bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutations in the organic cation/carnitine transporter OCTN2 in primary carnitine deficiency
Authors: Wang Yuhuan , Ye Jing , Ganapathy Vadivel , Longo Nicola
Published: July 2002

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease presents early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. The gene for this condition maps to 5q31.2–32 and OCTN2, an organic cation/carnitine transporter, also maps to the same chromosomal region. Here we test the causative role of OCTN2 in primary carnitine deficiency by searching for mutations in this gene in affected patients. Fibroblasts from patients with primary carnitine deficiency lacked mediated carnitine transport. Transfection of patient’s fibroblasts with the OCTN2 cDNA partially restored carnitine transport. Sequencing of the OCTN2 gene revealed different mutations in two unrelated patients. The first patient was homozygous (and both parents heterozygous) for a single base pair substitution converting the codon for Arg-282 to a STOP codon (R282X). The second patient was a compound heterozygote for a paternal 1-bp insertion producing a STOP codon (Y401X) and a maternal 1-bp deletion that produced a frameshift creating a subsequent STOP codon (458X). These mutations decreased the levels of mature OCTN2 mRNA and resulted in nonfunctional transporters, confirming that defects in the organic cation/carnitine transporter OCTN2 are responsible for primary carnitine deficiency.
In situ
atomic force microscopy study of Alzheimer’s β-amyloid peptide on different substrates: New insights into mechanism of β-sheet formation
Authors: Kowalewski Tomasz , Holtzman David M.
Published: July 2002

We have applied
in situ
atomic force microscopy to directly observe the aggregation of Alzheimer’s β-amyloid peptide (Aβ) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10–500 μM solutions of Aβ in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Aβ was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Aβ aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Aβ formed particulate, pseudomicellar aggregates, which at higher Aβ concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Aβ formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of β-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Aβ were oriented along three directions at 120° to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of β-sheets in comparison with α-helices. These studies show that
in situ
atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Aβ fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles
in vivo
.
Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis
Authors: Srivastava Rakesh K. , Mi Qing-Sheng , Hardwick J. Marie , Longo Dan L.
Published: July 2002

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the α1 and α2 helices (Δloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Δloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K→R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.
A single point mutation in the pore region of the epithelial Na
+
channel changes ion selectivity by modifying molecular sieving
Authors: Kellenberger Stephan , Gautschi Ivan , Schild Laurent
Published: July 2002

The epithelial Na
+
channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na
+
transport, mechanotransduction, and neurotransmission. The role of ENaC in Na
+
homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC β and γ subunits causing hypertension. The function of ENaC in Na
+
reabsorption depends critically on its ability to discriminate between Na
+
and other ions like K
+
or Ca
2+
. ENaC is virtually impermeant to K
+
ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC α subunit (αS589), which when mutated allows larger ions such as K
+
, Rb
+
, Cs
+
, and divalent cations to pass through the channel. The relative ion permeability of each of the αS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that αS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na
+
and Li
+
ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.
Direct observation of the (forbidden) S
1
state in carotenoids
Authors: Polívka Tomáš , Herek Jennifer L. , Zigmantas Donatas , Åkerlund Hans-Erik , Sundström Villy
Published: July 2002

Carotenoids are involved in a variety of biological functions, yet the underlying mechanisms are poorly understood, in part because of the long-standing difficulty in assigning the location of the first excited (S
1
) state. Here, we present a method for determining the energy of the forbidden S
1
state, on the basis of ultrafast spectroscopy of the short lived level. Femtosecond transient absorption spectra and kinetics of the S
1
→ S
2
transition revealed the location of the intermediate level in two carotenoid species involved in the xanthophyll cycle, zeaxanthin and violaxanthin, and yielded surprising implications regarding the mechanism of photoregulation in photosynthesis.
Analysis of microsatellite mutations in the mitochondrial DNA of
Saccharomyces cerevisiae
Authors: Sia Elaine Ayres , Butler Christine A. , Dominska Margaret , Greenwell Patricia , Fox Thomas D. , Petes Thomas D.
Published: July 2002

