bluuebunny/crossref_metadata_2025 huggingface dataset:
A corrinoid-dependent catabolic pathway for growth of aMethylobacteriumstrain with chloromethane
Authors: Vannelli Todd , Messmer Michael , Studer Alex , Vuilleumier Stéphane , Leisinger Thomas
Published: July 2002

Methylobacteriumsp. strain CM4, an aerobic methylotrophic α-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu:chloromethaneutilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described inMethylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.
Conservation of sequence and structure flanking the mouse and human β-globin loci: The β-globin genes are embedded within an array of odorant receptor genes
Authors: Bulger Michael , von Doorninck J. Hikke , Saitoh Noriko , Telling Agnes , Farrell Catherine , Bender M. A. , Felsenfeld Gary , Axel Richard , Groudine Mark
Published: July 2002

In mouse and human, the β-globin genes reside in a linear array that is associated with a positive regulatory element located 5′ to the genes known as the locus control region (LCR). The sequences of the mouse and human β-globin LCRs are homologous, indicating conservation of an essential function in β-globin gene regulation. We have sequenced regions flanking the β-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of ≈40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human β-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3′ of the β-globin locus in chicken. The data demonstrate an evolutionarily conserved genomic organization for the β-globin locus and suggest a possible role for the β-globin LCR in control of expression of these odorant receptor genes and/or the presence of mechanisms to separate regulatory signals in different tissues.
Reversibility of the pathological changes in the follicular dendritic cell network with treatment of HIV-1 infection
Authors: Zhang Zhi-Qiang , Schuler Troy , Cavert Winston , Notermans Daan W. , Gebhard Kristin , Henry Keith , Havlir Diane V. , Günthard Huldrych F. , Wong Joseph K. , Little Susan , Feinberg Mark B. , Polis Michael A. , Schrager Lewis K. , Schacker Timothy W. , Richman Douglas D. , Corey Lawrence , Danner Sven A. , Haase Ashley T.
Published: July 2002

Over the course of HIV-1 infection, the lymphoid follicles where the humoral immune response is generated initially increase in size and number and then progressively involute. In advanced disease, the network of the processes of follicular dendritic cells (FDCs) that serve as antigen repositories and anatomical substrate for B and T cells and antigen to interact is destroyed, contributing to the breakdown of the immune system. Because destruction of FDCs is associated with deposition of HIV-1, and much of the virus can be cleared from the network with antiretroviral therapy, we investigated the reversibility of damage. We measured the immunohistochemically stainable FDC compartment by quantitative image analysis, and we documented changes in this compartment at different stages of disease. We show that treatment, initiated even at advanced stages of HIV-1 disease, can slowly reverse pathological changes in the FDC network.
Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome
Authors: Almazán Fernando , González José M. , Pénzes Zoltan , Izeta Ander , Calvo Enrique , Plana-Durán Juan , Enjuanes Luis
Published: July 2002

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.
Purifying selection and birth-and-death evolution in the ubiquitin gene family
Authors: Nei Masatoshi , Rogozin Igor B. , Piontkivska Helen
Published: July 2002

Ubiquitin is a highly conserved protein that is encoded by a multigene family. It is generally believed that this gene family is subject to concerted evolution, which homogenizes the member genes of the family. However, protein homogeneity can be attained also by strong purifying selection. We therefore studied the proportion (pS) of synonymous nucleotide differences between members of the ubiquitin gene family from 28 species of fungi, plants, and animals. The results have shown thatpSis generally very high and is often close to the saturation level, although the protein sequence is virtually identical for all ubiquitins from fungi, plants, and animals. A small proportion of species showed a low level ofpSvalues, but these values appeared to be caused by recent gene duplication. It was also found that the number of repeat copies of the gene family varies considerably with species, and some species harbor pseudogenes. These observations suggest that the members of this gene family evolve almost independently by silent nucleotide substitution and are subjected to birth-and-death evolution at the DNA level.
Morphological castes in a vertebrate
Authors: O'Riain M. J. , Jarvis J. U. M. , Alexander R. , Buffenstein R. , Peeters C.
Published: July 2002

