bluuebunny/crossref_metadata_2025 huggingface dataset:
Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.
Authors: Verbruggen N , Hua X J , May M , Van Montagu M
Published: July 2002

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.
Structure and function in rhodopsin: high level expression of a synthetic bovine opsin gene and its mutants in stable mammalian cell lines.
Authors: Reeves P J , Thurmond R L , Khorana H G
Published: July 2002

Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.
Function of RNA secondary structures in transcriptional attenuation of the
Bacillus subtilis pyr
operon
Authors: Lu Yang , Turner Robert J. , Switzer Robert L.
Published: July 2002

The
Bacillus subtilis pyr
operon is regulated
by exogenous pyrimidines by a transcriptional attenuation mechanism.
Transcription
in vitro
from
pyr
DNA
templates specifying attenuation regions yielded terminated and
read-through transcripts of the expected lengths. Addition of the PyrR
regulatory protein plus UMP led to greatly increased termination.
Synthetic antisense deoxyoligonucleotides were used to probe possible
secondary structures in the
pyr
mRNA that were proposed
to play roles in controlling attenuation. Oligonucleotides
predicted to disrupt terminator structures suppressed termination,
whereas oligonucleotides predicted to disrupt the stem of
antiterminator stem-loops strongly promoted termination at the usual
termination site. Oligonucleotides that disrupt a previously
unrecognized stem-loop structure, called the anti-antiterminator, the
formation of which interferes with formation of the downstream
antiterminator, suppressed termination. We propose that transcriptional
attenuation of the
pyr
operon is governed by switching
between alternative antiterminator versus anti-antiterminator plus
terminator structures, and that PyrR acts by UMP-dependent binding to
and stabilization of the anti-antiterminator.
A compositional map of human chromosome band Xq28.
Authors: De Sario A , Geigl E M , Palmieri G , D'Urso M , Bernardi G
Published: July 2002

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.
Transcription termination factor La is also an initiation factor for RNA polymerase III.
Authors: Maraia R J
Published: July 2002

La RNA-binding protein is a transcription termination factor that facilitates recycling of template and RNA polymerase (pol) 111. Transcription complexes preassembled on immobilized templates were depleted of pol III after a single round of RNA synthesis in the presence of heparin and sarkosyl. The isolated complexes could then be complemented with highly purified pol III and/or recombinant La to test if La is required for transcription reinitiation. VA1, 7SL, and B1 transcription complexes cannot be transcribed by supplemental pol III in single or multiple-round transcription assays unless La is also provided. La mediates concentration-dependent activation of pol III initiation and thereby controls the use of preassembled stable transcription complexes. The initiation factor activity of La augments its termination factor activity to produce a novel mechanism of activated reinitiation. A model in which La serves pol III upon transcription initiation and again at termination is discussed.
The choroid plexus epithelium is the site of the organic anion transport protein in the brain
Authors: Angeletti Ruth Hogue , Novikoff Phyllis M. , Juvvadi Shailaja Rao , Fritschy Jean-Marc , Meier Peter J. , Wolkoff Allan W.
Published: July 2002

The mRNA for organic anion transport protein (oatp) was previously shown to be present in abundance in liver and kidney, and in small amounts in brain. Data obtained from experiments with reverse transcriptase–PCR techniques and
in situ
hybridization analysis showed that the oatp mRNA is present within the brain, localized to the choroid plexus. A sequence-specific antibody to the oatp polypeptide demonstrated the presence of the expected polypeptide with a molecular weight of 80,000 plus an immunoreactive species with a higher molecular weight in preparations of choroid plexus membranes. Examination of the choroid plexus by fluorescence confocal microscopy revealed that immunoreactive oatp polypeptide is localized to the apical surface of the choroid plexus epithelial cells, which contacts the cerebrospinal fluid. This localization of oatp is consistent with previous experiments showing vectorial transport of organic anions between the choroid plexus and the cerebrospinal fluid.
Trigger factor is induced upon cold shock and enhances viability of
Escherichia coli
at low temperatures
Authors: Kandror Olga , Goldberg Alfred L.
Published: July 2002