In the nuclear genome of
Saccharomyces cerevisiae
, simple, repetitive DNA sequences (microsatellites) mutate at rates much higher than nonrepetitive sequences. Most of these mutations are deletions or additions of repeat units. The yeast mitochondrial genome also contains many microsatellites. To examine the stability of these sequences, we constructed a reporter gene (
arg8
m
) containing out-of-frame insertions of either poly(AT) or poly(GT) tracts within the coding sequence. Yeast strains with this reporter gene inserted within the mitochondrial genome were constructed. Using these strains, we showed that poly(GT) tracts were considerably less stable than poly(AT) tracts and that alterations usually involved deletions rather than additions of repeat units. In contrast, in the nuclear genome, poly(GT) and poly(AT) tracts had similar stabilities, and alterations usually involved additions rather than deletions. Poly(GT) tracts were more stable in the mitochondria of diploid cells than in haploids. In addition, an
msh1
mutation destabilized poly(GT) tracts in the mitochondrial genome.
The oncogenic LIM-only transcription factor Lmo2 regulates angiogenesis but not vasculogenesis in mice
Authors: Yamada Yoshihiro , Pannell Richard , Forster Alan , Rabbitts Terence H.
Published: July 2002

The
LMO2
gene is activated by chromosomal translocations in human T cell acute leukemias, but in mouse embryogenesis, Lmo2 is essential for initiation of yolk sac and definitive hematopoiesis. The LMO2 protein comprises two LIM–zinc-finger-like protein interaction modules and functions by interaction with specific partners in DNA-binding transcription complexes. We have now investigated the role of Lmo2-associated transcription complexes in the formation of the vascular system by following the fate of
Lmo2
-null embryonic stem (ES) cells in mouse chimeras. Lmo2 is expressed in vascular endothelium, and
Lmo2
-null ES cells contributed to the capillary network normally until around embryonic day 9. However, after this time, marked disorganization of the vascular system was observed in those chimeric mice that have a high contribution of
Lmo2
-null ES cells. Moreover,
Lmo2
-null ES cells do not contribute to endothelial cells of large vessel walls of surviving chimeric mice after embryonic day 10. These results show that Lmo2 is not needed for
de novo
capillary formation from mesoderm but is necessary for angiogenic remodeling of the existing capillary network into mature vasculature. Thus, Lmo2-mediated transcription complexes not only regulate distinct phases of hematopoiesis but also angiogenesis, presumably by Lmo2 interacting with distinct partners in the different settings.
Substitutions in the aspartate transcarbamoylase domain of hamster CAD disrupt oligomeric structure
Authors: Qiu Yu , Davidson Jeffrey N.
Published: July 2002

Aspartate transcarbamoylase (ATCase; EC
2.1.3.2
) is one of three enzymatic domains of CAD, a protein whose native structure is usually a hexamer of identical subunits. Alanine substitutions for the ATCase residues Asp-90 and Arg-269 were generated in a bicistronic vector that encodes a 6-histidine-tagged hamster CAD. Stably transfected mammalian cells expressing high levels of CAD were easily isolated and CAD purification was simplified over previous procedures. The substitutions reduce the ATCase
V
max
of the altered CADs by 11-fold and 46-fold, respectively, as well as affect the enzyme's affinity for aspartate. At 25 mM Mg
2+
, these substitutions cause the oligomeric CAD to dissociate into monomers. Under the same dissociating conditions, incubating the altered CAD with the ATCase substrate carbamoyl phosphate or the bisubstrate analogue
N-
phosphonacetyl-
l
-aspartate unexpectedly leads to the reformation of hexamers. Incubation with the other ATCase substrate, aspartate, has no effect. These results demonstrate that the ATCase domain is central to hexamer formation in CAD and suggest that the ATCase reaction mechanism is ordered in the same manner as the
Escherichia coli
ATCase. Finally, the data indicate that the binding of carbamoyl phosphate induces conformational changes that enhance the interaction of CAD subunits.
Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43
Authors: Rash J. E. , Staines W. A. , Yasumura T. , Patel D. , Furman C. S. , Stelmack G. L. , Nagy J. I.
Published: July 2002

