bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential role of germinal vesicle material in the meiotic cell cycle of
Xenopus
oocytes
Authors: Iwashita Jun , Hayano Yoshimasa , Sagata Noriyuki
Published: July 2002

In almost all animal species, immature oocytes are arrested naturally in the first meiotic prophase, with a large nucleus called the germinal vesicle. A number of previous studies showed that both activation of maturation/M phase-promoting factor (MPF) (assayed by semiquantitative cytological methods) and some other maturational events occur essentially normally in enucleated oocytes from many amphibian species and mice. Hence, for nearly three decades, it has generally been believed that nuclear material is dispensable for MPF activation and the meiotic cell cycle in vertebrate oocytes. Here, we have challenged this view by examining the histone H1 kinase activities and the molecular forms of MPF in experimentally manipulated
Xenopus
oocytes. We show that oocytes injected with nuclear material undergo much more rapid MPF activation and maturation than uninjected control oocytes. Conversely, enucleated oocytes, unlike nucleated counterparts, undergo only weak MPF activation in meiosis I and no detectable MPF reactivation in meiosis II, the latter accompanying inhibitory tyrosine phosphorylation of cdc2 kinase, the catalytic subunit of MPF. These results argue strongly that nuclear material is indispensable for the meiotic cell cycle, particularly MPF reactivation (or cdc2 tyrosine dephosphorylation) on entry into meiosis II, in
Xenopus
oocytes. The classical and general view may thus need reconsideration.
Mapping of the ligand-selective domain of the
Xenopus laevis
corticotropin-releasing factor receptor 1: Implications for the ligand-binding site
Authors: Dautzenberg Frank M. , Wille Sandra , Lohmann Ragna , Spiess Joachim
Published: July 2002

The nonselective human corticotropin-releasing factor receptor 1 (hCRF-R1) and the ligand-selective
Xenopus
CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both high-affinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF-R1 followed by double or multiple point mutations were constructed. Binding studies and cAMP assays demonstrated that the N-terminal domain exhibited the complete ligand selectivity of xCRF-R1. The important region was mapped between amino acids 70 and 89; replacement of amino acids Arg
76
, Asn
81
, Gly
83
, Leu
88
, and Ala
89
in hCRF-R1 with the corresponding amino acids of xCRF-R1 (Gln
76
, Gly
81
, Val
83
, His
88
, and Leu
89
) resulted in a receptor that had ∼30-fold higher affinity for human/rat CRF than for sauvagine. Mutagenesis of these amino acids in xCRF-R1 to the human sequence completely abolished the ligand selectivity of xCRF-R1. Mutagenesis of amino acids 88 and 89 in hCRF-R1 or xCRF-R1 had only a minor (∼2.5-fold) effect on the ligand selectivity of the mutant receptor. Substitution of Arg
76
, Asn
81
, and Gly
83
in hCRF-R1 with the corresponding sequence of xCRF-R1 (Gln
76
, Gly
81
, and Val
83
) resulted in a receptor with ∼11-fold higher affinity for human/rat CRF compared with ovine CRF or sauvagine. When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70–89 of CRF-R1 are important for the ligand binding site.
Association of calcium channel α
1S
and β
1a
subunits is required for the targeting of β
1a
but not of α
1S
into skeletal muscle triads
Authors: Neuhuber Birgit , Gerster Uli , Döring Frank , Glossmann Hartmut , Tanabe Tsutomu , Flucher Bernhard E.
Published: July 2002

The skeletal muscle L-type Ca
2+
channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca
2+
release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α
1S
–β
1a
interactions in the incorporation of the functional channel complex into the triad, α
1S
and β
1a
[or a β
1a
-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α
1S
subunit was diffusely distributed in the cytoplasm. On coexpression with the α
1S
subunit βGFP distribution became clustered and colocalized with α
1S
immunofluorescence. Based on the colocalization of βGFP and α
1S
with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α
1S
with and without β
1a
restored Ca
2+
currents and depolarization-induced Ca
2+
release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α
1S
mutants in which the β interaction domain had been altered (α
1S
-Y366S) or deleted (α
1S
-Δ351–380). Although α
1S
-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca
2+
currents and depolarization-induced Ca
2+
release. Thus, β
1a
requires the association with the β interaction domain in the I–II cytoplasmic loop of α
1S
for its own incorporation into triad junctions, but stable α
1S
–β
1a
association is not necessary for the targeting of α
1S
into the triads or for its normal function in Ca
2+
conductance and excitation-contraction coupling.
Expression of the Na
+
/Ca
2+
exchanger emerges in hepatic stellate cells after activation in association with liver fibrosis
Authors: Nakamura Toshio , Arii Shigeki , Monden Kazunobu , Furutani Masaharu , Takeda Yoshihisa , Imamura Masayuki , Tominaga Makoto , Okada Yasunobu
Published: July 2002

