bluuebunny/crossref_metadata_2025 huggingface dataset:
The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties
Authors: Rovner Arthur S. , Fagnant Patricia M. , Lowey Susan , Trybus Kathleen M.
Published: July 2002

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.
Novel Vascular Molecule Involved in Monocyte Adhesion to Aortic Endothelium in Models of Atherogenesis
Authors: McEvoy Leslie M. , Sun Hailing , Tsao Philip S. , Cooke John P. , Berliner Judith A. , Butcher Eugene C.
Published: July 2002

Adhesion of monocytes to the endothelium in lesion-prone areas is one of the earliest events in fatty streak formation leading to atherogenesis. The molecular basis of increased monocyte adhesion is not fully characterized. We have identified a novel vascular monocyte adhesion-associated protein, VMAP-1, that plays a role in adhesion of monocytes to activated endothelium. Originally selected for its ability to block binding of a mouse monocyte-like cell line (WEHI78/24) to cytokine- or LPS-stimulated cultured mouse endothelial cells in vitro, antiVMAP-1 mAb LM151 cross-reacts with rabbit endothelium and blocks binding of human monocytes to cultured rabbit aortic endothelial cells stimulated with minimally modified low density lipoprotein, thought to be a physiologically relevant atherogenic stimulus. Most importantly, LM151 prevents adhesion of normal monocytes and monocytoid cells to intact aortic endothelium from cholesterol-fed rabbits in an ex vivo assay. VMAP-1 is a 50-kD protein. Immunohistology of vessels reveals focal constitutive expression in aorta and other large vessels. VMAP-1 is thus a novel vascular adhesion-associated protein that appears to play a critical role in monocyte adhesion to aortic endothelial cells in atherogenesis in vivo.
Increased Hypermutation at G and C Nucleotides in Immunoglobulin Variable Genes from Mice Deficient in the MSH2 Mismatch Repair Protein
Authors: Phung Quy H. , Winter David B. , Cranston Aaron , Tarone Robert E. , Bohr Vilhelm A. , Fishel Richard , Gearhart Patricia J.
Published: July 2002

Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2−/− mice were immunized with oxazolone, and B cells were analyzed for mutation in their VκOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G·C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.
Caspases Are Activated in a Branched Protease Cascade and Control Distinct Downstream Processes in Fas-induced Apoptosis
Authors: Hirata Hirokazu , Takahashi Atsushi , Kobayashi Susumu , Yonehara Shin , Sawai Hirofumi , Okazaki Toshiro , Yamamoto Kokichi , Sasada Masataka
Published: July 2002

Two novel synthetic tetrapeptides, VEID-CHO and DMQD-CHO, could selectively inhibit caspase-6 and caspase-3, respectively. We used these inhibitors to dissect the pathway of caspase activation in Fas-stimulated Jurkat cells and identify the roles of each active caspase in apoptotic processes. Affinity labeling techniques revealed a branched protease cascade in which caspase-8 activates caspase-3 and -7, and caspase-3, in turn, activates caspase-6. Both caspase-6 and -3 have major roles in nuclear apoptosis. Caspase-6 cleaves nuclear mitotic apparatus protein (NuMA) and mediates the shrinkage and fragmentation of nuclei. Caspase-3 cleaves NuMA at sites distinct from caspase-6, and mediates DNA fragmentation and chromatin condensation. It is also involved in extranuclear apoptotic events: cleavage of PAK2, formation of apoptotic bodies, and exposure of phosphatidylserine on the cell surface. In contrast, a caspase(s) distinct from caspase-3 or -6 mediates the disruption of mitochondrial membrane potential (permeability transition) and the shrinkage of cytoplasm. These findings demonstrate that caspases are organized in a protease cascade, and that each activated caspase plays a distinct role(s) in the execution of Fas-induced cell death.
Effector Cells of Both Nonhemopoietic and Hemopoietic Origin Are Required for Interferon (IFN)-γ– and Tumor Necrosis Factor (TNF)-α–dependent Host Resistance to the Intracellular Pathogen, Toxoplasma gondii
Authors: Yap George S. , Sher Alan
Published: July 2002