Physiological and ultrastructural evidence indicates that gap junctions link many classes of neurons in mammalian central nervous system (CNS), allowing direct electrical and metabolic communication. Among at least six gap junction-forming connexin proteins in adult rat brain, connexin- (Cx) 32, Cx36, and Cx43 have been reported to occur in neurons. However, no connexin has been documented at ultrastructurally defined neuronal gap junctions. To address this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular and regional localization of Cx36 in rat brain and spinal cord. Three antibodies were generated against different sequences in Cx36. By Western blotting, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx36 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive, spinal cord, and retina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43. Conversely, Cx43 but not Cx36 was detected in astrocyte and ependymocyte gap junctions. In >250 Cx32/Cx43 single- and double-labeled replicas from 10 CNS regions, no neuronal gap junctions were labeled for either Cx32 or Cx43. Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF, Cx36 labeling was widely distributed in neuropil, including along dendritic processes and within neuronal somata. On the basis of FRIL identification of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.
Genetically modified anthrax lethal toxin safely delivers whole HIV protein antigens into the cytosol to induce T cell immunity
Authors: Lu Yichen , Friedman Rachel , Kushner Nicholas , Doling Amy , Thomas Lawrence , Touzjian Neal , Starnbach Michael , Lieberman Judy
Published: July 2002

Bacillus anthraxlethal toxin can be engineered to deliver foreign proteins to the cytosol for antigen presentation to CD8 T cells. Vaccination with modified toxins carrying 8–9 amino acid peptide epitopes induces protective immunity in mice. To evaluate whether large protein antigens can be used with this system, recombinant constructs encoding several HIV antigens up to 500 amino acids were produced. These candidate HIV vaccines are safe in animals and induce CD8 T cells in mice. Constructs encoding gag p24 and nef stimulate gag-specific CD4 proliferation and a secondary cytotoxic T lymphocyte response in HIV-infected donor peripheral blood mononuclear cellsin vitro. These results lay the foundation for future clinical vaccine studies.
Genetic footprinting with
mariner
-based transposition in
Pseudomonas
aeruginosa
Authors: Wong Sandy M. , Mekalanos John J.
Published: July 2002

The complete DNA sequence of
Pseudomonas aeruginosa
provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in
P. aeruginosa
. Use of a highly efficient
in vivo
mariner transposition system in
P. aeruginosa
facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-
Sce
I homing endonuclease in conjunction with
in vitro
mariner mutagenesis to generate mutants within targeted regions of the
P. aeruginosa
chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed
in vivo
transposition systems. We tested this system with three genes in
P. aeruginosa
that have putative essential homologs in
Haemophilus influenzae
. We show that one of three
H. influenzae
essential gene homologs is needed for growth in
P. aeruginosa
, validating the practicality of this comparative genomics strategy to identify essential genes in
P. aeruginosa
.
A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes
Authors: Madin Kairat , Sawasaki Tatsuya , Ogasawara Tomio , Endo Yaeta
Published: July 2002

Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1–4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.
Extracellular protein kinase A as a cancer biomarker: Its expression by tumor cells and reversal by a myristate-lacking Cα and RIIβ subunit overexpression
Authors: Cho Yee Sook , Park Yun Gyu , Lee Youl Nam , Kim Meyoung-Kon , Bates Susan , Tan Langzhu , Cho-Chung Yoon S.
Published: July 2002

