bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance.
Authors: Jung J S , Bhat R V , Preston G M , Guggino W B , Baraban J M , Agre P
Published: May 2006

The aquaporins transport water through membranes of numerous tissues, but the molecular mechanisms for sensing changes in extracellular osmolality and regulating water balance in brain are unknown. We have isolated a brain aquaporin by homology cloning. Like aquaporin 1 (AQP1, also known as CHIP, channel-forming integral membrane protein of 28 kDa), the deduced polypeptide has six putative transmembrane domains but lacks cysteines at the known mercury-sensitive sites. Two initiation sites were identified encoding polypeptides of 301 and 323 amino acids; expression of each in Xenopus oocytes conferred a 20-fold increase in osmotic water permeability not blocked by 1 mM HgCl2, even after substitution of cysteine at the predicted mercury-sensitive site. Northern analysis and RNase protection demonstrated the mRNA to be abundant in mature rat brain but only weakly detectable in eye, kidney, intestine, and lung. In situ hybridization of brain localized the mRNA to ependymal cells lining the aqueduct, glial cells forming the edge of the cerebral cortex and brainstem, vasopressin-secretory neurons in supraoptic and paraventricular nuclei of hypothalamus, and Purkinje cells of cerebellum. Its distinctive expression pattern implicates this fourth mammalian member of the aquaporin water channel family (designated gene symbol, AQP4) as the osmoreceptor which regulates body water balance and mediates water flow within the central nervous system.
Plethystic Hopf algebras.
Authors: Rota G C , Stein J A
Published: May 2006

The notion of a plethystic algebra associated with a Hopf algebra endowed with a suitable bilinear form is defined. A special case is the Hopf algebra of symmetric functions.
An analogue of the DnaJ molecular chaperone in Escherichia coli.
Authors: Ueguchi C , Kakeda M , Yamada H , Mizuno T
Published: May 2006

Escherichia coli DnaJ functions as a typical molecular chaperone in coordination with other heat shock proteins such as DnaK and GrpE in a variety of cellular processes. In this study, it was found that E. coli possesses an analogue of DnaJ, as judged from not only its primary structure but also its possible function. This protein, named CbpA (for curved DNA-binding protein), was first identified as a DNA-binding protein that preferentially recognizes a curved DNA sequence. Cloning and nucleotide sequencing of the gene encoding CbpA revealed that the predicted product is very similar to DnaJ in amino acid sequence: overall identity is 39%. The cbpA gene functions as a multicopy suppressor for dnaJ mutations. The mutational lesions characteristic of a dnaJ null mutant--namely, temperature sensitivity for growth and defects in lambda phage and mini-F DNA replication--were all restored upon introduction of the cbpA gene on a multicopy plasmid. An insertional mutant of cbpA was also isolated, which showed no noticeable phenotype, particularly with regard to temperature sensitivity for growth. However, when this cbpA::kan allele was combined with the dnaJ null allele, the resultant strain was unable to grow at 37 degrees C, at which strains carrying each mutation alone could grow normally. These genetic results are interpreted as meaning that the function(s) of CbpA in E. coli is closely related to that of DnaJ.
Evolution of recombination among multiple selected loci: a generalized reduction principle.
Authors: Zhivotovsky L A , Feldman M W , Christiansen F B
Published: May 2006

Conditions for invasion by a new allele that controls the recombination pattern among an arbitrary number of genes under viability selection are studied. The recombination pattern may include interference. The new allele increases if its appropriately averaged marginal fitness is greater than the mean fitness prior to its introduction. Under weak additive-by-additive epistatic selection, this condition involves a weighted average of the changes in pairwise recombination rates relative to those prior to the introduction of the modifier. The weights here are positive functions of the epistatic selection components. In particular, the modifier allele may succeed even if it increases recombination among some pairs of loci, provided the overall average effect is one of reduction.
Proliferative responses and binding properties of hematopoietic cells transfected with low-affinity receptors for leukemia inhibitory factor, oncostatin M, and ciliary neurotrophic factor.
Authors: Gearing D P , Ziegler S F , Comeau M R , Friend D , Thoma B , Cosman D , Park L , Mosley B
Published: May 2006

Specific low-affinity receptors for leukemia inhibitory factor (LIF), oncostatin M (OSM; gp130), and ciliary neurotrophic factor (CNTF; receptor alpha, CNTFR alpha) may be utilized in various combinations to generate high-affinity binding sites and signal transduction. We have tested the ability of combinations of these receptors to transduce a proliferative signal in BAF-B03 cells. Coexpression of the LIF receptor and gp130 in these cells conferred high-affinity LIF and OSM binding and responsiveness to LIF and OSM. These cells also responded to CNTF in the absence of detectable binding. The further addition of CNTFR alpha conferred high-affinity CNTF binding and enhanced responsiveness to CNTF but did not modify responses to LIF or OSM. Coexpression of LIF receptor and CNTFR alpha resulted in a nonfunctional high-affinity binding site. These data are consistent with a role for the CNTFR alpha in enhancing CNTF action but the CNTFR alpha is not absolutely required for CNTF action and suggest a wider range of targets for CNTF.
Asynchronous pre- and postsynaptic activity induces associative long-term depression in area CA1 of the rat hippocampus in vitro.
Authors: Debanne D , Gähwiler B H , Thompson S M
Published: May 2006

