bluuebunny/crossref_metadata_2025 huggingface dataset:
Long term trends in the evolution of H(3) HA1 human influenza type A
Authors: Fitch Walter M. , Bush Robin M. , Bender Catherine A. , Cox Nancy J.
Published: July 2002

We have studied the HA1 domain of 254 human influenza A(H3N2) virus
genes for clues that might help identify characteristics of
hemagglutinins (HAs) of circulating strains that are predictive of that
strain’s epidemic potential. Our preliminary findings include the
following. (
i
) The most parsimonious tree found requires
1,260 substitutions of which 712 are silent and 548 are replacement
substitutions. (
ii
) The HA1 portion of the HA gene is
evolving at a rate of 5.7 nucleotide substitutions/year or 5.7
× 10
−3
substitutions/site per year.
(
iii
) The replacement substitutions are distributed
randomly across the three positions of the codon when allowance is made
for the number of ways each codon can change the encoded amino acid.
(
iv
) The replacement substitutions are not distributed
randomly over the branches of the tree, there being 2.2 times more
changes per tip branch than for non-tip branches. This result is
independent of how the virus was amplified (egg grown or kidney cell
grown) prior to sequencing or if sequencing was carried out directly on
the original clinical specimen by PCR. (
v
) These excess
changes on the tip branches are probably the result of a bias in the
choice of strains to sequence and the detection of deleterious
mutations that had not yet been removed by negative selection.
(
vi
) There are six hypervariable codons accumulating
replacement substitutions at an average rate that is 7.2 times that of
the other varied codons. (
vii
) The number of variable
codons in the trunk branches (the winners of the competitive race
against the immune system) is 47 ± 5, significantly fewer than in
the twigs (90 ± 7), which in turn is significantly fewer variable
codons than in tip branches (175 ± 8). (
viii
) A
minimum of one of every 12 branches has nodes at opposite ends
representing viruses that reside on different continents. This is,
however, no more than would be expected if one were to randomly
reassign the continent of origin of the isolates. (
ix
)
Of 99 codons with at least four mutations, 31 have ratios of non-silent
to silent changes with probabilities less than 0.05 of occurring by
chance, and 14 of those have probabilities <0.005. These observations
strongly support positive Darwinian selection. We suggest that the
small number of variable positions along the successful trunk lineage,
together with knowledge of the codons that have shown positive
selection, may provide clues that permit an improved prediction of
which strains will cause epidemics and therefore should be used for
vaccine production.
Mouse model of G
M2
activator deficiency manifests cerebellar pathology and motor impairment
Authors: Liu Yujing , Hoffmann Alexander , Grinberg Alexander , Westphal Heiner , McDonald Michael P. , Miller Katherine M. , Crawley Jacqueline N. , Sandhoff Konrad , Suzuki Kinuko , Proia Richard L.
Published: July 2002

The G
M2
activator deficiency (also known as the AB variant), Tay–Sachs disease, and Sandhoff disease are the major forms of the G
M2
gangliosidoses, disorders caused by defective degradation of G
M2
ganglioside. Tay–Sachs and Sandhoff diseases are caused by mutations in the genes (
HEXA
and
HEXB
) encoding the subunits of β-hexosaminidase A. The G
M2
activator deficiency is caused by mutations in the
GM2A
gene encoding the G
M2
activator protein. For degradation of G
M2
ganglioside by β-hexosamindase A, the G
M2
activator protein must participate by forming a soluble complex with the ganglioside. In each of the disorders, G
M2
ganglioside and related lipids accumulate to pathologic levels in neuronal lysosomes, resulting in clinically similar disorders with an onset in the first year of life, progressive neurodegeneration, and death by early childhood. We previously have described mouse models of Tay–Sachs (
Hexa
−/−) and Sandhoff (
Hexb
−/−) diseases with vastly different clinical phenotypes. The
Hexa
−/− mice were asymptomatic whereas the
Hexb
−/− mice were severely affected. Through gene disruption in embryonic stem cells we now have established a mouse model of the G
M2
activator deficiency that manifests an intermediate phenotype. The
Gm2a
−/− mice demonstrated neuronal storage but only in restricted regions of the brain (piriform, entorhinal cortex, amygdala, and hypothalamic nuclei) reminiscent of the asymptomatic Tay–Sachs model mice. However, unlike the Tay–Sachs mice, the
Gm2a
−/− mice displayed significant storage in the cerebellum and defects in balance and coordination. The abnormal ganglioside storage in the
Gm2a
−/− mice consisted of G
M2
with a low amount of G
A2
. The results demonstrate that the activator protein is required for G
M2
degradation and also may indicate a role for the G
M2
activator in G
A2
degradation.
Variations in frequencies of drug resistance in
Plasmodium falciparum
Authors: Rathod Pradipsinh K. , McErlean Tara , Lee Pei-Chieh
Published: July 2002

