bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure of heat shock locus V (HslV) from
Escherichia coli
Authors: Bochtler Matthias , Ditzel Lars , Groll Michael , Huber Robert
Published: July 2002

Heat shock locus V (HslV; also called ClpQ) is the proteolytic core of the ATP-dependent protease HslVU in
Escherichia coli
. It has sequence similarity with the β-type subunits of the eukaryotic and archaebacterial proteasomes. Unlike these particles, which display 72-point symmetry, it is a dimer of hexamers with 62-point symmetry. The crystal structure of HslV at 3.8-Å resolution, determined by isomorphous replacement and symmetry averaging, shows that in spite of the different symmetry of the particle, the fold and the contacts between subunits are conserved. A tripeptide aldehyde inhibitor, acetyl-Leu-Leu-norleucinal, binds to the N-terminal threonine residue of HslV, probably as a hemiacetal, relating HslV also functionally to the proteasomes of archaea and eukaryotes.
The distribution of genes in the genomes of Gramineae
Authors: Barakat Abdelali , Carels Nicolas , Bernardi Giorgio
Published: July 2002

Recent investigations showed that most maize genes are present in compositional fractions of nuclear DNA that cover only a 1–2% GC (molar fraction of guanosine plus cytosine in DNA) range and represent only 10–20% of the genome. These fractions, which correspond to compositional genome compartments that are distributed on all chromosomes, were collectively called the “gene space.” Outside the gene space, the maize genome appears to contain no genes, except for some zein genes and for ribosomal genes. Here, we investigated the distribution of genes in the genomes of two other Gramineae, rice and barley, and used a new set of probes to study further the gene distribution of maize. We found that the distribution of genes in these three genomes is basically similar in that all genes, except for ribosomal genes and some storage protein genes, were located in gene spaces that (
i
) cover GC ranges of 0.8%, 1.0%, and 1.6% and represent 12%, 17%, and 24% of the genomes of barley, maize, and rice, respectively; (
ii
) are due to a remarkably uniform base composition in the sequences surrounding the genes, which are now known to consist mainly of transposons; (
iii
) have sizes approximately proportional to genome sizes, suggesting that expansion–contraction phenomena proceed in parallel in the gene space and in the gene-empty regions of the genome; and (
iv
) only hybridize on the gene spaces (and not on the other DNA fractions) of other Gramineae.
Evolution of foraging behavior inDrosophilaby density-dependent selection
Authors: Sokolowski Marla B. , Pereira H. Sofia , Hughes Kimberley
Published: July 2002

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is theforaginglocus ofDrosophila melanogaster. Larvae with the rover allele,forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele,fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of theforsallele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations.
Use of intrinsic binding energy for catalysis by an RNA enzyme
Authors: Hertel Klemens J. , Peracchi Alessio , Uhlenbeck Olke C. , Herschlag Daniel
Published: July 2002

The contribution of several individual ribozyme⋅substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3′ or 5′ ends. The base pairs at positions 1.1–2.1 and 15.2–16.2, which flank the conserved core, each contribute 10
4
-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a “fraying model” in which each ribozyme⋅substrate helix can exist in either an unpaired (“open”) state or a helical (“closed”) state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of “intrinsic binding energy” from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.
Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits
Authors: Trapp Stefan , Tucker Stephen J. , Ashcroft Frances M.
Published: July 2002

The ATP-sensitive potassium channel (K-ATP channel) plays a key role in insulin secretion from pancreatic β-cells. It is closed by glucose metabolism, which stimulates secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and MgADP concentration, which inhibit and potentiate channel activity, respectively. The β-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. The site at which ATP mediates channel inhibition lies on Kir6.2, while the potentiatory action of MgADP involves the nucleotide-binding domains of SUR1. K-ATP channels are also activated by MgGTP and MgGDP. Furthermore, both nucleotides support the stimulatory actions of diazoxide. It is not known, however, whether guanine nucleotides mediate their effects by direct interaction with one or more of the K-ATP channel subunits or indirectly via a GTP-binding protein. We used a truncated form of Kir6.2, which expresses independently of SUR1, to show that GTP blocks K-ATP currents by interaction with Kir6.2 and that the potentiatory effects of GTP are endowed by SUR1. We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. This argues that the stimulatory effects of guanine nucleotides require the presence of both Walker A lysines.
Cloning and characterization of a plastidal and a mitochondrial isoform of tobacco protoporphyrinogen IX oxidase
Authors: Lermontova Inna , Kruse Elisabeth , Mock Hans-Peter , Grimm Bernhard
Published: July 2002