Associative long-term depression (LTD) was induced in hippocampal slice cultures with repeated low-frequency (0.3 Hz) stimulation of the Schaffer collateral pathway, only when such stimuli were preceded by intracellular injection of brief depolarizing current pulses in the postsynaptic CA1 pyramidal cell. The decrease in excitatory postsynaptic potential amplitude lasted > 30 min, could be reversed by induction of potentiation, could be induced at previously potentiated inputs, was input-specific, and did not require activation or potentiation of other inputs. The magnitude of the depression depended upon the time interval between depolarization and stimulation and upon the duration of the depolarization pulse. LTD was not observed in neurons impaled with electrodes containing a Ca2+ chelator. LTD could not be induced in the presence of an N-methyl-D-aspartate receptor antagonist, suggesting that voltage-dependent Ca2+ influx is necessary but not sufficient for LTD induction. We conclude that associative LTD results when synaptic activity follows postsynaptic depolarization within a circumscribed time window.
Evidence for a non-alpha-helical DNA-binding motif in the Rel homology region.
Authors: Liu J , Sodeoka M , Lane W S , Verdine G L
Published: May 2006

The Rel family of transcription factors serve as terminal messengers in a variety of developmental and receptor-mediated signaling pathways. These proteins are related by a domain of approximately 280 amino acids, the Rel homology region, which mediates dimerization and sequence-specific binding to DNA. Here we report the use of photocrosslinking and site-directed mutagenesis to identify specific contact partners in a Rel protein-DNA interface. Within the Rel homology region of NF-kappa B p50 (also known as KBF1), two amino acid residues were identified by photocrosslinking to adjacent bases in a beta-interferon regulatory element. Secondary structure analysis suggests that the DNA-binding motif of the Rel homology region comprises a beta-turn-beta structure, in contrast to the alpha-helical motifs so commonly observed in transcription factors.
Several mRNAs with variable 3' untranslated regions and different stability encode the human PR264/SC35 splicing factor.
Authors: Sureau A , Perbal B
Published: May 2006

We have recently established that several mRNAs encode the PR264/SC35 splicing factor and that their expression is related to that of c-Myb in human hematopoietic cells. We now report that the various PR264 mRNAs whose expression is transactivated by c-Myb proteins encode an identical PR264/SC35 polypeptide. These mRNAs differ only in their 3' untranslated sequences and exhibit significantly different half-lives. We also show that there is a direct correlation between the length of the 3' untranslated region and the stability of the mRNA species. Given that 5' and 3' splice site selection mediated by the PR264/SC35 splicing factor is concentration-dependent, we postulate that alternative splicing of the 3' untranslated sequences might represent another key element in regulating the levels of PR264/SC35 expression during hematopoietic differentiation.
Consequences of altered isoprenylation targets ona-factor export and bioactivity.
Authors: Caldwell G A , Wang S H , Naider F , Becker J M
Published: May 2006

Cysteine-containing amino acid sequences (CAAX,
CC, and CXC; C is cysteine, A is any aliphatic amino acid, and X is any amino
acid) are targets for the attachment of C15 (farnesyl) and C20 (geranylgeranyl)
isoprenoids to peptides and proteins by specific prenyltransferases. Although
much work has centered on the enzymatic mechanisms of these enzymes, the
biological consequences of the differential isoprenylation they catalyze remain
to be elucidated. Farnesylation of the a-factor mating pheromone of
Saccharomyces cerevisiae is a known prerequisite for its biological activity and
its secretion through a pathway utilizing the yeast STE6 protein, a homolog of
the mammalian multidrug resistance (MDR) P-glycoprotein. We generated specific
mutations in the a-factor gene to encode isoprenylation targets for
geranylgeranylation [Cys-Val-Ile-Leu (CVIL) and Ser-Val-Cys-Cys (SVCC)] in place
of the natural farnesylation motif [Cys-Val-Ile-Ala (CVIA)]. The a-factors
containing these modified prenylation sites were successfully exported by a
STE6-dependent mechanism. Furthermore, these peptides, as well as synthetic
geranylgeranyl a-factor, retained bioactivity. Chromatographic comparisons of
synthetic and biosynthetic pheromones suggest that, in vivo, a peptide substrate
containing the geranylgeranylation target CVIL can be both farnesylated and
geranylgeranylated. These results clearly demonstrate that in vivo (i) different
prenyltransferases may recognize the same substrate; (ii) both farnesylated and
geranylgeranylated a-factor peptides are substrates for export via STE6, a
MDR-like protein; and (iii) farnesylated and geranylgeranylated pheromones are
both biologically active.
Invariant tryptophan at a shielded site promotesfolding of the conformational unit of spectrin.
Authors: MacDonald R I , Musacchio A , Holmgren R A , Saraste M
Published: May 2006

