bluuebunny/crossref_metadata_2025 huggingface dataset:
Haploinsufficiency of
Sox9
results in defective cartilage primordia and premature skeletal mineralization
Authors: Bi Weimin , Huang Wendong , Whitworth Deanne J. , Deng Jian Min , Zhang Zhaoping , Behringer Richard R. , de Crombrugghe Benoit
Published: July 2002

In humans,
SOX9
heterozygous mutations cause the
severe skeletal dysmorphology syndrome campomelic dysplasia. Except for
clinical descriptions, little is known about the pathogenesis of this
disease. We have generated heterozygous
Sox9
mutant mice
that phenocopy most of the skeletal abnormalities of this syndrome. The
Sox9
+/−
mice died perinatally with cleft
palate, as well as hypoplasia and bending of many skeletal structures
derived from cartilage precursors. In embryonic day (E)14.5
heterozygous embryos, bending of radius, ulna, and tibia cartilages was
already prominent. In E12.5 heterozygotes, all skeletal elements
visualized by using Alcian blue were smaller. In addition, the overall
levels of
Col2a1
RNA at E10.5 and E12.5 were lower than
in wild-type embryos. We propose that the skeletal abnormalities
observed at later embryonic stages were caused by delayed or defective
precartilaginous condensations. Furthermore, in E18.5 embryos and in
newborn heterozygotes, premature mineralization occurred in many bones,
including vertebrae and some craniofacial bones. Because
Sox9
is not expressed in the mineralized portion of the
growth plate, this premature mineralization is very likely the
consequence of allele insufficiency existing in cells of the growth
plate that express
Sox9
. Because the hypertrophic zone
of the heterozygous
Sox9
mutants was larger than that of
wild-type mice, we propose that
Sox9
also has a role in
regulating the transition to hypertrophic chondrocytes in the growth
plate. Despite the severe hypoplasia of cartilages, the overall
organization and cellular composition of the growth plate were
otherwise normal. Our results suggest the hypothesis that two critical
steps of the chondrocyte differentiation pathway are sensitive to
Sox9
dosage. First, an early step presumably at the
stage of mesenchymal condensation of cartilage primordia, and
second, a later step preceding the transition of chondrocytes into
hypertrophic chondrocytes.
Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor
Authors: Lewis K. , Li C. , Perrin M. H. , Blount A. , Kunitake K. , Donaldson C. , Vaughan J. , Reyes T. M. , Gulyas J. , Fischer W. , Bilezikjian L. , Rivier J. , Sawchenko P. E. , Vale W. W.
Published: July 2002

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.
Increased sexual activity reduces male immune function inDrosophila melanogaster
Authors: McKean Kurt A. , Nunney Leonard
Published: July 2002

Despite the benefits of resistance, susceptibility to infectious disease is commonplace. Although specific susceptibility may be considered an inevitable consequence of the co-evolutionary arms race between parasite and host, a more general constraint may arise from the cost of an immune response. This “cost” hypothesis predicts a tradeoff between immune defense and other components of fitness. In particular, a tradeoff between immunity and sexually selected male behavior has been proposed. Here we provide experimental support for the direct phenotypic tradeoff between sexual activity and immunity by studying the antibacterial immune response inDrosophila melanogaster. Males exposed to more females showed a reduced ability to clear a bacterial infection, an effect that we experimentally link to changes in sexual activity. Our results suggest immunosuppression is an important cost of reproduction and that immune function and levels of disease susceptibility will be influenced by sexual selection.
CD40 stimulation accelerates deletion of tumor-specific CD8+T cells in the absence of tumor-antigen vaccination
Authors: Kedl Ross M. , Jordan Michael , Potter Terence , Kappler John , Marrack Philippa , Dow Steven
Published: July 2002

