bluuebunny/crossref_metadata_2025 huggingface dataset:
The histidine kinase-related domain participates in phytochrome B function but is dispensable
Authors: Krall Leonard , Reed Jason W.
Published: July 2002

Phytochromes are photoreceptors that control many plant light responses. Phytochromes have two carboxyl-terminal structural domains called the PAS repeat domain and the histidine kinase-related domain. These domains are each related to bacterial histidine kinase domains, and biochemical studies suggest that phytochromes are light-regulated kinases. The PAS repeat domain is important for proper phytochrome function and can interact with putative signaling partners. We have characterized several new phytochrome B mutants in
Arabidopsis
that express phyB protein, three of which affect the histidine kinase-related domain. Point mutations in the histidine kinase-related domain cause phenotypes similar to those of null mutants, indicating that this domain is important for phyB signaling. However, a truncation that removes most of the histidine kinase-related domain results in a phyB molecule with partial activity, suggesting that this domain is dispensable. These results suggest that phytochromes evolved in modular fashion. We discuss possible functions of the histidine kinase-related domain in phytochrome signaling.
Neurotensin-deficient mice show altered responses to antipsychotic drugs
Authors: Dobner Paul R. , Fadel Jim , Deitemeyer Nancy , Carraway Robert E. , Deutch Ariel Y.
Published: July 2002

The peptide transmitter neurotensin (NT) exerts diverse neurochemical effects that resemble those seen after acute administration of antipsychotic drugs (APDs). These drugs also induce NT expression in the striatum; this and other convergent findings have led to the suggestion that NT may mediate some APD effects. Here, we demonstrate that the ability of the typical APD haloperidol to induce Fos expression in the dorsolateral striatum is markedly attenuated in NT-null mutant mice. The induction of Fos and NT in the dorsolateral striatum in response to typical, but not atypical, APDs has led to the hypothesis that the increased expression of these proteins is mechanistically related to the production of extrapyramidal side effects (EPS). However, we found that catalepsy, which is thought to reflect the EPS of typical APDs, is unaffected in NT-null mutant mice, suggesting that NT does not contribute to the generation of EPS. We conclude that NT is required for haloperidol-elicited activation of a specific population of striatal neurons but not haloperidol-induced catalepsy. These results are consistent with the hypothesis that endogenous NT mediates a specific subset of APD actions.
The trimer-of-hairpins motif in membrane fusion: Visna virus
Authors: Malashkevich Vladimir N. , Singh Mona , Kim Peter S.
Published: July 2002

Structural studies of viral membrane fusion proteins suggest that a “trimer-of-hairpins” motif plays a critical role in the membrane fusion process of many enveloped viruses. In this motif, a coiled coil (formed by homotrimeric association of the N-terminal regions of the protein) is surrounded by three C-terminal regions that pack against the coiled coil in an oblique antiparallel manner. The resulting trimer-of-hairpins structure serves to bring the viral and cellular membranes together for fusion.
learncoil-vmf
, a computational program developed to recognize coiled coil-like regions that form the trimer-of-hairpins motif, predicts these regions in the membrane fusion protein of the Visna virus. Peptides corresponding to the computationally identified sequences were synthesized, and the soluble core of the Visna membrane fusion protein was reconstituted in solution. Its crystal structure at 1.5-Å resolution demonstrates that a trimer-of-hairpins structure is formed. Remarkably, despite less than 23% sequence identity, the ectodomains in Visna and HIV-1 envelope glycoproteins show detailed structural conservation, especially within the area of a hydrophobic pocket in the central coiled coil currently being targeted for the development of new anti-HIV drugs.
C
m
C(A/T)GG DNA methylation in mature B cell lymphoma gene silencing
Authors: Malone Cindy Sue , Miner Maurine D. , Doerr Jeanette R. , Jackson James P. , Jacobsen Steven E. , Wall Randolph , Teitell Michael
Published: July 2002

