bluuebunny/crossref_metadata_2025 huggingface dataset:
Combinatorial discovery of oxidative dehydrogenation catalysts within the Mo-V-Nb-O system
Authors: Cong Peijun , Dehestani Ahmad , Doolen Robert , Giaquinta Daniel M. , Guan Shenheng , Markov Victor , Poojary Damodara , Self Kyle , Turner Howard , Weinberg W. Henry
Published: July 2002

Combinatorial methodologies were used for the synthesis and screening of mixed metal oxide heterogeneous catalysts. Primary screening at low reactant conversions at a throughput of greater than 10,000 catalyst compositions per month was performed by using simultaneous MS and photothermal deflection spectroscopy on spatially separated thick film catalysts with ≈200 μg per catalyst prepared by using automated liquid dispensing. Secondary screening under realistic operating conditions was performed at a throughput of greater than 3,000 catalyst compositions per month on ≈50 mg of catalyst in an array of fixed bed microreactors with gas chromatograph detection. The approach was validated by the discovery of catalysts with superior performance to those previously described for the oxidative dehydrogenation of ethane to ethylene. We show the full implementation and integration of combinatorial methodologies for synthesis, screening, discovery, and optimization of multicomponent heterogeneous catalysts.
Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein
Authors: Vita Claudio , Drakopoulou Eugenia , Vizzavona Jean , Rochette Sandrine , Martin Loïc , Ménez André , Roumestand Christian , Yang Yin-Shan , Ylisastigui Loyda , Benjouad Abdelaziz , Gluckman Jean Claude
Published: July 2002

Protein–protein interacting surfaces are usually large and
intricate, making the rational design of small mimetics of these
interfaces a daunting problem. On the basis of a structural similarity
between the CDR2-like loop of CD4 and the β-hairpin region of a short
scorpion toxin, scyllatoxin, we transferred the side chains of nine
residues of CD4, central in the binding to HIV-1 envelope
glycoprotein (gp120), to a structurally homologous region of the
scorpion toxin scaffold. In competition experiments, the resulting
27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40
μM IC
50
. Structural analysis by NMR showed that both the
backbone of the chimeric β-hairpin and the introduced side chains
adopted conformations similar to those of the parent CD4. Systematic
single mutations suggested that most CD4 residues from the CDR2-like
loop were reproduced in the miniprotein, including the critical Phe-43.
The structural and functional analysis performed suggested five
additional mutations that, once incorporated in the miniprotein,
increased its affinity for gp120 by 100-fold to an IC
50
of
0.1–1.0 μM, depending on viral strains. The resulting mini-CD4
inhibited infection of CD4
+
cells by different virus
isolates. Thus, core regions of large protein–protein interfaces can
be reproduced in miniprotein scaffolds, offering possibilities for the
development of inhibitors of protein–protein interactions that may
represent useful tools in biology and in drug discovery.
A mitogen-activated protein kinase of the corn leaf pathogen
Cochliobolus heterostrophus
is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens
Authors: Lev Sophie , Sharon Amir , Hadar Ruthi , Ma Hong , Horwitz Benjamin A.
Published: July 2002

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen
Cochliobolus heterostrophus
to test its role as a component of the transduction pathways. The new gene,
CHK1
, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus
Magnaporthe grisea
and 59% identical to Fus3 of
Saccharomyces cerevisiae
. A series of
chk1
deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δ
chk1
mutants is attempted. The ability of Δ
chk1
mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of
pmk1
mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in
Cochliobolus heterostrophus
, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.
G
o
protein-dependent survival of primary accessory olfactory neurons
Authors: Tanaka Masaki , Treloar Helen , Kalb Robert G. , Greer Charles A. , Strittmatter Stephen M.
Published: July 2002

Extensive G protein-coupled receptor families in both the main and accessory olfactory systems have been implicated in axonal targeting, sensory function, and cell survival. Although sensory function seems to be mediated by G proteins, axonal guidance and cell survival may be G protein-independent processes. In the accessory olfactory system, the G
o
-containing neurons in the basal vomeronasal organ (VNO) project to the posterior accessory olfactory bulb (AOB), whereas more apically located VNO neurons contain G
i2
and project to the anterior AOB. Herein, we investigate the organization of the accessory olfactory system in mice with a targeted deletion in the G
o
α gene. The accessory olfactory system seems normal at birth; however, postnatally, the number of G
o
-receptor-containing VNO neurons decreases by half, and apoptotic neurons are detected. The axons of VNO neurons remain restricted to the posterior AOB. The posterior AOB is reduced in size but contains a synaptophysin-positive layer with the normal number of glomeruli. The posterior AOB has reduced mitral cell c-Fos immunoreactivity, consistent with decreased sensory activation of G
o
protein-coupled VNO receptor neurons. Thus, in the accessory olfactory system, receptor-coupled G proteins are required for cell survival.
Neandertal nasal structures and upper respiratory tract “specialization”
Authors: Franciscus Robert G.
Published: July 2002

