sanfilippo syndrome type B: 503del10, R297X, R626X, R643H, and R674H. The occurrence of a frameshift and a nonsense mutation in homozygous form confirms the identity of the NAGLU gene.
Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).
Authors: Hartmann C M , Hewson A S , Kos C H , Hilfiker H , Soumounou Y , Murer H , Tenenhouse H S
Published: July 2002

Na+-phosphate (Pi) cotransport across the renal brush border membrane is the rate limiting step in the overall reabsorption of filtered Pi. Murine and human renal-specific cDNAs (NaPi-7 and NaPi-3, respectively) related to this cotransporter activity (type II Na+-Pi cotransporter) have been cloned. We now report the cloning and characterization of the corresponding mouse (Npt2) and human (NPT2) genes. The genes were cloned by screening mouse genomic and human chromosome 5-specific libraries, respectively. Both genes are approximately 16 kb and are comprised of 13 exons and 12 introns, the junctions of which conform to donor and acceptor site consensus sequences. Putative CAAT and TATA boxes are located, respectively, at positions -147 and -40 of the Npt2 gene and -143 and -51 of the NPT2 gene, relative to nucleotide 1 of the corresponding cDNAs. The translation initiation site is within exon 2 of both genes. The first 220 bp of the mouse and human promoter regions exhibit 72% identity. Two transcription start sites (at positions -9 and - 10 with respect to nucleotide 1 of NaPi-7 cDNA) and two polyadenylylation signals were identified in the Npt2 gene by primer extension, 5' and 3' rapid amplification of cDNA ends (RACE). A 484-bp 5' flanking region of the Npt2 gene, comprising the CAAT box, TATA box, and exon 1, was cloned upstream of a luciferase reporter gene; this construct significantly stimulated luciferase gene expression, relative to controls, when transiently transfected into OK cells, a renal cell line expressing type II Na+ -Pi cotransporter activity. The present data provide a basis for detailed analysis of cis and trans elements involved in the regulation of Npt2/NPT2 gene transcription and facilitate screening for mutations in the NPT2 gene in patients with autosomally inherited disorders of renal Pi reabsorption.
Chronic, topical exposure to benzo[a]pyrene induces relatively high steady-state levels of DNA adducts in target tissues and alters kinetics of adduct loss.
Authors: Talaska G , Jaeger M , Reilman R , Collins T , Warshawsky D
Published: July 2002

Carcinogen-DNA adduct measurements may become useful biomarkers of effective dose and/or early effect. However, validation of this biomarker is required at several levels to ensure that human exposure and response are accurately reflected. Important in this regard is an understanding of the relative biomarker levels in target and nontarget organs and the response of the biomarker under the chronic, low-dose conditions to which humans are exposed. We studied the differences between single and chronic topical application of benzo[a]pyrene (BAP) on the accumulation and removal of BAP-DNA adducts in skin, lung, and liver. Animals were treated with BAP at 10, 25, or 50 nMol topically once or twice per week for as long as 15 weeks. Animals were sacrificed either at 24, 48, or 72 hr after the last dose at 1 and 30 treatments, and after 24 hr for all other treatment groups. Adduct levels increased with increasing dose, but the slope of the dose-response was different in each organ. At low doses, accumulation was linear in skin and lung, but at high doses the adduct levels in the lung increased dramatically at the same time when the levels in the skin reached apparent steady state. In the liver adduct, levels were lower than in target tissues and apparent steady-state adduct levels were reached rapidly, the maxima being independent of dose, suggesting that activating metabolism was saturated in this organ. Removal of adducts from skin, the target organ, was more rapid following single treatment than with chronic exposure. This finding is consistent with earlier data, indicating that some areas of the genome are more resistant to repair. Thus, repeated exposure and repair cycles would be more likely to cause an increase in the proportion of carcinogen-DNA adducts in repair-resistant areas of the genome. These findings indicate that single-dose experiments may underestimate the potential for carcinogenicity for compounds that follow this pattern.
Signature p53 mutation at DNA cross-linking sites in 8-methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancers.
Authors: Nataraj A J , Black H S , Ananthaswamy H N
Published: July 2002

A combination of psoralen and ultraviolet A radiation (PUVA) is widely used in the treatment of psoriasis. However, PUVA treatment increases the risk of developing skin cancer in psoriasis patients and induces skin cancer in mice. Since the DNA damage induced by PUVA is quite different from that induced by UV, we investigated whether PUVA-induced mouse skin cancers display carcinogen-specific mutations in the p53 tumor suppressor gene. The results indicated that 10 of 13 (77%) PUVA-induced skin tumors contained missense mutations predominantly at exons 6 and 7. In contrast, tumor-adjacent, PUVA-exposed skin from tumor-bearing animals did not exhibit p53 mutation in exons 4-8. Interestingly, about 40% of all mutations in PUVA-induced skin tumors occurred at 5'-TA sites, and an equal number of mutations occurred at one base flanking 5'TA or 5'-TAT sites. Since PUVA induces DNA cross-links exclusively at these sites and since UV "signature" mutations were rarely detected in PUVA-induced skin cancers, we can conclude that PUVA acts as a carcinogen by inducing unique PUVA signature mutations in p53. This finding may have implications for identifying the etiology of skin cancer in psoriasis patients who have undergone PUVA therapy.
A hyperphosphorylated form of the large subunit of RNA polymerase II is associated with splicing complexes and the nuclear matrix.
Authors: Mortillaro M J , Blencowe B J , Wei X , Nakayasu H , Du L , Warren S L , Sharp P A , Berezney R
Published: July 2002