Overexpression of cAMP-dependent protein kinase (PKA) type I isozyme is associated with cell proliferation and neoplastic transformation. The presence of PKA on the external surface of LS-174T human colon carcinoma cells has been shown. Here, we show that cancer cells of various cell types excrete PKA into the conditioned medium. This extracellular PKA (ECPKA) is present in active, free catalytic subunit (C subunit) form, and its activity is specifically inhibited by PKA inhibitory protein, PKI. Overexpression of the Cα or RIα subunit gene of PKA in an expression vector, which up-regulates intracellular PKA type I, markedly up-regulates ECPKA expression. In contrast, overexpression of the RIIβ subunit, which eliminates PKA type I, up-regulates PKA type II, and reverts the transformed phenotype, down-regulates ECPKA. A mutation in the Cα gene that prevents myristylation allows the intracellular PKA up-regulation but blocks the ECPKA increase, suggesting that the NH
2
-terminal myristyl group of Cα is required for the ECPKA expression. In serum of cancer patients, the ECPKA expression is up-regulated 10-fold as compared with normal serum. These results indicate that the ECPKA expression is an ordered cellular response of a living cell to actively exclude excess intracellular PKA molecules from the cell. This phenomenon is up-regulated in tumor cells and has an inverse relationship with the hormone dependency of breast cancer. Thus, the extracellular PKA may serve as a potential diagnostic and prognostic marker for cancer.
Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of
Escherichia coli
Authors: Kadokura Hiroshi , Bader Martin , Tian Hongping , Bardwell James C. A. , Beckwith Jon
Published: July 2002

The active-site cysteines of DsbA, the periplasmic
disulfide-bond-forming enzyme of
Escherichia coli
, are
kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn,
is oxidized by two kinds of quinones (ubiquinone for aerobic and
menaquinone for anaerobic growth) in the electron-transport chain. We
describe the isolation of
dsbB
missense mutations that
change a highly conserved arginine residue at position 48 to histidine
or cysteine. In these mutants, DsbB functions reasonably well
aerobically but poorly anaerobically. Consistent with this conditional
phenotype, purified R48H exhibits very low activity with menaquinone
and an apparent Michaelis constant (
K
m
) for
ubiquinone seven times greater than that of the wild-type DsbB, while
keeping an apparent
K
m
for DsbA similar to
that of wild-type enzyme. From these results, we propose that this
highly conserved arginine residue of DsbB plays an important role in
the catalysis of disulfide bond formation through its role in the
interaction of DsbB with quinones.
Peptide nucleic acids rather than RNA may have been the first genetic molecule
Authors: Nelson Kevin E. , Levy Matthew , Miller Stanley L.
Published: July 2002

Numerous problems exist with the current thinking of RNA as the first genetic material. No plausible prebiotic processes have yet been demonstrated to produce the nucleosides or nucleotides or for efficient two-way nonenzymatic replication. Peptide nucleic acid (PNA) is a promising precursor to RNA, consisting of
N
-(2-aminoethyl)glycine (AEG) and the adenine, uracil, guanine, and cytosine-
N
-acetic acids. However, PNA has not yet been demonstrated to be prebiotic. We show here that AEG is produced directly in electric discharge reactions from CH
4
, N
2
, NH
3
, and H
2
O. Electric discharges also produce ethylenediamine, as do NH
4
CN polymerizations. AEG is produced from the robust Strecker synthesis with ethylenediamine. The NH
4
CN polymerization in the presence of glycine leads to the adenine and guanine-
N
9
-acetic acids, and the cytosine and uracil-
N
1
-acetic acids are produced in high yield from the reaction of cyanoacetaldehyde with hydantoic acid, rather than urea. Preliminary experiments suggest that AEG may polymerize rapidly at 100°C to give the polypeptide backbone of PNA. The ease of synthesis of the components of PNA and possibility of polymerization of AEG reinforce the possibility that PNA may have been the first genetic material.
Attractin/Mahogany/Zitter plays a critical role in myelination of the central nervous system
Authors: Kuramoto Takashi , Kitada Kazuhiro , Inui Toshihide , Sasaki Yoshifumi , Ito Kazumi , Hase Takao , Kawagachi Saburo , Ogawa Yoshihiro , Nakao Kazuwa , Barsh Gregory S. , Nagao Minako , Ushijima Toshikazu , Serikawa Tadao
Published: July 2002

