bluuebunny/crossref_metadata_2025 huggingface dataset:
Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens
Authors: Fahey Jed W. , Zhang Yuesheng , Talalay Paul
Published: July 2002

Induction of phase 2 detoxication enzymes [e.g., glutathione
transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and
glucuronosyltransferases] is a powerful strategy for achieving
protection against carcinogenesis, mutagenesis, and other forms of
toxicity of electrophiles and reactive forms of oxygen. Since
consumption of large quantities of fruit and vegetables is associated
with a striking reduction in the risk of developing a variety of
malignancies, it is of interest that a number of edible plants contain
substantial quantities of compounds that regulate mammalian enzymes of
xenobiotic metabolism. Thus, edible plants belonging to the family
Cruciferae
and genus
Brassica
(e.g.,
broccoli and cauliflower) contain substantial quantities of
isothiocyanates (mostly in the form of their glucosinolate precursors)
some of which (e.g., sulforaphane or 4-methylsulfinylbutyl
isothiocyanate) are very potent inducers of phase 2 enzymes.
Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers
including broccoli and cauliflower contain 10–100 times higher levels
of glucoraphanin (the glucosinolate of sulforaphane) than do the
corresponding mature plants. Glucosinolates and isothiocyanates can be
efficiently extracted from plants, without hydrolysis of glucosinolates
by myrosinase, by homogenization in a mixture of equal volumes of
dimethyl sulfoxide, dimethylformamide, and acetonitrile at −50°C.
Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin
or sulforaphane as the principal enzyme inducer) were highly effective
in reducing the incidence, multiplicity, and rate of development of
mammary tumors in dimethylbenz(
a
)anthracene-treated
rats. Notably, sprouts of many broccoli cultivars contain negligible
quantities of indole glucosinolates, which predominate in the mature
vegetable and may give rise to degradation products (e.g.,
indole-3-carbinol) that can enhance tumorigenesis. Hence, small
quantities of crucifer sprouts may protect against the risk of cancer
as effectively as much larger quantities of mature vegetables of the
same variety.
Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa
Authors: Pipe Steven W. , Kaufman Randal J.
Published: July 2002

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced
in vitro
stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min
in vitro
, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.
Intron self-complementarity enforces exon inclusion in a yeast pre-mRNA
Authors: Howe Kenneth James , Ares Manuel
Published: July 2002

Skipping of internal exons during removal of introns from pre-mRNA must be avoided for proper expression of most eukaryotic genes. Despite significant understanding of the mechanics of intron removal, mechanisms that ensure inclusion of internal exons in multi-intron pre-mRNAs remain mysterious. Using a natural two-intron yeast gene, we have identified distinct RNA–RNA complementarities within each intron that prevent exon skipping and ensure inclusion of internal exons. We show that these complementarities are positioned to act as intron identity elements, bringing together only the appropriate 5′ splice sites and branchpoints. Destroying either intron self-complementarity allows exon skipping to occur, and restoring the complementarity using compensatory mutations rescues exon inclusion, indicating that the elements act through formation of RNA secondary structure. Introducing new pairing potential between regions near the 5′ splice site of intron 1 and the branchpoint of intron 2 dramatically enhances exon skipping. Similar elements identified in single intron yeast genes contribute to splicing efficiency. Our results illustrate how intron secondary structure serves to coordinate splice site pairing and enforce exon inclusion. We suggest that similar elements in vertebrate genes could assist in the splicing of very large introns and in the evolution of alternative splicing.
The absence of effect of
gid
or
mioC
transcription on the initiation of chromosomal replication in
Escherichia coli
Authors: Bates David B. , Boye Erik , Asai Tsuneaki , Kogoma Tokio
Published: July 2002

