bluuebunny/crossref_metadata_2025 huggingface dataset:
A lymphocyte-activating monoclonal antibody induces regression of human tumors in severe combined immunodeficient mice
Authors: Hardy Britta , Kovjazin Riva , Raiter Annat , Ganor Nirit , Novogrodsky Abraham
Published: July 2002

Monoclonal antibodies were raised against Daudi B-lymphoblastoid cell line membranes. An mAb (BAT) was selected for its ability to stimulate human and murine lymphocyte proliferation. BAT induced cytotoxicity in human and murine lymphocytes against natural killer cell-sensitive and -resistant tumor cell lines. A single intravenous administration of BAT to mice that had been inoculated with various murine tumors (e.g., B16 melanoma, 3LL carcinoma, and methylcholanthrene fibrosarcoma) resulted in striking antitumor effects as manifested by complete tumor regression and prolonged survival of the treated mice. BAT exhibited a diminished but significant antitumor effect in athymic nude mice, which are deficient in T lymphocytes, and in beige mice, which are deficient in NK cells. Furthermore, selective depletion of T or NK cells in mice reduced the response to the antitumor effect of BAT. These data indicate a dual role for T and NK cells in mediating the antitumor activity of BAT. We report here on the antitumor activity of BAT mAb on human tumor xenografts in mice. BAT demonstrated an antitumor effect in nude mice bearing human colon carcinoma (HT29) xenografts. It failed, however, to inhibit established lung metastases in severe combined immunodeficient (SCID) mice that had been inoculated (i.v.) with SK28 human melanoma. Engraftment of human lymphocytes into SCID mice bearing human melanoma xenografts rendered them responsive to the antitumor effect of BAT. The efficacy of BAT in the regression of human tumors by activation of human lymphocytes indicates its potential clinical use.
Appendicular robusticity and the paleobiology of modern human emergence
Authors: Trinkaus Erik
Published: July 2002

The emergence of modern humans in the Late Pleistocene, whatever its phylogenetic history, was characterized by a series of behaviorally important shifts reflected in aspects of human hard tissue biology and the archeological record. To elucidate these shifts further, diaphyseal cross-sectional morphology was analyzed by using cross-sectional areas and second moments of area of the mid-distal humerus and midshaft femur. The humeral diaphysis indicates a gradual reduction in habitual load levels from Eurasian late archaic, to Early Upper Paleolithic early modern, to Middle Upper Paleolithic early modern hominids, with the Levantine Middle Paleolithic early modern humans being a gracile anomalous outlier. The femoral diaphysis, once variation in ecogeographically patterned body proportions is taken into account, indicates no changes across the pre-30,000 years B.P. samples in habitual locomotor load levels, followed by a modest decrease through the Middle Upper Paleolithic.
Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments
Authors: Van Ewijk Willem , de Kruif John , Germeraad Wilfred T. V. , Berendes Paul , Röpke Carsten , Platenburg Peter Paul , Logtenberg Ton
Published: July 2002

In the murine thymus, the stroma forms microenvironments that control different steps in T cell development. To study the architecture of such microenvironments and more particularly the nature of communicative signals in lympho–stromal interaction during T cell development, we have employed the phage antibody display technology, with the specific aim of isolating thymic stromal cell-specific single-chain antibodies from a semisynthetic phage library. A subtractive approach using intact, mildly fixed thymic fragments as target tissue and lymphocytes as absorber cells generated monoclonal phages (MoPhabs) detecting subsets of murine thymic stromal cells. In the present paper we report on the reactivity of single-chain antibodies derived from three MoPhabs, TB4–4, TB4–20, and TB4–28. While TB4–4 and TB4–20 are both epithelium specific, TB4–28 detects an epitope expressed on both epithelial- and mesenchymal-derived stromal cells. TB4–4 reacts with all cortical epithelial cells and with other endoderm-derived epithelia, but this reagent leaves the majority of medullary epithelial cells unstained. In contrast, MoPhab TB4–20 detects both cortical and medullary thymic epithelial cells, as well as other endoderm- and ectoderm-derived epithelial cells. Cross-reaction of single-chain antibodies to human thymic stromal cells shows that our semisynthetic phage antibody display library, in combination with the present subtractive approach, permits detection of evolutionary conserved epitopes expressed on subsets of thymic stromal cells.
Solution structure of the cytohesin-1 (B2–1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1
Authors: Betz Stephen F. , Schnuchel Arndt , Wang Hong , Olejniczak Edward T. , Meadows Robert P. , Lipsky Brian P. , Harris Edith A. S. , Staunton Donald E. , Fesik Stephen W.
Published: July 2002