Morphological specialization for a specific role has, until now,
been assumed to be restricted to social invertebrates. Herein we show
that complete physical dimorphism has evolved between reproductives and
helpers in the eusocial naked mole-rat. Dimorphism is a consequence of
the lumbar vertebrae lengthening after the onset of reproduction in
females. This is the only known example of morphological castes in a
vertebrate and is distinct from continuous size variation between
breeders and helpers in other species of cooperatively breeding
vertebrates. The evolution of castes in a mammal and insects represents
a striking example of convergent evolution for enhanced fecundity in
societies characterized by high reproductive skew. Similarities in the
selective environment between naked mole-rats and eusocial insect
species highlight the selective conditions under which queen/worker
castes are predicted to evolve in animal societies.
UL82 virion protein activates expression of immediate early viral genes in human cytomegalovirus-infected cells
Authors: Bresnahan Wade A. , Shenk Thomas E.
Published: July 2002

The human cytomegalovirus UL82 gene encodes a protein (pp71) that
is localized in the tegument domain of the virus particle. The UL82
gene product is delivered to the nucleus at the time of infection, and
it is believed to function in gene activation. We have constructed a
human cytomegalovirus mutant, AD
sub
UL82, that lacks a
substantial portion of the UL82 coding region. It was propagated on
human diploid fibroblasts expressing the UL82 gene product, and it was
possible to produce a mutant virus lacking the UL82 protein by
passaging virus stocks for one cycle of growth on normal,
noncomplementing fibroblasts. The UL82-deficient mutant displays a
multiplicity-dependent growth defect in normal human fibroblasts. The
growth of AD
sub
UL82 is severely restricted at low input
multiplicities (0.01–0.1 plaque-forming units per cell), producing a
yield that is reduced by a factor of about 10
5
in
comparison to wild-type virus. At higher input multiplicities (10
plaque-forming units per cell), AD
sub
UL82 grew nearly as
well as the wild-type virus. By using a human cytomegalovirus gene
array, we demonstrated that UL82 functions to facilitate virus mRNA
accumulation very early during the human cytomegalovirus replication
cycle. The growth phenotype associated with the UL82 mutant seems to
result from its inability to efficiently activate human cytomegalovirus
immediate early genes.
Twist fields, the elliptic genus, and hidden symmetry
Authors: Jaffe Arthur
Published: July 2002

We combine infinite dimensional analysis (in particular
a priori
estimates and twist positivity) with classical geometric structures, supersymmetry, and noncommutative geometry. We establish the existence of a family of examples of two-dimensional, twist quantum fields. We evaluate the elliptic genus in these examples. We demonstrate a hidden SL(2,ℤ) symmetry of the elliptic genus, as suggested by Witten.
Inward Investment and Economic Regeneration: Listening to Workers in Rhondda-Cynon-Taff
Authors: Cato Molly Scott
Published: July 2002

The paper presents the results of a two-year research study conducted in the Rhondda-Cynon-Taff local authority area of South Wales. The research focused on assessing the effect of the inward-investment strategy on local economic regeneration and local people's reaction to the policy. The paper reports survey questionnaire results an d the interview responses of local employees. A summary of the recent employment history of the area is followed by a discussion of policies introduced to tackle unemployment. Local people express cynicism about foreign companies, suggesting that the grants they receive are more important to them than genuine commitment to the regeneration of the local economy. The feeling was expressed that the balance of fun ding priorities works against local entrepreneurs, while the concentration on ‘foreign saviours’ also undermines their confidence. The paper concludes by recommending several new approaches to tackling unemployment in RCT: focusing on Tower Colliery as a ‘local champion’; emphasizing ‘associative entrepreneurship’ that prioritizes the needs of the community rather than the success of the individual; and introducing a Citizen s' In come scheme to tackle the problem of economic inactivity.
Two-Generation Reproduction Study and Immunotoxicity Screen in Rats Dosed with Phenol via the Drinking Water
Authors: Ryan B. M. , Selby R. , Gingell R. , Waechter J. M. , Butala J. H. , Dimond S. S. , Dunn B. J. , House R. , Morrissey R.
Published: July 2002

