bluuebunny/crossref_metadata_2025 huggingface dataset:
An abundant cell-surface polypeptide is required for swimming by the nonflagellated marine cyanobacterium Synechococcus.
Authors: Brahamsha B
Published: July 2002

Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique and mysterious form of motility characterized by the ability to swim in liquid in the absence of flagella. An abundant cell-surface-associated polypeptide that is required for swimming motility by Synechococcus sp. strain WH8102 has been identified, and the gene encoding it, swmA, has been cloned and sequenced. The predicted SwmA protein contains a number of Ca2+-binding motifs as well as several potential N-glycosylation sites. Insertional inactivation of swmA in Synechococcus sp. strain WH8102 results in a loss of the ability to translocate, although the mutant strain, Swm-1, generates torque. This suggests that SwmA functions in the generation of thrust.
Glutamate is required to maintain the steady-state potassium pool in Salmonella typhimurium.
Authors: Yan D , Ikeda T P , Shauger A E , Kustu S
Published: July 2002

In many bacteria, accumulation of K+ at high external osmolalities is accompanied by accumulation of glutamate. To determine whether there is an obligatory relationship between glutamate and K+ pools, we studied mutant strains of Salmonella typhimurium with defects in glutamate synthesis. Enteric bacteria synthesize glutamate by the combined action of glutamine synthetase and glutamate synthase (GS/GOGAT cycle) or the action of biosynthetic glutamate dehydrogenase (GDH). Activity of the GS/GOGAT cycle is required under nitrogen-limiting conditions and is decreased at high external ammonium/ammonia ((NH4)+) concentrations by lowered synthesis of GS and a decrease in its catalytic activity due to covalent modification (adenylylation by GS adenylyltransferase). By contrast, GDH functions efficiently only at high external (NH4)+ concentrations, because it has a low affinity for (NH4)+. When grown at low concentrations of (NH4)+ (< or = 2 mM), mutant strains of S. typhimurium that lack GOGAT and therefore are dependent on GDH have a low glutamate pool and grow slowly; we now demonstrate that they have a low K+ pool. When subjected to a sudden (NH4)+ upshift, strains lacking GS adenylyltransferase drain their glutamate pool into glutamine and grow very slowly; we now find that they also drain their K+ pool. Restoration of the glutamate pool in these strains at late times after shift was accompanied by restoration of the K+ pool and a normal growth rate. Taken together, the results indicate that glutamate is required to maintain the steady-state K+ pool -- apparently no other anion can substitute as a counter-ion for free K+ -- and that K+ glutamate is required for optimal growth.
Naturally occurring variation in copia expression is due to both element (cis) and host (trans) regulatory variation.
Authors: Matyunina L V , Jordan I K , McDonald J F
Published: July 2002

Significant differences in levels of copia [Drosophila long terminal repeat (LTR) retrotransposon] expression exist among six species representing the Drosophila melanogaster species complex (D. melanogaster, Drosophila mauritiana, Drosophila simulans, Drosophila sechellia, Drosophila yakuba, and Drosophila erecta) and a more distantly related species (Drosophila willistoni). These differences in expression are correlated with major size variation mapping to putative regulatory regions of the copia 5' LTR and adjacent untranslated leader region (ULR). Sequence analysis indicates that these size variants were derived from a series of regional duplication events. The ability of the copia LTR-ULR size variants to drive expression of a bacterial chloramphenicol acetyltransferase reporter gene was tested in each of the seven species. The results indicate that both element-encoded (cis) and host-genome-encoded (trans) genetic differences are responsible for the variability in copia expression within and between Drosophila species. This finding indicates that models purporting to explain the dynamics and distribution of retrotransposons in natural populations must consider the potential impact of both element-encoded and host-genome-encoded regulatory variation to be valid. We propose that interelement selection among retrotransposons may provide a molecular drive mechanism for the evolution of eukaryotic enhancers which can be subsequently distributed throughout the genome by retrotransposition.
Interleukin 7 receptor-deficient mice lack gammadelta T cells.
Authors: Maki K , Sunaga S , Komagata Y , Kodaira Y , Mabuchi A , Karasuyama H , Yokomuro K , Miyazaki J I , Ikuta K
Published: July 2002