Continual exposure of malarial parasite populations to different drugs may have selected not only for resistance to individual drugs but also for genetic traits that favor initiation of resistance to novel unrelated antimalarials. To test this hypothesis, different
Plasmodium falciparum
clones having varying numbers of preexisting resistance mechanisms were treated with two new antimalarial agents: 5-fluoroorotate and atovaquone. All parasite populations were equally susceptible in small numbers. However, when large populations of these clones were challenged with either of the two compounds, significant variations in frequencies of resistance became apparent. On one extreme, clone D6 from West Africa, which was sensitive to all traditional antimalarial agents, failed to develop resistance under simple nonmutagenic conditions
in vitro
. In sharp contrast, the Indochina clone W2, which was known to be resistant to all traditional antimalarial drugs, independently acquired resistance to both new compounds as much as a 1,000 times more frequently than D6. Additional clones that were resistant to some (but not all) traditional antimalarial agents acquired resistance to atovaquone at high frequency, but not to 5-fluoroorotate. These findings were unexpected and surprising based on current views of the evolution of drug resistance in
P. falciparum
populations. Such new phenotypes, named accelerated resistance to multiple drugs (ARMD), raise important questions about the genetic and biochemical mechanisms related to the initiation of drug resistance in malarial parasites. Some potential mechanisms underlying ARMD phenotypes have public health implications that are ominous.
Expression and regulation of the neuropeptide Y Y2 receptor in sensory and autonomic ganglia
Authors: Zhang Xu , Shi Tiejun , Holmberg Kristina , Landry Marc , Huang Wenjin , Xiao Huasheng , Ju Gong , Hökfelt Tomas
Published: July 2002

The Y2 subtype of neuropeptide tyrosine (NPY) receptors (Y2R) and some neuropeptides have been studied with
in situ
hybridization in sensory and autonomic neurons of rat and monkey. Between 10% and 20% of the lumbar dorsal root ganglion (DRG) neuron profiles (NPs) contain Y2R mRNA in the rat and monkey. In rat DRGs Y2R mRNA is expressed in calcitonin gene-related peptide (CGRP)-positive, medium-sized, and large neurons, that is in a complementary fashion to the Y1R that is located in small CGRP neurons. In monkey DRGs Y2R mRNA is expressed mainly in small neurons. Peripheral axotomy up-regulates the Y2R in small and large DRG neurons in both species. Y2R and NPY mRNAs are colocalized in many large neurons in axotomized rat DRGs. Y2R mRNA is expressed in 50% of the NPs in the nodose ganglion with a modest increase after axotomy. Y2R mRNA is detected in a few NPs in normal rat superior cervical ganglia, with a marked increase after transection of the carotid nerves. No Y2R mRNA-positive, but many (≈30%) weakly Y1R mRNA-positive NPs were found in the sphenopalatine ganglion. Finally, Y2R mRNA levels increase in rat spinal motoneurons after axotomy. Thus, under normal circumstances NPY may act on Y1 and Y2Rs expressed, respectively, in small and large CGRP-positive DRG neurons in the rat. Y2R may be an important receptor in the viscero-sensory neurons. Y2Rs may be particularly important after axotomy serving as presynaptic and/or autoreceptors on rat DRG, superior cervical ganglion, and nodose ganglion neurons and as presynaptic receptors in monkey DRG neurons.
Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil
Authors: Fidock David A. , Wellems Thomas E.
Published: July 2002

Increasing resistance of
Plasmodium falciparum
malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the
P. falciparum
bifunctional DHFR–thymidylate synthase enzyme, we have transformed
P. falciparum
with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that
P. falciparum
drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of
P. falciparum
genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.
Cleft palate in mice with a targeted mutation in the γ-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67
Authors: Condie Brian G. , Bain Gerard , Gottlieb David I. , Capecchi Mario R.
Published: July 2002

The functions of neurotransmitters in fetal development are poorly understood. Genetic observations have suggested a role for the inhibitory amino acid neurotransmitter γ-aminobutyric acid (GABA) in the normal development of the mouse palate. Mice homozygous for mutations in the β-3 GABA
A
receptor subunit develop a cleft secondary palate. GABA, the ligand for this receptor, is synthesized by the enzyme glutamic acid decarboxylase. We have disrupted one of the two mouse Gad genes by gene targeting and also find defects in the formation of the palate. The striking similarity in phenotype between the receptor and ligand mutations clearly demonstrates a role for GABA signaling in normal palate development.
A retro-inverso peptide corresponding to the GH loop of foot-and-mouth disease virus elicits high levels of long-lasting protective neutralizing antibodies
Authors: Briand Jean-Paul , Benkirane Nadia , Guichard Gilles , Newman John F. E. , Van Regenmortel Marc H. V. , Brown Fred , Muller Sylviane
Published: July 2002

