bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of acute in vivo human immunodeficiency virus infection by human interleukin 10 treatment of SCID mice implanted with human fetal thymus and liver.
Authors: Kollmann T R , Pettoello-Mantovani M , Katopodis N F , Hachamovitch M , Rubinstein A , Kim A , Goldstein H
Published: July 2002

To improve the usefulness of in vivo mode for the investigation of the pathophysiology of human immunodeficiency virus (HIV) infection, we modified the construction of SCID mice implanted with human fetal thymus and liver (thy/liv-SCID-hu mice) so that the peripheral blood of the mice contained significant numbers of human monocytes and T cells. After inoculation with HIV-1(59), a primary patient isolate capable of infecting monocytes and T cells, the modified thy/liv-SCID-hu mice developed disseminated HIV infection that was associated with plasma viremia. The development of plasma viremia and HIV infection in thy/liv-SCID-hu mice inoculated with HIV-1(59) was inhibited by acute treatment with human interleukin (IL) 10 but not with human IL-12. The human peripheral blood mononuclear cells in these modified thy/liv-SCID-hu mice were responsive to in vivo treatment with exogenous cytokines. Human interferon gamma expression in the circulating human peripheral blood mononuclear cells was induced by treatment with IL-12 and inhibited by treatment with IL-10. Thus, these modified thy/liv-SCID-hu mice should prove to be a valuable in vivo model for examining the role of immunomodulatory therapy in modifying HIV infection. Furthermore, our demonstration of the vivo inhibitory effect of IL-10 on acute HIV infection suggests that further studies may be warranted to evaluate whether there is a role for IL-10 therapy in preventing HIV infection in individuals soon after exposure to HIV such as for children born to HIV-infected mothers.
Thiocalsin: a thioredoxin-linked, substrate-specific protease dependent on calcium.
Authors: Besse I , Wong J H , Kobrehel K , Buchanan B B
Published: July 2002

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.
Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.
Authors: Jensen L G , Olsen O , Kops O , Wolf N , Thomsen K K , von Wettstein D
Published: July 2002

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.
Expression of Fas ligand in liver metastases of human colonic adenocarcinomas
Authors: Shiraki Katsuya , Tsuji Naoki , Shioda Toshi , Isselbacher Kurt J. , Takahashi Hiroshi
Published: July 2002

Fas ligand (FasL) plays a pivotal role in lymphocyte cytotoxicity and the maintenance of immunological homeostasis. Since FasL has been implicated in the existence of immunologically privileged body sites by inducing apoptosis of activated T lymphocytes, we investigated the expression of FasL in human colon cancers. We found that two out of seven primary tumors and all four hepatic metastatic tumors of surgically obtained colonic adenocarcinoma expressed FasL mRNA and protein, detected by reverse transcription-coupled PCR and by immunohistochemical staining, respectively. Expression of FasL was not detected in normal colonic epithelial cells. FasL mRNA was also expressed in some human colonic adenocarcinoma cell lines including SW480, SW1116, and LS180 cells. Cell-surface-associated FasL was detected in these human colon cancer cells by fluorescence immunocytochemical staining. In addition, the expressed FasL was demonstrated to be functional, since coculture experiments using FasL-expressing SW480 cells resulted in apoptosis of Jurkat T leukemia cells that are sensitive to Fas-mediated apoptosis, and this process was specifically inhibited by the neutralizing anti-human FasL antibody. Thus, our findings and other data suggest an alternative mechanism that enables tumors to evade immune destruction by inducing apoptosis in activated T lymphocytes. Furthermore, constitutive expression of FasL in hepatic metastatic tumors suggests that FasL may also be important in their colonization in the liver through induction of apoptosis in the surrounding Fas-expressing hepatocytes.
Mathematical approaches to comparative linguistics
Authors: Warnow Tandy
Published: July 2002

The inference of the evolutionary history of a set of languages is
a complex problem. Although some languages are known to be related
through descent from common ancestral languages, for other languages
determining whether such a relationship holds is itself a difficult
problem. In this paper we report on new methods, developed by linguists
Johanna Nichols (University of California, Berkeley), Donald Ringe and
Ann Taylor (University of Pennsylvania, Philadelphia), and me, for
answering some of the most difficult questions in this domain. These
methods and the results of the analyses based on these methods were
presented in November 1995 at the Symposium on the Frontiers of Science
held by the National Academy of Sciences.
Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p
Authors: Aalto Markku K. , Jäntti Jussi , Östling Jonas , Keränen Sirkka , Ronne Hans
Published: July 2002