Although interferon (IFN)-γ–activated, mononuclear phagocytes are considered to be the major effectors of resistance to intracellular pathogens, it is unclear how they control the growth of microorganisms that reside in nonhemopoietic cells. Pathogens within such cells may be killed by metabolites secreted by activated macrophages or, alternatively, directly controlled by cytokine-induced microbicidal mechanisms triggered within infected nonphagocytic cells. To distinguish between these two basic mechanisms of cell-mediated immunity, reciprocal bone marrow chimeras were constructed between wild-type and IFN-γ receptor–deficient mice and their survival assessed following infection with Toxoplasma gondii, a protozoan parasite that invades both hemopoietic and nonhemopoietic cell lineages. Resistance to acute and persistent infection was displayed only by animals in which IFN-γ receptors were expressed in both cellular compartments. Parallel chimera experiments performed with tumor necrosis factor (TNF) receptor–deficient mice also indicated a codependence on hemopoietic and nonhemopoietic lineages for optimal control of the parasite. In contrast, in mice chimeric for inducible nitric oxide synthase (iNOS), an enzyme associated with IFN-γ–induced macrophage microbicidal activity, expression by cells of hemopoietic origin was sufficient for host resistance. Together, these findings suggest that, in concert with bone marrow–derived effectors, nonhemopoietic cells can directly mediate, in the absence of endogenous iNOS, IFN-γ– and TNF-α–dependent host resistance to intracellular infection.
Cd2 Sets Quantitative Thresholds in T Cell Activation
Authors: Bachmann Martin F. , Barner Marijke , Kopf Manfred
Published: July 2002

It has been proposed that CD2, which is highly expressed on T cells, serves to enhance T cell–antigen presenting cell (APC) adhesion and costimulate T cell activation. Here we analyzed the role of CD2 using CD2-deficient mice crossed with transgenic mice expressing a T cell receptor specific for lymphocytic choriomeningitis virus (LCMV)-derived peptide p33. We found that absence of CD2 on T cells shifted the p33-specific dose–response curve in vitro by a factor of 3–10. In comparison, stimulation of T cells in the absence of lymphocyte function–associated antigen (LFA)-1–intercellular adhesion molecule (ICAM)-1 interaction shifted the dose–response curve by a factor of 10, whereas absence of both CD2–CD48 and LFA-1–ICAM-1 interactions shifted the response by a factor of ∼100. This indicates that CD2 and LFA-1 facilitate T cell activation additively. T cell activation at low antigen density was blocked at its very first steps, as T cell APC conjugate formation, TCR triggering, and Ca2+ fluxes were affected by the absence of CD2. In vivo, LCMV-specific, CD2-deficient T cells proliferated normally upon infection with live virus but responded in a reduced fashion upon cross-priming. Thus, CD2 sets quantitative thresholds and fine-tunes T cell activation both in vitro and in vivo.
Innate Recognition of Bacteria in Human Milk Is Mediated by a Milk-Derived Highly Expressed Pattern Recognition Receptor, Soluble Cd14
Authors: Labéta Mario O. , Vidal Karine , Nores Julia E. Rey , Arias Mauricio , Vita Natalio , Morgan B. Paul , Guillemot Jean Claude , Loyaux Denis , Ferrara Pascual , Schmid Daniel , Affolter Michael , Borysiewicz Leszek K. , Donnet-Hughes Anne , Schiffrin Eduardo J.
Published: July 2002

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.
Efficient Generation of a Hepatitis B Virus Cytotoxic T Lymphocyte Epitope Requires the Structural Features of Immunoproteasomes
Authors: Sijts Alice J.A.M. , Ruppert Thomas , Rehermann Barbara , Schmidt Marion , Koszinowski Ulrich , Kloetzel Peter-M.
Published: July 2002