The tryptophan that is highly conserved among
repeating structural units of spectrin is reported to promote the conformational
stability of one such unit of chicken brain alpha-spectrin. Four constructs were
inserted into pET vectors for overexpression in Escherichia coli of the
following spectrin peptides: (i) two adjacent but separately expressed
"conformationally phased" repeating units, R16 and R17, one of which
(R17) contains a single tryptophan; (ii) a mutant, M17, of the single
tryptophan-containing unit with alanine substituted for the tryptophan; and
(iii) a conformationally unphased unit, 1617, composed of half of each of the
phased units. Both the mutant unit and the unphased unit were much more readily
digested by chymotrypsin and by elastase than the phased units and exhibited
only 38% and 54% as much alpha-helical structure, respectively, as the phased
units by their far UV CD spectra; 90 degrees light scattering measurements
revealed the folded peptides to be predominantly monomeric in solution, whereas
the unfolded, protease-sensitive peptides consisted of dimers and/or trimers.
This trend was corroborated by their dynamic light scattering. Both the
blue-shifted wavelength of maximal emission and the relative inaccessibility to
acrylamide of the single tryptophan in the folded unit indicate that the
invariant tryptophan occupies a site that is shielded from the aqueous
phase.
Dual promoter activation by the human beta-globinlocus control region.
Authors: Bresnick E H , Felsenfeld G
Published: May 2006

The human beta-globin locus control region (LCR)
is necessary for high-level and position-independent expression of globin genes
in erythroid cells. A variety of mechanisms have been proposed for the
cis-activation of individual members of the beta-globin gene family by the LCR
located 10-50 kilobases upstream. It is not known, however, whether a given LCR
can activate all developmentally appropriate globin family members on its
chromosome or whether, within a given chromosome, the LCR must be committed to
activating only a single gene. We have devised an experiment to distinguish
between these possibilities. This experiment takes advantage of the fact that if
two genes in a cluster are transcriptionally active and their promoters,
therefore, are in a conformation hypersensitive to nucleases, restriction
enzymes that cleave the promoters will excise the intervening chromatin
fragment. The Apa I sites on human fetal G gamma- and A gamma-globin gene
promoters are accessible to cleavage in nuclei from the human erythroleukemia
cell line K562, which expresses these genes, but not in HeLa cells. We find that
Apa I digestion leads to excision in high yield of the fragment spanning these
promoters, showing that a LCR element is capable of sharing its activating
function among members of a gene cluster on a single chromosome.
Polioviruses containing picornavirus type 1 and/ortype 2 internal ribosomal entry site elements: genetic hybrids and theexpression of a foreign gene.
Authors: Alexander L , Lu H H , Wimmer E
Published: May 2006

A picornavirus hybrid genome was constructed in
which the internal ribosomal entry site (IRES) of encephalomyocarditis virus was
inserted between the 5' non-translated region and the open reading frame of
poliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybrid
RNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could be
removed from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) in
which the translation of the PV open reading frame normally promoted by the type
1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. This
result indicates that these elements are not likely to contain cis-acting
elements necessary for PV replication or encapsidation. A foreign gene
(bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPO
cDNA between the PV and encephalomyocarditis virus IRES elements. The
dicistronic RNA replicated in HeLa cells and yielded a derivative of PV
(W1-DICAT) with a genome 17% longer than that of wild-type PV. CAT assays and
immunoblot analyses showed that the viral RNA efficiently expressed the foreign
gene in cell culture. The CAT activity diminished somewhat with each passage of
the dicistronic virus, an observation which suggested that the inserted gene had
a deleterious effect on viral replication. However, even after five virus
passages, a significant quantity of the foreign gene was still expressed.
Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNA
species that replicated and expressed luciferase for up to 20 hr after
transfection. However, this elongated RNA was not encapsidated.
Disomic locus content mapping.
Authors: Morton N E
Published: May 2006

A linear genetic map may be constructed from
segregation of markers in DNA fragments broken by radiation or shearing and/or
incorporated into a vector: this is locus content mapping. Theory for monosomic
locus content mapping is extended to disomy, making the phase of heterozygous
markers informative for linkage and each clone potentially informative for every
chromosome. Because there is no interference among breaks and phase may be
reliable inferred for all heterozygous loci, multiple pairwise mapping is
especially appropriate.
Liver-directed gene therapy: quantitative evaluationof promoter elements by using in vivo retroviral transduction.
Authors: Rettinger S D , Kennedy S C , Wu X , Saylors R L , Hafenrichter D G , Flye M W , Ponder K P
Published: May 2006

Liver-directed gene therapy will be applicable to
many inherited diseases. Although various protocols have been devised for in
vivo delivery of retrovirus, comparison of hepatocyte transduction frequencies
has been difficult due to variations in retroviral titer and a paucity of DNA
data. We have previously reported an in vivo rat hepatocyte transduction
technique which involves 70% hepatectomy followed 24 hr later by portal vein
injection of retrovirus during hepatic in-flow occlusion. In this study, we
employed this method and concentrated retroviral preparations to achieve
transduction of up to 15% of hepatocytes as determined by a quantitative PCR
assay. As an initial step toward identifying promoters which lead to high-level
long-term expression of retroviral transduced genes, we used our in vivo
delivery system to compare the Moloney murine leukemia virus long terminal
repeat (LTR) promoter with the promoter for the large subunit of murine RNA
polymerase II (Pol-II). Human alpha 1-antitrypsin (hAAT) was used as the
reporter gene to facilitate long-term analysis of expression. Serum hAAT levels
were higher for the Pol-II promoter (143 ng/ml) than for the LTR promoter (50
ng/ml). This difference was consistent with the higher transduction frequency
observed for the Pol-II-hAAT vector. Although serum hAAT expression was
sustained for up to 1 year in six of eight Pol-II-hAAT-transduced rats and three
of five LTR-hAAT-transduced rats and was proportional to hAAT mRNA level and
proviral DNA frequency, in vivo expression was significantly lower than in
transduced tissue culture cells. We conclude that a high frequency of in vivo
transduction can be achieved by using retroviral vectors and our rapid
transduction protocol, but transduced gene expression remains a serious problem.
The quantitative assays described herein will facilitate in vivo comparisons of
gene regulatory elements.
Regulation of human insulin receptor RNA splicing invivo.
Authors: Norgren S , Zierath J , Wedell A , Wallberg-Henriksson H , Luthman H
Published: May 2006

