bluuebunny/crossref_metadata_2025 huggingface dataset:
BLNK mediates Syk-dependent Btk activation
Authors: Baba Yoshihiro , Hashimoto Shoji , Matsushita Masato , Watanabe Dai , Kishimoto Tadamitsu , Kurosaki Tomohiro , Tsukada Satoshi
Published: July 2002

Btk is a critical molecule in B cell antigen receptor (BCR)-coupled signaling, and its activity is regulated by Lyn and Syk. Although the molecular mechanism of Lyn-dependent Btk activation has been investigated, that of Syk-dependent Btk activation has remained unidentified. We have demonstrated that BLNK mediates Syk-dependent Btk activation. In a reconstitution cell system, coexpression of BLNK allows Syk to phosphorylate Btk on its tyrosine 551, leading to the enhancement of Btk activity. This phosphorylation depends on the interaction of Btk and BLNK by means of the Btk-Src homology 2 domain. The existence of such an activation mechanism is supported by the observation that the BCR-induced Btk phosphorylation and activation are significantly reduced in BLNK-deficient B cells as well as in Syk-deficient B cells. Although previous observations have identified the function of BLNK as the linker that integrates the action of Btk and Syk into downstream effectors such as phospholipase Cγ2, our present study indicates another function of BLNK that connects the activity of Syk to that of Btk.
Cluster self-assembly of di[gold(I)]halonium cations
Authors: Schmidbaur Hubert , Hamel Armin , Mitzel Norbert W. , Schier Annette , Nogai Stefan
Published: July 2002

Treatment of gold(I) halide complexes of the type L-Au-X [where L = PPh
3
, PEt
3
with X = Cl, Br, I, or L = 2,6-(MeO)
2
C
6
H
3
PPh
2
with X = Cl] with AgSbF
6
in the molar ratio 2:1 in dichloromethane/tetrahydrofuran at −78°C affords high yields of di[gold(I)]halonium salts of the formula {X[Au(PR
3
)]
2
}
+
SbF

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(2-8). A determination of the crystal structures of the four triarylphosphine complexes (2-4, 8) revealed the presence of novel
tetra
nuclear
di
cations with a highly symmetrical structure (point group S
4
) that arises from self-assembly of the
di
nuclear
mono
cations through a set of four equivalent aurophilic Au–Au interactions. A comparison with two reference structures of corresponding chloronium perchlorate and bromonium tetrafluoroborate salts with monomeric, dinuclear cations shows that the geometry of the latter is greatly altered on dimerization to optimize the interactions between the closed-shell metal centers (Au: 5d
10
). Weak metallophilic bonding clearly becomes significant only in crystal lattices where anions with a larger ionic radius (SbF

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vs. BF

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, ClO

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) reduce the otherwise dominant role of strong interionic Coulomb forces. The results indicate that aurophilic bonding is indeed an ubiquitous, quite dependable mode of intermetallic interactions provided that the right environment is chosen to allow the weak forces to become operative.
Distinct Hox protein sequences determine specificity in different tissues
Authors: Chauvet Sophie , Merabet Samir , Bilder David , Scott Matthew P. , Pradel Jacques , Graba Yacine
Published: July 2002

Hox
genes encode evolutionarily conserved transcription factors that control the morphological diversification along the anteroposterior (A/P) body axis. Expressed in precise locations in the ectoderm, mesoderm, and endoderm, Hox proteins have distinct regulatory activities in different tissues. How Hox proteins achieve tissue-specific functions and why cells lying at equivalent A/P positions but in different germ layers have distinctive responses to the same Hox protein remains to be determined. Here, we examine this question by identifying parts of Hox proteins necessary for Hox function in different tissues. Available genetic markers allow the regulatory effects of two Hox proteins, Abdominal-A (AbdA) and Ultrabithorax (Ubx), to be distinguished in the
Drosophila
embryonic epidermis and visceral mesoderm (VM). Chimeric Ubx/AbdA proteins were tested in both tissues and used to identify protein sequences that endow AbdA with a different target gene specificity from Ubx. We found that distinct protein sequences define AbdA, as opposed to Ubx, function in the epidermis vs. the VM. These sequences lie mostly outside the homeodomain (HD), emphasizing the importance of non-HD residues for specific Hox activities. Hox tissue specificity is therefore achieved by sensing distinct Hox protein structures in different tissues.
Mutation of melanosome protein RAB38 in
chocolate
mice
Authors: Loftus Stacie K. , Larson Denise M. , Baxter Laura L. , Antonellis Anthony , Chen Yidong , Wu Xufeng , Jiang Yuan , Bittner Michael , Hammer John A. , Pavan William J.
Published: July 2002