Interferon (IFN)-γ–induced cells express the proteasome subunits low molecular weight protein (LMP)2, LMP7, and MECL-1 (multicatalytic endopeptidase complex–like 1), leading to the formation of immunoproteasomes. Although these subunits are thought to optimize MHC class I antigen processing, the extent of their role and the mechanistic aspects involved remain unclear. Herein, we study the proteolytic generation of an human histocompatibility leukocyte antigen (HLA)-Aw68–restricted hepatitis B virus core antigen (HBcAg) cytotoxic T lymphocyte (CTL) epitope that is recognized by peripheral blood lymphocytes from patients with acute self-limited but not chronic hepatitis B virus (HBV). Immunological data suggest that IFN-γ–induced rather than uninduced HeLa cells process and present the HBV CTL epitope upon infection with HBcAg-expressing vaccinia viruses. Analyses of 20S proteasome digests of synthetic polypeptides covering the antigenic HBcAg peptide demonstrate that only immunoproteasomes efficiently perform the cleavages needed for the liberation of this HBV CTL epitope. Although the concerted presence of the three immunosubunits appears essential, we find that both catalytically active LMP7 and inactive LMP7 T1A support CTL epitope generation. We conclude that LMP7 influences the structural features of 20S proteasomes, thereby enhancing the activity of the LMP2 and MECL-1 catalytic sites, which provide cleavage specificity. Thus, LMP7 incorporation is of greater functional importance for the generation of an HBV CTL epitope than cleavage specificity.
High Frequency of Autoreactive Myelin Proteolipid Protein–Specific T Cells in the Periphery of Naive Mice
Authors: Anderson Ana C. , Nicholson Lindsay B. , Legge Kevin L. , Turchin Vadim , Zaghouani Habib , Kuchroo Vijay K.
Published: July 2002

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2s) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139–151 and PLP 178–191) bind to IAs with similar affinity; however, the immune response to the PLP 139–151 epitope is always dominant. The immunodominance of the PLP 139–151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4+ cells) reactive to PLP 139–151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139–151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139–151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139–151, as the DM20 isoform of PLP (which lacks residues 116–150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139–151 in the embryonic thymus results in a significant reduction of PLP 139–151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139–151-reactive T cells.
Perinatal Blockade of B7-1 and B7-2 Inhibits Clonal Deletion of Highly Pathogenic Autoreactive T Cells
Authors: Gao Jian-Xin , Zhang Huiming , Bai Xue-Feng , Wen Jing , Zheng Xincheng , Liu Jinqing , Zheng Pan , Liu Yang
Published: July 2002

A number of in vitro studies have suggested that costimulatory molecules B7-1 and B7-2 and their receptor CD28 can promote clonal deletion, and limited in vivo studies have indicated that CD28 is involved in the clonal deletion of some T cells. However, the significance of B7-mediated clonal deletion in preventing autoimmune diseases has not been studied systematically. Here we report that the perinatal blockade of B7-1 and B7-2 substantially inhibits the clonal deletion of T cells in the thymus and leads to an accumulation of T cells capable of inducing fatal multiorgan inflammation. These results reveal a critical role for costimulatory molecules B7-1 and B7-2 in deleting pathogenic autoreactive T cells in the thymus. The critical role of B7-1 and B7-2 in T cell clonal deletion may explain, at least in part, the paradoxical increase of autoimmune disease in mice deficient for this family of costimulatory molecules, such as cytotoxic T lymphocyte associated molecule 4, CD28, and B7-2. The strong pathogenicity of the self-reactive T cells supports a central hypothesis in immunology, which is that clonal deletion plays an important role in preventing autoimmune diseases.
Anomalous Effect of Permeant Ion Concentration on Peak Open Probability of Cardiac Na+ Channels
Authors: Townsend Claire , Hartmann Hali A. , Horn Richard
Published: July 2002

Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. Decreasing extracellular permeant ion concentration decreases outward Na+ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na+, Li+, or hydrazinium) is substituted for a relatively impermeant cation (K+, Rb+, Cs+, N -methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na+ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to −140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.
NO3−-induced pH Changes in Mammalian Cells
Authors: Chow Chung-Wai , Kapus Andras , Romanek Robert , Grinstein Sergio
Published: July 2002