The rat zitter (
zi
) mutation induces hypomyelination
and vacuolation in the central nervous system (CNS), which result in
early-onset tremor and progressive flaccid paresis. By positional
cloning, we found a marked decrease in Attractin (
Atrn
)
mRNA in the brain of the
zi
/
zi
rat and
identified
zi
as an 8-bp deletion at a splice donor site
of
Atrn. Atrn
has been known to play multiple roles in
regulating physiological processes that are involved in monocyte–T
cell interaction,
agouti
-related hair pigmentation, and
control of energy homeostasis. Rat
Atrn
gene encoded two
isoforms, a secreted and a membrane form, as a result of alternative
splicing. The
zi
mutation at the
Atrn
locus darkened coat color when introduced into agouti rats, as also
described in mahogany (
mg
) mice, carrying the homozygous
mutation at the
Atrn
locus. Transgenic rescue
experiments showed that the membrane-type
Atrn
complemented both neurological alteration and abnormal pigmentation in
zi
/
zi
rats, but that the secreted-type
Atrn
complemented neither mutant phenotype. Furthermore,
we discovered that
mg
mice exhibited hypomyelination and
vacuolation in the CNS associated with body tremor. We conclude from
these results that the membrane
Atrn
has a critical role
in normal myelination in the CNS and would provide insights into the
physiology of myelination as well as the etiology of myelin diseases.
Polar side chains drive the association of model transmembrane peptides
Authors: Gratkowski Holly , Lear James D. , DeGrado William F.
Published: July 2002

The forces stabilizing the three-dimensional structures of membrane
proteins are currently not well understood. Previously, it was shown
that a single Asn side chain in a transmembrane segment can mediate the
dimerization and trimerization of a variety of hydrophobic helices.
Here, we examine the tendencies of a representative set of amino acids
(Asn, Gln, Asp, Glu, Lys, Ala, Val, Leu, Ser, Thr) to direct the
oligomerization of a model transmembrane helix. The model peptide is
entirely hydrophobic throughout a 20-residue segment and contains a
single central site for the introduction of various amino acid
“guests.” Analytical ultracentrifugation and gel electrophoresis
were used to determine the stoichiometry and free energy of association
of the entire set of peptides within micelles. Variants with two polar
atoms at the guest site—Asn, Gln, Asp, and Glu—formed stable trimers,
whereas residues with one or fewer polar atoms showed a much weaker
tendency to associate. The data are examined in light of the
frequencies of occurrence of various amino acid side chains in membrane
proteins and provide insight into the role of polar interactions in
directing transmembrane helix association. These data also suggest an
approach to the design of variants of natural single-span transmembrane
proteins with various potentials to associate in the bilayer.
A human myeloma cell line suitable for the generation of human monoclonal antibodies
Authors: Karpas Abraham , Dremucheva Alla , Czepulkowski Barbara H.
Published: July 2002

Ever since monoclonal antibodies were produced in 1975 with mouse
myeloma cells there has been interest in developing human myeloma
cultures for the production of monoclonal antibodies. However, despite
multiple attempts, no human myeloma line suitable for hybridoma
production has been described. Here we report the derivation of a
hypoxanthine–aminopterin–thymidine-sensitive and ouabain-resistant
human myeloma cell line (Karpas 707H) that contains unique genetic
markers. We show that this line is useful for the generation of stable
human hybridomas. It can easily be fused with ouabain-sensitive
Epstein–Barr virus-transformed cells as well as with fresh tonsil
and blood lymphocytes, giving rise to stable hybrids that continuously
secrete very large quantities of human immunoglobulins. The derived
hybrids do not lose immunoglobulin secretion over many months of
continuous growth. The availability of this cell line should enable the
in vitro
immortalization of human antibody-producing B
cells that are formed
in vivo
. The monoclonal antibodies
produced may have advantages in immunotherapy.
Serum levels of pro‐ and anti‐inflammatory cytokines in non‐pregnant women, during pregnancy, labour and abortion
Authors: Vassiliadis S. , Ranella A. , Papadimitriou L. , Makrygiannakis A. , Athanassakis I.
Published: July 2002