Mutations of genes needed for melanocyte function can result in oculocutaneous albinism. Examination of similarities in human gene expression patterns by using microarray analysis reveals that
RAB38
, a small GTP binding protein, demonstrates a similar expression profile to melanocytic genes. Comparative genomic analysis localizes human
RAB38
to the mouse chocolate (
cht
) locus. A G146T mutation occurs in the conserved GTP binding domain of RAB38 in
cht
mice.
Rab38

cht

/Rab38

cht

mice exhibit a brown coat similar in color to mice with a mutation in tyrosinase-related protein 1 (
Tyrp1
), a mouse model for oculocutaneous albinism. The targeting of TYRP1 protein to the melanosome is impaired in
Rab38

cht

/Rab38

cht

melanocytes. These observations, and the fact that green fluorescent protein-tagged RAB38 colocalizes with end-stage melanosomes in wild-type melanocytes, suggest that RAB38 plays a role in the sorting of TYRP1. This study demonstrates the utility of expression profile analysis to identify mammalian disease genes.
Elucidating the biosynthesis of 2-carboxyarabinitol 1-phosphate through reduced expression of chloroplastic fructose 1,6-bisphosphate phosphatase and radiotracer studies with
14
CO
2
Authors: Andralojc P. John , Keys Alfred J. , Kossmann Jens , Parry Martin A. J.
Published: July 2002

2-Carboxyarabinitol 1-phosphate limits photosynthetic CO
2
assimilation at low light because it is a potent, naturally occurring inhibitor of ribulose 1,5-bisphosphate carboxylase/oxygenase. Evidence is presented that this inhibitor is derived from chloroplastic fructose 1,6-bisphosphate. First, transgenic plants containing decreased amounts of chloroplastic fructose 1,6-bisphosphate phosphatase contained increased amounts of fructose 1,6-bisphosphate and 2-carboxyarabinitol 1-phosphate and greatly increased amounts of the putative intermediates hamamelose and 2-carboxyarabinitol, which in some cases were as abundant as sucrose. Second, French bean leaves in the light were shown to incorporate
14
C from
14
CO
2
sequentially into fructose 1,6-bisphosphate, hamamelose bisphosphate, hamamelose monophosphate, hamamelose, and 2-carboxyarabinitol. As shown previously,
14
C assimilated by photosynthesis was also incorporated into 2-carboxyarabinitol 1-phosphate during subsequent darkness.
Configurationally homogeneous diastereomers of a linear hexa(tertiary phosphine): Enantioselective self-assembly of a double-stranded parallel helicate of the type (P)-[Cu3(hexaphos)2](PF6)3
Authors: Bowyer Paul K. , Cook Vernon C. , Gharib-Naseri Nahid , Gugger Paul A. , Rae A. David , Swiegers Gerhard F. , Willis Anthony C. , Zank Johann , Wild S. Bruce
Published: July 2002

Three configurationally homogeneous diastereomers of the linear hexa(tertiary phosphine) Ph2PCH2CH2P(Ph)CH2CH2P(Ph)CH2CH2P(Ph)CH2CH2P(Ph)CH2CH2PPh2(hexaphos) have been isolated in enantiomerically pure form, namely (R,S,S,R)-, (R,S,S,S)-, and (S,S,S,S)-hexaphos. The strongly helicating (R,S,S,R)-(−) form of the ligand combines with copper(I) ions to generate by stereoselective self-assembly thePenantiomer of a parallel helicate of the type [Cu3(hexaphos)2](PF6)3, which has been characterized by x-ray crystallography. Theoretical modeling of the cation indicates that it is the relationship between the helicities of the two 10-membered rings containing the three copper ions, each of which has the twist-boat–chair–boat conformation, and the configurations of the three chiral, tetrahedral copper stereocenters ofPconfiguration that determines the stereochemistry of the parallel and double α-helix conformers of the double-stranded trinuclear metal helicate.
Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)
Authors: Giraldo Rafael , Díaz-Orejas Ramón
Published: July 2002

The proteins responsible for the initiation of DNA replication are
thought to be essentially unrelated in bacteria and
archaea/eukaryotes. Here we show that RepA, the initiator from the
Pseudomonas
plasmid pPS10, and the C-terminal domain of
ScOrc4p, a subunit of
Saccharomyces cerevisiae
(Sc)
origin recognition complex (ORC), share sequence similarities. Based on
biochemical and spectroscopic evidence, these similarities include
common structural elements, such as a winged-helix domain and a
leucine-zipper dimerization motif. We have also found that ScOrc4p, as
previously described for RepA-type initiators, interacts with
chaperones of the Hsp70 family both
in vitro
and
in vivo
, most probably to regulate the assembly of
active ORC. In evolutionary terms, our results are compatible with the
recruitment of the same protein module for initiation of DNA
replication by the ancestors of present-day Gram-negative bacteria
plasmids, archaea, and eukaryotes.
The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability
Authors: Hung Li-Wei , Holbrook Elizabeth L. , Holbrook Stephen R.
Published: July 2002