The interleukin 7 receptor (IL-7R) plays a crucial role in early B- and T-cell development. It consists of a unique a chain and a common gamma chain [IL-2 receptor gamma chain (IL-2Rgamma)]. Gene inactivation of IL-7, IL-7R, and IL-2Rgamma resulted in severe impairment of B and T lymphopoiesis in mice. In addition, IL-2Rgamma-deficient mice lack gammadelta T cells in the skin and have the impaired development of natural killer (NK) cells and intraepithelial lymphocytes. To explore the role of IL-7/IL-7R system in gammadelta T- and NK-cell development, we have generated and analyzed IL-7R-deficient mice. gammadelta T cells were absent from skin, gut, liver, and spleen in the deficient mice. In contrast, alphabeta T and B cells were detected in reduced, but certain, numbers, and NK cells developed normally. The gammadelta T-cell development in fetal and adult thymus was also completely blocked. These results clearly demonstrate that the signal from IL-7R is indispensable for gammadelta T-cell development in both thymic and extrathymic pathways. On the contrary, it is suggested that NK-cell development requires cytokine(s) other than IL-7.
Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit.
Authors: Sun J B , Rask C , Olsson T , Holmgren J , Czerkinsky C
Published: July 2002

Oral administration of autoantigens can prevent and partially suppress autoimmune diseases in a number of experimental models, Depending on the dose of antigen fed, this approach appears to involve distinct yet reversible and short-lasting mechanisms (anergy/deletion and suppression) and usually requires repeated feeding of large (suppression) to massive (anergy/deletion) amounts of autoantigens to be effective. Most importantly, this approach is relatively less effective in animals already systemically sensitized to the fed antigen, such as in animals already harboring autoreactive T cells and, thus, presumably also in humans suffering from an autoimmune disorder. We have previously shown that feeding a single dose of minute amounts of antigens conjugated to cholera toxin B subunit (CTB) can effectively suppress delayed-type hypersensitivity reactions in systemically immune animals. We now report that feeding small amounts of myelin basic protein (MBP) conjugated to CTB either before or after disease induction protected rats from experimental autoimmune encephalomyelitis. Such treatment was as effective in suppressing interleukin 2 production and proliferative responses of lymph node cells to MBP as treatment involving repeated feeding with much larger (50- to 100-fold) doses of free MBP. Different from the latter treatment, which led to decreased production of interferon-gamma in lymph nodes, low-dose oral CTB-MBP treatment was associated with increased interferon-gamma production. Most importantly, low-dose oral CTB-MBP treatment greatly reduced the level of leukocyte infiltration into spinal cord tissue compared with treatment with repeated feeding of large doses of MBP. These results suggest that the protection from experimental autoimmune encephalomyelitis achieved by feeding CTB-conjugated myelin autoantigen involves immunomodulating mechanisms that are distinct from those implicated by conventional protocols of oral tolerance induction.
Psychophysical evidence for fast region-based segmentation processes in motion and color.
Authors: Møller P , Hurlbert A C
Published: July 2002

Theories of image segmentation suggest that the human visual system may use two distinct processes to segregate figure from background: a local process that uses local feature contrasts to mark borders of coherent regions and a global process that groups similar features over a larger spatial scale. We performed psychophysical experiments to determine whether and to what extent the global similarity process contributes to image segmentation by motion and color. Our results show that for color, as well as for motion, segmentation occurs first by an integrative process on a coarse spatial scale, demonstrating that for both modalities the global process is faster than one based on local feature contrasts. Segmentation by motion builds up over time, whereas segmentation by color does not, indicating a fundamental difference between the modalities. Our data suggest that segmentation by motion proceeds first via a cooperative linking over space of local motion signals, generating almost immediate perceptual coherence even of physically incoherent signals. This global segmentation process occurs faster than the detection of absolute motion, providing further evidence for the existence of two motion processes with distinct dynamic properties.
In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains.
Authors: Fernandes-Alnemri T , Armstrong R C , Krebs J , Srinivasula S M , Wang L , Bullrich F , Fritz L C , Trapani J A , Tomaselli K J , Litwack G , Alnemri E S
Published: July 2002