Despite the widely accepted view that transcription of
gid
and
mioC
is required for efficient initiation of cloned
oriC
, we show that these transcriptions have very little effect on initiation of chromosome replication at wild-type chromosomal
oriC
. Furthermore, neither
gid
nor
mioC
transcription is required in cells deficient in the histone-like proteins Fis or IHF. However,
oriC
that is sufficiently impaired for initiation by deletion of DnaA box R4 requires transcription of at least one of these genes. We conclude that transcription of
mioC
and especially
gid
is needed to activate
oriC
only under suboptimal conditions. We suggest that either the rifampicin-sensitive step of initiation is some other transcription occurring from promoter(s) within
oriC
, or the original inference of transcriptional activation derived from the rifampicin experiments is incorrect.
Thermodynamic stability of wild-type and mutant p53 core domain
Authors: Bullock Alex N. , Henckel Julia , DeDecker Brian S. , Johnson Christopher M. , Nikolova Penka V. , Proctor Mark R. , Lane David P. , Fersht Alan R.
Published: July 2002

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn
2+
ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25°C and 9.8 kcal/mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.
Volatile signals of the major histocompatibility complex in male mouse urine
Authors: Singer Alan G. , Beauchamp Gary K. , Yamazaki Kunio
Published: July 2002

Variation in the genes of the major histocompatibility complex (MHC) contributes to unique individual odors (odortypes) in mice, as demonstrated by the ability of trained mice in a Y-maze olfactometer to discriminate nearly identical inbred mice that differ genetically only at the MHC (MHC congenic mice), while they cannot distinguish genetically identical inbred mice. Similar distinctions are possible with urine, a substance that is involved in many facets of mouse chemical communication. This paper reports results supporting the hypothesis that the MHC-determined urinary odor is composed of a mixture of volatile carboxylic acids occurring in relative concentrations that are characteristic of the odortype. Y-maze behavioral testing of urine fractions from anion exchange chromatography indicates that volatile acids are necessary and sufficient to convey MHC odortype information. Diethyl ether extracts, which are expected to contain the more volatile, less polar organic acids, were also discriminable in the Y-maze olfactometer. Ether extracts of 12 different urine samples from each of two panels of MHC congenic mice were analyzed by gas chromatography. No compounds unique to urine of either genotype were detected, but compounds did appear to occur in characteristic ratios in most of the samples of each type. Nonparametric statistical analysis of the gas chromatographic data showed that eight of the peaks occurred in significantly different relative concentrations in the congenic samples. One of the peaks was shown to represent phenylacetic acid, which has implications for the mechanism of the MHC specification of odortype.
Binary transgenic mouse model for studying the trans control of globin gene switching: Evidence that GATA-1 is an
in vivo
repressor of human ɛ gene expression
Authors: Li Qiliang , Clegg Christopher , Peterson Kenneth , Shaw Sara , Raich Natacha , Stamatoyannopoulos George
Published: July 2002

To test whether human GATA-1 (hGATA-1) is involved in the transcriptional control of globin gene switching, we produced transgenic mice overexpressing hGATA-1, crossed them with mice carrying a human β-globin locus yeast artificial chromosome (βYAC), and analyzed globin gene expression in their progeny. Mice carrying both the hGATA-1 and the βYAC transgenes had normal levels of γ- and β-globin mRNA and no distortion in the rate or in the timing of γ-to-β switch, indicating that hGATA-1 is not involved in the developmental control of γ- and β-globin genes. In contrast, mice carrying the hGATA-1 and the βYAC transgenes had 5- to 6-fold lower expression of the human ɛ globin gene compared with βYAC mice lacking the hGATA-1 transgene. These results provide direct
in vivo
evidence that hGATA-1 is a specific repressor of human ɛ gene expression. Our findings also suggest that binary transgenic mouse systems based on overexpression of transcriptional factors can be used to investigate the trans control of human globin gene switching. Systems as the one we describe here should be useful in the study of any developmentally controlled human gene for which transgenic mice are available.
Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2
Authors: Winter David B. , Phung Quy H. , Umar Asad , Baker Sean M. , Tarone Robert E. , Tanaka Kiyoji , Liskay R. Michael , Kunkel Thomas A. , Bohr Vilhelm A. , Gearhart Patricia J.
Published: July 2002