The crystal and molecular structure of an RNA duplex corresponding to the high affinity Rev protein binding element (RBE) has been determined at 2.1-Å resolution. Four unique duplexes are present in the crystal, comprising two structural variants. In each duplex, the RNA double helix consists of an annealed 12-mer and 14-mer that form an asymmetric internal loop consisting of G-G and G-A noncanonical base pairs and a flipped-out uridine. The 12-mer strand has an A-form conformation, whereas the 14-mer strand is distorted to accommodate the bulges and noncanonical base pairing. In contrast to the NMR model of the unbound RBE, an asymmetric G-G pair with N2-N7 and N1-O6 hydrogen bonding, is formed in each helix. The G-A base pairing agrees with the NMR structure in one structural variant, but forms a novel water-mediated pair in the other. A backbone flip and reorientation of the G-G base pair is required to assume the RBE conformation present in the NMR model of the complex between the RBE and the Rev peptide.
The crystal structure of a heptameric archaeal Sm protein: Implications for the eukaryotic snRNP core
Authors: Mura Cameron , Cascio Duilio , Sawaya Michael R. , Eisenberg David S.
Published: July 2002

Sm proteins form the core of small nuclear ribonucleoprotein
particles (snRNPs), making them key components of several
mRNA-processing assemblies, including the spliceosome. We report the
1.75-Å crystal structure of SmAP, an Sm-like archaeal protein that
forms a heptameric ring perforated by a cationic pore. In addition to
providing direct evidence for such an assembly in eukaryotic snRNPs,
this structure (
i
) shows that SmAP homodimers are
structurally similar to human Sm heterodimers, (
ii
)
supports a gene duplication model of Sm protein evolution, and
(
iii
) offers a model of SmAP bound to single-stranded
RNA (ssRNA) that explains Sm binding-site specificity. The pronounced
electrostatic asymmetry of the SmAP surface imparts directionality to
putative SmAP–RNA interactions.
Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene
kal-1
Authors: Bülow Hannes E. , Berry Katherine L. , Topper Liat H. , Peles Elior , Hobert Oliver
Published: July 2002

Kallmann syndrome is a neurological disorder characterized by various behavioral and neuroanatomical defects. The X-linked form of this disease is caused by mutations in the
KAL-1
gene, which codes for a secreted molecule that is expressed in restricted regions of the brain. Its molecular mechanism of action has thus far remained largely elusive. We show here that expression of the
Caenorhabditis elegans
homolog of
KAL-1
in selected sensory and interneuron classes causes a highly penetrant, dosage-dependent, and cell autonomous axon-branching phenotype. In a different cellular context, heterologous
C. elegans kal-1
expression causes a highly penetrant axon-misrouting phenotype. The axon-branching and -misrouting activities require different domains of the KAL-1 protein. In a genetic modifier screen we isolated several loci that either suppress or enhance the
kal-1
-induced axonal defects, one of which codes for an enzyme that modifies specific residues in heparan sulfate proteoglycans, namely heparan-6
O
-sulfotransferase. We hypothesize that KAL-1 binds by means of a heparan sulfate proteoglycan to its cognate receptor or other extracellular cues to induce axonal branching and axon misrouting.
Painting of fourth, a chromosome-specific protein inDrosophila
Authors: Larsson Jan , Chen J. Don , Rasheva Vanya , Rasmuson-Lestander Åsa , Pirrotta Vincenzo
Published: July 2002

Chromosome-specific gene regulation is known thus far only as a mechanism to equalize the transcriptional activity of the single male X chromosome with that of the two female X chromosomes. InDrosophila melanogaster, a complex including the five Male-Specific Lethal (MSL) proteins, “paints” the male X chromosome, mediating its hypertranscription. Here, with the molecular cloning ofPainting of fourth(Pof), we describe a previously uncharacterized gene encoding a chromosome-specific protein inDrosophila. Unlike the MSL proteins, POF paints an autosome, the fourth chromosome ofDrosophila melanogaster. Chromosome translocation analysis shows that the binding depends on an initiation site in the proximal region of chromosome 4 and spreads in cis to involve the entire chromosome. The spreading depends on sequences or structures specific to chromosome 4 and cannot extend to parts of other chromosomes translocated to the fourth. Spreading can also occur in trans to a paired homologue that lacks the initiation region. In the related speciesDrosophila busckii, POF paints the entire X chromosome exclusively in males, suggesting relationships between the fourth chromosome and the X and between POF complexes and dosage-compensation complexes.
Incipient speciation by sexual isolation in
Drosophila
: Concurrent evolution at multiple loci
Authors: Ting Chau-Ti , Takahashi Aya , Wu Chung-I
Published: July 2002