DNA methylation has been linked to gene silencing in cancer. Primary effusion lymphoma (PEL) and myeloma are lymphoid malignancies that arise from terminally differentiated B cells. Interestingly, PEL do not express immunoglobulins or most B lineage-specific genes. The B cell-specific
B29
(Igβ/CD79b) gene is silenced in PEL and some myelomas but is expressed in other normal and malignant B cells.
B29
expression was reactivated in PEL by demethylating and histone deacetylase inhibiting treatments. Bisulfite sequencing revealed two types of DNA methylation in silenced
B29
promoters: at conventional CpG and at CC(A/T)GG
B29
promoter sites. The pattern of methylated CpG (
m
CpG) and C
m
C(A/T)GG
B29
promoter methylation observed was similar to that recently reported for epigenetic silencing of an integrated retrovirus. Methylation of C
m
C(A/T)GG sites in the
B29
promoter significantly repressed
in vivo
transcriptional activity. Also, methylation of a central conserved C
m
CTGG
B29
promoter site blocked the binding of early B cell factor. This methylated motif formed DNA–protein complexes with nuclear extracts from all cell types examined. Therefore, C
m
C(A/T)GG methylation may represent an important type of epigenetic marker on mammalian DNA that impacts transcription by altering DNA–protein complex formation.
Plastic neural changes and reading improvement caused by audiovisual training in reading-impaired children
Authors: Kujala T. , Karma K. , Ceponiene R. , Belitz S. , Turkkila P. , Tervaniemi M. , Näätänen R.
Published: July 2002

This study aimed at determining whether audiovisual training without linguistic material has a remediating effect on reading skills and central auditory processing in dyslexic children. It was found that this training resulted in plastic changes in the auditory cortex, indexed by enhanced electrophysiological mismatch negativity and faster reaction times to sound changes. Importantly, these changes were accompanied by improvement in reading skills. The results indicate that reading difficulties can be ameliorated by special training programs and, further, that the training effects can be observed in brain activity. Moreover, the fact that the present training effects were obtained by using a program including no linguistic material indicates that dyslexia is at least partly based on a general auditory perceptual deficit.
On the failure of de novo-designed peptides as biocatalysts.
Authors: Corey M J , Corey E
Published: July 2002

While the elegance and efficiency of enzymatic catalysis have long tempted chemists and biochemists with reductionist leanings to try to mimic the functions of natural enzymes in much smaller peptides, such efforts have only rarely produced catalysts with biologically interesting properties. However, the advent of genetic engineering and hybridoma technology and the discovery of catalytic RNA have led to new and very promising alternative means of biocatalyst development. Synthetic chemists have also had some success in creating nonpeptide catalysts with certain enzyme-like characteristics, although their rates and specificities are generally much poorer than those exhibited by the best novel biocatalysts based on natural structures. A comparison of the various approaches from theoretical and practical viewpoints is presented. It is suggested that, given our current level of understanding, the most fruitful methods may incorporate both iterative selection strategies and rationally chosen small perturbations, superimposed on frameworks designed by nature.
A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange.
Authors: Lin X , Barber D L
Published: July 2002

Activation of the ubiquitously expressed Na-H exchanger, NHE1, results in an increased efflux of intracellular H+. The increase in intracellular pH associated with this H+ efflux may contribute to regulating cell proliferation, differentiation, and neoplastic transformation. Although NHE1 activity is stimulated by growth factors and hormones acting through multiple GTPase-mediated pathways, little is known about how the exchanger is directly regulated. Using expression library screening, we identified a novel protein that specifically binds to NHE1 at a site that is critical for growth factor stimulation of exchange activity. This protein is homologous to calcineurin B and calmodulin and is designated CHP for calcineurin B homologous protein. Like NHE1, CHP is widely expressed in human tissues. Transient overexpression of CHP inhibits serum- and GTP-ase-stimulated NHE1 activity. CHP is a phosphoprotein and expression of constitutively activated GTPases decreases CHP phosphorylation. The phosphorylation state of CHP may therefore be an important signal controlling mitogenic regulation of NHE1.
Altered recognition mutants of the response regulator PhoB: A new genetic strategy for studying protein–protein interactions
Authors: Haldimann Andreas , Prahalad Murali K. , Fisher Stewart L. , Kim Soo-Ki , Walsh Christopher T. , Wanner Barry L.
Published: July 2002