Peptides corresponding to the immunodominant loop located at residues 135–158 on capsid protein VP1 of foot-and-mouth disease virus (FMDV) generally elicit high levels of anti-peptide and virus-neutralizing antibodies. In some instances, however, the level of neutralizing antibodies is low or even negligible, even though the level of anti-peptide antibodies is high. We have shown previously that the antigenic activity of peptide 141–159 of VP1 of a variant of serotype A can be mimicked by a retro-inverso (all-dretro or retroenantio) peptide analogue. This retro-inverso analogue induced greater and longer-lasting antibody titers than did the correspondingl-peptide. We now show that a single inoculation of the retro-inverso analogue elicits high levels of neutralizing antibodies that persist longer than those induced against the correspondingl-peptide and confer substantial protection in guinea pigs challenged with the cognate virus. In view of the high stability to proteases of retro-inverso peptide analogues and their enhanced immunogenicity, these results have practical relevance in designing potential peptide vaccines.
Dendritic cell ontogeny: A human dendritic cell lineage of myeloid origin
Authors: Olweus Johanna , BitMansour Andrew , Warnke Roger , Thompson Peter A. , Carballido Jose , Picker Louis J. , Lund-Johansen Fridtjof
Published: July 2002

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor α chain (IL-3Rαhi). The CD34+IL-3RαhiDC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cellsin vitro. The IL-3RαhiDC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.
Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide
Authors: Schipani E. , Lanske B. , Hunzelman J. , Luz A. , Kovacs C. S. , Lee K. , Pirro A. , Kronenberg H. M. , Jüppner H.
Published: July 2002

Mice in which the genes encoding the parathyroid hormone (PTH)-related peptide (PTHrP) or the PTH/PTHrP receptor have been ablated by homologous recombination show skeletal dysplasia due to accelerated endochondral bone formation, and die at birth or
in utero
, respectively. Skeletal abnormalities due to decelerated chondrocyte maturation are observed in transgenic mice where PTHrP expression is targeted to the growth plate, and in patients with Jansen metaphyseal chondrodysplasia, a rare genetic disorder caused by constitutively active PTH/PTHrP receptors. These and other findings thus indicate that PTHrP and its receptor are essential for chondrocyte differentiation. To further explore the role of the PTH/PTHrP receptor in this process, we generated transgenic mice in which expression of a constitutively active receptor, HKrk-H223R, was targeted to the growth plate by the rat α1 (II) collagen promoter. Two major goals were pursued: (
i
) to investigate how constitutively active PTH/PTHrP receptors affect the program of chondrocyte maturation; and (
ii
) to determine whether expression of the mutant receptor would correct the severe growth plate abnormalities of PTHrP-ablated mice (PTHrP−/−). The targeted expression of constitutively active PTH/PTHrP receptors led to delayed mineralization, decelerated conversion of proliferative chondrocytes into hypertrophic cells in skeletal segments that are formed by the endochondral process, and prolonged presence of hypertrophic chondrocytes with delay of vascular invasion. Furthermore, it corrected at birth the growth plate abnormalities of PTHrP−/− mice and allowed their prolonged survival. “Rescued” animals lacked tooth eruption and showed premature epiphyseal closure, indicating that both processes involve PTHrP. These findings suggest that rescued PTHrP−/− mice may gain considerable importance for studying the diverse, possibly tissue-specific role(s) of PTHrP in postnatal development.
Midbrain injection of recombinant adeno-associated virus encoding rat glial cell line-derived neurotrophic factor protects nigral neurons in a progressive 6-hydroxydopamine-induced degeneration model of Parkinson’s disease in rats
Authors: Mandel R. J. , Spratt S. K. , Snyder R. O. , Leff S. E.
Published: July 2002