Trigger factor (TF) in
Escherichia coli
is a molecular chaperone with remarkable properties: it has prolyl-isomerase activity, associates with nascent polypeptides on ribosomes, binds to GroEL, enhances GroEL’s affinity for unfolded proteins, and promotes degradation of certain polypeptides. Because the latter effects appeared larger at 20°C, we studied the influence of temperature on TF expression. Unlike most chaperones (e.g., GroEL), which are heat-shock proteins (hsps), TF levels increased progressively as growth temperature decreased from 42°C to 16°C and even rose in cells stored at 4°C. Upon temperature downshift from 37°C to 10°C or exposure to chloramphenicol, TF synthesis was induced, like that of many cold-shock proteins. We therefore tested if TF expression might be important for viability at low temperatures. When stored at 4°C,
E. coli
lose viability at exponential rates. Cells with reduced TF content die faster, while cells overexpressing TF showed greater viability. Although TF overproduction protected against cold, it reduced viability at 50°C, while TF deficiency enhanced viability at this temperature. By contrast, overproduction of GroEL/ES, or hsps generally, while protective against high temperatures, reduced viability at 4°C, which may explain why expression of hsps is suppressed in the cold. Thus, TF represents an example of an
E. coli
protein which protects cells against low temperatures. Moreover, the differential induction of TF at low temperatures and hsps at high temperatures appears to provide selective protection against these opposite thermal extremes.
Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function
Authors: Westfall Margaret V. , Rust Elizabeth M. , Metzger Joseph M.
Published: July 2002

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca
2+
-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca
2+
relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca
2+
activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca
2+
-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca
2+
activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.
Construction of a catalytically active iron superoxide dismutase by rational protein design
Authors: Pinto Ann L. , Hellinga Homme W. , Caradonna John P.
Published: July 2002

The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein,
Escherichia coli
thioredoxin (Trx), a protein that does not naturally contain a transition metal-binding site. Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site. The electronic spectra of the holoprotein and its N
3
−
and F
−
adducts are analogous to those previously reported for native {Fe
3+
}SOD. Activity assays showed that {Fe
3+
}Trx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type
E. coli
iron SOD indicated that {Fe
3+
}Trx-SOD catalyzes the dismutation reaction at a rate on the order of 10
5
M
−1
s
−1
. The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.
Gene replacement by homologous recombination in the multicellular green alga
Volvox carteri
Authors: Hallmann Armin , Rappel Annette , Sumper Manfred
Published: July 2002

With only two different cell types, the haploid green alga
Volvox
represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of
Volvox
reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in
Volvox
. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (
nit
A) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in
Volvox
to apply the powerful tool of gene disruption for functional studies of novel genes.
Functional and nonfunctional mutations distinguished by random recombination of homologous genes
Authors: Zhao Huimin , Arnold Frances H.
Published: July 2002

We describe a convenient method for distinguishing functional from nonfunctional or deleterious mutations in homologous genes. High fidelity
in vitro
gene recombination (“DNA shuffling”) coupled with sequence analysis of a small sampling of the shuffled library exhibiting the evolved behavior allows identification of those mutations responsible for the behavior in a background of neutral and deleterious mutations. Functional mutations are expected to occur in 100% of the sequenced screened sample; neutral mutations are found in 50% on average, and deleterious mutations do not appear at all. When used to analyze 10 mutations in a laboratory-evolved gene encoding a thermostable subtilisin E, this method rapidly identified the two responsible for the observed protease thermostability; the remaining eight were neutral with respect to thermostability, within the precision of the screening assay. A similar approach, coupled with selection for growth and survival of the host organism, could be used to distinguish adaptive from neutral mutations.
Temporal variation, dormancy, and coexistence: A field test of the storage effect
Authors: Cáceres Carla E.
Published: July 2002

