bluuebunny/crossref_metadata_2025 huggingface dataset:
Amyloid precursor protein processing and Aβ
42
deposition in a transgenic mouse model of Alzheimer disease
Authors: Johnson-Wood K. , Lee M. , Motter R. , Hu K. , Gordon G. , Barbour R. , Khan K. , Gordon M. , Tan H. , Games D. , Lieberburg I. , Schenk D. , Seubert P. , McConlogue L.
Published: July 2002

The PDAPP transgenic mouse, which overexpresses human amyloid precursor protein (APP717V→F), has been shown to develop much of the pathology associated with Alzheimer disease. In this report, levels of APP and its amyloidogenic metabolites were measured in brain regions of transgenic mice between 4 and 18 months of age. While absolute levels of APP expression likely contribute to the rate of amyloid β-peptide (Aβ) deposition, regionally specific factors also seem important, as homozygotic mice express APP levels in pathologically unaffected regions in excess of that measured in certain amyloid plaque-prone regions of heterozygotic mice. Regional levels of APP and APP-β were nearly constant at all ages, while Aβ levels dramatically and predictably increased in brain regions undergoing histochemically confirmed amyloidosis, most notably in the cortex and hippocampus. In hippocampus, Aβ concentrations increase 17-fold between the ages of 4 and 8 months, and by 18 months of age are over 500-fold that at 4 months, reaching an average level in excess of 20 nmol of Aβ per g of tissue. Aβ
1–42
constitutes the vast majority of the depositing Aβ species. The similarities observed between the PDAPP mouse and human Alzheimer disease with regard to Aβ
42
deposition occurring in a temporally and regionally specific fashion further validate the use of the model in understanding processes related to the disease.
Transgenic elite
indica
rice plants expressing CryIAc ∂-endotoxin of
Bacillus thuringiensis
are resistant against yellow stem borer (
Scirpophaga incertulas
)
Authors: Nayak Pritilata , Basu Debabrata , Das Sampa , Basu Asitava , Ghosh Dipankar , Ramakrishnan Neeliyath A. , Ghosh Maloy , Sen Soumitra K.
Published: July 2002

Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of
Bacillus thuringiensis
(
Bt
) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate
Bt
-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB),
Scirpophaga incertulas
, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that
cryIAb
and
cryIAc
are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop
Bt
-transgenic rice with these ICP genes independently in a single cultivar. A
cryIAc
gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize
ubiquitin 1
gene, and the
nos
terminator. The gene construct was delivered to embryogenic calli of IR64, an elite
indica
rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic
cryIAc
gene was expressed stably in the T
2
generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.
Regulation of insulin preRNA splicing by glucose
Authors: Wang Juehu , Shen Luping , Najafi Habiba , Kolberg Janice , Matschinsky Franz M. , Urdea Mickey , German Michael
Published: July 2002

Glucose tightly regulates the synthesis and secretion of insulin by β cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single β cell (βTC3 cells or mouse islets), whereas 1 million non-insulin-producing α cells (αTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from β cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up ≈2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription.
A single receptor encoded by
vzg-1
/
lp
A1
/
edg-
2
couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid
Authors: Fukushima Nobuyuki , Kimura Yuka , Chun Jerold
Published: July 2002

Extracellular lysophosphatidic acid (LPA) produces diverse cellular responses in many cell types. Recent reports of several molecularly distinct G protein-coupled receptors have raised the possibility that the responses to LPA stimulation could be mediated by the combination of several uni-functional receptors. To address this issue, we analyzed one receptor encoded by
ventricular zone gene-1
(
vzg-1
) (also referred to as
lp
A1
/
edg-2
) by using heterologous expression in a neuronal and nonneuronal cell line. VZG-1 expression was necessary and sufficient in mediating multiple effects of LPA: [
3
H]-LPA binding, G protein activation, stress fiber formation, neurite retraction, serum response element activation, and increased DNA synthesis. These results demonstrate that a single receptor, encoded by
vzg-1
, can activate multiple LPA-dependent responses in cells from distinct tissue lineages.
An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription
Authors: Yokomori Kyoko , Verrijzer C. Peter , Tjian Robert
Published: July 2002

The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated. It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting. Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors. The zinc-finger domain of E56, required for transcriptional activity, is critical for this function. In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly. These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.
Mitogenic and oncogenic properties of the small G protein Rap1b
Authors: Altschuler Daniel L. , Ribeiro-Neto Fernando
Published: July 2002