Activation of hepatic stellate (Ito) cells is a final common pathway of liver fibrosis. The findings presented in this paper indicate that expression of Na
+
/Ca
2+
exchanger (NCX) emerges in rat hepatic stellate cells after activation
in vitro
during primary culture or
in vivo
in response to intoxication with CCl
4
. NCX mRNA became detectable by Northern blot analysis in cultured stellate cells on day 3, as was α-smooth muscle actin, an indicator not only of smooth muscle differentiation but also of stellate cell activation. Western blot analysis showed expression of the exchanger protein in the activated stellate cells. Functional expression of the exchanger, monitored by Ni
2+
-sensitive, verapamil-insensitive intracellular free Ca
2+
increases in response to reduction of extracellular Na
+
concentration, became sizable by using Fura-2 in stellate cells by 7 days in culture. Furthermore, increased expression of the exchanger mRNA was found predominantly in stellate cells freshly isolated from the CCl
4
model rat of hepatic fibrosis. Thus, it is concluded that NCX expression is closely associated with activation of hepatic stellate cells
in vitro
and
in vivo
. Because, even at the whole liver level, increased expression of NCX mRNA became observable after induction of liver fibrosis, it is suggested that NCX expression serves a useful diagnostic marker of liver fibrosis or cirrhosis.
Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA
Authors: Buschta-Hedayat Neda , Buterin Tonko , Hess Martin T. , Missura Miriam , Naegeli Hanspeter
Published: July 2002

Nondistorting C4′ backbone adducts serve as molecular tools to analyze the strategy by which a limited number of human nucleotide excision repair (NER) factors recognize an infinite variety of DNA lesions. We have constructed composite DNA substrates containing a noncomplementary site adjacent to a nondistorting C4′ adduct to show that the loss of hydrogen bonding contacts between partner strands is an essential signal for the recruitment of NER enzymes. This specific conformational requirement for excision is mediated by the affinity of xeroderma pigmentosum group A (XPA) protein for nonhybridizing sites in duplex DNA. XPA recognizes defective Watson–Crick base pair conformations even in the absence of DNA adducts or other covalent modifications, apparently through detection of hydrophobic base components that are abnormally exposed to the double helical surface. This recognition function of XPA is enhanced by replication protein A (RPA) such that, in combination, XPA and RPA constitute a potent molecular sensor of denatured base pairs. Our results indicate that the XPA–RPA complex may promote damage recognition by monitoring Watson–Crick base pair integrity, thereby recruiting the human NER system preferentially to sites where hybridization between complementary strands is weakened or entirely disrupted.
Corticotropin-releasing hormone deficiency unmasks the proinflammatory effect of epinephrine
Authors: Karalis Katia P. , Kontopoulos Effie , Muglia Louis J. , Majzoub Joseph A.
Published: July 2002

Traditionally, the adrenal gland has been considered an important endocrine component of the pathway to inhibit acute inflammation via hypothalamic corticotropin-releasing hormone (CRH)-mediated secretion of glucocorticoid. Immunoreactive CRH found in inflamed tissues is a potent proinflammatory factor. Using genetic and pharmacological models of CRH deficiency, we now show that CRH deficiency unmasks a major proinflammatory effect of epinephrine secreted from the adrenal medulla. Together, epinephrine and peripheral CRH stimulate inflammation, and glucocorticoid acts as a counterbalancing force in this regard. Our findings suggest that stimulation of the acute inflammatory response should be included with the other “fight-or-flight” actions of epinephrine.
Response tuning in bacterial chemotaxis
Authors: Jasuja Ravi , Yu-Lin  , Trentham David R. , Khan Shahid
Published: July 2002