Previous work has established a role for CD40-mediated signals in eliciting helper-dependent CD8+T cell responses. Here we investigated the effects ofin vivoCD40 stimulation on the survival and function of tumor-specific CD8+T cells in a mouse melanoma model system. We found that agonistic anti-CD40 antibody treatment alone of tumor-bearing mice accelerated the deletion of tumor-antigen-specific T cells. However, long-term survival and function of tumor-antigen-specific T cells could be achieved when viral immunization with tumor antigen and anti-CD40 treatment were combined. This rescue of CD8+T cells could not be easily replicated by inflammatory or antigen-specific stimuli alone, demonstrating the specificity of signals that regulate the deletion or survival of tumor-specific T cells. These results demonstrate that opposing effects can be elicited by CD40 stimulationin vivoand suggest the need for caution in using this treatment for cancer patients.
Prostitution and the sex discrepancy in reported number of sexual partners
Authors: Brewer Devon D. , Potterat John J. , Garrett Sharon B. , Muth Stephen Q. , Roberts John M. , Kasprzyk Danuta , Montano Daniel E. , Darrow William W.
Published: July 2002

One of the most reliable and perplexing findings from surveys of
sexual behavior is that men report substantially more sexual partners
than women do. We use data from national sex surveys and studies of
prostitutes and their clients in the United States to examine sampling
bias as an explanation for this disparity. We find that prostitute
women are underrepresented in the national surveys. Once their
undersampling and very high numbers of sexual partners are factored in,
the discrepancy disappears. Prostitution's role in the discrepancy is
not readily apparent because men are reluctant to acknowledge that
their reported partners include prostitutes.
Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene
SEMA3B
Authors: Tomizawa Yoshio , Sekido Yoshitaka , Kondo Masashi , Gao Boning , Yokota Jun , Roche Joëlle , Drabkin Harry , Lerman Michael I. , Gazdar Adi F. , Minna John D.
Published: July 2002

Semaphorins
SEMA3B
and its homologue
SEMA3F
are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of
SEMA3B
and
SEMA3F
, we transfected them into lung cancer NCI-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type
SEMA3B
compared with the control vector. By contrast, only 30–40% reduction in colony formation was seen after the transfection of
SEMA3F
or
SEMA3B
variants carrying lung cancer-associated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant
SEMA3B
underwent apoptosis. We found that lung cancers (
n
= 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with
SEMA3B
and
SEMA3F
and found that medium from wild-type
SEMA3B
transfectants reduced the growth of several lung cancer lines 30–90%, whereas
SEMA3B
mutants or
SEMA3F
had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the
SEMA3B
5′ region of lung cancers not expressing S
EMA3B
but no methylation in
SEMA3B
-expressing tumors. These results are consistent with
SEMA3B
functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.
Activation of the epsilon isoform of protein kinase C in the mammalian nerve terminal
Authors: Saitoh Naoto , Hori Tetsuya , Takahashi Tomoyuki
Published: July 2002

Activation of protein kinase C (PKC) by phorbol ester facilitates hormonal secretion and transmitter release, and phorbol ester-induced synaptic potentiation (PESP) is a model for presynaptic facilitation. A variety of PKC isoforms are expressed in the central nervous system, but the isoform involved in the PESP has not been identified. To address this question, we have applied immunocytochemical and electrophysiological techniques to the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of rat auditory brainstem. Western blot analysis indicated that both the Ca
2+
-dependent “conventional” PKC and Ca
2+
-independent “novel” PKC isoforms are expressed in the MNTB. Denervation of afferent fibers followed by organotypic culture, however, selectively decreased “novel” ɛPKC isoform expressed in this region. The afferent calyx terminal was clearly labeled with the ɛPKC immunofluorescence. On stimulation with phorbol ester, presynaptic ɛPKC underwent autophosphorylation and unidirectional translocation toward the synaptic side. Chelating presynaptic Ca
2+
, by using membrane permeable EGTA analogue or high concentration of EGTA directly loaded into calyceal terminals, had only a minor attenuating effect on the PESP. We conclude that the Ca
2+
-independent ɛPKC isoform mediates the PESP at this mammalian central nervous system synapse.
Long-term microclimatic stress causes rapid adaptive radiation of
kaiABC
clock gene family in a cyanobacterium,
Nostoc linckia
, from “Evolution Canyons” I and II, Israel
Authors: Dvornyk Volodymyr , Vinogradova Oxana , Nevo Eviatar
Published: July 2002