A recombinant adeno-associated virus (rAAV) vector capable of infecting cells and expressing rat glial cell line-derived neurotrophic factor (rGDNF), a putative central nervous system dopaminergic survival factor, under the control of a potent cytomegalovirus (CMV) immediate/early promoter (AAV-MD-rGDNF) was constructed. Two experiments were performed to evaluate the time course of expression of rAAV-mediated GDNF protein expression and to test the vector in an animal model of Parkinson’s disease. To evaluate the ability of rAAV-rGDNF to protect nigral dopaminergic neurons in the progressive Sauer and Oertel 6-hydroxydopamine (6-OHDA) lesion model, rats received perinigral injections of either rAAV-rGDNF virus or rAAV-
lacZ
control virus 3 weeks prior to a striatal 6-OHDA lesion and were sacrificed 4 weeks after 6-OHDA. Cell counts of back-labeled fluorogold-positive neurons in the substantia nigra revealed that rAAV-MD-rGDNF protected a significant number of cells when compared with cell counts of rAAV-CMV-
lacZ
-injected rats (94% vs. 51%, respectively). In close agreement, 85% of tyrosine hydroxylase-positive cells remained in the nigral rAAV-MD-rGDNF group vs. only 49% in the
lacZ
group. A separate group of rats were given identical perinigral virus injections and were sacrificed at 3 and 10 weeks after surgery. Nigral GDNF protein expression remained relatively stable over the 10 weeks investigated. These data indicate that the use of rAAV, a noncytopathic viral vector, can promote delivery of functional levels of GDNF in a degenerative model of Parkinson’s disease.
Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis
Authors: Elmore Lynne W. , Hancock Amy R. , Chang Shau-Feng , Wang Xin W. , Chang Seung , Callahan Christiana P. , Geller David A. , Will Hans , Harris Curtis C.
Published: July 2002

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association.
In vitro
binding studies indicate that the
ayw
and
adr
viral subtypes of HBx bind similar amounts of glutathione
S
-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.
Antisense oligonucleotides against rat brain α
1E
DNA and its atrial homologue decrease T-type calcium current in atrial myocytes
Authors: Piedras-Rentería Erika S. , Chen Chien-Chang , Best Philip M.
Published: July 2002

Low voltage-activated, or T-type, calcium currents are important regulators of neuronal and muscle excitability, secretion, and possibly cell growth and differentiation. The gene (or genes) coding for the pore-forming subunit of low voltage-activated channel proteins has not been unequivocally identified. We have used reverse transcription–PCR to identify partial clones from rat atrial myocytes that share high homology with a member of the E class of calcium channel genes. Antisense oligonucleotides targeting one of these partial clones (raE1) specifically block the increase in T-current density that normally results when atrial myocytes are treated with insulin-like growth factor 1 (IGF-1). Antisense oligonucleotides targeting portions of the neuronal rat α
1E
sequence, which are not part of the clones detected in atrial tissue, also block the IGF-1-induced increase in T-current, suggesting that the high homology to α
1E
seen in the partial clone may be present in the complete atrial sequence. The basal T-current expressed in these cells is also blocked by antisense oligonucleotides, which is consistent with the notion that IGF-1 up-regulates the same gene that encodes the basal current. These results support the hypothesis that a member of the E class of calcium channel genes encodes a low voltage-activated calcium channel in atrial myocytes.
Transcriptional activation of mucin by
Pseudomonas aeruginosa
lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease
Authors: Li Jian-Dong , Dohrman Austin F. , Gallup Marianne , Miyata Susumu , Gum James R. , Kim Young S. , Nadel Jay A. , Prince Alice , Basbaum Carol B.
Published: July 2002

An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of
Pseudomonas aeruginosa
infection in the airways, and here we provide evidence directly linking
P. aeruginosa
infection to mucus overproduction. We show that
P. aeruginosa
lipopolysaccharide profoundly up-regulates transcription of the mucin gene
MUC 2
in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyrphostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.
A cholera toxoid-insulin conjugate as an oral vaccine against spontaneous autoimmune diabetes
Authors: Bergerot Isabelle , Ploix Corinne , Petersen Jacob , Moulin Valerie , Rask Carola , Fabien Nicole , Lindblad Marianne , Mayer Anne , Czerkinsky Cecil , Holmgren Jan , Thivolet Charles
Published: July 2002