Two-component regulatory systems require highly specific
interactions between histidine kinase (transmitter) and response
regulator (receiver) proteins. We have developed a novel genetic
strategy that is based on tightly regulated synthesis of a given
protein to identify domains and residues of an interacting protein that
are critical for interactions between them. Using a reporter strain
synthesizing the nonpartner kinase VanS under tight arabinose control
and carrying a promoter-
lacZ
fusion activated by
phospho-PhoB, we isolated altered recognition (AR) mutants of PhoB
showing enhanced activation (phosphorylation) by VanS as
arabinose-dependent Lac
+
mutants. Changes in the
PhoB
AR
mutants cluster in a “patch” near the proposed
helix 4 of PhoB based on the CheY crystal structure (a homolog of the
PhoB receiver domain) providing further evidence that helix 4 lies in
the kinase-regulator interface. Based on the CheY structure, one mutant
has an additional change in a region that may propagate a
conformational change to helix 4. The overall genetic strategy
described here may also be useful for studying interactions of other
components of the vancomycin resistance and P
i
signal
transduction pathways, other two-component regulatory systems, and
other interacting proteins. Conditionally replicative
oriR
R6Kγ
attP
“genome
targeting” suicide plasmids carrying mutagenized
phoB
coding regions were integrated into the chromosome of a reporter strain
to create mutant libraries; plasmids encoding mutant PhoB proteins were
subsequently retrieved by P1-Int-Xis cloning. Finally, the use of
similar genome targeting plasmids and P1-Int-Xis cloning should be
generally useful for constructing genomic libraries from a wide array
of organisms.
Truncated elongation factor G lacking the G domain promotes translocation of the 3' end but not of the anticodon domain of peptidyl-tRNA.
Authors: Borowski C , Rodnina M V , Wintermeyer W
Published: July 2002

The mechanism by which elongation factor G (EF-G) catalyzes the translocation of tRNAs and mRNA on the ribosome is not known. The reaction requires GTP, which is hydrolyzed to GDP. Here we show that EF-G from Escherichia coli lacking the G domain still catalyzed partial translocation in that it promoted the transfer of the 3' end of peptidyl-tRNA to the P site on the 50S ribosomal subunit into a puromycin-reactive state in a slow-turnover reaction. In contrast, it did not bring about translocation on the 30S subunit, since (i) deacylated tRNA was not released from the P site and (ii) the A site remained blocked for aminoacyl-tRNA binding during and after partial translocation. The reaction probably represents the first EF-G-dependent step of translocation that follows the spontaneous formation of the A/P state that is not puromycin-reactive [Moazed, D. & Noller, H. F. (1989) Nature (London) 342, 142-148]. In the complete system--i.e., with intact EF-G and GTP--the 50S phase of translocation is rapidly followed by the 30S phase during which the tRNAs together with the mRNA are shifted on the small ribosomal subunit, and GTP is hydrolyzed. As to the mechanism of EF-G function, the results show that the G domain has an important role, presumably exerted through interactions with other domains of EF-G, in the promotion of translocation on the small ribosomal subunit. The G domain's intramolecular interactions are likely to be modulated by GTP binding and hydrolysis.
Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells
Authors: Coulson Barbara S. , Londrigan Sarah L. , Lee Debra J.
Published: July 2002

Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the α2β1 integrin ligand site DGE. In VP7, the αxβ2 integrin ligand site GPR and the α4β1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the
tie
receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express α2β1 and β2 integrins. Rotavirus VP4-mediated cell entry may involve the α2β1 integrin, whereas VP7 appears to interact with αxβ2 and α4β1 integrins.
Ancient origin of the complement lectin pathway revealed by molecular cloning of mannan binding protein-associated serine protease from a urochordate, the Japanese ascidian,Halocynthia roretzi
Authors: Ji Xin , Azumi Kaoru , Sasaki Makoto , Nonaka Masaru
Published: July 2002