Cyanobacteria are the only prokaryotes known thus far possessing regulation of physiological functions with approximate daily periodicity, or circadian rhythms, that are controlled by a cluster of three genes,
kaiA
,
kaiB
, and
kaiC
. Here we demonstrate considerably higher genetic polymorphism and extremely rapid evolution of the
kaiABC
gene family in a filamentous cyanobacterium,
Nostoc linckia
, permanently exposed to the acute natural environmental stress in the two microsite evolutionary models known as “Evolution Canyons,” I (Mount Carmel) and II (Upper Galilee) in Israel. The family consists of five distinct subfamilies (
kaiI–kaiV
) comprising at least 20 functional genes and pseudogenes. The obtained data suggest that the duplications of
kai
genes have adaptive significance, and some of them are evolutionarily quite recent (≈80,000 years ago). The observed patterns of within- and between-subfamily polymorphisms indicate that positive diversifying, balancing, and purifying selections are the principal driving forces of the
kai
gene family's evolution.
Conscious recollection and the human hippocampal formation: evidence from positron emission tomography.
Authors: Schacter D L , Alpert N M , Savage C R , Rauch S L , Albert M S
Published: July 2002

We used positron emission tomography (PET) to examine the role of the hippocampal formation in implicit and explicit memory. Human volunteers studied a list of familiar words, and then they either provided the first word that came to mind in response to three-letter cues (implicit memory) or tried to recall studied words in response to the same cues (explicit memory). There was no evidence of hippocampal activation in association with implicit memory. However, priming effects on the implicit memory test were associated with decreased activity in extrastriate visual cortex. On the explicit memory test, subjects recalled many target words in one condition and recalled few words in a second condition, despite trying to remember them. Comparisons between the two conditions showed that blood-flow increases in the hippocampal formation are specifically associated with the conscious recollection of studied words, whereas blood-flow increases in frontal regions are associated with efforts to retrieve target words. Our results help to clarify some puzzles concerning the role of the hippocampal formation in human memory.
Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.
Authors: Kutchera W , Jones D A , Matsunami N , Groden J , McIntyre T M , Zimmerman G A , White R L , Prescott S M
Published: July 2002

Evidence from epidemiological studies, clinical trials, and animal experiments indicates that inhibitors of prostaglandin synthesis lower the risk of colon cancer. We tested the hypothesis that abnormal expression of prostaglandin H synthase 2 (PHS-2), which can be induced by oncogenes and tumor promoters, occurs during colon carcinogenesis by examining its level in colon tumors. Human colon cancers were found to have an increased expression of PHS-2 mRNA compared with normal colon specimens from the same patient (n = 5). In situ hybridization showed that the neoplastic colonocytes had increased expression of PHS-2 (n = 4). Additionally, five colon cancer cell lines were shown to express high levels of PHS-2 mRNA even in the absence of a known inducer of PHS-2. To study the basis for this increased gene expression, we transfected a colon cancer cell line, HCT-116, with a reporter gene containing 2.0 kb of the 5' regulatory sequence of the PHS-2 gene. Constitutive transcription of the reporter gene was observed, whereas normal control cell lines transcribed the reporter only in response to an exogenous agonist. We conclude that PHS-2 is transcribed abnormally in human colon cancers and that this may be one mechanism by which prostaglandins or related compounds that support carcinogenesis are generated.
Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.
Authors: Morimoto K , Patel M , Corisdeo S , Hooper D C , Fu Z F , Rupprecht C E , Koprowski H , Dietzschold B
Published: July 2002

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.
Disruption of the MacMARCKS gene prevents cranial neural tube closure and results in anencephaly.
Authors: Chen J , Chang S , Duncan S A , Okano H J , Fishell G , Aderem A
Published: July 2002

MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. Biochemical evidence demonstrates that these proteins integrate calcium and PKC-dependent signals to regulate actin structure at the membrane. We report here that deletion of the MacMARCKS gene prevents cranial neural tube closure in the developing brain, resulting in anencephaly. This suggests a central role for MacMARCKS and the PKC signal transduction pathway in the folding of the anterior neural plate during the early phases of brain formation, and supports the hypothesis that actin-based motility directs cranial neural tube closure.
IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.
Authors: Lan M S , Wasserfall C , Maclaren N K , Notkins A L
Published: July 2002

IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 50 from age-matched normal controls. Sixty-six percent of the sera from patients, but none of the sera from controls, reacted with IA-2. The same diabetic sera tested for autoantibodies to islet cells (ICA) by indirect immunofluorescence and glutamic acid decarboxylase (GAD65Ab) by depletion ELISA showed 68% and 52% positivity, respectively. Up to 86% of the IDDM patients had autoantibodies to IA-2 and/or GAD65. Moreover, greater than 90% (14 of 15) of the ICA-positive but GAD65Ab-negative sera had autoantibodies to IA-2. Absorption experiments showed that the immunofluorescence reactivity of ICA-positive sera was greatly reduced by prior incubation with recombinant IA-2 or GAD65 when the respective antibody was present. A little over one-half (9 of 16) of the IDDM sera that were negative for ICA were found to be positive for autoantibodies to IA-2 and/or GAD65, arguing that the immunofluorescence test for ICA is less sensitive than the recombinant tests for autoantibodies to IA-2 and GAD65. It is concluded that IA-2 is a major islet cell autoantigen in IDDM, and, together with GAD65, is responsible for much of the reactivity of ICA with pancreatic islets. Tests for the detection of autoantibodies to recombinant IA-2 and GAD65 may eventually replace ICA immunofluorescence for IDDM population screening.
Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice.
Authors: Reiss K , Cheng W , Ferber A , Kajstura J , Li P , Li B , Olivetti G , Homcy C J , Baserga R , Anversa P
Published: July 2002

Transgenic mice were generated in which the cDNA for the human insulin-like growth factor 1B (IGF-1B) was placed under the control of a rat alpha-myosin heavy chain promoter. In mice heterozygous for the transgene, IGF-1B mRNA was not detectable in the fetal heart at the end of gestation, was present in modest levels at 1 day after birth, and increased progressively with postnatal maturation, reaching a peak at 75 days. Myocytes isolated from transgenic mice secreted 1.15 +/- 0.25 ng of IGF-1 per 10(6) cells per 24 hr versus 0.27 +/- 0.10 ng in myocytes from homozygous wild-type littermates. The plasma level of IGF-1 increased 84% in transgenic mice. Heart weight was comparable in wild-type littermates and transgenic mice up to 45 days of age, but a 42%, 45%, 62%, and 51% increase was found at 75, 135, 210, and 300 days, respectively, after birth. At 45, 75, and 210 days, the number of myocytes in the heart was 21%, 31%, and 55% higher, respectively, in transgenic animals. In contrast, myocyte cell volume was comparable in transgenic and control mice at all ages. In conclusion, overexpression of IGF-1 in myocytes leads to cardiomegaly mediated by an increased number of cells in the heart.
Computational models of cortical visual processing.
Authors: Heeger D J , Simoncelli E P , Movshon J A
Published: July 2002

The visual responses of neurons in the cerebral cortex were first adequately characterized in the 1960s by D. H. Hubel and T. N. Wiesel [(1962) J. Physiol. (London) 160, 106-154; (1968) J. Physiol. (London) 195, 215-243] using qualitative analyses based on simple geometric visual targets. Over the past 30 years, it has become common to consider the properties of these neurons by attempting to make formal descriptions of these transformations they execute on the visual image. Most such models have their roots in linear-systems approaches pioneered in the retina by C. Enroth-Cugell and J. R. Robson [(1966) J. Physiol. (London) 187, 517-552], but it is clear that purely linear models of cortical neurons are inadequate. We present two related models: one designed to account for the responses of simple cells in primary visual cortex (V1) and one designed to account for the responses of pattern direction selective cells in MT (or V5), an extrastriate visual area thought to be involved in the analysis of visual motion. These models share a common structure that operates in the same way on different kinds of input, and instantiate the widely held view that computational strategies are similar throughout the cerebral cortex. Implementations of these models for Macintosh microcomputers are available and can be used to explore the models' properties.
Channel behavior in a gamma-aminobutyrate transporter.
Authors: Cammack J N , Schwartz E A
Published: July 2002