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating
Escherichia coli hemG
mutant from heme auxotrophy. The two sequences show similarity to the recently published
Arabidopsis
PPOX,
Bacillus subtilis
hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first
in vitro
translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steady-state RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in
E. coli
. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.
Identification of genes required for
Drosophila
eye development using a phenotypic enhancer-trap
Authors: Pignoni Francesca , Hu Birong , Zipursky S. Lawrence
Published: July 2002

A novel method of
P
-element mutagenesis is described for the isolation of mutants affecting the development of the
Drosophila
compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a
boss
cDNA transgene, in an otherwise
boss
mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the
boss
reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the
boss
reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the
marbles, glass, gap1,
and
fasciclin II
genes were isolated. This phenotypic enhancer-trap facilitates (
i
) the isolation of enhancer-traps with a specific expression pattern, and (
ii
) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.
Deltorphin transport across the blood–brain barrier
Authors: Fiori Anna , Cardelli Patrizia , Negri Lucia , Savi Maria Rosaria , Strom Roberto , Erspamer Vittorio
Published: July 2002

In vivo
antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood–brain barrier penetration rate.
In vitro
, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na
+
ions from the incubation medium decreases the carrier affinity for deltorphins (−25%), but does not affect the
V
max
value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective δ-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for δ- or μ-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that μ-, δ-, and κ-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with
l
-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.
Nuclear localization signal binding protein from
Arabidopsis
mediates nuclear import of
Agrobacterium
VirD2 protein
Authors: Ballas Nurit , Citovsky Vitaly
Published: July 2002

T-DNA nuclear import is a central event in genetic transformation of plant cells by
Agrobacterium
. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5′ end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from
Arabidopsis
. The identified protein, designated AtKAPα, specifically bound VirD2
in vivo
and
in vitro
. VirD2–AtKAPα interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPα was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin α family. Indeed, AtKAPα efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPα specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.
Inhibition of NF-κB DNA binding and nitric oxide induction in human T cells and lung adenocarcinoma cells by selenite treatment
Authors: Kim Ick Young , Stadtman Thressa C.
Published: July 2002

NF-κB is a major transcription factor consisting of 50(p50)- and 65(p65)-kDa proteins that controls the expression of various genes, among which are those encoding cytokines, cell adhesion molecules, and inducible NO synthase (iNOS). After initial activation of NF-κB, which involves release and proteolysis of a bound inhibitor, essential cysteine residues are maintained in the active reduced state through the action of thioredoxin and thioredoxin reductase. In the present study, activation of NF-κB in human T cells and lung adenocarcinoma cells was induced by recombinant human tumor necrosis factor α or bacterial lipopolysaccharide. After lipopolysaccharide activation, nuclear extracts were treated with increasing concentrations of selenite, and the effects on DNA-binding activity of NF-κB were examined. Binding of NF-κB to nuclear responsive elements was decreased progressively by increasing selenite levels and, at 7 μM selenite, DNA-binding activity was completely inhibited. Selenite inhibition was reversed by addition of a dithiol, DTT. Proportional inhibition of iNOS activity as measured by decreased NO products in the medium (NO
2
−
and NO
3
−
) resulted from selenite addition to cell suspensions. This loss of iNOS activity was due to decreased synthesis of NO synthase protein. Selenium at low essential levels (nM) is required for synthesis of redox active selenoenzymes such as glutathione peroxidases and thioredoxin reductase, but in higher toxic levels (>5–10 μM) selenite can react with essential thiol groups on enzymes to form RS–Se–SR adducts with resultant inhibition of enzyme activity. Inhibition of NF-κB activity by selenite is presumed to be the result of adduct formation with the essential thiols of this transcription factor.
Primate species richness is determined by plant productivity: Implications for conservation
Authors: Kay Richard F. , Madden Richard H. , Van Schaik Carel , Higdon David
Published: July 2002

