bluuebunny/crossref_metadata_2025 huggingface dataset:
Neural BC1 RNA as an evolutionary marker: guinea pig remains a rodent.
Authors: Martignetti J A , Brosius J
Published: May 2006

The traditional morphologically grounded placement of South American guinea pig-like rodents (Caviomorpha) within one of the two rodent suborders, Hystricognathi, has been disputed by recent analysis of protein and nucleic acid sequence data. The Caviomorpha and possibly all Hystricognathi would be considered a separate order, distinct from the other rodent suborder, Sciurognathi, and thus of the order Rodentia, and would be placed closer phylogenetically to other mammals [Graur, D., Hide, W. A. & Li, W.-H. (1991) Nature (London) 351, 649-652]. To address the discrepancy between morphological comparisons and sequence analyses, we have applied an alternative form of molecular analysis. We demonstrate that BC1 RNA, a neural-specific small cytoplasmic RNA that is the product of a retropositionally generated gene (a gene derived by reverse transcription of RNA followed by insertion of the DNA copy into the genome), is present in Sciurognathi and guinea pig but not in other mammalian orders including Lagomorpha, Artiodactyla, and Primates. The species-confined, tissue-specific expression of a retroposed sequence therefore supports the morphological evidence for monophyly of Rodentia inclusive of guinea pig and demonstrates the usefulness of such molecular genetic markers. Furthermore, the conservation and tissue-specific expression of the BC1 RNA gene in the two divergent rodent suborders suggests that this macromolecule has been exapted into a functional role (i.e., coopted into a variant or novel function) in the rodent nervous system.
The role of complement component C3b and its receptors in sperm-oocyte interaction.
Authors: Anderson D J , Abbott A F , Jack R M
Published: May 2006

Previous studies have shown that human sperm that have undergone the acrosome reaction express a unique tissue-specific variant of the complement component 3 (C3)-binding molecule membrane cofactor protein (MCP, CD46) and that damaged or dead sperm activate the alternative pathway of complement and bind C3 catabolites. In this study we provide evidence that MCP on sperm that have undergone the acrosome reaction specifically binds dimeric C3b and that human sperm acrosomal proteases released during the acrosome reaction directly cleave C3, facilitating its binding to MCP. Furthermore, human and hamster oocytes can activate the alternative pathway of complement and also bind human C3 fragments. Monoclonal antibodies specific for complement receptors type 1 (CD35) and type 3 (CD11b/CD18) bind to the human oocyte plasma membrane, indicating that specific complement-binding molecules may play a role in the attachment of C3 catabolites to oocytes. Subsaturating concentrations of dimeric C3b (0.01-1 microM) promoted penetration of hamster oocytes by human sperm, whereas saturating doses (> 10 microM) inhibited this process. In addition, antibodies to both MCP and C3 significantly inhibited penetration of hamster oocytes by human sperm. These data provide evidence that regulated gamete-induced generation of C3 fragments and the binding of these fragments by selectively expressed receptors on sperm and oocytes may be an initial step in gamete interaction, leading to membrane fusion and fertilization.
Disruption of mitotic spindle orientation in a yeast dynein mutant.
Authors: Li Y Y , Yeh E , Hays T , Bloom K
Published: May 2006

Dynein motor isoforms have been implicated as potential kinetochore-associated motors that power chromosome-to-pole movements during mitosis. The recent identification and sequence determination of genes encoding dynein isoforms has now permitted the in vivo analysis of dynein function in mitosis. In this report we describe the identification and mutational analysis of the gene, DHC1, encoding a dynein heavy chain isoform in Saccharomyces cerevisiae. Sequence analysis of a 9-kb genomic fragment of the DHC1 gene predicts a polypeptide highly homologous to dynein sequences characterized from sea urchin, Dictyostelium, Drosophila, and rat. Mutations in the yeast dynein gene disrupt the normal movement of the spindle into budding daughter cells but have no apparent effect on spindle assembly, spindle elongation, or chromosome segregation. Our results suggest that, in yeast, a dynein microtubule motor protein has a nonessential role in spindle assembly and chromosome movement but is involved in establishing the proper spindle orientation during cell division.
Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression.
Authors: Schulte S , Stoffel W
Published: May 2006

