bluuebunny/crossref_metadata_2025 huggingface dataset:
Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress
Authors: Jenck Francois , Moreau Jean-Luc , Martin James R. , Kilpatrick Gavin J. , Reinscheid Rainer K. , Monsma Frederick J. , Nothacker Hans-Peter , Civelli Olivier
Published: July 2002

Orphanin FQ (OFQ, Nociceptin) is a recently discovered 17-amino acid neuropeptide that is structurally related to the opioid peptides but does not bind opioid receptors. OFQ has been proposed to act as an anti-opioid peptide, but its widespread sites of action in the brain suggest that it may have more general functions. Here we show that OFQ plays an important role in higher brain functions because it can act as an anxiolytic to attenuate the behavioral inhibition of animals acutely exposed to stressful/anxiogenic environmental conditions. OFQ anxiolytic-like effects were consistent across several behavioral paradigms generating different types of anxiety states in animals (light-dark preference, elevated plus-maze, exploratory behavior of an unfamiliar environment, pharmacological anxiogenesis, operant conflict) and were observed at low nonsedating doses (0.1–3 nmol, intracerebroventricular). Like conventional anxiolytics, OFQ interfered with regular sensorimotor function at high doses (>3 nmol). Our results show that an important role of OFQ is to act as an endogenous regulator of acute anxiety responses. OFQ, probably in concert with other major neuropeptides, exerts a modulatory role on the central integration of stressful stimuli and, thereby, may modulate anxiety states generated by acute stress.
Wheat grain hardness results from highly conserved mutations in the friabilin components puroindoline a and b
Authors: Giroux Michael J. , Morris Craig F.
Published: July 2002

“Soft” and “hard” are the two main market classes of wheat (
Triticum aestivum
L.) and are distinguished by expression of the
Hardness
gene. Friabilin, a marker protein for grain softness (
Ha
), consists of two proteins, puroindoline a and b (pinA and pinB, respectively). We previously demonstrated that a glycine to serine mutation in pinB is linked inseparably to grain hardness. Here, we report that the pinB serine mutation is present in 9 of 13 additional randomly selected hard wheats and in none of 10 soft wheats. The four exceptional hard wheats not containing the serine mutation in pinB express no pinA, the remaining component of the marker protein friabilin. The absence of pinA protein was linked inseparably to grain hardness among 44 near-isogenic lines created between the soft variety Heron and the hard variety Falcon. Both pinA and pinB apparently are required for the expression of grain softness. The absence of pinA protein and transcript and a glycine-to-serine mutation in pinB are two highly conserved mutations associated with grain hardness, and these friabilin genes are the suggested tightly linked components of the
Hardness
gene. A previously described grain hardness related gene termed “
GSP-1
” (grain softness protein) is not controlled by chromosome 5D and is apparently not involved in grain hardness. The association of grain hardness with mutations in both pinA or pinB indicates that these two proteins alone may function together to effect grain softness. Elucidation of the molecular basis for grain hardness opens the way to understanding and eventually manipulating this wheat endosperm property.
The incompatible interaction betweenPhytophthora parasiticavar.nicotianaerace 0 and tobacco is suppressed in transgenic plants expressing antisense lipoxygenase sequences
Authors: Rancé Iann , Fournier Joëlle , Esquerré-Tugayé Marie-Thérèse
Published: July 2002

Nicotiana tabacum46-8 cultivar displays an incompatible interaction with race 0 ofPhytophthora parasiticavar.nicotianae(Ppn), a fungal pathogen of most tobacco cultivars. At the plant level, incompatibility is characterized by the induction of lipoxygenase (LOX, EC = 1.13.11.12) activity and localized hypersensitive cell death before defense gene activation. To evaluate the involvement of LOX in the onset of plant defense, tobacco 46-8 plants were genetically engineered using full-length or partial-length antisense (AS) tobacco LOX cDNA constructs. AS expression strongly reduced elicitor- and pathogen-induced LOX activity. Eight independent AS-LOX lines were selected and assayed for their response toPpn. After root or stem inoculation with race 0, all AS-LOX lines but one displayed a compatible phenotype whereas control transformed plants, not containing the AS-LOX cassette, showed the typical incompatible reaction. The presence of the fungus in transgenic lines was demonstrated by PCR amplification of aPpn-specific genomic sequence. A linear relationship was found between the extent of LOX suppression and the size of the lesion caused by the fungus. The AS-LOX plants also showed enhanced susceptibility toward the compatible fungusRhizoctonia solani. The results demonstrate the strong involvement of LOX in the establishment of incompatibility in plant–microorganism interactions, consistent with its role in the defense of host plants.
Isolation of a myoglobin molten globule by selective cobalt(III)-induced unfolding
Authors: Blum Ofer , Haiek Abed , Cwikel Dory , Dori Zvi , Meade Thomas J. , Gray Harry B.
Published: July 2002

