bluuebunny/crossref_metadata_2025 huggingface dataset:
A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface
Authors: Gong Xiaohua , Chang Amy
Published: July 2002

Pma1 is a plasma membrane H
+
-ATPase whose activity at the cell surface is essential for cell viability. In this paper we describe a temperature-sensitive
pma1
allele,
pma1
-
10
(with two point mutations in the first cytoplasmic loop of Pma1), in which the newly synthesized mutant protein fails to remain stable at the cell surface at 37°C. Instead, Pma1-10 appears to undergo internalization for vacuolar degradation in a manner dependent on End4, Vps27, Doa4, and Pep4. By contrast with wild-type Pma1, mutant Pma1-10 is hypophosphorylated and fails to associate with a Triton-insoluble fraction at 37°C, suggesting failure to enter lipid rafts. Kinetic analysis reveals that, at the permissive temperature, newly synthesized Pma1-10 acquires Triton-insolubility before becoming stabilized. We suggest that phosphorylation and lipid raft association may play important roles in maintaining protein stability at the plasma membrane.
Myosin I mutants with only 1% of wild-type actin-activated MgATPase activity retain essentialin vivofunction(s)
Authors: Liu Xiong , Osherov Nir , Yamashita Roxanne , Brzeska Hanna , Korn Edward D. , May Gregory S.
Published: July 2002

The single class I myosin (MYOA) ofAspergillus nidulansis essential for hyphal growth. It is generally assumed that the functions of all myosins depend on their actin-activated MgATPase activity. Here we show that MYOA mutants with no more than 1% of the actin-activated MgATPase activity of wild-type MYOAin vitroand no detectablein vitromotility activity can support fungal cell growth, albeit with a delay in germination time and a reduction in hyphal elongation. From these and other data, we conclude that the essential role(s) of myosin I inA. nidulansis probably structural, requiring little, if any, actin-activated MgATPase or motor activity, which have long been considered the defining characteristics of the myosin family.
The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle
Authors: Duensing Stefan , Lee Lily Y. , Duensing Anette , Basile John , Piboonniyom Siribang-on , Gonzalez Sonia , Crum Christopher P. , Münger Karl
Published: July 2002

Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.
Ethanol hypersensitivity and olfactory discrimination defect in mice lacking a homolog of
Drosophila neuralized
Authors: Ruan Youlin , Tecott Laurence , Jiang Ming-Ming , Jan Lily Yeh , Jan Yuh Nung
Published: July 2002

Neurogenic genes in the Notch receptor-mediated signaling pathway
play important roles in neuronal cell fate specification as well as
neuronal differentiation. The
Drosophila neuralized
gene
is one of the neurogenic genes. We have cloned a mouse homolog of
Drosophila neuralized
,
m
-neu1, and found
that the
m
-neu1 transcript is expressed in
differentiated neurons. Mice deficient for
m
-neu1 are
viable and morphologically normal, but exhibit specific defects in
olfactory discrimination and hypersensitivity to ethanol. These
findings reveal an essential role of
m
-neu1 in ensuring
proper processing of certain information in the adult brain.
X-ray structure of sensory rhodopsin II at 2.1-Å resolution
Authors: Royant Antoine , Nollert Peter , Edman Karl , Neutze Richard , Landau Ehud M. , Pebay-Peyroula Eva , Navarro Javier
Published: July 2002

Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound HtrI and HtrII transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here, we present the x-ray structure of SRII from
Natronobacterium pharaonis
(pSRII) at 2.1-Å resolution, revealing a unique molecular architecture of the retinal-binding pocket. In particular, the structure of pSRII exhibits a largely unbent conformation of the retinal (as compared with bacteriorhodopsin and halorhodopsin), a hydroxyl group of Thr-204 in the vicinity of the Schiff base, and an outward orientation of the guanidinium group of Arg-72. Furthermore, the structure reveals a putative chloride ion that is coupled to the Schiff base by means of a hydrogen-bond network and a unique, positively charged surface patch for a probable interaction with HtrII. The high-resolution structure of pSRII provides a structural basis to elucidate the mechanisms of phototransduction and color tuning.
DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells
Authors: Nasrin Nargis , Ogg Scott , Cahill Catherine M. , Biggs William , Nui Simin , Dore Justin , Calvo Dominica , Shi Yang , Ruvkun Gary , Alexander-Bridges Maria C.
Published: July 2002