Mucosally induced immunological tolerance is an attractive strategy for preventing or treating illnesses resulting from untoward inflammatory immune reactions against self- or non-self-antigens. Oral administration of relevant autoantigens and allergens has been reported to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, the approach often requires repeated feeding of large amounts of tolerogens over long periods and is only partly effective in animals already systemically sensitized to the ingested antigen such as in animals already harboring autoreactive T cells, and thus presumably also in humans with an autoimmune disease. We have recently shown that oral administration of microgram amounts of antigen coupled to cholera toxin B subunit (CTB), can effectively suppress systemic T cell reactivity in naive as well as in immune animals. We now report that feeding small amounts (2–20 μg) of human insulin conjugated to CTB can effectively suppress beta cell destruction and clinical diabetes in adult nonobese diabetic (NOD) mice. The protective effect could be transferred by T cells from CTB-insulin-treated animals and was associated with reduced lesions of insulitis. Furthermore, adoptive co-transfer experiments involving injection of Thy-1,2 recipients with diabetogenic T cells from syngeneic mice and T cells from congenic Thy-1,1 mice fed with CTB-insulin demonstrated a selective recruitment of Thy-1,1 donor cells in the peripancreatic lymph nodes concomitant with reduced islet cell infiltration. These results suggest that protection against autoimmune diabetes can be achieved by feeding minute amounts of a pancreas islet cell autoantigen linked to CTB and appears to involve the selective migration and retention of protective T cells into lymphoid tissues draining the site of organ injury.
Relationship between “proto-splice sites” and intron phases: Evidence from dicodon analysis
Authors: Long Manyuan , de Souza Sandro J. , Rosenberg Carl , Gilbert Walter
Published: July 2002

The coding sequence at the boundaries of exons flanking nuclear introns shows some degree of conservation. To the extent that such sequences might be recognized by the splicing machinery, this conservation may be a derived result of evolution for efficient splicing. Alternatively, such conserved sequences might be remnants of proto-splice sites, which might have existed early in eukaryotic genes and served as the targets for the insertion of introns, as has been proposed by the introns-late theory. The distribution of intron phases, the position of the intron within a codon, is biased with an over-representation of phase 0 introns. Could any distribution of proto-splice sites account for today’s intron phase distribution? Here, we examine the dicodon usage in six model organisms, based on current sequences in the GenBank database, and predict the phase distribution that would be expected if introns had been inserted into proto-splice sites. However, these predictions differ between the various model organisms and disagree with the observed intron phase distributions. Thus, we reject the hypothesis that introns are inserted into hypothetical proto-splice sites. Finally, we analyze the sequences around the splice sites of introns in all six of the species to show that the actual conservation of sequence in exon regions near introns is very small and differs considerably between these species, which is inconsistent with a general proto-splice sites model.
PD-loop: A complex of duplex DNA with an oligonucleotide
Authors: Bukanov Nikolay O. , Demidov Vadim V. , Nielsen Peter E. , Frank-Kamenetskii Maxim D.
Published: July 2002

A stable complex between duplex DNA and an oligonucleotide is assembled with the aid of a DNA synthetic mimic, peptide nucleic acid (PNA). Homopyrimidine PNAs are known to invade into short homopurine tracts in duplex DNA forming P-loops. We have found that P-loops, formed at two closely located purine tracts in the same DNA strand separated by a mixed purine–pyrimidine sequence, merge and open the double helix between them. The opposite DNA strand, which is not bound with PNA, exposes and becomes accessible for complexing with an oligonucleotide via Watson–Crick pairing. As a result, the PD-loop emerges, which consists of locally open duplex DNA, PNA “openers,” and an oligonucleotide. The PD-loop stability and sequence specificity are demonstrated by affinity capture of duplex DNAs by using biotinylated oligonucleotides and streptavidin-covered magnetic beads. The type of complex formed by PNAs, an oligonucleotide and duplex DNA we describe, opens ways for development of various
in vitro
and
in situ
hybridization techniques with duplex DNA and may find applications in DNA nanotechnology and genomics.
Evidence of high levels of methylglyoxal in cultured Chinese hamster ovary cells
Authors: Chaplen Frank W. R. , Fahl William E. , Cameron Douglas C.
Published: July 2002

Methylglyoxal is an α-ketoaldehyde and dicarbonyl formed in cells as a side product of normal metabolism. Endogenously produced dicarbonyls, such as methylglyoxal, are involved in numerous pathogenic processes
in vivo
, including carcinogenesis and advanced glycation end-product formation; advanced glycation end-products are contributors to the pathophysiology of aging and chronic diabetes. Despite recent advances in understanding of the systemic effects of methylglyoxal, the full significance of this compound remains unknown. Herein we provide evidence that the majority of the methylglyoxal present
in vivo
is bound to biological ligands. The basis for our finding is an experimental approach that provides a measure of the bound methylglyoxal present in living systems, in this instance Chinese hamster ovary cells; with our approach, as much as 310 μM methylglyoxal was detected, 100- to 1,000-fold more than observed previously in biological systems. Several artifacts were considered before concluding that the methylglyoxal was associated with cellular structures, including phosphate elimination from triose phosphates, carbohydrate degradation under the assay conditions, and interference from the derivatizing agent used as part of the assay procedure. A major source of the recovered methylglyoxal is most probably modified cellular proteins. With methylglyoxal at about 300 μM, 0.02% of cellular amino acid residues could be modified. As few as one or two “hits” with methylglyoxal per protein molecule have previously been reported to be sufficient to cause protein endocytosis and subsequent degradation. Thus, 5–10% of cellular proteins may be modified to physiologically significant levels.
Cyclic AMP regulates potassium channel expression in C6 glioma by destabilizing Kv1.1 mRNA
Authors: Allen Margaret L. , Koh Duk-Su , Tempel Bruce L
Published: July 2002