The explanation of patterns in species richness ranks among the most important tasks of ecology. Current theories emphasize the interaction between historical and geographical factors affecting the size of the regional species pool and of locally acting processes such as competitive exclusion, disturbance, productivity, and seasonality. Local species richness, or alpha diversity, of plants and primary consumers has been claimed to peak in habitats of low and intermediate productivity, which, if true, has major implications for conservation. Here, by contrast, we show that local richness of Neotropical primates (platyrrhines) is influenced by both historical biogeography and productivity but not by tree species richness or seasonality. This pattern indicates that habitats with the highest plant productivity are also the richest for many important primary consumers. We show further that fragmentation of Amazonian rain forests in the Pleistocene, if it occurred, appears to have had a negligible influence on primate alpha species richness.
The Ti plasmid increases the efficiency of
Agrobacterium tumefaciens
as a recipient in
virB
-mediated conjugal transfer of an IncQ plasmid
Authors: Bohne Jutta , Yim Andy , Binns Andrew N.
Published: July 2002

The T-DNA transfer apparatus of
Agrobacterium tumefaciens
mediates the delivery of the T-DNA into plant cells, the transfer of the IncQ plasmid RSF1010 into plant cells, and the conjugal transfer of RSF1010 between
Agrobacteria
. We show in this report that the
Agrobacterium
-to-
Agrobacterium
conjugal transfer efficiencies of RSF1010 increase dramatically if the recipient strain, as well as the donor strain, carries a wild-type Ti plasmid and is capable of
vir
gene expression. Investigation of possible mechanisms that could account for this increased efficiency revealed that the VirB proteins encoded by the Ti plasmid were required. Although, with the exception of VirB1, all of the proteins that form the putative T-DNA transfer apparatus (VirB1–11, VirD4) are required for an
Agrobacterium
strain to serve as an RSF1010 donor, expression of only a subset of these proteins is required for the increase in conjugal transfer mediated by the recipient. Specifically, VirB5, 6, 11, and VirD4 are essential donor components but are dispensable for the increased recipient capacity. Defined point mutations in
virB9
affected donor and recipient capacities to the same relative extent, suggesting that similar functions of VirB9 are important in both of these contexts.
Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine
Authors: Sedegah Martha , Jones Trevor R. , Kaur Manjit , Hedstrom Richard , Hobart Peter , Tine John A. , Hoffman Stephen L.
Published: July 2002

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressingPlasmodium yoelii(Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P< 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.
Inhibitor binding changes domain mobility in the iron–sulfur protein of the mitochondrial
bc
1 complex from bovine heart
Authors: Kim Hoeon , Xia Di , Yu Chang-An , Xia Jia-Zhi , Kachurin Anatoly M. , Zhang Li , Yu Linda , Deisenhofer Johann
Published: July 2002

We have analyzed crystal structures of cytochrome
bc
1 complexes with electron transfer inhibitors bound to the ubiquinone binding pockets Q
i
and/or Q
o
in the cytochrome
b
subunit. The presence or absence of the Q
i
inhibitor antimycin A did not affect the binding of the Q
o
inhibitors. Different subtypes of Q
o
inhibitors had dramatically different effects on the mobility of the extramembrane domain of the iron–sulfur protein (ISP): Binding of 5-undecyl-6-hydroxy-4,7-dioxobenzothiazol and stigmatellin (subtype Q
o
–II and Q
o
–III, respectively) led to a fixation of the ISP domain on the surface of cytochrome
b
, whereas binding of myxothiazol and methoxyacrylate-stilbene (subtype Q
o
–I) favored release of this domain. The native structure has an empty Q
o
pocket and is intermediate between these extremes. On the basis of these observations we propose a model of quinone oxidation in the
bc
1 complex, which incorporates fixed and loose states of the ISP as features important for electron transfer and, possibly, also proton transport.
Neuroblast pattern formation: Regulatory DNA that confers the
vnd/NK-2
homeobox gene pattern on a reporter gene in transgenic lines of
Drosophila
Authors: Saunders H.-M. H. , Koizumi K. , Odenwald W. , Nirenberg M.
Published: July 2002