The effect of NO3− on intracellular pH (pHi) was assessed microfluorimetrically in mammalian cells in culture. In cells of human, hamster, and murine origin addition of extracellular NO3− induced an intracellular acidification. This acidification was eliminated when the cytosolic pH was clamped using ionophores or by perfusing the cytosol with highly buffered solutions using patch-pipettes, ruling out spectroscopic artifacts. The NO3−- induced pH change was not due to modulation of Na+/H+ exchange, since it was also observed in Na+/H+ antiport-deficient mutants. Though NO3− is known to inhibit vacuolar-type (V) H+-ATPases, this effect was not responsible for the acidification since it persisted in the presence of the potent V-ATPase inhibitor bafilomycin A1. NO3−/HCO3− exchange as the underlying mechanism was ruled out because acidification occurred despite nominal removal of HCO3−, despite inhibition of the anion exchanger with disulfonic stilbenes and in HEK 293 cells, which seemingly lack anion exchangers (Lee, B.S., R.B. Gunn, and R.R. Kopito. 1991. J. Biol. Chem. 266:11448– 11454). Accumulation of intracellular NO3−, measured by the Greiss method after reduction to NO2−, indicated that the anion is translocated into the cells along with the movement of acid equivalents. The simplest model to explain these observations is the cotransport of NO3− with H+ (or the equivalent counter-transport of NO3− for OH−). The transporter appears to be bi-directional, operating in the forward as well as reverse directions. A rough estimate of the fluxes of NO3− and acid equivalents suggests a one-to-one stoichiometry. Accordingly, the rate of transport was unaffected by sizable changes in transmembrane potential. The cytosolic acidification was a saturable function of the extracellular concentration of NO3− and was accentuated by acidification of the extracellular space. The putative NO3−-H+ cotransport was inhibited markedly by ethacrynic acid and by α-cyano-4-hydroxycinnamate, but only marginally by 4,4′-diisothiocyanostilbene-2,2′ disulfonate or by p-chloromercuribenzene sulfonate. The transporter responsible for NO3−-induced pH changes in mammalian cells may be related, though not identical, to the NO3−-H+ cotransporter described in Arabidopsis and Aspergillus. The mammalian cotransporter may be important in eliminating the products of NO metabolism, particularly in cells that generate vast amounts of this messenger. By cotransporting NO3− with H+ the cells would additionally eliminate acid equivalents from activated cells that are metabolizing actively, without added energetic investment and with minimal disruption of the transmembrane potential, inasmuch as the cotransporter is likely electroneutral.
Properties of an Inwardly Rectifying ATP-sensitive K+ Channel in the Basolateral Membrane of Renal Proximal Tubule
Authors: Mauerer Ulrich R. , Boulpaep Emile L. , Segal Alan S.
Published: July 2002

The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells. Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (γslope, in = 24.5 ± 0.6 pS, γchord, out = 3.7 ± 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding–unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+, Cs+, Li+, NH4+, or Cl−. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0.2 mM ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.
Light-dependent Changes in Outer Segment Free-Ca2+ Concentration in Salamander Cone Photoreceptors
Authors: Sampath A.P. , Matthews H.R. , Cornwall M.C. , Bandarchi J. , Fain G.L.
Published: July 2002

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 ± 2.4 and 640 ± 55 ms (mean ± SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 ± 30 and 1,400 ± 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 ± 37 nM in the dark-adapted outer segment and 5.5 ± 2.4 nM after saturating illumination (mean ± SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.
Interaction between the Pore and a Fast Gate of the Cardiac Sodium Channel
Authors: Townsend Claire , Horn Richard
Published: July 2002

Permeant ions affect a fast gating process observed in human cardiac sodium channels (Townsend, C., H.A. Hartmann, and R. Horn. 1997. J. Gen. Physiol. 110:11–21). Removal of extracellular permeant ions causes a reduction of open probability at positive membrane potentials. These results suggest an intimate relationship between the ion-conducting pore and the gates of the channel. We tested this hypothesis by three sets of manipulations designed to affect the binding of cations within the pore: application of intracellular pore blockers, mutagenesis of residues known to contribute to permeation, and chemical modification of a native cysteine residue (C373) near the extracellular mouth of the pore. The coupling between extracellular permeant ions and this fast gating process is abolished both by pore blockers and by a mutation that severely affects selectivity. A more superficial pore mutation or chemical modification of C373 reduces single channel conductance while preserving both selectivity of the pore and the modulatory effects of extracellular cations. Our results demonstrate a modulatory gating role for a region deep within the pore and suggest that the structure of the permeation pathway is largely preserved when a channel is closed.
Functional and Morphological Correlates of Connexin50 Expressed in Xenopus laevis Oocytes
Authors: Zampighi Guido A. , Loo Donald D.F. , Kreman Michael , Eskandari Sepehr , Wright Ernest M.
Published: July 2002

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 ± 3 helices, (b) when their density reached 300–400/μm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1–2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in ∼0.1-μm diameter “coated” and in larger 0.2–0.5-μm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5–40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80,000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60–500 μm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in ∼24 h.
Multiple symmetric positive solutions for a second order boundary value problem
Authors: Henderson Johnny , Thompson H.
Published: July 2002

For the second order boundary value problem,

y
+
f
(
y
)
=
0

y+f(y)=0

,

0
≤
t
≤
1

0\leq t\leq 1

,

y
(
0
)
=
0
=
y
(
1
)

y(0)=0=y(1)