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins,SSO1andSSO2, as suppressors of the temperature-sensitivesec1–1mutation. We now describe a third suppressor ofsec1–1, which we callMSO1. UnlikeSSO1andSSO2,MSO1is specific forsec1and does not suppress mutations in any otherSECgenes.MSO1encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lackMSO1are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss ofMSO1shows synthetic lethality with mutations inSEC1,SEC2, andSEC4, and other synthetic phenotypes with mutations in several other late-actingSECgenes. We further found that Mso1p interacts with Sec1p bothin vitroand in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.
Steroid receptor induction of gene transcription: A two-step model
Authors: Jenster Guido , Spencer Thomas E. , Burcin Mark M. , Tsai Sophia Y. , Tsai Ming-Jer , O’Malley Bert W.
Published: July 2002

Coactivators, such as steroid receptor coactivator 1 (SRC-1A) and CREB (cAMP response element binding protein)-binding protein (CBP), are required for efficient steroid receptor transactivation. Using an
in vitro
transcription assay, we found that progesterone receptor (PR)-driven transcription is inhibited by a dominant negative PR ligand-binding domain-interacting region of SRC-1A, indicating that SRC-1A is required for actual transcriptional processes. In addition, these coactivators also possess intrinsic histone acetyltransferase (HAT) activity and bind to each other and another HAT, p300/CBP-associated factor. Here we show that the human PR also interacts with p300/CBP-associated factor
in vitro
. Recruitment of multiple HATs to target promoters suggests an important role for chromatin remodeling in transcriptional activation of genes by steroid receptors. In transient transfection assays, we found that addition of a histone deacetylase inhibitor, trichostatin A, strongly potentiated PR-driven transcription. In contrast, directing histone deacetylase-1 (HD1) to a promoter using the GAL4 DNA binding domain inhibited transcription. Furthermore, PR transactivation was repressed by recruiting HD1 into the PR-DNA complex by fusing HD1 to a PR ligand-binding domain-interacting portion of SRC-1. Collectively, these results suggest that targeted histone acetylation by recruited HAT cofactors and histone deacetylation are important factors affecting PR transactivation. Recruitment of coactivators and HATs by the liganded PR
in vivo
may result in (
i
) remodeling of transcriptionally repressed chromatin to facilitate assembly and (
ii
) enhanced stabilization of the preinitiation complex by the activation functions of coactivators and the liganded PR itself.
Molecular cloning of a peroxisomal Ca
2+
-dependent member of the mitochondrial carrier superfamily
Authors: Weber Franz E. , Minestrini Gianluca , Dyer James H. , Werder Moritz , Boffelli Dario , Compassi Sabina , Wehrli Ernst , Thomas Richard M. , Schulthess Georg , Hauser Helmut
Published: July 2002

A cDNA from a novel Ca
2+
-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in
Escherichia coli
, and it was demonstrated that it bound Ca
2+
, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca
2+
which was bound with high affinity (
K
d
≈ 0.37 μM), the apparent Hill coefficient for Ca
2+
-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca
2+
binding N-terminal half of the transporter faces the cytosol.
Immunotargeting of liposomes to activated vascular endothelial cells: A strategy for site-selective delivery in the cardiovascular system
Authors: Spragg David D. , Alford Dennis R. , Greferath Ruth , Larsen Charles E. , Lee Kyung-Dall , Gurtner Geoffrey C. , Cybulsky Myron I. , Tosi P. Francois , Nicolau Claude , Gimbrone Michael A.
Published: July 2002