DNA fragments −0.57, −2.2, −2.9, −5.3, and −8.4 kb in length from the upstream regulatory region of the
vnd/NK-2
gene were cloned in the 5′-flanking region of a β-galactosidase (β-gal) reporter gene in the P-element pCaSpeR-AUG-β-gal, and the effects of the DNA on the pattern and time of expression of β-gal were determined in transgenic embryos. Embryos from 11 lines transformed with −8.4 kb of
vnd/NK-2
regulatory DNA expressed β-gal patterns that closely resemble those of
vnd/NK-2
. In embryos from four lines transformed with −5.3 kb of
vnd/NK-2
DNA, β-gal was found in the normal
vnd/NK-2
pattern in the nerve cord but not in part of the cephalic region. β-Gal patterns in embryos from transgenic lines containing −0.57, −2.2, or −2.9 kb of
vnd/NK-2
DNA did not resemble
vnd/NK-2
. Null
vnd/NK-2
mutant embryos containing the homozygous P-element p[−8.4 to +0.34 β-gal] expressed little β-gal in contrast to siblings with a wild-type
vnd/NK-2
gene. We conclude that (
i
) the 8.4-kb DNA fragment from the
vnd/NK-2
gene contains the nucleotide sequences required to generate the normal pattern of
vnd/NK-2
gene expression, sequences that may be involved in the switch between neuroblast vs. epidermoblast pathways of development, (
ii
) the 5′-flanking region of the
vnd/NK-2
gene between −5.3 and −8.4 kb is required for
vnd/NK-2
gene expression in the most dorsoanterior part of the cephalic region, and (
iii
) vnd/NK-2 protein is required, directly or indirectly, for maintenance of
vnd/NK-2
gene expression.
Telomerase reverse transcriptase genes identified in
Tetrahymena thermophila
and
Oxytricha trifallax
Authors: Bryan Tracy M. , Sperger Jamie M. , Chapman Karen B. , Cech Thomas R.
Published: July 2002

Telomerase reverse transcriptase (TERT) has been identified as the catalytic subunit of the chromosome end-replicating enzyme in
Euplotes
, yeasts, and mammals. However, it was not reported among the protein components of purified
Tetrahymena
telomerase, the first telomerase identified and the most thoroughly studied. It therefore seemed possible that
Tetrahymena
used an alternative telomerase that lacked a TERT protein. We now report the cloning and sequencing of a
Tetrahymena thermophila
gene whose encoded protein has the properties expected for a TERT, including large size (133 kDa), basicity (calculated
pI
= 10.0), and reverse transcriptase sequence motifs with telomerase-specific features. The expression of mRNA from the
Tetrahymena TERT
gene increases dramatically at 2–5 h after conjugation, preceding
de novo
addition of telomeres to macronuclear DNA molecules. We also report the cloning and sequencing of the ortholog from
Oxytricha trifallax
. The
Oxytricha
macronuclear
TERT
gene has no introns, whereas that of
Tetrahymena
has 18 introns. Sequence comparisons reveal a new amino acid sequence motif (CP), conserved among the ciliated protozoan TERTs, and allow refinement of previously identified motifs. A phylogenetic tree of the known TERTs follows the phylogeny of the organisms in which they are found, consistent with an ancient origin rather than recent transposition. The conservation of TERTs among eukaryotes supports the model that telomerase has a conserved core (TERT plus the RNA subunit), with other subunits of the holoenzyme being more variable among species.
Up-regulation of inducible nitric oxide synthase expression in cancer-prone p53 knockout mice
Authors: Ambs Stefan , Ogunfusika Mofolusara O. , Merriam William G. , Bennett William P. , Billiar Timothy R. , Harris Curtis C.
Published: July 2002