A hyperphosphorylated form of the largest subunit of RNA polymerase II (pol IIo) is associated with the pre-mRNA splicing process. Pol IIo was detected in association with a subset of small nuclear ribonucleoprotein particle and Ser-Arg protein splicing factors and also with pre-mRNA splicing complexes assembled in vitro. A subpopulation of pol IIo was localized to nuclear "speckle" domains enriched in splicing factors, indicating that it may also be associated with RNA processing in vivo. Moreover, pol IIo was retained in a similar pattern following in situ extraction of cells and was quantitatively recovered in the nuclear matrix fraction. The results implicate nuclear matrix-associated hyperphosphorylated pol IIo as a possible link in the coordination of transcription and splicing processes.
Insulin-secreting non-islet cells are resistant to autoimmune destruction.
Authors: Lipes M A , Cooper E M , Skelly R , Rhodes C J , Boschetti E , Weir G C , Davalli A M
Published: July 2002

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus.
Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.
Authors: Warnecke J M , Fürste J P , Hardt W D , Erdmann V A , Hartmann R K
Published: July 2002

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.
Differential inhibition of HIV-1 preintegration complexes and purified integrase protein by small molecules.
Authors: Farnet C M , Wang B , Lipford J R , Bushman F D
Published: July 2002

To replicate, HIV-1 must integrate a cDNA copy of the viral RNA genome into a chromosome of the host. The integration system is a promising target for antiretroviral agents, but to date no clinically useful integration inhibitors have been identified. Previous screens for integrase inhibitors have assayed inhibition of reactions containing HIV-1 integrase purified from an Escherichia coli expression system. Here we compare action of inhibitors in vitro on purified integrase and on subviral preintegration complexes (PICs) isolated from lymphoid cells infected with HIV-1. We find that many inhibitors active against purified integrase are inactive against PICs. Using PIC assays as a primary screen, we have identified three new anthraquinone inhibitors active against PICs and also against purified integrase. We propose that PIC assays are the closest in vitro match to integration in vivo and, as such, are particularly appropriate for identifying promising integration inhibitors.
Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.
Authors: Stryhn A , Andersen P S , Pedersen L O , Svejgaard A , Holm A , Thorpe C J , Fugger L , Buus S , Engberg J
Published: July 2002

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.
Cloning and characterization of four murine homeobox genes.
Authors: Rovescalli A C , Asoh S , Nirenberg M
Published: July 2002

Four novel murine homeobox genes, Uncx-4.1, OG-2, OG-9, and OG-12, were cloned and partially sequenced. The amino acid sequence of the mouse Uncx-4.1 homeodomain is closely related to the sequence of the unc-4 homeodomain of Caenorhabditis elegans. However, the OG-2, OG-9, and OG-12 homeodomains are relatively diverged and are not closely related to any previously described homeodomain. Northern blot analyses revealed multiple bands of Uncx-4.1, OG-2, OG-9, and OG-12 poly(A)+ RNA in RNA from mouse embryos and adults that change during development and showed that each gene is expressed in a tissue-specific manner. OG-12 cDNAs were cloned that correspond to two alternatively spliced species of OG-12 mRNA. Three major bands of Uncx-4.1 poly(A)+ RNA were found only in RNA from adult mouse brain, but an additional band was observed in RNA from all of the other tissues tested. Major bands of OG-9 and OG-2 poly(A)+ RNA were found only in RNA from striated muscle; however, trace bands were detected in RNA from other tissues.
NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.
Authors: Rebatchouk D , Daraselia N , Narita J O
Published: July 2002

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.
Essential requirement of an invariant V alpha 14 T cell antigen receptor expression in the development of natural killer T cells.
Authors: Taniguchi M , Koseki H , Tokuhisa T , Masuda K , Sato H , Kondo E , Kawano T , Cui J , Perkes A , Koyasu S , Makino Y
Published: July 2002

NK1.1+ T [natural killer (NK) T] cells express an invariant T cell antigen receptor alpha chain (TCR alpha) encoded by V alpha 14 and J alpha 281 segments in association with a limited number of V betas, predominantly V beta 8.2. Expression of the invariant V alpha 14/J alpha 281, but not V alpha 1, TCR in transgenic mice lacking endogenous TCR alpha expression blocks the development of conventional T alpha beta cells and leads to the preferential development of V alpha 14 NK T cells, suggesting a prerequisite role of invariant V alpha 14 TCR in NK T cell development. In V beta 8.2 but not B beta 3 transgenic mice, two NK T cells with different CD3 epsilon expressions, CD3 epsilon(dim) and CD3 epsilon(high), can be identified. CD3 epsilon(high) NK T cells express surface V alpha 14/V beta 8 TCR, indicating a mature cell type, whereas CD3 epsilon(dim) NK T cells express V beta 8 without V alpha 14 TCR and no significant CD3 epsilon expression (CD3 epsilon(dim)) on the cell surface. However, the latter are positive for recombination activating gene (RAG-1 and RAG-2) mRNA, which are only expressed in the precursor or immature T cell lineage, and also possess CD3 epsilon mRNA in their cytoplasm, suggesting that CD3 epsilon(dim) NK T cells are the precursor of V alpha 14 NK T cells.