It has been widely reported that the small GTP-binding protein Rap1 has an anti-Ras and anti-mitogenic activity. Thus, it is generally accepted that a normal physiological role of Rap1 proteins is to antagonize Ras mitogenic signals, presumably by forming nonproductive complexes with proteins that are typically effectors or modulators of Ras. Rap1 is activated by signals that raise intracellular levels of cAMP, a molecule that has long been known to exert both inhibitory and stimulatory effects on cell growth. We have now tested the intriguing hypothesis that Rap1 could have mitogenic effects in systems in which cAMP stimulates cell proliferation. The result of experiments addressing this possibility revealed that Rap1 has full oncogenic potential. Expression of Rap1 in these cells results in a decreased doubling time, an increased saturation density, and an unusual anchorage-dependent morphological transformation. Most significantly, however, Rap1-expressing cells formed tumors when injected into nude mice. Thus, we propose that the view that holds Rap1 as an antimitogenic protein should be restricted and conclude that Rap1 is a conditional oncoprotein.
Promoter escape limits the rate of RNA polymerase II transcription and is enhanced by TFIIE, TFIIH, and ATP on negatively supercoiled DNA
Authors: Kugel Jennifer F. , Goodrich James A.
Published: July 2002

To measure rate constants for discrete steps of single-round transcription (preinitiation complex formation, promoter escape, and transcript elongation), kinetic studies were performed in a well defined human RNA polymerase II transcription system. These experiments revealed that promoter escape limits the rate of transcription from the adenovirus major late promoter (AdMLP) contained on negatively supercoiled DNA. TFIIE and TFIIH were found to significantly increase fractional template usage during a single round of transcription in an ATP-dependent reaction. The observed rate constant for promoter escape, however, was not greatly affected by TFIIE and TFIIH. Our results are explained by a model in which transcription branches into at least two pathways: one that results in functional promoter escape and full-length RNA synthesis, and another in which preinitiation complexes abort during promoter escape and do not produce full-length RNA transcripts. These results with negatively supercoiled templates agree with our earlier conclusion that TFIIE, TFIIH, and ATP direct promoter escape and support a model in which the TFIIH helicases stimulate promoter escape in an ATP-dependent reaction.
The location of the carboxy-terminal region of γ chains in fibrinogen and fibrin D domains
Authors: Mosesson Michael W. , Siebenlist Kevin R. , Meh David A. , Wall Joseph S. , Hainfeld James F.
Published: July 2002

Elongated fibrinogen molecules are comprised of two outer “D” domains, each connected through a “coiled-coil” region to the central “E” domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of γ chains in each D domain (the γ
XL
site) by incorporating intermolecular ɛ-(γ-glutamyl)lysine bonds between amine donor γ406 lysine of one γ chain and a glutamine acceptor at γ398 or γ399 of another. Several lines of evidence show that crosslinked γ chains extend “transversely” between the strands of each fibril, but other data suggest instead that crosslinked γ chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the γ
XL
site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au
11
). Fibrinogen was examined by scanning transmission electron microscopy to locate Au
11
-cadaverine-labeled γ398/399 D domain sites. Seventy-nine percent of D domain Au
11
clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au
11
clusters in a distal position. In fibrin fibrils, D domain Au
11
clusters were located in middle to proximal positions. These findings show that most C-terminal γ chains in fibrinogen or fibrin are oriented toward the central domain and indicate that γ
XL
sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.
Δ5-Androstenediol is a natural hormone with androgenic activity in human prostate cancer cells
Authors: Miyamoto Hiroshi , Yeh Shuyuan , Lardy Henry , Messing Edward , Chang Chawnshang
Published: July 2002