Schwartz and Tattersall [Schwartz, J. H. & Tattersall, I.
(1996)
Proc. Natl. Acad. Sci. USA
93, 10852–10854]
have argued for a previously unrecognized suite of autapomorphies in
the internal nasal region of Neandertals that make them unique, not
only among hominids, but possibly among all other terrestrial mammals.
These purported autapomorphies include (
i
) the
development of an internal nasal margin bearing a well developed and
vertically oriented medial projection; (
ii
) a pronounced
medial swelling of the lateral nasal wall into the posterior nasal
cavity; and (
iii
) the lack of an ossified roof over the
lacrimal groove. In addition, Laitman
et al
. [Laitman,
J. T., Reidenberg, J. S., Marquez, S. & Gannon, P. J.
(1996)
Proc. Natl. Acad. Sci. USA
93, 10543–10545]
pointed to these features as evidence for upper respiratory tract
specializations among the Neandertals, indicating potential differences
in behavior compared with modern humans. Critically reviewing the
anatomical basis for Schwartz and Tattersall’s contentions reveals
several serious problems with their analysis, including
(
i
) reliance on specimens with damaged, incomplete, or,
in some cases, entirely absent relevant anatomy; (
ii
)
failure to consider primary vs. secondary spatial consequences in nasal
trait conceptualization; and (
iii
) failure to consider
actual ranges of variation in these traits in both fossil and recent
humans. Accordingly, the unique phylogenetic and adaptive
“specializations” attributed to Neandertal internal nasal
structures are unwarranted.
Stable transduction of quiescent CD34+CD38−human hematopoietic cells by HIV-1-based lentiviral vectors
Authors: Case Scott S. , Price Mary A. , Jordan Craig T. , Yu Xiao Jin , Wang Lijun , Bauer Gerhard , Haas Dennis L. , Xu Dakun , Stripecke Renata , Naldini Luigi , Kohn Donald B. , Crooks Gay M.
Published: July 2002

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringentin vitroassays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34+cells (45.5% GFP+) and in CD34+CD38−cells in G0(12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34+cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34+cells. The MLV vector did not transduce more primitive, quiescent CD34+CD38−cells (n= 8). In contrast, stable transduction of CD34+CD38−cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 ± 5.2%,n= 7). GFP expression in clones from single CD34+CD38−cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34+and CD34+CD38−cells (13.5 ± 2.5%,n= 11 and 12.2 ± 9.7%,n= 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.
Phthalascidin, a synthetic antitumor agent with potency and mode of action comparable to ecteinascidin 743
Authors: Martinez Eduardo J. , Owa Takashi , Schreiber Stuart L. , Corey E. J.
Published: July 2002

A series of totally synthetic molecules that are structurally related to the marine natural product ecteinascidin 743 (Et 743) has been prepared and evaluated as antitumor agents. The most active of these, phthalascidin, is very similar to Et 743 with regard to
in vitro
potency and mode of action across a variety of cell types. The antiproliferative activity of phthalascidin (IC
50
= 0.1–1 nM) is greater than that of the agents Taxol, camptothecin, adriamycin, mitomycin C, cisplatin, bleomycin, and etoposide by 1–3 orders of magnitude, and the mechanism of action is clearly different from these currently used drugs. Phthalascidin and Et 743 induce DNA–protein cross-linking and, although they seem to interact with topoisomerase (topo) I (but not topo II), topo I may not be the primary protein target of these agents. Phthalascidin and Et 743 show undiminished potency in camptothecin- and etoposide-resistant cells. Phthalascidin is more readily synthesized and more stable than Et 743, which is currently undergoing clinical trials. The relationship of chemical structure and antitumor activity for this class of molecules has been clarified by this study.
Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis
Authors: Hornung Ellen , Walther Matthias , Kühn Hartmut , Feussner Ivo
Published: July 2002

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme/substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L+H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13
S
)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and/or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.
Bicoid functions without its TATA-binding protein-associated factor interaction domains
Authors: Schaeffer Valérie , Janody Florence , Loss Céline , Desplan Claude , Wimmer Ernst A.
Published: July 2002