Disturbance of the cytokine equilibrium has been accused for many pathological disorders. Microbial infections, autoimmune diseases, graft rejection have been correlated to over‐ or under‐production of specific cytokines which are produced as responder molecules to the various immune stimuli. The sole naturally occurring immune reaction in the organism is developed during the gestational period where, despite the presence of a semi‐allogeneic graft, maternal immunoreactivity is driven to support fetal growth. The successful embryo development has been attributed to the important intervention of cytokines where some have been characterized as indispensable and others deleterious to fetal growth. However, the physiological levels of many factors during the gestational process have not been determined. Thus, in the present study we have measured and established the values of IL‐1α, IL‐2, IL‐3, IL‐4, IL‐6, IL‐10, IL‐12, GM‐CSF, TNF‐ α and IFN‐γ during all phases of human pregnancy (first, second and third trimester of pregnancy, labour, abortions of the first trimester) as well as in the non‐pregnant control state. This is an attempt to assess serum protein concentrations and present the physiological levels of these cytokines at certain time intervals providing thus a diagnostic advantage in pregnancy cases where the mother cannot immunologically support the fetus. Exploitation of this knowledge and further research may be useful for therapeutic interventions in the future.
IRES Elements in Connexin Genes: A Hypothesis Explaining the Need for Connexins to Be Regulated at the Translational Level
Authors: Werner Rudolf
Published: July 2002

The fact that stem cells do not express gap junctions while most differentiated cells do suggests that gap junctions serve a vital function in the maintenance of the differentiated state. This would require that connexin genes be expressed even in conditions where cap‐mediated translation is impaired. It is hypothesized that IRES elements in the 5'‐UTRs of the mRNAs of most connexins are required for this purpose.
Architecture and Function of the Human Endonucleases RNase P and RNase MRP
Authors: Van Eenennaam Hans , Jarrous Nayef , van Venrooij Walther J. , Pruijn Ger J. M.
Published: July 2002

In the past decade, important advances have been made in our knowledge of the composition of human RNase MRP and RNase P complexes. Both ribonucleoprotein particles function as endonucleases and contain RNA components that are structurally related. RNase MRP has been suggested to be involved in the processing of precursor rRNA; RNase P, in the maturation of tRNA. Here we give an overview of current data on the structure and function of human RNase MRP and RNase P particles, with emphasis on their molecular composition. At present, seven protein subunits, probably all associated with both ribonucleoprotein particles, have been isolated and their corresponding cDNAs cloned. Although no known structural motifs can be identified in the amino acid sequences of these proteins, the majority is clearly rich in basic residues. For two protein subunits, a cluster of basic amino acids have been shown to be involved in nucleolar accumulation, whereas another protein, which lacks such a region, probably enters the nucleolus by way of a piggyback mechanism. The binding regions for several of the protein subunits on the RNA have been identified, and the data have been used to create a putative structural model for the RNase MRP particle. The rather obscure situation concerning the association of the autoantigenic Th‐40 protein and its possible relationship with one of the subunits, Rpp38, is discussed.
Newt Myotubes Reenter the Cell Cycle by Phosphorylation of the Retinoblastoma Protein
Authors: Tanaka Elly M. , Gann Alexander A.F. , Gates Phillip B. , Brockes Jeremy P.
Published: July 2002

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb−/−mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467– 1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb−/− mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.
Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells
Authors: IJzendoorn Sven C.D. van , Zegers Mirjam M.P. , Kok Jan Willem , Hoekstra Dick
Published: July 2002

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chainlabeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37°C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer.

The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi–related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.
Tim23, a Protein Import Component of the Mitochondrial Inner Membrane, Is Required for Normal Activity of the Multiple Conductance Channel, MCC
Authors: Lohret Timothy A. , Jensen Robert E. , Kinnally Kathleen W.
Published: July 2002