Reaction of the Schiff-base complex [Co(acetylacetonate-ethylenediimine)(NH
3
)
2
]
+
with metmyoglobin at pH 6.5 yields a partially folded protein containing six Co(III) complexes. Although half of its α-helical secondary structure is retained, absorption and CD spectra indicate that the tertiary structure in both B-F and AGH domains is disrupted in the partially folded protein. In analogy to proton-induced unfolding, it is likely that the loss of tertiary structure is triggered by metal-ion binding to histidines. Cobalt(III)-induced unfolding of myoglobin is unique in its selectivity (other proteins are unaffected) and in allowing the isolation of the partially folded macromolecule (the protein does not refold or aggregate upon removal of free denaturant).
Molecular determinants of bacterial adhesion monitored by atomic force microscopy
Authors: Razatos Anneta , Ong Yea-Ling , Sharma Mukul M. , Georgiou George
Published: July 2002

Bacterial adhesion and the subsequent formation of biofilm are major concerns in biotechnology and medicine. The initial step in bacterial adhesion is the interaction of cells with a surface, a process governed by long-range forces, primarily van der Waals and electrostatic interactions. The precise manner in which the force of interaction is affected by cell surface components and by the physiochemical properties of materials is not well understood. Here, we show that atomic force microscopy can be used to analyze the initial events in bacterial adhesion with unprecedented resolution. Interactions between the cantilever tip and confluent monolayers of isogenic strains of
Escherichia coli
mutants exhibiting subtle differences in cell surface composition were measured. It was shown that the adhesion force is affected by the length of core lipopolysaccharide molecules on the
E. coli
cell surface and by the production of the capsular polysaccharide, colanic acid. Furthermore, by modifying the atomic force microscope tip we developed a method for determining whether bacteria are attracted or repelled by virtually any biomaterial of interest. This information will be critical for the design of materials that are resistant to bacterial adhesion.
Genetic code origins: tRNAs older than their synthetases?
Authors: de Pouplana Lluís Ribas , Turner Robert J. , Steer Brian A. , Schimmel Paul
Published: July 2002

We present a phylogenetic analysis to determine whether a given tRNA molecule was established in evolution before its cognate aminoacyl-tRNA synthetase. The earlier appearance of tRNA versus their metabolically related enzymes is a prediction of the RNA world theory, but the available synthetase and tRNA sequences previously had not allowed a formal comparison of their relative time of appearance. Using data recently obtained from the emerging genome projects, our analysis points to the extant forms of lysyl-tRNA synthetase being preceded in evolution by the establishment of the identity of lysine tRNA.
The ORF47 and ORF66 putative protein kinases of varicella-zoster virus determine tropism for human T cells and skin in the SCID-hu mouse
Authors: Moffat Jennifer F. , Zerboni Leigh , Sommer Marvin H. , Heineman Thomas C. , Cohen Jeffrey I. , Kaneshima Hideto , Arvin Ann M.
Published: July 2002

The varicella-zoster virus (VZV) genes ORF47 and ORF66 are predicted to encode serine/threonine protein kinases, which are homologs of herpes simplex virus 1 (HSV-1) UL13, and US3. When mutants were constructed by inserting stop codons into ORF47 and ORF66, the recombinants ROka47S and ROka66S, as well as intact ROka replicated in tissue culture. In contrast, inoculation of human thymus/liver or skin implants in SCID-hu mice showed that ORF47 protein was required for viral growth in human T cells and skin. Eliminating ORF66 expression inhibited VZV infectivity for T cells partially but did not impair replication in skin compared with ROka. Infectivity for T cells and skin was restored when ROka47S virus was complemented by insertion of ORF47 into a distant, noncoding site. The ORF47 gene product is the first VZV protein identified as necessary for T cell tropism. It also is essential for skin infectivity
in vivo
, as is glycoprotein C. Expression of ORF66 did not compensate for the absence of the ORF47 protein. The requirement for ORF47 expression in T cells and skin indicates that this gene product, which is dispensable
in vitro
, has a critical role within differentiated cells that are essential targets for VZV pathogenesis
in vivo
.
Specific intercellular binding of the β-amyloid precursor protein to the presenilins induces intercellular signaling: Its significance for Alzheimer’s disease
Authors: Dewji Nazneen N. , Singer S. J.
Published: July 2002