This study evaluated the potential reproductive toxicity of phenol in a rat two-generation reproduction study, which included additional study endpoints, such as sperm count and motility, developmental landmarks, histological evaluation of suspect target organs (liver, kidneys, spleen, and thymus), weanling reproductive organ weights, and an immunotoxicity screening plaque assay. Phenol was administered to 30 Sprague-Dawley rats/sex/group in the drinking water at concentrations of 0, 200, 1000, or 5000 ppm. Parental (P1) animals were treated for 10 weeks prior to mating, during mating, gestation, lactation, and until sacrifice. The F1 generation (P1 offspring) was treated using a similar regimen, while the F2 generation was not treated. After mating, 10 P1 males/group were evaluated using standard clinical pathology parameters and an immunotoxicity screening plaque assay. Significant reductions in water and food consumption were observed in the 5000-ppm group in both generations; corollary reductions in body weight/body weight gain were also observed. Mating performance and fertility in both generations were similar to controls, and no adverse effects on vaginal cytology or male reproductive function were observed. Vaginal opening and preputial separation were delayed in the 5000-ppm group, and were considered to be secondary to the reduction in F1 body weight. Litter survival of both generations was reduced in the 5000-ppm group. Absolute uterus and prostate weights were decreased in the F1 generation at all dose levels; however, no underlying pathology was observed and there was no functional deficit in reproductive performance. Therefore, these findings were not considered to be adverse. No evidence of immunotoxicity was noted in the 5000-ppm group. The effects noted at the high concentration were presumed to be associated with flavor aversion to phenol in the drinking water. Based on a comprehensive examination of all parameters, the no-observable-adverse-effect level (NOAEL) for reproductive toxicity of phenol administered in drinking water to rats is 1000 ppm. The corresponding daily intake of phenol for an adult rat at the NOAEL of 1000 ppm is equivalent to about 70 mg/kg/day for males and 93 mg/kg/day for females.
Animal Models Used in Identifying Gender-Related Differences
Authors: Curry Bishop B.
Published: July 2002

There is general agreement in the scientific community on the need to identify appropriate animal models that can be used to screen for gender-based differences. At the same time, there is a growing expectation for data from these models to mimic or be more predictive of the human experience. The species in this review will include nonhuman primates, rats, mice, rabbits, swine, hamsters, gerbils, quail, and fish. Although some of the models are unique, the gender-related differences, in most instances, may be correlated with man, due to the conservation of biological systems across species.
Multiple Chemical Sensitivities: Psychogenic or Toxicodynamic Origins
Authors: Gots Ronald E. , Pirages Suellen W.
Published: July 2002

The multiple chemical sensitivity (MCS) phenomenon can cause significant dysfunction and symptomatology and presents a difficult challenge for patient management. Central to the MCS debate is whether this phenomenon results from a primary emotional response to perceived chemical exposures or from pathological interactions between chemicals and biological systems. Those who believe the latter argue that toxic interactions result in physiological impairment and that subsequent emotional problems derive from such impairment. Distinguishing between psychogenic (emotional) or a toxicodynamic (chemical toxicity) origin is essential to the medical management of an MCS patient. A psychogenic basis requires treatment with appropriate behavioral therapies; in contrast, a belief in a strictly toxicodynamic etiology argues for avoidance and often precludes treatments that address the psychological responses. Current scientific evidence strongly suggests that behavioral or psychogenic explanations predominate for reported MCS symptoms. Acceptance of a purely toxic origination (i.e., pathological abnormalities result from a low level chemical exposure) defies known toxicological and medical principles; whereas psychogenic explanations are consistent with these principles. Because symptoms are the end points of many diseases with many causes, both physical and emotional, modern medicine is charged with and expected to consider both when treating MCS patients. The argument can be made that insufficient information exists about the causal nature of many diseases, and future research may provide support for a strict toxicodynamic cause. However, the practice of medicine must be based upon current knowledge, not future possibilities. Proper care of MCS patients requires identifying the existence of both psychological and organic pathological dysfunction. The rejection of a psychological aspect of the MCS phenomenon and appropriate behavioral treatments is both illogical and detrimental to MCS sufferers.
Differential Targeting of Vesicular Stomatitis Virus G Protein and Influenza Virus Hemagglutinin Appears During Myogenesis of L6 Muscle Cells
Authors: Rahkila P. , Luukela V. , Väänänen K. , Metsikkö K.
Published: July 2002