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartate specific cysteine proteases (ASCPs) is responsible for the apoptotic changes observed in mammalian cells undergoing programmed cell death. Here we describe the cloning of two novel ASCPs from human Jurkat T-lymphocytes. Like other ASCPs, the new proteases, named Mch4 and Mch5, are derived from single chain proenzymes. However, their putative active sites contain a QACQG pentapeptide instead of the QACRG present in ail known ASCPs. Also, their N termini contain FADD-like death effector domains, suggesting possible interaction with FADD. Expression of Mch4 in Escherichia coli produced an active protease that, like other ASCPs, was potently inhibited (Kj = 14 nM) by the tetrapeptide aldehyde DEVD-CHO. Interestingly, both Mch4 and the serine protease granzyme B cleave recombinant proCPP32 and proMch3 at a conserved IXXD-S sequence to produce the large and small subunits of the active proteases. Granzyme B also cleaves proMch4 at a homologous IXXD-A processing sequence to produce mature Mch4. These observations suggest that CPP32 and Mch3 are targets of mature Mch4 protease in apoptotic cells. The presence of the FADD-like domains in Mch4 and Mch5 suggests a role for these proteases in the Fas-apoptotic pathway. In addition, these proteases could participate in the granzyme B apoptotic pathways.
Substitution rate calibration of small subunit ribosomal RNA identifies chlorarachniophyte endosymbionts as remnants of green algae.
Authors: Van de Peer Y , Rensing S A , Maier U G , De Wachter R
Published: July 2002

Chlorarachniophytes are amoeboid algae with chlorophyll a and b containing plastids that are surrounded by four membranes instead of two as in plants and green algae. These extra membranes form important support for the hypothesis that chlorarachniophytes have acquired their plastids by the ingestion of another eukaryotic plastid-containing alga. Chlorarachniophytes also contain a small nucleus-like structure called the nucleomorph situated between the two inner and the two outer membranes surrounding the plastid. This nucleomorph is a remnant of the endosymbiont's nucleus and encodes, among other molecules, small subunit ribosomal RNA. Previous phylogenetic analyses on the basis of this molecule provided unexpected and contradictory evidence for the origin of the chlorarachniophyte endosymbiont. We developed a new method for measuring the substitution rates of the individual nucleotides of small subunit ribosomal RNA. From the resulting substitution rate distribution, we derived an equation that gives a more realistic relationship between sequence dissimilarity and evolutionary distance than equations previously available. Phylogenetic trees constructed on the basis of evolutionary distances computed by this new method clearly situate the chlorarachniophyte nucleomorphs among the green algae. Moreover, this relationship is confirmed by transversion analysis of the Chlorarachnion plastid small subunit ribosomal RNA.
Apoptosis and APC in colorectal tumorigenesis.
Authors: Morin P J , Vogelstein B , Kinzler K W
Published: July 2002

Tumors result from disruptions in the homeostatic mechanisms that regulate cell birth and cell death. In colon cancer, one of the earliest manifestation of this imbalance is the formation of polyps, caused by somatic and inherited mutations of the adenomatous polyposis coli (APC) tumor suppressor gene in both humans and mice. While the importance of APC in tumorigenesis is well documented, how it functions to prevent tumors remains a mystery. Using a novel inducible expression system, we show that expression of APC in human colorectal cancer cells containing endogenous inactive APC alleles results in a substantial diminution of cell growth. Further evaluation demonstrated that this was due to the induction of cell death through apoptosis. These results suggest that apoptosis plays a role not only in advanced tumors but also at the very earliest stages of neoplasia.
Gene recognition via spliced sequence alignment.
Authors: Gelfand M S , Mironov A A , Pevzner P A
Published: July 2002

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.
Operant conditioning of H-reflex changes synaptic terminals on primate motoneurons.
Authors: Feng-Chen K C , Wolpaw J R
Published: July 2002