Drosophila melanogaster
from Zimbabwe and nearby
regions shows strong but asymmetric sexual isolation from its
cosmopolitan counterparts. By creating stable chromosome-substitution
lines, earlier studies were able to show that the two major autosomes
have very large effects on both male mating success and female mating
preference. In this study, we genetically dissect this sexual isolation
by recombination analysis between a whole-chromosome substitution line
(which carries a Zimbabwe-derived third chromosome) and a strain with
seven visible markers on that chromosome. Four loci are responsible for
male mating success and three others are found to control female mating
preference. Because male and female traits are not closely linked,
their strong association among isofemale lines is most likely a
reflection of sexual selection in nature. The results suggest that a
large number of behavioral loci may evolve concurrently in the
incipient stage of speciation before other aspects of reproductive
isolation (such as hybrid sterility) have become evident. The results
shed light on the population genetic processes underlying the formation
of nascent species, as well as modes of speciation.
Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate
Authors: Navazio Lorella , Bewell Michael A. , Siddiqua Ashia , Dickinson George D. , Galione Antony , Sanders Dale
Published: July 2002

Higher plants share with animals a responsiveness to the Ca
2+
mobilizing agents inositol 1,4,5-trisphosphate (InsP
3
) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular
45
Ca
2+
flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca
2+
-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca
2+
with a
K
1/2
= 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca
2+
pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca
2+
pathway distinguishes it from the InsP
3
- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca
2+
release is insensitive to heparin and 8-NH
2
-cADPR, specific inhibitors of the InsP
3
- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca
2+
release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca
2+
release pathway is independent of cytosolic free Ca
2+
and therefore incapable of operating Ca
2+
-induced Ca
2+
release. In contrast to the sea urchin system, the NAADP-gated Ca
2+
release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca
2+
mobilization pathways and Ca
2+
release sites might contribute to the generation of stimulus-specific Ca
2+
signals in plant cells.
The nuclear factor kappa B (NF-κB): A potential therapeutic target for estrogen receptor negative breast cancers
Authors: Biswas Debajit K. , Dai Sun-Chun , Cruz Antonio , Weiser Barbara , Graner Edgard , Pardee Arthur B.
Published: July 2002

The effect of a kinase inhibitor Go6796 on growth of epidermal growth factor (EGF)-stimulated estrogen receptor negative (ER−) breast cancer cells
in vivo
and role of nuclear factor kappa B (NF-κB) on tumorogenesis have been investigated. This was studied in an animal model by implanting ER− mouse mammary epithelial tumor cells (CSMLO) in syngeneic A-J mice. (
i
) Local administration of Go6976 an inhibitor of protein kinases C alpha and beta inhibited growth of tumors and caused extensive necrotic degeneration and regression of the tumors without causing any microscopically detectable damage to the vital organs liver and lung. (
ii
) Stable expression of dominant-negative mutants of the beta subunit (dnIkkβ) of the inhibitory kappa B (IκB) kinase (dnIkk) that selectively blocked activation of NF-κB caused loss of tumorigenic potential of CSMLO cells. Stable expression of dnIkkβ also blocked phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-κB and overexpression of cyclin D1, concomitantly with the loss or reduced tumorigenic potential of these cells. Thus, results from
in vivo
and
in vitro
experiments strongly suggest the involvement of NF-κB in ER− mammary epithelial cell-mediated tumorigenesis. We propose that blocking NF-κB activation not only inhibits cell proliferation, but also antagonizes the antiapoptotic role of this transcription factor in ER− breast cancer cells. Thus, NF-κB is a potential target for therapy of EGFR family receptor-overexpressing ER− breast cancers.
A distinctive role for the
Yersinia
protein kinase: Actin binding, kinase activation, and cytoskeleton disruption
Authors: Juris Stephen J. , Rudolph Amy E. , Huddler Don , Orth Kim , Dixon Jack E.
Published: July 2002

The bacterial pathogens of the genus
Yersinia
deliver several virulence factors into target cells using a type III secretion system. We demonstrate that
Yersinia
protein kinase A (YpkA), an essential bacterial virulence factor, is produced as an inactive serine/threonine kinase. The inactive kinase is activated within the host cell by a cytosolic eukaryotic activator. Using biochemical purification techniques, we demonstrate that actin is a cellular activator of YpkA. This stimulation of YpkA kinase activity by actin depends on the presence of the C-terminal twenty amino acids of YpkA, because deletion of these 20 aa not only obliterates YpkA activity, but it also destroys the interaction between YpkA and actin. Activated YpkA functions within cultured epithelial cells to disrupt the actin cytoskeleton. The disruption of the actin cytoskeleton by YpkA would be expected to inhibit macrophage function and phagocytosis of
Yersinia
.
A trithorax-group complex purified from
Saccharomyces cerevisiae
is required for methylation of histone H3
Authors: Nagy Peter L. , Griesenbeck Joachim , Kornberg Roger D. , Cleary Michael L.
Published: July 2002