Theoretical models suggest that overlapping generations, in combination with a temporally fluctuating environment, may allow the persistence of competitors that otherwise would not coexist. Despite extensive theoretical development, this “storage effect” hypothesis has received little empirical attention. Herein I present the first explicit mathematical analysis of the contribution of the storage effect to the dynamics of competing natural populations. In Oneida Lake, NY, data collected over the past 30 years show a striking negative correlation between the water-column densities of two species of suspension-feeding zooplankton,Daphnia galeata mendotaeandDaphnia pulicaria. I have demonstrated competition between these two species and have shown that both possess long-lived eggs that establish overlapping generations. Moreover, recruitment to this long-lived stage varies annually, so that both daphnids have years in which they are favored (for recruitment) relative to their competitor. When the long-term population growth rates are calculated both with and without the effects of a variable environment, I show thatD. galeata mendotaeclearly cannot persist without the environmental variation and prolonged dormancy (i.e., storage effect) whereasD. pulicariapersists through consistently high per capita recruitment to the long-lived stage.
Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system
Authors: Coen Laurent , Osta Rosario , Maury Martine , Brûlet Philippe
Published: July 2002

The nontoxic proteolytic C fragment of tetanus toxin (TTC peptide) has the same ability to bind nerve cells and be retrogradely transported through a synapse as the native toxin. We have investigated its potential use as an
in vivo
neurotropic carrier. In this work we show that a hybrid protein encoded by the
lacZ
–TTC gene fusion retains the biological functions of both proteins
in vivo
—i.e., retrograde transynaptic transport of the TTC fragment and β-galactosidase enzymatic activity. After intramuscular injection, enzymatic activity could be detected in motoneurons and connected neurons of the brainstem areas. This strategy could be used to deliver a biological activity to neurons from the periphery to the central nervous system. Such a hybrid protein could also be used to map synaptic connections between neural cells.
Anti-idiotype RNA selected with an anti-nuclear export signal antibody is actively transported in oocytes and inhibits Rev- and cap-dependent RNA export
Authors: Hamm Jörg , Huber Jochen , Lührmann Reinhard
Published: July 2002

The anti-idiotype approach is based on the assumption that an antibody specific for a receptor-binding domain of a ligand could be structurally related to the receptor. Therefore, a structural mimic of a receptor-binding domain, selected with an anti-ligand antibody, might be a functional substrate for the receptor. This hypothesis was addressed here by generating antibodies recognizing the Rev-nuclear export signal (NES). A functional NES is required for active export, presumably by interacting directly or indirectly with the nuclear pore complex. Anti-NES antibodies were used to isolate RNA mimics of the NES peptide from combinatorial RNA libraries. The RNA-mimics are exported actively, block Rev-dependent export of a reporter RNA, and inhibit cap-dependent U1 snRNA export in
Xenopus
oocytes, properties previously reported for NES-peptide conjugates.
Identification of a thiamin-dependent synthase in
Escherichia coli
required for the formation of the 1-deoxy-
d
-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol
Authors: Sprenger Georg A. , Schörken Ulrich , Wiegert Thomas , Grolle Sigrid , de Graaf Albert A. , Taylor Sean V. , Begley Tadhg P. , Bringer-Meyer Stephanie , Sahm Hermann
Published: July 2002

In
Escherichia coli
, 1-deoxy-
d
-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996)
Chem. Commun
. 1187–1188]. Here we show that an open reading frame at 9 min on the chromosomal map of
E. coli
encodes an enzyme (
d
eoxy
x
ylulose-5-
p
hosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (
dxs
), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by
1
H and
31
P NMR spectroscopy. Although DXP synthase of
E. coli
shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.
Functional separation of pre-rRNA processing steps revealed by truncation of the U3 small nucleolar ribonucleoprotein component, Mpp10
Authors: Lee Sarah J. , Baserga Susan J.
Published: July 2002