Operant conditioning of the primate triceps surae H-reflex, the electrical analog of the spinal stretch reflex, creates a memory trace that includes changes in the spinal cord. To define the morphological correlates of this plasticity, we analyzed the synaptic terminal coverage of triceps surae motoneurons from animals in which the triceps surae H-reflex in one leg had been increased (HRup mode) or decreased (HRdown mode) by conditioning and compared them to each other and to motoneurons from unconditioned animals. Motoneurons were labeled by intramuscular injection of cholera toxin-horseradish peroxidase. A total of 5055 terminals on the cell bodies and proximal dendrites of 114 motoneurons from 14 animals were studied by electron microscopy. Significant differences were found between HRup and HRdown animals and between HRup and naive (i.e., unconditioned) animals. F terminals (i.e., putative inhibitory terminals) were smaller and their active zone coverage on the cell body was lower on motoneurons from the conditioned side of HRup animals than on motoneurons from the conditioned side of HRdown animals. C terminals (i.e., terminals associated with postsynaptic cisterns and rough endoplasmic reticulum) were smaller and the number of C terminals in each C complex (i.e., a group of contiguous C terminals) was larger on motoneurons from the conditioned side of HRup animals than on motoneurons either from the conditioned side of HRdown animals or from naive animals. Because the treatment of HRup and HRdown animals differed only in the reward contingency, the results imply that the two contingencies had different effects on motoneuron synaptic terminals. In combination with other recent data, they show that H-reflex conditioning produces a complex pattern of spinal cord plasticity that includes changes in motoneuron physiological properties as well as in synaptic terminals. Further delineation of this pattern should reveal the contribution of the structural changes described here to the learned change in behavior.
Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.
Authors: Mouz N , Tricot C , Ebel C , Petillot Y , Stalon V , Dideberg O
Published: July 2002

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).
Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure.
Authors: Suzuki M , Baskin D , Hood L , Loeb L A
Published: July 2002

Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.
Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.
Authors: Halpert I , Sires U I , Roby J D , Potter-Perigo S , Wight T N , Shapiro S D , Welgus H G , Wickline S A , Parks W C
Published: July 2002

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.
A minimal gene set for cellular life derived by comparison of complete bacterial genomes.
Authors: Mushegian A R , Koonin E V
Published: July 2002

The recently sequenced genome of the parasitic bacterium Mycoplasma genitalium contains only 468 identified protein-coding genes that have been dubbed a minimal gene complement [Fraser, C.M., Gocayne, J.D., White, O., Adams, M.D., Clayton, R.A., et al. (1995) Science 270, 397-403]. Although the M. genitalium gene complement is indeed the smallest among known cellular life forms, there is no evidence that it is the minimal self-sufficient gene set. To derive such a set, we compared the 468 predicted M. genitalium protein sequences with the 1703 protein sequences encoded by the other completely sequenced small bacterial genome, that of Haemophilus influenzae. M. genitalium and H. influenzae belong to two ancient bacterial lineages, i.e., Gram-positive and Gram-negative bacteria, respectively. Therefore, the genes that are conserved in these two bacteria are almost certainly essential for cellular function. It is this category of genes that is most likely to approximate the minimal gene set. We found that 240 M. genitalium genes have orthologs among the genes of H. influenzae. This collection of genes falls short of comprising the minimal set as some enzymes responsible for intermediate steps in essential pathways are missing. The apparent reason for this is the phenomenon that we call nonorthologous gene displacement when the same function is fulfilled by nonorthologous proteins in two organisms. We identified 22 nonorthologous displacements and supplemented the set of orthologs with the respective M. genitalium genes. After examining the resulting list of 262 genes for possible functional redundancy and for the presence of apparently parasite-specific genes, 6 genes were removed. We suggest that the remaining 256 genes are close to the minimal gene set that is necessary and sufficient to sustain the existence of a modern-type cell. Most of the proteins encoded by the genes from the minimal set have eukaryotic or archaeal homologs but seven key proteins of DNA replication do not. We speculate that the last common ancestor of the three primary kingdoms had an RNA genome. Possibilities are explored to further reduce the minimal set to model a primitive cell that might have existed at a very early stage of life evolution.
A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.
Authors: Maestrini E , Tamagnone L , Longati P , Cremona O , Gulisano M , Bione S , Tamanini F , Neel B G , Toniolo D , Comoglio P M
Published: July 2002