Alternative splicing involving the inclusion or
exclusion of exon 11 in insulin receptor mRNA results in two isoforms of the
alpha subunit. The two subunits display tissue-specific variation in relative
abundance at both RNA and protein levels and discrete differences in biological
properties. We have previously reported a small decrease in the relative level
of RNA molecules lacking exon 11 (Ex 11-) in skeletal muscle of
non-insulin-dependent diabetes mellitus (NIDDM) patients. In the present study,
we describe a drastically altered ratio in favor of Ex 11- RNA in a NIDDM
patient with markedly impaired insulin-mediated glucose utilization. The ratio
between the splice variants changed from 74% to 48% Ex 11- RNA after initiation
of insulin treatment, which considerably improved his blood glucose
concentrations and insulin-stimulated glucose utilization rate. This shows that
splicing can be regulated by metabolic and/or hormonal factors in response to
changes in the in vivo milieu. No genomic deletion or base substitution in
either the coding regions or exon-intron borders was found that explains the
altered splicing. Heterozygous mutations were excluded in sequences of putative
importance for splicing outside the analyzed regions as both alleles were
expressed and spliced in an identical fashion. Furthermore, these results
suggest that this patient fails to regulate alternative splicing of exon 11 in
the manner observed in most NIDDM patients and that this defect is associated
with the extreme impairment in insulin action.
The human CAN protein, a putative oncogene productassociated with myeloid leukemogenesis, is a nuclear pore complex protein thatfaces the cytoplasm.
Authors: Kraemer D , Wozniak R W , Blobel G , Radu A
Published: May 2006

We have carried out partial amino acid sequence
analysis of a putative nuclear pore complex protein (nucleoporin) of rat that
reacts with wheat germ agglutinin and with the polyspecific monoclonal antibody
414. Surprisingly, these partial amino acid sequence data revealed a high degree
of similarity with the human CAN protein, the complete cDNA-derived primary
structure of which was reported by Von Lindern et al. [Von Lindern, M.,
Fornerod, M., van Baal, S., Jaegle, M., de Wit, T., Buijs, A. & Grosveld, G.
(1992) Mol. Cell. Biol. 12, 1687-1697]. The CAN protein has been proposed to be
a putative oncogene product associated with myeloid leukemogenesis. Its
subcellular localization was not established. To confirm that the putative rat
nucleoporin is indeed a homolog of the human CAN protein and to determine its
subcellular localization, we expressed a 39-kDa internal segment of the
213,790-Da human CAN protein in Escherichia coli and raised monospecific
antibodies, which reacted with the putative rat nucleoporin. Immunofluorescence
microscopy of HeLa cells gave a punctate nuclear surface staining pattern
characteristic of nucleoporins, and immunoelectron microscopy yielded specific
decoration of the cytoplasmic side of the nuclear pore complex. This suggests
that the protein is part of the short fibers that emanate from the cytoplasmic
aspect of the nuclear pore complex. In agreement with previously proposed
nomenclature for nucleoporins, we propose the alternative term nup214
(nucleoporin of 214 kDa) for the CAN protein.
Stiffness changes of the cupula associated with themechanics of hair cells in the fish lateral line.
Authors: van Netten S M , Khanna S M
Published: May 2006

Cupular vibration in the lateral-line canal of
fish was measured in response to motion of the fluid in the canal by
laser-heterodyne interferometry. The results show that the mechanical
output/input ratio of the cupula depends on the stimulus amplitude; the cupula
thus behaves nonlinearly. The nonlinearity is due to the hair bundles, since it
disappears when the cupula is uncoupled from the underlying hair cells. A model
of cupular dynamics in which the behavior of the gating springs of the hair
cells is incorporated predicts nonlinear responses that are similar to the
measurements, suggesting that the nonlinear behavior of the cupula may be
attributed to the opening and closing of the transduction channels of the hair
cells.
Single-channel currents trigger action potentials insmall cultured hippocampal neurons.
Authors: Johansson S , Arhem P
Published: May 2006