Cytohesin-1 (B2–1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the β2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein’s guanine nucleotide exchange factor function and β2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 α-helices that form a unique tertiary fold. The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining
1
H-
15
N and
1
H-
13
C chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-1 occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the β2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.
Footprints on the viral DNA ends in Moloney murine leukemia virus preintegration complexes reflect a specific association with integrase
Authors: Wei Shui-Qing , Mizuuchi Kiyoshi , Craigie Robert
Published: July 2002

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3′ end processing.
Activation of microhelix charging by localized helix destabilization
Authors: Alexander Rebecca W. , Nordin Brian E. , Schimmel Paul
Published: July 2002

We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNA
Gln
revealed an uncoupling of the first (1⋅72) base pair of tRNA
Gln
, and tRNA
Met
was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1⋅72 distortion is probably effected by the synthetase–anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1⋅72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.
Preplay contracting in the Prisoners’ Dilemma
Authors: Andreoni James , Varian Hal
Published: July 2002

We consider a modified Prisoners’ Dilemma game in which each agent can offer to pay the other agent to cooperate. The subgame perfect equilibrium of this two-stage game is Pareto efficient. We examine experimentally whether subjects actually manage to achieve this efficient outcome. We find an encouraging level of support for the mechanism, but also find some evidence that subjects’ tastes for cooperation and equity may have significant interactions with the incentives provided by the mechanism.
3-Hydroxy-3-methylglutaryl CoA reductase inhibitors up-regulate transforming growth factor-β signaling in cultured heart cells via inhibition of geranylgeranylation of RhoA GTPase
Authors: Park Ho-Jin , Galper Jonas B.
Published: July 2002

Transforming growth factor-β (TGFβ) signaling has been shown to play a role in cardiac development as well as in the pathogenesis of cardiovascular disease. Prior studies have suggested a relationship between cholesterol metabolism and TGFβ signaling. Here we demonstrate that induction of the cholesterol metabolic pathway by growth of embryonic chicken atrial cells in medium supplemented with lipoprotein-depleted serum coordinately decreased the expression of the TGFβ type II receptor (TGFβRII), TGFβ
1
, and TGFβ signaling as measured by plasminogen activator inhibitor-1 (PAI-1) promoter activity. Inhibition of the cholesterol metabolic pathway by the hydrophobic 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase inhibitors, simvastatin and atorvastatin, reversed the effect of lipoprotein-depleted serum and up-regulated TGFβRII expression, whereas the hydrophilic HMGCoA reductase inhibitor, pravastatin, had no effect. Simvastatin stimulated the expression of TGFβRII, TGFβ
1
, and PAI-1 at the level of transcription. Experiments using specific inhibitors of different branches of the cholesterol metabolic pathway demonstrated that simvastatin exerted its effect on TGFβ signaling by inhibition of the geranylgeranylation pathway. C3 exotoxin, which specifically inactivates geranylgeranylated Rho GTPases, mimicked the effect of simvastatin on PAI-1 promoter activity. Cotransfection of cells with a PAI-1 promoter-reporter and a dominant-negative RhoA mutant increased PAI-1 promoter activity, whereas cotransfection with a dominant-active RhoA mutant decreased PAI-1 promoter activity. These data support the conclusion that TGFβ signaling is regulated by RhoA GTPase and demonstrate a relationship between cholesterol metabolism and TGFβ signaling. Our data suggest that in patients treated with HMGCoA reductase inhibitors, these agents may exert effects independent of cholesterol lowering on TGFβ signaling in the heart.
Simulation of biomimetic recognition between polymers and surfaces
Authors: Golumbfskie Aaron J. , Pande Vijay S. , Chakraborty Arup K.
Published: July 2002