In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.
p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts.
Authors: Dietrich C , Bartsch T , Schanz F , Oesch F , Wieser R J
Published: July 2002

Proteases are known to play important roles in cell growth control, although the underlying mechanisms are still poorly understood. Here we show that the protease inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal induced cell cycle arrest in platelet-derived growth factor-stimulated human fibroblasts at the G1/S boundary of the cell cycle by inhibiting the proteasome. Inhibition of the proteasome resulted in accumulation of the tumor suppressor p53, which was followed by an increase in the amount of the cyclin-dependent kinase-inhibitor p21. As a consequence, both phosphorylation and activity of the cyclin-dependent kinase 2/cyclin E complex were inhibited. We further observed that the retinoblastoma gene product, pRb, remained in the hypophosphorylated state, thus preventing cells from progression into the S-phase. These studies strongly support the hypothesis that the proteasome is a key regulator in the G1-phase of cell cycle progression.
Active transposition in zebrafish.
Authors: Lam W L , Lee T S , Gilbert W
Published: July 2002

We have identified a new family of Tc1-like transposons in the zebrafish, Danio rerio. The sequence of a candidate active transposon, deduced from sample Tzf elements, shows limited resemblance to the previously described Tdr1 elements of zebrafish. Both the Tzf and the Tdr elements are extremely abundant in zebrafish. We describe here a general strategy for detecting transposition events in a complex genome and demonstrate its utility by selectively monitoring hundreds of potentially active Tzf copies in the zebrafish genome against a background of other related elements. We have followed members of a zebrafish pedigree, using this two-dimensional transposon display strategy, to identify the first examples of active transposition of such elements in vertebrates.
An iterative method for extracting energy-like quantities from protein structures.
Authors: Thomas P D , Dill K A
Published: July 2002

We present a method (ENERGI) for extracting energy-like quantities from a data base of protein structures. In this paper, we use the method to generate pairwise additive amino acid "energy" scores. These scores are obtained by iteration until they correctly discriminate a set of known protein folds from decoy conformations. The method succeeds in lattice model tests and in the gapless threading problem as defined by Maiorov and Crippen [Maiorov, V. N. & Crippen, G. M. (1992) J. Mol. Biol. 227, 876-888]. A more challenging test of threading a larger set of test proteins derived from the representative set of Hobohm and Sander [Hobohm, U. & Sander, C. (1994) Protein Sci. 3, 522-524] is used as a "workbench" for exploring how the ENERGI scores depend on their parameter sets.
Specific transcellular binding between membrane proteins crucial to Alzheimer disease.
Authors: Dewji N N , Singer S J
Published: July 2002

Molecular genetic studies of families suffering from genetic forms of early onset Alzheimer disease (AD) have identified three genes and their protein products as being crucially involved in the etiology of AD. The three proteins are all integral membrane proteins. One of them is beta-APP, the precursor of the beta-amyloid found in the characteristic neuritic plaques present in the brains of AD patients. The other two, S182 and STM2, are homologous in amino acid sequence to one another but are unrelated to beta-APP. It is shown here, using cultured cells transfected for each of these proteins, that beta-APP binds specifically and transcellularly to either S182 or STM2. We propose that this transcellular binding may not only be important in normal neuronal physiology and development but may be directly involved in the process of formation of beta-amyloid from beta-APP.
Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble lymphotoxin-β receptor–IgG1 fusion protein
Authors: Ettinger Rachel , Browning Jeffrey L. , Michie Sara A. , van Ewijk Willem , McDevitt Hugh O.
Published: July 2002