The U3 small nucleolar ribonucleoprotein (snoRNP) is required for three cleavage events that generate the mature 18S rRNA from the pre-rRNA. In
Saccharomyces cerevisiae
, depletion of Mpp10, a U3 snoRNP-specific protein, halts 18S rRNA production and impairs cleavage at the three U3 snoRNP-dependent sites: A0, A1, and A2. We have identified truncation mutations of Mpp10 that affect 18S rRNA synthesis and confer cold-sensitivity and slow growth. However, distinct from yeast cells depleted of Mpp10, the mutants carrying these truncated Mpp10 proteins accumulate a novel precursor, resulting from cleavage at only A0. The Mpp10 truncations do not alter association of Mpp10 with the U3 snoRNA, nor do they affect snoRNA or protein stability. Thus, the role in processing of the U3 snoRNP can be separated into cleavage at the A0 site, which occurs in the presence of truncated Mpp10, and cleavage at the A1/A2 sites, which occurs only with intact Mpp10. These results strongly argue for a role for Mpp10 in processing at the A1/A2 sites.
Very short-term plasticity in hippocampal synapses
Authors: Dobrunz Lynn E. , Huang Emily P. , Stevens Charles F.
Published: July 2002

Hippocampal pyramidal neurons often fire in bursts of action potentials with short interspike intervals (2–10 msec). These high-frequency bursts may play a critical role in the functional behavior of hippocampal neurons, but synaptic plasticity at such short times has not been carefully studied. To study synaptic modulation at very short time intervals, we applied pairs of stimuli with interpulse intervals ranging from 7 to 50 msec to CA1 synapses isolated by the method of minimal stimulation in hippocampal slices. We have identified three components of short-term paired-pulse modulation, including (
i
) a form of synaptic depression manifested after a prior exocytotic event, (
ii
) a form of synaptic depression that does not depend on a prior exocytotic event and that we postulate is based on inactivation of presynaptic N-type Ca
2+
channels, and (
iii
) a dependence of paired-pulse facilitation on the exocytotic history of the synapse.
Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: Implications for CD40–CD40 ligand signaling in atherosclerosis
Authors: Mach François , Schönbeck Uwe , Sukhova Galina K. , Bourcier Todd , Bonnefoy Jean-Yves , Pober Jordan S. , Libby Peter
Published: July 2002

Increasing evidence supports involvement of inflammation and immunity in atherogenesis. We report here that CD40 ligand (CD40L), an immunoregulatory signaling molecule heretofore considered largely restricted to recently activated CD4+T lymphocytes, is expressed by human vascular endothelial cells (EC), smooth muscle cells (SMC), and human macrophagesin vitro, and is coexpressed with its receptor CD40 on all three cells types in human atherosclerotic lesionsin situ. Cultured human vascular EC, SMC, and human macrophages all constitutively expressed CD40L mRNA as well as protein. Stimulation with interleukin 1β, tumor necrosis factor α, or interferon γ increased surface levels andde novosynthesis of CD40L on all three cell types. CD40L expressed on EC, SMC, and macrophages exhibited biological activity, as it induced B7.2 expression on B cells. Human vascular SMC also constitutively expressed CD40, the receptor for CD40L, and through CD40 signaling, human recombinant CD40L induced expression of proinflammatory cytokines in these cells, identifying SMC as a target for CD40L. Human atherosclerotic lesions (n= 8) showed expression of immunoreactive CD40L on EC, SMC, and macrophages, while normal arterial tissues (n= 5) contained no CD40L. In atheroma CD40L+cells often also expressed CD40. These observations establish human vascular EC, SMC, and human macrophages as a novel source of CD40L, and point to T cell-independent CD40 signaling, and a broader function of this pathway in regulation of nonimmune cells, as illustrated here by potential autocrine and paracrine activation during atherogenesis.
Phospholipase A
2
-mediated activation of mitogen-activated protein kinase by angiotensin II
Authors: Dulin Nickolai O. , Alexander Larry D. , Harwalkar Subash , Falck John R. , Douglas Janice G.
Published: July 2002