Many biological processes, such as transmembrane signaling and pathogen–host interactions, are initiated by a protein recognizing a specific pattern of binding sites on part of a membrane or cell surface. By recognition, we imply that the polymer quickly finds and then adsorbs strongly on the pattern-matched region and not on others. The development of synthetic systems that can mimic such recognition between polymers and surfaces could have significant impact on advanced applications such as the development of sensors, molecular-scale separation processes, and synthetic viral inhibition agents. Attempting to affect recognition in synthetic systems by copying the detailed chemistries to which nature has been led over millenia of evolution does not seem practical for most applications. This leads us to the following question: Are there any universal strategies that can affect recognition between polymers and surfaces? Such generic strategies may be easier to implement in abiotic applications. We describe results that suggest that biomimetic recognition between synthetic polymers and surfaces is possible by exploiting certain generic strategies, and we elucidate the kinetic mechanisms by which this occurs. Our results suggest convenient model systems for experimental studies of dynamics in free energy landscapes characteristic of frustrated systems.
Controlled unzipping of a bacterial surface layer with atomic force microscopy
Authors: Müller Daniel J. , Baumeister Wolfgang , Engel Andreas
Published: July 2002

We have combined high-resolution atomic force microscopy (AFM) imaging and force spectroscopy to gain insight into the interaction forces between the individual protomers of the hexagonally packed intermediate (HPI) layer of
Deinococcus radiodurans
. After imaging the HPI layer, the AFM stylus was attached to individual protomers by enforced stylus-sample contact to allow force spectroscopy experiments. Imaging of the HPI layer after recording force-extension curves allowed adhesion forces to be correlated with structural alterations. By using this approach, individual protomers of the HPI layer were found to be removed at pulling forces of ≈300 pN. Furthermore, it was possible to sequentially unzip entire bacterial pores formed by six HPI protomers. The combination of high-resolution AFM imaging of individual proteins with the determination of their intramolecular forces is a method of studying the mechanical stability of supramolecular structures at the level of single molecules.
GA-binding protein factors, in concert with the coactivator CREB binding protein/p300, control the induction of the interleukin 16 promoter in T lymphocytes
Authors: Bannert Norbert , Avots Andris , Baier Michael , Serfling Edgar , Kurth Reinhard
Published: July 2002

Interleukin 16 (IL-16) is a chemotactic cytokine that binds to the
CD4 receptor and affects the activation of T cells and replication of
HIV. It is expressed as a large 67-kDa precursor protein (pro-IL-16) in
lymphocytes, macrophages, and mast cells, as well as in airway
epithelial cells from asthmatics after challenge with allergen. This
pro-IL-16 is subsequently processed to the mature cytokine of 13 kDa.
To study the expression of IL-16 at the transcriptional level, we
cloned the human chromosomal IL-16 gene and analyzed its promoter. The
human IL-16 gene consists of seven exons and six introns. The 5′
sequences up to nucleotide −120 of the human and murine IL-16 genes
share >84% sequence homology and harbor promoter elements for
constitutive and inducible transcription in T cells. Although both
promoters lack any TATA box, they contain two CAAT box-like motifs and
three binding sites of GA-binding protein (GABP) transcription factors.
Two of these motifs are part of a highly conserved and inducible dyad
symmetry element shown previously to control a remote IL-2 enhancer and
the CD18 promoter. In concert with the coactivator CREB binding
protein/p300, which interacts with GABPα, the binding of GABPα
and -β to the dyad symmetry element controls the induction of IL-16
promoter in T cells. Supplementing the data on the processing of
pro-IL-16, our results indicate the complexity of IL-16 expression,
which is tightly controlled at the transcriptional and
posttranslational levels in T lymphocytes.
Crystal structure of a thermostable type B DNA polymerase from
Thermococcus gorgonarius
Authors: Hopfner Karl-Peter , Eichinger Andreas , Engh Richard A. , Laue Frank , Ankenbauer Waltraud , Huber Robert , Angerer Bernhard
Published: July 2002

Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 Å resolution crystal structure of a DNA polymerase from the Archaea
Thermococcus gorgonarius
and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA–protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this “closed” conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.
Control of fertilization-independent endosperm development by the
MEDEA
polycomb gene in
Arabidopsis
Authors: Kiyosue Tomohiro , Ohad Nir , Yadegari Ramin , Hannon Mike , Dinneny Jose , Wells Derek , Katz Anat , Margossian Linda , Harada John J. , Goldberg Robert B. , Fischer Robert L.
Published: July 2002

Higher plant reproduction is unique because two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, a tissue that supports embryo development. To understand mechanisms that initiate reproduction, we isolated a mutation in
Arabidopsis
,
f644
, that allows for replication of the central cell and subsequent endosperm development without fertilization. When mutant
f644
egg and central cells are fertilized by wild-type sperm, embryo development is inhibited, and endosperm is overproduced. By using a map-based strategy, we cloned and sequenced the
F644
gene and showed that it encodes a SET-domain polycomb protein. Subsequently, we found that
F644
is identical to
MEDEA
(
MEA
), a gene whose maternal-derived allele is required for embryogenesis [Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M. A. & Gagliano, W. B. (1998)
Science
280, 446–450]. Together, these results reveal functions for plant polycomb proteins in the suppression of central cell proliferation and endosperm development. We discuss models to explain how polycomb proteins function to suppress endosperm and promote embryo development.
A novel p

H
2

control on the expression of flagella in the hyperthermophilic strictly hydrogenotrophic methanarchaeaon
Methanococcus jannaschii
Authors: Mukhopadhyay Biswarup , Johnson Eric F. , Wolfe Ralph S.
Published: July 2002

The methanarchaeon,
Methanococcus jannaschii
, a
hyperthermophilic, autotrophic, and strictly hydrogenotrophic
inhabitant of submarine hydrothermal vents, was cultivated in a reactor
at two hydrogen partial pressure (p

H
2

) values, 178 kPa
(high) and 650 Pa (ultralow), and the cells were subjected to a
comparative proteome analysis. From these studies, it was discovered
that, when p

H
2

was high and the cell density was low
(a combination representing a hydrogen-excess condition), the cells
possessed very low or undetectable levels of four flagella-related
polypeptides (FlaB2, FlaB3, FlaD, and FlaE); electron microscopic
examination showed that most of these cells were devoid of flagella.
Flagella synthesis occurred when hydrogen became limiting either at
high cell density under high p

H
2

or at low cell
density under low p

H
2

. The results from a
p

H
2

-shift experiment corroborated the above
observations. The p

H
2

-dependent changes in the levels
of two methanogenic enzymes (MTD and HMDX) were as expected, and thus
they served as internal controls. To our knowledge, this is the first
example for the regulation of expression of flagella by hydrogen in any
domain of life and for a control of any kind on flagella synthesis in
the archaea. Our work also provides the only known example for each of
the following: (
i
) the pure culture cultivation of a
methanogen at an ultralow, near ecologically relevant
p

H
2

; (
ii
) experimental functional
genomics for
M. jannaschii
; and (
iii
) the
use of proteomics with
M. jannaschii
.
Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion
Authors: Coller Hilary A. , Grandori Carla , Tamayo Pablo , Colbert Trent , Lander Eric S. , Eisenman Robert N. , Golub Todd R.
Published: July 2002

MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21
Cip1
was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl
cis-trans
isomerase F and FKBP52, the latter of which plays a role in cell division in
Arabidopsis
, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous
Myc
in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.
Systematic Analysis of Verbalizations Produced in Comparing Musical Timbres
Authors: Samoylenko Elena , McAdams Stephen , Nosulenko Valery
Published: July 2002

A method of verbal protocol analysis is presented in which verbal units used to compare sound events are extracted from the protocols and analysed from three general points of view: (1) their logical sense, (2) their stimulus‐relatedness, and (3) their semantic content. At each level, several analysis steps progressively label the verbal units from hierarchically superior to inferior levels (with increasing differentiation as one descends the hierarchy). The technique shows great reliability across analyses by independent experts. It was applied to an investigation of timbre comparisons in which Russian nonmusician subjects rated the dissimilarity of timbres presented in pairs and described verbally their similarities and differences. The technique demonstrates an effectiveness in revealing differences in focus on perceptual attributes of timbre that contribute to inter‐ and intra‐subject discrepancies in dissimilarity ratings. Further, as concerns identification of the sound sources producing the events, there is evidence that basic categories are very general in nature in these nonmusician subjects.
Differential Diagnosis of Byssinosis by Blood Histamine and Pulmonary Function Test: A Review and an Appraisal
Authors: Venkatakrishna-Bhatt H. , Mohan-Rao N. , Panchal G. M.
Published: July 2002

A study was conducted on byssinotic ( N = 8) and nonbyssinotic ( N = 16) mill workers exposed to cotton dust and on controls ( N = 15) from a cotton dust-free zone. They were examined for chest tightness and breathlessness during successive days from Mondays to Fridays. In addition to monitoring the workers only on Mondays after shift work, their blood histamine levels and pulmonary function tests such as FEV1, PEFR, and FEF25–75% (PEFR represents airflow of larger airways, FEF25–75% reflects airflow in smaller airways, and FEV1 represents airflow in both central and peripheral airways) were screened, but Monday data were only counted to appraise a contrast between these two parameters. The results showed that the histamine levels were significantly higher in the cotton dust-exposed workers in association with significantly decreased FEV1, PEFR, and FEF25–75%, indicating bronchoconstriction of the central, larger, and peripheral airways. Histamine can cause a severe constriction of the central as well as peripheral airways in cotton dust-exposed workers, enabling a paradoxical effect for a differential diagnosis of byssinosis.
Chromium Toxic Effect Monitoring Using Ozonation Method
Authors: Poznyak T. , Puga J. M. , Kiseleva E. , Martinez L.
Published: July 2002

The hexavalent chromium toxicity (in vitro) to plasma, erythrocytes, and semen lipids was evaluated. The ozonation technique is suggested to realize the rapid measurements of the lipid peroxidation (LPO) by means of the double bond indexes (DB-index and DBcell-index) calculation. The obtained experimental results permit to conclude that it is possible to detect the chromium effect on LPO by ozonation. The DB-index and DBcell-index determination in the plasma, erythrocytes, and sperm can be considered as a measure of this effect Ozonation, suggested in this work, can provide the useful preliminary information for specialists and is a quantitative, fast, inexpensive, and simple method. In view of these comments, we conclude that the suggested ozonation method can be considered as the acceptable modern technique for the chromium toxic effect monitoring in vitro.
Tissue Distribution of125 l-LabeIed Albumin in Rats, and Whole Blood and Exhaled Elimination Kinetics of Octafluoropropane in Anesthetized Canines, Following Intravenous Administration of Optison® (FS069)
Authors: Killam Anne L. , Mehlhaff Paige M. , Zavorskas Paul A. , Greener Yigal , McFerran Britta A. , Miller Jeff J. , Burrascano Cindy , Jablonski Edward G. , Anderson Leslie , Dittrich Howard C.
Published: July 2002