Recent identification of a C3-like gene in sea urchins revealed the presence of a complement system in invertebrates. To elucidate further the components and function of the pre-vertebrate complement system, we attempted to isolate an ascidian (urochordata) C3 convertase. After identification of C3 cDNA fromHalocynthia roretzi, a Japanese ascidian, reverse transcriptase–PCR amplification of hepatopancreas RNA was performed using primers encoding highly conserved amino acid sequences of the vertebrate Bf and C2 serine protease domain. Two candidate sequences were identified, and the corresponding cDNA clones were isolated from a hepatopancreas library. Surprisingly, neither clone is related to Bf/C2 but rather share the same domain structure of mammalian C1r/C1s/MASP (mannan binding protein-associated serine protease), and are more related evolutionarily to mammalian MASP than to mammalian C1r or C1s. The identification of the tunicate MASP clones, amplified with primers designed to amplify Bf or C2, suggests that the lectin pathway antedated the classical and alternative pathways of complement activation.
Enhanced proteolysis of thiopurine
S
-methyltransferase (TPMT) encoded by mutant alleles in humans (
TPMT
∗
3
A
,
TPMT
∗
2
): Mechanisms for the genetic polymorphism of TPMT activity
Authors: Tai Hung-Liang , Krynetski Eugene Y. , Schuetz Erin G. , Yanishevski Yuri , Evans William E.
Published: July 2002

TPMT is a cytosolic enzyme that catalyzes the
S
-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (
TPMT
∗
2
and
TPMT
∗
3A
), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units/ml pRBC (
r
s
= 0.99;
P
< 0.001). Similarly, heterologous expression of wild-type (
TPMT
∗
1
) and mutant (
TPMT
∗
2
and
TPMT
∗
3A
) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with
in vitro
translation in rabbit reticulocyte lysates. In contrast, pulse–chase experiments revealed significantly shorter degradation half-lives for
TPMT
∗
2
and
TPMT
∗
3A
(∼0.25 hr) compared with wild-type
TPMT
∗
1
(18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (
pre-1
strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by
TPMT
∗
2
and
TPMT
∗
3A
as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human
TPMT
locus.
The human cytomegalovirus US6 glycoprotein inhibits transporter associated with antigen processing-dependent peptide translocation
Authors: Lehner Paul J. , Karttunen Jaana T. , Wilkinson Gavin W. G. , Cresswell Peter
Published: July 2002

In its attempt to evade cytotoxic T cell recognition, human cytomegalovirus encodes several genes that target MHC class I molecules at different points in their assembly pathway. We show here that the human cytomegalovirus US6 gene encodes a 22-kDa glycoprotein that binds the transporter-associated with antigen processing (TAP)/class I complex and inhibits translocation of peptide from the cytosol to the endoplasmic reticulum. Major histocompatibility complex class I molecules are therefore unable to load TAP-dependent peptides, resulting in the retention of MHC class I molecules in the endoplasmic reticulum, with a consequent reduction in class I at the cell surface. Interferon-γ treatment of US6 transfected cells overcomes this inhibition of peptide translocation and restores class I at the cell surface to wild type levels. The functional consequence of TAP inhibition is that US6 transfected cells are unable to present endogenous antigen to cytotoxic T lymphocytes and are therefore resistant to cytotoxic T lymphocyte lysis.
The U5 RNA of trypanosomes deviates from the canonical U5 RNA: The
Leptomonas collosoma
U5 RNA and its coding gene
Authors: Xu Yu-xin , Ben-Shlomo Herzel , Michaeli Shulamit
Published: July 2002

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid
Leptomonas collosoma
revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5′ phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of
Trypanosoma brucei
[Dungan, J. D., Watkins, K. P. & Agabian, N. (1996)
EMBO J
. 15, 4016–4029]. The
L. collosoma
U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6⋅U5 tri-small nuclear RNP complex. In contrast to
T. brucei
U5 RNA (62 nt), the
L. collosoma
homologue is longer (80 nt) and possesses a second stem–loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNA
Cys
was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5′ splice site region (positions −1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.
An intercalation inhibitor altering the target selectivity of DNA damaging agents: Synthesis of site-specific aflatoxin B
1
adducts in a p53 mutational hotspot
Authors: Kobertz William R. , Wang David , Wogan Gerald N. , Essigmann John M.
Published: July 2002