Histone methylation has emerged as an important mechanism for
regulating the transcriptional accessibility of chromatin. Several
methyltransferases have been shown to target histone amino-terminal
tails and mark nucleosomes associated with either euchromatic or
heterochromatic states. However, the biochemical machinery
responsible for regulating histone methylation and integrating it with
other cellular events has not been well characterized. We report here
the purification, molecular identification, and genetic and biochemical
characterization of the Set1 protein complex that is necessary for
methylation of histone H3 at lysine residue 4 in
Saccharomyces
cerevisiae
. The seven-member 363-kDa complex contains homologs
of
Drosophila melanogaster
proteins Ash2 and Trithorax
and
Caenorhabditis
elegans
protein DPY-30, which are implicated in
the maintenance of
Hox
gene expression and regulation of
X chromosome dosage compensation, respectively. Mutations of Set1
protein comparable to those that disrupt developmental function of its
Drosophila
homolog Trithorax abrogate histone
methylation in yeast. These studies suggest that epigenetic regulation
of developmental and sex-specific gene expression are species-specific
readouts for a common chromatin remodeling machinery associated
mechanistically with histone methylation.
Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the
Dutt1
/
Robo1
gene
Authors: Xian Jian , Clark Katherine J. , Fordham Rosalyn , Pannell Richard , Rabbitts Terence H. , Rabbitts Pamela H.
Published: July 2002

Chromosome 3 allele loss in preinvasive bronchial
abnormalities and carcinogen-exposed, histologically normal
bronchial epithelium indicates that it is an early, possibly the first,
somatic genetic change in lung tumor development. Candidate tumor
suppressor genes have been isolated from within distinct 3p regions
implicated by heterozygous and homozygous allele loss. We have proposed
that
DUTT1,
nested within homozygously deleted regions
at 3p12–13, is the tumor suppressor gene that deletion-mapping and
tumor suppression assays indicate is located in proximal 3p. The same
gene,
ROBO1
(accession number
AF040990
), was
independently isolated as the human homologue of the
Drosophila
gene,
Roundabout
. The gene,
coding for a receptor with a domain structure of the neural-cell
adhesion molecule family, is widely expressed and has been
implicated in the guidance and migration of axons, myoblasts, and
leukocytes in vertebrates. A deleted form of the gene, which mimics a
naturally occurring, tumor-associated human homozygous deletion of exon
2 of
DUTT1
/
ROBO1
, was introduced into
the mouse germ line. Mice homozygous for this targeted mutation, which
eliminates the first Ig domain of Dutt1/Robo1, frequently die
at birth of respiratory failure because of delayed lung maturation.
Lungs from these mice have reduced air spaces and increased mesenchyme,
features that are present some days before birth. Survivors acquire
extensive bronchial epithelial abnormalities including hyperplasia,
providing evidence of a functional relationship between a 3p gene and
the development of bronchial abnormalities associated with early lung
cancer.
Two mutant prion proteins expressed in cultured cells acquire biochemical properties reminiscent of the scrapie isoform.
Authors: Lehmann S , Harris D A
Published: July 2002

Prion diseases are a group of fatal neurodegenerative disorders that are unique in being infectious, genetic, and sporadic in origin. Infectious cases are caused by prions, which are composed primarily of PrPSc, a posttranslationally modified isoform of the normal cellular prion protein PrPC. Inherited cases are linked to insertional or point mutations in the host gene encoding PrPC. To investigate the molecular mechanisms underlying inherited prion diseases, we have constructed stably transfected Chinese hamster ovary cells that express mouse PrPs homologous to two human PrPs associated with familial Creutzfeldt-Jakob disease. One mouse PrP molecule carries a Glu-->Lys substitution at codon 199, and the other carries an insertion of six additional octapeptide repeats between codons 51 and 90. We find that both of these mutant PrPs display several biochemical hallmarks of PrPSc when synthesized in cell culture. Unlike wild-type PrP, the mutant proteins are detergent insoluble and are relatively resistant to digestion by proteinase K, yielding an N-terminally truncated core fragment of 27-30 kDa. Pulse-chase labeling experiments demonstrate that these properties are acquired posttranslationally, and are accompanied by increased metabolic stability of the protein. Our results provide the first evidence that a molecule with properties reminiscent of PrPSc can be generated de novo in cultured cells.
A JAK-STAT pathway regulates wing vein formation in Drosophila.
Authors: Yan R , Luo H , Darnell J E , Dearolf C R
Published: July 2002