Mutations are introduced into rearranged Ig variable genes at a frequency of 10−2mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair geneXpaor the mismatch repair genePms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not byPms2−/−human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.
Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia
Authors: Sogawa Kazuhiro , Numayama-Tsuruta Keiko , Ema Masatsugu , Abe Manabu , Abe Hisaku , Fujii-Kuriyama Yoshiaki
Published: July 2002

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O
2
radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors,
S
-nitroso-
l
-glutathione and 3-morpholinosydnonimine, giving IC
50
values of 7.8, 211, and 490 μM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC
50
= 6.6 μM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC
50
= 2.5 μM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1α or HIF-1α-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1α to a DNA-binding form.
A second major native von Hippel–Lindau gene product, initiated from an internal translation start site, functions as a tumor suppressor
Authors: Schoenfeld Alan , Davidowitz Eliot J. , Burk Robert D.
Published: July 2002

The von Hippel–Lindau (VHL) tumor suppressor gene is inactivated in both sporadic and inherited clear cell renal carcinoma associated with VHL disease. We have identified two distinct native products of the human VHL gene, with apparent molecular masses of 24 and 18 kDa. The 18-kDa VHL protein was more abundant in nearly all cell lines examined. Reintroduction of the 18-kDa
VHL
gene product into renal carcinoma cells lacking wild-type VHL protein led to down-regulation of vascular endothelial growth factor (VEGF) mRNA and glucose transporter GLUT1 protein and suppressed tumor formation in nude mice. The 18-kDa VHL protein also demonstrated binding to elongins B and C. In an
in vitro
assay, the second in-frame AUG codon present in VHL mRNA was shown to be necessary and sufficient for production of the 18-kDa VHL protein, consistent with an internal translation mechanism. These data provide evidence for a second major VHL gene product, which contains the functional domains of the VHL gene. Moreover, these results indicate that internal translation initiation is an important mechanism for production of the major VHL protein.
Protection against lipoapoptosis of β cells through leptin-dependent maintenance of Bcl-2 expression
Authors: Shimabukuro Michio , Wang May-Yun , Zhou Yan-Ting , Newgard Christopher B. , Unger Roger H.
Published: July 2002

Obesity causes its complications through functional and morphologic damage to remotely situated tissues via undetermined mechanisms. In one rodent model of obesity, the Zucker diabetic fatty
fa
/
fa
rat, overaccumulation of triglycerides in the pancreatic islets may be responsible for a gradual depletion of β cells, leading to the most common complication of obesity, non-insulin-dependent diabetes mellitus. At the onset of non-insulin-dependent diabetes mellitus, the islets from
fa
/
fa
rats contain up to 100 times the fat content of islets from normal lean rats. Ultimately, about 75% of the β cells disappear from these fat-laden islets as a consequence of apoptosis induced by long-chain fatty acids (FA). Here we quantify Bcl-2, the anti-apoptosis factor in these islets, and find that Bcl-2 mRNA and protein are, respectively, 85% and 70% below controls. In normal islets cultured in 1 mM FA, Bcl-2 mRNA declined by 68% and completely disappeared in
fa/fa
islets cultured in FA. In both groups, suppression was completely blocked by the fatty acyl-CoA synthetase inhibitor, triacsin C, evidence of its mediation by fatty acyl-CoA. To determine whether leptin action blocked FA-induced apoptosis, we cultured normal and
fa/fa
islets in 1 mM FA with or without leptin. Leptin completely blocked FA-induced Bcl-2 suppression in normal islets but had no effect on islets from
fa/fa
rats, which are unresponsive to leptin because of a mutation in their leptin receptors (OB-R). However, when wild-type OB-R is overexpressed in
fa/fa
islets, leptin completely prevented FA-induced Bcl-2 suppression and DNA fragmentation.
Lagging strand replication of rolling-circle plasmids: Specific recognition of the
ssoA
-type origins in different gram-positive bacteria
Authors: Kramer M. Gabriela , Espinosa Manuel , Misra Tapan K. , Khan Saleem A.
Published: July 2002