High concentrations of nitric oxide (NO) cause DNA damage and apoptosis in many cell types. Thus, regulation of NO synthase (NOS) activity is essential for minimizing effects of cytotoxic and genotoxic nitrogen oxide species. We have shown previously that NO-induced p53 protein accumulation down-regulates basal and cytokine-modulated inducible NOS (NOS2) expression in human cellsin vitro. To further characterize the feedback loop between NOS2 and p53, we have investigated NO production, i.e., urinary nitrate plus nitrite excretion, and NOS2 expression in homozygous p53 knockout (KO) mice. We report here that untreated p53 KO mice excreted 70% more nitrite plus nitrate than mice with wild-type (wt) p53. NOS2 protein expression was constitutively detected in the spleen of untreated p53 KO mice, whereas it was undetectable in the spleen of wt p53 controls. Upon treatment with heat-inactivatedCorynebacterium parvum, urinary nitrite plus nitrate excretion of p53 KO mice exceeded that of wt controls by approximately 200%.C. parvumtreatment also induced p53 accumulation in the liver. Splenectomy reduced the NO output ofC. parvum-treated p53 KO mice but not of wt p53 controls. Although NO production and NOS2 protein expression were increased similarly in KO and wt p53 mice 10 days after injection ofC. parvum, NOS2 expression returned to baseline levels only in wt p53 controls while remaining up-regulated in p53 KO mice. These genetic and functional data indicate that p53 is an important transrepressor of NOS2 expressionin vivoand attenuates excessive NO production in a regulatory negative feedback loop.
Aminophosphinic inhibitors as transition state analogues of enkephalin-degrading enzymes: A class of central analgesics
Authors: Chen Huixiong , Noble Florence , Coric Pascale , Fournie-Zaluski Marie-Claude , Roques Bernard P.
Published: July 2002

Inhibition of aminopeptidase N and neutral endopeptidase-24.11, two zinc metallopeptidases involved in the inactivation of the opioid peptides enkephalins, produces potent physiological analgesic responses, without major side-effects, in all animal models of pain in which morphine is active. Dual inhibitors of both enzymes could fill the gap between opioid analgesics and antalgics. Until now, attempts to find a compound with high affinity both for neutral endopeptidase and aminopeptidase N have failed. We report here the design of dual competitive inhibitors of both enzymes with KIvalues in the nanomolar range. These have been obtained by selecting R1, R2,and R3determinants in aminophosphinic-containing inhibitors: NH2—CH(R1)P(O)—(OH)CH2—CH(R2)CONH—CH(R3)COOH, for optimal recognition of the two enkephalin inactivating enzymes, whose active site peculiarities, determined by site-directed mutagenesis, have been taken into account. The best inhibitors were 10× more potent than described dual inhibitors in alleviating acute and inflammatory nociceptive stimuli in mice, thus providing a basis for the development of a family of analgesics devoid of opioid side effects.
Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase
Authors: Nissley Dwight V. , Boyer Paul L. , Garfinkel David J. , Hughes Stephen H. , Strathern Jeffrey N.
Published: July 2002

We previously demonstrated that hybrid retrotransposons composed of the yeast Ty
1
element and the reverse transcriptase (RT) of HIV-1 are active in the yeast
Saccharomyces cerevisiae.
The RT activity of these hybrid Ty
1
/HIV-1 (
his3AI
/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (−)-
(S)
-8-Chloro-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-
jk
][1,4]-benzodiazepin-2(1
H
)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses.
Carbohydrate binding and resistance to proteolysis control insecticidal activity of
Griffonia simplicifolia
lectin II
Authors: Zhu-Salzman Keyan , Shade Richard E. , Koiwa Hisashi , Salzman Ron A. , Narasimhan Meena , Bressan Ray A. , Hasegawa Paul M. , Murdock Larry L.
Published: July 2002

Griffonia simplicifolia
leaf lectin II (GSII), a plant defense protein against certain insects, consists of an
N
-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid,
Callosobruchus maculatus
(F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.
An essential, multicomponent signal transduction pathway required for cell cycle regulation in
Caulobacter
Authors: Wu Jianguo , Ohta Noriko , Newton Austin
Published: July 2002