Genetic evidence has implicated three proteins, the β-amyloid precursor protein (β-APP) and the two homologous presenilins (PS-1 and PS-2), in the etiology of Alzheimer’s disease (AD). How these three proteins jointly contribute to AD, however, is not clear. Nor is any of their normal physiological functions known. Herein, we demonstrate, confirming a prediction made earlier, that β-APP and either PS-1 or PS-2 act as a specific membrane-bound ligand binding intercellularly with either of its two membrane receptors. This results in a cell–cell adhesion, after which rapid transient increases in protein tyrosine kinase activity and protein tyrosine phosphorylation occur coordinately inside one or both of the two adherent cells. The spectrum of proteins modified by tyrosine phosphorylation differs depending on whether PS-1 or PS-2 is involved in the specific intercellular binding to β-APP, which implies that PS-1 and PS-2 have distinct, rather than redundant, functions in normal physiology. The relevance of this intercellular interaction and signaling process to AD is discussed.
Self-incompatibility alleles from
Physalis
: Implications for historical inference from balanced genetic polymorphisms
Authors: Richman Adam D. , Kohn Joshua R.
Published: July 2002

Balanced genetic polymorphism has been proposed as a source from which to infer population history complementary to that of neutral genetic polymorphism, because genetic polymorphism maintained by balancing selection permits inferences about population size over much longer spans of time. However, empirical data for both
S
genes and major histocompatibility complex genes do not fit expectations of coalescent theory. Species-specific gene genealogies have longer terminal branches than expected, indicating an apparent slowdown in the origination of new alleles. Here, we present evidence that divergent
S
alleles were selectively maintained in
Physalis cinerascens
during a reduction in population size, generating longer terminal branches in the
S
gene genealogy relative to the congener
Physalis
crassifolia
. Retention of divergent alleles during reduction in the number of alleles violates assumptions of the coalescent model used to estimate effective population size. Recent theoretical and empirical results are consistent with the proposition that nonrandom sorting is a general property of balanced genetic polymorphisms, suggesting that studies of balanced polymorphism that infer the absence of population bottlenecks may overestimate effective population size.
A continuous topological change during phase transitions in amphiphile/water systems
Authors: Funari Sérgio S. , Rapp Gert
Published: July 2002

Amphiphiles are molecules such as surfactants or lipids that have a polar head group (hydrophilic) attached to nonpolar hydrophobic alkyl chains. Because of this characteristic they self-assemble in water and give rise to a wide range of phases with different structures and properties. Aqueous dispersions of amphiphiles are present in every aspect of day-to-day life—e.g., forming biological cell membranes, stabilizing emulsified food, or being used as soap. Time-resolved x-ray diffraction has been used to study the hexadecylhexa(oxyethylene glycol) ether (C
16
EO
6
)/water system, which shows an intermediate phase whose structure depends on the thermal path between lamellar and hexagonal structures. Heating the hexagonal phase from room temperature leads to a lamellar phase via an Ia3d cubic structure. Cooling from the lamellar phase initially leads epitaxially to an intermediate R3̄m before the hexagonal phase is reached. Both cubic and R3̄m phases are formed by very similar rod units, but the overall structures differ because of their spatial distribution and they both bridge morphologically the hexagonal and lamellar phases. The Ia3d does so on heating, whereas the R3̄m does on cooling. The structural path during the phase transitions is determined by topological similarities between the forming phase and the one from which it originates. Although the estimated curvature energies for these two phases are similar, on cooling, kinetics and topology are initial factors determining the path for the phase transitions, whereas on heating energy is the dominant factor.
A potential mechanism underlying the increased susceptibility of individuals with a polymorphism in NAD(P)H:quinone oxidoreductase 1 (NQO1) to benzene toxicity
Authors: Moran Julie L. , Siegel David , Ross David
Published: July 2002

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a two-electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. A polymorphism in NQO1, a C609T substitution, has been identified, and individuals homozygous for this change (T/T) have no detectable NQO1. Exposed workers with a T/T genotype have an increased risk of benzene hematotoxicity. This finding suggests NQO1 is protective against benzene toxicity, which is difficult to reconcile with the lack of detectable NQO1 in human bone marrow. The human promyeloblastic cell line, KG-1a, was used to investigate the ability of the benzene metabolite hydroquinone (HQ) to induce NQO1. A concentration-dependent induction of NQO1 protein and activity was observed in KG-1a cells cultured with HQ. Multiple detoxification systems, including NQO1 and glutathione protect against benzene metabolite-induced toxicity. Indeed, exposure to a noncytotoxic concentration of HQ induced both NQO1 and soluble thiols and protected against HQ-induced apoptosis. NQO1 protein and activity increased in wild-type human bone marrow cells (C/C) exposed to HQ, whereas no NQO1 was induced by HQ in bone marrow cells with the T/T genotype. Intermediate induction of NQO1 by HQ was observed in heterozygous bone marrow cells (C/T). NQO1 also was induced by HQ in wild-type (C/C) human bone marrow CD34
+
progenitor cells. Our data suggest that failure to induce functional NQO1 may contribute to the increased risk of benzene poisoning in individuals homozygous for the NQO1 C609T substitution (T/T).
Mapping the molecular determinant of pathogenicity in a hammerhead viroid: A tetraloop within the
in vivo
branched RNA conformation
Authors: De la Peña Marcos , Navarro Beatriz , Flores Ricardo
Published: July 2002