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed
ssoA
, which have conserved sequence and structural features, function efficiently only in their natural hosts
in vivo
. To study the host specificity of sso sequences, we have analyzed the functions of two closely related
ssoA
s belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in
Staphylococcus aureus
. The pLS1
ssoA
functioned poorly
in vivo
in
S. aureus
as evidenced by accumulation of high levels of ssDNA but supported efficient replication
in vitro
in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in
S. aureus
cell-free extracts may be limiting
in vivo
. Mapping of the initiation points of lagging strand synthesis
in vivo
and
in vitro
showed that DNA synthesis initiates from specific sites within the pLS1
ssoA
. These results demonstrate that specific initiation of replication can occur from the pLS1
ssoA
in
S. aureus
although it plays a minimal role in lagging strand synthesis
in vivo
. Therefore, the poor functionality of the pLS1
in vivo
in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the
S. aureus
and
Bacillus subtilis
RNA polymerases from the pE194 and pLS1
ssoA
s. The
S. aureus
RNA polymerase bound more efficiently to the native pE194
ssoA
as compared with the pLS1
ssoA
, suggesting that the strength of RNA polymerase–
ssoA
interaction may play a major role in the functionality of the
ssoA
sequences in Gram-positive bacteria.
Syntaxin 1A inhibits CFTR chloride channels by means of domain-specific protein–protein interactions
Authors: Naren Anjaparavanda P. , Quick Michael W. , Collawn James F. , Nelson Deborah J. , Kirk Kevin L.
Published: July 2002

Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein–protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing
Xenopus
oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl
−
currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant ΔF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.
Natural trans
-
splicing in carnitine octanoyltransferase pre-mRNAs in rat liver
Authors: Caudevilla Concha , Serra Dolors , Miliar Angel , Codony Carles , Asins Guillermina , Bach Montserrat , Hegardt Fausto G.
Published: July 2002

Carnitine octanoyltransferase (COT) transports medium-chain fatty acids through the peroxisome. During isolation of a COT clone from a rat liver library, a cDNA in which exon 2 was repeated, was characterized. Reverse transcription-PCR amplifications of total RNAs from rat liver showed a three-band pattern. Sequencing of the fragments revealed that, in addition to the canonical exon organization, previously reported [Choi, S. J.
et al.
(1995)
Biochim. Biophys. Acta
1264, 215–222], there were two other forms in which exon 2 or exons 2 and 3 were repeated. The possibility of this exonic repetition in the COT gene was ruled out by genomic Southern blot. To study the gene expression, we analyzed RNA transcripts by Northern blot after RNase H digestion of total RNA. Three different transcripts were observed. Splicing experiments also were carried out
in vitro
with different constructs that contain exon 2 plus the 5′ or the 3′ adjacent intron sequences. Our results indicate that accurate joining of two exons 2 occurs by a trans-splicing mechanism, confirming the potential of these structures for this process in nature. The trans-splicing can be explained by the presence of three exon-enhancer sequences in exon 2. Analysis by Western blot of the COT proteins by using specific antibodies showed that two proteins corresponding to the expected
M
r
are present in rat peroxisomes. This is the first time that a natural trans-splicing reaction has been demonstrated in mammalian cells.
Natural genetic exchange between
Haemophilus
and
Neisseria
: Intergeneric transfer of chromosomal genes between major human pathogens
Authors: Kroll J. Simon , Wilks Kathryn E. , Farrant Jayne L. , Langford Paul R.
Published: July 2002