In renal proximal tubule epithelial cells, a membrane-associated phospholipase A
2
(PLA
2
) is a major signaling pathway linked to angiotensin II (Ang II) type 2 receptor (AT
2
). The current studies were designed to test the hypothesis that membrane-associated PLA
2
-induced release of arachidonic acid (AA) and/or its metabolites may serve as an upstream mediator of Ang II-induced mitogen-activated protein kinase (MAPK) activation. Ang II stimulated transient dose-dependent phosphorylation of MAPK with a maximum at 1 μM (10 min). Inhibition of PLA
2
by mepacrine diminished both AA release and MAPK phosphorylation, induced by Ang II. Furthermore, AA itself induced time- and dose-dependent phosphorylation of MAPK, supporting the importance of PLA
2
as a mediator of Ang II signaling. The effects of both Ang II and AA on MAPK phosphorylation were protein kinase C independent and abolished by the inhibitor of cytochrome P450 isoenzyme, ketoconazole. Moreover, 5,6-epoxyeicosatrienoic acid and 14,15-epoxyeicosatrienoic acid, the cytochrome P450-dependent metabolites of AA, significantly stimulated MAPK activity in renal proximal tubule epithelial cells. These observations document a mechanism of Ang II-induced MAPK phosphorylation, mediated by PLA
2
-dependent release of AA and cytochrome P450-dependent production of epoxy derivatives of AA.
The m2 form of the
Helicobacter pylori
cytotoxin has cell type-specific vacuolating activity
Authors: Pagliaccia Cristina , de Bernard Marina , Lupetti Pietro , Ji Xuhuai , Burroni Daniela , Cover Timothy L. , Papini Emanuele , Rappuoli Rino , Telford John L. , Reyrat Jean-Marc
Published: July 2002

The
Helicobacter pylori
toxin VacA causes vacuolar degeneration in mammalian cell lines
in vitro
and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the
vacA
gene have been described, and the m2 cytotoxin always has been described as inactive in the
in vitro
HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.
Nociceptin/orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice
Authors: Inoue Makoto , Kobayashi Motomasa , Kozaki Shunji , Zimmer Andreas , Ueda Hiroshi
Published: July 2002

We have studied the
in vivo
signaling mechanisms involved in nociceptin/orphanin FQ (Noci)-induced pain responses by using a flexor-reflex paradigm. Noci was 10,000 times more potent than substance P (SP) in eliciting flexor responses after intraplantar injection into the hind limb of mice, but the action of Noci seems to be mediated by SP. Mice pretreated with an NK1 tachykinin receptor antagonist or capsaicin, or mice with a targeted disruption of the tachykinin 1 gene no longer respond to Noci. The action of Noci appears to be mediated by the Noci receptor, a pertussis toxin-sensitive G protein–coupled receptor that stimulates inositol trisphosphate receptor and Ca
2+
influx. These findings suggest that Noci indirectly stimulates nerve endings of nociceptive primary afferent neurons through a local SP release.
Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway
Authors: Solomon Vered , Baracos Vickie , Sarraf Pasha , Goldberg Alfred L.
Published: July 2002

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin–proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of
125
I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of
125
I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T
3
) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3α, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3α-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover,
125
I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.
A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells
Authors: Garabedian Emily M. , Humphrey Peter A. , Gordon Jeffrey I.
Published: July 2002

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.
The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO
2
permeability of the red blood cell membrane
Authors: Forster R. E. , Gros G. , Lin L. , Ono Y. , Wunder M.
Published: July 2002