Aflatoxin B
1
(AFB
1
) is a potent human carcinogen implicated in the etiology of hepatocellular carcinoma. Upon metabolic activation to the reactive epoxide, AFB
1
forms DNA adducts primarily at the N7 position of guanines. To elucidate more fully the molecular mechanism of AFB
1
-induced mutagenesis, an intercalation inhibitor was designed to probe the effects of intercalation by AFB
1
epoxide on its reaction with DNA. DNA duplexes were prepared consisting of a target strand containing multiple potentially reactive guanines and a nontarget strand containing a
cis
-syn thymidine-benzofuran photoproduct. Because the covalently linked benzofuran moiety physically occupies an intercalation site, we reasoned that such a site would be rendered inaccessible to AFB
1
epoxide. By strategic positioning of this intercalation inhibitor in the intercalation site 5′ to a specific guanine, the adduct yield at that site was greatly diminished, indicating that intercalation by AFB
1
epoxide contributes favorably to adduct formation. Using this approach it has been possible to simplify the production of site-specifically modified oligonucleotides containing AFB
1
adducts in the sequence context of a p53 mutational hotspot. Moreover, we report herein isolation of site-specifically AFB
1
-modified oligonucleotides in sequences containing multiple guanines. Use of intercalation inhibitors will facilitate both investigation of the ability of other carcinogens to intercalate into DNA and the synthesis of specific carcinogen-DNA adducts.
Search for the pathogenesis of the differing phenotype in two compound heterozygote Hungarian brothers with the same genotypic triosephosphate isomerase deficiency
Authors: Hollán Susan , Magócsi Mária , Fodor Elfrieda , Horányi Margit , Harsányi Veronika , Farkas Tibor
Published: July 2002

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethylcholinesterase and calmodulin induced Ca
2+
ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.
A multimeric complex and the nuclear targeting of the
Drosophila
Rel protein Dorsal
Authors: Yang Jun , Steward Ruth
Published: July 2002

The intracellular part of the Rel signal transduction pathway in
Drosophila
is encoded by
Toll
,
tube
,
pelle
,
dorsal
, and
cactus
, and it functions to form the dorsal–ventral axis in the
Drosophila
embryo. Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus. Tube and Pelle are required to relay the signal from Toll to the Dorsal–Cactus complex. In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal. We confirmed these interactions in an
in vitro
binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube–Dorsal interaction is necessary for normal signal transduction. We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction.
Generation of cDNA expression libraries enriched for in-frame sequences
Authors: Davis Claytus A. , Benzer Seymour
Published: July 2002

Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60–80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.
Trans receptor inhibition of human glioblastoma cells by erbB family ectodomains
Authors: O’Rourke Donald M. , Qian Xiaolan , Zhang Hong-Tao , Davis James G. , Nute Eric , Meinkoth Judith , Greene Mark I.
Published: July 2002

Our aim has been to understand the features of erbB receptor homo- and heterodimer assembly to develop approaches to disrupt receptor activation. We have developed a general approach to cause erbB receptor-specific trans inhibition of human neoplasia. The clonal progression of human astrocytomas to a more malignant phenotype often involves the amplification and overexpression of the epidermal growth factor receptor (EGFr) gene. We have selectively targeted the EGFr in human glioblastoma cells with kinase-deficient mutants of the erbB family derived from the ectodomain of the Neu oncogene that are able to form heterodimers with EGFr and inhibit EGFr-dependent phenotypes. In EGFr-positive U87MG human glioblastoma cells, expression of the Neu ectodomain inhibits EGF-, but not platelet-derived growth factor-, induced DNA synthesis; inhibits cell proliferation in the presence of EGF, but not platelet-derived growth factor; inhibits the ability of U87MG to form colonies in soft agar; and inhibits transforming efficiency in athymic mice. These studies establish that EGFr-mediated signal transduction is important in the maintenance of malignant glioma, and that trans receptor inhibition is a novel way to abrogate abnormal growth of these tumors. Neu ectodomains will be useful in determining the manner in which the EGFr contributes to glial tumorigenesis and in the design of pharmaceuticals that disable erbB family oncoproteins. In addition, these studies provide a rationale for the application of the Neu ectodomain in gene therapy approaches to human malignant glioma and, potentially, to other systemic epithelial malignancies expressing erbB family receptors.
COS1
, a two-component histidine kinase that is involved in hyphal development in the opportunistic pathogen
Candida albicans
Authors: Alex Lisa A. , Korch Christopher , Selitrennikoff Claude P. , Simon Melvin I.
Published: July 2002