The tissue distributions and physiological properties of a variety of cloned voltage-gated potassium channel genes have been characterized extensively, yet relatively little is known about the mechanisms controlling expression of these genes. Here, we report studies on the regulation of Kv1.1 expressed endogenously in the C6 glioma cell line. We demonstrate that elevation of intracellular cAMP leads to the accelerated degradation of Kv1.1 RNA. The cAMP-induced decrease in Kv1.1 RNA is followed by a decrease in Kv1.1 protein and a decrease in the whole cell sustained K+current amplitude. Dendrotoxin-I, a relatively specific blocker of Kv1.1, blocks 96% of the sustained K+current in glioma cells, causing a shift in the resting membrane potential from −40 mV to −7 mV. These data suggest that expression of Kv1.1 contributes to setting the resting membrane potential in undifferentiated glioma cells. We therefore suggest that receptor-mediated elevation of cAMP reduces outward K+current density by acting at the translational level to destabilize Kv1.1 RNA, an additional mechanism for regulating potassium channel gene expression.
Cell division regulates the T cell cytokine repertoire, revealing a mechanism underlying immune class regulation
Authors: Gett Amanda V. , Hodgkin Philip D.
Published: July 2002

Naive T lymphocytes have the potential to differentiate and produce a range of cytokines crucial for appropriate immune responses. How T lymphocytes vary their cytokine output during differentiation is unknown, although they are clearly influenced by the cytokines already present in the environment. Here we show that the number of divisions taken by the cells after activation is a critical element in T cell differentiation. Our experiments used the dye 5-(and 6-)carboxyfluorescein diacetate, succinimidyl ester to track cells in different divisions after activation by anti-CD3 in the presence of the differentiating cytokine interleukin (IL)-4. The patterns of acquisition or loss of secretion of IL-2, IL-3, IL-4, IL-5, IL-10, and interferon γ all varied markedly with division number. These relationships were consistent regardless of the time-dependent variation in distribution of T cells among divisions. Thus, the observed combination of complex asynchronous T cell growth, overlaying a fixed probability of acquisition or loss of a cytokine at each division can explain why T cell differentiation displays the contradictory features of being both highly stochastic and highly controlled. Furthermore, these data reveal that T cells share a common regulatory strategy with B cells, whereby changes in the class of immune response are linked to the process of clonal expansion.
Characteristics of acute and chronic kainate excitotoxic damage to the optic nerve
Authors: Matute Carlos
Published: July 2002

Macroglial cells express ionotropic glutamate receptors. In the adult optic nerve, reverse transcription–PCR showed that the native receptors are formed by subunits belonging to the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate classes. Because activation of AMPA and kainate receptors can be toxic to oligodendrocytes
in vitro
, I examined the nature of the damage caused by kainate, an agonist of both receptor classes, applied directly onto the optic nerve. Acute infusion or chronic slow delivery of the agonist caused massive nerve damage that was more extensive in the latter. Interestingly, chronic delivery also produced inflammation and demyelination in well circumscribed areas of the nerve, together with other pathological features that closely resemble those observed in multiple sclerosis patients. Acute and chronic kainate lesions were both prevented by the non-
N
-methyl-
d
-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. However, GYKI53655, a specific AMPA receptor antagonist, did not significantly reduce the size of the lesion, suggesting that the kainate toxicity was mainly mediated through activation of kainate-preferring glutamate receptors. These observations suggest that alterations in glutamate signaling may be detrimental to oligodendrocytes and may be involved in the pathogenesis of multiple sclerosis and other demyelinating diseases.
Fok
I dimerization is required for DNA cleavage
Authors: Bitinaite Jurate , Wah David A. , Aggarwal Aneel K. , Schildkraut Ira
Published: July 2002