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of
12
C
18
O
16
O from a red cell suspension as the
18
O exchanges between labeled CO
2
+ HCO
3
−
and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or
A
) inside the cell and of the membrane self-exchange permeability to HCO
3
−
(
P

m,HCO
−

3

). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO
3
−
/Cl
−
transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced
P

m,HCO
−

3

as expected, but also appeared to reduce intracellular
A
, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in
P

m,CO
2

would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO
2
and HCO
3
−
, we found that DIDS inhibited both
P

m,HCO
−

3

and
P

m,CO
2

, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO
2
permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO
2
exchange.
Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals
Authors: Ranga Udaykumar , Woffendin Clive , Verma Sunita , Xu Ling , June Carl H. , Bishop D. Keith , Nabel Gary J.
Published: July 2002

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4
+
cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with ≈3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4
+
cells in HIV infection.
Biochemical evolution. I. Polymerization on internal, organophilic silica surfaces of dealuminated zeolites and feldspars
Authors: Smith Joseph V.
Published: July 2002

Catalysis at mineral surfaces might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and photochemical gas reactions. Many ideas are implausible in detail because the proposed mineral surfaces strongly prefer water and other ionic species to organic ones. The molecular sieve silicalite (Union Carbide; = Al-free Mobil ZSM-5 zeolite) has a three-dimensional, 10-ring channel system whose electrically neutral Si-O surface strongly adsorbs organic species over water. Three -O-Si tetrahedral bonds lie in the surface, and the fourth Si-O points inwards. In contrast, the outward Si-OH of simple quartz and feldspar crystals generates their ionic organophobicity. The ZSM-5-type zeolite mutinaite occurs in Antarctica with boggsite and tschernichite (Al-analog of Mobil Beta). Archean mutinaite might have become de-aluminated toward silicalite during hot/cold/wet/dry cycles. Catalytic activity of silicalite increases linearly with Al-OH substitution for Si, and Al atoms tend to avoid each other. Adjacent organophilic and catalytic Al-OH regions in nanometer channels might have scavenged organic species for catalytic assembly into specific polymers protected from prompt photochemical destruction. Polymer migration along weathered silicic surfaces of micrometer-wide channels of feldspars might have led to assembly of replicating catalytic biomolecules and perhaps primitive cellular organisms. Silica-rich volcanic glasses should have been abundant on the early Earth, ready for crystallization into zeolites and feldspars, as in present continental basins. Abundant chert from weakly metamorphosed Archaean rocks might retain microscopic clues to the proposed mineral adsorbent/catalysts. Other framework silicas are possible, including ones with laevo/dextro one-dimensional channels. Organic molecules, transition-metal ions, and P occur inside modern feldspars.
Tight clustering and hemizygosity of apomixis-linked molecular markers in
Pennisetum squamulatum
implies genetic control of apospory by a divergent locus that may have no allelic form in sexual genotypes
Authors: Ozias-Akins Peggy , Roche Dominique , Hanna Wayne W.
Published: July 2002

Apomixis is a naturally occurring mode of reproduction that results in embryo formation without the involvement of meiosis or fertilization of the egg. Seed-derived progeny of an apomictic plant are genetically identical to the maternal parent. We are studying a form of apomixis called apospory that occurs in the genus
Pennisetum
, a taxon in the grass family. A cultivated member of this genus, pearl millet (
Pennisetum glaucum
), reproduces sexually. A wild relative of pearl millet,
Pennisetum squamulatum
, that is an obligate aposporous species, is cross-compatible with pearl millet when used as a pollen donor in the interspecific cross. We present herein the genetic mapping of 13 molecular markers in an interspecific hybrid population of 397 individuals that segregates for apomixis and sexuality. Surprisingly, 12 of the 13 markers strictly cosegregated with aposporous embryo sac development, clearly defining a contiguous apospory-specific genomic region in which no genetic recombination was detected. Lack of or suppression of recombination may be coincidentally associated with the chromosomal context of the apomixis locus or it may be a consequence of its evolution that is essential for preservation of gene function as has been previously shown in studies of complex loci in both plant and animal species.
How vertebrate and invertebrate visual pigments differ in their mechanism of photoactivation
Authors: Nakagawa Masashi , Iwasa Tatsuo , Kikkawa Satoshi , Tsuda Motoyuki , Ebrey Thomas G.
Published: July 2002