We present evidence that the JAK-STAT signal transduction pathway regulates multiple developmental processes in Drosophila. We screened for second-site mutations that suppress the phenotype of the hyperactive hopTum-1 Jak kinase, and recovered a mutation that meiotically maps to the known chromosomal position of D-Stat, a Drosophila stat gene. This hypomorphic mutation, termed statHJ contains a nucleotide substitution in the first D-Stat intron, resulting in a reduction in the number of correctly processed transcripts. Further, the abnormally processed mRNA encodes a truncated protein that has a dominant negative effect on transcriptional activation by the wild-type cDNA in cell culture. statHJ mutants exhibit patterning defects that include the formation of ectopic wing veins, similar to those seen in mutants of the epidermal growth factor/receptor pathway. Abnormalities in embryonic and adult segmentation and in tracheal development were also observed. The hopTum-1 and statHJ mutations can partially compensate for each other genetically, and Hop overexpression can increase D-Stat transcriptional activity in vitro, indicating that the gene products interact in a common regulatory pathway.
Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activated oncogenes to determine transformation and malignant potential
Authors: Fan Huizhou , Villegas Cristy , Wright Jim A.
Published: July 2002

Ribonucleotide reductase is a highly regulated cell
cycle-controlled activity that is essential for DNA synthesis and
repair. A retroviral vector for the R2 component of mammalian
ribonucleotide reductase, the rate-limiting protein for enzyme activity
and DNA synthesis in proliferating cells, was constructed and
introduced into mammalian cells. Expression of Myc epitope-tagged R2
protein in benign BALB/c 3T3 and NIH 3T3 cells leads to a greatly
increased frequency of focus formation in cooperation with
H-
ras
transformation. Four lines of
H-
ras
-transformed mouse 10T½ fibroblasts showed
increased growth efficiency in soft agar after infection with the
recombinant R2 expression virus vector. Furthermore, cells with altered
R2 expression also exhibited significantly reduced subcutaneous tumor
latency and increased tumor growth rates in syngeneic mice, and showed
markedly elevated metastatic potential in lung metastasis assays. The
results indicate that altered R2 gene expression cooperates with
ras
in mechanisms of malignant progression. A major Ras
pathway involves the Raf-1 protein, which is recruited to the plasma
membrane for activation. We show that recombinant R2 expression leads
to significant increases in membrane-associated Raf-1 protein and
mitogen-activating protein kinase-2 activity suggesting a mechanism for
the observed Ras/R2 synergism. In support of this finding, we observed
that activated Rac-1, which operates parallel to Raf-1 and cooperates
with Raf-1 in Ras activated pathways, also cooperates with R2 in
cellular transformation. These studies demonstrate that the R2 protein
can participate in other critical cellular functions in addition to
ribonucleotide reduction, and that deregulated R2 is a novel tumor
progressor determinant that cooperates in oncogene-mediated mechanisms,
which control malignant potential.
Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K
+
channel  subunit
Authors: Chan Kim W. , Sui Jin-Liang , Vivaudou Michel , Logothetis Diomedes E.
Published: July 2002

G protein-gated inwardly rectifying K
+
(GIRK) channels,
which are important regulators of membrane excitability both in heart
and brain, appear to function as heteromultimers. GIRK1 is unique in
the GIRK channel family in that although it is by itself inactive, it
can associate with the other family members (GIRK2–GIRK5) to enhance
their activity and alter their single-channel characteristics. By
generating a series of chimeras, we identified a phenylalanine residue,
F137, in the pore region of GIRK1 that critically controls channel
activity. F137 is found only in GIRK1, while the remaining GIRK
channels possess a conserved serine residue in the analogous position.
The single-point mutant GIRK4(S143F) behaved as a GIRK1 analog, forming
multimers with GIRK2, GIRK4, or GIRK5 channels that exhibited
prolonged single-channel open-time duration and enhanced activity
compared with that of homomultimers. Expression of the corresponding
GIRK1(F137S) mutant alone resulted in appreciable channel activity with
novel characteristics that was further enhanced upon coexpression with
other GIRK subunits. Thus, although the F137 residue renders the GIRK1
subunit inactive, when combined with other GIRK heteromeric partners it
alters their gating and contributes to their enhanced activity.
Symmetries throughout organic evolution
Authors: García-Bellido Antonio
Published: July 2002

The biological realm has inherited symmetries from the
physicochemical realm, but with the increasing complexity at higher
phenomenological levels of life, some inherited symmetries are broken
while novel symmetries appear. These symmetries are of two types,
structural and operational. Biological novelties result from breaking
operational symmetries. They are followed by acquisition of regularity
and stability, in a recurrent process throughout complexity levels.
Biosynthesis of major histocompatibility complex molecules and generation of T cells in Ii TAP1 double-mutant mice.
Authors: Tourne S , van Santen H M , van Roon M , Berns A , Benoist C , Mathis D , Ploegh H
Published: July 2002