It is known that androst-5-ene-3β,17β-diol (Adiol), a precursor of testosterone (T), can activate estrogen target genes. The androgenic activity of Adiol itself, however, is poorly understood. Using a transient transfection assay, we here demonstrate in human prostate cancer cells that Adiol can activate androgen receptor (AR) target genes in the presence of AR, and that AR coactivator ARA70 can further enhance this Adiol-induced AR transcriptional activity. In contrast to this finding, an active metabolite of dehydroepiandrosterone, 7-oxo-dehydroepiandrosterone, does not activate AR target gene in the absence or presence of ARA70. Thin layer chromatography analysis reveals that T, dihydrotestosterone, and 17β-estradiol are undetectable in human prostate cancer DU145 cells after treatment with Adiol. Additionally, a proteolysis assay shows that a distinct ligand-receptor conformational difference exists between T-AR and Adiol-AR. Together, the above findings and the fact that T, but not Adiol, can induce transcriptional activity in a mutant AR (mtAR708), suggest that, without being metabolized into T, Adiol itself may represent a natural hormone with androgenic activity in human prostate cancer cells. Because two potent antiandrogens, hydroxyflutamide (Eulexin), and bicalutamide (casodex), that are widely used for the treatment of prostate cancer, fail to block Adiol-mediated induction of AR transcriptional activity in prostate cancer cells, the effectiveness of so-called “total androgen blockage,” a standard treatment for prostate cancer, may need to be reevaluated.
A double-headed Gly-Pro-Arg-Pro ligand mimics the functions of the E domain of fibrin for promoting the end-to-end crosslinking of γ chains by factor XIII
a
Authors: Lorand Laszlo , Parameswaran Kumarapuram N. , Murthy S. N. Prasanna
Published: July 2002

The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its α chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIII
a
-catalyzed end-to-end ligation of the γ chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIII
a
reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of α chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.
Discrimination of the closely related A and D genomes of the hexaploid oat
Avena sativa
L.
Authors: Linares Concha , Ferrer Esther , Fominaya Araceli
Published: July 2002

A satellite DNA sequence, As120a, specific to the A-genome chromosomes in the hexaploid oat,
Avena sativa
L., was isolated by subcloning a fragment with internal tandem repeats from a plasmid, pAs120, that had been obtained from an
Avena strigosa
(As genome) genomic library. Southern and
in situ
hybridization showed that sequences with homology to sequences within pAs120 were dispersed throughout the genome of diploid (A and C genomes), tetraploid (AC genomes), and hexaploid (ACD genomes)
Avena
species. In contrast, sequences homologous to As120a were found in two A-genome species (
A. strigosa
and
Avena longiglumis
) and in the hexaploid
A. sativa
whereas this sequence was little amplified in the tetraploid
Avena murphyi
and was absent in the remaining A- and C-genome diploid species.
In situ
hybridization of pAs120a to hexaploid oat species revealed the distribution of elements of the As120a repeated family over both arms of 14 of 42 chromosomes of this species. By using double
in situ
hybridization with pAs120a and a C genome-specific probe, three sets of 14 chromosomes were revealed corresponding to the A, C, and D genomes of the hexaploid species. Simultaneous
in situ
hybridizations with pAs120a and ribosomal probes were used to assign the SAT chromosomes of hexaploid species to their correct genomes. This work reports a sequence able to distinguish between the closely related A and D genomes of hexaploid oats. This sequence offers new opportunities to analyze the relationships of
Avena
species and to explore the possible evolution of various polyploid oat species.
Dispersal of first “workers” in social wasps: Causes and implications of an alternative reproductive strategy
Authors: Reeve H. Kern , Peters John M. , Nonacs Peter , Starks Philip T.
Published: July 2002

Many “workers” in north temperate colonies of the eusocial paper waspPolistes fuscatusdisappear within a few days of eclosion. We provide evidence that these females are pursuing an alternative reproductive strategy, i.e., dispersing to overwinter and become nest foundresses the following spring, instead of helping to rear brood on their natal nests. A female is most likely to stay and help at the natal nest (i.e., least likely to disperse) when it is among the first workers to emerge and when it emerges on a nest with more pupae (even though worker-brood relatedness tends to be lower in such colonies). The latter cause may result from the fact that pupae-laden nests are especially likely to survive, and thus any direct or indirect reproductive payoffs for staying and working are less likely to be lost. Disappearing females are significantly smaller than predicted if dispersal tendency was independent of body size (emergence order-controlled), suggesting that the females likely to be most effective at challenging for reproductive rights within the natal colony (i.e., the largest females) are also most likely to stay. Thus, early dispersal is conditional on a female’s emergence order, the maturity of its natal nest, and its body size. Finally, we present evidence that foundresses may actively limit the sizes of first-emerging females, perhaps to decrease the probability that the latter can effectively challenge foundresses for reproductive rights. The degree to which foundresses limit the size of first-emerging females accords well with the predictions of the theory of staying incentives.
Differential use of the signal recognition particle translocase targeting pathway for inner membrane protein assembly in
Escherichia coli
Authors: de Gier Jan-Willem L. , Scotti Pier A. , Sääf Annika , Valent Quido A. , Kuhn Andreas , Luirink Joen , von Heijne Gunnar
Published: July 2002