Chemotaxis of enteric bacteria in spatial gradients toward a source of chemoattractant is accomplished by increases in the length of swimming runs up the gradient. Biochemical components of the intracellular signal pathway have been identified, but mechanisms for achieving the high response sensitivity remain unknown. Binding of attractant ligand to its receptor inactivates a receptor-associated histidine kinase, CheA, which phosphorylates the signal protein CheY. The reduction in phospho-CheY, CheY-P, levels prolongs swimming runs. Here, the stimulus–response relation has been determined by measurement of excitation responses mediated by the Tar receptor to defined concentration jumps of the attractant, aspartate, administered within milliseconds by photolysis of a photolabile precursor. The bacteria responded to <1% changes in Tar occupancy when adapted to aspartate over concentrations spanning three orders of magnitude. Response amplitudes increased approximately logarithmically with stimulus strength, extending responsiveness over a greater stimulus range. The extent and form of this relation indicates that, in contrast to mechanisms for adaptive recovery, excitation signal generation involves amplification based on cooperative interactions. These interactions could entail inactivation of multiple receptor–CheA signaling complexes and/or simultaneous activation of CheY-P dephosphorylation.
Neutral endopeptidase (EC 3.4.24.11) terminates colitis by degrading substance P
Authors: Sturiale S. , Barbara G. , Qiu B. , Figini M. , Geppetti P. , Gerard N. , Gerard C. , Grady E. F. , Bunnett N. W. , Collins S. M.
Published: July 2002

Neurogenic inflammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inflammation, the mechanisms that terminate inflammation are unknown. We examined whether neutral endopeptidase (NEP), a cell-surface enzyme that degrades SP in the extracellular fluid, terminates neurogenic inflammation in the colon. In NEP knockout mice, the SP concentration in the colon was ≈2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was ≈4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inflammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inflammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular fluid under basal conditions and terminates its proinflammatory effects. Because we have previously shown that intestinal inflammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inflammation.
Long-term doxycycline-controlled expression of human tyrosine hydroxylase after direct adenovirus-mediated gene transfer to a rat model of Parkinson’s disease
Authors: Corti Olga , Sánchez-Capelo Amelia , Colin Philippe , Hanoun Naïma , Hamon Michel , Mallet Jacques
Published: July 2002

Developments of technologies for delivery of foreign genes to the central nervous system are opening the field to promising treatments for human neurodegenerative diseases. Gene delivery vectors need to fulfill several criteria of efficacy and safety before being applied to humans. The ability to drive expression of a therapeutic gene in an adequate number of cells, to maintain long-term expression, and to allow exogenous control over the transgene product are essential requirements for clinical application. We describe the use of an adenovirus vector encoding human tyrosine hydroxylase (TH) 1 under the negative control of the tetracycline-sensitive gene regulatory system for direct injection into the dopamine-depleted striatum of a rat model of Parkinson’s disease. This vector mediated synthesis of TH in numerous striatal cells and transgene expression was observed in a large proportion of them for at least 17 weeks. Furthermore, doxycyline, a tetracycline analog, allowed efficient and reversible control of transgene expression. Thus, the insertion of a tetracycline-sensitive regulatory cassette into a single adenovirus vector provides a promising system for the development of successful and safe therapies for human neurological diseases. Our results also confirm that future effective gene replacement approaches to Parkinson’s disease will have to consider the concomitant transfer of TH and GTP-cyclohydrolase transgenes because the synthesis of the TH cofactor tetrahydrobiopterin may be crucial for restoration of the dopaminergic deficit.
The dimensions of the protein import channels in the outer and inner mitochondrial membranes
Authors: Schwartz Michael P. , Matouschek Andreas
Published: July 2002

Most mitochondrial proteins are imported into mitochondria through transmembrane channels composed largely, and perhaps exclusively, of proteins. We have determined the effective internal diameter of the protein import channel in the mitochondrial outer membrane to be between 20 Å and 26 Å during translocation. The diameter of the import channel in the inner membrane is smaller than the diameter of the outer membrane import channel. These results were obtained by measuring the effect of rigid steric bulk introduced into precursor proteins on import.
Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules
Authors: Yin Hong , Artsimovitch Irina , Landick Robert , Gelles Jeff
Published: July 2002