Endothelial-selective delivery of therapeutic agents, such as drugs or genes, would provide a useful tool for modifying vascular function in various disease states. A potential molecular target for such delivery is E-selectin, an endothelial-specific cell surface molecule expressed at sites of activationin vivoand inducible in cultured human umbilical vein endothelial cells (HUVEC) by treatment with cytokines such as recombinant human interleukin 1β (IL-1β). Liposomes of various types (classical, sterically stabilized, cationic, pH-sensitive), each conjugated with mAb H18/7, a murine monoclonal antibody that recognizes the extracellular domain of E-selectin, bound selectively and specifically to IL-1β-activated HUVEC at levels up to 275-fold higher than to unactivated HUVEC. E-selectin-targeted immunoliposomes appeared in acidic, perinuclear vesicles 2–4 hr after binding to the cell surface, consistent with internalization via the endosome/lysosome pathway. Activated HUVEC incubated with E-selectin-targeted immunoliposomes, loaded with the cytotoxic agent doxorubicin, exhibited significantly decreased cell survival, whereas unactivated HUVEC were unaffected by such treatment. These results demonstrate the feasibility of exploiting cell surface activation markers for the endothelial-selective delivery of biologically active agents via immunoliposomes. Application of this targeting approachin vivomay lead to novel therapeutic strategies in the treatment of cardiovascular disease.
Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage
Authors: Chan S. J. , Plisetskaya E. M. , Urbinati E. , Jin Y. , Steiner D. F.
Published: July 2002

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes
in vitro
to synthesize new cartilage matrix, measured by enhanced uptake of
35
S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of
35
S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed
in toto
in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.
A direct electrode-driven P450 cycle for biocatalysis
Authors: Reipa Vytautas , Mayhew Martin P. , Vilker Vincent L.
Published: July 2002

The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC
1.14.15.1
) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-
exo
-hydroxycamphor/nmol of CYP101 per min.
The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation
Authors: Visvader Jane E. , Mao Xiaohong , Fujiwara Yuko , Hahm Kyungmin , Orkin Stuart H.
Published: July 2002

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.
Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD
Authors: Claesson-Welsh Lena , Welsh Michael , Ito Nobuyuki , Anand-Apte Bela , Soker Shay , Zetter Bruce , O’Reilly Michael , Folkman Judah
Published: July 2002

Angiostatin, a fragment of plasminogen, has been identified and characterized as an endogenous inhibitor of neovascularization. We show that angiostatin treatment of endothelial cells in the absence of growth factors results in an increased apoptotic index whereas the proliferation index is unchanged. Angiostatin also inhibits migration and tube formation of endothelial cells. Angiostatin treatment has no effect on growth factor-induced signal transduction but leads to an RGD-independent induction of the kinase activity of focal adhesion kinase, suggesting that the biological effects of angiostatin relate to subversion of adhesion plaque formation in endothelial cells.
Anomalies in coral reef community metabolism and their potential importance in the reef CO
2
source-sink debate
Authors: Chisholm John R. M. , Barnes David J.
Published: July 2002

It is not certain whether coral reefs are sources of or sinks for atmospheric CO
2
. Air–sea exchange of CO
2
over reefs has been measured directly and inferred from changes in the seawater carbonate equilibrium. Such measurements have provided conflicting results. We provide community metabolic data that indicate that large changes in CO
2
concentration can occur in coral reef waters via biogeochemical processes not directly associated with photosynthesis, respiration, calcification, and CaCO
3
dissolution. These processes can significantly distort estimates of reef calcification and net productivity and obscure the contribution of coral reefs to global air–sea exchange of CO
2
. They may, nonetheless, explain apparent anomalies in the metabolic performance of reefs close to land and reconcile the differing experimental findings that have given rise to the CO
2
debate.
Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors
Authors: Jiang Zhi-hong , Zhang Wan-jiang , Rao Yi , Wu Jane Y.
Published: July 2002

The importance of alternative splicing in regulating apoptosis has been suggested by findings of functionally antagonistic proteins generated by alternative splicing of several genes involved in apoptosis. Among these, Ich-1 (also named as caspase-2) encodes a member of the caspase family of proteases. Two forms of Ich-1 are produced as a result of alternative splicing: Ich-1L, which causes apoptosis, and Ich-1S, which prevents apoptosis. The precise nature of Ich-1 alternative splicing and its regulation remain unknown. Here, we show that the production of Ich-1L and Ich-1S transcripts results from alternative exclusion or inclusion of a 61-bp exon. Several splicing factors can regulate Ich-1 splicing. Serine-arginine-rich proteins SC35 and ASF/SF2 promote exon skipping, decreasing the ratio of Ich-1S to Ich-1L transcripts; whereas heterogeneous nuclear ribonucleoprotein A1 facilitates exon inclusion, increasing this ratio. Furthermore, in cultured cells, SC35 overexpression increases apoptosis; whereas heterogeneous nuclear ribonucleoprotein A1 overexpression decreases apoptosis. These results provide the first direct evidence that splicing factors can regulate Ich-1 alternative splicing and suggest that alternative splicing may be an important regulatory mechanism for apoptosis.
Enzymatic reaction with unnatural substrates: DNA photolyase (
Escherichia coli
) recognizes and reverses thymine [2+2] dimers in the DNA strand of a DNA/PNA hybrid duplex
Authors: Ramaiah Danaboyina , Kan Yongzhi , Koch Troels , Ørum Henrik , Schuster Gary B.
Published: July 2002