Four maternal systems are known to pattern the early
Drosophila
embryo. The key component of the anterior system is the homeodomain protein Bicoid (Bcd). Bcd needs the contribution of another anterior morphogen, Hunchback (Hb), to function properly: Bcd and Hb synergize to organize anterior development. A molecular mechanism for this synergy has been proposed to involve specific interactions of Bcd and Hb with TATA-binding protein-associated factors (TAF
II
s) that are components of the general transcription machinery. Bcd contains three putative activation domains: a glutamine-rich region, which interacts
in vitro
with TAF
II
110; an alanine-rich domain, which targets TAF
II
60; and a C-terminal acidic region, which has an unknown role. We have generated flies carrying
bcd
transgenes lacking one or several of these domains to test their function
in vivo
. Surprisingly, a
bcd
transgene that lacks all three putative activation domains is able to rescue the
bcd
E1
null phenotype to viability. Moreover, the development of these embryos is not affected by the presence of dominant negative mutations in TAF
II
110 or TAF
II
60. This means that the interactions observed
in vitro
between Bcd and TAF
II
60 or TAF
II
110 aid transcriptional activation but are dispensable for normal development.
Specific molecular recognition of mixed nucleic acid sequences: An aromatic dication that binds in the DNA minor groove as a dimer
Authors: Wang Lei , Bailly Christian , Kumar Arvind , Ding Daoyuan , Bajic Miroslav , Boykin David W. , Wilson W. David
Published: July 2002

Phenylamidine cationic groups linked by a furan ring (furamidine) and related compounds bind as monomers to AT sequences of DNA. An unsymmetric derivative (DB293) with one of the phenyl rings of furamidine replaced with a benzimidazole has been found by quantitative footprinting analyses to bind to GC-containing sites on DNA more strongly than to pure AT sequences. NMR structural analysis and surface plasmon resonance binding results clearly demonstrate that DB293 binds in the minor groove at specific GC-containing sequences of DNA in a highly cooperative manner as a stacked dimer. Neither the symmetric bisphenyl nor bisbenzimidazole analogs of DB293 bind significantly to the GC containing sequences. DB293 provides a paradigm for design of compounds for specific recognition of mixed DNA sequences and extends the boundaries for small molecule-DNA recognition.
Leptin, troglitazone, and the expression of sterol regulatory element binding proteins in liver and pancreatic islets
Authors: Kakuma Tetsuya , Lee Young , Higa Moritake , Wang Zhuo-wei , Pan Wentong , Shimomura Iichiro , Unger Roger H.
Published: July 2002

Overaccumulation of lipids in nonadipose tissues of obese rodents may lead to lipotoxic complications such as diabetes. To assess the pathogenic role of the lipogenic transcription factor, sterol regulatory element binding protein 1 (SREBP-1), we measured its mRNA in liver and islets of obese, leptin-unresponsive
fa
/
fa
Zucker diabetic fatty rats. Hepatic SREBP-1 mRNA was 2.4 times higher than in lean +/+ controls, primarily because of increased SREBP-1c expression. mRNA of lipogenic enzymes ranged from 2.4- to 4.6-fold higher than lean controls, and triacylglycerol (TG) content was 5.4 times higher. In pancreatic islets of
fa
/
fa
rats, SREBP-1c was 3.4 times higher than in lean +/+ Zucker diabetic fatty rats. The increase of SREBP-1 in liver and islets of untreated
fa
/
fa
rats was blocked by 6 weeks of troglitazone therapy, and the diabetic phenotype was prevented. Up-regulation of SREBP-1 also occurred in livers of Sprague–Dawley rats with diet-induced obesity. Hyperleptinemia, induced in lean +/+ rats by adenovirus gene transfer, lowered hepatic SREBP-1c by 74% and the lipogenic enzymes from 35 to 59%. In conclusion, overnutrition increases and adenovirus-induced hyperleptinemia decreases SREBP-1c expression in liver and islets. SREBP-1 overexpression, which is prevented by troglitazone, may play a role in the ectopic lipogenesis and lipotoxicity complicating obesity in Zucker diabetic fatty rats.
Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors
Authors: Oberwinkler Johannes , Stavenga Doekele G.
Published: July 2002

During illumination, Ca
2+
enters fly photoreceptor cells through light-activated channels that are located in the rhabdomere, the compartment specialized for phototransduction. From the rhabdomere, Ca
2+
diffuses into the cell body. We visualize this process by rapidly imaging the fluorescence in a cross section of a photoreceptor cell injected with a fluorescent Ca
2+
indicator
in vivo
. The free Ca
2+
concentration in the rhabdomere shows a very fast and large transient shortly after light onset. The free Ca
2+
concentration in the cell body rises more slowly and displays a much smaller transient. After ≈400 ms of light stimulation, the Ca
2+
concentration in both compartments reaches a steady state, indicating that thereafter an amount of Ca
2+
, equivalent to the amount of Ca
2+
flowing into the cell, is extruded. Quantitative analysis demonstrates that during the steady state, the free Ca
2+
concentration in the rhabdomere and throughout the cell body is the same. This shows that Ca
2+
extrusion takes place very close to the location of Ca
2+
influx, the rhabdomere, because otherwise gradients in the steady-state distribution of Ca
2+
should be measured. The close colocalization of Ca
2+
influx and Ca
2+
extrusion ensures that, after turning off the light, Ca
2+
removal from the rhabdomere is faster than from the cell body. This is functionally significant because it ensures rapid dark adaptation.
Structure and function in rhodopsin: Destabilization of rhodopsin by the binding of an antibody at the N-terminal segment provides support for involvement of the latter in an intradiscal tertiary structure
Authors: Cha Kiweon , Reeves Philip J. , Khorana H. Gobind
Published: July 2002