Two-component histidine kinases recently have been found in eukaryotic organisms including fungi, slime molds, and plants. We describe the identification of a gene,
COS1
, from the opportunistic pathogen
Candida albicans
by using a PCR-based screening strategy. The sequence of
COS1
indicates that it encodes a homolog of the histidine kinase Nik-1 from the filamentous fungus
Neurospora crassa. COS1
is also identical to a gene called
CaNIK1
identified in
C. albicans
by low stringency hybridization using
CaSLN1
as a probe [Nagahashi, S., Mio, T., Yamada-Okabe, T., Arisawa, M., Bussey, H. & Yamada-Okabe, H. (1998)
Microbiol.
44, 425–432]. We assess the function of
COS1/CaNIK1
by constructing a diploid deletion mutant. Mutants lacking both copies of
COS1
appear normal when grown as yeast cells; however, they exhibit defective hyphal formation when placed on solid agar media, either in response to nutrient deprivation or serum. In constrast to the
Δnik-1
mutant, the
Δcos1/Δcos1
mutant does not demonstrate deleterious effects when grown in media of high osmolarity; however both
Δnik-1
and
Δcos1/Δcos1
mutants show defective hyphal formation. Thus, as predicted for Nik-1, Cos1p may be involved in some aspect of hyphal morphogenesis and may play a role in virulence properties of the organism.
Role of p38 mitogen-activated protein kinase in HIV type 1 production
in vitro
Authors: Shapiro Leland , Heidenreich Kim A. , Meintzer Mary Kay , Dinarello Charles A.
Published: July 2002

The proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) promote HIV type 1 viral replication
in vitro
. In the present studies, HIV production was increased in the macrophagic U1 cell line expressing the HIV genome after exposure to IL-1β, osmotic stress, or surface adhesion, suggesting a confluence of signaling pathways for proinflammatory cytokines and cell stressors. The p38 mitogen-activated protein kinase (MAPK) mediates both cytokine and stress responses; thus the role of this kinase in HIV production was investigated. HIV production as measured by p24 antigen correlated with changes in the expression of a specific (non-alpha) isoform of p38 MAPK. In the presence of a specific p38 MAPK inhibitor (p38 inh), IL-1β-induced HIV production was suppressed by more than 90% and IL-1β-induced IL-8 production was suppressed completely, both with IC
50
of 0.01 μM. p38 inhibition blocked cell-associated p24 antigen and secreted virus to a similar extent. The p38 inh also decreased constitutive HIV production in freshly infected peripheral blood mononuclear cells by up to 50% (
P
< 0.05). Interruption of p38 MAPK activity represents a viable target for inhibition of HIV.
Arabinogalactan-proteins from
Nicotiana alata
and
Pyrus communis
contain glycosylphosphatidylinositol membrane anchors
Authors: Youl Joelian J. , Bacic Antony , Oxley David
Published: July 2002