Fok
I is a type IIs restriction endonuclease comprised of a DNA recognition domain and a catalytic domain. The structural similarity of the
Fok
I catalytic domain to the type II restriction endonuclease
Bam
HI monomer suggested that the
Fok
I catalytic domains may dimerize. In addition, the
Fok
I structure, presented in an accompanying paper in this issue of
Proceedings
, reveals a dimerization interface between catalytic domains. We provide evidence here that
Fok
I catalytic domain must dimerize for DNA cleavage to occur. First, we show that the rate of DNA cleavage catalyzed by various concentrations of
Fok
I are not directly proportional to the protein concentration, suggesting a cooperative effect for DNA cleavage. Second, we constructed a
Fok
I variant,
Fok
N13Y, which is unable to bind the
Fok
I recognition sequence but when mixed with wild-type
Fok
I increases the rate of DNA cleavage. Additionally, the
Fok
I catalytic domain that lacks the DNA binding domain was shown to increase the rate of wild-type
Fok
I cleavage of DNA. We also constructed an
Fok
I variant,
Fok
D483A, R487A, which should be defective for dimerization because the altered residues reside at the putative dimerization interface. Consistent with the
Fok
I dimerization model, the variant
Fok
D483A, R487A revealed greatly impaired DNA cleavage. Based on our work and previous reports, we discuss a pathway of DNA binding, dimerization, and cleavage by
Fok
I endonuclease.
[3Fe-4S] to [4Fe-4S] cluster conversion in
Desulfovibrio fructosovorans
[NiFe] hydrogenase by site-directed mutagenesis
Authors: Rousset Marc , Montet Yael , Guigliarelli Bruno , Forget Nicole , Asso Marcel , Bertrand Patrick , Fontecilla-Camps Juan C. , Hatchikian E. Claude
Published: July 2002

The role of the high potential [3Fe-4S]
1+,0
cluster of [NiFe] hydrogenase from
Desulfovibrio
species located halfway between the proximal and distal low potential [4Fe-4S]
2+,1+
clusters has been investigated by using site-directed mutagenesis. Proline 238 of
Desulfovibrio fructosovorans
[NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of
D. fructosovorans
hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.
Genetic instability of cancer cells is proportional to their degree of aneuploidy
Authors: Duesberg Peter , Rausch Charlotte , Rasnick David , Hehlmann Ruediger
Published: July 2002

Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell lines, recently analyzed by Lengnaueret al. [Lengnauer, C., Kinzler, K. W. & Vogelstein, B. (1997)Nature (London)386, 623–627], also corresponds exactly to their degree of aneuploidy. We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.
Bacteroides fragilis
enterotoxin cleaves the zonula adherens protein, E-cadherin
Authors: Wu Shaoguang , Lim Kuei-Cheng , Huang Julie , Saidi Roxan F. , Sears Cynthia L.
Published: July 2002

Strains of
Bacteroides fragilis
associated with diarrheal disease (enterotoxigenic
B. fragilis
) produce a 20-kDa zinc-dependent metalloprotease toxin
(B. fragilis
enterotoxin; BFT) that reversibly stimulates chloride secretion and alters tight junctional function in polarized intestinal epithelial cells. BFT alters cellular morphology and physiology most potently and rapidly when placed on the basolateral membrane of epithelial cells, suggesting that the cellular substrate for BFT may be present on this membrane. Herein, we demonstrate that BFT specifically cleaves within 1 min the extracellular domain of the zonula adherens protein, E-cadherin. Cleavage of E-cadherin by BFT is ATP-independent and essential to the morphologic and physiologic activity of BFT. However, the morphologic changes occurring in response to BFT are dependent on target-cell ATP. E-cadherin is shown here to be a cellular substrate for a bacterial toxin and represents the identification of a mechanism of action, cell-surface proteolytic activity, for a bacterial toxin.
Rolipram, a type IV-specific phosphodiesterase inhibitor, facilitates the establishment of long-lasting long-term potentiation and improves memory
Authors: Barad Mark , Bourtchouladze Roussoudan , Winder Danny G. , Golan Hava , Kandel Eric
Published: July 2002

In an attempt to improve behavioral memory, we devised a strategy to amplify the signal-to-noise ratio of the cAMP pathway, which plays a central role in hippocampal synaptic plasticity and behavioral memory. Multiple high-frequency trains of electrical stimulation induce long-lasting long-term potentiation, a form of synaptic strengthening in hippocampus that is greater in both magnitude and persistence than the short-lasting long-term potentiation generated by a single tetanic train. Studies using pharmacological inhibitors and genetic manipulations have shown that this difference in response depends on the activity of cAMP-dependent protein kinase A. Genetic studies have also indicated that protein kinase A and one of its target transcription factors, cAMP response element binding protein, are important in memory
in vivo
. These findings suggested that amplification of signals through the cAMP pathway might lower the threshold for generating long-lasting long-term potentiation and increase behavioral memory. We therefore examined the biochemical, physiological, and behavioral effects in mice of partial inhibition of a hippocampal cAMP phosphodiesterase. Concentrations of a type IV-specific phosphodiesterase inhibitor, rolipram, which had no significant effect on basal cAMP concentration, increased the cAMP response of hippocampal slices to stimulation with forskolin and induced persistent long-term potentiation in CA1 after a single tetanic train. In both young and aged mice, rolipram treatment before training increased long- but not short-term retention in freezing to context, a hippocampus-dependent memory task.
A lineage-specific protein kinase crucial for myeloid maturation
Authors: Semizarov Dimitry , Glesne David , Laouar Amale , Schiebel Katrin , Huberman Eliezer
Published: July 2002