Chrysanthemum chlorotic mottle viroid (CChMVd) is an RNA of 398–399 nt that can adopt hammerhead structures in both polarity strands. We have identified by Northern-blot hybridization a nonsymptomatic strain (CChMVd-NS) that protects against challenge inoculation with the symptomatic strain (CChMVd-S). Analysis of CChMVd-NS cDNA clones has revealed a size and sequence very similar to those of the CChMVd-S strain. Some of the mutations observed in CChMVd-NS molecular variants were previously identified in CChMVd-S RNA, but others were never found in this RNA. When bioassayed in chrysanthemum, cDNA clones containing the CChMVd-NS specific mutations were infectious but nonsymptomatic. Site-directed mutagenesis showed that one of the CChMVd-NS-specific mutations, a UUUC → GAAA substitution, was sufficient to change the symptomatic phenotype into the nonsymptomatic one without altering the final accumulation level of the viroid RNA. The pathogenicity determinant–to our knowledge, a determinant of this class has not been described previously in hammerhead viroids–is located in a tetraloop of the computer-predicted branched conformation for CChMVd RNA. Analysis of the sequence heterogeneity found in CChMVd-S and -NS variants strongly supports the existence of such a conformation
in vivo
, showing that the rod-like or quasi-rod-like secondary structure is not a universal paradigm for viroids.
Evidence for the function of an exonic splicing enhancer after the first catalytic step of pre-mRNA splicing
Authors: Chew Shern L. , Liu Hong-Xiang , Mayeda Akila , Krainer Adrian R.
Published: July 2002

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. Thein vitrosplicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3′ exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3′ exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.
Trapping of megabase-sized DNA molecules during agarose gel electrophoresis
Authors: Gurrieri Sergio , Smith Steven B. , Bustamante Carlos
Published: July 2002

Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated λ-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting that both processes occur through nick melting near the vertex of the U-shape. Consistently, molecules nicked by exposure to UV radiation trap more readily than unexposed ones. The critical trapping tension at the vertex is estimated to be 15 pN, a force sufficient to melt nicks bent around gel fibers, and, according to our model, trap a molecule. Strategies to reduce molecular tension and avoid trapping are discussed.
Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis
Authors: Shiraishi Masahiko , Chuu Ying H. , Sekiya Takao
Published: July 2002

We have constructed a library of DNA fragments heavily methylated in human adenocarcinomas of the lung to permit the comprehensive isolation of methylated CpG islands in cancer. Heavily methylated genomic DNA fragments from tumors of nine male patients were enriched using a methylated DNA binding column and used for construction of the library. From this library, DNA fragments having properties of CpG islands were isolated on the basis of their reduced rate of strand dissociation during denaturing gradient gel electrophoresis. Approximately 1,000 clones, corresponding to 0.3% of the library were analyzed, and nine DNA fragments were identified as being associated with CpG islands that were methylated in tumor DNA. One CpG island was methylated specifically in tumor DNA, whereas the remaining eight CpG islands were methylated both in normal and tumor DNA derived from the same patients. Our results suggest that the number of CpG islands methylated specifically in tumors is not large. The library, which contains DNA fragments from methylated CpG islands comprehensively, is expected to be valuable when elucidating epigenetic processes involved in carcinogenesis.
Nontropic actions of neurotrophins: Subcortical nerve growth factor gene delivery reverses age-related degeneration of primate cortical cholinergic innervation
Authors: Conner J. M. , Darracq M. A. , Roberts Jeff , Tuszynski M. H.
Published: July 2002

Normal aging is associated with a significant reduction in
cognitive function across primate species. However, the structural and
molecular basis for this age-related decline in neural function has yet
to be defined clearly. Extensive cell loss does not occur as a
consequence of normal aging in human and nonhuman primate species. More
recent studies have demonstrated significant reductions in functional
neuronal markers in subcortical brain regions in primates as a
consequence of aging, including dopaminergic and cholinergic systems,
although corresponding losses in cortical innervation from these
neurons have not been investigated. In the present study, we report
that aging is associated with a significant 25% reduction in cortical
innervation by cholinergic systems in rhesus monkeys
(
P
< 0.001). Further, these age-related reductions
are ameliorated by cellular delivery of human nerve growth factor to
cholinergic somata in the basal forebrain, restoring levels of
cholinergic innervation in the cortex to those of young monkeys
(
P
= 0.89). Thus, (
i
) aging is
associated with a significant reduction in cortical cholinergic
innervation; (
ii
) this reduction is reversible by
growth-factor delivery; and (
iii
) growth factors can
remodel axonal terminal fields at a distance, representing a nontropic
action of growth factors in modulating adult neuronal structure and
function (i.e., administration of growth factors to cholinergic somata
significantly increases axon density in terminal fields). These
findings are relevant to potential clinical uses of growth factors to
treat neurological disorders.
Similarities in the Clinical Characteristics Related to Alcohol Dependence in Two Populations
Authors: Schuckit Marc A. , Smith Tom L. , Danko George P. , Reich Theodore , Bucholz Kathleen K. , Bierut Laura J.
Published: July 2002