Major histocompatibility complex (MHC) class I and II molecules are loaded with peptides in distinct subcellular compartments. The transporter associated with antigen processing (TAP) is responsible for delivering peptides derived from cytosolic proteins to the endoplasmic reticulum, where they bind to class I molecules, while the invariant chain (Ii) directs class II molecules to endosomal compartments, where they bind peptides originating mostly from exogenous sources. Mice carrying null mutations of the TAP1 or Ii genes (TAP10) or Ii0, respectively) have been useful tools for elucidating the two MHC/peptide loading pathways. To evaluate to what extent these pathways functionally intersect, we have studied the biosynthesis of MHC molecules and the generation of T cells in Ii0TAP10 double-mutant mice. We find that the assembly and expression of class II molecules in Ii0 and Ii0TAP10 animals are indistinguishable and that formation and display of class I molecules is the same in TAP10 and Ii0TAP10 animals. Thymic selection in the double mutants is as expected, with reduced numbers of both CD4+ CD8- and CD4- CD8+ thymocyte compartments. Surprisingly, lymph node T-cell populations look almost normal; we propose that population expansion of peripheral T cells normalizes the numbers of CD4+ and CD8+ cells in Ii0TAP10 mice.
Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure.
Authors: Phillip D , Ruban A V , Horton P , Asato A , Young A J
Published: July 2002

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n </= 9 conjugated double bonds (e.g., violaxanthin) had no effect on fluorescence. When quenching in the light-harvesting complex of photosystem II was induced by a lowering of pH to 5.5, carotenoids with n </= 9 conjugated double bonds (including violaxanthin) caused a noticeable inhibition of fluorescence quenching relative to the control. Of the 10 carotenoids tested, quenching induced by the addition of the tertiary amine compound, dibucaine, to isolated light-harvesting complex of photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.
Purification and physical properties of the male and female double sex proteins of Drosophila.
Authors: Cho S , Wensink P C
Published: July 2002

The double sex gene (dsx) encodes two proteins, DSX(M) and DSX(F), that regulate sex-specific transcription in Drosophila. These proteins bind target sites in DNA from which the male-specific DSX(M) represses and the female-specific DSX(F) activates transcription of yolk protein (Yp) genes. We investigated the physical properties of these DSX proteins, which are identical in their amino-terminal 397 residues but are entirely different in their carboxyl-terminal sequences (DSX(F), 30 amino acids; DSX(M), 152 amino acids). DSX(M) and DSX(F) were overexpressed in cultured insect cells and purified to near homogeneity. Gel filtration chromatography and glycerol gradient sedimentation showed that at low concentrations both proteins are dimers of highly asymmetrical shape. The axial ratios are approximately 18:1 (DSX(M), 860 X 48 angstroms; DSX(F), 735 X 43 angstroms). At higher concentrations, the proteins form tetramers. Through use of a novel, double crosslinking assay (protein-DNA plus protein-protein), we demonstrated that a DNA regulatory site binds to both monomers of the DSX dimer and to only two monomers of the tetramer. Furthermore, binding another DNA molecule to what we presume is the second and identical site in the tetramer dramatically shifts the equilibrium from tetramers to dimers. These oligomerization and DNA binding properties are indistinguishable between the male and female proteins.
Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.
Authors: Miller M M , Goto R M , Taylor R L , Zoorob R , Auffray C , Briles R W , Briles W E , Bloom S E
Published: July 2002

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.
Toward a mechanism for GroEL.GroES chaperone activity: an ATPase-gated and -pulsed folding and annealing cage.
Authors: Corrales F J , Fersht A R
Published: July 2002

Free GroEL binds denatured proteins very tightly: it retards the folding of barnase 400-fold and catalyzes unfolding fluctuations in native barnase and its folding intermediate. GroEL undergoes an allosteric transition from its tight-binding T-state to a weaker binding R-state on the cooperative binding of nucleotides (ATP/ADP) and GroES. The preformed GroEL.GroES.nucleotide complex retards the folding of barnase by only a factor of 4, and the folding rate is much higher than the ATPase activity that releases GroES from the complex. Binding of GroES and nucleotides to a preformed GroEL.denatured-barnase complex forms an intermediately fast-folding complex. We propose the following mechanism for the molecular chaperone. Denatured proteins bind to the resting GroEL.GroES.nucleotide complex. Fast-folding proteins are ejected as native structures before ATP hydrolysis. Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis. This step causes transient release of GroES and formation of the GroEL.denatured-protein complexes with higher annealing potential. The intermediately fast-folding complex is formed on subsequent rebinding of GroES. The ATPase activity of GroEL.GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster-folding R-states to the tighter-binding/stronger annealing T-states. The molecular chaperone acts as a combination of folding cage and an annealing machine.
Memory B lymphocytes migrate to bone marrow in humans
Authors: Paramithiotis Eustache , Cooper Max D.
Published: July 2002