Assembly of several inner membrane proteins—leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep—has been studied
in vitro
and in
Escherichia coli
strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the
E. coli
signal recognition particle (SRP), or SecE, an essential core component of the
E. coli
preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.
Thyroid hormone receptor β-dependent expression of a potassium conductance in inner hair cells at the onset of hearing
Authors: Rüsch Alfons , Erway Lawrence C. , Oliver Dominik , Vennström Björn , Forrest Douglas
Published: July 2002

To elucidate the role of thyroid hormone receptors (TRs) α1 and β in the development of hearing, cochlear functions have been investigated in mice lacking TRα1 or TRβ. TRs are ligand-dependent transcription factors expressed in the developing organ of Corti, and loss of TRβ is known to impair hearing in mice and in humans. Here, TRα1-deficient (TRα1
−/−
) mice are shown to display a normal auditory-evoked brainstem response, indicating that only TRβ, and not TRα1, is essential for hearing. Because cochlear morphology was normal in TRβ
−/−
mice, we postulated that TRβ regulates functional rather than morphological development of the cochlea. At the onset of hearing, inner hair cells (IHCs) in wild-type mice express a fast-activating potassium conductance,
I
K,f
, that transforms the immature IHC from a regenerative, spiking pacemaker to a high-frequency signal transmitter. Expression of
I
K,f
was significantly retarded in TRβ
−/−
mice, whereas the development of the endocochlear potential and other cochlear functions, including mechanoelectrical transduction in hair cells, progressed normally. TRα1
−/−
mice expressed
I
K,f
normally, in accord with their normal auditory-evoked brainstem response. These results establish that the physiological differentiation of IHCs depends on a TRβ-mediated pathway. When defective, this may contribute to deafness in congenital thyroid diseases.
Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5
Authors: Schatz Octavian , Oft Martin , Dascher Christiane , Schebesta Michael , Rosorius Olaf , Jaksche Herbert , Dobrovnik Marika , Bevec Dorian , Hauber Joachim
Published: July 2002

It has previously been shown that interaction of eukaryotic initiation factor 5A (eIF-5A) with the Rev trans-activator protein of HIV-1 mediates the transport of unspliced or incompletely spliced viral mRNAs across the nuclear envelope. Consequently, mutants of eIF-5A block Rev function and thereby replication of HIV-1 in trans, indicating that eIF-5A is a crucial protein that connects the viral Rev regulator with cellular RNA transport systems. Here we show that the ribosomal protein L5, which is the central protein component of the 5S rRNA export system, is a cellular interaction partner of eIF-5A. Functional studies demonstrate that overexpression of L5 protein significantly enhances Rev activity. Furthermore, Rev nuclear export activity is inhibited in human somatic cells by antibodies that recognize eIF-5A or L5. Our data suggest that the Rev export pathway shares components of a cellular transport system involved in the intracellular trafficking of polymerase III (5S rRNA) transcripts.
Identification of a silencing element in the human 15q11-q13 imprinting center by using transgenicDrosophila
Authors: Lyko Frank , Buiting Karin , Horsthemke Bernhard , Paro Renato
Published: July 2002

Prader–Willi syndrome (PWS) and Angelman syndrome are neurogenetic disorders caused by the lack of a paternal or a maternal contribution from human chromosome 15q11-q13, respectively. Deletions in the transcription unit of the imprintedSNRPNgene have been found in patients who have PWS or Angelman syndrome because of a parental imprint switch failure in this chromosomal domain. It has been suggested that theSNRPNexon 1 region, which is deleted in the PWS patients, contains an imprint switch element from which the maternal and paternal epigenotypes of the 15q11-q13 domain originate. Using the model organismDrosophila, we show here that a fragment from this region can function as a silencer in transgenic flies. Repression was detected specifically from this element and could not be observed with control human sequences. Additional experiments allowed the delineation of the silencer to a fragment of 215 bp containing theSNRPNpromoter region. These results provide an additional link between genomic imprinting and an evolutionary conserved silencing mechanism. We suggest that the identified element participates in the long range regulation of the imprinted 15q11-q13 domain or locally repressesSNRPNexpression from the maternal allele.
Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or Leber congenital amaurosis
Authors: Morimura Hiroyuki , Fishman Gerald A. , Grover Sandeep A. , Fulton Anne B. , Berson Eliot L. , Dryja Thaddeus P.
Published: July 2002