Arabinogalactan-proteins (AGPs) are a class of proteoglycans found in cell secretions and plasma membranes of plants. Attention is currently focused on their structure and their potential role in growth and development. We present evidence that two members of a major class of AGPs, the classical AGPs, AGP
Na
1 from styles of
Nicotiana alata
and AGP
Pc
1 from cell suspension cultures of
Pyrus communis
, undergo C-terminal processing involving glycosylphosphatidylinositol membrane anchors. The evidence is that (
i
) the transmembrane helix at the C terminus predicted from the cDNA encoding these proteins is not present—the C-terminal amino acid is Asn
87
and Ser
97
for AGP
Na
1 and AGP
Pc
1, respectively; (
ii
) both AGP protein backbones are substituted with ethanolamine at the C-terminal amino acid; and (
iii
) inositol, glucosamine, and mannose are present in the native AGPs. An examination of the deduced amino acid sequences of other classical AGP protein backbones shows that glycosylphosphatidylinositol-anchors may be a common feature of this class of AGPs.
High level transactivation by a modified
Bombyx
ecdysone receptor in mammalian cells without exogenous retinoid X receptor
Authors: Suhr Steven T. , Gil Elad B. , Senut Marie-Claude , Gage Fred H.
Published: July 2002

Our studies of the
Bombyx mori
ecdysone receptor (BE) revealed that, unlike the
Drosophila melanogaster
ecdysone receptor (DE), treatment of BE with the ecdysone agonist tebufenozide stimulated high level transactivation in mammalian cells without adding an exogenous heterodimer partner. Gel mobility shift and transfection assays with both the
ultraspiracle
gene product (Usp) and retinoid X receptor heterodimer partners indicated that this property of BE stems from significantly augmented heterodimer complex formation and concomitant DNA binding. We have mapped this “gain of function” to determinants within the D and E domains of BE and demonstrated that, although the D domain determinant is sufficient for high affinity heterodimerization with Usp, both determinants are necessary for high affinity interaction with retinoid X receptor. Modified BE receptors alone used as replication-defective retroviruses potently stimulated separate “reporter” viruses in all cell types examined, suggesting that BE has potentially broad utility in the modulation of transgene expression in mammalian cells.
A very large protein with diverse functional motifs is deficient in
rjs
(runty, jerky, sterile) mice
Authors: Lehman Anne L. , Nakatsu Yoshimichi , Ching Ada , Bronson Roderick T. , Oakey Rebecca J. , Keiper-Hrynko Natalie , Finger Joshua N. , Durham-Pierre Donna , Horton Daniel B. , Newton J Michael , Lyon Mary F. , Brilliant Murray H.
Published: July 2002

Three radiation-induced alleles of the mouse
p
locus,

p
6H

,

p
25H

, and

p
bs

, cause defects in growth, coordination, fertility, and maternal behavior in addition to
p
gene-related hypopigmentation. These alleles are associated with disruption of the
p
gene plus an adjacent gene involved in the disorders listed. We have identified this adjacent gene, previously named
rjs
(
r
unty
j
erky
s
terile), by positional cloning. The
rjs
cDNA is very large, covering 15,264 nucleotides. The predicted
rjs
-encoded protein (4,836 amino acids) contains several sequence motifs, including three RCC1 repeats, a structural motif in common with cytochrome
b
5
, and a HECT domain in common with E6-AP ubiquitin ligase. On the basis of sequence homology and conserved synteny, the
rjs
gene is the single mouse homolog of a previously described five- or six-member human gene family. This family is represented by at least two genes, HSC7541 and KIAA0393, from human chromosome 15q11–q13. HSC7541 and KIAA0393 lie close to, or within, a region commonly deleted in most Prader–Willi syndrome patients. Previous work has suggested that the multiple phenotypes in
rjs
mice might be due to a common neuroendocrine defect. In addition to this proposed mode of action, alternative functions of the
rjs
gene are evaluated in light of its known protein homologies.
The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides
Authors: Arnould Sylvain , Camadro Jean-Michel
Published: July 2002

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the
K
m
for protoporphyrinogen and the
K
i
for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.
RNA polymerase subunit RPB5 plays a role in transcriptional activation
Authors: Miyao Takenori , Woychik Nancy A.
Published: July 2002