Exocytic organelles undergo profound reorganization during myoblast differentiation and fusion. Here, we analyzed whether glycoprotein processing and targeting changed during this process by using vesicular stomatitis virus (VSV) G protein and influenza virus hemagglutinin (HA) as models. After the induction of differentiation, the maturation and transport of the VSV G protein changed dramatically. Thus, only half of the G protein was processed and traveled through the Golgi, whereas the other half remained unprocessed. Experiments with the VSV tsO45 mutant indicated that the unprocessed form folded and trimerized normally and then exited the ER. It did not, however, travel through the Golgi since brefeldin A recalled it back to the ER. Influenza virus HA glycoprotein, on the contrary, acquired resistance to endoglycosidase H and insolubility in Triton X-100, indicating passage through the Golgi. Biochemical and morphological assays indicated that the HA appeared at the myotube surface. A major fraction of the Golgi-processed VSV G protein, however, did not appear at the myotube surface, but was found in intracellular vesicles that partially colocalized with the regulatable glucose transporter. Taken together, the results suggest that, during early myogenic differentiation, the VSV G protein was rerouted into developing, muscle-specific membrane compartments. Influenza virus HA, on the contrary, was targeted to the myotube surface.
The UDP-Glc:Glycoprotein Glucosyltransferase Is Essential for Schizosaccharomyces pombe Viability under Conditions of Extreme Endoplasmic Reticulum Stress
Authors: Fanchiotti Sandra , Fernández Fabiana , D'Alessio Cecilia , Parodi Armando J.
Published: July 2002

Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28°C. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28°C and did not grow at 37°C. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37°C and had, when grown at 28°C, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation–deglucosylation catalyzed by GT and GII.
The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL)
Authors: West Kip A. , Zhang Huaye , Brown Michael C. , Nikolopoulos Sotiris N. , Riedy M.C. , Horwitz Alan F. , Turner Christopher E.
Published: July 2002

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinΔLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin–PKL interaction. In cells overexpressing paxillinΔLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLΔPBS2) induced phenotypic changes reminiscent of paxillinΔLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.
Centromere identity in Drosophila is not determined in vivo by replication timing
Authors: Sullivan Beth , Karpen Gary
Published: July 2002

Centromeric chromatin is uniquely marked by the centromere-specific histone CENP-A. For assembly of CENP-A into nucleosomes to occur without competition from H3 deposition, it was proposed that centromeres are among the first or last sequences to be replicated. In this study, centromere replication in Drosophila was studied in cell lines and in larval tissues that contain minichromosomes that have structurally defined centromeres. Two different nucleotide incorporation methods were used to evaluate replication timing of chromatin containing CID, a Drosophila homologue of CENP-A. Centromeres in Drosophila cell lines were replicated throughout S phase but primarily in mid S phase. However, endogenous centromeres and X-derived minichromosome centromeres in vivo were replicated asynchronously in mid to late S phase. Minichromosomes with structurally intact centromeres were replicated in late S phase, and those in which centric and surrounding heterochromatin were partially or fully deleted were replicated earlier in mid S phase. We provide the first in vivo evidence that centromeric chromatin is replicated at different times in S phase. These studies indicate that incorporation of CID/CENP-A into newly duplicated centromeres is independent of replication timing and argue against determination of centromere identity by temporal sequestration of centromeric chromatin replication relative to bulk genomic chromatin.
The replication timing program of the Chinese hamster β-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase
Authors: Li Feng , Chen Jianhua , Izumi Masako , Butler Mark C. , Keezer Susan M. , Gilbert David M.
Published: July 2002

We have examined the dynamics of nuclear repositioning and the establishment of a replication timing program for the actively transcribed dihydrofolate reductase (DHFR) locus and the silent β-globin gene locus in Chinese hamster ovary cells. The DHFR locus was internally localized and replicated early, whereas the β-globin locus was localized adjacent to the nuclear periphery and replicated during the middle of S phase, coincident with replication of peripheral heterochromatin. Nuclei were prepared from cells synchronized at various times during early G1 phase and stimulated to enter S phase by introduction into Xenopus egg extracts, and the timing of DHFR and β-globin replication was evaluated in vitro. With nuclei isolated 1 h after mitosis, neither locus was preferentially replicated before the other. However, with nuclei isolated 2 or 3 h after mitosis, there was a strong preference for replication of DHFR before β-globin. Measurements of the distance of DHFR and β-globin to the nuclear periphery revealed that the repositioning of the β-globin locus adjacent to peripheral heterochromatin also took place between 1 and 2 h after mitosis. These results suggest that the CHO β-globin locus acquires the replication timing program of peripheral heterochromatin upon association with the peripheral subnuclear compartment during early G1 phase.
High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis
Authors: Beqaj Safedin , Jakkaraju Sandhya , Mattingly Raymond R. , Pan Desi , Schuger Lucia
Published: July 2002

Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621–2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027–3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321–1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341–1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase–PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.
Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium
Authors: Miyoshi Keiko , Shillingford Jonathan M. , Smith Gilbert H. , Grimm Sandra L. , Wagner Kay-Uwe , Oka Takami , Rosen Jeffrey M. , Robinson Gertraud W. , Hennighausen Lothar
Published: July 2002

Functional development of mammary epithelium during pregnancy depends on prolactin signaling. However, the underlying molecular and cellular events are not fully understood. We examined the specific contributions of the prolactin receptor (PrlR) and the signal transducers and activators of transcription 5a and 5b (referred to as Stat5) in the formation and differentiation of mammary alveolar epithelium. PrlR- and Stat5-null mammary epithelia were transplanted into wild-type hosts, and pregnancy-mediated development was investigated at a histological and molecular level. Stat5-null mammary epithelium developed ducts but failed to form alveoli, and no milk protein gene expression was observed. In contrast, PrlR-null epithelium formed alveoli-like structures with small open lumina. Electron microscopy revealed undifferentiated features of organelles and a perturbation of cell–cell contacts in PrlR- and Stat5-null epithelia. Expression of NKCC1, an Na-K-Cl cotransporter characteristic for ductal epithelia, and ZO-1, a protein associated with tight junction, were maintained in the alveoli-like structures of PrlR- and Stat5-null epithelia. In contrast, the Na-Pi cotransporter Npt2b, and the gap junction component connexin 32, usually expressed in secretory epithelia, were undetectable in PrlR- and Stat5-null mice. These data demonstrate that signaling via the PrlR and Stat5 is critical for the proliferation and differentiation of mammary alveoli during pregnancy.
Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis
Authors: Heese Maren , Gansel Xavier , Sticher Liliane , Wick Peter , Grebe Markus , Granier Fabienne , Jürgens Gerd
Published: July 2002

Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants. In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane. Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen. AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate. A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype. atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering. In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal. Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay. Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.
Muscle-specific RING finger-1 interacts with titin to regulate sarcomeric M-line and thick filament structure and may have nuclear functions via its interaction with glucocorticoid modulatory element binding protein-1
Authors: McElhinny Abigail S. , Kakinuma Kazumi , Sorimachi Hiroyuki , Labeit Siegfried , Gregorio Carol C.
Published: July 2002

The COOH-terminal A168–170 region of the giant sarcomeric protein titin interacts with muscle-specific RING finger-1 (MURF-1). To investigate the functional significance of this interaction, we expressed green fluorescent protein fusion constructs encoding defined fragments of titin's M-line region and MURF-1 in cardiac myocytes. Upon expression of MURF-1 or its central region (containing its titin-binding site), the integrity of titin's M-line region was dramatically disrupted. Disruption of titin's M-line region also resulted in a perturbation of thick filament components, but, surprisingly, not of the NH2-terminal or I-band regions of titin, the Z-lines, or the thin filaments. This specific phenotype also was caused by the expression of titin A168–170. These data suggest that the interaction of titin with MURF-1 is important for the stability of the sarcomeric M-line region.

MURF-1 also binds to ubiquitin-conjugating enzyme-9 and isopeptidase T-3, enzymes involved in small ubiquitin-related modifier–mediated nuclear import, and with glucocorticoid modulatory element binding protein-1 (GMEB-1), a transcriptional regulator. Consistent with our in vitro binding data implicating MURF-1 with nuclear functions, endogenous MURF-1 also was detected in the nuclei of some myocytes. The dual interactions of MURF-1 with titin and GMEB-1 may link myofibril signaling pathways (perhaps including titin's kinase domain) with muscle gene expression.
Nuclear and cytoplasmic shuttling of TRADD induces apoptosis via different mechanisms
Authors: Morgan Michael , Thorburn Jacqueline , Pandolfi Pier Paolo , Thorburn Andrew
Published: July 2002