, where

f
:

R

→
[
0
,

∞
)
,

f:\, \mathbb {R}\rightarrow [0,\,\infty ),

growth conditions are imposed on

f
f

which yield the existence of at least three symmetric positive solutions.
Some Lie superalgebras associated to the Weyl algebras
Authors: Musson Ian
Published: July 2002

Let

g

{\mathfrak {g}}

be the Lie superalgebra

o
s
p
(
1
,
2
r
)

osp(1,2r)

. We show that there is a surjective homomorphism from

U
(

g

)

U({\mathfrak {g}})

to the

r

t
h

r^{th}

Weyl algebra

A

r

A_{r}

, and we use this to construct an analog of the Joseph ideal. We also obtain a decomposition of the adjoint representation of

g

{\mathfrak {g}}

on

A
r

A_r

and use this to show that if

A

r

A_{r}

is made into a Lie superalgebra using its natural

Z

2

{\mathbb Z}_{2}

-grading, then

A

r

=
k
⊕
[

A

r

,

A

r

]

A_{r} = k \oplus [A_{r}, A_{r}]

. In addition, we show that if

[

A
r

,

A
r

]

[A_r, A_r]

and

[

A
s

,

A
s

]

[A_s, A_s]

are isomorphic as Lie superalgebras, then

r
=
s

r=s

. This answers a question of S. Montgomery.
Rates of mixing for potentials of summable variation
Authors: Pollicott Mark
Published: July 2002

It is well known that for subshifts of finite type and equilibrium measures associated to Hölder potentials we have exponential decay of correlations. In this article we derive explicit rates of mixing for equilibrium states associated to more general potentials.
Density of Carmichael numbers with three prime factors
Authors: Balasubramanian R. , Nagaraj S.
Published: July 2002

We get an upper bound of

O
(

x

5

/

14
+
o
(
1
)

)

O(x^{5/14+o(1)})

on the number of Carmichael numbers

≤
x

\leq x

with exactly three prime factors.
Solving constrained Pell equations
Authors: Kedlaya Kiran
Published: July 2002

Consider the system of Diophantine equations

x
2

−
a

y
2

=
b

x^2 - ay^2 = b

,

P
(
x
,
y
)
=

z

2

P(x,y) = z^{2}

, where

P
P

is a given integer polynomial. Historically, such systems have been analyzed by using Baker’s method to produce an upper bound on the integer solutions. We present a general elementary approach, based on an idea of Cohn and the theory of the Pell equation, that solves many such systems. We apply the approach to the cases

P
(
x
,
y
)
=
c

y
2

+
d

P(x, y) = cy^2 + d

and

P
(
x
,
y
)
=
c
x
+
d

P(x, y) = cx + d

, which arise when looking for integer points on an elliptic curve with a rational 2-torsion point.
Mammalian Homolog of Drosophila Tumor Suppressor Lethal (2) Giant Larvae Interacts with Basolateral Exocytic Machinery in Madin-Darby Canine Kidney Cells
Authors: Mu∸sch Anne , Cohen David , Yeaman Charles , Nelson W. James , Rodriguez-Boulan Enrique , Brennwald Patrick J.
Published: July 2002

The Drosophila tumor suppressor protein lethal (2) giant larvae [l(2)gl] is involved in the establishment of epithelial cell polarity during development. Recently, a yeast homolog of the protein has been shown to interact with components of the post-Golgi exocytic machinery and to regulate a late step in protein secretion. Herein, we characterize a mammalian homolog of l(2)gl, called Mlgl, in the epithelial cell line Madin-Darby canine kidney (MDCK). Consistent with a role in cell polarity, Mlgl redistributes from a cytoplasmic localization to the lateral membrane after contact-naive MDCK cells make cell-cell contacts and establish a polarized phenotype. Phosphorylation within a highly conserved region of Mlgl is required to restrict the protein to the lateral domain, because a recombinant phospho-mutant is distributed in a nonpolar manner. Membrane-bound Mlgl from MDCK cell lysates was coimmunoprecipitated with syntaxin 4, a component of the exocytic machinery at the basolateral membrane, but not with other plasma membrane solubleN-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are either absent from or not restricted to the basolateral membrane domain. These data suggest that Mlgl contributes to apico-basolateral polarity by regulating basolateral exocytosis.
IMAGEne I: clustering and ranking of I.M.A.G.E. cDNA clones
corresponding to known genes
Authors: Cariaso M. , Folta P. , Wagner M. , Kuczmarski T. , Lennon G.
Published: July 2002

Motivation: To enhance the usefulness of the I.M.A.G.E. Consortium (Lennon et al. , 1996, Genomics , 33, 151–152) cDNA clone collection by directed analysis and organization of their associated Expressed Sequence Tags (ESTs), thus enabling effective mining of the immense amounts of public cDNA information.