Cerebrosides and sulfatides are major glycosphingolipids of the lipid bilayer of the myelin sheath assembled by oligodendrocytes and Schwann cells during myelination. Cerebrosides are synthesized by ceramide UDPgalactosyltransferase [CGT; 2-hydroxyacylsphinogosine 1-beta-galactosyl-transferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-beta-D-galactosyltransferase; UDPgalactose:2-(2-hydroxyacyl)sphingosine 1-beta-D-galactosyltransferase, EC 2.4.1.45] with UDPgalactose and ceramide as substrates. Here we describe a purification method from microsomes of myelinating rat brains that includes ion exchange, dye ligand, and lectin affinity chromatography. The enzyme was identified as a 64-kDa high-mannose glycoprotein. A CGT-specific cDNA clone was isolated from a rat brain cDNA library using CGT oligonucleotides derived from peptide sequences. The cDNA insert encodes a polypeptide of 541 amino acid residues with a molecular weight of 61,126. The polypeptide has three putative glycosylation sites and one hydrophobic domain at the C terminus. A 20-residue N-terminal signal sequence is lost during cotranslational translocation. Northern blot analysis demonstrates that CGT expression is restricted to brain tissue and is time dependent, correlating with myelin basic protein expression. In situ hybridization reveals that CGT expression is restricted to the oligodendrocyte-containing cell layers of cerebrum and cerebellum, which also express myelin basic protein. The amino acid sequence of CGT shows significant homology to mammalian UDPglucuronyltransferases, which suggests a common evolutionary origin of these enzymes.
Nonlinear processes in visual pattern discrimination.
Authors: Wilson H R
Published: May 2006

Visual performance on many simple pattern discrimination tasks can be accurately predicted by quasi-linear models composed of oriented linear filters followed by simple contrast nonlinearities. However, many complex discrimination tasks require highly nonlinear processes for their explanation. Evidence is provided for two nonlinear processes in pattern discrimination: (i) one process involves a sequence of filtering, rectification, and subsequent filtering to extract texture boundaries; (ii) the second process results from contrast gain-control processes. It is suggested that quasi-linear processes and nonlinear texture-boundary processes, each with an appropriate contrast gain control, may operate in parallel to provide the basis for all higher-level visual analyses.
Cell-free pool of CD14 mediates activation of transcription factor NF-kappa B by lipopolysaccharide in human endothelial cells.
Authors: Read M A , Cordle S R , Veach R A , Carlisle C D , Hawiger J
Published: May 2006

Lipopolysaccharide (LPS), a major envelope component of Gram-negative bacteria, is the most frequent causative agent of septic shock and disseminated intravascular coagulation. LPS activates both CD14-positive (monocytes, macrophages, polymorphonuclear leukocytes) and CD14-negative (B-cell lines, endothelial cells) cells. CD14, a 55-kDa glycosyl-phosphatidylinositol-anchored membrane protein present on mature myeloid cells, serves as a receptor for LPS in complex with a soluble (serum-derived) LPS-binding protein (LBP). In this report, we show that human umbilical vein endothelial cells (HUVEC), which do not express measurable CD14 protein, become 3000-fold more sensitive to LPS-induced activation in the presence of serum, as measured by activation of the transcription factor NF-kappa B and expression of mRNA encoding tissue factor, a procoagulant molecule. This enhanced responsiveness of HUVEC is specifically mediated by the cell-free pool of CD14 (soluble CD14, sCD14) found in serum. The role of sCD14 in HUVEC activation by LPS was established by (i) the blocking effect of monoclonal anti-CD14 antibodies which discriminate between cell-bound and sCD14, (ii) the lack of the serum-enhancing effect after immunodepletion of sCD14, and (iii) establishing a reconstituted system in which recombinant sCD14 was sufficient to enhance the effects of LPS in the absence of serum and without a requirement for LBP. Thus, this mechanism of endothelial cell activation by LPS involves a cell-free pool of sCD14 most likely shed from CD14-positive cells of the monocytic lineage.
Preformed dimeric state of the sensor protein VirA is involved in plant--Agrobacterium signal transduction.
Authors: Pan S Q , Charles T , Jin S , Wu Z L , Nester E W
Published: May 2006

Plant signal molecules such as acetosyringone and certain monosaccharides induce the expression of Agrobacterium tumefaciens virulence (vir) genes, which are required for the processing, transfer, and possibly integration of a piece of the bacterial plasmid DNA (T-DNA) into the plant genome. Two fo the vir genes, virA and virG, belonging to the bacterial two-component regulatory system family, control the induction of vir genes by plant signals. virA encodes a membrane-bound sensor kinase protein and virG encodes a cytoplasmic regulator protein. Although it is well established from in vitro studies that the signal transduction process involves VirA autophosphorylation and subsequent phosphate transfer to VirG, the structural state of the VirA protein involved in signal transduction is not understood. In this communication, we describe an in vivo crosslinking approach which provides physical evidence that VirA exists as a homodimer in its native configuration. The dimerization of VirA neither requires nor is stimulated by the plant signal molecule acetosyringone. We also present genetic data which support the hypothesis that VirA exists as a homodimer which is the functional state transducing the plant signal in an intersubunit mechanism. To our knowledge, this report provides the first evidence that a bacterial membrane-bound sensor kinase exists and functions as a homodimer in vivo.
Interaction of glucocorticosteroid receptor and wild-type or mutated 90-kDa heat shock protein coexpressed in baculovirus-infected Sf9 cells.
Authors: Cadepond F , Binart N , Chambraud B , Jibard N , Schweizer-Groyer G , Segard-Maurel I , Baulieu E E
Published: May 2006