OPTISON, an agent being developed as an intravenous (IV) ultrasound contrast agent, consists of a suspension of octafluoro-propane(OFP)-containing albumin microspheres. The distribution and elimination of the albumin component and the elimination kinetics of the OFP component of OPTISON (FS069) were studied in the conscious rat and anesthetized canine models, respectively. Radioiodinated OPTISON at 0.25 ml/kg (average dose 5.4 × 107 DPM/rat) and nonradioactive OPTISON, at dosages of 0.3, 0.6, and 1.0 ml/kg, was administered intravenously to conscious rats or to sodium pentobarbital-anesthetized and ventilated canines, respectively. A separate group of rats was housed in metabolism cages for 24 hours to capture excreted radioactivity. The tissue distribution data for the radiolabeled albumin in rats showed that the 125I activity recovered in the liver was the highest of all the tissues at each timepoint (peak liver radioactivity at 5 minutes with 50.4% of the dose), suggesting that the major route of uptake and metabolism of the radiolabeled albumin shell and its fragments occurred in the liver. The 125I activity was excreted in the urine, where most of the recovered radioactivity (58.3%) was found at the end of 24 hours. In the anesthetized canine study, simultaneous venous blood samples and exhaled air samples plus additional exhaled air samples were analyzed by gas chromatography. OFP was rapidly exhaled through the lungs after an IV injection such that a maximum of less than 10% of the total dose appeared in the venous blood samples. Statistical moment analysis showed rapid OFP elimination with mean residence times of 46, 41, and 38 seconds for the three dosages, and mean total recoveries for the exhaled OFP were 111%, 100.5%, and 121.6%, respectively. OFP was rapidly exhaled through the lungs after OPTISON injection with short mean residence times from statistical moment analysis. Exhaled OFP displayed one-compartment model kinetics with a measurable distribution phase in the blood using classical pharmacokinetic modeling. The albumin component appeared to be cleared primarily by the liver and radioactivity was excreted in the urine.
Relevance Theory And Pragmatic Impairment
Authors: Leinonen Eeva , Kerbel Debra
Published: July 2002

This paper summarizes aspects of relevance theory that are useful for exploring impairment of pragmatic comprehension in children. It explores data from three children with pragmatic language diffi culties within this framework. Relevance theory is seen to provide a means of explaining why, in a given context, a particular utterance is problematic. It thus enables one to move on from mere description of problematic behaviours towards their explanation. The theory provides a clearer delineation between the explicit and the implicit, and hence between semantics and pragmatics. This enables one to place certain difficulties more firmly within semantics and others within pragmatics. Relevance, and its maximization in communication, are squarely placed within human cognition, which suggests a close connection between pragmatic and cognitive (dis)functioning. Relevance theory thus emerges as a powerful tool in the exploration and understanding of pragmatic language difficulties in children and offers therapeutically valuable insight into the nature of interactions involving individuals with such impairments.
Splicing Factors Associate with Hyperphosphorylated RNA Polymerase II in the Absence of Pre-mRNA
Authors: Kim Euikyung , Du Lei , Bregman David B. , Warren Stephen L.
Published: July 2002

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains multiple tandem copies of the consensus heptapeptide, TyrSerProThrSerProSer. Concomitant with transcription initiation the CTD is phosphorylated. Elongating polymerase has a hyperphosphorylated CTD, but the role of this modification is poorly understood. A recent study revealed that some hyperphosphorylated polymerase molecules (Pol IIo) are nonchromosomal, and hence transcriptionally unengaged (Bregman, D.B., L. Du, S. van der Zee, S.L. Warren. 1995. J. Cell Biol. 129: 287–298). Pol IIo was concentrated in discrete splicing factor domains, suggesting a possible relationship between CTD phosphorylation and splicing factors, but no evidence beyond immunolocalization data was provided to support this idea. Here, we show that Pol IIo co-immunoprecipitates with members of two classes of splicing factors, the Sm snRNPs and non-snRNP SerArg (SR) family proteins. Significantly, Pol IIo's association with splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged. We also provide definitive evidence that hyperphosphorylation of Pol II's CTD is poorly correlated with its transcriptional activity. Using monoclonal antibodies (mAbs) H5 and H14, which are shown here to recognize phosphoepitopes on Pol II's CTD, we have quantitated the level of Pol IIo at different stages of the cell cycle. The level of Pol IIo is similar in interphase and mitotic cells, which are transcriptionally active and inactive, respectively. Finally, complexes containing Pol IIo and splicing factors can be prepared from mitotic as well as interphase cells. The experiments reported here establish that hyperphosphorylation of the CTD is a good indicator of polymerase's association with snRNP and SR splicing factors, but not of its transcriptional activity. Most importantly, the present study suggests that splicing factors may associate with the polymerase via the hyperphosphorylated CTD.
Human Cytomegalovirus Immediate Early Interaction with Host Nuclear Structures: Definition of an Immediate Transcript Environment
Authors: Ishov Alexander M. , Stenberg Richard M. , Maul Gerd G.
Published: July 2002