Spontaneous neuronal impulse activity appears to
play a key role in some neural processes, such as the normal establishment of
interneuronal connections during development. In addition, spontaneous impulses
may be essential for the functional operation of neuronal networks. Mechanisms
of spontaneous non-pacemaker impulse generation are, however, not well known. In
this work, spontaneous electrical activity in small cultured hippocampal neurons
from rat was studied with tight-seal recording techniques. The results
demonstrate that spontaneous individual openings of single ion channels can
trigger impulse generation in these high-resistance cells. First, impulses
recorded in the whole-cell mode were apparently induced by spontaneous
plateau-potential events showing the characteristics expected from individual
openings and closures of ion channels. Second, patch-clamp recordings in the
cell-attached configuration showed that openings of single ion channels in the
patch membrane could trigger cellular impulses, detected as biphasic current
deflections. These findings suggest that the random gating of ion channel
molecules can be used as a mechanism for stochastic triggering of spontaneous
impulses in mammalian central neurons.
Osmotin overexpression in potato delays development ofdisease symptoms.
Authors: Liu D , Raghothama K G , Hasegawa P M , Bressan R A
Published: May 2006

Transgenic potato and tobacco plants carrying the
osmotin gene under the control of the cauliflower mosaic virus 35S promoter
constitutively overexpressed osmotin to a level of approximately 2% of total
cellular protein. Leaves of transgenic potato plants exhibited delayed
development of disease symptoms after inoculation with spore suspensions of
Phytophthora infestans, which is the cause of late blight disease of potato. In
contrast, transgenic tobacco plants did not display any change in the
development of disease symptoms when challenged with either spore suspensions or
fungal mycelia of Phytophthora parasitica var. nicotianae. Using in vitro
assays, purified osmotin was found to be more effective against P. infestans.
Some inhibition of P. parasitica also was observed in vitro even though no in
vivo effect could be established.
Phosphorus-31 magnetic resonance spectra revealprolonged intracellular acidosis in the brain following subarachnoidhemorrhage.
Authors: Brooke N S , Ouwerkerk R , Adams C B , Radda G K , Ledingham J G , Rajagopalan B
Published: May 2006

Subarachnoid hemorrhage may be complicated by
cerebral ischemia which, though reversible initially, can progress to an
irreversible neurological deficit. 31P magnetic resonance spectroscopy, which
can determine intracellular pH and thus detect areas of ischemia noninvasively,
was applied to 10 patients on 30 occasions, at various times after subarachnoid
hemorrhage. In 5 of them, there were focal areas of the brain in which the
intracellular pH was reduced to < 6.8 compared with the normal range of 7.05
+/- 0.05. Consciousness was impaired in 4 of these patients. Repeat studies in
these 4 patients showed that intracellular pH remained abnormally low for
several days but eventually returned toward normal. The return of intracellular
pH to normal paralleled an improvement in clinical condition in each case. In
the fifth patient with lowered regions of intracellular pH, there had been an
impaired level of consciousness and a transient focal deficit prior to the
single study. In the other 5 patients there were no areas of reduced pHi even
though in 3 of them there was intraventricular or cisternal blood shown on brain
computerized tomography. In 2 of these 3 patients there were no abnormal
neurological signs at the time of the magnetic resonance study. The third
patient had a dense and persistent hemiparesis. The remaining two patients had
no abnormal neurological signs at any stage. We suggest that the areas of
acidosis may reflect ischemia which is potentially reversible. Since the
technique is noninvasive, sequential 31P magnetic resonance spectroscopy of the
brain offers a method of detecting cerebral ischemia and, more importantly, of
assessing methods of treatment.
Stimulation with specific antigen can block superantigen-mediated deletion of T cells in vivo.
Authors: McCormack J E , Kappler J , Marrack P
Published: May 2006

The T-cell response to pigeon cytochrome c peptide, residues 88-104 (pcytC), in B10.BR mice is mediated largely by cells bearing both V beta 3 and V alpha 11 variable regions of the T-cell antigen receptor. These cells are, therefore, reactive with the superantigen staphylococcal enterotoxin A (SEA). Recent reports have shown that in vivo exposure to superantigen can lead to deletion of superantigen-reactive T cells from the pool of mature T cells in the periphery. Here we show that upon cotreatment of animals with both SEA and pcytC, bulk deletion of the population of SEA-reactive cells is maintained, while the subpopulation of SEA-reactive T cells that also responds to pcytC is not deleted but instead proliferates in response to pcytC. These results are discussed with regard to mechanisms regulating the balance between T-cell tolerance and T-cell activation in vivo.
Organization of the human CD40L gene: implications for molecular defects in X chromosome-linked hyper-IgM syndrome and prenatal diagnosis.
Authors: Villa A , Notarangelo L D , Di Santo J P , Macchi P P , Strina D , Frattini A , Lucchini F , Patrosso C M , Giliani S , Mantuano E
Published: May 2006