RPE65 is a protein of unknown function expressed specifically by the retinal pigment epithelium. We examined all 14 exons of this gene in 147 unrelated patients with autosomal recessive retinitis pigmentosa (RP), in 15 patients with isolate RP, and in 45 patients with Leber congenital amaurosis (LCA). Sequence anomalies that were likely to be pathogenic were found in two patients with recessive RP, in one patient with isolate RP recategorized as recessive, and in seven patients with LCA. Cosegregation analysis in each available family showed that all affected individuals were either homozygotes or compound heterozygotes and that all unaffected individuals were either heterozygote carriers or homozygous wild type. In one family, there was one instance of a new mutation not present in either parent of the affected individual. In another family, affected members with recessive RP in three branches (i.e., three distinct pairs of parents) were compound heterozygotes for the same two mutations or homozygous for one of them. Based on our results, mutations in the
RPE65
gene appear to account for ≈2% of cases of recessive RP and ≈16% of cases of LCA.
Mechanism of Ca
2+
-dependent nuclear accumulation of calmodulin
Authors: Liao Birong , Paschal Bryce M. , Luby-Phelps Katherine
Published: July 2002

The intracellular Ca
2+
receptor calmodulin (CaM) coordinates responses to extracellular stimuli by modulating the activities of its various binding proteins. Recent reports suggest that, in addition to its familiar functions in the cytoplasm, CaM may be directly involved in rapid signaling between cytoplasm and nucleus. Here we show that Ca
2+
-dependent nuclear accumulation of CaM can be reconstituted in permeabilized cells. Accumulation was blocked by M13, a CaM antagonist peptide, but did not require cytosolic factors or an ATP regenerating system. Ca
2+
-dependent influx of CaM into nuclei was not blocked by inhibitors of nuclear localization signal-mediated nuclear import in either permeabilized or intact cells. Fluorescence recovery after photobleaching studies of CaM in intact cells showed that influx is a first-order process with a rate constant similar to that of a freely diffusible control molecule (20-kDa dextran). Studies of CaM efflux from preloaded nuclei in permeablized cells revealed the existence of three classes of nuclear binding sites that are distinguished by their Ca
2+
-dependence and affinity. At high [Ca
2+
], efflux was enhanced by addition of a high affinity CaM-binding protein outside the nucleus. These data suggest that CaM diffuses freely through nuclear pores and that CaM-binding proteins in the nucleus act as a sink for Ca
2+
-CaM, resulting in accumulation of CaM in the nucleus on elevation of intracellular free Ca
2+
.
Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood–brain barrier and specifically reduce gene expression
Authors: Tyler Beth M. , Jansen Karen , McCormick Daniel J. , Douglas Christopher L. , Boules Mona , Stewart Jennifer A. , Zhao Lihong , Lacy Benjamin , Cusack Bernadette , Fauq Abdul , Richelson Elliott
Published: July 2002

Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood–brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i.p. delivery by crossing the normal blood–brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown
in vivo
to be a potentially effective therapeutic treatment.
Purification of the yeast U4/U6⋅U5 small nuclear ribonucleoprotein particle and identification of its proteins
Authors: Stevens Scott W. , Abelson John
Published: July 2002

The yeast U4/U6⋅U5 pre-mRNA splicing small nuclear ribonucleoprotein (snRNP) is a 25S small nuclear ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6⋅U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6⋅U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6⋅U5 snRNP from yeast and the powerful sequencing methodologies using small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.
MADS-box genes reveal that gnetophytes are more closely related to conifers than to flowering plants
Authors: Winter Kai-Uwe , Becker Annette , Münster Thomas , Kim Jan T. , Saedler Heinz , Theissen Günter
Published: July 2002