A monoclonal anti-rhodopsin antibody (B6–30N), characterized by
Hargrave and coworkers [Adamus, G., Zam, Z. S., Arendt, A.,
Palczewski, K., McDowell, J. M. & Hargrave, P. (1991)
Vision Res.
31, 17–31] as recognizing a short peptide
sequence at the N terminus, failed to bind to rhodopsin when the latter
was solubilized in dodecylmaltoside (DM). Of the detergents tested thus
far, DM affords maximum stability to rhodopsin. Solubilization of
rhodopsin in cholate allowed binding of the antibody, but the binding
caused destabilization as evidenced by the accelerated loss of
absorbance at 500 nm. The result provides support for the earlier
conclusion that the N-terminal segment is an integral part of a
tertiary structure in the intradiscal domain of native rhodopsin
coupled to a tertiary structure in the transmembrane domain. Additional
comparative studies on the stability of rhodopsin in different
detergents were carried out after direct solubilization from rod outer
segments and after extensive treatments to remove the
endogenous phospholipids. Purification of rhodopsin in DM
resulted in essentially quantitative removal of endogenous
phospholipids. When rhodopsin thus purified was treated with the above
antibody in DM and in cholate, enhanced destabilization (5-fold) was
observed in the latter detergent.
The occurrence of Jovian planets and the habitability of planetary systems
Authors: Lunine Jonathan I.
Published: July 2002

Planets of mass comparable to or larger than Jupiter's have been detected around over 50 stars, and for one such object a definitive test of its nature as a gas giant has been accomplished with data from an observed planetary transit. By virtue of their strong gravitational pull, giant planets define the dynamical and collisional environment within which terrestrial planets form. In our solar system, the position and timing of the formation of Jupiter determined the amount and source of the volatiles from which Earth's oceans and the source elements for life were derived. This paper reviews and brings together diverse observational and modeling results to infer the frequency and distribution of giant planets around solar-type stars and to assess implications for the habitability of terrestrial planets.
Factors associated with continued smoking during pregnancy: analysis of socio‐demographic, pregnancy and smoking‐related factors
Authors: PENN G. , OWEN L.
Published: July 2002

This study investigated the characteristics associated with smoking during pregnancy. A total of 7836 pregnant women were surveyed between 1992 and 1999 in England. Of these 27% were smoking during pregnancy. Pregnant women were more likely to smoke if they were less educated, living in rented accommodation, in unskilled manual or unemployed groups, and single or had a partner who smokes. Analysed by logistic regression, whether or not a pregnant women has a partner and, if so, his smoking status, was by far the biggest predictor of the pregnant woman's current smoking status. Thus, compared with women with partners who never smoke, the odds ratio (OR) of smoking during pregnancy for women with a partner who smokes was 2.3 (1.9–2.7) while those with no partner had an OR of 4.8 (3.8–6.0). For women exposed to passive smoke at home or at work the OR was 2.5 (2.1–3.0). Housing tenure was the most important socio‐demographic predictor. Thus pregnant women living in rented council housing were nearly twice as likely (OR 1.93;1.63–2.29) as those buying their own home to be current smokers. The number of women who continued to smoke during pregnancy increased 10‐fold from the least to the most deprived group. These findings highlight the importance of tobacco control strategies that address pregnant women's life circumstances and broader inequalities as well as those that focus on individual smoking behaviour.
A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle
Authors: Lübeck Jens , Heins Lisa , Soll Jürgen
Published: July 2002

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.
Mammalian p55CDC Mediates Association of the Spindle Checkpoint Protein Mad2 with the Cyclosome/Anaphase-promoting Complex, and is Involved in Regulating Anaphase Onset and Late Mitotic Events
Authors: Kallio Marko , Weinstein Jasminder , Daum John R. , Burke Daniel J. , Gorbsky Gary J.
Published: July 2002