This report evaluates whether the characteristics associated with alcohol abuse and dependence are similar in two groups of men despite their enrollment in different research projects and the resulting differences in education and related background variables. Data regarding demographic and substance use characteristics and problems were gathered using similar research instruments from the 15‐year follow‐up of 108 highly educated DSM‐III‐R alcohol‐dependent men from the San Diego Prospective Study (Group 1) and 306 similarly diagnosed men with lower education from the Collaborative Study on the Genetics of Alcoholism (COGA) (Group 2). Both groups of alcoholics reported high rates of alcohol‐related problems, a similar maximum number of drinks per day, and similar proportions of drug use/non‐use, although Group 1 subjects were less likely to report alcohol withdrawal and items associated with loss of control. Thus, teachers and clinicians may be able to generalize their knowledge and experience across different groups of alcoholics, using common sense modifications of their expectations based on the general characteristics of the groups involved despite differences in background variables, including education, income, marital status, and employment.
Regulation of Endosome Sorting by a Specific PP2A Isoform
Authors: Molloy Sean S. , Thomas Laurel , Kamibayashi Craig , Mumby Marc C. , Thomas Gary
Published: July 2002

The regulated sorting of proteins within the trans-Golgi network (TGN)/endosomal system is a key determinant of their biological activity in vivo. For example, the endoprotease furin activates of a wide range of proproteins in multiple compartments within the TGN/endosomal system. Phosphorylation of its cytosolic domain by casein kinase II (CKII) promotes the localization of furin to the TGN and early endosomes whereas dephosphorylation is required for efficient transport between these compartments (Jones, B.G., L. Thomas, S.S. Molloy, C.D. Thulin, M.D. Fry, K.A. Walsh, and G. Thomas. 1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:5869–5883). Here we show that phosphorylated furin molecules internalized from the cell surface are retained in a local cycling loop between early endosomes and the plasma membrane. This cycling loop requires the phosphorylation state-dependent furin-sorting protein PACS-1, and mirrors the trafficking pathway described recently for the TGN localization of furin (Wan, L., S.S. Molloy, L. Thomas, G. Liu, Y. Xiang, S.L. Ryback, and G. Thomas. 1998. Cell. 94:205–216). We also demonstrate a novel role for protein phosphatase 2A (PP2A) in regulating protein localization in the TGN/endosomal system. Using baculovirus recombinants expressing individual PP2A subunits, we show that the dephosphorylation of furin in vitro requires heterotrimeric phosphatase containing B family regulatory subunits. The importance of this PP2A isoform in directing the routing of furin from early endosomes to the TGN was established using SV-40 small t antigen as a diagnostic tool in vivo. The role of both CKII and PP2A in controlling multiple sorting steps in the TGN/endosomal system indicates that the distribution of itinerant membrane proteins may be acutely regulated via signal transduction pathways.
Smads, Tak1, and Their Common Target Atf-2 Play a Critical Role in Cardiomyocyte Differentiation
Authors: Monzen Koshiro , Hiroi Yukio , Kudoh Sumiyo , Akazawa Hiroshi , Oka Toru , Takimoto Eiki , Hayashi Doubun , Hosoda Toru , Kawabata Masahiro , Miyazono Kohei , Ishii Shunsuke , Yazaki Yoshio , Nagai Ryozo , Komuro Issei
Published: July 2002

We previously demonstrated that bone morphogenetic proteins (BMPs) induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1. Transcription factors Smads mediate transforming growth factor-β signaling and the ATF/CREB family transcription factor ATF-2 has recently been shown to act as a common target of the Smad and the TAK1 pathways. We here examined the role of Smads and ATF-2 in cardiomyocyte differentiation of P19CL6, a clonal derivative of murine P19 cells. Although P19CL6 efficiently differentiates into cardiomyocytes when treated with dimethyl sulfoxide, P19CL6noggin, a P19CL6 cell line constitutively overexpressing the BMP antagonist noggin, did not differentiate into cardiomyocytes. Cooverexpression of Smad1, a ligand-specific Smad, and Smad4, a common Smad, restored the ability of P19CL6noggin to differentiate into cardiomyocytes, whereas stable overexpression of Smad6, an inhibitory Smad, completely blocked differentiation of P19CL6, suggesting that the Smad pathway is necessary for cardiomyocyte differentiation. ATF-2 stimulated the βMHC promoter activity by the synergistic manner with Smad1/4 and TAK1 and promoted terminal cardiomyocyte differentiation of P19CL6noggin, whereas overexpression of the dominant negative form of ATF-2 reduced the promoter activities of several cardiac-specific genes and inhibited differentiation of P19CL6. These results suggest that Smads, TAK1, and their common target ATF-2 cooperatively play a critical role in cardiomyocyte differentiation.
Role of PI 3-kinase, Akt and Bcl-2–related proteins in sustaining the survival of neurotrophic factor–independent adult sympathetic neurons
Authors: Orike Nina , Middleton Gayle , Borthwick Emma , Buchman Vladimir , Cowen Timothy , Davies Alun M.
Published: July 2002