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson–Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a
cis, sy
n-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.
Ultrasonic tagging of light: Theory
Authors: Mahan G. D. , Engler W. E. , Tiemann J. J. , Uzgiris E.
Published: July 2002

A theory is provided for the detection efficiency of diffuse light whose frequency is modulated by an acoustical wave. We derive expressions for the speckle pattern of the modulated light, as well as an expression for the signal-to-noise ratio for the detector. The aim is to develop a new imaging technology for detection of tumors in humans. The acoustic wave is focused into a small geometrical volume, which provides the spatial resolution for the imaging. The wavelength of the light wave can be selected to provide information regarding the kind of tumor.
Electrophoresis in lyotropic polymer liquid crystals
Authors: Rill Randolph L. , Locke Bruce R. , Liu Yingjie , Van Winkle David H.
Published: July 2002

Excellent electrophoretic separations of a variety of biological molecules can be accomplished by using uncharged, triblock copolymers as the “gel” media. These copolymers form uncrosslinked, lyotropic liquid crystalline phases of large micelles between which molecules must travel. Unlike crosslinked hydrogels in common use, these alternative media have highly ordered internal structures. Pluronic F127, representative of the copolymer class, contains poly(ethylene oxide) (EO) and poly(propylene oxide) (PO) units with an approximate molecular formula (EO)
106
(PO)
70
(EO)
106
. Concentrated (18–30%) solutions of Pluronic F127 are freely flowing liquids at low temperature (0–5°C) but form gel-like, cubic liquid crystals of large, spherical micelles when warmed. The utility of these media is illustrated by separations of linear, double-stranded DNA up to 3,000 bp long by conventional electrophoresis, and of single-stranded DNAs from 4 to 60 nt long by capillary electrophoresis. Extraordinary separations of supercoiled DNAs were also obtained by capillary electrophoresis. The versatility, availability, and ease of use of Pluronic polymers offer major advantages over conventional media for preparative and high performance analytical separations of nucleic acids and other biomolecules. Mechanisms of molecular transport and separation operating in polymer liquid crystals must differ in fundamental ways from those in crosslinked gels. Lyotropic polymer liquid crystals are unique systems for elucidating mechanisms of macromolecule migration in ordered, dense media, and provide opportunities in separations science.
Prevention of Th2-mediated murine allergic airways disease by soluble antigen administration in the neonate
Authors: Hogan Simon P. , Foster Paul S. , Charlton Brett , Slattery Robyn M.
Published: July 2002

It has been demonstrated recently that neonatal antigen administration in the mouse can lead to priming for Th2-mediated immune responses. This observation has important implications for the development of vaccination strategies in humans, particularly for individuals who may be predisposed to atopy or asthma. In this paper it is shown that although i.p. administration of antigen (100 μg) in adjuvant to the neonate does indeed prime for Th2-mediated disease in mice [allergic airways disease (AAD)], when the same relatively low dose of antigen is given in soluble form no priming occurs. Further, administration of a larger dose of soluble antigen (1 mg) actually prevents the ability to prime for a Th2 response subsequently and so prevents the induction of AAD. Protection from disease was associated with evidence of functional inactivation of both Th1 and Th2 ovalbumin-specific T cells. In contrast, administration of a very low dose of antigen (10 μg) primed for a Th2 response in a similar fashion to antigen in adjuvant. We suggest that the adjuvant lowers the “effective” dose of antigen administered in the neonate and thereby primes for Th2-type immune responses. These findings demonstrate that neonatal antigen administration can inhibit Th2-mediated diseases, such as AAD, but the dose of antigen may be critical to avoid predisposition to disease.
A protein with protective properties against the cellular defense reactions in insects
Authors: Asgari Sassan , Theopold Ulrich , Wellby Craig , Schmidt Otto
Published: July 2002