We have investigated the function of p55CDC, a mammalian protein related to Cdc20 and Hct1/Cdh1 in Saccharomyces cerevisiae, and Fizzy and Fizzy-related in Drosophila. Immunofluorescence studies and expression of a p55CDC-GFP chimera demonstrate that p55CDC is concentrated at the kinetochores in M phase cells from late prophase to telophase. Some p55CDC is also associated with the spindle microtubules and spindle poles, and some is diffuse in the cytoplasm. At anaphase, the concentration of p55CDC at the kinetochores gradually diminishes, and is gone by late telophase. In extracts prepared from M phase, but not from interphase HeLa cells, p55CDC coimmunoprecipitates with three important elements of the M phase checkpoint machinery: Cdc27, Cdc16, and Mad2. p55CDC is required for binding Mad2 with the Cdc27 and Cdc16. Thus, it is likely that p55CDC mediates the association of Mad2 with the cyclosome/anaphase-promoting complex. Microinjection of anti-p55CDC antibody into mitotic mammalian cells induces arrest or delay at metaphase, and impairs progression of late mitotic events. These studies suggest that mammalian p55CDC may be part of a regulatory and targeting complex for the anaphase-promoting complex.
A Role for NIMA in the Nuclear Localization of Cyclin B in Aspergillus nidulans
Authors: Wu L. , Osmani S.A. , Mirabito P.M.
Published: July 2002

NIMA promotes entry into mitosis in late G2 by some mechanism that is after activation of the Aspergillus nidulans G2 cyclin-dependent kinase, NIMXCDC2/NIMECyclin B. Here we present two independent lines of evidence which indicate that this mechanism involves control of NIMXCDC2/NIMECyclin B localization. First, we found that NIMECyclin B localized to the nucleus and the nucleus-associated organelle, the spindle pole body, in a NIMA-dependent manner. Analysis of cells from asynchronous cultures, synchronous cultures, and cultures arrested in S or G2 showed that NIMECyclin B was predominantly nuclear during interphase, with maximal nuclear accumulation in late G2. NIMXCDC2 colocalized with NIMECyclin B in G2 cells. Although inactivation of NIMA using either the nimA1 or nimA5 temperature-sensitive mutations blocked cells in G2, NIMXCDC2/NIMECyclin B localization was predominantly cytoplasmic rather than nuclear. Second, we found that nimA interacts genetically with sonA, which is a homologue of the yeast nucleocytoplasmic transporter GLE2/RAE1. Mutations in sonA were identified as allele-specific suppressors of nimA1. The sonA1 suppressor alleviated the nuclear division and NIMECyclin B localization defects of nimA1 cells without markedly increasing NIMXCDC2 or NIMA kinase activity. These results indicate that NIMA promotes the nuclear localization of the NIMXCDC2/ NIMECyclin B complex, by a process involving SONA. This mechanism may be involved in coordinating the functions of NIMXCDC2 and NIMA in the regulation of mitosis.
Ca2+-dependent Muscle Dysfunction Caused by Mutation of the Caenorhabditis elegans Troponin T-1 Gene
Authors: McArdle Kristen , Allen Taylor StC. , Bucher Elizabeth A.
Published: July 2002

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca2+]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061–1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca2+-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca2+ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca2+ signaling support that CeTnT-1 phenotypes are dependent on Ca2+ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca2+] does not require TnT.
Daip1, a Dictyostelium Homologue of the Yeast Actin-Interacting Protein 1, Is Involved in Endocytosis, Cytokinesis, and Motility
Authors: Konzok Angelika , Weber Igor , Simmeth Evelyn , Hacker Ulrike , Maniak Markus , Müller-Taubenberger Annette
Published: July 2002

The 64-kD protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum, and higher eukaryotes.

We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia, and other pseudopodia. In cells expressing green fluorescent protein (GFP)-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions.

Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid-phase uptake, phagocytosis, and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results obtained by DAip1 overexpression and effects of latrunculin-A treatment, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork.
Protein Kinase C–Dependent Mobilization of the α6β4 Integrin from Hemidesmosomes and Its Association with Actin-Rich Cell Protrusions Drive the Chemotactic Migration of Carcinoma Cells
Authors: Rabinovitz Isaac , Toker Alex , Mercurio Arthur M.
Published: July 2002

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the α6β4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express α6β4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the α6β4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis (∼1 ng/ml), the α6β4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained α6β4 in association with F-actin. Importantly, we demonstrate that this mobilization of α6β4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-α and that it is associated with the phosphorylation of the β4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin–rich cell protrusions that mediate α6β4-dependent cell movement but also the disruption of α6β4-containing hemidesmosomes by protein kinase C.
Mutations in the α-Tubulin 67C Gene Specifically Impair Achiasmate Segregation in Drosophila melanogaster
Authors: Matthies Heinrich J.G. , Messina Lisa G. , Namba Ruria , Greer Kimberly J. , Walker M.Y. , Hawley R. Scott
Published: July 2002