The evolutionary origin of the angiosperms (flowering plants
sensu stricto
) is still enigmatic. Answers to the question of angiosperm origins are intimately connected to the identification of their sister group among extinct and extant taxa. Most phylogenetic analyses based on morphological data agree that among the groups of extant seed plants, the gnetophytes are the sister group of the angiosperms. According to this view, angiosperms and gnetophytes are the only extant members of a clade called “anthophytes” to emphasize their shared possession of flower-like reproductive structures. However, most phylogeny reconstructions based on molecular data so far did not support an anthophyte clade, but also could not clarify the case because support for alternative groupings has been weak or controversial. We have isolated 13 different homologs of MADS-type floral homeotic genes from the gnetophyte
Gnetum gnemon
. Five of these genes fall into monophyletic gene clades also comprising putatively orthologous genes from flowering plants and conifers, among them orthologs of floral homeotic B and C function genes. Within these clades the
Gnetum
genes always form distinct subclades together with the respective conifer genes, to the exclusion of the angiosperm genes. This provides strong molecular evidence for a sister-group relationship between gnetophytes and conifers, which is in contradiction to widely accepted interpretations of morphological data for almost a century. Our phylogeny reconstructions and the outcome of expression studies suggest that complex features such as flower-like reproductive structures and double-fertilization arose independently in gnetophytes and angiosperms.
Structural basis of chaperone self-capping in P pilus biogenesis
Authors: Hung Danielle L. , Pinkner Jerome S. , Knight Stefan D. , Hultgren Scott J.
Published: July 2002

PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of
Escherichia coli
. It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm. We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface. Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2–D2 and F1–G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface. An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits.
Expression cloning of a human α1,4-N-acetylglucosaminyltransferase that forms GlcNAcα1→4Galβ→R, a glycan specifically expressed in the gastric gland mucous cell-type mucin
Authors: Nakayama Jun , Yeh Jiunn-Chern , Misra Anup K. , Ito Susumu , Katsuyama Tsutomu , Fukuda Minoru
Published: July 2002

Among mucus-secreting cells, the gastric gland mucous cells, Brunner’s glands, accessory glands of pancreaticobiliary tract, and pancreatic ducts exhibiting gastric metaplasia are unique in that they express class III mucin identified by paradoxical Con A staining composed of periodate oxidation, sodium borohydride reduction, Con A, and horseradish peroxidase reaction. Recently it was shown that these mucous cells secrete glycoproteins having GlcNAcα1→4Galβ→R at nonreducing terminals of the carbohydrate moieties. Herein we describe the expression cloning of a cDNA encoding a human α1,4-N-acetylglucosaminyltransferase (α4GnT), a key enzyme for the formation of GlcNAcα1→4Galβ1→R. COS-1 cells were thus cotransfected with a stomach cDNA library and a leukosialin cDNA. Transfected COS-1 cells were screened by using monoclonal antibodies specific for GlcNAcα1→4Galβ→R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAcα1→4Galβ→R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The α4GnT gene is located at chromosome 3p14.3, and its transcripts are expressed in the stomach and pancreas. Anin vitroGlcNAc transferase assay by using a soluble α4GnT revealed that α1,4-linked GlcNAc residues are transferred most efficiently to core 2 branchedO-glycans (Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNAc), forming GlcNAcα1→4Galβ1→4GlcNAcβ1→6(GlcNAcα1→4Galβ1→3)GalNAc. Transfection of α4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that α4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the α4GnT is a glycosyltransferase that forms α1,4-linked GlcNAc residues, preferentially inO-glycans.
The
Xanthomonas
Hrp type III system secretes proteins from plant and mammalian bacterial pathogens
Authors: Rossier Ombeline , Wengelnik Kai , Hahn Karoline , Bonas Ulla
Published: July 2002

Studies of essential pathogenicity determinants in Gram-negative bacteria have revealed the conservation of type III protein secretion systems that allow delivery of virulence factors into host cells from plant and animal pathogens. Ten of 21 Hrp proteins of the plant pathogen
Xanthomonas campestris
pv.
vesicatoria
have been suggested to be part of a type III machinery. Here, we report the
hrp
-dependent secretion of two avirulence proteins, AvrBs3 and AvrRxv, by
X. campestris
pv.
vesicatoria
strains that constitutively express
hrp
genes. Secretion occurred without leakage of a cytoplasmic marker in minimal medium containing BSA, at pH 5.4. Secretion was strictly
hrp
-dependent because a mutant carrying a deletion in
hrcV
, a conserved
hrp
gene, did not secrete AvrBs3 and AvrRxv. Moreover, the Hrp system of
X. campestris
pv.
vesicatoria
was able to secrete proteins from two other plant pathogens: PopA, a protein secreted via the Hrp system in
Ralstonia solanacearum
, and AvrB, an avirulence protein from
Pseudomonas syringae
pv.
glycinea
. Interestingly,
X. campestris
pv.
vesicatoria
also secreted YopE, a type III-secreted cytotoxin of the mammalian pathogen
Yersinia pseudotuberculosis
in a
hrp
-dependent manner. YerA, a YopE-specific chaperone, was required for YopE stability but not for secretion in
X. campestris
pv.
vesicatoria
. Our results demonstrate the functional conservation of the type III system of
X. campestris
for secretion of proteins from both plant and mammalian pathogens and imply recognition of their respective secretion signals.
Thrombospondin-2: A potent endogenous inhibitor of tumor growth and angiogenesis
Authors: Streit Michael , Riccardi Lucia , Velasco Paula , Brown Lawrence F. , Hawighorst Thomas , Bornstein Paul , Detmar Michael
Published: July 2002