Results: This paper introduces the IMAGEne suite of tools, which clusters ESTs around known genes, then ranks each clone within a cluster. IMAGEne filters data from known gene sequence databases and the GenBank’s EST database (Boguski and Shuler, 1995, Nature Genet., 10, 369–371). It applies biological criteria in connection with judicious use of the BLAST (Altschul et al. , 1990, J. Mol. Biol., 215), FASTA (Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA , 85, 2444–2448); (Pearson, 1990, Methods Enzymol., 183, 63–98); (Gusfield, 1997, Algorithms on Strings, Trees, and Sequences , Cambridge University Press), and SIM (Huang et al. , 1990, Comput. Appl. Biosci., 6, 373–381) tools to form known gene clusters. It then applies criteria derived from experienced biologists to select the best representative I.M.A.G.E. clone for a gene. The tool provides an intuitive Java interface for query and display of the gene and its associated clones, thus directing researchers in selecting a clone that will best enhance their research. An important product is a listing of clones that best represent all known genes. The listing will be used for re-arraying clones into minimally redundant Master Arrays. Both the listings and Master Arrays will be made available to the public, which will be a valuable resource to the genomic community in furthering discovery in the area of gene function.

Availability: IMAGEne can be accessed free of charge through the I.M.A.G.E. Consortium web page at http://bbrp.llnl.gov/image/image.html

Contact: folta2@llnl.gov
Ca2+‐activated Cl− channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion
Authors: ZSEMBERY Ákos , Strazzabosco Mario , Graf JÜrg
Published: July 2002

This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC‐1) and CFTR‐deficient (CFPAC‐1; ΔF508 mutation) cell lines and measured HCO3− extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC‐1 cells, cAMP causes parallel activation of Cl− channels and of HCO3− extrusion by DIDS‐sensitive and Na+‐independent Cl‐/HCO3− exchange, both effects being inhibited by Cl− channel blockers NPPB and glibenclamide. 2) In CFPAC‐1 cells, cAMP fails to stimulate Cl−/ HCO3− exchange and Cl− channels, except after promoting surface expression of ΔF508‐CFTR by glycerol treatment. Instead, raising intracellular Ca2+ concentration to 1 μmol/l or stimulating purinergic receptors with ATP (10 and 100 μmol/l) leads to parallel activation of Cl− channels and HCO3− extrusion. 3) K+ channel function is required for coupling cAMP‐ and Ca2+‐dependent Cl− channel activation to effective stimulation of Cl−/HCO3− exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl−/ HCO3− exchange is directly correlated to activation of apical membrane Cl− channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP‐activated Cl− channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca2+‐dependent Cl− channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.—Zsembery, A., Strazzabosco, M., Graf, J. Ca2+‐activated Cl− channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion. FASEB J. 14, 2345–2356 (2000)
Vector rational interpolation algorithms of Burlisch-Stoer-Neville form
Authors: Sweatman Winston L.
Published: July 2002

We present algorithms of the Bulirsch–Stoer–Neville form for calculating the value of the vector rational interpolant. The derivation parallels that of the Bulirsch–Stoer algorithm for the usual scalar rational interpolation algorithm of Stoer and Bulirsch, using the Clifford algebra foundations for vector rational interpolants of Roberts.
Norepinephrine exocytosis stimulated by α–latrotoxin requires both external and stored Ca
2+
and is mediated by latrophilin, G proteins and phospholipase C
Authors: Rahman M. Atiqur , Ashton Anthony C. , Meunier Frédéric A. , Davletov Bazbek A. , Oliver Dolly J. , Ushkaryov Yuri A.
Published: July 2002