Drosophila melanogaster oocytes heterozygous for mutations in the α-tubulin 67C gene (αtub67C) display defects in centromere positioning during prometaphase of meiosis I. The centromeres do not migrate to the poleward edges of the chromatin mass, and the chromatin fails to stretch during spindle lengthening. These results suggest that the poleward forces acting at the kinetochore are compromised in the αtub67C mutants. Genetic studies demonstrate that these mutations also strongly and specifically decrease the fidelity of achiasmate chromosome segregation. Proper centromere orientation, chromatin elongation, and faithful segregation can all be restored by a decrease in the amount of the Nod chromokinesin. These results suggest that the accurate segregation of achiasmate chromosomes requires the proper balancing of forces acting on the chromosomes during prometaphase.
Actin Bound to the Heterogeneous Nuclear Ribonucleoprotein Hrp36 Is Associated with Balbiani Ring mRNA from the Gene to Polysomes
Authors: Percipalle Piergiorgio , Zhao Jian , Pope Brian , Weeds Alan , Lindberg Uno , Daneholt Bertil
Published: July 2002

In the salivary glands of the dipteran Chironomus tentans, a specific messenger ribonucleoprotein (mRNP) particle, the Balbiani ring (BR) granule, can be visualized during its assembly on the gene and during its nucleocytoplasmic transport. We now show with immunoelectron microscopy that actin becomes associated with the BR particle concomitantly with transcription and is present in the particle in the nucleoplasm. DNase I affinity chromatography experiments with extracts from tissue culture cells indicate that both nuclear and cytoplasmic actin are bound to the heterogeneous RNP (hnRNP) protein hrp36, but not to the hnRNP proteins hrp23 and hrp45. The interaction is likely to be direct as purified actin binds to recombinant hrp36 in vitro. Furthermore, it is demonstrated by cross linking that nuclear as well as cytoplasmic actin are bound to hrp36 in vivo. It is known that hrp36 is added cotranscriptionally along the BR mRNA molecule and accompanies the RNA through the nuclear pores and into polysomes. We conclude that actin is likely to be bound to the BR transcript via hrp36 during the transfer of the mRNA from the gene all the way into polysomes.
Polo boxes and Cut23 (Apc8) mediate an interaction between polo kinase and the anaphase-promoting complex for fission yeast mitosis
Authors: May Karen M. , Reynolds Nicola , Cullen C. Fiona , Yanagida Mitsuhiro , Ohkura Hiroyuki
Published: July 2002

The fission yeast plo1+ gene encodes a polo-like kinase, a member of a conserved family of kinases which play multiple roles during the cell cycle. We show that Plo1 kinase physically interacts with the anaphase-promoting complex (APC)/cyclosome through the noncatalytic domain of Plo1 and the tetratricopeptide repeat domain of the subunit, Cut23. A new cut23 mutation, which specifically disrupts the interaction with Plo1, results in a metaphase arrest. This arrest can be rescued by high expression of Plo1 kinase. We suggest that this physical interaction is crucial for mitotic progression by targeting polo kinase activity toward the APC.
Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration
Authors: Maheshwari Gargi , Wiley H. Steven , Lauffenburger Douglas A.
Published: July 2002

Cell responses to soluble regulatory factors may be strongly influenced by the mode of presentation of the factor, as in matrix-bound versus diffusible modes. The possibly diverse effect of presenting a growth factor in autocrine as opposed to exogenous (or paracrine) mode is an especially important issue in cell biology. We demonstrate here that migration behavior of human mammary epithelial cells in response to stimulation by epidermal growth factor (EGF) is qualitatively different for EGF presented in exogenous (paracrine), autocrine, and intracrine modes. When EGF is added as an exogenous factor to the medium of cells that express EGF receptor (EGFR) but not EGF, cell migration speed increases while directional persistence decreases. When these EGFR-expressing cells are made to also express via retroviral transfection EGF in protease-cleaveable transmembrane form on the plasma membrane, migration speed similarly increases, but directional persistence increases as well. Addition of exogenous EGF to these cells abrogates their enhanced directional persistence, reducing their directionality to a level similar to wild-type cells. If the EGFR-expressing cells are instead transduced with a gene encoding EGF in a soluble form, migration speed and directional persistence were unaffected. Thus, autocrine presentation of EGF at the plasma membrane in a protease-cleavable form provides these cells with an enhanced ability to migrate persistently in a given direction, consistent with their increased capability for organizing into gland-like structures. In contrast, an exogenous/paracrine mode of EGF presentation generates a “scattering” response by the cells. These findings emphasize the functional importance of spatial restriction of EGFR signaling, and suggest critical implications for growth factor–based therapeutic treatments.
A New Mechanism of NK Cell Cytotoxicity Activation: The CD40–CD40 Ligand Interaction
Authors: Carbone Ennio , Ruggiero Giuseppina , Terrazzano Giuseppe , Palomba Carmen , Manzo Ciro , Fontana Silvia , Spits Hergen , Kärre Klas , Zappacosta Serafino
Published: July 2002