The molecular mechanism of how insects recognize intruding microorganisms and parasites and distinguish them from own body structures is not well known. We explored evolutionary adaptations in an insect parasitoid host interaction to identify components that interfere with the recognition of foreign objects and cellular encapsulation. Because some parasitoids provide protection for the developing wasp in the absence of an overt suppression of the insect host defense, we analyzed the surface of eggs and symbiotic viruses for protective properties. Here we report on the molecular cloning of a 32-kDa protein (Crp32) that is one of the major protective components. It is produced in the calyx cells of the female wasp ovaries and attached to the surface of the egg and other particles including polydnaviruses. The recombinant protein confers protection to coated objects in a cellular encapsulation assay suggesting that a layer of Crp32 may prevent cellular encapsulation reactions by a local inactivation of the host defense system.
Tissue factor is required for uterine hemostasis and maintenance of the placental labyrinth during gestation
Authors: Erlich Jonathan , Parry Graham C. N. , Fearns Colleen , Muller Martin , Carmeliet Peter , Luther Thomas , Mackman Nigel
Published: July 2002

We employed a novel mouse line that expresses low levels of human tissue factor (TF) in the absence of murine TF to analyze the role of TF in gestation. Low-TF female mice had a 14–18% incidence of fatal postpartum uterine hemorrhage, suggesting that TF plays an important role in uterine hemostasis. Low-TF female mice mated with low-TF male mice had a 42% incidence of fatal midgestational hemorrhage (
n
= 41), whereas no fatal midgestational hemorrhages were observed in low-TF female mice mated with wild-type male mice (
n
= 43). Placentas of low-TF embryos from both low-TF and normal (+/−) TF females were abnormal and contained numerous maternal blood pools in the labyrinth. Placentas of TF null embryos surviving beyond embryonic day 10.5 exhibited similar defects. The mouse maternal–embryonic placental barrier consists of four cellular layers (layers I, II, and III and endothelial cells), where layer I lines the maternal lacunae. Comparison of TF-deficient placentas with control placentas by immunohistochemical and ultrastructural analyses revealed thinning of layer I and a reduction in the number of cellular contacts of layer I trophoblasts spanning the maternal blood space between adjacent trabeculae. These structural changes in low-TF and TF null placentas result in enlarged maternal lacunae, as determined by morphometric analysis, and placental hemorrhage, which leads to midgestational death of low-TF female mice. This study demonstrated that TF is required for uterine hemostasis and revealed an unexpected role of TF in the maintenance of the placental labyrinth.
The mouse bagpipe gene controls development of axial skeleton, skull, and spleen
Authors: Lettice Laura A. , Purdie Lorna A. , Carlson Geoffrey J. , Kilanowski Fiona , Dorin Julia , Hill Robert E.
Published: July 2002

The mouse
Bapx1
gene is homologous to the
Drosophila
homeobox containing
bagpipe
(
bap
) gene. A shared characteristic of the genes in these two organisms is expression in gut mesoderm. In
Drosophila, bap
functions to specify the formation of the musculature of the midgut. To determine the function of the mammalian cognate, we targeted a mutation into the
Bapx1
locus. Bapx1, similar to
Drosophila
, does have a conspicuous role in gut mesoderm; however, this appears to be restricted to development of the spleen. In addition,
Bapx1
has a major role in the development of the axial skeleton. Loss of
Bapx1
affects the distribution of sclerotomal cells, markedly reducing the number that appear ventromedially around the notochord. Subsequently, the structures in the midaxial region, the intervertebral discs, and centra of the vertebral bodies, fail to form. Abnormalities are also found in those bones of the basal skull (basioccipital and basisphenoid bones) associated with the notochord. We postulate that
Bapx1
confers the capacity of cells to interact with the notochord, effecting inductive interactions essential for development of the vertebral column and chondrocranium.
A direct role for arrestins in desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes
Authors: Mukherjee Sutapa , Palczewski Krzysztof , Gurevich Vsevolod , Benovic Jeffrey L. , Banga J. Paul , Hunzicker-Dunn Mary
Published: July 2002