Cessation of transcription at specific terminator DNA sequences is used by viruses, bacteria, and eukaryotes to regulate the expression of downstream genes, but the mechanisms of transcription termination are poorly characterized. To elucidate the kinetic mechanism of termination at the intrinsic terminators of enteric bacteria, we observed, by using single-molecule light microscopy techniques, the behavior of surface-immobilized
Escherichia coli
RNA polymerase (RNAP) molecules
in vitro
. An RNAP molecule remains at a canonical intrinsic terminator for ≈64 s before releasing DNA, implying the formation of an elongation-incompetent (paused) intermediate by transcription complexes that terminate but not by those that read through the terminator. Analysis of pause lifetimes establishes a complete minimal mechanism of termination in which paused intermediate formation is both necessary and sufficient to induce release of RNAP at the terminator. The data suggest that intrinsic terminators function by a nonequilibrium process in which terminator effectiveness is determined by the relative rates of nucleotide addition and paused state entry by the transcription complex.
Nerve growth factor is an autocrine factor essential for the survival of macrophages infected with HIV
Authors: Garaci Enrico , Caroleo Maria Cristina , Aloe Luigi , Aquaro Stefano , Piacentini Mauro , Costa Nicola , Amendola Alessandra , Micera Alessandra , Caliò Raffaele , Perno Carlo-Federico , Levi-Montalcini Rita
Published: July 2002

Nerve growth factor (NGF) is a neurotrophin with the ability to exert specific effects on cells of the immune system. Human monocytes/macrophages (M/M) infected
in vitro
with HIV type 1 (HIV-1) are able to produce substantial levels of NGF that are associated with enhanced expression of the high-affinity NGF receptor (p140 trkA) on the M/M surface. Treatment of HIV-infected human M/M with anti-NGF Ab blocking the biological activity of NGF leads to a marked decrease of the expression of p140 trkA high-affinity receptor, a concomitant increased expression of p75
NTR
low-affinity receptor for NGF, and the occurrence of apoptotic death of M/M. Taken together, these findings suggest a role for NGF as an autocrine survival factor that rescues human M/M from the cytopathic effect caused by HIV infection.
Mineral surfaces and bioavailability of heavy metals: A molecular-scale perspective
Authors: Brown Gordon E. , Foster Andrea L. , Ostergren John D.
Published: July 2002

There is a continual influx of heavy metal contaminants and
pollutants into the biosphere from both natural and anthropogenic
sources. A complex variety of abiotic and biotic processes affects
their speciation and distribution, including adsorption onto and
desorption from mineral surfaces, incorporation in precipitates or
coprecipitates, release through the dissolution of minerals, and
interactions with plants and microbes. Some of these processes can
effectively isolate heavy metals from the biosphere, whereas others
cause their release or transformation to different species that may be
more (or less) bioavailable and/or toxic to organisms. Here we focus on
abiotic adsorption and precipitation or coprecipitation processes
involving the common heavy metal contaminant lead and the
metalloids arsenic and selenium in mine tailings and
contaminated soils. We have used extremely intense x-rays from
synchrotron sources and a structure-sensitive method known as x-ray
absorption fine structure (XAFS) spectroscopy to determine the
molecular-level speciation of these elements at concentrations of 50 to
several thousand ppm in the contaminated environmental samples as well
as in synthetic sorption samples. Our XAFS studies of As and Pb in the
mine tailings show that up to 50% of these contaminants in the samples
studied may be present as adsorbed species on mineral surfaces, which
makes them potentially more bioavailable than when present in sparingly
soluble solid phases. Our XAFS studies of Se(VI) sorption on
Fe
2+
-containing sulfates show that this element undergoes
redox reactions that transform it into less bioavailable and less toxic
species. This type of information on molecular-level speciation of
heavy metal and metalloid contaminants in various environmental
settings is needed to prioritize remediation efforts and to assess
their potential hazard to humans and other organisms.
Ion channel genes and human neurological disease: Recent progress, prospects, and challenges
Authors: Cooper Edward C. , Jan Lily Yeh
Published: July 2002

What do epilepsy, migraine headache, deafness, episodic ataxia,
periodic paralysis, malignant hyperthermia, and generalized myotonia
have in common? These human neurological disorders can be caused by
mutations in genes for ion channels. Many of the channel diseases are
“paroxysmal disorders” whose principal symptoms occur
intermittently in individuals who otherwise may be healthy and active.
Some of the ion channels that cause human neurological disease are old
acquaintances previously cloned and extensively studied by channel
specialists. In other cases, however, disease-gene hunts have led the
way to the identification of new channel genes. Progress in the study
of ion channels has made it possible to analyze the effects of human
neurological disease-causing channel mutations at the level of the
single channel, the subcellular domain, the neuronal network, and the
behaving organism.
Homologs of the yeast Sec complex subunits Sec62p and Sec63p are abundant proteins in dog pancreas microsomes
Authors: Tyedmers Jens , Lerner Monika , Bies Christiane , Dudek Johanna , Skowronek Markus H. , Haas Ingrid G. , Heim Nicole , Nastainczyk Wolfgang , Volkmer Jörg , Zimmermann Richard
Published: July 2002