Coexpression of the human glucocorticosteroid receptor (hGR) and chicken 90-kDa heat shock protein alpha (chsp90) in recombinant baculovirus-infected Sf9 cells is a system that provides a large quantity of wild-type chsp90-hGR complexes able to bind hormone ([3H]triamcinolone acetonide; TA), sedimenting at 8 S, and displaceable to 11 S by BF4 and D7 alpha anti-chsp90 monoclonal antibodies. Thus, we were able to examine the effects of selective chsp90 mutations on hetero-oligomeric complex formation. Two deletions involved hydrophilic regions, A between amino acids 221 and 290 and B between amino acids 530 and 581, and the third, Z, removed a central leucine heptad repeat region (amino acids 392-419). When these chsp90 mutants were expressed, the lack of displacement of [3H]TA receptor complexes on sucrose gradient by specific chsp90 antibodies was consistent with the formation of [3H]TA receptor complexes containing only endogenous insect hsp90. By using an immunoadsorption method and sedimentation analysis, we found that the deletion of region A precluded the interaction of chsp90 with the hGR, while B and Z deletions led to formation of abnormal complexes with the hGR, which displayed large forms (> 10 S), were unable to bind hormone, and apparently formed only small amounts of tightly bound nuclei hGR upon in vivo hormone treatment. As a whole, the data are consistent with distinct roles of hsp90 regions in hGR function.
Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients.
Authors: Dumaz N , Drougard C , Sarasin A , Daya-Grosjean L
Published: May 2006

The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. We have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a good target for studying mutation spectra since there are > 100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze > 40 XP skin tumors (mainly basal and squamous cell carcinomas), we have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC-->TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues.
Simple models for reading neuronal population codes.
Authors: Seung H S , Sompolinsky H
Published: May 2006

In many neural systems, sensory information is distributed throughout a population of neurons. We study simple neural network models for extracting this information. The inputs to the networks are the stochastic responses of a population of sensory neurons tuned to directional stimuli. The performance of each network model in psychophysical tasks is compared with that of the optimal maximum likelihood procedure. As a model of direction estimation in two dimensions, we consider a linear network that computes a population vector. Its performance depends on the width of the population tuning curves and is maximal for width, which increases with the level of background activity. Although for narrowly tuned neurons the performance of the population vector is significantly inferior to that of maximum likelihood estimation, the difference between the two is small when the tuning is broad. For direction discrimination, we consider two models: a perceptron with fully adaptive weights and a network made by adding an adaptive second layer to the population vector network. We calculate the error rates of these networks after exhaustive training to a particular direction. By testing on the full range of possible directions, the extent of transfer of training to novel stimuli can be calculated. It is found that for threshold linear networks the transfer of perceptual learning is nonmonotonic. Although performance deteriorates away from the training stimulus, it peaks again at an intermediate angle. This nonmonotonicity provides an important psychophysical test of these models.
beta-Amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease.
Authors: Roher A E , Lowenson J D , Clarke S , Woods A S , Cotter R J , Gowing E , Ball M J
Published: May 2006

Reinvestigation of the chemical structure of beta-amyloid peptide (A beta) deposits in the vascular tissue of Alzheimer disease brains revealed that the 42-residue form A beta-(1-42), rather than the more soluble A beta-(1-40) form, is the predominant peptide. Following removal of the surrounding tissue with SDS and collagenase, A beta was solubilized in formic acid and purified by Superose 12 chromatography. Peptides generated by enzymatic and chemical digestion of the A beta were purified by HPLC and characterized by amino acid analysis, sequence analysis, and mass spectrometry. In the leptomeningeal vessels, the average ratio of A beta-(1-42)/A beta-(1-40) was 58:42, whereas in the parenchymal vessels this ratio was 75:25. Interestingly, vascular A beta contains considerably less isomerized and racemized aspartyl residues than does neuritic plaque A beta, suggesting that the vascular amyloid is "younger." The discrete nature of the bands and spherical deposits of A beta associated with arterioles and capillaries, respectively, suggests that this amyloid arises from the vascular tissue itself. Increasing A beta deposition appears to lead to the distortion and occlusion of capillaries, which may contribute significantly to the pathology of Alzheimer disease.
spoT-dependent accumulation of guanosine tetraphosphate in response to fatty acid starvation in Escherichia coli.
Authors: Seyfzadeh M , Keener J , Nomura M
Published: May 2006