α–latrotoxin (LTX) stimulates massive release of neurotransmitters by binding to a heptahelical transmembrane protein, latrophilin. Our experiments demonstrate that latrophilin is a G–protein–coupled receptor that specifically associates with heterotrimeric G proteins. The latrophilin–G protein complex is very stable in the presence of GDP but dissociates when incubated with GTP, suggesting a functional interaction. As revealed by immunostaining, latrophilin interacts with Gα
q/11
and Gα
o
but not with Gα
s
, Gα
i
or Gα
z
, indicating that this receptor may couple to several G proteins but it is not promiscuous. The mechanisms underlying LTX–evoked norepinephrine secretion from rat brain nerve terminals were also studied. In the presence of extracellular Ca
2+
, LTX triggers vesicular exocytosis because botulinum neurotoxins E, C1 or tetanus toxin inhibit the Ca
2+
–dependent component of the toxin–evoked release. Based on (i) the known involvement of Gα
q
in the regulation of inositol–1,4,5–triphosphate generation and (ii) the requirement of Ca
2+
in LTX action, we tested the effect of inhibitors of Ca
2+
mobilization on the toxin–evoked norepinephrine release. It was found that aminosteroid U73122, which inhibits the coupling of G proteins to phospholipase C, blocks the Ca
2+
–dependent toxin's action. Thapsigargin, which depletes intracellular Ca
2+
stores, also potently decreases the effect of LTX in the presence of extracellular Ca
2+
. On the other hand, clostridial neurotoxins or drugs interfering with Ca
2+
metabolism do not inhibit the Ca
2+
–independent component of LTX–stimulated release. In the absence of Ca
2+
, the toxin induces in the presynaptic membrane non–selective pores permeable to small fluorescent dyes; these pores may allow efflux of neurotransmitters from the cytoplasm. Our results suggest that LTX stimulates norepinephrine exocytosis only in the presence of external Ca
2+
provided intracellular Ca
2+
stores are unperturbed and that latrophilin, G proteins and phospholipase C may mediate the mobilization of stored Ca
2+
, which then triggers secretion.
Transgenic models of Huntington'sdisease
Authors: Sathasivam Kirupa , Hobbs Carl , Mangiarini Laura , Mahal Amarbirpal , Turmaine Mark , Doherty Pat , Davies Stephen W. , Bates Gillian P.
Published: July 2002

Huntington'sdisease (HD) is an inherited neurodegenerative disorder caused by a CAG–polyglutamine repeat expansion. A mouse model of this disease has been generated by the introduction of exon 1 of the human HD gene carrying highly expanded CAG repeats into the mouse germ line (R6 lines). Transgenic mice develop a progressive neurological phenotype with a movement disorder and weight loss similar to that in HD. We have previously identified neuronal inclusions in the brains of these mice that have subsequently been established as the pathological hallmark of polyglutamine disease. Inclusions are present before symptoms, which in turn occur long before any selective neuronal cell death can be identified. We have extended the search for inclusions to skeletal muscle, which, like brain, contains terminally differentiated cells. We have conducted an investigation into the skeletal muscle atrophy that occurs in the R6 lines, (i) to provide possible insights into the muscle bulk loss observed in HD patients, and (ii) to conduct a parallel analysis into the consequence of inclusion formation to that being performed in brain. The identification of inclusions in skeletal muscle might be additionally useful in monitoring the ability of drugs to prevent inclusion formation
in vivo
.
Effects of nitrogen and phosphorus fertilization in a lowland evergreen rainforest
Authors: Mirmanto E. , Proctor J. , Green J. , Nagy L. , Suriantata
Published: July 2002

A nutrient addition experiment was set up in August 1993 in a species–rich primary lowland dipterocarp forest in Barito Ulu, Central Kalimantan, Indonesia. The following treatments were applied: control, +N, +P and +NP. There were five blocks of four 50 m times 50 m plots with a separate treatment for each plot. Fine litterfall was measured on all the plots from l May 1994 for 12 months. Litterfall mass and phosphorus concentrations were significantly higher in all the fertilizer treatments compared with the controls. All trees (greater than or equal to 10 cm dbh) were measured in August 1993 and in August 1998, and there was no significant girth increment response to fertilization in dipterocarps or non–dipterocarps. Dipterocarps of the red meranti group showed a doubling of girth increment in the +NP treatment, however, the difference from the control fell short of significance.
The origin and early evolution of tracheids in vascular plants: integration of palaeobotanical and neobotanical data
Authors: Friedman William E. , Cook Martha E.
Published: July 2002