Members of the bacterial families
Haemophilus
and
Neisseria
, important human pathogens that commonly colonize the nasopharynx, are naturally competent for DNA uptake from their environment. In each genus this process is discriminant in favor of its own and against foreign DNA through sequence specificity of DNA receptors. The
Haemophilus
DNA uptake apparatus binds a 29-bp oligonucleotide domain containing a highly conserved 9-bp core sequence, whereas the neisserial apparatus binds a 10-bp motif. Each motif (“uptake sequence”, US) is highly over-represented in the chromosome of the corresponding genus, particularly concentrated with core sequences in inverted pairs forming gene terminators. Two
Haemophilus
core USs were unexpectedly found forming the terminator of
sodC
in
Neisseria meningitidis
(meningococcus), and sequence analysis strongly suggests that this virulence gene, located next to
IS
1106, arose through horizontal transfer from
Haemophilus
. By using USs as search strings in a computer-based analysis of genome sequence, it was established that while USs of the “wrong” genus do not occur commonly in
Neisseria
or
Haemophilus
, where they do they are highly likely to flag domains of chromosomal DNA that have been transferred from
Haemophilus
. Three independent domains of
Haemophilus
-like DNA were found in the meningococcal chromosome, associated respectively with the virulence gene
sodC
, the
bio
gene cluster, and an unidentified
orf
. This report identifies intergenerically transferred DNA and its source in bacteria, and further identifies transformation with heterologous chromosomal DNA as a way of establishing potentially important chromosomal mosaicism in these pathogenic bacteria.
A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells
Authors: Holaska James M. , Paschal Bryce M.
Published: July 2002

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with
N-
ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.
A protein required for nuclear-protein import, Mog1p, directly interacts with GTP–Gsp1p, the
Saccharomyces cerevisiae
Ran homologue
Authors: Oki Masaya , Nishimoto Takeharu
Published: July 2002

We previously isolated 25 temperature-sensitive
gsp1
alleles of
Saccharomyces cerevisiae
Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors—
PDE2, NTF2,
and a gene designated
MOG1
—all of which rescued a growth defect of these
gsp1
strains. The
gsp1
suppression occurred even in the absence of
GSP2
, another
S. cerevisiae GSP1
-like gene. Previously,
NTF2
was reported to suppress
gsp1
but not
PDE2
. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both
MOG1
and
NTF2
suppressed a series of
gsp1
alleles with similar efficiency, and both suppressed
gsp1
even with a single gene dose. Consistent with the high efficiency of
gsp1
suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of
MOG1
made yeast temperature-sensitive for growth. Δmog1, which was suppressed by overexpression of
NTF2
, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that
PDE2
suppressed both
gsp1
and
rna1–1
indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.
A highly conserved lysine residue on the head domain of type II keratins is essential for the attachment of keratin intermediate filaments to the cornified cell envelope through isopeptide crosslinking by transglutaminases
Authors: Candi Eleonora , Tarcsa Edit , Digiovanna John J. , Compton John G. , Elias Peter M. , Marekov Lyuben N. , Steinert Peter M.
Published: July 2002

We have addressed the question of how keratin intermediate filaments are associated with the cell envelope at the periphery of cornified epidermal cells. Many peptides from human epidermal cell envelopes containing isopeptide crosslinks inserted by transglutaminases
in vivo
have been characterized. A major subset involves the type II keratin chains keratin 1, 2e, 5, or 6 crosslinked to several protein partners through a lysine residue located in a conserved region of the V1 subdomain of their head domains. This sequence specificity was confirmed in
in vitro
crosslinking experiments. Previously the causative mutation in a family with diffuse nonepidermolytic palmar-plantar keratoderma was shown to be the loss in one allele of the same lysine residue of the keratin 1 chain. Ultrastructural studies of affected palm epidermis have revealed abnormalities in the organization of keratin filaments subjacent to the cell envelope and in the shape of the cornified cells. Together, these data suggest a mechanism for the coordination of cornified cell structure by permanent covalent attachment of the keratin intermediate filament cytoskeleton to the cell envelope by transglutaminase crosslinking. Furthermore, these studies identify the essential role of a conserved lysine residue on the head domains of type II keratins in the supramolecular organization of keratin filaments in cells.
Protein hydration in solution: Experimental observation by x-ray and neutron scattering
Authors: Svergun D. I. , Richard S. , Koch M. H. J. , Sayers Z. , Kuprin S. , Zaccai G.
Published: July 2002