We previously isolated a mutant of Escherichia coli that is preferentially affected in the synthesis of rRNA and has a mutation in the gene (accD) encoding a subunit of acetyl-CoA carboxylase. Using this mutant and other mutants of the pathway for fatty acid and phospholipid biosynthesis as well as cerulenin, a specific inhibitor of fatty acid synthesis, we show that (i) inhibition of fatty acid synthesis in the presence of both a carbon source and all 20 amino acids stimulates the accumulation of guanosine tetraphosphate (ppGpp) and leads to preferential inhibition of rRNA synthesis, (ii) this ppGpp accumulation is spoT dependent, and (iii) the generation of the metabolic signal that stimulates this spoT-mediated response probably does not depend on either phospholipid starvation or a significant reduction in the level of ATP.
Costimulation of T-cell activation and virus production by B7 antigen on activated CD4+ T cells from human immunodeficiency virus type 1-infected donors.
Authors: Haffar O K , Smithgall M D , Bradshaw J , Brady B , Damle N K , Linsley P S
Published: May 2006

Infection with the human immunodeficiency virus type 1 (HIV-1) requires T-cell activation. Recent studies have shown that interactions of the T-lymphocyte receptors CD28 and CTLA-4 with their counter receptor, B7, on antigen-presenting cells are required for optimal T-cell activation. Here we show that HIV-1 infection is associated with decreased expression of CD28 and increased expression of B7 on CD4+ T-cell lines generated from seropositive donors by alloantigen stimulation. Loss of CD28 expression was not seen on CD4+ T-cell lines from seronegative donors, but up-regulation of B7 expression was observed upon more prolonged culture. Both T-cell proliferation and interleukin 2 mRNA accumulation in HIV-1-infected cultures required costimulation with exogenous B7 because these events were blocked by CTLA4Ig, a soluble form of CTLA-4 that binds B7 with high avidity. In contrast, levels of HIV-1 RNA were not affected by CTLA4Ig, indicating that regulation of virus transcription in these cultures did not depend upon CD28-B7 engagement. Infected T cells could present alloantigen to fresh, uninfected CD4+ T cells, leading to increased proliferation and virus spread to the activated cells. Both of these events were blocked by CTLA4Ig. Thus, chronic activation of HIV-1-infected CD4+ T cells reduces expression of CD28 and increases expression of B7, thereby enabling these T cells to become antigen-presenting cells for uninfected CD4+ T cells; this might be another mechanism for HIV-1 transmission via T-cell-T-cell contact.
Dimer formation by an N-terminal coiled coil in the APC protein.
Authors: Joslyn G , Richardson D S , White R , Alber T
Published: May 2006

Mutations in the human APC gene are associated with an inherited predisposition to colon cancer. APC codes for polypeptides of approximately 2800 amino acids, with sequence homologies to coiled-coil proteins in the first 900 residues. To determine the oligomerization properties of the APC protein, we used genetic and biochemical approaches to examine the ability of APC fragments to self-associate. A subdomain comprising the first 55 amino acids of APC was found to form a stable, parallel, helical dimer, as expected for a coiled coil. The location of a key dimerization element at the N terminus of the protein supports models in which mutations in APC exert effects through dimerization of the mutant gene products.
Activation of phosphotyrosine phosphatase activity by reduction of cell-substrate adhesion.
Authors: Maher P A
Published: May 2006

Treatment of chicken embryo fibroblasts (CEFs) with trypsin results in a dose- and time-dependent loss of phosphotyrosine from cellular proteins. A similar, but less marked, reduction in protein tyrosine phosphorylation occurs upon incubation of CEFs in phosphate-buffered saline (PBS). The decrease in the phosphotyrosine content of proteins following treatment with trypsin or PBS, as determined by immunoblotting of cell extracts with anti-phosphotyrosine antibodies, corresponds with a loss of phosphotyrosine antibody immunoreactivity at focal contacts, as detected by immunofluorescence microscopy. The recovery of phosphotyrosine in cellular proteins occurs within 30 min following removal of trypsin, even in the presence of the protein synthesis inhibitor cycloheximide, indicating that the loss of phosphotyrosine-containing proteins is not due to their degradation by trypsin. Pretreatment of CEFs with inhibitors of protein-tyrosine-phosphatases greatly reduces the loss of phosphotyrosine from proteins brought about by trypsin. In addition, phosphotyrosine phosphatase activity is increased in extracts prepared from trypsin-treated CEFs. The loss of phosphotyrosine from proteins following treatment with trypsin or PBS is not specific to CEFs but is also observed in established fibroblast lines. Taken together these results suggest that the activity of one or more phosphotyrosine phosphatases is regulated by cell-substrate adhesion.
Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.
Authors: Lobkovsky E , Moews P C , Liu H , Zhao H , Frere J M , Knox J R
Published: May 2006