Although there is clear evidence for the establishment of terrestrial plant life by the end of the Ordovician, the fossil record indicates that land plants remained extremely small and structurally simple until the Late Silurian. Among the events associated with this first major radiation of land plants is the evolution of tracheids, complex water–conducting cells defined by the presence of lignified secondary cell wall thickenings. Recent palaeobotanical analyses indicate that Early Devonian tracheids appear to possess secondary cell wall thickenings composed of two distinct layers: a degradation–prone layer adjacent to the primary cell wall and a degradation–resistant (possibly lignified) layer next to the cell lumen. In order to understand better the early evolution of tracheids, developmental and comparative studies of key basal (and potentially plesiomorphic) extant vascular plants have been initiated. Ultra–structural analysis and enzyme degradation studies of wall structure (to approximate diagenetic alterations of fossil tracheid structure) have been conducted on basal members of each of the two major clades of extant vascular plants:
Huperzia
(Lycophytina) and
Equisetum
(Euphyllophytina). This research demonstrates that secondary cell walls of extant basal vascular plants include a degradation–prone layer (‘template layer’) and a degradation–resistant layer (‘resistant layer’). This pattern of secondary cell wall formation in the water–conducting cells of extant vascular plants matches the pattern of wall thickenings in the tracheids of early fossil vascular plants and provides a key evolutionary link between tracheids of living vascular plants and those of their earliest fossil ancestors. Further studies of tracheid development and structure among basal extant vascular plants will lead to a more precise reconstruction of the early evolution of water–conducting tissues in land plants, and will add to the current limited knowledge of spatial, temporal and cytochemical aspects of cell wall formation in tracheary elements of vascular plants.
Thermodynamics of interactions of urea and guanidinium salts with protein surface: Relationship between solute effects on protein processes and changes in water‐accessible surface area
Authors: Courtenay Elizabeth S. , Capp Michael W. , Record M. Thomas
Published: July 2002

To interpret effects of urea and guanidinium (GuH+) salts on processes that involve large changes in protein water‐accessible surface area (ASA), and to predict these effects from structural information, a thermodynamic characterization of the interactions of these solutes with different types of protein surface is required. In the present work we quantify the interactions of urea, GuHCl, GuHSCN, and, for comparison, KCl with native bovine serum albumin (BSA) surface, using vapor pressure osmometry (VPO) to obtain preferential interaction coefficients (Γμ3) as functions of nondenaturing concentrations of these solutes (0–1 molal). From analysis of Γμ3 using the local‐bulk domain model, we obtain concentration‐independent partition coefficients KnatP that characterize the accumulation of these solutes near native protein (BSA) surface: KnatP,urea= 1.10 ± 0.04, Knat = 2.4 ± 0.2, Knat = 1.60 ± 0.08, relative to Knat ≡ 1 and Knat = 1.0 ± 0.08. The relative magnitudes of KnatP are consistent with the relative effectiveness of these solutes as perturbants of protein processes. From a comparison of partition coefficients for these solutes and native surface (KnatP) with those determined by us previously for unfolded protein and alanine‐based peptide surface KunfP, we dissect KP into contributions from polar peptide backbone and other types of protein surface. For globular protein‐urea interactions, we find KnatP,urea = KunfP,urea. We propose that this equality arises because polar peptide backbone is the same fraction (0.13) of total ASA for both classes of surface. The analysis presented here quantifies and provides a physical basis for understanding Hofmeister effects of salt ions and the effects of uncharged solutes on protein processes in terms of KP and the change in protein ASA.
A model for the lateral penumbra in water of a 200‐MeV proton beam devoted to clinical applications
Authors: Oozeer R. , Mazal A. , Rosenwald J. C. , Belshi R. , Nauraye C. , Ferrand R. , Biensan S.
Published: July 2002

An experimental approach for modeling the lateral penumbra of a proton beam has been investigated. Measurements were made with a silicon diode in a water tank. Several geometrical configurations (phantom position, collimator‐to‐surface distance, collimator diameter, bolus thickness, air gap, etc.) and beam characteristics (range, modulation, etc.) have been studied. The results show that the lateral penumbra is almost independent of the beam modulation and the diameter of the collimator. The use of scaled variables for depth and penumbra allows us to represent the increase in penumbra with depth for any configuration with a second order polynomial function, provided that the penumbra at the entrance of the medium and at the depth of the range are known.