In vertebrate visual pigments, a glutamic acid serves as a negative counterion to the positively charged chromophore, a protonated Schiff base of retinal. When photoisomerization leads to the Schiff base deprotonating, the anionic glutamic acid becomes protonated, forming a neutral species that activates the visual cascade. We show that in octopus rhodopsin, the glutamic acid has no anionic counterpart. Thus, the “counterion” is already neutral, so no protonated form of an initially anionic group needs to be created to activate. This helps to explain another observation—that the active photoproduct of octopus rhodopsin can be formed without its Schiff base deprotonating. In this sense, the mechanism of light activation of octopus rhodopsin is simpler than for vertebrates, because it eliminates one of the steps required for vertebrate rhodopsins to achieve their activating state.
Stretch-activated single K
+
channels account for whole-cell currents elicited by swelling
Authors: Vanoye Carlos G. , Reuss Luis
Published: July 2002

Functionally significant stretch-activated ion channels have been clearly identified in excitable cells. Although single-channel studies suggest their expression in other cell types, their activity in the whole-cell configuration has not been shown. This discrepancy makes their physiological significance doubtful and suggests that their mechanical activation is artifactual. Possible roles for these molecules in nonexcitable cells are acute cell-volume regulation and, in epithelial cells, the complex adjustment of ion fluxes across individual cell membranes when the rate of transepithelial transport changes. We report the results of experiments on isolated epithelial cells expressing in the basolateral membrane stretch-activated K
+
channels demonstrable by the cell-attached patch-clamp technique. In these cells, reversible whole-cell currents were elicited by both isosmotic and hyposmotic cell swelling. Cation selectivity and block by inorganic agents were the same for single-channel and whole-cell currents, indicating that the same entity underlies single-channel and whole-cell currents and that the single-channel events are not artifactual. In these cells, when the rate of apical-membrane NaCl entry increases, the cell Na
+
content and volume also increase, stimulating the Na
+
,K
+
-ATPase at the basolateral membrane, i.e., both Na
+
extrusion and K
+
uptake increase. We speculate that, under these conditions, the parallel activation of basolateral K
+
channels (by the swelling) elevates conductive K
+
loss, tending to maintain the cell K
+
content constant (“pump-leak parallelism”). This study describes a physiologically relevant stretch-activated channel, at both the single-channel and whole-cell levels, in a nonneural cell type.
Unexpected abundance of self-splicing introns in the genome of bacteriophage Twort: Introns in multiple genes, a single gene with three introns, and exon skipping by group I ribozymes
Authors: Landthaler Markus , Shub David A.
Published: July 2002

Analysis of RNA that can be labeled with GTP indicates the existence of group I introns in genes of at least three transcriptional classes in the genome of
Staphylococcus aureus
bacteriophage Twort. A single ORF of 142 amino acids (Orf142) is interrupted by three self-splicing group I introns, providing the first example of a phage gene with multiple intron insertions. Twort Orf142 is encoded in a message that is abundant 15–20 min after infection and is highly similar to a late gene product (Orf8) of the morphologically related
Listeria
phage A511. The introns in
orf142
are spliced
in vivo
and contain all the conserved features of primary sequence and secondary structure of group I introns in subgroup IA2, which includes the introns in
Escherichia coli
phage T4 and the
Bacillus
phages β22 and SPO1. Introns I2 and I3 in
orf142
are highly similar, and their intron insertion sites are closely spaced. The presence of transcripts with a skipped exon between these introns indicates that they may fold into a single active ribozyme resulting in alternative splicing. Alternatively, the cleaved 5′ exon preceding I2 may undergo
trans
splicing to the 3′ exon that follows I3. Regardless of the detailed mechanism, these results demonstrate a new means whereby a single gene can give rise to multiple messenger RNAs.
Cancer-specific chromosome alterations in the constitutive fragile region
FRA3
B
Authors: Mimori Koshi , Druck Teresa , Inoue Hiroshi , Alder Hansjuerg , Berk Lori , Mori Masaki , Huebner Kay , Croce Carlo M.
Published: July 2002