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61αp monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.
Measuring the rate of intramolecular contact formation in polypeptides
Authors: Lapidus Lisa J. , Eaton William A. , Hofrichter James
Published: July 2002

Formation of a specific contact between two residues of a polypeptide chain is an important elementary process in protein folding. Here we describe a method for studying contact formation between tryptophan and cysteine based on measurements of the lifetime of the tryptophan triplet state. With tryptophan at one end of a flexible peptide and cysteine at the other, the triplet decay rate is identical to the rate of quenching by cysteine. We show that this rate is also close to the diffusion-limited rate of contact formation. The length dependence of this end-to-end contact rate was studied in a series of Cys-(Ala-Gly-Gln)

k

-Trp peptides, with
k
varying from 1 to 6. The rate decreases from ∼1/(40 ns) for
k
= 1 to ∼1/(140 ns) for
k
= 6, approaching the length dependence expected for a random coil (
n
−3/2
) for the longest peptides.
Ca
2+
-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin
Authors: Quetglas Stephanie , Leveque Christian , Miquelis Raymond , Sato Kazuki , Seagar Michael
Published: July 2002

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca
2+
could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca
2+
-dependent calmodulin binding (
K
d
= 500 nM) to glutathione
S-
transferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP
77–94
, immediately C-terminal to the tetanus toxin cleavage site (Q
76
–F
77
). In isolated synaptic vesicles, Ca
2+
/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a
35
S-SNAP-25, syntaxin 1 GST-VAMP
1–96
complex was inhibited by Ca
2+
/calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP
77–94
and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in αhelical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca
2+
may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.
Genetic modification of survival in tissue-specific knockout mice with mitochondrial cardiomyopathy
Authors: Li Hong , Wang Jianming , Wilhelmsson Hans , Hansson Anna , Thorén Peter , Duffy John , Rustin Pierre , Larsson Nils-Göran
Published: July 2002

We recently described a mouse model that reproduces important pathophysiological features of mitochondrial DNA (mtDNA) mutation diseases. The gene for mouse mitochondrial transcription factor A,
Tfam
(also called mtTFA), a nucleus-encoded key regulator of mtDNA expression, was targeted with
loxP
sites (
Tfam
loxP
) and disrupted
in vivo
by transgenic expression of
cre
-recombinase from the muscle creatinine kinase (
Ckmm
) promoter. This promoter is active from embryonic day 13, and the knockouts had normal respiratory chain function in the heart at birth and developed mitochondrial cardiomyopathy postnatally. In this paper, we describe a heart-knockout strain obtained by mating
Tfam
loxP
mice to animals expressing
cre
-recombinase from the α-myosin heavy chain (
Myhca
) promoter. This promoter is active from embryonic day 8, and the knockouts had onset of mitochondrial cardiomyopathy during embryogenesis. The age of onset of cardiac respiratory chain dysfunction can thus be controlled by temporal regulation of
cre
-recombinase expression. Further characterization demonstrated that ≈75% of the knockouts died in the neonatal period, whereas, surprisingly, ≈25% survived for several months before dying from dilated cardiomyopathy with atrioventricular heart conduction blocks. Modifying gene(s) affect the life span of the knockouts, because ≈95% of the knockout offspring from an intercross of the longer-living knockouts survived the neonatal period. Thus, the tissue-specific knockouts we describe here not only reproduce important pathophysiological features of mitochondrial cardiomyopathy but also provide a powerful system by which to identify modifying genes of potential therapeutic value.
CD8+T cells recognize an inclusion membrane-associated protein from the vacuolar pathogenChlamydia trachomatis
Authors: Fling Steven P. , Sutherland R. Alec , Steele Lisa N. , Hess Bruce , D'Orazio Sarah E. F. , Maisonneuve Jean-François , Lampe Mary F. , Probst Peter , Starnbach Michael N.
Published: July 2002