Recent evidence suggests a potential role for thrombospondin-2 (TSP-2), a matricellular glycoprotein, in the regulation of primary angiogenesis. To directly examine the biological effect of TSP-2 expression on tumor growth and angiogenesis, human A431 squamous cell carcinoma cells, which do not express TSP-2, were stably transfected with a murine TSP-2 expression vector or with vector alone. A431 cells expressing TSP-2 did not show an altered growth rate, colony-forming ability, or susceptibility to induction of apoptosis
in vitro.
However, injection of TSP-2-transfected clones into the dermis of nude mice resulted in pronounced inhibition of tumor growth that was significantly stronger than the inhibition observed in A431 clones stably transfected with a thrombospondin-1 (TSP-1) expression vector, and combined overexpression of TSP-1 and TSP-2 completely prevented tumor formation. Extensive areas of necrosis were observed in TSP-2-expressing tumors, and both the density and the size of tumor vessels were significantly reduced, although tumor cell expression of the major tumor angiogenesis factor, vascular endothelial growth factor, was maintained at high levels. These findings establish TSP-2 as a potent endogenous inhibitor of tumor growth and angiogenesis.
Identification of a cell protein (FIP-3) as a modulator of NF-κB activity and as a target of an adenovirus inhibitor of tumor necrosis factor α-induced apoptosis
Authors: Li Yongan , Kang Jian , Friedman Joshua , Tarassishin Leonid , Ye Jianjiang , Kovalenko Andrei , Wallach David , Horwitz Marshall S.
Published: July 2002

FIP-3 (14.7K interacting protein) was discovered during a search for cell proteins that could interact with an adenovirus protein (Ad E3–14.7K) that had been shown to prevent tumor necrosis factor (TNF)-α-induced cytolysis. FIP-3, which contains leucine zippers and a zinc finger domain, inhibits both basal and induced transcriptional activity of NF-κB and causes a late-appearing apoptosis with unique morphologic manifestations. Ad E3–14.7K can partially reverse apoptotic death induced by FIP-3. FIP-3 also was shown to bind to other cell proteins, RIP and NIK, which previously had been described as essential components of TNF-α-induced NF-κB activation. In addition, FIP-3 inhibited activation of NF-κB induced by TNF-α, the TNFR-1 receptor, RIP, NIK, and IKKβ, as well as basal levels of endogenous NF-κB in 293 cells. Because the activation of NF-κB has been shown to inhibit apoptosis, FIP-3 appears both to activate a cell-death pathway and to inhibit an NF-κB-dependent survival mechanism.
Interferon γ expressed by a recombinant respiratory syncytial virus attenuates virus replication in mice without compromising immunogenicity
Authors: Bukreyev Alexander , Whitehead Stephen S. , Bukreyeva Natalya , Murphy Brian R. , Collins Peter L.
Published: July 2002