IgM-bearing B lymphocytes with mature phenotype (CD10−CD24loIgD+) are acquired after birth in the bone marrow of humans. These B cells are defined here as relatively large, nondividing lymphocytes, variable proportions of which express cell surface molecules indicative of relatively recent activation. Analysis of VH52(heavy chain variable region) gene transcripts indicated point mutations throughout the Ig variable region from the mature IgM+B population but not from the immature B cells in the bone marrow. The mutations were concentrated in the complementarity determining regions, and amino acid substitutions were favored over silent mutations, findings indicative of antigen selection within germinal centers in peripheral lymphoid tissues. The VHsequence analysis also revealed the existence of clonal relatives in individual bone marrow samples. These antigen-experienced lymphocytes did not secrete Ig spontaneously but could be induced to do soin vitro. The data suggest that a subpopulation of memory B lymphocytes generated during antigen responses recirculates to the bone marrow in humans.
Incipient species formation in salamanders of the
Ensatina
complex
Authors: Wake David B.
Published: July 2002

The
Ensatina eschscholtzii
complex of plethodontid
salamanders, a well-known “ring species,” is thought to
illustrate stages in the speciation process. Early research, based on
morphology and coloration, has been extended by the incorporation of
studies of protein variation and mitochondrial DNA sequences. The new
data show that the complex includes a number of geographically and
genetically distinct components that are at or near the species level.
The complex is old and apparently has undergone instances of range
contraction, isolation, differentiation, and then expansion and
secondary contact. While the hypothesis that speciation is retarded by
gene flow around the ring is not supported by molecular data, the
general biogeographical hypothesis is supported. There is evidence of a
north to south range expansion along two axes, with secondary contact
and completion of the ring in southern California. Current research
targets regions once thought to show primary intergradation, but which
molecular markers reveal to be zones of secondary contact. Here
emphasis is on the subspecies
E. e. xanthoptica
, which
is involved in four distinct secondary contacts in central California.
There is evidence of renewed genetic interactions upon recontact, with
greater genetic differentiation within
xanthoptica
than
between it and some of the interacting populations. The complex
presents a full array of intermediate conditions between well-marked
species and geographically variable populations. Geographically
differentiated segments represent a diversity of depths of time of
isolation and admixture, reflecting the complicated geomorphological
history of California.
Ensatina
illustrates the
continuing difficulty in making taxonomic assignments in complexes
studied during species formation.
Xenopus
Cdc6 confers sperm binding competence to oocytes without inducing their maturation
Authors: Tian Jingdong , Thomsen Gerald H. , Gong Hui , Lennarz William J.
Published: July 2002

Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)
+
mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)
+
mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode
Xenopus
Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.
Evolution of the folding ability of proteins through functional selection
Authors: Saito Seiji , Sasai Masaki , Yomo Tetsuya
Published: July 2002

An evolutionary process is simulated with a simple spin-glass-like model of proteins to examine the origin of folding ability. At each generation, sequences are randomly mutated and subjected to a simulation of the folding process based on the model. According to the frequency of local configurations at the active sites, sequences are selected and passed to the next generation. After a few hundred generations, a sequence capable of folding globally into a native conformation emerges. Moreover, the selected sequence has a distinct energy minimum and an anisotropic funnel on the energy surface, which are the imperative features for fast folding of proteins. The proposed model reveals that the functional selection on the local configurations leads a sequence to fold globally into a conformation at a faster rate.
The ribosome-in-pieces: Binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA
Authors: Munishkin Alexander , Wool Ira G.
Published: July 2002

An oligoribonucleotide (a 27-mer) that mimics the sarcin/ricin (S/R) domain of
Escherichia coli
23S rRNA binds elongation factor EF-G; the
K
d
is 6.9 μM, whereas for binding to ribosomes it is 0.7 μM. Binding saturates when EF-G and the S/R RNA are equimolar; at saturation 70% of the input RNA is in complexes with EF-G. Binding of EF-G to S/R RNA does not require GTP but is inhibited by GDP; the inhibition by GDP is overcome by GTP. The effects of mutations of the S/R domain nucleotides G2655, A2660, and G2661 suggest that EF-G recognizes the conformation of the RNA rather than the identity of the nucleotides. EF-G also binds to an oligoribonucleotide (an 84-mer) that has the thiostrepton region of 23S rRNA; however, EF-G binds independently to S/R and thiostrepton oligoribonucleotides.
Loss of HMW1 and HMW3 in noncytadhering mutants of
Mycoplasma pneumoniae
occurs post-translationally
Authors: Popham Phillip L. , Hahn Tae-Wook , Krebes Kathleen A. , Krause Duncan C.
Published: July 2002

The genomic sequence of
Mycoplasma pneumoniae
establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication. However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species. This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia. Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation. However, the organization, regulation, and assembly of the attachment organelle in
M. pneumoniae
are poorly understood, and no counterparts have been identified among the walled bacteria.
M. pneumoniae
possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle. The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle. Disruptions in
hmw2
result in the loss of HMW1 and HMW3. However, the
hmw1
and
hmw3
genes were transcribed and translated at wild-type levels in
hmw2
mutants. HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period. Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover. Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW
−
mutants.