Early in ontogeny, the secondary lymphoid organs become populated with numerous cells of mesodermal origin which forms both the lymphoid and stromal elements. The critical receptor/ligand interactions necessary for lymphoid organogenesis to occur are for the most part unknown. Although lymphotoxin-α (LTα) has been shown to be required for normal lymph node, Peyer’s patch, and splenic development, it is unclear if soluble LTα3, and/or cell-bound lymphotoxin-αβ (LTαβ) mediate these developmental events. Here we report that blocking LTαβ/lymphotoxin-β receptor (LTβR) interactionin vivoby generating mice which express a soluble LTβR–Fc fusion protein driven by the human cytomegalovirus promoter results in an array of anatomic abnormalities affecting both the spleen and Peyer’s patches, but not the lymph nodes. These results demonstrate that surface LTαβ ligand plays a critical role in normal lymphoid organ development.
Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate
Authors: Werner Eric D. , Brodsky Jeffrey L. , McCracken Ardythe A.
Published: July 2002

Until recently, the degradation of aberrant and unassembled proteins retained in the endoplasmic reticulum (ER) was thought to involve unidentified ER-localized proteases. We now show that the ER-associated degradation (ERAD) of two mutant proteins that accumulate in the ER lumen is inhibited in a proteasome-defective yeast strain and when cytosol from this mutant is used in anin vitroassay. In addition, ERAD is limitedin vitroin the presence of the proteasome inhibitors, 3,4-dichloroisocoumarin and lactacystin. Furthermore, we find that an ERAD substrate is exported from ER-derived microsomes, and the accumulation of exported substrate is 2-fold greater when proteasome mutant cytosol is used in place of wild-type cytosol. We conclude that lumenal ERAD substrates are exported from the yeast ER to the cytoplasm for degradation by the proteasome complex.
Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: Evidence suggesting multiple cholesterol esterification enzymes in mammals
Authors: Meiner Vardiella L. , Cases Sylvaine , Myers Heather M. , Sande Eric R. , Bellosta Stefano , Schambelan Morris , Pitas Robert E. , McGuire James , Herz Joachim , Farese Robert V.
Published: July 2002

The microsomal enzyme acyl-CoA:cholesterol acyltransferase (ACAT; EC2.3.1.26) catalyzes the esterification of cellular cholesterol with fatty acids to form cholesterol esters. ACAT activity is found in many tissues, including macrophages, the adrenal glands, and the liver. In macrophages, ACAT is thought to participate in foam cell formation and thereby to contribute to atherosclerotic lesion development. Disruption of the gene for ACAT (Acact) in mice resulted in decreased cholesterol esterification in ACAT-deficient fibroblasts and adrenal membranes, and markedly reduced cholesterol ester levels in adrenal glands and peritoneal macrophages; the latter finding will be useful in testing the role of ACAT and macrophage foam cell formation in atherosclerosis. In contrast, the livers of ACAT-deficient mice contained substantial amounts of cholesterol esters and exhibited no reduction in cholesterol esterification activity. These tissue-specific reductions in cholesterol esterification provide evidence that in mammals this process involves more than one form of esterification enzyme.
Biosynthesis of vitamin B
12
: Factor IV, a new intermediate in the anaerobic pathway
Authors: Scott A. Ian , Stolowich Neal J. , Wang Jianji , Gawatz Oliver , Fridrich Erwin , Müller Gerhard
Published: July 2002

The structure of a novel tetradehydrocorrin, factor IV, isolated
from
Propionibacterium shermanii
has been established by
multidimensional NMR spectroscopy. Incorporation of radiolabeled factor
IV into cobyrinic acid established the biointermediacy of this cobalt
complex, whose structure has implications for the mechanisms of the
anaerobic pathway to B
12
.
BAX-induced cell death may not require interleukin 1β-converting enzyme-like proteases
Authors: Xiang Jialing , Chao Debra T. , Korsmeyer Stanley J.
Published: July 2002

Expression of BAX, without another death stimulus, proved
sufficient to induce a common pathway of apoptosis. This
included the activation of interleukin 1β-converting enzyme
(ICE)-like proteases with cleavage of the endogenous
substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation
inhibitor for the rho family), as well as the fluorogenic peptide
acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The
inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk)
successfully blocked this protease activity and prevented FAS-induced
death but not BAX-induced death. Blocking ICE-like protease activity
prevented the cleavage of nuclear and cytosolic substrates and
the DNA degradation that followed BAX induction. However, the fall in
mitochondrial membrane potential, production of reactive oxygen
species, cytoplasmic vacuolation, and plasma membrane permeability that
are downstream of BAX still occurred. Thus, BAX-induced alterations in
mitochondrial function and subsequent cell death do not apparently
require the known ICE-like proteases.
Network of tangential pathways for neuronal migration in adult mammalian brain
Authors: Doetsch Fiona , Alvarez-Buylla Arturo
Published: July 2002