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.
Filipin-dependent Inhibition of Cholera Toxin: Evidence for Toxin Internalization and Activation through Caveolae-like Domains
Authors: Orlandi Palmer A. , Fishman Peter H.
Published: July 2002

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A1 peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-β-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit–mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.
Claudin-11/OSP-based Tight Junctions of Myelin Sheaths in Brain and Sertoli Cells in Testis
Authors: Morita Kazumasa , Sasaki Hiroyuki , Fujimoto Kazushi , Furuse Mikio , Tsukita Shoichiro
Published: July 2002

Members of the newly identified claudin gene family constitute tight junction (TJ) strands, which play a pivotal role in compartmentalization in multicellular organisms. We identified oligodendrocyte-specific protein (OSP) as claudin-11, a new claudin family member, due to its sequence similarity to claudins as well as its ability to form TJ strands in transfected fibroblasts. Claudin-11/OSP mRNA was expressed in the brain and testis. Immunofluorescence microscopy with anti–claudin-11/OSP polyclonal antibody (pAb) and anti-neurofilament mAb revealed that in the brain claudin-11/OSP-positive linear structures run in a gentle spiral around neurofilament-positive axons. At the electron microscopic level, these linear structures were identified as the so-called interlamellar strands in myelin sheaths of oligodendrocytes. In testis, well-developed TJ strands of Sertoli cells were specifically labeled with anti–claudin-11/OSP pAb both at immunofluorescence and electron microscopic levels. These findings indicated that the interlamellar strands of oligodendrocyte myelin sheaths can be regarded as a variant of TJ strands found in many other epithelial cells, and that these strands share a specific claudin species, claudin-11/OSP, with those in Sertoli cells to create and maintain the repeated compartments around axons by oligodendrocytes.
Connexin43 Deficiency Causes Delayed Ossification, Craniofacial Abnormalities, and Osteoblast Dysfunction
Authors: Lecanda Fernando , Warlow Pamela M. , Sheikh Sharmin , Furlan Federico , Steinberg Thomas H. , Civitelli Roberto
Published: July 2002

Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell–cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987–995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249–2258). Here, using the Cx43-null mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43-null animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.
Hypoxia–Reoxygenation Triggers Coronary Vasospasm in Isolated Bovine Coronary Arteries via Tyrosine Nitration of Prostacyclin Synthase
Authors: Zou Ming-Hui , Bachschmid Markus
Published: July 2002