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the
Caenorhabditis elegans
protein DAF-16 binds the IGFBP-1⋅IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1⋅IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in
C. elegans
fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1⋅IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.
Length-dependent stability and strand length limits in antiparallel β-sheet secondary structure
Authors: Stanger Heather E. , Syud Faisal A. , Espinosa Juan F. , Giriat Izabela , Muir Tom , Gellman Samuel H.
Published: July 2002

Designed peptides that fold autonomously to specific conformations in aqueous solution are useful for elucidating protein secondary structural preferences. For example, autonomously folding model systems have been essential for establishing the relationship between α-helix length and α-helix stability, which would be impossible to probe with α-helices embedded in folded proteins. Here, we use designed peptides to examine the effect of strand length on antiparallel β-sheet stability. α-Helices become more stable as they grow longer. Our data show that a two-stranded β-sheet (“β-hairpin”) becomes more stable when the strands are lengthened from five to seven residues, but that further strand lengthening to nine residues does not lead to further β-hairpin stabilization for several extension sequences examined. (In one case, all-threonine extension, there may be an additional stabilization on strand lengthening from seven to nine residues.) These results suggest that there may be an intrinsic limit to strand length for most sequences in antiparallel β-sheet secondary structure.
Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering
Authors: Fassett Marlys S. , Davis Daniel M. , Valter Markus M. , Cohen George B. , Strominger Jack L.
Published: July 2002

Natural killer (NK) cell cytotoxicity is determined by a balance of positive and negative signals. Negative signals are transmitted by NK inhibitory receptors (killer immunoglobulin-like receptors, KIR) at the site of membrane apposition between an NK cell and a target cell, where inhibitory receptors become clustered with class I MHC ligands in an organized structure known as an inhibitory NK immune synapse. Immune synapse formation in NK cells is poorly understood. Because signaling by NK inhibitory receptors could be involved in this process, the human NK tumor line YTS was transfected with signal-competent and signal-incompetent KIR2DL1. The latter were generated by truncating the KIR2DL1 cytoplasmic tail or by introducing mutations in the immunoreceptor tyrosine-based inhibition motifs. The KIR2DL1 mutants retained their ability to cluster class I MHC ligands on NK cell interaction with appropriate target cells. Therefore, receptor–ligand clustering at the inhibitory NK immune synapse occurs independently of KIR2DL1 signal transduction. However, parallel examination of NK cell membrane lipid rafts revealed that KIR2DL1 signaling is critical for blocking lipid raft polarization and NK cell cytotoxicity. Moreover, raft polarization was inhibited by reagents that disrupt microtubules and actin filaments, whereas synapse formation was not. Thus, NK lipid raft polarization and inhibitory NK immune synapse formation occur by fundamentally distinct mechanisms.
The human decatenation checkpoint
Authors: Deming Paula B. , Cistulli Cheryl A. , Zhao Hui , Graves Paul R. , Piwnica-Worms Helen , Paules Richard S. , Downes C. Stephen , Kaufmann William K.
Published: July 2002