NK recognition is regulated by a delicate balance between positive signals initiating their effector functions, and inhibitory signals preventing them from proceeding to cytolysis. Knowledge of the molecules responsible for positive signaling in NK cells is currently limited. We demonstrate that IL-2–activated human NK cells can express CD40 ligand (CD40L) and that recognition of CD40 on target cells can provide an activation pathway for such human NK cells. CD40-transfected P815 cells were killed by NK cell lines expressing CD40L, clones and PBLderived NK cells cultured for 18 h in the presence of IL-2, but not by CD40L-negative fresh NK cells. Cross-linking of CD40L on IL-2–activated NK cells induced redirected cytolysis of CD40-negative but Fc receptor-expressing P815 cells. The sensitivity of human TAP-deficient T2 cells could be blocked by anti-CD40 antibodies as well as by reconstitution of TAP/MHC class I expression, indicating that the CD40-dependent pathway for NK activation can be downregulated, at least in part, by MHC class I molecules on the target cells. NK cell recognition of CD40 may be important in immunoregulation as well as in immune responses against B cell malignancies.
The Recognition of the Nonclassical Major Histocompatibility Complex (MHC) Class I Molecule, T10, by the γδ T Cell, G8
Authors: Crowley Michael P. , Reich Ziv , Mavaddat Nasim , Altman John D. , Chien Yueh-hsiu
Published: July 2002

Recent studies have shown that many nonclassical major histocompatibility complex (MHC) (class Ib) molecules have distinct antigen-binding capabilities, including the binding of nonpeptide moieties and the binding of peptides that are different from those bound to classical MHC molecules. Here, we show that one of the H-2T region–encoded molecules, T10, when produced in Escherichia coli, can be folded in vitro with β2-microglobulin (β2m) to form a stable heterodimer in the absence of peptide or nonpeptide moieties. This heterodimer can be recognized by specific antibodies and is stimulatory to the γδ T cell clone, G8. Circular dichroism analysis indicates that T10/β2m has structural features distinct from those of classical MHC class I molecules. These results suggest a new way for MHC-like molecules to adopt a peptide-free structure and to function in the immune system.
Transferable Anergy: Superantigen Treatment Induces CD4+ T Cell Tolerance That Is Reversible and Requires CD4−CD8− Cells and Interferon γ
Authors: Cauley Linda S. , Cauley Keith A. , Shub Fillipa , Huston Gail , Swain Susan L.
Published: July 2002

Bacterial superantigens induce peripheral unresponsiveness in CD4+ T cell populations that express appropriate Vβ chains. We have used Vβ3/Vα11 T cell receptor transgenic (Tg) mice and the Vβ3-specific superantigen staphylococcal enterotoxin A (SEA) to further investigate the mechanisms that contribute to such unresponsiveness. As in other models, in vivo exposure to SEA rendered the Tg CD4+ cells unresponsive to subsequent restimulation in vitro with antigen or mitogens. However, when the SEA-treated CD4+ cells were completely purified away from all other contaminating cells, they regained the ability to proliferate and secrete cytokines. Moreover, enriched CD4−CD8− cells from the SEA-treated mice suppressed the responses of fresh control CD4+ cells in mixed cultures indicating that the apparent “anergy” was both transferable and reversible. Further analysis demonstrated that interferon γ, but not the Fas receptor, played a critical role in the suppression.
Visualization of CD4/CD8 T Cell Commitment
Authors: Chan Susan , Correia-Neves Margarida , Dierich Andrée , Benoist Christophe , Mathis Diane
Published: July 2002

A system to innocuously visualize T cell lineage commitment is described. Using a “knock-in” approach, we have generated mice expressing a β-galactosidase reporter in place of CD4; expression of β-galactosidase in these animals appears to be an accurate and early indicator of CD4 gene transcription. We have exploited this knock-in line to trace CD4/CD8 lineage commitment in the thymus, avoiding important pitfalls of past experimental approaches. Our results argue in favor of a selective model of thymocyte commitment, demonstrating a fundamentally symmetrical process: engagement of either class of major histocompatibility complex (MHC) molecule by a differentiating CD4+CD8+ cell can give rise to T cell antigen receptor (TCR)hi thymocytes of either lineage. Key findings include (a) direct demonstration of a substantial number of CD4-committed, receptor/coreceptor-mismatched cells in MHC class II– deficient mice, a critical prediction of the selective model; (b) highly efficient rescue of such “mismatched” intermediates by forced expression of CD8 in a TCR transgenic line, and an explanation of why previous experiments of this nature were less successful—a major past criticism of the selective model; (c) direct demonstration of an analogous, though smaller, population of CD8-committed mismatched intermediates in class I–deficient animals. Finally, we found no evidence of a CD4 default pathway.
Chlamydia Inhibits Interferon γ–inducible Major Histocompatibility Complex Class II Expression by Degradation of  Upstream Stimulatory Factor 1
Authors: Zhong Guangming , Fan Tao , Liu Li
Published: July 2002