Cell differentiation and division in
Caulobacter crescentus
are regulated by a signal transduction pathway mediated by the histidine kinase DivJ and the essential response regulator DivK. Here we report genetic and biochemical evidence that the DivJ and DivK proteins function to control the activity of CtrA, a response regulator required for multiple cell cycle events, including flagellum biosynthesis, DNA replication, and cell division. Temperature-sensitive
sokA
(
s
uppressor
o
f
div

K

) alleles were isolated as extragenic suppressors of a cold-sensitive
divK
mutation and mapped to the C terminus of the CtrA protein. The
sokA
alleles also suppress the lethal phenotype of a
divK
gene disruption and the cold-sensitive cell division phenotype of
divJ
mutants. The relationship between these signal transduction components and their target was further defined by demonstrating that the purified DivJ kinase phosphorylates CtrA, as well as DivK. Our studies also showed that phospho-CtrA activates transcription
in vitro
from the class II flagellar genes and that their promoters are recognized by the principal
C. crescentus
sigma factor σ
73
. We propose that an essential signal transduction pathway mediated by DivJ, DivK, and CtrA coordinates cell cycle and developmental events in
C. crescentus
by regulating the level of CtrA phosphorylation and transcription from σ
73
-dependent class II gene promoters. Our results suggest that an unidentified phosphotransfer protein or kinase (X) is responsible for phosphoryl group transfer to CtrA in the proposed DivJ ⇒ DivK ⇒ X ⇒ CtrA phosphorelay pathway.
Evolutionary matches of enzyme and transporter capacities to dietary substrate loads in the intestinal brush border
Authors: Weiss Stacey L. , Lee Eric A. , Diamond Jared
Published: July 2002

Safety factors of enzymes and transporters are defined as the ratio of
V
max
(maximal reaction rates at high substrate concentrations) to the reaction rate under actual physiological conditions. Although corresponding safety factors have been measured for macroscopic biological structures and for human-engineered structures, safety factors have been little studied at the molecular level. Some evolutionary considerations suggest that safety factors should be modestly in excess of 1.0 (“enough but not too much”) and should tend to be similar for the various steps of a pathway consisting of two or more elements arranged in series. Hence we used a preparation of intact mouse small intestine to measure
V
max
values (capacities) of brush-border sucrase (yielding glucose plus fructose) and of the brush-border glucose transporter, for comparison with each other and with dietary sucrose loads. Load was manipulated by varying dietary sucrose level or by studying lactating mice with increased energy requirements. Capacities both of sucrase and the glucose transporter increased with sucrose load (i.e., both proteins are inducible) and remained approximately matched to each other except on a carbohydrate-free diet. Their safety factors decreased from
ca.
2.7 at low load to 1.0 at high load. Thus, neither sucrase nor the glucose transporter is
the
rate-limiting step for sucrose digestion; both steps are equally limiting. The modest safety factors and matched capacities must be genetically programmed through natural selection, with benefits of excess capacities being balanced against costs of biosynthetic energy and limited membrane space.
Overexpression of theSUP45gene encoding a Sup35p-binding protein inhibits the induction of thede novoappearance of the[PSI+]prion
Authors: Derkatch Irina L. , Bradley Michael E. , Liebman Susan W.
Published: July 2002

[PSI+], a non-Mendelian element found in some strains ofSaccharomyces cerevisiae, is presumed to be the manifestation of a self-propagating prion conformation of eRF3 (Sup35p). Translation termination factor eRF3 enhances the activity of release factor eRF1 (Sup45p). As predicted by the prion model, overproduction of Sup35p induces thede novoappearance of[PSI+]. However, another non-Mendelian determinant,[PIN+], is required for this induction. We now show thatSUP45overexpression inhibits the induction of[PSI+]by Sup35p overproduction in[PIN+]strains, but has no effect on the propagation of[PSI+]or on the[PIN]status of the cells. We also show thatSUP45overexpression counteracts the growth inhibition usually associated with overexpression ofSUP35in[PSI+]strains. We argue that excess Sup45p inhibits[PSI+]seed formation. Because Sup45p complexes with Sup35p, we hypothesize that excess Sup45p may sequester Sup35p, thereby reducing the opportunity for Sup35p conformational flips and/or self-interactions leading to prion formation. Thisin vivoyeast result is reminiscent of thein vitrofinding by investigators of Alzheimer disease that apolipoprotein E inhibits amyloid nucleation, but does not reduce seeded growth of amyloid.
Vaccination with an attenuated Creutzfeldt–Jakob disease strain prevents expression of a virulent agent
Authors: Manuelidis Laura
Published: July 2002