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.
Regulation by the neuropeptide cholecystokinin (CCK-8S) of protein phosphorylation in the neostriatum.
Authors: Snyder G L , Fisone G , Morino P , Gundersen V , Ottersen O P , Hökfelt T , Greengard P
Published: May 2006

Despite physiological evidence that cholecystokinin (CCK) is an excitatory neurotransmitter in the brain, little is known about its mechanism of action. CCK immunoreactivity in the brain, including projections to the striatum, is primarily attributable to the sulfated octapeptide CCK-8S. We report here that CCK-8S abolishes cAMP-dependent phosphorylation of a dopamine- and cAMP-regulated 32-kDa phosphoprotein (DARPP-32) in striatal neurons. The effect of CCK-8S is prevented by antagonists of CCKB and N-methyl-D-aspartate receptors. Our results support a model in which CCK-8S, originating from CCK or CCK/glutamate corticostriatal neurons, promotes the release of an excitatory neurotransmitter that causes the dephosphorylation and inactivation of DARPP-32, a potent protein phosphatase inhibitor, thereby modulating neuronal excitability.
Transgenic mice expressing the human GLUT4/muscle-fat facilitative glucose transporter protein exhibit efficient glycemic control.
Authors: Liu M L , Gibbs E M , McCoid S C , Milici A J , Stukenbrok H A , McPherson R K , Treadway J L , Pessin J E
Published: May 2006

To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (approximately 70 mg/dl) compared to wild-type mice (approximately 130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (approximately 90 mg/dl), whereas wild-type mice displayed a typical 2- to 3-fold increase (approximately 250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were approximately 2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.
Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice.
Authors: Gaensler K M , Kitamura M , Kan Y W
Published: May 2006

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.
The gamma chain of the high-affinity receptor for IgE is a major functional subunit of the T-cell antigen receptor complex in gamma delta T lymphocytes.
Authors: Qian D , Sperling A I , Lancki D W , Tatsumi Y , Barrett T A , Bluestone J A , Fitch F W
Published: May 2006

T-cell activation is a consequence of the clonotypic T-cell antigen receptor (TCR) binding to an antigen followed by signal transduction via the invariant subunits of the TCR/CD3 complex. gamma delta TCR cells are a small subset of T cells that populate both the epithelial and lymphoid tissues and have unique antigen specificity and function. However, the composition of invariant chains within the gamma delta TCR/CD3 complex has not been well characterized. Here we report that, unlike the majority of alpha beta T cell, gamma delta T cells isolated from spleen and intestinal epithelial tissue express high levels of the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) as one invariant subunit of their TCR/CD3 complex. Fc epsilon RI gamma exists as both a homodimer and a heterodimer associated with the TCR zeta chain. Moreover, stimulation of the gamma delta TCR results in rapid tyrosine phosphorylation of Fc epsilon RI gamma. Our results suggest that utilization of distinct receptor signaling components may enable the coupling of antigen stimulation to the activation of different signal transduction pathways in alpha beta and gamma delta T cells.
Correlations between isochores and chromosomal bands in the human genome.
Authors: Saccone S , De Sario A , Wiegant J , Raap A K , Della Valle G , Bernardi G
Published: May 2006

The human genome is made up of long DNA segments, the isochores, which are compositionally homogeneous and can be subdivided into a small number of families characterized by different G+C levels. Chromosome in situ suppression hybridization (in which excess unlabeled human DNA is added to suppress hybridization of repeated sequences present in the probe, enabling enhanced observation of single-copy sequences) of DNA fractions characterized by an increasing G+C level was carried out to determine the distribution of "single-copy" sequences corresponding to isochore families L1+L2, H1, H2, and H3 on metaphase chromosomes. This produced a banding pattern progressing from a relatively diffuse staining to an R-banding, to a T-banding. More specifically, our results showed that (i) T-bands are formed by the G+C-richest isochores of the H3 family and by part of the G+C-rich isochores of the H1 and H2 families (with a predominance of the latter); (ii) R'-bands (namely, R-bands exclusive of T-bands) are formed to almost equal extents by G+C-rich isochores of the H1 families (with a minor contribution of the H2 and H3 families) and by G+C-poor isochores of the L1+L2 families; (iii) G-bands essentially consist of G+C-poor isochores from the L1+L2 families, with a minor contribution of isochores from the H1 family. These results not only clarify the correlations between DNA base composition and chromosomal bands but also provide information on the distribution of genes in chromosomes, gene concentration increasing with the G+C levels of isochores.
Aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains.
Authors: Yang Y R , Schachman H K
Published: May 2006