Cells in the brains of adult mammals continue to proliferate in the
subventricular zone (SVZ) throughout the lateral wall of the lateral
ventricle. Here we show, using whole mount dissections of this wall
from adult mice, that the SVZ is organized as an extensive network of
chains of neuronal precursors. These chains are immunopositive to the
polysialylated form of NCAM, a molecule present at sites of plasticity,
and TuJ1, an early neuronal marker. The majority of the chains are
oriented along the rostrocaudal axis and many join the rostral
migratory stream that terminates in the olfactory bulb. Using focal
microinjections of DiI and transplantation of SVZ cells carrying a
neuron-specific reporter gene, we demonstrate that cells originating at
different rostrocaudal levels of the SVZ migrate rostrally and reach
the olfactory bulb where they differentiate into neurons. Our results
reveal an extensive network of pathways for the tangential chain
migration of neuronal precursors throughout the lateral wall of the
lateral ventricle in the adult mammalian brain.
Stimulation of growth factor receptor signal transduction by activation of voltage-sensitive calcium channels.
Authors: Rosen L B , Greenberg M E
Published: July 2002

To understand the mechanisms by which electrical activity may generate long-term responses in the nervous system, we examined how activation of voltage-sensitive calcium channels (VSCCs) can stimulate the Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Calcium influx through L-type VSCCs leads to tyrosine phosphorylation of the adaptor protein Shc and its association with the adaptor protein Grb2, which is bound to the guanine nucleotide exchange factor Sos1. In response to calcium influx, Shc, Grb2, and Sos1 inducibly associate with a 180-kDa tyrosine-phosphorylated protein, which was determined to be the epidermal growth factor receptor (EGFR). Calcium influx induces tyrosine phosphorylation of the EGFR to levels that can activate the MAPK signaling pathway. Thus, ion channel activation stimulates growth factor receptor signal transduction.
Isolation of an oxygen-sensitive FNR protein of Escherichia coli: interaction at activator and repressor sites of FNR-controlled genes.
Authors: Melville S B , Gunsalus R P
Published: July 2002

The Escherichia coli fnr gene product, FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state. To investigate this process, a fnr mutant that encodes an altered FNR protein with three amino acid substitutions in the N-terminal domain was constructed by site-directed mutagenesis. In vivo, the mutant behaved like a wild-type strain under anaerobic conditions but had a 14-fold elevated level of transcriptional activation of a reporter gene during aerobic cell growth. The altered fur gene was overexpressed in E. coli and the resultant FNR protein was purified to near homogeneity by using anaerobic chromatography procedures. An in vitro Rsa I restriction site protection assay was developed that allowed for the assessment of oxygen-dependent DNA binding of the mutant FNR protein. The FNR protein was purified as a monomer of M(r) 28,000 that contained nonheme iron at 2.05 +/- 0.34 mol of Fe per FNR monomer. In vitro DNase I protection studies were performed to establish the locations of the FNR-binding sites at the narG, narK, dmsA, and hemA promoters that are regulated by either activation or repression of their transcription. The sizes of the DNA footprints are consistent with the binding of two monomers of FNR that protect the symmetrical FNR-recognition sequence TTGAT-nnnnATCAA. Exposure of the FNR protein or protein-DNA complex to air for even short periods of time (approximately 5 min) led to the complete loss of DNA protection at a consensus FNR recognition site. A model whereby the FNR protein exists in the cell as a monomer that assembles on the DNA under anaerobic conditions to form a dimer is discussed.
Short-term synaptic enhancement and long-term potentiation in neocortex.
Authors: Castro-Alamancos M A , Connors B W
Published: July 2002