Although slow and persistent viruses often escape host defenses infection may be prevented by live vaccines. To determine whether an attenuated “slow” strain of the Creutzfeldt–Jakob disease agent (SY) could block expression of a virulent “fast” strain (FU), outbred CD-1 mice were inoculated intracerebrally with low infectious doses of SY and challenged 80 days later with higher doses of FU. For comparison, the same SY and FU samples were inoculated in two parallel control groups. All 18 superinfected mice showed incubation times identical to those inoculated with only the SY strain, yielding clinical disease >110 days later than predicted for the FU strain. Neurological signs, such as scratching and an extended clinical phase, were also characteristic for SY but not FU infection. Moreover, the widespread cortical pathology of FU was not detectable in superinfected mice. Western blot analyses further showed no strain-specific differences in prion protein (PrP) band profiles for all experimental groups, although there was ∼10-fold more protease-resistant PrP (PrP-res) in FU brains during terminal disease. In contrast, infectivity assays revealed an ∼10,000-fold difference between SY and FU at terminal stages, indicating that PrP-res content does not correlate with infectivity. In summary, an attenuated strain of the Creutzfeldt–Jakob disease agent evokes substantial interference against a virulent agent. Because superinfected mice had little PrP-res just before the onset of clinical disease and retained abundant cellular PrP, cellular PrP was not the factor limiting FU replication. The mechanisms underlying SY interference are not understood but could be based on host recognition of foreign molecular features shared by this class of invasive agents involving antibody production, and possibly involve defective viral particles produced by attenuated variants.
A cell cycle-regulated adenine DNA methyltransferase from
Caulobacter crescentus
processively methylates GANTC sites on hemimethylated DNA
Authors: Berdis Anthony J. , Lee Irene , Coward James K. , Stephens Craig , Wright Rachel , Shapiro Lucy , Benkovic Stephen J.
Published: July 2002

The kinetic properties of an adenine DNA methyltransferase involved in cell cycle regulation of
Caulobacter crescentus
have been elucidated by using defined unmethylated or hemimethylated DNA (DNA
HM
) substrates. Catalytic efficiency is significantly enhanced with a DNA
HM
substrate. Biphasic kinetic behavior during methyl incorporation is observed when unmethylated or DNA
HM
substrates are used, indicating that a step after chemistry limits enzyme turnover and is most likely the release of enzyme from methylated DNA product. The enzyme is thermally inactivated at 30°C within 20 min; this process is substantially decreased in the presence of saturating concentrations of DNA
HM
, suggesting that the enzyme preferentially binds DNA before
S
-adenosylmethionine. The activity of the enzyme shows an unusual sensitivity to salt levels, apparently dissociating more rapidly from methylated DNA product as the salt level is decreased. The enzyme acts processively during methylation of specific DNA sequences, indicating a preferred order of product release in which
S
-adenosylhomocysteine is released from enzyme before fully methylated DNA. The kinetic behavior and activity of the enzyme are consistent with the temporal constraints during the cell cycle-regulated methylation of newly replicated chromosomal DNA.
Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation
Authors: Deussing Jan , Roth Wera , Saftig Paul , Peters Christoph , Ploegh Hidde L. , Villadangos José A.
Published: July 2002