Based on the demonstration that active enzyme is formed in vitro and in vivo from polypeptide fragments of the catalytic chains of aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and the evidence that NH2 and COOH termini of wild-type chains are in close proximity, we constructed altered genes to determine whether circularly permuted catalytic chains could fold and assemble into active catalytic trimers. Two slightly different genetic constructs led to the expression in good yield of circularly permuted catalytic chains, which associated in vivo into active trimers. They, in turn, combined in vitro with wild-type regulatory dimers to form ATCase-like molecules. Both polypeptide chains began at residue 235 in a different domain from the NH2 terminus of wild type and had an overlapping sequence of eight residues at the COOH terminus. One had a six-amino-acid linker, and the other had a deletion of four residues. Enzymes containing rearranged chains were similar to their wild-type counterparts in physical properties. Whereas values of Vmax were close to those of wild-type trimers and ATCase, the Km values were more than 10-fold greater. Also the allosteric properties characteristic of wild-type ATCase were lacking in the enzymes containing permuted chains. Denaturation of trimers by urea was reversible, and recovery of activity in both rate and yield was comparable to that of wild-type trimers. The experiments demonstrate that folding of chains into clearly defined domains and the assembly of active, thermodynamically stable oligomers are not dependent on the positions of NH2 and COOH termini; the folded structures are a consequence of the final sequence and not the order of biosynthetic addition of amino acids.
apl-1, a Caenorhabditis elegans gene encoding a protein related to the human beta-amyloid protein precursor.
Authors: Daigle I , Li C
Published: May 2006

The major component of senile plaques found in the brains of Alzheimer disease patients is the beta-amyloid peptide, which is derived from a larger amyloid precursor protein (APP). Recently, a number of APP and APP-related proteins have been identified in different organisms and constitute the family of APP proteins. We have isolated several cDNAs encoding an APP-related protein in the nematode Caenorhabditis elegans and have designated the corresponding gene as apl-1. The apl-1 transcripts undergo two forms of posttranscriptional modification: trans-splicing and alternative polyadenylylation. In vitro translation of an apl-1 cDNA results in a protein of approximately the expected size. Similar to the Drosophila, human, and mouse APP-related proteins, APL-1 does not appear to contain the beta-amyloid peptide. Because APP-related proteins seem to be conserved through evolution, the apl-1 gene from C. elegans should be important for determining the normal function of human APP.
Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C.
Authors: Dahlbäck B , Carlsson M , Svensson P J
Published: May 2006

Although patients with thromboembolic disease frequently have family histories of thrombosis, well-defined defects such as inherited deficiencies of anticoagulant proteins are found only in a minority of cases. Based on the hypothesis that a poor anticoagulant response to activated protein C (APC) would predispose to thrombosis, a set of new coagulation assays was developed that measure the anticoagulant response in plasma to APC. A middle-aged man with a history of multiple thrombotic events was identified. The addition of APC to his plasma did not result in a normal anticoagulant response as measured by prolongation of clotting time in an activated partial thromboplastin time (APTT) assay. Four of the proband's relatives had medical histories of multiple thrombotic events, and they and several other family members responded poorly to APC in the APTT-based assay. Subnormal anticoagulant responses to APC were also found in factor IXa- and Xa-based assays. Several possible mechanisms for the observed phenomenon were ruled out, such as functional protein S deficiency, a protein C-inhibitory antibody, or a fast-acting protease inhibitor against APC. Moreover, restriction fragment-length polymorphism analysis excluded possible linkage of the underlying molecular defect to factor VIII and von Willebrand factor genes. We now describe a previously unrecognized mechanism for familial thromboembolic disease that is characterized by poor anticoagulant response to APC. This would appear to be explained best by a hypothesized inherited deficiency of a previously unrecognized cofactor to APC. As we have identified two additional, unrelated cases with thrombosis and inherited poor anticoagulant response to APC, this may constitute an important cause for familial thrombophilia.
Regulation of serine protease activity by aluminum: implications for Alzheimer disease.
Authors: Clauberg M , Joshi J G
Published: May 2006