Repetitive stimuli reliably induce long-term potentiation (LTP) of synapses in the upper layers of the granular somatosensory cortex but not the agranular motor cortex of rats. Herein we examine, in these same cortical areas, short-term changes in synaptic strength that occur during the LTP induction period. theta-Burst stimulation produced a strong short-term enhancement of synapses in the granular area but only weak enhancement in the agranular area. The magnitude of enhancement during stimulation was strongly correlated with the magnitude of LTP subsequently expressed. Short-term enhancement was abolished by an antagonist of N-methyl-D-aspartate (NMDA) receptors but remained in the presence of a non-NMDA receptor antagonist. Inhibitory postsynaptic potentials of the granular and agranular areas displayed similar frequency sensitivity, but the frequency sensitivity of NMDA receptor-dependent excitatory postsynaptic potentials differed significantly between areas. We propose that pathway-specific differences in short-term enhancement are due to variations in the frequency dependence of NMDA currents; different capacities for short-term enhancement may explain why repetitive stimulation more readily induces LTP in the somatosensory cortex than in the motor cortex.
Improved antitumor activity of a recombinant anti-Lewis(y) immunotoxin not requiring proteolytic activation.
Authors: Kuan C T , Pastan I
Published: July 2002

B1(dsFv)-PE33 is a recombinant immunotoxin composed of a mutant form of Pseudomonas exotoxin (PE) that does not need proteolytic activation and a disulfide-stabilized Fv fragment of the anti-Lewis(y) monoclonal antibody B1, which recognizes a carbohydrate epitope on human carcinoma cells. In this molecule, amino acids 1-279 of PE are deleted and domain Ib (amino acids 365-394) is replaced by the heavy chain variable region (VH) domain of monoclonal antibody B1. The light chain (VL) domain is connected to the VH domain by a disulfide bond. This recombinant toxin, termed B1(dsFv)-PE33, does not require proteolytic activation and it is smaller than other immunotoxins directed at Lewis(y), all of which require proteolytic activation. Furthermore, it is more cytotoxic to antigen-positive cell lines. B1(dsFv)-PE38 has the highest antitumor activity of anti-Lewis(y) immunotoxins previously constructed. B1(dsFv)-PE33 caused complete regression of tumors when given at 12 micrograms/kg (200 pmol/kg) every other day for three doses, whereas B1(dsFv)-PE38 did not cause regressions at 13 micrograms/kg (200 pmol/kg). By bypassing the need for proteolytic activation and decreasing molecular size we have enlarged the therapeutic window for the treatment of human cancers growing in mice, so that complete remissions are observed at 2.5% of the LD50.
Anticoagulant repertoire of the hookworm Ancylostoma caninum.
Authors: Stassens P , Bergum P W , Gansemans Y , Jespers L , Laroche Y , Huang S , Maki S , Messens J , Lauwereys M , Cappello M , Hotez P J , Lasters I , Vlasuk G P
Published: July 2002

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.
Spontaneous avoidance behavior in Drosophila null for calmodulin expression.
Authors: Heiman R G , Atkinson R C , Andruss B F , Bolduc C , Kovalick G E , Beckingham K
Published: July 2002

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.
Pentameric subunit stoichiometry of a neuronal glutamate receptor.
Authors: Ferrer-Montiel A V , Montal M
Published: July 2002

Ionotropic glutamate receptors, neurotransmitter-activated ion channels that mediate excitatory synaptic transmission in the central nervous system, are oligomeric membrane proteins of unknown subunit stoichiometry. To determine the subunit stoichiometry we have used a functional assay based on the blockade of two alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor subunit 1 (GluR1) mutant subunits selectively engineered to exhibit differential sensitivity to the open channel blockers phencyclidine and dizolcipine (MK-801). Coinjection into amphibian oocytes of weakly sensitive with highly sensitive subunit complementary RNAs produces functional heteromeric channels with mixed blocker sensitivities. Increasing the fraction of the highly sensitive subunit augmented the proportion of drug-sensitive receptors. Analysis of the data using a model based on random aggregation of receptor subunits allowed us to determine a pentameric stoichiometry for GluR1. This finding supports the view that a pentameric subunit organization underlies the structure of the neuronal ionotropic glutamate receptor gene family.