During infection withChlamydia trachomatis, CD8+T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8+T cells recognize antigens processed from cytosolic proteins, theChlamydiaantigens recognized by these CD8+T cells very likely have access to the host cell cytoplasm during infection. The identity of theseC. trachomatisproteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein fromC. trachomatisrecognized by protective CD8+T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the humanC. trachomatisserovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against allC. trachomatisserovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction ofC. trachomatiswith its host cell.
Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation
Authors: Wolf Dana G. , Courcelle Charmain Tan , Prichard Mark N. , Mocarski Edward S.
Published: July 2002

The human cytomegalovirus UL97 kinase, an important target of
antiviral therapy, has an impact on at least two distinct phases of
viral replication. Compared with wild-type virus, the UL97 deletion
mutant exhibits an early replication defect that reduces DNA
accumulation by 4- to 6-fold, as well as a late capsid maturation
defect responsible for most of the observed 100- to 1000-fold reduction
in replication. Block-release experiments with the antiviral
2-bromo-5,6-dichloro-1-(β-
d
-ribofuranosyl)-benzimidazole
revealed an important role for UL97 kinase in capsid assembly. Although
cleavage of concatemeric DNA intermediates to unit-length genomes
remained unaffected, progeny mutant virus maturation was delayed, with
accumulation of progeny at significantly reduced levels compared with
wild type after release of this block. Transmission electron microscopy
confirmed the aberrant accumulation of empty A-like capsids containing
neither viral DNA nor an internal scaffold structure, consistent with a
failure to stably package DNA in mutant virus-infected cells. The
function of UL97 in DNA synthesis as well as capsid assembly suggests
that protein phosphorylation mediated by this herpesvirus-conserved
kinase increases the efficiency of these two distinct phases of virus
replication.
The Nature and Functions of Meta-tags: Covert Infringement of Trademarks and Other Issues
Authors: Ramiscal Noel
                      Guivani
Published: July 2002

Meta-tags are one of the 'associational tools' considered essential to the operation of the World Wide Web. Their significance has been highlighted by recent controversies in the United States which focus on one of their important functions, i.e. as indices of websites which search engines read and rely upon in looking for the appropriate content relative to queries submitted to them. This article deals with the effect of meta-tags on the intellectual property rights of website owners. The author presents a critical survey of the American meta-tag cases and offers an Australian perspective.
Cultural Diversity and Australian Commercial Television Drama: Policy, Industry and Recent Research Contexts
Authors: May Harvey
Published: July 2002

Media studies research in the 1990s illustrated an incongruity between cultural diversity in the Australian community and the representation of that diversity on commercial television screens. Australian drama in particular received much criticism for its seemingly 'Anglo' portrayal of Australian society. Most of this former research was based on program content analysis and critical approaches to studying the media. From the mid-1990s, anecdotal evidence suggested an improvement in the casting of actors from culturally diverse backgrounds. This paper contextualises policy and industry developments in the 1990s related to cultural diversity and presents new research undertaken within the commercial television drama industry. In order to determine the status of cultural diversity and commercial television drama at the end of the 1990s, a casting survey of all Australian commercial drama programs broadcast in 1999 was carried out. This was complemented by interviews with industry personnel and a two-week content analysis of programming. The research establishes the degree of casting for actors from culturally diverse backgrounds and offers explanations for both improvements made over the previous years and the continuing obstacles faced by some groups in gaining a place in our popular drama programs.
An Information Infrastructure for Development
Authors: Lamberton Donald M.
Published: July 2002

The aim of this paper is to bring the challenge of an information perspective to bear on development issues and in particular on the role of telecommunications. In a too quiet revolution, information economics has lagged in the race for popular interest behind the brash 'new economy' rhetoric which argues that the intellectual problem is one of catching up with a new technology economy, i.e. a CISCO online economy that can exist miraculously and independently of supporting non-online activities, institutions and culture, rather than coping with the deeper socioeconomic issues that have long been eroding the theoretical foundations of economic theory, its touchstone of economic efficiency, and myriad policy prescriptions.
Responsible alcohol service: lessons from evaluations of server training and policing initiatives
Authors: STOCKWELL TIM
Published: July 2002