Recently, CD40L has been identified as the gene responsible for X chromosome-linked hyper-IgM syndrome (HIGM1). CD40L on activated T cells from HIGM1 patients fails to bind B-cell CD40 molecules, and subsequent analysis of CD40L transcripts by reverse transcription PCR demonstrated coding region mutations in these patients. This approach, however, is of limited use for prenatal diagnosis of HIGM1 in the early-gestation fetus. In this report, we have defined the genomic structure of the CD40L gene, which is composed of five exons and four intervening introns. With this information, we have defined at the genomic level the CD40L gene abnormalities for three previously described HIGM1 patients who demonstrated clustered deletions in the CD40L coding region. These different deletions arose from three distinct mechanisms, including (i) a splice donor mutation with exon skipping, (ii) a splice acceptor mutation with utilization of a cryptic splice site, and (iii) a deletion/insertion event with the creation of a new splice acceptor site. In addition, we have performed prenatal evaluation of an 11-week-old fetus at risk for HIGM1. CD40L genomic clones provide a starting point for further studies of the genetic elements that control CD40L expression. Our knowledge of the CD40L gene structure will prove useful for the identification of additional mutations in HIGM1 and for performing genetic counseling about this disease.
Multiple substitutions in the von Willebrand factor gene that mimic the pseudogene sequence.
Authors: Eikenboom J C , Vink T , Briët E , Sixma J J , Reitsma P H
Published: May 2006

We have analyzed a type IIB and a type I von Willebrand disease family for the presence of mutations in the region coding for the glycoprotein Ib binding domain of the von Willebrand factor. Since this sequence is also present in the highly homologous von Willebrand factor pseudogene, we have studied genomic DNA as well as cDNA, which was produced from RNA isolated from endothelial cells or platelets. In both families, we have detected multiple consecutive nucleotide substitutions in the 5' end of exon 28 that result in a sequence identical to the von Willebrand factor pseudogene. These substitutions were also found in cDNA, which proves that they are present in the active gene. The occurrence of multiple adjacent substitutions that exactly reflect a part of the sequence of the von Willebrand factor pseudogene is difficult to reconcile with sequential single mutational events. We therefore hypothesize that each of these multiple substitutions arose from one recombinational event between gene and pseudogene.
Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin.
Authors: Block T M , Lu X , Platt F M , Foster G R , Gerlich W H , Blumberg B S , Dwek R A
Published: May 2006

The imino sugar N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor of the oligosaccharide-trimming enzyme alpha-glucosidase I. Hepatitis B virus (HBV) contains three surface proteins (HBs proteins) of different sizes that are singly or doubly N-glycosylated and are essential for the formation of infectious virus. Therefore, the replication and secretion of HBV in the human hepatoma cell line HepG2 were studied in the presence of NBDNJ. In the stably HBV-transfected HepG 2.2.15 cells and in HBV-infected HepG2 cells, NBDNJ suppressed secretion of HBV particles and caused intracellular retention of HBV DNA. The secretion of subviral particles was less affected. These data suggest that inhibitors of oligosaccharide trimming may be useful for antiviral therapy of hepatitis B and for the study of the intracellular transport of the viral glycoproteins.
Sexual origins of British Aspergillus nidulans isolates.
Authors: Geiser D M , Arnold M L , Timberlake W E
Published: May 2006

Aspergillus nidulans is a holomorphic fungus, capable of producing both meiotically and mitotically derived spores. Meiosis may be an evolutionary relic in this species because it is potentially capable of mitotic recombination and because most Aspergilli lack the ability to produce meiotic spores. We tested the null hypothesis that meiosis has been a major factor in the origin of strains of A. nidulans from Great Britain by estimating linkage disequilibrium among restriction fragment length polymorphisms. These strains belong to different heterokaryon compatibility groups and are thus incapable of undergoing mitotic recombination with one another, so any recombination evidenced by linkage equilibrium is assumed to be the result of meiosis. Eleven cosmid clones of known chromosomal origin were used to generate multilocus genotypes based on restriction-pattern differences for each heterokaryon compatibility group. Low levels of genetic variation and little linkage disequilibrium were found, indicating that the heterokaryon compatibility groups represent recently diverged lineages that arose via meiotic recombination. The null hypothesis that loci are independent could not be rejected. Additionally, low levels of electrophoretic karyotype variation were indicative of meiosis. We conclude that although A. nidulans probably propagates in a primarily clonal fashion, recombination events are frequent enough to disrupt the stable maintenance of clonal genotypes. We further conclude that the British heterokaryon compatibility groups arose via recombination and not through novel mutation.
Ca2+ influx through stretch-activated cation channels activates maxi K+ channels in porcine endocardial endothelium.
Authors: Hoyer J , Distler A , Haase W , Gögelein H
Published: May 2006

The endocardial endothelium is an important modulator of myocardial function. The present study demonstrates the existence of a stretch-activated Ca(2+)-permeable cation channel and of a Ca(2+)-activated K+ channel in the endocardial endothelium of the porcine right atrium. The stretch-activated channel is permeable for K+, Na+, Ca2+, and Ba2+, with mean conductances of approximately 32 pS for the monovalent cations and approximately 13 pS for divalent cations. The Ca(2+)-activated K+ channel has a mean conductance of 192 pS in symmetrical KCl. solution. Channel activity is strongly dependent on membrane potential and the cytosolic Ca2+ concentration. Half-maximal activation occurs at a cytosolic Ca2+ concentration of approximately 5 microM. The influx of Ca2+ through the stretch-activated channel is sufficient to activate the Ca(2+)-activated K+ channel in cell-attached patches. Upon activation of the stretch-activated channel, the cytosolic Ca2+ concentration increases, at least locally, to values of approximately 0.5 microM, as deduced from the open probability of the Ca(2+)-dependent K+ channel that was activated simultaneously. The stretch-activated channels are capable of inducing an intracellular Ca2+ signal and may have a role as mechanosensors in the atrial endothelium, possibly activated by atrial overload.
Population migration and the spread of types 1 and 2 human immunodeficiency viruses.
Authors: Quinn T C
Published: May 2006