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.
CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae
Authors: Lee Shaun W. , Bonnah Robert A. , Higashi Dustin L. , Atkinson John P. , Milgram Sharon L. , So Magdalene
Published: July 2002

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.
CD161 (NKR-P1A) Costimulation of CD1d-dependent Activation of Human T Cells Expressing Invariant Vα24JαQ T Cell Receptor α Chains
Authors: Exley Mark , Porcelli Steven , Furman Margo , Garcia Jorge , Balk Steven
Published: July 2002

A population of human T cells expressing an invariant Vα24JαQ T cell antigen receptor (TCR) α chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other CD161+ T cells, the major histocompatibility complex–like nonpolymorphic CD1d molecule is the target for the TCR expressed by these T cells (Vα24invt T cells) and by the homologous murine NK1 (NKR-P1C)+ T cell population. In this report, CD161 was shown to act as a specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by Vα24invt T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in Vα24invt T cell activation, and costimulation through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited Vα24invt T cell proliferation and cytokine secretion in response to CD1d+ target cells, demonstrating a physiological accessory molecule function for CD161. However, CD1d-restricted target cell lysis by activated Vα24invt T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in Vα24invt T cell costimulation through CD161 did not appear to involve stable association with tyrosine kinase p56Lck. These results demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human Vα24invt T cells.
The Immunogenicity of a New Human Minor Histocompatibility Antigen Results from Differential Antigen Processing
Authors: Brickner Anthony G. , Warren Edus H. , Caldwell Jennifer A. , Akatsuka Yoshiki , Golovina Tatiana N. , Zarling Angela L. , Shabanowitz Jeffrey , Eisenlohr Laurence C. , Hunt Donald F. , Engelhard Victor H. , Riddell Stanley R.
Published: July 2002

Minor histocompatibility antigens (mHAgs) present a significant impediment to organ and bone marrow transplantation between HLA-identical donor and recipient pairs. Here we report the identification of a new HLA-A*0201–restricted mHAg, HA-8. Designation of this mHAg as HA-8 is based on the nomenclature of Goulmy (Goulmy, E. 1996. Curr. Opin. Immunol. 8:75–81). This peptide, RTLDKVLEV, is derived from KIAA0020, a gene of unknown function located on chromosome 9. Polymorphic alleles of KIAA0020 encode the alternative sequences PTLDKVLEV and PTLDKVLEL. Genotypic analysis demonstrated that the HA-8–specific cytotoxic T lymphocyte (CTL) clone SKH-13 recognized only cells that expressed the allele encoding R at P1. However, when PTLDKVLEV was pulsed onto cells, or when a minigene encoding this sequence was used to artificially translocate this peptide into the endoplasmic reticulum, it was recognized by CTLs nearly as well as RTLDKVLEV. This indicates that the failure of CTLs to recognize cells expressing the PTLDKVLEV-encoding allele of KIAA0020 is due to a failure of this peptide to be appropriately proteolyzed or transported. Consistent with the latter possibility, PTLDKVLEV and its longer precursors were transported poorly compared with RTLDKVLEV by transporter associated with antigen processing (TAP). These studies identify a new human mHAg and provide the first evidence that minor histocompatibility differences can result from the altered processing of potential antigens rather than differences in interaction with the relevant major histocompatibility complex molecule or T cell receptor.
Dendritic Cells Cross-Present Latency Gene Products from Epstein-Barr Virus–Transformed B Cells and Expand Tumor-Reactive Cd8+ Killer T Cells
Authors: Subklewe Marion , Paludan Casper , Tsang Ming L. , Mahnke Karsten , Steinman Ralph M. , Münz Christian
Published: July 2002

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8+ T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I–negative LCLs, when presented by DCs, also could elicit responses to MHC class II–negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8+ T cell response, in both lytic and interferon γ secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8+ T cells recognized targets at low doses, 1–10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.
Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons
Authors: Ogden David , Capiod Thierry
Published: July 2002