Interferon γ (IFN-γ) has pleiotropic biological effects, including intrinsic antiviral activity as well as stimulation and regulation of immune responses. An infectious recombinant human respiratory syncytial virus (rRSV/mIFN-γ) was constructed that encodes murine (m) IFN-γ as a separate gene inserted into the G-F intergenic region. Cultured cells infected with rRSV/mIFN-γ secreted 22 μg mIFN-γ per 106cells. The replication of rRSV/mIFN-γ, but not that of a control chimeric rRSV containing the chloramphenicol acetyl transferase (CAT) gene as an additional gene, was 63- and 20-fold lower than that of wild-type (wt) RSV in the upper and lower respiratory tract, respectively, of mice. Thus, the attenuation of rRSV/mIFN-γin vivocould be attributed to the activity of mIFN-γ and not to the presence of the additional geneper se. The mice were completely resistant to subsequent challenge with wt RSV. Despite its growth restriction, infection of mice with rRSV/mIFN-γ induced a level of RSV-specific antibodies that, on day 56, was comparable to or greater than that induced by infection with wt RSV. Mice infected with rRSV/mIFN-γ developed a high level of IFN-γ mRNA and an increased amount of interleukin 12 p40 mRNA in their lungs, whereas other cytokine mRNAs tested were unchanged compared with those induced by wt RSV. Because attenuation of RSV typically is accompanied by a reduction in immunogenicity, expression of IFN-γ by an rRSV represents a method of attenuation in which immunogenicity can be maintained rather than be reduced.
Ubiquitin-dependent degradation of IκBα is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1
Authors: Hatakeyama Shigetsugu , Kitagawa Masatoshi , Nakayama Keiko , Shirane Michiko , Matsumoto Masaki , Hattori Kimihiko , Higashi Hideaki , Nakano Hiroyasu , Okumura Ko , Onoé Kazunori , Good Robert A. , Nakayama Kei-ichi
Published: July 2002

Activation of the transcription factor nuclear factor kappa B (NF-κB) is controlled by proteolysis of its inhibitory subunit (IκB) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of IκBα by a large multisubunit complex containing IκB kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/βTrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with IκBα only when IκBα is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of IκBα in concert with IκB kinases, resulting in nuclear translocation of NF-κB. In addition, FWD1 strikingly evoked the ubiquitination of IκBα in the
in vitro
system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of IκBα. These results suggest that the substrate-specific degradation of IκBα is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated IκBα. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-κB through control of IκB protein stability.
Optical imaging of functional domains in the cortex of the awake and behaving monkey
Authors: Vnek Norbert , Ramsden Benjamin M. , Hung Chou P. , Goldman-Rakic Patricia S. , Roe Anna Wang
Published: July 2002

As demonstrated by anatomical and physiological studies, the cerebral cortex consists of groups of cortical modules, each comprising populations of neurons with similar functional properties. This functional modularity exists in both sensory and association neocortices. However, the role of such cortical modules in perceptual and cognitive behavior is unknown. To aid in the examination of this issue we have applied the high spatial resolution optical imaging methodology to the study of awake, behaving animals. In this paper, we report the optical imaging of orientation domains and blob structures, approximately 100–200 μm in size, in visual cortex of the awake and behaving monkey. By overcoming the spatial limitations of other existing imaging methods, optical imaging will permit the study of a wide variety of cortical functions at the columnar level, including motor and cognitive functions traditionally studied with positron-emission tomography or functional MRI techniques.
The baculovirus antiapoptotic p35 gene also functions via an oxidant-dependent pathway
Authors: Sah Nand K. , Taneja Tarvinder K. , Pathak Niteen , Begum Rasheedunnisa , Athar Mohammad , Hasnain Seyed E.
Published: July 2002

Cellular imbalance in the levels of antioxidants and reactive oxygen species resulting in apoptosis is directly associated with a number of parasitic infections, aging, and several genetic and multifactorial diseases. The baculovirusAcNPV-encoded antiapoptoticp35gene prevents apoptosis induced by a variety of apoptotic agents in different systems. We demonstrate the ability of thep35gene to inhibit oxidative stress-induced apoptosis.In vitroculturedSpodoptera frugiperda (Sf9) insect cells infected with wild-typeAcNPV carrying the antiapoptoticp35gene did not undergo apoptosis when subjected to oxidative stress generated by the exogenous application of oxidants orin vivogeneration of reactive oxygen species or on direct exposure of cells to UV radiations. AnAcNPV mutant carrying a deletion of thep35gene failed to arrest cell death. Transfection of cells with a recombinant plasmid containing thep35gene under the transcriptional control of a stress promoter (Drosophila hsp70) was also able to rescue cells from oxidative stress-induced cell death, demonstrating the direct involvement of P35. ESR spin-trapping studies conductedin vitroandin vivodemonstrated that P35 functions directly as an antioxidant by mopping out free radicals and consequently prevents cell death by acting at an upstream step in the reactive oxygen species-mediated cell death pathway.