Over 14 million people are estimated to be infected with the human immunodeficiency viruses (HIV), with nearly three-fourths of the infected persons residing in developing countries. One factor responsible for dissemination of both HIV-1 and HIV-2 worldwide was the intense migration of individuals, from rural to urban centers with subsequent return migration and internationally due to civil wars, tourism, business purposes, and the drug trade. In sub-Saharan Africa, between 1960 and 1980, urban centers with more than 500,000 inhabitants increased from 3 to 28, and more than 75 military coups occurred in 30 countries. The result was a massive migration of rural inhabitants to urban centers concomitant with the spread of HIV-1 to large population centers. With the associated demographic, economic, and social changes, an epidemic of sexually transmitted diseases and HIV-1 was ignited. Migratory patterns were also responsible for the spread of endemic HIV-2 to neighboring West African countries and eventually to Europe, the Americans, and India. Although Southeast Asia was the last region in which HIV-1 was introduced, it has the greatest potential for rapid spread due to population density and inherent risk behaviors. Thus, the migration of poor, rural, and young sexually active individuals to urban centers coupled with large international movements of HIV-infected individuals played a prominent role in the dissemination of HIV globally. The economic recession has aggravated the transmission of HIV by directly increasing the population at risk through increased urban migration, disruption of rural families and cultural values, poverty, and prostitution and indirectly through a decrease in health care provision. Consequently, social and economic reform as well as sexual behavior education need to be intensified if HIV transmission is to be controlled.
Human ecology and behavior and sexually transmitted bacterial infections.
Authors: Holmes K K
Published: May 2006

The three direct determinants of the rate of spread of sexually transmitted diseases (STDs) are sexual behaviors, the mean duration of infectiousness, and the mean efficiency of sexual transmission of each STD. Underlying ecological and behavioral factors that operate through one or more of these direct determinants lie on a continuum, ranging from those most proximate back to those more remote (in time or mechanism) from the direct determinants. Most remote and least modifiable are the historical stages of economic development that even today conspicuously influence patterns of sexual behavior. Next are the distribution and changing patterns of climate, hygiene, and population density; the global population explosion and stages of the demographic transition; and ongoing changes in human physiology (e.g., menarche at younger age) and culture (e.g., later marriage). More proximate on the continuum are war, migration, and travel; and current policies for economic development and social welfare. Most recent or modifiable are technologic and commercial product development (e.g., oral contraceptives); circumcision, condom, spermicide, and contraception practices; patterns of illicit drug use that influence sexual behaviors; and the accessibility, quality, and use of STD health care. These underlying factors help explain why the curable bacterial STDs are epidemic in developing countries and why the United States is the only industrialized country that has failed to control bacterial STDs during the AIDS era.
Direct selection for a catalytic mechanism from combinatorial antibody libraries.
Authors: Janda K D , Lo C H , Li T , Barbas C F , Wirsching P , Lerner R A
Published: May 2006

Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementarity-determining regions. Libraries were panned with an alpha-phenethyl pyridyl disulfide that undergoes disulfide interchange. Out of 10 randomly picked clones, two contained an unpaired cysteine, one of which was studied. The antibody catalyzed the hydrolysis of the corresponding thioester where the electrophilic carbonyl occupies the three-dimensional space that was defined by the reactive sulfur atom during selection. The reaction operates by covalent catalysis. Although the steady-state rate enhancement relative to the activated thiol ester substrate is modest, hydrolysis of the acylated cysteine intermediate is remarkably efficient with a catalytic advantage of about four orders of magnitude. The results suggest that iterative mechanism-based selection procedures can recapitulate the enzymatic mechanisms refined through evolution.
Ctk: a protein-tyrosine kinase related to Csk that defines an enzyme family.
Authors: Klages S , Adam D , Class K , Fargnoli J , Bolen J B , Penhallow R C
Published: May 2006

We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.
Mutation detection by mismatch binding protein, MutS, in amplified DNA: application to the cystic fibrosis gene.
Authors: Lishanski A , Ostrander E A , Rine J
Published: May 2006

An experimental strategy for detecting heterozygosity in genomic DNA has been developed based on preferential binding of Escherichia coli MutS protein to DNA molecules containing mismatched bases. The binding was detected by a gel mobility-shift assay. This approach was tested by using as a model the most commonly occurring mutations within the cystic fibrosis (CFTR) gene. Genomic DNA samples were amplified with 5'-end-labeled primers that bracket the site of the delta F508 3-bp deletion in exon 10 of the CFTR gene. The renatured PCR products from homozygotes produced homoduplexes; the PCR products from heterozygotes produced heteroduplexes and homoduplexes (1:1). MutS protein bound more strongly to heteroduplexes that correspond to heterozygous carriers of delta F508 and contain a CTT or a GAA loop in one of the strands than to homoduplexes corresponding to homozygotes. The ability of MutS protein to detect heteroduplexes in PCR-amplified DNA extended to fragments approximately 500 bp long. The method was also able to detect carriers of the point mutations in exon 11 of the CFTR gene by a preferential binding of MutS to single-base mismatches in PCR-amplified DNA.
Primary structure and function of a second essential member of the heterooligomeric TCP1 chaperonin complex of yeast, TCP1 beta.
Authors: Miklos D , Caplan S , Mertens D , Hynes G , Pitluk Z , Kashi Y , Harrison-Lavoie K , Stevenson S , Brown C , Barrell B
Published: May 2006