To identify genes involved in macrophage development, we used the differential display technique and compared the gene expression profiles for human myeloid HL-60 leukemia cell lines susceptible and resistant to macrophage maturation. We identified a gene coding for a protein kinase, protein kinase X (PRKX), which was expressed in the maturation-susceptible, but not in the resistant, cell line. The expression of the PRKX gene was found to be induced during monocyte, macrophage, and granulocyte maturation of HL-60 cells. We also studied the expression of the PRKX gene in 12 different human tissues and transformed cell lines and found that, among these tissues and cell types, the PRKX gene is expressed only in blood. Among the blood cell lineages, the PRKX gene is specifically expressed in macrophages and granulocytes. Antisense inhibition of PRKX expression blocked terminal development in both the leukemic HL-60 cells and normal peripheral blood monocytes, implying that PRKX is a key mediator of macrophage and granulocyte maturation. Using the HL-60 cell variant deficient in protein kinase C-β (PKC-β) and several stable PKC-β transfectants, we found that PRKX gene expression is under control of PKC-β; hence PRKX is likely to act downstream of this PKC isozyme in the same signal transduction pathway leading to macrophage maturation.
Hsp70 accumulation in chondrocytic cells exposed to high continuous hydrostatic pressure coincides with mRNA stabilization rather than transcriptional activation
Authors: Kaarniranta K. , Elo M. , Sironen R. , Lammi M. J. , Goldring M. B. , Eriksson J. E. , Sistonen L. , Helminen H. J.
Published: July 2002

In response to various stress stimuli, heat shock genes are induced to express heat shock proteins (Hsps). Previous studies have revealed that expression of heat shock genes is regulated both at transcriptional and posttranscriptional level, and the rapid transcriptional induction of heat shock genes involves activation of the specific transcription factor, heat shock factor 1 (HSF1). Furthermore, the transcriptional induction can vary in intensity and kinetics in a signal- and cell-type-dependent manner. In this study, we demonstrate that mechanical loading in the form of hydrostatic pressure increases heat shock gene expression in human chondrocyte-like cells. The response to continuous high hydrostatic pressure was characterized by elevated mRNA and protein levels of Hsp70, without activation of HSF1 and transcriptional induction of hsp70 gene. The increased expression of Hsp70 was mediated through stabilization of hsp70 mRNA molecules. Interestingly, in contrast to static pressurization, cyclic hydrostatic loading did not result in the induction of heat shock genes. Our findings show that hsp70 gene expression is regulated posttranscriptionally without transcriptional induction in chondrocyte-like cells upon exposure to high continuous hydrostatic pressure. We suggest that the posttranscriptional regulation in the form of hsp70 mRNA stabilization provides an additional mode of heat shock gene regulation that is likely to be of significant importance in certain forms of stress.
Probing the genetic population structure of
Trypanosoma cruzi
with polymorphic microsatellites
Authors: Oliveira Riva P. , Broude Natalia E. , Macedo Andrea M. , Cantor Charles R. , Smith Cassandra L. , Pena Sérgio D. J.
Published: July 2002

We describe here the identification of eight polymorphic microsatellite loci with (CA)

n

repeats in the
Trypanosoma cruzi
genome based on the affinity capture of fragments using biotinylated (CA)
12
attached to streptavidin-coated magnetic beads. The presence of two peaks in PCR amplification products from individual clones confirmed that
T. cruzi
is diploid. Hardy–Weinberg and linkage disequilibrium analyses suggested that sexual reproduction is rare or absent and that the population structure is clonal. Several strains, especially those isolated from nonhuman sources, showed more than two alleles in many loci demonstrating that they were multiclonal. The phylogenetic analysis of
T. cruzi
based on microsatellites revealed a great genetic distance among strains, although the strain dispersion profile in the Wagner network was in general agreement with the species dimorphism found by PCR amplification of the divergent region of the rRNA 24Sα gene.