Chromatid catenation is actively monitored in human cells, with progression from G
2
to mitosis being inhibited when chromatids are insufficiently decatenated. Mitotic delay was quantified in normal and checkpoint-deficient human cells during treatment with ICRF-193, a topoisomerase II catalytic inhibitor that prevents chromatid decatenation without producing topoisomerase-associated DNA strand breaks. Ataxia telangiectasia (A-T) cells, defective in DNA damage checkpoints, showed normal mitotic delay when treated with ICRF-193. The mitotic delay in response to ICRF-193 was ablated in human fibroblasts expressing an ataxia telangiectasia mutated- and rad3-related (ATR) kinase-inactive
ATR
allele (ATR
ki
). BRCA1-mutant HCC1937 cells also displayed a defect in ICRF-193-induced mitotic delay, which was corrected by expression of wild-type BRCA1. Phosphorylations of hCds1 or Chk1 and inhibition of Cdk1 kinase activity, which are elements of checkpoints associated with DNA damage or replication, did not occur during ICRF-193-induced mitotic delay. Over-expression of cyclin B1 containing a dominant nuclear localization signal, and inhibition of Crm1-mediated nuclear export, reversed ICRF-193-induced mitotic delay. In combination, these results imply that ATR and BRCA1 enforce the decatenation G
2
checkpoint, which may act to exclude cyclin B1/Cdk1 complexes from the nucleus. Moreover, induction of ATR
ki
produced a 10-fold increase in chromosomal aberrations, further emphasizing the vital role for ATR in genetic stability.
Associative memory Hamiltonians for structure prediction without homology: Alpha-helical proteins
Authors: Hardin Corey , Eastwood Michael P. , Luthey-Schulten Zaida , Wolynes Peter G.
Published: July 2002

Energy landscape theory is used to obtain optimized energy
functions for predicting protein structure, without using homology
information. At short sequence separation the energy functions are
associative memory Hamiltonians constructed from a database of folding
patterns in nonhomologous proteins and at large separations they have
the form of simple pair potentials. The lowest energy minima provide
reasonably accurate tertiary structures even though no homologous
proteins are included in the construction of the Hamiltonian. We also
quantify the funnel-like nature of these energy functions by using free
energy profiles obtained by the multiple histogram method.
Degradation of the kinesin Kip1p at anaphase onset is mediated by the anaphase-promoting complex and Cdc20p
Authors: Gordon Donna M. , Roof David M.
Published: July 2002

Kip1p of
Saccharomyces cerevisiae
is a bipolar kinesin in the conserved bimC kinesin subfamily that mediates mitotic spindle–pole separation. Here, we show that Kip1p is regulated immediately after anaphase initiation by its rapid degradation. Degradation required the ubiquitin protein ligase called the anaphase-promoting complex, the anaphase-promoting complex activating protein Cdc20, and a unique 43-aa sequence in Kip1p. Degradation also required import of Kip1p into the nucleus, but occurred independently of spindle association. A mutation that stabilized Kip1p impaired anaphase progression. The timing of degradation suggests that Kip1p functions primarily during spindle assembly and metaphase, and that Kip1p degradation facilitates structural changes in the mitotic spindle as anaphase progresses.
Physical reasons for the unusual α-helix stabilization afforded by charged or neutral polar residues in alanine-rich peptides
Authors: Vila Jorge A. , Ripoll Daniel R. , Scheraga H. A.
Published: July 2002

We have carried out conformational energy calculations on
alanine-based copolymers with the sequence
Ac-AAAAAXAAAA-NH
2
in water, where X stands for lysine or
glutamine, to identify the underlying source of stability of
alanine-based polypeptides containing charged or highly soluble polar
residues in the absence of charge–charge interactions. The results
indicate that ionizable or neutral polar residues introduced into the
sequence to make them soluble sequester the water away from the CO and
NH groups of the backbone, thereby enabling them to form internal
hydrogen bonds. This solvation effect dictates the conformational
preference and, hence, modifies the
conformational
propensity
of alanine residues. Even though we carried out
simulations for specific amino acid sequences, our results provide an
understanding of some of the basic principles that govern the process
of folding of these short sequences independently of the kind of
residues introduced to make them soluble. In addition, we have
investigated through simulations the effect of the bulk dielectric
constant on the conformational preferences of these peptides. Extensive
conformational Monte Carlo searches on terminally blocked 10-mer and
16-mer homopolymers of alanine in the absence of salt were carried out
assuming values for the dielectric constant of the solvent ɛ of 80,
40, and 2. Our simulations show a clear tendency of these oligopeptides
to augment the α-helix content as the bulk dielectric constant of the
solvent is lowered. This behavior is due mainly to a loss of exposure
of the CO and NH groups to the aqueous solvent. Experimental evidence
indicates that the
helical propensity
of the amino acids
in water shows a dramatic increase on addition of certain alcohols,
such us trifluoroethanol. Our results provide a possible explanation of
the mechanism by which alcohol/water mixtures affect the free energy
of helical alanine oligopeptides relative to nonhelical ones.
An unusual vitamin E constituent (α-tocomonoenol) provides enhanced antioxidant protection in marine organisms adapted to cold-water environments
Authors: Yamamoto Yorihiro , Fujisawa Akio , Hara Akihiko , Dunlap Walter C.
Published: July 2002