Antigen presentation by major histocompatibility complex (MHC) class II molecules requires the participation of different proteases in the endocytic route to degrade endocytosed antigens as well as the MHC class II-associated invariant chain (Ii). Thus far, only the cysteine protease cathepsin (Cat) S appears essential for complete destruction of Ii. The enzymes involved in degradation of the antigens themselves remain to be identified. Degradation of antigensin vitroand experiments using protease inhibitors have suggested that Cat B and Cat D, two major aspartyl and cysteine proteases, respectively, are involved in antigen degradation. We have analyzed the antigen-presenting properties of cells derived from mice deficient in either Cat B or Cat D. Although the absence of these proteases provoked a modest shift in the efficiency of presentation of some antigenic determinants, the overall capacity of Cat B−/−or Cat D−/−antigen-presenting cells was unaffected. Degradation of Ii proceeded normally in Cat B−/−splenocytes, as it did in Cat D−/−cells. We conclude that neither Cat B nor Cat D are essential for MHC class II-mediated antigen presentation.
A small number of residues in the class II molecule I-Auconfer the ability to bind the myelin basic protein peptide Ac1-11
Authors: Pearson Cecelia I. , Gautam Anand M. , Rulifson Ingrid C. , Liblau Roland S. , McDevitt Hugh O.
Published: July 2002

The N-terminal peptide Ac1-11 of myelin basic protein induces experimental autoimmune encephalomyelitis in H-2uand (H-2u× H-2s) mice but does not in H-2smice. Ac1-11 binds weakly to the class II major histocompatibility complex (MHC) molecule I-Aubut not at all to I-As. We have studied the interaction of Ac1-11 and I-Auas a model system for therapeutic intervention in the autoimmune response seen in experimental autoimmune encephalomyelitis. Two polymorphic residues that differ between I-Auand I-As, Y26β and T28β, and one conserved residue, E74β, confer specific binding of Ac1-11 to I-Au. A fourth residue, R70β in I-Au, affects both peptide binding and T cell recognition. These results are consistent with a model that places arginine at position five of Ac1-11 in pockets 4 and 7 of the MHC groove, which is formed in part by residues 26, 28, 70, and 74 of Aβuand places lysine at position four of Ac1-11, previously shown to be a major MHC contact, in hydrophobic pocket 6. The data indicate that the primary region of I-Authat confers specific binding of Ac1-11 lies in the center of the peptide binding groove rather than in the region that contacts the N terminus of the peptide, as has been shown for HLA DR and the homologous I-E molecules.
Toward functional genomics in bacteria: Analysis of gene expression in
Escherichia coli
from a bacterial artificial chromosome library of
Bacillus cereus
Authors: Rondon Michelle R. , Raffel Sandra J. , Goodman Robert M. , Handelsman Jo
Published: July 2002

As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in
Escherichia coli
from genomic DNA of the Gram-positive bacterium
Bacillus cereus
. This library provides 5.75-fold coverage of the
B. cereus
genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of
B. cereus
genes in the library, we screened it for expression of several
B. cereus
activities in the
E. coli
host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific
B. cereus
loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.
Identification and characterization of a human tRNA-specific adenosine deaminase related to the ADAR family of pre-mRNA editing enzymes
Authors: Maas Stefan , Gerber André P. , Rich Alexander
Published: July 2002

The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA
Ala
. It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998)
EMBO J.
17, 4780–4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a
Drosophila melanogaster
ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins.
In vitro
, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A
37
in the anticodon loop of tRNA
Ala
from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem–loop of tRNA
Ala
alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA
Ala
, which is conserved from yeast to man.
Molecular dynamics simulations of unfolding and refolding of a β-hairpin fragment of protein G
Authors: Pande Vijay S. , Rokhsar Daniel S.
Published: July 2002

We have studied the unfolding and refolding pathway of a β-hairpin fragment of protein G by using molecular dynamics. Although this fragment is small, it possesses several of the qualities ascribed to small proteins: cooperatively formed β-sheet secondary structure and a hydrophobic “core” of packed side chains. At high temperatures, we find that the β-hairpin unfolds through a series of sudden, discrete conformational changes. These changes occur between states that are identified with the folded state, a pair of partially unfolded kinetic intermediates, and the unfolded state. To study refolding at low temperatures, we perform a series of short simulations starting from the transition states of the discrete transitions determined by the unfolding simulations.