The structure of the protein–solvent interface is the subject of controversy in theoretical studies and requires direct experimental characterization. Three proteins with known atomic resolution crystal structure (lysozyme,
Escherichia coli
thioredoxin reductase, and protein R1 of
E. coli
ribonucleotide reductase) were investigated in parallel by x-ray and neutron scattering in H
2
O and D
2
O solutions. The analysis of the protein–solvent interface is based on the significantly different contrasts for the protein and for the hydration shell. The results point to the existence of a first hydration shell with an average density ≈10% larger than that of the bulk solvent in the conditions studied. Comparisons with the results of other studies suggest that this may be a general property of aqueous interfaces.
Chimeric anti-angiogenin antibody cAb 26–2F inhibits the formation of human breast cancer xenografts in athymic mice
Authors: Piccoli Renata , Olson Karen A. , Vallee Bert L. , Fett James W.
Published: July 2002

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26–2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26–2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26–2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26–2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26–2F. Furthermore, the capacities of cAb 26–2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26–2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.
HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A
Authors: Lee Ni , Llano Manuel , Carretero Marta , Ishitani Akiko , Navarro Francisco , López-Botet Miguel , Geraghty Daniel E.
Published: July 2002

We previously showed that the availability of a nonamer peptide derived from certain HLA class I signal sequences is a necessary requirement for the stabilization of endogenous HLA-E expression on the surface of 721.221 cells. This led us to examine the ability of HLA-E to protect HLA class I transfectants from natural killer (NK) cell-mediated lysis. It was possible to implicate the CD94/NKG2A complex as an inhibitory receptor recognizing this class Ib molecule by using as target a .221 transfectant selectively expressing surface HLA-E. HLA-E had no apparent inhibitory effect mediated through the identified Ig superfamily (Ig-SF) human killer cell inhibitory receptors or ILT2/LIR1. Further studies of CD94/NKG2+ NK cell-mediated recognition of .221 cells transfected with different HLA class I allotypes (i.e., -Cw4, -Cw3, -B7) confirmed that the inhibitory interaction was mediated by CD94/NKG2A recognizing the surface HLA-E molecule, because only antibodies directed against either HLA-E, CD94, or CD94/NKG2A specifically restored lysis. Surface stabilization of HLA-E in cold-treated .221 cells loaded with appropriate peptides was sufficient to confer protection, resulting from recognition of the HLA class Ib molecule by the CD94/NKG2A inhibitory receptor. Consistent with the prediction that the ligand for CD94/NKG2A is expressed ubiquitously, our examination of HLA-E antigen distribution indicated that it is detectable on the surface of a wide variety of cell types.
Conjugation of the ubiquitin-like protein NEDD8 to cullin-2 is linked to von Hippel–Lindau tumor suppressor function
Authors: Liakopoulos Dimitris , Büsgen Tanja , Brychzy Alexander , Jentsch Stefan , Pause Arnim
Published: July 2002

The von Hippel–Lindau tumor suppressor protein pVHL assembles with cullin-2 (hCUL-2) and elongin B/C forming a protein complex, CBC
VHL
, that resembles SKP1–CDC53–F-box protein ubiquitin ligases. Here, we show that hCUL-2 is modified by the conserved ubiquitin-like protein NEDD8 and that NEDD8–hCUL-2 conjugates are part of CBC
VHL
complexes
in vivo
. Remarkably, the formation of these conjugates is stimulated by the pVHL tumor suppressor. A tumorigenic pVHL variant, however, is essentially deficient in this activity. Thus, ligation of NEDD8 to hCUL-2 is linked to pVHL activity and may be important for pVHL tumor suppressor function.
Evidence on the origin of cassava: Phylogeography of
Manihot esculenta
Authors: Olsen Kenneth M. , Schaal Barbara A.
Published: July 2002