We previously showed that the conductance of a mitochondrial inner membrane channel, called MCC, was specifically blocked by peptides corresponding to mitochondrial import signals. To determine if MCC plays a role in protein import, we examined the relationship between MCC and Tim23p, a component of the protein import complex of the mitochondrial inner membrane. We find that antibodies against Tim23p, previously shown to inhibit mitochondrial protein import, inhibit MCC activity. We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation. In contrast to wild-type MCC, we find that the conductance of MCC from the tim23-1 mutant is not significantly blocked by mitochondrial presequence peptides. Tim23 antibodies and the tim23-1 mutation do not, however, alter the activity of PSC, a presequence-peptide sensitive channel in the mitochondrial outer membrane. Our results show that Tim23p is required for normal MCC activity and raise the possibility that precursors are translocated across the inner membrane through the pore of MCC.
Type IV Collagen Is Detectable in Most, but Not All, Basement Membranes of Caenorhabditis elegans and Assembles on Tissues That Do Not Express It
Authors: Graham Patricia L. , Johnson Jeffrey J. , Wang Shaoru , Sibley Marion H. , Gupta Malini C. , Kramer James M.
Published: July 2002

Type IV collagen in Caenorhabditis elegans is produced by two essential genes, emb-9 and let-2, which encode α1- and α2-like chains, respectively. The distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is detected in all basement membranes except those on the pseudocoelomic face of body wall muscle and on the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were shown to be expressed in the same cells, primarily body wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with basement membranes that contain type IV collagen, these tissues do not express either type IV collagen gene. Using an epitope-tagged emb-9 construct, we show that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes. Additionally, we show that expression of functional type IV collagen only in body wall muscle cells is sufficient for C. elegans to complete development and be partially fertile. Since type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface–associated molecule(s) is required to facilitate type IV collagen assembly.
CDO: An Oncogene-, Serum-, and Anchorage-regulated Member of the Ig/Fibronectin Type III Repeat Family
Authors: Kang Jong-Sun , Gao Min , Feinleib Jessica L. , Cotter Philip D. , Guadagno Sarah N. , Krauss Robert S.
Published: July 2002

Cell adhesion molecules of the Ig superfamily are implicated in a wide variety of biological processes, including cell migration, axon guidance and fasciculation, and growth control and tumorigenesis. Expression of these proteins can be highly dynamic and cell type specific, but little is known of the signals that regulate such specificity. Reported here is the molecular cloning and characterization of rat CDO, a novel cell surface glycoprotein of the Ig superfamily that contains five Ig-like repeats, followed by three fibronectin type III–like repeats in its extracellular region, and a 256-amino acid intracellular region that does not resemble other known proteins. In rat embryo fibroblasts, cdo mRNA expression is maximal in confluent, quiescent cells. It is rapidly and transiently down-regulated by serum stimulation of such cells, and is constitutively down-regulated in oncogene-transformed derivatives of these cells. CDO protein levels are also dramatically regulated by cell–substratum adhesion, via a mechanism that is independent of cdo mRNA expression. The amount of CDO produced at the surface of a cell may therefore be governed by a complex balance of signals, including mitogenic stimuli that regulate cdo mRNA levels, and substratum-derived signals that regulate CDO protein production. cdo mRNA is expressed at low levels in most adult rat tissues. A closely related human gene maps to chromosome 11q23–24, a region that displays frequent loss of heterozygosity in human lung, breast, and ovarian tumors. Taken together, these data suggest that loss of CDO function could play a role in oncogenesis.
The Number and Location of Glycans on Influenza Hemagglutinin Determine Folding and Association with Calnexin and Calreticulin
Authors: Hebert Daniel N. , Zhang Jian-Xin , Chen Wei , Foellmer Brigitte , Helenius Ari
Published: July 2002