The repetitive spiking of free cytosolic [Ca2+] ([Ca2+]i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP3-evoked Ca2+ release. The kinetics of both processes were studied with flash photolytic release of InsP3 and time resolved measurements of [Ca2+]i in single cells. InsP3 evoked Ca2+ flux into the cytosol was measured as d[Ca2+]i/dt, and the kinetics of Ca2+ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP3 concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP3 suggests that metabolism of InsP3 is important in determining the minimal concentration needed to produce Ca2+ release. A distinct latency or delay of several hundred milliseconds after release of low InsP3 concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca2+]i, suggesting a cooperative action of Ca2+ in InsP3 receptor activation. InsP3-evoked flux and peak [Ca2+]i increased with InsP3 concentration up to 5–10 μM, with large variation from cell to cell at each InsP3 concentration. The duration of InsP3-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca2+]i/dt and much less cell to cell variation than the dependence of flux on InsP3 concentration, suggesting that the rate of termination of the Ca2+ flux depends on the free Ca2+ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca2+ flux, up to 50 μM · s−1 and in Purkinje neurons at high flux up to 1,400 μM · s−1. Experiments in which [Ca2+]i was controlled at resting or elevated levels support a mechanism in which InsP3-evoked Ca2+ flux is inhibited by Ca2+ inactivation of closed receptor/channels due to Ca2+ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP3-evoked Ca2+ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP3 receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP3 receptor density promotes fast rising, rapidly inactivating InsP3-evoked [Ca2+]i transients.
Weak amenability of triangular Banach algebras
Authors: Forrest B. , Marcoux L.
Published: July 2002

Let

A

\mathcal {A}

and

B

\mathcal {B}

be unital Banach algebras, and let

M

\mathcal {M}

be a Banach

A

,

B

\mathcal {A},\mathcal {B}

-module. Then

T

=

[

A

a
m
p
;

M

0

a
m
p
;

B

]

\mathcal {T} = \begin {bmatrix}\mathcal {A} & \mathcal {M}\\ 0 & \mathcal {B} \end {bmatrix}

becomes a triangular Banach algebra when equipped with the Banach space norm
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="Start 2 By 2 Matrix 1st Row 1st Column a 2nd Column a m p semicolon m 2nd Row 1st Column 0 2nd Column a m p semicolon b EndMatrix equals double-vertical-bar a double-vertical-bar Subscript script upper A Baseline plus double-vertical-bar m double-vertical-bar Subscript script upper M Baseline plus double-vertical-bar b double-vertical-bar Subscript script upper B">

‖

[

a

a
m
p
;
m

0

a
m
p
;
b

]

‖

=
‖
a

‖

A

+
‖
m

‖

M

+
‖
b

‖

B

\left \Vert \begin {bmatrix} a & m\\ 0 & b \end {bmatrix} \right \Vert = \Vert a \Vert _{\mathcal {A}} + \Vert m \Vert _{\mathcal {M}} + \Vert b \Vert _{\mathcal {B}}

. A Banach algebra

T

\mathcal {T}

is said to be

n
n

-weakly amenable if all derivations from

T

\mathcal {T}

into its

n

t
h

n^{\mathrm {th}}

dual space

T

(
n
)

\mathcal {T}^{(n)}

are inner. In this paper we investigate Arens regularity and

n
n

-weak amenability of a triangular Banach algebra

T

\mathcal {T}

in relation to that of the algebras

A

\mathcal {A}

,

B

\mathcal {B}

and their action on the module

M

\mathcal {M}

.
On perturbations of matrix pencils with real spectra. II
Authors: Bhatia Rajendra , Li Ren-Cang
Published: July 2002

A well-known result on spectral variation of a Hermitian matrix due to Mirsky is the following: Let

A
A

and

A
~

\widetilde A

be two

n
×
n

n\times n

Hermitian matrices, and let

λ
1

\lambda _1

, …,

λ

n

\lambda _{n}

and

λ
~

1

\widetilde \lambda _1

, …,

λ
~

n

\widetilde \lambda _{n}

be their eigenvalues arranged in ascending order. Then
<mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" alttext="triple-vertical-bar-delimiter d i a g left-parenthesis lamda 1 minus lamda overTilde Subscript 1 Baseline comma ellipsis comma lamda Subscript n Baseline minus lamda overTilde Subscript n Baseline right-parenthesis triple-vertical-bar-delimiter less-than-or-equal-to triple-vertical-bar-delimiter upper A minus upper A overTilde triple-vertical-bar-delimiter">