The adapter protein tumor necrosis factor receptor (TNFR)1–associated death domain (TRADD) plays an essential role in recruiting signaling molecules to the TNFRI receptor complex at the cell membrane. Here we show that TRADD contains a nuclear export and import sequence that allow shuttling between the nucleus and the cytoplasm. In the absence of export, TRADD is found within nuclear structures that are associated with promyelocytic leukemia protein (PML) nuclear bodies. In these structures, the TRADD death domain (TRADD-DD) can activate an apoptosis pathway that is mechanistically distinct from its action at the membrane-bound TNFR1 complex. Apoptosis by nuclear TRADD-DD is promyelocytic leukemia protein dependent, involves p53, and is inhibited by Bcl-xL but not by caspase inhibitors or dominant negative FADD (FADD-DN). Conversely, apoptosis induced by TRADD in the cytoplasm is resistant to Bcl-xL, but sensitive to caspase inhibitors and FADD-DN. These data indicate that nucleocytoplasmic shuttling of TRADD leads to the activation of distinct apoptosis mechanisms that connect the death receptor apparatus to nuclear events.
Novel Genetic Regulation of  T Helper 1 (Th1)/Th2 Cytokine Production and Encephalitogenicity in Inbred Mouse Strains
Authors: Conboy Irina M. , DeKruyff Rosemarie H. , Tate Keri M. , Cao Zhu A. , Moore Tom A. , Umetsu Dale T. , Jones Patricia P.
Published: July 2002

Development of T helper cell (Th)1 or Th2 cytokine responses is essential for effector and regulatory functions of T helper cells. We have compared cytokine profiles of myelin basic protein (MBP) Ac1-16 peptide-specific T helper cells from inbred mouse strains expressing identical k haplotype-derived MHC class II molecules B10.A and B10.BR. B10.BR T cell lines (TCL) produced Th1 cytokines (including high levels of TNF-α) and induced experimental autoimmune encephalomyelitis after adoptive transfer. In contrast, B10.A TCL produced Th2 cytokines (including low levels of TNF-α) and were poorly encephalitogenic. The contributions of the genetic origin of the T cells and the APC were explored. Serial restimulations of the B10.BR TCL with B10.A or (B10.A × B10.BR) F1 splenic antigen presenting cells (APC) during the establishment of TCL markedly reduced both Th1 cytokine production and encephalitogenicity. In addition, a single restimulation with B10.A splenic APC reduced IFN-γ and TNF-α production by established Th1 MBP-specific Ak-restricted B10.BR TCL and by a Th1 KLH-specific, Ek-restricted B10.BR T cell clone. These studies suggest that B10.A and B10.BR APC differ in their ability to stimulate IFN-γ and TNF-α production by mature Th1 cells and also influence their Th1/Th2 commitment in vivo. The nature of the downregulatory activity of B10.A APC on IFN-γ and TNF-α production was explored. 2-hour supernatants from antigen-activated B10.A APC/TCL cultures or from B10.A APC activated by LPS had the same inhibitory effects on IFN-γ and TNF-α production by B10.BR TCL. The downregulatory effects of B10.A APC are independent of TNF-α, IL-4, IL-10, IL-12p40, IFN-γ, IL-13, TGF-β, and PGE2. Thus, genetic difference(s) between B10.A and B10.BR APC appear(s) to control the production or activity of a novel soluble cytokine regulatory factor that influences Th1/Th2 commitment and controls production of IFN-γ and TNF-α by mature Th1 cells.
Molecular and Functional Characterization of  Two Novel Human C-C Chemokines as Inhibitors of  Two Distinct Classes of Myeloid Progenitors
Authors: Patel Vikram P. , Kreider Brent L. , Li Yuling , Li Haodong , Leung Kam , Salcedo Theodora , Nardelli Bernardetta , Pippalla Vani , Gentz Solange , Thotakura Rao , Parmelee David , Gentz Reiner , Garotta Gianni
Published: July 2002