We have sequenced 870 kilobases of the
FHIT
/
FRA3B
locus, from
FHIT
intron 3 to intron 7. The locus is AT rich (61.5%) and Alu poor (6.2%), and it apparently does not harbor other genes. In a detailed analysis of the 308-kilobase region between
FHIT
exon 5 and the telomeric end of intron 3, a region known to encompass a human papillomavirus-16 integration site and two clusters of aphidicolin-induced chromosome 3p14.2 breakpoints, we have precisely mapped 10 deletion and translocation endpoints in cancer-derived cell lines relative to positions of specific repetitive elements, regions of high genome flexibility and aphidicolin-induced breakpoints. Conclusions are (
i
) that aphidicolin-induced breakpoint clusters fall close to high-flexibility sequences, suggesting that these sequences contribute directly to aphidicolin-induced fragility; (
ii
) that 9 of the 10
FHIT
allelic deletions in cancer cell lines resulted in loss of exons, with 7 deletion endpoints near long interspersed nuclear elements or long terminal repeat elements; and (
iii
) that cancer-specific deletions encompass multiple high-flexibility genomic regions, suggesting that fragile breaks may occur at these regions, whereas repair of the breaks involves homologous pairing of flanking sequences with concomitant deletion of the damaged fragile sequence.
Kinase interaction domain of kinase-associated protein phosphatase, a phosphoprotein-binding domain
Authors: Li Jia , Smith George P. , Walker John C.
Published: July 2002

Kinase-associated protein phosphatase interacts specifically with plant receptor-like protein kinases. This interaction is thought to be a key step in signal perception and transduction. The minimal kinase interaction (KI) domain of kinase-associated protein phosphatase was mapped to a 119-aa segment spanning residues 180 to 298. A forkhead-associated (FHA) homology region resides in this minimal KI domain. Site-directed mutagenesis of four highly conserved sites in this FHA homology region abolishes the KI domain’s interaction with receptor-like protein kinases, indicating that the FHA region is essential for binding. Serial deletion analysis indicates that 30 aa on each side of the FHA region are also needed for binding; this minimal functional unit is designated as the KI domain. Kinetic studies using surface plasmon resonance indicate that the binding between the KI domain and receptor-like protein kinases has a dissociation constant (
K
D
) of about 25–100 nM, which is similar to the binding affinity of two other well characterized phosphorylation-dependent protein-binding domains (14-3-3 and Src homology 2) and their high-affinity phosphopeptide ligands.
Identification and characterization of a negative regulator of FtsZ ring formation in
Bacillus subtilis
Authors: Levin Petra Anne , Kurtser Iren G. , Grossman Alan D.
Published: July 2002

During the bacterial cell cycle, the tubulin-like cell-division protein FtsZ polymerizes into a ring structure that establishes the location of the nascent division site. We have identified a regulator of FtsZ ring formation in
Bacillus subtilis
. This protein, EzrA, modulates the frequency and position of FtsZ ring formation. The loss of
ezrA
resulted in cells with multiple FtsZ rings located at polar as well as medial sites. Moreover, the critical concentration of FtsZ required for ring formation was lower in
ezrA
null mutants than in wild-type cells. EzrA was associated with the cell membrane and also colocalized with FtsZ to the nascent septal site. We propose that EzrA interacts either with FtsZ or with one of its binding partners to promote depolymerization.