The luteinizing hormone/choriogonadotropin (LH/CG) receptor (R) is a heptahelical R that, upon agonist binding, activates the stimulatory guanine nucleotide-binding protein (G
s
) and the downstream effector adenylyl cyclase (AC). Like other G protein-coupled Rs, the LH/CG R subsequently exhibits reduced agonist-dependent effector activity, or desensitization, in response to saturating agonist. Unlike desensitization of many other G protein-coupled Rs, the
in vivo
desensitization response of LH/CG R-stimulated AC activity of ovarian follicles to the preovulatory surge of LH can be mimicked under cell-free conditions. Based on evidence that porcine ovarian follicular membranes unexpectedly contained β-arrestin-1, the role of arrestins in desensitization of the LH/CG R was investigated. Results showed that neutralizing arrestin antibodies blocked the development of desensitization and that desensitization was rescued with a synthetic peptide corresponding to the antibody-binding epitope on β-arrestin-1. These results suggest that endogenous β-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R. Addition of recombinant purified β-arrestin-1 mimicked human chorionic gonadotrophin to promote desensitization of human chorionic gonadotrophin-stimulated AC activity, in the presence of the ATP phosphorylation antagonist adenylyl-imidodiphosphate, with an ED
50
of ≈0.1 nM. Increased levels of an 87-kDa protein reactive with glycoprotein hormone R-reactive antibody, consistent with the LH/CG R, coimmunoprecipitated with follicular membrane β-arrestin-1 in response to LH/CG R activation compared with unactivated R. Taken together, these results show that ovarian follicles contain membrane-associated β-arrestin-1, that β-arrestin-1 participates in agonist-dependent desensitization of the LH/CG R, and that the trigger for β-arrestin-1 binding to the LH/CG R appears to be R activation.
Mechanisms for induction of acquired host immunity by neutrophil peptide defensins
Authors: Lillard James W. , Boyaka Prosper N. , Chertov Oleg , Oppenheim Joost J. , McGhee Jerry R.
Published: July 2002

Human neutrophil peptide (HNP) defensins were studied to determine their potential effects on adaptive mucosal immunity. Intranasal delivery of HNPs plus ovalbumin (OVA) enhanced OVA-specific serum IgG antibody (Ab) responses. However, OVA-specific IgA Abs were not induced in mucosal secretions or in serum. CD4+T cells of intranasally immunized mice displayed higher OVA-specific proliferative responses and elevated production of interferon γ, interleukin (IL) 5, IL-6, and IL-10 when compared with control groups receiving OVA alone.In vitro, HNPs also enhanced both proliferative responses and T helper (Th) cytokine secretion profiles of CD3ɛ-stimulated spleen- and Peyer’s patch-derived naive CD4+T cells. HNPs modulated the expression of costimulatory molecules by lipopolysaccharide- or CD3ɛ-stimulated splenic and Peyer’s patch B or T cell populations, respectively. These studies show that defensins enhance systemic IgG, but not IgA, Ab responses through help provided by CD4+Th1- and Th2-type cytokines and foster B and T cell interactions to link innate immunity with the adaptive immune system.
On the role of the weather in the deaths of R. F. Scott and his companions
Authors: Solomon Susan , Stearns Charles R.
Published: July 2002

Robert Falcon Scott and his companions reached the South Pole in January of 1912, only to die on their return journey at a remote site on the Ross Ice Shelf, about 170 miles from their base camp on the coast. Numerous contributing causes for their deaths have been proposed, but it has been assumed that the cold temperatures they reported encountering on the Ross Ice Shelf, near 82–80°S during their northward trek toward safety, were not unusual. The weather in the region where they perished on their unassisted trek by foot from the Pole remained undocumented for more than half a century, but it has now been monitored by multiple automated weather stations for more than a decade. The data recorded by Scott and his men from late February to March 19, 1912, display daily temperature minima that were on average 10 to 20°F below those obtained in the same region and season since routine modern observations began in 1985. Only 1 year in the available 15 years of measurements from the location where Scott and his men perished displays persistent cold temperatures at this time of year close to those reported in 1912. These remarkably cold temperatures likely contributed substantially to the exhaustion and frostbite Scott and his companions endured, and their deaths were therefore due, at least in part, to the unusual weather conditions they endured during their cold march across the Ross Ice Shelf of Antarctica.
A role for FEN-1 in nonhomologous DNA end joining: The order of strand annealing and nucleolytic processing events
Authors: Wu Xiantuo , Wilson Thomas E. , Lieber Michael R.
Published: July 2002