Calnexin and calreticulin are homologous molecular chaperones that promote proper folding, oligomeric assembly, and quality control of newly synthesized glycoproteins in the endoplasmic reticulum (ER). Both are lectins that bind to substrate glycoproteins that have monoglucosylated N-linked oligosaccharides. Their binding to newly translated influenza virus hemagglutinin (HA), and various mutants thereof, was analyzed in microsomes after in vitro translation and expression in live CHO cells. A large fraction of the HA molecules was found to occur in ternary HA– calnexin–calreticulin complexes. In contrast to calnexin, calreticulin was found to bind primarily to early folding intermediates. Analysis of HA mutants with different numbers and locations of N-linked glycans showed that although the two chaperones share the same carbohydrate specificity, they display distinct binding properties; calreticulin binding depends on the oligosaccharides in the more rapidly folding top/hinge domain of HA whereas calnexin is less discriminating. Calnexin's binding was reduced if the HA was expressed as a soluble anchor-free protein rather than membrane bound. When the co- and posttranslational folding and trimerization of glycosylation mutants was analyzed, it was observed that removal of stem domain glycans caused accelerated folding whereas removal of the top domain glycans (especially the oligosaccharide attached to Asn81) inhibited folding. In summary, the data established that individual N-linked glycans in HA have distinct roles in calnexin/calreticulin binding and in co- and posttranslational folding.
A Single Immunoglobulin-like Domain of the Human Neural Cell Adhesion Molecule L1 Supports Adhesion by Multiple Vascular and Platelet Integrins
Authors: Felding-Habermann Brunhilde , Silletti Steve , Mei Fang , Siu Chi-Hung , Yip Paul M. , Brooks Peter C. , Cheresh David A. , O'Toole Timothy E. , Ginsberg Mark H. , Montgomery Anthony M.P.
Published: July 2002

The neural cell adhesion molecule L1 has been shown to function as a homophilic ligand in a variety of dynamic neurological processes. Here we demonstrate that the sixth immunoglobulin-like domain of human L1 (L1-Ig6) can function as a heterophilic ligand for multiple members of the integrin superfamily including αvβ3, αvβ1, α5β1, and αIIbβ3. The interaction between L1-Ig6 and αIIbβ3 was found to support the rapid attachment of activated human platelets, whereas a corresponding interaction with αvβ3 and αvβ1 supported the adhesion of umbilical vein endothelial cells. Mutation of the single Arg-Gly-Asp (RGD) motif in human L1-Ig6 effectively abrogated binding by the aforementioned integrins. A L1 peptide containing this RGD motif and corresponding flanking amino acids (PSITWRGDGRDLQEL) effectively blocked L1 integrin interactions and, as an immobilized ligand, supported adhesion via αvβ3, αvβ1, α5β1, and αIIbβ3. Whereas β3 integrin binding to L1-Ig6 was evident in the presence of either Ca2+, Mg2+, or Mn2+, a corresponding interaction with the β1 integrins was only observed in the presence of Mn2+. Furthermore, such Mn2+-dependent binding by α5β1 and αvβ1 was significantly inhibited by exogenous Ca2+. Our findings suggest that physiological levels of calcium will impose a hierarchy of integrin binding to L1 such that αvβ3 or active αIIbβ3 > αvβ1 > α5β1. Given that L1 can interact with multiple vascular or platelet integrins it is significant that we also present evidence for de novo L1 expression on blood vessels associated with certain neoplastic or inflammatory diseases. Together these findings suggest an expanded and novel role for L1 in vascular and thrombogenic processes.
A Calcium Signaling Cascade Essential for Myosin Thick Filament Assembly in Xenopus Myocytes
Authors: Ferrari Michael B. , Ribbeck Katharina , Hagler Donald J. , Spitzer Nicholas C.
Published: July 2002

Spontaneous calcium release from intracellular stores occurs during myofibrillogenesis, the process of sarcomeric protein assembly in striated muscle. Preventing these Ca2+ transients disrupts sarcomere formation, but the signal transduction cascade has not been identified. Here we report that specific blockade of Ca2+ release from the ryanodine receptor (RyR) activated Ca2+ store blocks transients and disrupts myosin thick filament (A band) assembly. Inhibition of an embryonic Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) by blocking the ATP-binding site, by allosteric phosphorylation, or by intracellular delivery of a pseudosubstrate peptide, also disrupts sarcomeric organization. The results indicate that both RyRs and MLCK, which have well-described calcium signaling roles in mature muscle contraction, have essential developmental roles during construction of the contractile apparatus.
ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes
Authors: Ooi Chean Eng , Dell'Angelica Esteban C. , Bonifacino Juan S.
Published: July 2002

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.