Cassava (
Manihot esculenta
subsp.
esculenta
) is a staple crop with great economic importance worldwide, yet its evolutionary and geographical origins have remained unresolved and controversial. We have investigated this crop’s domestication in a phylogeographic study based on the single-copy nuclear gene glyceraldehyde 3-phosphate dehydrogenase (
G3pdh
). The
G3pdh
locus provides high levels of noncoding sequence variation in cassava and its wild relatives, with 28 haplotypes identified among 212 individuals (424 alleles) examined. These data represent one of the first uses of a single-copy nuclear gene in a plant phylogeographic study and yield several important insights into cassava’s evolutionary origin: (
i
) cassava was likely domesticated from wild
M. esculenta
populations along the southern border of the Amazon basin; (
ii
) the crop does not seem to be derived from several progenitor species, as previously proposed; and (
iii
) cassava does not share haplotypes with
Manihot pruinosa
, a closely related, potentially hybridizing species. These findings provide the clearest picture to date on cassava’s origin. When considered in a genealogical context, relationships among the
G3pdh
haplotypes are incongruent with taxonomic boundaries, both within
M. esculenta
and at the interspecific level; this incongruence is probably a result of lineage sorting among these recently diverged taxa. Although phylogeographic studies in animals have provided many new evolutionary insights, application of phylogeography in plants has been hampered by difficulty in obtaining phylogenetically informative intraspecific variation. This study demonstrates that single-copy nuclear genes can provide a useful source of informative variation in plants.
Cross-linked protein crystals for vaccine delivery
Authors: St. Clair Nancy , Shenoy Bhami , Jacob Lawrence D. , Margolin Alexey L.
Published: July 2002

The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present paper, we propose the use of cross-linked protein crystals (CLPCs) as antigens. We compare the immunogenicity of CLPCs of human serum albumin with that of soluble protein and conclude that there are marked differences in the immune response to the different forms of human serum albumin. Relative to the soluble protein, crystalline forms induce and sustain over almost a 6-month study a 6- to 10-fold increase in antibody titer for highly cross-linked crystals and an approximately 30-fold increase for lightly cross-linked crystals. We hypothesize that the depot effect, the particulate structure of CLPCs, and highly repetitive nature of protein crystals may play roles in the enhanced production of circulating antibodies. Several features of CLPCs, such as their remarkable stability, purity, biodegradability, and ease of manufacturing, make them highly attractive for vaccine formulations. This work paves the way for a systematic study of protein crystallinity and cross-linking on enhancement of humoral and T cell responses.
New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase
Authors: Humphreys John M. , Hemm Matthew R. , Chapple Clint
Published: July 2002

The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in
Arabidopsis
. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the
K
m
values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant
Arabidopsis
caffeic acid/5-hydroxyferulic acid
O-
methyltransferase and [
14
C]
S
-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in
Arabidopsis
. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect.
Intracellular gene transfer in action: Dual transcription and multiple silencings of nuclear and mitochondrial
cox2
genes in legumes
Authors: Adams Keith L. , Song Keming , Roessler Philip G. , Nugent Jacqueline M. , Doyle Jane L. , Doyle Jeff J. , Palmer Jeffrey D.
Published: July 2002

The respiratory gene
cox2
, normally present in the mitochondrion, was previously shown to have been functionally transferred to the nucleus during flowering plant evolution, possibly during the diversification of legumes. To search for novel intermediate stages in the process of intracellular gene transfer and to assess the evolutionary timing and frequency of
cox2
transfer, activation, and inactivation, we examined nuclear and mitochondrial (mt)
cox2
presence and expression in over 25 legume genera and mt
cox2
presence in 392 genera. Transfer and activation of
cox2
appear to have occurred during recent legume evolution, more recently than previously inferred. Many intermediate stages of the gene transfer process are represented by
cox2
genes in the studied legumes. Nine legumes contain intact copies of both nuclear and mt
cox2
, although transcripts could not be detected for some of these genes. Both
cox2
genes are transcribed in seven legumes that are phylogenetically interspersed with species displaying only nuclear or mt
cox2
expression. Inactivation of
cox2
in each genome has taken place multiple times and in a variety of ways, including loss of detectable transcripts or transcript editing and partial to complete gene loss. Phylogenetic evidence shows about the same number (3–5) of separate inactivations of nuclear and mt
cox2
, suggesting that there is no selective advantage for a mt vs. nuclear location of
cox2
in plants. The current distribution of
cox2
presence and expression between the nucleus and mitochondrion in the studied legumes is probably the result of chance mutations silencing either
cox2
gene.
Vibrational population relaxation of carbon monoxide in the heme pocket of photolyzed carbonmonoxy myoglobin: Comparison of time-resolved mid-IR absorbance experiments and molecular dynamics simulations
Authors: Sagnella Diane E. , Straub John E. , Jackson Timothy A. , Lim Manho , Anfinrud Philip A.
Published: July 2002