The role of peroxynitrite in hypoxia–reoxygenation-induced coronary vasospasm was investigated in isolated bovine coronary arteries. Hypoxia–reoxygenation selectively blunted prostacyclin (PGI2)-dependent vasorelaxation and elicited a sustained vasoconstriction that was blocked by a cyclooxygenase inhibitor, indomethacin, and SQ29548, a thromboxane (Tx)A2/prostaglandin H2 receptor antagonist, but not by CGS13080, a TxA2 synthase blocker. The inactivation of PGI2 synthase, as evidenced by suppressed 6-keto-PGF1α release and a decreased conversion of 14C-prostaglandin H2 into 6-keto-PGF1α, was paralleled by an increased nitration in both vascular endothelium and smooth muscle of hypoxia–reoxygenation-exposed vessels. The administration of the nitric oxide (NO) synthase inhibitors as well as polyethylene-glycolated superoxide dismutase abolished the vasospasm by preventing the inactivation and nitration of PGI2 synthase, suggesting that peroxynitrite was implicated. Moreover, concomitant administration to the organ baths of the two precursors of peroxynitrite, superoxide, and NO mimicked the effects of hypoxia–reoxygenation, although none of them were effective when given separately. We conclude that hypoxia–reoxygenation elicits the formation of superoxide, which causes loss of the vasodilatory action of NO and at the same time yields peroxynitrite. Subsequently, peroxynitrite nitrates and inactivates PGI2 synthase, leaving unmetabolized prostaglandin H2, which causes vasospasm, platelet aggregation, and thrombus formation via the TxA2/prostaglandin H2 receptor.
T Cells Can Use Either T Cell Receptor or Cd28 Receptors to Absorb and Internalize Cell Surface Molecules Derived from Antigen-Presenting Cells
Authors: Hwang Inkyu , Huang Jing-Feng , Kishimoto Hidehiro , Brunmark Anders , Peterson Per A. , Jackson Michael R. , Surh Charles D. , Cai Zeling , Sprent Jonathan
Published: July 2002

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)–peptide–major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell–APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.
Granzyme B–Mediated Cytochrome C Release Is Regulated by the Bcl-2 Family Members Bid and Bax
Authors: Heibein Jeffrey A. , Goping Ing Swie , Barry Michele , Pinkoski Michael J. , Shore Gordon C. , Green Douglas R. , Bleackley R. Chris
Published: July 2002

Cytotoxic T lymphocytes (CTLs) destroy target cells through a mechanism involving the exocytosis of cytolytic granule components including granzyme B (grB) and perforin, which have been shown to induce apoptosis through caspase activation. However, grB has also been linked with caspase-independent disruption of mitochondrial function. We show here that cytochrome c release requires the direct proteolytic cleavage of Bid by grB to generate a 14-kD grB-truncated product (gtBid) that translocates to mitochondria. In turn, gtBid recruits Bax to mitochondria through a caspase-independent mechanism where it becomes integrated into the membrane and induces cytochrome c release. Our results provide evidence for a new pathway by which CTLs inflict damage and explain the caspase-independent mechanism of mitochondrial dysfunction.
Self-similar sets in complete metric spaces
Authors: Schief Andreas
Published: July 2002

We develop a theory for Hausdorff dimension and measure of self-similar sets in complete metric spaces. This theory differs significantly from the well-known one for Euclidean spaces. The open set condition no longer implies equality of Hausdorff and similarity dimension of self-similar sets and that

K
K

has nonzero Hausdorff measure in this dimension. We investigate the relationship between such properties in the general case.
Frames, Riesz bases, and discrete Gabor/wavelet expansions
Authors: Christensen Ole
Published: July 2002

This paper is a survey of research in discrete expansions over the last 10 years, mainly of functions in

L
2

(

R

)

L^2(\mathbb R)

. The concept of an orthonormal basis

{

f
n

}

\{f_n\}

, allowing every function

f
∈

L
2

(

R

)

f \in L^2(\mathbb R)

to be written

f
=
∑

c
n

f
n

f=\sum c_nf_n

for suitable coefficients

{

c
n

}

\{c_n\}

, is well understood. In separable Hilbert spaces, a generalization known as frames exists, which still allows such a representation. However, the coefficients

{

c
n

}

\{c_n\}

are not necessarily unique. We discuss the relationship between frames and Riesz bases, a subject where several new results have been proved over the last 10 years. Another central topic is the study of frames with additional structure, most important Gabor frames (consisting of modulated and translated versions of a single function) and wavelets (translated and dilated versions of one function). Along the way, we discuss some possible directions for future research.