A new vitamin E constituent having an unusual methylene unsaturation at the isoprenoid-chain terminus of α-tocopherol (α-Toc) was isolated from chum salmon eggs and was found to have identical antioxidant activity as does α-Toc in methanol or liposomal suspension at 37°C. Here we report that this marine-derived tocopherol (MDT) is broadly distributed with α-Toc in the tissue of marine fish, and that the MDT composition of total vitamin E is greater in the flesh of cold-water salmon (12–20%) than in that of tropical fish (≤2.5%). Vitamin E analysis of cultured masu salmon maintained on a MDT-deplete diet showed substantially less MDT content than native masu salmon, suggesting a trophic origin of MDT. This contention is supported by the finding of MDT in marine plankton from the cold waters of Hokkaido. We found that MDT inhibited peroxidation of cholesterol-containing phosphatidylcholine liposomes to a greater extent than did α-Toc at 0°C. Furthermore, the ratios of the rate constants for MDT and α-Toc to scavenge peroxyl radicals increased with decreasing rates of radical flux in liposomes and fish oil at 0°C, indicating that the enhanced activity of MDT at low temperature is attributed to its greater rate of diffusion in viscous lipids. These results suggest that MDT production, or its trophic accumulation, may reduce lipid peroxidation in marine organisms functionally adapted to cold-water environments.
A targeted dominant negative mutation of the thyroid hormone α1 receptor causes increased mortality, infertility, and dwarfism in mice
Authors: Kaneshige Masahiro , Suzuki Hideyo , Kaneshige Kumiko , Cheng Jun , Wimbrow Heather , Barlow Carrolee , Willingham Mark C. , Cheng Sheue-yann
Published: July 2002

Mutations in the thyroid hormone receptor β (TRβ) gene
result in resistance to thyroid hormone. However, it is unknown
whether mutations in the TRα gene could lead to a similar disease. To
address this question, we prepared mutant mice by targeting mutant
thyroid hormone receptor kindred PV (PV) mutation to the TRα gene
locus by means of homologous recombination (TRα1PV mice). The PV
mutation was derived from a patient with severe resistance to thyroid
hormone that has a frameshift of the C-terminal 14 aa of TRβ1. We
knocked in the same PV mutation to the corresponding TRα gene locus
to compare the phenotypes of TRα1
PV/+
mice with those of
TRβ
PV/+
mice. TRα1
PV/+
mice were viable,
indicating that the mutation of the TRα gene is not embryonic lethal.
In drastic contrast to the TRβ
PV/+
mice, which do not
exhibit a growth abnormality, TRα1
PV/+
mice were dwarfs.
These dwarfs exhibited increased mortality and reduced fertility. In
contrast to TRβ
PV/+
mice, which have a hyperactive
thyroid, TRα1
PV/+
mice exhibited mild thyroid failure.
The
in vivo
pattern of abnormal regulation of T3 target
genes in TRα1
PV/+
mice was unique from those of
TRβ
PV/+
mice. The distinct phenotypes exhibited by
TRα1
PV/+
and TRβ
PV/+
mice indicate that the
in vivo
functions of TR mutants are isoform-dependent.
The TRα1
PV/+
mice may be used as a tool to uncover human
diseases associated with mutations in the TRα gene and, furthermore,
to understand the molecular mechanisms by which TR isoforms exert their
biological activities.
Cell behavior in traveling wave patterns of myxobacteria
Authors: Welch Roy , Kaiser Dale
Published: July 2002