Two novel human β-chemokines, Ckβ-8 or myeloid progenitor inhibitory factor 1 (MPIF-1), and Ckβ-6 or MPIF-2, were discovered as part of a large scale cDNA sequencing effort. The MPIF-1 and MPIF-2 cDNAs were isolated from aortic endothelium and activated monocyte libraries, respectively. Both of the cDNAs were cloned into a baculovirus vector and expressed in insect cells. The mature recombinant MPIF-1 protein consists of 99 amino acids and is most homologous to macrophage inflammatory protein (MIP)-1α, showing 51% identity. It displays chemotactic activity on resting T lymphocytes and monocytes, a minimal but significant activity on neutrophils, and is negative on activated T lymphocytes. MPIF-1 is also a potent suppressor of bone marrow low proliferative potential colony-forming cells, a committed progenitor that gives rise to granulocyte and monocyte lineages. The mature recombinant MPIF-2 has 93 amino acid residues and shows 39 and 42% identity with monocyte chemoattractant protein (MCP)-3 and MIP-1α, respectively. It displays chemotactic activity on resting T lymphocytes, a minimal activity on neutrophils, and is negative on monocytes and activated T lymphocytes. On eosinophils, MPIF-2 produces a transient rise of cytosolic Ca2+ and uses the receptor for eotaxin and MCP-4. In hematopoietic assays, MPIF-2 strongly suppressed the colony formation by the high proliferative potential colony-forming cell (HPP-CFC), which represents a multipotential hematopoietic progenitor.
High Levels of a Major Histocompatibility Complex II–Self Peptide Complex on Dendritic Cells from the T Cell Areas of Lymph Nodes
Authors: Inaba Kayo , Pack Maggie , Inaba Muneo , Sakuta Hiraki , Isdell Frank , Steinman Ralph M.
Published: July 2002

T lymphocytes recirculate continually through the T cell areas of peripheral lymph nodes. During each passage, the T cells survey the surface of large dendritic cells (DCs), also known as interdigitating cells. However, these DCs have been difficult to release from the lymph node. By emphasizing the use of calcium-free media, as shown by Vremec et al. (Vremec, D., M. Zorbas, R. Scollay, D.J. Saunders, C.F. Ardavin, L. Wu, and K. Shortman. 1992. J. Exp. Med. 176:47–58.), we have been able to release and enrich DCs from the T cell areas. The DCs express the CD11c leukocyte integrin, the DEC-205 multilectin receptor for antigen presentation, the intracellular granule antigens which are recognized by monoclonal antibodies M342, 2A1, and MIDC-8, very high levels of MHC I and MHC II, and abundant accessory molecules such as CD40, CD54, and CD86. When examined with the Y-Ae monoclonal which recognizes complexes formed between I-Ab and a peptide derived from I-Eα, the T cell area DCs expressed the highest levels. The enriched DCs also stimulated a T-T hybridoma specific for this MHC II–peptide complex, and the hybridoma underwent apoptosis. Therefore DCs within the T cell areas can be isolated. Because they present very high levels of self peptides, these DCs should be considered in the regulation of self reactivity in the periphery.
Interleukin 6 Is Required for the Development of Collagen-induced Arthritis
Authors: Alonzi Tonino , Fattori Elena , Lazzaro Domenico , Costa Patrizia , Probert Lesley , Kollias George , De Benedetti Fabrizio , Poli Valeria , Ciliberto Gennaro
Published: July 2002

Interleukin-6 (IL-6) is overproduced in the joints of patients with rheumatoid arthritis (RA) and, based on its multiple stimulatory effects on cells of the immune system and on vascular endothelia, osteoclasts, and synovial fibroblasts, is believed to participate in the development and clinical manifestations of this disease. In this study we have analysed the effect of ablating cytokine production in two mouse models of arthritis: collagen-induced arthritis (CIA) in DBA/1J mice and the inflammatory polyarthritis of tumor necrosis factor α (TNF-α) transgenic mice. IL-6 was ablated by intercrossing an IL-6 null mutation into both arthritis-susceptible genetic backgrounds and disease development was monitored by measuring clinical, histological, and biochemical parameters. Two opposite responses were observed; while arthritis in TNF-α transgenic mice was not affected by inactivation of the IL-6 gene, DBA/1J, IL-6−/− mice were completely protected from CIA, accompanied by a reduced antibody response to type II collagen and the absence of inflammatory cells and tissue damage in knee joints. These results are discussed in the light of the present knowledge of cytokine networks in chronic inflammatory disorders and suggest that IL-6 receptor antagonists might be beneficial for the treatment of RA.
Rapid Secretion of Prestored Interleukin 8 from Weibel-Palade Bodies of Microvascular Endothelial Cells
Authors: Utgaard Jon Olav , Jahnsen Frode L. , Bakka Arne , Brandtzaeg Per , Haraldsen Guttorm
Published: July 2002

Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils. Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We found that cultured human microvascular endothelial cells from the intestine (HIMEC) and from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf  ). This observation was corroborated by the immunohistochemical observation of double-positive granules (IL-8+vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min, whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8–containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8–containing granules were inducible by treatment with recombinant human IL-1β for 24 h; additional histamine treatment doubled IL-8 secretion from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.
Locally Up-regulated Lymphotoxin α, Not Systemic Tumor Necrosis Factor α, Is the Principle Mediator of Murine Cerebral Malaria
Authors: Engwerda Christian R. , Mynott Tracey L. , Sawhney Sanjeet , De Souza J. Brian , Bickle Quentin D. , Kaye Paul M.
Published: July 2002

Cerebral malaria (CM) causes death in children and nonimmune adults. TNF-α has been thought to play a key role in the development of CM. In contrast, the role of the related cyto-kine lymphotoxin α (LTα) in CM has been overlooked. Here we show that LTα, not TNFα, is the principal mediator of murine CM. Mice deficient in TNFα (B6.TNFα−/−) were as susceptible to CM caused by Plasmodium berghei (ANKA) as C57BL/6 mice, and died 6 to 8 d after infection after developing neurological signs of CM, associated with perivascular brain hemorrhage. Significantly, the development of CM in B6.TNFα−/− mice was not associated with increased intracellular adhesion molecule (ICAM)-1 expression on cerebral vasculature and the intraluminal accumulation of complement receptor 3 (CR3)-positive leukocytes was moderate. In contrast, mice deficient in LTα (B6.LTα−/−) were completely resistant to CM and died 11 to 14 d after infection with severe anemia and hyperparasitemia. No difference in blood parasite burden was found between C57BL/6, B6.TNFα−/−, and B6.LTα−/− mice at the onset of CM symptoms in the two susceptible strains. In addition, studies in bone marrow (BM) chimeric mice showed the persistence of cerebral LTα mRNA after irradiation and engraftment of LTα-deficient BM, indicating that LTα originated from a radiation-resistant cell population.
The Number of Subunits Comprising the Channel Formed by the T Domain of Diphtheria Toxin
Authors: Gordon Michael , Finkelstein Alan
Published: July 2002

In the presence of a low pH environment, the channel-forming T domain of diphtheria toxin undergoes a conformational change that allows for both its own insertion into planar lipid bilayers and the translocation of the toxin's catalytic domain across them. Given that the T domain contributes only three transmembrane segments, and the channel is permeable to ions as large as glucosamine+ and NAD−, it would appear that the channel must be a multimer. Yet, there is substantial circumstantial evidence that the channel may be formed from a single subunit. To test the hypothesis that the channel formed by the T domain of diphtheria toxin is monomeric, we made mixtures of two T domain constructs whose voltage-gating characteristics differ, and then observed the gating behavior of the mixture's single channels in planar lipid bilayers. One of these constructs contained an NH2-terminal hexahistidine (H6) tag that blocks the channel at negative voltages; the other contained a COOH-terminal H6 tag that blocks the channel at positive voltages. If the channel is constructed from multiple T domain subunits, one expects to see a population of single channels from this mixture that are blocked at both positive and negative voltages. The observed single channels were blocked at either negative or positive voltages, but never both. Therefore, we conclude that the T domain channel is monomeric.
The Bergman kernel function: Explicit formulas and zeroes
Authors: Boas Harold , Fu Siqi , Straube Emil
Published: July 2002

We show how to compute the Bergman kernel functions of some special domains in a simple way. As an application of the explicit formulas, we show that the Bergman kernel functions of some convex domains, for instance the domain in 

C

3

\mathbb {C}^3

defined by the inequality

|

z
1

|

+

|

z
2

|

+

|

z
3

|

>
1

|z_1|+|z_2|+|z_3|>1

, have zeroes.
Geometric Isomorphisms between Infinite Dimensional Teichmüller Spaces
Authors: Earle Clifford , Gardiner Frederick
Published: July 2002

Let

X
X

and

Y
Y

be the interiors of bordered Riemann surfaces with finitely generated fundamental groups and nonempty borders. We prove that every holomorphic isomorphism of the Teichmüller space of

X
X

onto the Teichmüller space of

Y
Y

is induced by a quasiconformal homeomorphism of

X
X

onto

Y
Y

. These Teichmüller spaces are not finite dimensional and their groups of holomorphic automorphisms do not act properly discontinuously, so the proof presents difficulties not present in the classical case. To overcome them we study weak continuity properties of isometries of the tangent spaces to Teichmüller space and special properties of Teichmüller disks.