The brain of Alzheimer disease patients contains plaques that are diagnostic for the disease. The plaques also contain beta-amyloid peptide, alpha 1-antichymotrypsin, and the element aluminum. We present indirect evidence that can relate all three components of plaques to each other in such a way as to suggest their involvement in the etiology of the disease. The beta-amyloid peptide is derived by proteolytic processing from beta-amyloid precursor proteins and some of these proteins contain a domain that is highly homologous to bovine pancreatic trypsin inhibitor. Bovine pancreatic trypsin inhibitor also inhibits alpha-chymotrypsin and we show that aluminum affects both the activity and the inhibition of this enzyme. At pH 6.5, in the presence of aluminum, the enzyme activity is doubled, and the inhibitor is only 1% as effective as in the absence of the metal ion. The inhibition by BX-9, a protease inhibitor prepared from protein components of amyloid plaques, is also reduced by aluminum; so too is that by alpha 1-antichymotrypsin but to a lesser degree. In the Alzheimer brain, we propose that aluminum may accelerate proteolytic processing of the beta-amyloid precursor protein by suppression of the inhibitor domain. Thus, the beta-amyloid peptide may accumulate and initiate plaque formation.
Molecular, functional, and evolutionary analysis of sequences specific to Salmonella.
Authors: Groisman E A , Sturmoski M A , Solomon F R , Lin R , Ochman H
Published: May 2006

In that salmonellae have been implicated in an unprecedented array of diseases, sequences found to be specific to this species are often thought to be involved in the virulence attributes not seen in other enteric bacteria. To identify the molecular, genetic, and phenotypic characteristics that differentiate bacterial species, we analyzed five cloned DNA fragments that were originally described as being confined to Salmonella. Most of these segments mapped to unique positions on the Salmonella typhimurium chromosome indicative of independent evolutionary events, and three had G+C contents considerably lower than that of the Salmonella genome, suggesting that they arose through horizontal transfer. The nucleotide sequence was determined for one of the clones exhibiting an atypical base composition. This 4.9-kb fragment contained an open reading frame with structural similarity to the LysR family of transcriptional regulators. Strains harboring deletions in this region were tested for > 120 phenotypic characteristics including the effects on a collection of environmentally regulated lac gene fusions. In addition, all deletion strains behaved like the wild-type parent when tested for virulence in mice.
Enhanced interaction of L-selectin with the high endothelial venule ligand via selectively oxidized sialic acids.
Authors: Norgard K E , Han H , Powell L , Kriegler M , Varki A , Varki N M
Published: May 2006

The selectins are adhesion receptors that play key roles in leukocyte trafficking. Each has an N-terminal C-type lectin domain that binds to specific carbohydrates in a calcium-dependent manner. L-selectin recognizes sulfated, sialylated ligands on lymph node high endothelial venules. This recognition is abolished by strong periodate oxidation under conditions that destroy oligosaccharides. In contrast, mild periodate oxidation, which selectively oxidizes the side chain of sialic acid residues without affecting the underlying oligosaccharide, markedly enhances this interaction. The enhancement is calcium dependent, indicating that lectin recognition is maintained. Reduction of the sialic acid aldehydes generated by mild periodate to alcohol groups abolishes this effect. Covalent cross-linking of the oxidized ligand to L-selectin can be demonstrated, suggesting Schiff base formation between lysine residues of the selectin and the newly formed aldehydes. Such selectively oxidized sialylated ligands could be used to probe the lectin domains of the selectins and to identify lysine residues near the binding site. Also, this approach could be used to design drugs for disrupting leukocyte-endothelial interactions leading to pathological inflammation.
A simple direct method for finding persistence times of populations and application to conservation problems.
Authors: Mangel M , Tier C
Published: May 2006

The computation of persistence times of populations has become a central focus in conservation biology. We describe a simple, direct method for finding the statistics of persistence times by assuming that there is a maximum population size. Thus, even though the population dynamics may be very complex for population sizes below the maximum, it is possible to write a finite set of equations from which the mean and second moment of the persistence time can be found by using simple, algebraic methods. We apply the method to compute the mean and coefficient of variation of persistence times of populations that suffer large decrements (catastrophes). Our results show that in the presence of catastrophes, the increase in mean persistence time with large populations is not nearly as rapid as other theories suggest and that catastrophes occurring at even modest rates can considerably increase the risk of extinction.
Multiple mechanisms are implicated in the regulation of NF-kappa B activity during human cytomegalovirus infection.
Authors: Kowalik T F , Wing B , Haskill J S , Azizkhan J C , Baldwin A S , Huang E S
Published: May 2006