Eukaryotic repair of double-strand DNA breaks can occur
either by homologous recombination or by nonhomologous DNA end joining
(NHEJ). NHEJ relies on Ku70/86, XRCC4, DNA ligase IV, and
DNA-dependent protein kinase. NHEJ involves a synapsis step in which
the two ends are maintained in proximity, processing steps in which
nucleases and polymerases act on the ends, an alignment step in which a
few nucleotides of terminal homology guide the ends into preferred
alignments, and a ligation step. Some of the steps, such as ligation,
rely on a single enzymatic component. However, the processing steps
begin and end with a wide array of alternative substrates and products,
respectively, and likely involve multiple nucleases and polymerases.
Given the alternative pathways that can be catalyzed by the remaining
nucleases and polymerases, no one of these processing enzymes is likely
to be essential. The only requirement for the processing enzymes, as a
collective, is to generate a ligatable configuration, namely a
ligatable nick on each strand. Here, we have tested the two major known
5′-specific nucleases of
Saccharomyces cerevisiae
for
involvement in NHEJ. Whereas
EXO1
does not appear to be
involved to any detectable level, deleting
RAD27
(FEN-1
of yeast) leads to a 4.4-fold reduction specifically of those NHEJ
events predicted to proceed by means of 5′ flap intermediates. Because
Rad27/FEN-1 acts specifically at 5′ flap structures, these results
suggest that the NHEJ alignment step precedes nucleolytic processing
steps in a significant fraction of NHEJ events.
Transforming growth factor β stimulation of colorectal cancer cell lines: Type II receptor bypass and changes in adhesion molecule expression
Authors: Ilyas Mohammad , Efstathiou Jason A. , Straub Josef , Kim Hee C. , Bodmer Walter F.
Published: July 2002

The type II transforming growth factor (TGF)-β receptor gene (
TGFBR2
) is often mutated in nucleotide repeat sequences in colorectal cancers that are replication error positive (RER+). These mutations are thought to be selected for escape from growth inhibition by TGF-β rather than representing bystander events because of an increased mutation rate. We investigated the role of
TGFBR2
mutations in 12 colorectal cancer cell lines. Six of these were RER+, and these were shown to have homozygous
TGFBR2
mutations. All cell lines then were tested for changes in proliferation in response to TGF-β stimulation. Despite homozygous mutation of the type II TGF-β receptor, two RER+ cell lines, Lovo and SW48, showed statistically significant growth inhibition when stimulated by TGF-β1 in serum-free conditions. This shows that the type II TGF-β receptor can be bypassed in certain cases to maintain growth inhibition. We next investigated whether there was any alternative mode through which
TGFBR2
mutation may give a selective advantage, such as a change in adhesion molecule expression. All cell lines were stimulated with TGF-β1 and adhesion molecules detected by ELISA. No consistent changes were identified between the RER+ and the RER− cell lines, although changes in E-cadherin, β-catenin, and γ-catenin were identified in individual cell lines. We conclude that (
i
) type II TGF-β receptor activity can be bypassed and thus
TGFBR2
mutations in RER+ cancers may, at least sometimes, be just “bystander” events and (
ii
) TGF-β can affect adhesion molecule expression so that
TGFBR2
mutation may give rise to a selective advantage through an effect other escape from growth inhibition.
Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4
Authors: Schatzle John D. , Sheu Shane , Stepp Susan E. , Mathew Porunelloor A. , Bennett Michael , Kumar Vinay
Published: July 2002

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and γ-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.
Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages by production of peroxynitrite
Authors: Hickman-Davis Judy , Gibbs-Erwin Julie , Lindsey J. Russell , Matalon Sadis
Published: July 2002

We have previously shown that surfactant protein A (SP-A) mediatesin vitrokilling of mycoplasmas by alveolar macrophages (AMs) from resistant C57BL/6 mice through a nitric oxide (⋅NO)-dependent mechanism. Herein, SP-A-deficient [SP-A(−/−)] and inducible ⋅NO synthase-deficient [iNOS(−/−)] mice were infected intranasally with 105or 107colony-forming units ofMycoplasma pulmonis. SP-A(−/−) mice were as susceptible to mycoplasmal infection as highly susceptible C3H/He mice, and far more susceptible than resistant C57BL/6 mice. iNOS(−/−) mice had significantly greater numbers of mycoplasmas and severity of lung lesions than iNOS(+/+) controls.In vitro, AMs isolated from C57BL/6 mice, activated with IFN-γ, incubated with SP-A (25 μg/ml), and infected with 1010colony-forming units ofM. pulmonis, killed mycoplasmas within 6 h. Mycoplasmal killing was abrogated by 1,000 units/ml of copper-zinc superoxide dismutase. In the absence of AMs, incubation ofM. pulmoniswith the peroxynitrite generator 3-morpholinosynodiomine⋅HCl (SIN-1) effected complete killing of mycoplasmas by 90 min in a dose-dependent manner. Addition of copper-zinc superoxide dismutase (3,000 units/ml), which converts SIN-1 to a ⋅NO donor, prevented this killing. Neither of the reactive oxygen species generated by xanthine oxidase (10 milliunits/ml, plus 500 μM xanthine and 100 μM FeCl3), nor ⋅NO generated by 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA NONOate) (100 μM) killed mycoplasmas. These data establish that peroxynitrite generation by AMs is necessary for the killing of a pathogenin vitroandin vivo.
Rapid antibody responses by low-dose, single-step, dendritic cell-targeted immunization
Authors: Wang Hui , Griffiths Michelle N. , Burton Dennis R. , Ghazal Peter
Published: July 2002