Cells in the early stages of starvation-induced fruiting body development migrate in a highly organized periodic pattern of equispaced accumulations that move as traveling waves. Two sets of waves are observed moving in opposite directions with the same wavelength and speed. To learn how the behavior of individual cells contributes to the wave pattern, fluorescent cells were tracked within a rippling population. These cells exhibit at least three types of organized behavior. First, most cell movement occurs along the same axis as the rippling movement. Second, there is a high degree of cell alignment parallel to the direction of rippling, as indicated by the biased movement. Third, by controlling the reversal frequency, cell movement becomes periodic in a rippling field. The periodicity of individual cells matches the period of macroscopic rippling. This last behavior is unique to a rippling population and, on the basis ofMyxococcus xanthusgenetic data, we conclude that this periodicity is linked to the C signal, a nondiffusible cell contact-mediated signaling molecule. When two cells moving in opposite directions meet end to end, they transmit the C signal to each other and in response reverse their gliding direction. This model of traveling waves represents a new mode of biological pattern formation that depends on cell-contact interactions rather than reaction diffusion.
Receptor-mediated gene transfer into macrophages.
Authors: Ferkol T , Perales J C , Mularo F , Hanson R W
Published: July 2002

Gene transfer systems targeting various receptors have been developed to introduce functional genes into cells in culture and into intact animals. A synthetic molecular conjugate, consisting of mannosylated polylysine that exploits endocytosis via the macrophage mannose receptor, was constructed and complexed to expression plasmids containing either the Photinus pyralis luciferase or Escherichia coli beta-galactosidase (lacZ) reporter genes. The DNA complexes were used to transfect murine macrophages isolated from peritoneal exudates in vitro. Luciferase and beta-galactosidase activity was found in transfected cells in culture, whereas complexes consisting of an irrelevant plasmid bound to mannosylated polylysine or the expression plasmid bound to galactosylated polylysine resulted in no detectable transgene expression. Gene transfer was inhibited by the addition of excess mannosylated bovine serum albumin to the culture medium before transfection. Reporter genes were also transferred into macrophages residing in the spleen and liver of adult animals using this system. Luciferase activity was maximal at 4 days after transfection and decreased to lower levels by 16 days. Transgene expression conformed to the distribution of cells that had nonspecific esterase, a cytochemical marker for macrophages. Thus, this system can be used to introduce functional genes into macrophages and may be an approach to the treatment of storage diseases that affect the reticuloendothelial system.
In vitro reconstitution of human replication factor C from its five subunits.
Authors: Uhlmann F , Cai J , Flores-Rozas H , Dean F B , Finkelstein J , O'Donnell M , Hurwitz J
Published: July 2002

Replication factor C (RFC, also called Activator I) is part of the processive eukaryotic DNA polymerase holoenzymes. The processive elongation of DNA chains requires that DNA polymerases are tethered to template DNA at primer ends. In eukaryotes the ring-shaped homotrimeric protein, proliferating cell nuclear antigen (PCNA), ensures tight template-polymerase interaction by encircling the DNA strand. Proliferating cell nuclear antigen is loaded onto DNA through the action of RFC in an ATP-dependent reaction. Human RFC is a protein complex consisting of five distinct subunits that migrate through SDS/polyacrylamide gels as protein bands of 140, 40, 38, 37, and 36 kDa. All five genes encoding the RFC subunits have been cloned and sequenced. A functionally identical RFC complex has been isolated from Saccharomyces cerevisiae and the deduced amino acid sequences among the corresponding human and yeast subunits are homologous. Here we report the expression of the five cloned human genes using an in vitro coupled transcription/translation system and show that the gene products form a complex resembling native RFC that is active in supporting an RFC-dependent replication reaction. Studies on the interactions between the five subunits suggest a cooperative mechanism in the assembly of the RFC complex. A three-subunit core complex, consisting of p36, p37, and p40, was identified and evidence is presented that p38 is essential for the interaction between this core complex and the large p140 subunit.
Interactions between tRNA identity nucleotides and their recognition sites in glutaminyl-tRNA synthetase determine the cognate amino acid affinity of the enzyme.
Authors: Ibba M , Hong K W , Sherman J M , Sever S , Söll D
Published: July 2002