Responsible alcohol service programmes have evolved in many countries alongside a general increase in the availability of alcohol and a greater focus on the prevention of alcohol‐related road crashes. They also recognize the reality that a great deal of high‐risk drinking and preventable harm occurs in and around licensed premises or as drinkers make their way home. Early US efficacy studies of programmes which trained managers and barstaff to limit customers' levels of intoxication and prevent drink driving showed promise. Studies of effectiveness of these programmes in the wider community, and in the absence of the enforcement of liquor laws, found little benefit. The data will be interpreted as suggesting that, in reality, skills deficits in the serving of alcohol are not a significant problem compared with the motivational issue for a commercial operation of abiding by laws that are rarely enforced and which are perceived as risking the goodwill of their best customers. Australian, UK and US experiences with liquor law enforcement by police will be discussed along with outcomes from the Australian invention of Alcohol Accords, informal agreements between police, licensees and local councils to trade responsibly. It will be concluded that the major task involved in lifting standards of service and preventing harm is to institutionalize legal and regulatory procedures which impact most on licensed premises. A number of strategies are suggested also for creating a political and social climate which supports the responsible service of alcohol and thereby supports the enactment and enforcement of appropriate liquor laws.
Predictors of Nonadherence to HIV‐Related Medication Regimens During Methadone Stabilization
Authors: Avants S. Kelly , Margolin Arthur , Warburton Lara A. , Hawkins Keith A. , Shi Julia
Published: July 2002

Nonadherence to HIV‐related medication regimens among drug‐abusing patients decreases therapeutic effectiveness and may limit patient access to newer, highly active antiretroviral therapies (HAART). A number of factors have been associated with medication nonadherence; however, few studies have examined predictors of nonadherence specifically in HIV‐positive drug abusers. In the current study, a comprehensive assessment battery was administered to 42 HIV‐positive, injection drug users beginning methadone maintenance. HIV‐related medication adherence was assessed weekly by self‐report during the 4‐week methadone stabilization phase. Thirty‐six percent of patients reported less than 80% adherence to their medication regimen at entry into methadone. Medication adherence increased significantly during the 4‐week stabilization phase. Significant zero‐order correlations were found between nonadherence during stabilization and viral load, low educational attainment, depression, and neuropsychological tests of problem solving ability and cognitive flexibility. Independent predictors of nonadherence were low levels of education and poor emotional functioning. Implications for early intervention are discussed.
Monitoring the crack epidemic through urine testing: establishment of routine detection methods
Authors: Riley K. Jack , Lu Natalie T. , Meeker James E. , Lo Peter , Fortner Neil , Taylor Bruce G.
Published: July 2002

Abuse of crack continues to be of major concern. Because of limits to biological testing, self‐report has been the only mechanism for distinguishing crack use from powder use. Researchers have reported that smoking crack generates unique pyrolysis products that are detectable in urine, but no study has addressed how these products could be used as the marker for crack use, neither has any study addressed how reliable is the detection technology. The National Institute of Justice has developed a project to address these issues. The project consisted of (1) interviews conducted with and urine specimens collected from 2327 adult arrestees; (2) development and validation of procedures for routine GC/MS confirmation of crack use; and (3) establishment of standard profiles for two crack pyrolysis products, anhydroecgonine methylester (AEME) and ecgonidine (ECD). We found that AEME and ECD could be detected in urine specimens for periods of up to 40 hours. Most importantly, we demonstrated that to accurately measure crack use both AEME and ECD are necessary. Our results indicated that nearly 31% of the specimens were positive for undifferentiated cocaine, of which more than 88% were positive for crack. This resulted in crack prevalence rates of nearly 31% for females and 27% for males. These results will be used to further monitor the crack epidemic and to provide information that can inform the development of public policy as it relates to this drug.
COPI-independent Anterograde Transport: Cargo-selective ER to Golgi Protein Transport in Yeast COPI Mutants
Authors: Gaynor Erin C. , Emr Scott D.
Published: July 2002

The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.
Absence of the Mid-sized Neurofilament Subunit Decreases Axonal Calibers, Levels of Light Neurofilament (NF-L), and Neurofilament Content
Authors: Elder Gregory A. , Friedrich Victor L. , Bosco Paolo , Kang Chulho , Gourov Andrei , Tu Pang-Hsien , Lee Virginia M.-Y. , Lazzarini Robert A.
Published: July 2002

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.
minifly, A Drosophila Gene Required for Ribosome Biogenesis
Authors: Giordano Ennio , Peluso Ivana , Senger Stefania , Furia Maria
Published: July 2002