A mutation in RPB5
(
rpb5–9
)
, an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon
in vitro
transcription with mutant whole-cell extracts.
In vivo
experiments using inducible reporter plasmids and Northern analysis support the
in vitro
data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with
rpb5–9
are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.
Abnormality in catalase import into peroxisomes leads to severe neurological disorder
Authors: Sheikh Faruk G. , Pahan Kalipada , Khan Mushfiquddin , Barbosa Ernest , Singh Inderjit
Published: July 2002

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H
2
O
2
or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the
in situ
levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H
2
O
2
toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease.
Structural characterization of an engineered tandem repeat contrasts the importance of context and sequence in protein folding
Authors: Sagermann Martin , Baase Walter A. , Matthews Brian W.
Published: July 2002

To test a different approach to understanding the relationship between the sequence of part of a protein and its conformation in the overall folded structure, the amino acid sequence corresponding to an α-helix of T4 lysozyme was duplicated in tandem. The presence of such a sequence repeat provides the protein with “choices” during folding. The mutant protein folds with almost wild-type stability, is active, and crystallizes in two different space groups, one isomorphous with wild type and the other with two molecules in the asymmetric unit. The fold of the mutant is essentially the same in all cases, showing that the inserted segment has a well-defined structure. More than half of the inserted residues are themselves helical and extend the helix present in the wild-type protein. Participation of additional duplicated residues in this helix would have required major disruption of the parent structure. The results clearly show that the residues within the duplicated sequence tend to maintain a helical conformation even though the packing interactions with the remainder of the protein are different from those of the original helix. It supports the hypothesis that the structures of individual α-helices are determined predominantly by the nature of the amino acids within the helix, rather than the structural environment provided by the rest of the protein.
Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry
Authors: Griffin Timothy J. , Hall Jeff G. , Prudent James R. , Smith Lloyd M.
Published: July 2002

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.
Ligand-receptor binding measured by laser-scanning imaging
Authors: Zuck Paul , Lao Zhege , Skwish Stephen , Glickman J. Fraser , Yang Ke , Burbaum Jonathan , Inglese James
Published: July 2002

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.
X-ngnr-1
and
Xath3
promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina
Authors: Perron Muriel , Opdecamp Karin , Butler Karen , Harris William A. , Bellefroid Eric J.
Published: July 2002

Xath3
encodes a
Xenopus
neuronal-specific basic helix–loop–helix transcription factor related to the
Drosophila
proneural factor atonal. We show here that
Xath3
acts downstream of
X-ngnr-1
during neuronal differentiation in the neural plate and retina and that its expression and activity are modulated by Notch signaling. X-ngnr-1 activates
Xath3
and
NeuroD
by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm,
X-ngnr-1
and
Xath3
have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina,
X-ngnr-1
and
Xath3
overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that
X-ngnr-1
and
Xath3
regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity.
Interpretation of the spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle
Authors: Dér András , Oroszi László , Kulcsár Ágnes , Zimányi László , Tóth-Boconádi Rudolf , Keszthelyi Lajos , Stoeckenius Walther , Ormos Pál
Published: July 2002

We have recently introduced a method, made possible by an improved orienting technique using a combination of electric and magnetic fields, that allows the three-dimensional detection of the intramolecular charge displacements during the photocycle of bacteriorhodopsin. This method generates electric asymmetry, a prerequisite for the detection of electric signal on the macroscopic sample, in all three spatial dimensions. Purple membrane fragments containing bacteriorhodopsin were oriented so that their permanent electric dipole moment vectors were perpendicular to the membrane plane and pointed in the same direction. The resulting cylindrical symmetry was broken by photoselection,
i. e.
, by flash excitation with low intensity linearly polarized light. From the measured electric signals, the three-dimensional motion of the electric charge center in the bacteriorhodopsin molecules was calculated for the first 400 μs. Simultaneous absorption kinetic recording provided the time-dependent concentrations of the intermediates. Combining the two sets of data, we determined the discrete dipole moments of intermediates up to M. When compared with the results of current molecular dynamics calculations, the data provided a decisive experimental test for selecting the optimal theoretical model for the proton transport and should eventually lead to a full description of the mechanism of the bacteriorhodopsin proton pump.