Infection-induced activation of the human cytomegalovirus major immediate early enhancer/promoter has been shown to be regulated primarily by transcription factor NF-kappa B cis elements. However, the mechanism(s) by which human cytomegalovirus induces NF-kappa B activity is unknown. A study was therefore undertaken to determine how this virus would affect normal NF-kappa B regulation. Viral infection of fibroblasts resulted in the specific stimulation of promoters containing major histocompatibility complex NF-kappa B cis elements fused upstream of the chloramphenicol acetyltransferase reporter gene. Electrophoretic mobility shift assays of nuclear extracts derived from mock- and virus-infected cells showed dramatic and sustained increases in DNA-binding proteins specific for these NF-kappa B sequences. Experiments using MAD-3 I kappa B, a specific inhibitor of NF-kappa B, and antibodies directed against rel family members demonstrated that the induced binding activities contained p50 and p65 proteins but not c-rel. Northern analysis indicated maximal levels of p50 mRNA by 4 h postinfection, whereas p65 and MAD-3 I kappa B mRNA accumulation peaked at 48-72 h postinfection, suggesting different regulatory mechanisms for p50 and p65/I kappa B genes. Electrophoretic mobility shift assays with deoxycholate-treated cytoplasmic extracts demonstrated a 3- to 4-fold decrease in the cytosolic stores of NF-kappa B binding activity by 4 h postinfection. Western blots probed with antibodies directed against MAD-3 I kappa B or pp40 (a protein isolated from chicken with sequence and biochemical properties similar to those of MAD-3 I kappa B) indicated that a cross-reactive peptide of 39 kDa was no longer detectable after 24 h postinfection. These results demonstrate that the activation and maintenance of nuclear NF-kappa B DNA binding and enhancer activities upon human cytomegalovirus infection occurs by multiple mechanisms.
Mutational analysis of the structure and biogenesis of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803.
Authors: Smart L B , Warren P V , Golbeck J H , McIntosh L
Published: May 2006

We have utilized the unicellular cyanobacterium Synechocystis sp. PCC 6803 to incorporate site-directed amino acid substitutions into the photosystem I (PSI) reactioncenter protein PsaB. A cysteine residue (position 565 of PsaB) proposed to serve as a ligand to the [4Fe-4S] center Fx was changed to serine, histidine, and aspartate. These three mutants--C565S, C565H, and C565D--all exhibited greatly reduced accumulation of PSI reaction-center proteins and failed to grow autotrophically, indicating that this cysteine most likely does coordinate Fx, which is crucial for PSI biogenesis. Interestingly, the strain C565S accumulated significantly more PSI than the other two cysteine mutants and displayed photoreduction of the [4Fe-4S] terminal electron acceptors FA and FB. Mutations were also introduced into a leucine zipper motif of PsaB, proposed to participate in reaction-center dimerization. The mutants L522V, L536M, and L522V/L536M all exhibited wild-type characteristics and grew autotrophically, whereas the L522P mutation prevented PSI accumulation. These data do not provide support for a major structural role of the leucine zipper in reaction-center dimerization or in assembly of Fx. However, the amino acid substitutions incorporated were conservative and might not have perturbed the leucine zipper.
Apoptosis in C3H/10T1/2 mouse embryonic cells: evidence for internucleosomal DNA modification in the absence of double-strand cleavage.
Authors: Tomei L D , Shapiro J P , Cope F O
Published: May 2006

Apoptosis in embryonic C3H/10T1/2 (clone 8) cells is marked by specific changes in morphology and DNA fragmentation that differ from those found in apoptotic thymocytes. These results demonstrate that ultrastructural changes within the nucleus associated with endonucleolytic degradation are linked with structural degradation at higher levels of chromatin organization. Strand modifications within the internucleosomal linker region are shown to involve alkaline-sensitive sites that appear to be sensitive to S1 endonuclease. Our results suggest that apoptosis is not dependent upon internucleosomal cleavage and may reveal the penultimate step and the nature of the metabolic cascade that leads to cell death.
Clonal expansions of activated gamma/delta T cells in recent-onset multiple sclerosis.
Authors: Shimonkevitz R , Colburn C , Burnham J A , Murray R S , Kotzin B L
Published: May 2006

Multiple sclerosis (MS) is a chronic disease characterized by focal demyelination of the white matter of the brain and spinal cord. Central nervous system damage appears to be mediated by infiltrating T lymphocytes and macrophages, and a central role for autoreactive CD4+ T cells has been proposed. However, the initial immune events that lead to the chronic process of MS remain unidentified. We now present evidence that a subset of T lymphocytes bearing gamma/delta T-cell antigen receptors has been activated in patients with recent-onset disease. Cells recovered from the cerebrospinal fluid of subjects with MS were cultured for short periods of time in medium supplemented with T-cell growth factors. Expansions of V delta 1 and V delta 2 T-cell receptor-bearing lymphocytes were found only in cell populations obtained from subjects with recent-onset disease. Similar populations were not expanded in subjects with chronic MS or other neurological diseases. Junctional region sequencing showed the expanded gamma/delta T cells to be oligoclonal in nature, suggestive of specific stimulation by antigen. These results reveal a fundamental difference in the immunopathogenesis of acute vs. chronic disease and provide additional insight into the autoimmune nature of MS.