⦀
diag
⁡
(

λ
1

−

λ
~

1

,
…
,

λ
n

−

λ
~

n

)
⦀

≤

⦀
A
−

A
~

⦀

\left \Vvert \operatorname {diag} (\lambda _1- \widetilde \lambda _1,\ldots ,\lambda _n- \widetilde \lambda _n) \right \Vvert \le \left \Vvert A-\widetilde A \right \Vvert

for any unitarily invariant norm

⦀
⋅
⦀

\Vvert \cdot \Vvert

. In this paper, we generalize this to the perturbation theory for diagonalizable matrix pencils with real spectra. The much studied case of definite pencils is included in this.
Calculus of embeddings
Authors: Weiss Michael
Published: July 2002

Let

M
M

and

N
N

be smooth manifolds, where

M
⊂
N

M\subset N

and

dim
⁡
(
N
)
−
dim
⁡
(
M
)
≥
3

\dim (N)-\dim (M)\ge 3

. A disjunction lemma for embeddings proved recently by Goodwillie leads to a calculation up to extension problems of the base point component of the space of smooth embeddings of

M
M

in

N
N

. This is mostly in terms of

i
m
m

(
M
,
N
)

\mathbf {imm}(M,N)

, the space of smooth immersions, which is well understood, and embedding spaces

e
m
b

(
S
,
N
)

\mathbf {emb}(S,N)

for finite subsets

S
S

of

M
M

with few elements. The meaning of few depends on the precision desired.
VE-Cadherin Regulates Endothelial Actin Activating Rac and Increasing Membrane Association of Tiam
Authors: Lampugnani Maria Grazia , Zanetti Adriana , Breviario Ferruccio , Balconi Giovanna , Orsenigo Fabrizio , Corada Monica , Spagnuolo Raffaella , Betson Martha , Braga Vania , Dejana Elisabetta
Published: July 2002

Previously published reports support the concept that, besides promoting homotypic intercellular adhesion, cadherins may transfer intracellular signals. However, the signaling pathways triggered by cadherin clustering and their biological significance are still poorly understood. We report herein that transfection of VE-cadherin (VEC) cDNA in VEC null endothelial cells induces actin rearrangement and increases the number of vinculin positive adhesion plaques. VEC expression augments the level of active Rac but decreases active Rho. Microinjection of a dominant negative Rac mutant altered stress fiber organization, whereas inhibition of Rho was ineffective. VEC expression increased protein and mRNA levels of the Rac-specific guanosine exchange factor Tiam-1 and induced its localization at intercellular junctions. In addition, in the presence of VEC, the amounts of Tiam, Rac, and the Rac effector PAK as well as the level of PAK phosphorylation were found increased in the membrane/cytoskeletal fraction. These observations are consistent with a role of VEC in localizing Rac and its signaling partners in the same membrane compartment, facilitating their reciprocal interaction. Through this mechanism VEC may influence the constitutive organization of the actin cytoskeleton.
Automated management of gene discovery projects.
Authors: Taylor S , Hodgman T C , Littler C B , Sayle R A , Gill R W , Oxer M D , O'Donnell C H , Lyall A
Published: July 2002

SUMMARY: The paper reports on programs, organization and Hypertext Mark-up Language (HTML) presentation provided by the Hierarchical Automated Gene Identification System (HAGIS) designed for managing sequencing projects. AVAILABILITY: On request from the authors. CONTACT: st23646@ggr.co.uk
New agents for the treatment of infarcted myocardium
Authors: Yamauchi Hidetoshi , Desgranges Pascal , Lecerf Laure , Papy Dulce , Tournaire Marie‐Claude , Moczar Madeleine , Loisance Daniel , Barritault Denis
Published: July 2002

Local delivery of angiogenic growth factors for the treatment of myocardial ischemia has been well documented in various animal models, and clinical trials are now in progress. Our strategy was radically different, based on selective protection of some of the growth factors naturally present within the injured tissue. This protection was obtained by applying a chemically defined substitute for Dextran called RGTA11 (for ReGeneraTing Agent). RGTA is a family of agents, which has properties mimicking those of heparan sulfates toward heparin‐binding growth factors (HBGF) and which stimulate tissue repair and protection. Indeed, we have previously shown that RGTA prevents most of the damage resulting from acute skeletal muscle ischemia [FASEB J. (1999) 13, 761–766]. We now show that the same agent can be used for the treatment of myocardial infarction. Acute myocardial infarction was induced in pigs by ligation of the left circumflex artery. One hour later, a single injection of 10 μg of RGTA11 was made in the center of the infarcted area. Three weeks later we observed 1) recovery of 84% of the initial left ventricular ejection fraction (only 55% in saline‐treated controls), 2) an almost 50% reduction in the infarct size, 3) a reduction in fibrotic tissue formation, 4) significant preservation of myocytes, and 5) an increase in the number of blood vessels. The treatment of ischemic heart disease with RGTA would have clear advantages over other therapies such as growth factor, gene, or cell transplants, based on a stable, simple, and easy‐to‐develop chemical product.