We report that chlamydiae, which are obligate intracellular bacterial pathogens, can inhibit interferon (IFN)-γ–inducible major histocompatibility complex (MHC) class II expression. However, the IFN-γ–induced IFN regulatory factor-1 (IRF-1) and intercellular adhesion molecule 1 (ICAM-1) expression is not affected, suggesting that chlamydia may selectively target the IFN-γ signaling pathways required for MHC class II expression. Chlamydial inhibition of MHC class II expression is correlated with degradation of upstream stimulatory factor (USF)-1, a constitutively and ubiquitously expressed transcription factor required for IFN-γ induction of class II transactivator (CIITA) but not of  IRF-1 and ICAM-1. CIITA is an obligate mediator of IFN-γ–inducible MHC class II expression. Thus, diminished CIITA expression as a result of USF-1 degradation may account for the suppression of the IFN-γ–inducible MHC class II in chlamydia-infected cells. These results reveal a novel immune evasion strategy used by the intracellular bacterial pathogen chlamydia that improves our understanding of the molecular basis of pathogenesis.
A Single Gene (tts) Located outside the cap Locus Directs the Formation of Streptococcus pneumoniae Type 37 Capsular Polysaccharide
Authors: Llull Daniel , Muñoz Rosario , López Rubens , García Ernesto
Published: July 2002

The molecular aspects of the type 37 pneumococcal capsular biosynthesis, a homopolysaccharide composed of sophorosyl units (β-d-Glc-(1→2)-β-d-Glc) linked by β-1,3 bonds, have been studied. Remarkably, the biosynthesis of the type 37 capsule is driven by a single gene (tts) located far apart from the cap locus responsible for capsular formation in all of the types characterized to date in Streptococcus pneumoniae. However, a cap37 locus virtually identical to the cap33f cluster has been found in type 37 strains, although some of its genes are inactivated by mutations. The tts gene has been sequenced and its transcription start point determined. Tts shows sequence motifs characteristic of cellulose synthases and other β-glycosyltransferases. Insertion of the tts gene into the pneumococcal DNA causes a noticeable genome reorganization in such a way that genes normally separated by more than 350 kb in the chromosome are located together in clinical isolates of type 37. Encapsulated pneumococcal strains belonging to 10 different serotypes (or serogroups) transformed with tts synthesized type 37 polysaccharide, leading to the formation of strains that display the binary type of capsule. Type 37 pneumococcus constitutes the first case of a natural, genetically binary strain and represents a novel alternative to the mechanisms of intertype transformation.
B Cells of HIV-1–Infected Patients Bind Virions through Cd21–Complement Interactions and Transmit Infectious Virus to Activated T Cells
Authors: Moir Susan , Malaspina Angela , Li Yuexia , Chun Tae-Wook , Lowe Tomeka , Adelsberger Joseph , Baseler Michael , Ehler Linda A. , Liu Shuying , Davey Richard T. , Mican Jo Ann M. , Fauci Anthony S.
Published: July 2002

The impact of HIV-associated immunopathogenesis on B cells has been largely associated with indirect consequences of viral replication. This study demonstrates that HIV interacts directly with B cells in both lymphoid tissues and peripheral blood. B cells isolated from lymph node and peripheral blood mononuclear cells (PBMCs) of 4 and 23 chronically infected patients, respectively, demonstrated similar capacities to pass virus to activated HIV-negative PBMCs when compared with CD4+ cells from the same patients. However, in contrast to T cells, virus associated with B cells was surface bound, as shown by its sensitivity to pronase and the staining pattern revealed by in situ amplification of HIV-1 RNA. Cell sorting and ligand displacing approaches established that CD21 was the HIV-binding receptor on B cells, and that this association was mediated through complement-opsonized virus. These B cells were also found to express significantly lower levels of CD21 compared with HIV-negative individuals, suggesting a direct perturbing effect of HIV on B cells. These findings suggest that B cells, although they themselves are not readily infected by HIV, are similar to follicular dendritic cells in their capacity to serve as extracellular reservoirs for HIV-1. Furthermore, B cells possess the added capability of circulating in peripheral blood and migrating through tissues where they can potentially interact with and pass virus to T cells.