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs both ensure accurate RNA recognition and prevent the binding of noncognate substrates. Here we show for Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) that the accuracy of tRNA recognition also determines the efficiency of cognate amino acid recognition. Steady-state kinetics revealed that interactions between tRNA identity nucleotides and their recognition sites in the enzyme modulate the amino acid affinity of GlnRS. Perturbation of any of the protein-RNA interactions through mutation of either component led to considerable changes in glutamine affinity with the most marked effects seen at the discriminator base, the 10:25 base pair, and the anticodon. Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that its constituents can be differentiated based upon biochemical function and their contribution to the apparent Gibbs' free energy of tRNA binding. Interactions with the acceptor stem act as strong determinants of tRNA specificity, with the discriminator base positioning the 3' end. The 10:25 base pair and U35 are apparently the major binding sites to GlnRS, with G36 contributing both to binding and recognition. Furthermore, we show that E. coli tryptophanyl-tRNA synthetase also displays tRNA-dependent changes in tryptophan affinity when charging a noncognate tRNA. The ability of tRNA to optimize amino acid recognition reveals a novel mechanism for maintaining translational fidelity and also provides a strong basis for the coevolution of tRNAs and their cognate synthetases.
X-ray structure determination of a metastable state of carbonmonoxy myoglobin after photodissociation.
Authors: Hartmann H , Zinser S , Komninos P , Schneider R T , Nienhaus G U , Parak F
Published: July 2002

The x-ray structure of carbon monoxide (CO)-ligated myoglobin illuminated during data collection by a laser diode at the wavelength lambda = 690 nm has been determined to a resolution of 1.7 A at T = 36 K. For comparison, we also measured data sets of deoxymyoglobin and CO-ligated myoglobin. In the photon-induced structure the electron density associated with the CO ligand can be described by a tube extending from the iron into the heme pocket over more than 4 A. This density can be interpreted by two discrete positions of the CO molecule. One is close to the heme iron and can be identified to be bound CO. In the second, the CO is dissociated from the heme iron and lies on top of pyrrole ring C. At our experimental conditions the overall structure of myoglobin in the metastable state is close to the structure of a CO-ligated molecule. However, the iron has essentially relaxed into the position of deoxymyoglobin. We compare our results with those of Schlichting el al. [Schlichting, I., Berendzen, J., Phillips, G. N., Jr., & Sweet, R. M. (1994) Nature 317, 808-812], who worked with the myoglobin mutant (D122N) that crystallizes in the space group P6 and Teng et al. [Teng, T. Y., Srajer, V. & Moffat, K. (1994) Nat. Struct. Biol. 1, 701-705], who used native myoglobin crystals of the space group P2(1). Possible reasons for the structural differences are discussed.
Targeted disruption of the arylsulfatase B gene results in mice resembling the phenotype of mucopolysaccharidosis VI.
Authors: Evers M , Saftig P , Schmidt P , Hafner A , McLoghlin D B , Schmahl W , Hess B , von Figura K , Peters C
Published: July 2002

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase B (ASB), which is involved in degradation of dermatan sulfate and chondroitin 4-sulfate. A MPS VI mouse model was generated by targeted disruption of the ASB gene. Homozygous mutant animals exhibit ASB enzyme deficiency and elevated urinary secretion of dermatan sulfate. They develop progressive symptoms resembling those of MPS VI in humans. Around 4 weeks of age facial dysmorphia becomes overt, long bones are shortened, and pelvic and costal abnormalities are observed. Major alterations in bone formation with perturbed cartilaginous tissues in newborns and widened, perturbed, and persisting growth plates in adult animals are seen. All major parenchymal organs show storage of glycosaminoglycans preferentially in interstitial cells and macrophages. Affected mice are fertile and mortality is not elevated up to 15 months of age. This mouse model will be a valuable tool for studying pathogenesis of MPS VI and may help to evaluate therapeutical approaches for lysosomal storage diseases.
Rad51 expression and localization in B cells carrying out class switch recombination.
Authors: Li M J , Peakman M C , Golub E I , Reddy G , Ward D C , Radding C M , Maizels N
Published: July 2002

Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast. In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced. Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells. Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies. Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur. Germinal center B cells are not stained by anti-Rad51 antibodies. Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation. These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.
Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.
Authors: Chatterjee B , Song C S , Jung M H , Chen S , Walter C A , Herbert D C , Weaker F J , Mancini M A , Roy A K
Published: July 2002

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.
Cation-pi interactions in aromatics of biological and medicinal interest: electrostatic potential surfaces as a useful qualitative guide.
Authors: Mecozzi S , West A P , Dougherty D A
Published: July 2002

The cation-pi interaction is an important, general force for molecular recognition in biological receptors. Through the sidechains of aromatic amino acids, novel binding sites for cationic ligands such as acetylcholine can be constructed. We report here a number of calculations on prototypical cation-pi systems, emphasizing structures of relevance to biological receptors and prototypical heterocycles of the type often of importance in medicinal chemistry. Trends in the data can be rationalized using a relatively simple model that emphasizes the electrostatic component of the cation-pi interaction. In particular, plots of the electrostatic potential surfaces of the relevant aromatics provide useful guidelines for predicting cation-pi interactions in new systems.
Threonine-for-leucine mutation within domain M2 of the neuronal alpha(7) nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist.
Authors: Palma E , Mileo A M , Eusebi F , Miledi R
Published: July 2002

A study was made of the effects of 5-hydroxytryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA. In contrast, 5HT elicited membrane currents (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.
Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding.
Authors: Hagen S J , Hofrichter J , Szabo A , Eaton W A
Published: July 2002

How fast can a protein fold? The rate of polypeptide collapse to a compact state sets an upper limit to the rate of folding. Collapse may in turn be limited by the rate of intrachain diffusion. To address this question, we have determined the rate at which two regions of an unfolded protein are brought into contact by diffusion. Our nanosecond-resolved spectroscopy shows that under strongly denaturing conditions, regions of unfolded cytochrome separated by approximately 50 residues diffuse together in 35-40 microseconds. This result leads to an estimate of approximately (1 microsecond)-1 as the upper limit for the rate of protein folding.
G protein-coupled cholecystokinin-B/gastrin receptors are responsible for physiological cell growth of the stomach mucosa in vivo.
Authors: Nagata A , Ito M , Iwata N , Kuno J , Takano H , Minowa O , Chihara K , Matsui T , Noda T
Published: July 2002

Many peptide hormone and neurotransmitter receptors belonging to the seven membrane-spanning G protein-coupled receptor family have been shown to transmit ligand-dependent mitogenic signals in vitro. However, the physiological roles of the mitogenic activity through G protein-coupled receptors in vivo remain to be elucidated. Here we have generated G protein-coupled cholecystokinin (CCK)-B/gastrin receptor deficient-mice by gene targeting. The homozygous mice showed a remarkable atrophy of the gastric mucosa macroscopically, even in the presence of severe hypergastrinemia. The atrophy was due to a decrease in parietal cells and chromogranin A-positive enterochromaffin-like cells expressing the H+,K(+)-ATPase and histidine decarboxylase genes, respectively. Oral administration of a proton pump inhibitor, omeprazole, which induced hypertrophy of the gastric mucosa with hypergastrinemia in wild-type littermates, did not eliminate the gastric atrophy of the homozygotes. These results clearly demonstrated that the G protein-coupled CCK-B/gastrin receptor is essential for the physiological as well as pathological proliferation of gastric mucosal cells in vivo.
Flows of knowledge from universities and federal laboratories: Modeling the flow of patent citations over time and across institutional and geographic boundaries
Authors: Jaffe Adam B. , Trajtenberg Manuel
Published: July 2002

The extent to which new technological knowledge flows across
institutional and national boundaries is a question of great importance
for public policy and the modeling of economic growth. In this paper we
develop a model of the process generating subsequent citations to
patents as a lens for viewing knowledge diffusion. We find that the
probability of patent citation over time after a patent is granted fits
well to a double-exponential function that can be interpreted as the
mixture of diffusion and obsolescense functions. The results indicate
that diffusion is geographically localized. Controlling for other
factors, within-country citations are more numerous and come more
quickly than those that cross country boundaries.
Ultrafast exciton relaxation in the B850 antenna complex ofRhodobacter sphaeroides
Authors: Nagarajan V. , Alden R. G. , Williams J. C. , Parson W. W.
Published: July 2002

Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacteriumRhodobacter sphaeroides. Absorption anisotropy decays also were measured. We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay. We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate.
CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins
Authors: Chen Tie , Gotschlich Emil C.
Published: July 2002

Neisseria gonorrhoeae
(GC) or
Escherichia
coli
expressing phase-variable opacity (Opa) protein
(Opa
+
GC or Opa
+
E. coli
) adhere
to human neutrophils and stimulate phagocytosis, whereas their
counterparts not expressing Opa protein (Opa
−
GC or
Opa
−
E. coli
) do not. Opa
+
GC
or
E. coli
do not adhere to human lymphocytes and
promyelocytic cell lines such as HL-60 cells. The adherence of
Opa
+
GC to the neutrophils can be enhanced dramatically if
the neutrophils are preactivated. These data suggest that the
components binding the Opa
+
bacteria might exist in the
granules. CGM1a antigen, a transmembrane protein of the
carcinoembryonic antigen family, is exclusively expressed in the
granulocytic lineage. The predicted molecular weight of CGM1a is ≈30
kDa. We observed specific binding of OpaI
+
E.
coli
to a 30-kDa band of polymorphonuclear leukocytes lysates.
To prove the hypothesis that the 30-kDa CGM1a antigen from neutrophils
was the receptor of Opa
+
bacteria, we showed that a HeLa
cell line expressing human CGM1a antigen (HeLa-CGM1a) bound Opa
+
E. coli
and subsequently engulfed the bacteria.
Monoclonal antibodies (COL-1) against CGM1 blocked the interaction
between Opa
+
E. coli
and HeLa-CGM1a. These
results demonstrate that HeLa cells when expressing the CGM1a antigens
bind and internalize OpaI
+
bacteria.
Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.
Authors: Li B , Subleski M , Fusaki N , Yamamoto T , Copeland T , Princler G L , Kung H , Kamata T
Published: July 2002

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.
Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.
Authors: Coppin C M , Finer J T , Spudich J A , Vale R D
Published: July 2002

Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.
Pre-mRNA splicing in plants: characterization of Ser/Arg splicing factors.
Authors: Lopato S , Mayeda A , Krainer A R , Barta A
Published: July 2002

The fact that animal introns are not spliced out in plants suggests that recognition of pre-mRNA splice sites differs between the two kingdoms. In plants, little is known about proteins required for splicing, as no plant in vitro splicing system is available. Several essential splicing factors from animals, such as SF2/ASF and SC-35, belong to a family of highly conserved proteins consisting of one or two RNA binding domain(s) (RRM) and a C-terminal Ser/Arg-rich (SR or RS) domain. These animal SR proteins are required for splice site recognition and spliceosome assembly. We have screened for similar proteins in plants by using monoclonal antibodies specific for a phosphoserine epitope of the SR proteins (mAb1O4) or for SF2/ASF. These experiments demonstrate that plants do possess SR proteins, including SF2/ASF-like proteins. Similar to the animal SR proteins, this group of proteins can be isolated by two salt precipitations. However, compared to the animal SR proteins, which are highly conserved in size and number, SR proteins from Arabidopsis, carrot, and tobacco exhibit a complex pattern of intra- and interspecific variants. These plant SR proteins are able to complement inactive HeLa cell cytoplasmic S1OO extracts that are deficient in SR proteins, yielding functional splicing extracts. In addition, plant SR proteins were active in a heterologous alternative splicing assay. Thus, these plant SR proteins are authentic plant splicing factors.
The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively
Authors: Sata Masataka , Stafford Walter F. , Mabuchi Katsuhide , Ikebe Mitsuo
Published: July 2002

While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal/smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met
1
–Gly
773
) and the C-terminal long α-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly
773
–Ser
1104
) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chain-associated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.