We have compared the kinetics of antibody responses in conventional and dendritic cell-targeted immunization by using a model antigen in mice. Targeting was achieved by linking the reporter antigen (polyclonal goat anti-hamster antibody) to N418, a hamster mAb that binds to the CD11c molecule on the surface of murine dendritic cells. Intradermal injection of submicrogram quantities of goat anti-hamster antibody complexed to mAb N418 elicited goat antibody-specific serum IgG in mice. Antigen-specific IgG titers were detectable by day 5, with titers that ranged from 1:1000 to 1:100,000 by day 7. In contrast, when the goat antigen was injected alone or in the presence of a hamster antibody control to form nontargeted complexes, goat-specific serum IgG was undetectable at day 7. Additional control experiments showed that the interaction between the model antigen and mAb N418 is required for amplification of the serum antibody response. These studies demonstrate that a single-step, facilitated-delivery of small amounts of protein antigen to dendritic cellsin vivocan give very rapid and high antibody responses. The approach may be particularly useful for vaccination immediately before or just after exposure to a pathogen and may enhance the utility of subunit antigens as immunogens.
Heteroduplex mobility assay-guided sequence discovery: Elucidation of the small subunit (18S) rDNA sequences of
Pfiesteria piscicida
and related dinoflagellates from complex algal culture and environmental sample DNA pools
Authors: Oldach David W. , Delwiche Charles F. , Jakobsen Kjetill S. , Tengs Torstein , Brown Ernest G. , Kempton Jason W. , Schaefer Eric F. , Bowers Holly A. , Glasgow Howard B. , Burkholder JoAnn M. , Steidinger Karen A. , Rublee Parke A.
Published: July 2002

The newly described heterotrophic estuarine dinoflagellate
Pfiesteria piscicida
has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in
Pfiesteria
research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect
Pfiesteria piscicida
18S rDNA “signature” sequences in complex pools of DNA and used those data as the basis for determination of the complete
P. piscicida
18S rDNA sequence. Specific PCR assays for
P. piscicida
and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.
Harnessing Community Self-Help: Some Lessons from Rural England
Authors: Williams Colin C.
Published: July 2002

Attempts to harness community self-help currently tend to follow the ‘third sector’ route of developing existing community-based groups. The aim of this paper is to evaluate critically this approach. Drawing upon case study evidence from rural England, existing community-based groups are displayed to be primarily sociability vehicles for higher-income populations. In consequence, if community self-help is to be harnessed to improve the material circumstances of lower-income populations, a ‘third sector’ approach is inappropriate. Instead, a ‘fourth sector’ approach is shown to be required that develops acts of one-to-one reciprocity. The paper concludes by outlining some possible initiatives to implement this policy approach.
New Products of the 1980s and 1990s: The Diffusion of Household Technology in the Decade 1985-1995
Authors: Ironmonger D. S. , Lloyd-Smith C. W. , Soupourmas F.
Published: July 2002

The management of our households and the way we spend our leisure time has been greatly influenced by the introduction of various household technologies in the 1980s and 1990s. This paper compares the extent and rate of adoption of selected household products in Australian households. The diffusion of colour television sets, video cassette recorders, compact disc players, microwave ovens and personal computers into various types of households during the 1980s and 1990s was examined. This study found that different types of Australian households adopted new technology at different rates. The levels of ownership of various household technologies were highest for households with children. In general, one-adult households were slower to adopt new household technology when compared to other types of households. This paper speculates as to why one-person households are slower to purchase the latest in cooking and entertainment technology.