A role for heterooligomeric TCP1 complex as a chaperonin in the eukaryotic cytosol has recently been suggested both by structural similarities with other chaperonins and by in vitro experiments showing it to mediate ATP-dependent folding of actin, tubulin, and luciferase. Here we present the primary structure of a second subunit of the complex and present genetic and functional analyses. The TCP1 beta amino acid sequence, predicted from the cloned gene, bears 35% identity to TCP1, termed here TCP1 alpha, containing the same highly conserved residues found in the collective sequence of chaperonins. The predicted product was identified as the fastest-migrating species of the TCP1 complex purified from soluble extracts of yeast. The TCP1 beta gene, like TCP1 alpha, is essential. Strains containing lethal disruptions of either gene could not be rescued by additional copies of the other. Spores bearing disruption of either gene germinated as single, large-budded cells. Similarly, large-budded cells were observed following shift to 37 degrees C of strains carrying temperature-sensitive mutations in either TCP1 alpha or TCP1 beta. The arrested cells contained replicated DNA present in single nuclear masses, associated with abnormal tubulin staining patterns, supporting the assertion that mitotic spindle formation and function are impaired. We conclude that TCP1 beta supplies an essential function that partially overlaps with that of TCP1 alpha in acting as a molecular chaperone in tubulin and spindle biogenesis.
Amphiregulin as an autocrine growth factor for c-Ha-ras- and c-erbB-2-transformed human mammary epithelial cells.
Authors: Normanno N , Selvam M P , Qi C F , Saeki T , Johnson G , Kim N , Ciardiello F , Shoyab M , Plowman G , Brandt R
Published: May 2006

Amphiregulin (AR), a member of the epidermal growth factor (EGF) family, was found to be as potent as EGF in stimulating the anchorage-dependent growth (ADG) of immortalized, nontransformed human mammary epithelial MCF-10A cells. MCF-10A cells transformed by either an activated human c-Ha-ras protooncogene (MCF-10A ras) or by overexpression of a nonactivated rat c-neu gene (MCF-10A neu) exhibited a 35% reduction in the response to AR in ADG when compared to MCF-10A cells, but AR was still as potent as EGF in these transformants. Exogenous AR exhibited only 15-20% of the activity of EGF in stimulating the anchorage-independent growth, a response that is normally dependent upon exogenous EGF, of the oncogene-transformed MCF-10A cells. MCF-10A cells express low levels of a 1.4-kb AR mRNA transcript, while MCF-10A ras and MCF-10A neu cells display a 15- to 30-fold increase in the levels of AR mRNA and endogenous AR protein as determined by Western blot analysis. Exogenous EGF was found to induced both the AR mRNA and protein in the MCF-10A parental and transformed cells. A 20-mer phosphorothioate antisense deoxyoligonucleotide complementary to the 5' sequence of AR mRNA was able to significantly reduce the levels of endogenous AR protein and to inhibit the EGF-stimulated ADG and anchorage-independent growth of MCF-10A ras and MCF-10A neu cells. These data suggest that AR may function as an EGF-dependent autocrine growth factor in mammary epithelial cells that have been transformed by either a point-mutated c-Ha-ras or c-neu.
A primate T-lymphotropic virus, PTLV-L, different from human T-lymphotropic viruses types I and II, in a wild-caught baboon (Papio hamadryas).
Authors: Goubau P , Van Brussel M , Vandamme A M , Liu H F , Desmyter J
Published: May 2006

Searching for clues to the evolution of the primate T-lymphotropic viruses (PTLVs), which include the human and the simian T-lymphotropic viruses (HTLV and STLV), we have identified another PTLV, which differs sufficiently from the known PTLV-I and PTLV-II types to be designated here PTLV-L. The virus was isolated from a wild-born baboon (Papio hamadryas) from Eritrea. In a cDNA library a 1802-bp-long fragment was identified that extends from the env region, including the complete transmembrane protein gene, to part of the tax/rex gene. Homologies at the nucleotide sequence level of PTLV-L, prototype simian T-lymphotropic virus-PH969, with HTLV-I and -II, respectively, were 62% and 64% overall, 65% and 70% in the env region, and 80% and 80% in the partial tax/rex sequence. In the 5' part of the pX region a significant homology was seen only with HTLV-II (52%). Phylogenetic analysis based on the gene encoding the transmembrane protein indicates that PTLV-L represents a PTLV type with a long independent evolution, longer than any strain within the PTLV-I or PTLV-II groups. The finding of another PTLV type in African baboons is further evidence of the wide variety of PTLV found on this continent. Whether PTLV-L resembles PTLV-I and PTLV-II in the extension of its host range to other primates, including humans, remains to be seen.