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.
A Role for Caveolin and the Urokinase Receptor in Integrin-mediated Adhesion and Signaling
Authors: Wei Ying , Yang Xiuwei , Liu Qiumei , Wilkins John A. , Chapman Harold A.
Published: July 2002

The assembly of signaling molecules surrounding the integrin family of adhesion receptors remains poorly understood. Recently, the membrane protein caveolin was found in complexes with β1 integrins. Caveolin binds cholesterol and several signaling molecules potentially linked to integrin function, e.g., Src family kinases, although caveolin has not been directly implicated in integrin-dependent adhesion. Here we report that depletion of caveolin by antisense methodology in kidney 293 cells disrupts the association of Src kinases with β1 integrins resulting in loss of focal adhesion sites, ligand-induced focal adhesion kinase (FAK) phosphorylation, and adhesion. The nonintegrin urokinase receptor (uPAR) associates with and stabilizes β1 integrin/caveolin complexes. Depletion of caveolin in uPAR-expressing 293 cells also disrupts uPAR/integrin complexes and uPAR-dependent adhesion. Further, β1 integrin/caveolin complexes could be disassociated by uPAR-binding peptides in both uPAR-transfected 293 cells and human vascular smooth muscle cells. Disruption of complexes by peptides in intact smooth muscle cells blocks the association of Src family kinases with β1 integrins and markedly impairs their migration on fibronectin. We conclude that ligand-induced signaling necessary for normal β1 integrin function requires caveolin and is regulated by uPAR. Caveolin and uPAR may operate within adhesion sites to organize kinase-rich lipid domains in proximity to integrins, promoting efficient signal transduction.
The Mitotic Phosphorylation Cycle of the Cis-Golgi Matrix Protein Gm130
Authors: Lowe Martin , Gonatas Nicholas K. , Warren Graham
Published: July 2002

The cis-Golgi matrix protein GM130 is phosphorylated in mitosis on serine 25. Phosphorylation inhibits binding to p115, a vesicle-tethering protein, and has been implicated as an important step in the mitotic Golgi fragmentation process. We have generated an antibody that specifically recognizes GM130 phosphorylated on serine 25, and used this antibody to study the temporal regulation of phosphorylation in vivo. GM130 is phosphorylated in prophase as the Golgi complex starts to break down, and remains phosphorylated during further breakdown and partitioning of the Golgi fragments in metaphase and anaphase. In telophase, GM130 is dephosphorylated as the Golgi fragments start to reassemble. The timing of phosphorylation and dephosphorylation correlates with the dissociation and reassociation of p115 with Golgi membranes. GM130 phosphorylation and p115 dissociation appear specific to mitosis, since they are not induced by several drugs that trigger nonmitotic Golgi fragmentation. The phosphatase responsible for dephosphorylation of mitotic GM130 was identified as PP2A. The active species was identified as heterotrimeric phosphatase containing the Bα regulatory subunit, suggesting a role for this isoform in the reassembly of mitotic Golgi membranes at the end of mitosis.
Coordinate Regulation of Cadherin and Integrin Function by the Chondroitin Sulfate Proteoglycan Neurocan
Authors: Li Hedong , Leung Tin-Chung , Hoffman Stanley , Balsamo Janne , Lilien Jack
Published: July 2002

N-cadherin and β1-integrins play decisive roles in morphogenesis and neurite extension and are often present on the same cell. Therefore, the function of these two types of adhesion systems must be coordinated in time and space to achieve the appropriate cell and tissue organization. We now show that interaction of the chondroitin sulfate proteoglycan neurocan with its GalNAcPTase receptor coordinately inhibits both N-cadherin– and β1-integrin–mediated adhesion and neurite outgrowth. Furthermore, the inhibitory activity is localized to an NH2-terminal fragment of neurocan containing an Ig loop and an HA-binding domain. The effect of neurocan on β1-integrin function is dependent on a signal originating from the cadherin cytoplasmic domain, possibly mediated by the nonreceptor protein tyrosine kinase Fer, indicating that cadherin and integrin engage in direct cross-talk. In the developing chick, neural retina neurocan is present in the inner plexiform layer from day 7 on, and the GalNAcPTase receptor becomes restricted to the inner nuclear layer and the ganglion cell layer (as well as the fiber layer), the two forming a sandwich. These data suggest that the coordinate inhibition of cadherin and integrin function on interaction of neurocan with its receptor may prevent cell and neurite migration across boundaries.