The vibrational energy relaxation of carbon monoxide in the heme pocket of sperm whale myoglobin was studied by using molecular dynamics simulation and normal mode analysis methods. Molecular dynamics trajectories of solvated myoglobin were run at 300 K for both the δ- and ɛ-tautomers of the distal His-64. Vibrational population relaxation times of 335 ± 115 ps for the δ-tautomer and 640 ± 185 ps for the ɛ-tautomer were estimated by using the Landau–Teller model. Normal mode analysis was used to identify those protein residues that act as the primary “doorway” modes in the vibrational relaxation of the oscillator. Although the CO relaxation rates in both the ɛ- and δ-tautomers are similar in magnitude, the simulations predict that the vibrational relaxation of the CO is faster in the δ-tautomer with the distal His playing an important role in the energy relaxation mechanism. Time-resolved mid-IR absorbance measurements were performed on photolyzed carbonmonoxy hemoglobin (Hb
13
CO). From these measurements, a T
1
time of 600 ± 150 ps was determined. The simulation and experimental estimates are compared and discussed.
Genomic analysis of human and mouse
TCL1
loci reveals a complex of tightly clustered genes
Authors: Hallas Cora , Pekarsky Yuri , Itoyama Takahiro , Varnum James , Bichi Roberta , Rothstein Jay L. , Croce Carlo M.
Published: July 2002

TCL1
and
TCL1b
genes on human chromosome 14q23.1 are activated in T cell leukemias by translocations and inversions at 14q32.1, juxtaposing them to regulatory elements of T cell receptor genes. In this report we present the cloning, mapping, and expression analysis of the human and murine
TCL1/Tcl1
locus. In addition to
TCL1
and
TCL1b
, the human locus contains two additional genes,
TCL1
-neighboring genes (
TNG
) 1 and 2, encoding proteins of 141 and 110 aa, respectively. Both genes show no homology to any known genes, but their expression profiles are very similar to those of
TCL1
and
TCL1b. TNG1
and
TNG2
also are activated in T cell leukemias with rearrangements at 14q32.1. To aid in the development of a mouse model we also have characterized the murine
Tcl1
locus and found five genes homologous to human
TCL1b. Tcl1b1
–
Tcl1b5
proteins range from 117 to 123 aa and are 65–80% similar, but they show only a 30–40% similarity to human
TCL1b
. All five mouse
Tcl1b
and murine
Tcl1
mRNAs are abundant in mouse oocytes and two-cell embryos but rare in various adult tissues and lymphoid cell lines. These data suggest a similar or complementary function of these proteins in early embryogenesis.
Analysis of three structurally related antiviral compounds in complex with human rhinovirus 16
Authors: Hadfield Andrea T. , Diana Guy D. , Rossmann Michael G.
Published: July 2002

Rhinoviruses are a frequent cause of the common cold. A series of antirhinoviral compounds have been developed that bind into a hydrophobic pocket in the viral capsid, stabilizing the capsid and interfering with cell attachment. The structures of a variety of such compounds, complexed with rhinovirus serotypes 14, 16, 1A, and 3, previously have been examined. Three chemically similar compounds, closely related to a drug that is undergoing phase III clinical trials, were chosen to determine the structural impact of the heteroatoms in one of the three rings. The compounds were found to have binding modes that depend on their electronic distribution. In the compound with the lowest efficacy, the terminal ring is displaced by 1 Å and rotated by 180° relative to the structure of the other two. The greater polarity of the terminal ring in one of the three compounds leads to a small displacement of its position relative to the other compounds in the hydrophobic end of the antiviral compound binding pocket to a site where it makes fewer interactions. Its lower efficacy is likely to be the result of the reduced number of interactions. A region of conserved residues has been identified near the entrance to the binding pocket where there is a corresponding conservation of the mode of binding of these compounds to different serotypes. Thus, variations in residues lining the more hydrophobic end of the pocket are primarily responsible for the differences in drug efficacies.