Current produced by a gamma-aminobutyrate (GABA) transporter stably transfected into a mammalian cell line was observed in cell-attached and excised membrane patches. When GABA was absent, a fraction of the transporters produced cation-permeable channels. When GABA plus Na+ was on either side of the membrane, the majority of transporters produced a high-frequency current noise attributed to the movement of ions in an occluded pore.
An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.
Authors: Anderson J S , Teutsch M , Dong Z , Wortis H H
Published: July 2002

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.
A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.
Authors: Mebius R E , Streeter P R , Michie S , Butcher E C , Weissman I L
Published: July 2002

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.
Peptide nucleic acid pre-gel hybridization: An alternative to Southern hybridization
Authors: Perry-O’Keefe Heather , Yao Xian-Wei , Coull James M. , Fuchs Martin , Egholm Michael
Published: July 2002

We have found that it is possible to use labeled peptide nucleic
acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as
an alternative for Southern hybridization. In this technique, the PNA
probe is hybridized to a denatured DNA sample at low ionic strength and
the mixture is loaded directly on to an electrophoresis system for size
separation. Ensuing gel electrophoresis separates the single-stranded
DNA fragments by length. The neutral backbone of PNA allows for
hybridization at low ionic strength and imparts very low mobility to
excess PNA. Detection of the bound PNA is possible by direct
fluorescence detection with capillary electrophoresis, or the DNA/PNA
hybrids can be blotted onto a membrane and detected with standard
chemiluminescent techniques. Efficient single bp discrimination was
achieved routinely using both capillary and slab-gel electrophoresis.
Iron-sulfur cluster disassembly in the FNR protein of
Escherichia coli
by O
2
: [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity
Authors: Khoroshilova Natalia , Popescu Codrina , Münck Eckard , Beinert Helmut , Kiley Patricia J.
Published: July 2002

The transcription factor FNR (
f
umarate
n
itrate
r
eduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in
Escherichia coli
when oxygen becomes scarce. To define the oxidation state and type of Fe-S cluster present in the active form of FNR, we have studied anaerobically purified FNR with Mössbauer spectroscopy. Our data showed that this form of FNR contained a [4Fe-4S]
2+
cluster (δ = 0.45 mm/s; Δ
E
Q
= 1.22 mm/s) and that the [4Fe-4S]
2+
cluster was rapidly destroyed on exposure of FNR to air. Under these conditions, the yellow–green active form of FNR turned deep red; analysis of sulfide indicated that 70% of the labile sulfide was still present, suggesting that the Fe-S cluster had been converted into a different form. Little [3Fe-4S] cluster was, however, detected by EPR. According to Mössbauer spectroscopy, the [4Fe-4S]
2+
cluster was converted in about 60% yield to a [2Fe-2S]
2+
cluster (δ = 0.28 mm/s; Δ
E
Q
= 0.58 mm/s) following 17 min of exposure to air. The [2Fe-2S]
2+
cluster form of FNR was much more stable to oxygen, but was unable to sustain biological activity (e.g., DNA binding). However, DNA binding and the absorption spectrum characteristic of the [4Fe-4S]
2+
cluster could be largely restored from the [2Fe-2S]
2+
form when Cys, Fe, DTT, and the NifS protein were added. It has yet to be determined whether the form of FNR containing the [2Fe-2S]
2+
cluster has any biological significance, e.g., as an
in vivo
intermediate that is more rapidly converted to the active form than the apoprotein.
Factors controlling the competition among rotational and vibrational energy transfer channels in glyoxal
Authors: Parmenter Charles S. , Clegg Samuel M. , Krajnovich Douglas J. , Lu Shao-ping
Published: July 2002

The state-to-state transfer of rotational and vibrational energy has been studied for
S
1
glyoxal (CHOCHO) in collisions with D
2
, N
2
, CO and C
2
H
4
using crossed molecular beams. A laser is used to pump glyoxal seeded in He to its
S
1
zero point level with zero angular momentum about its top axis (
K
′ = 0). The inelastic scattering to each of at least 26
S
1
glyoxal rotational and rovibrational levels is monitored by dispersed
S
1
–
S
0
fluorescence. Various collision partners are chosen to investigate the relative influences of reduced mass and the collision pair interaction potential on the competition among the energy transfer channels. When the data are combined with that obtained previously from other collision partners whose masses range from 2 to 84 amu, it is seen that the channel competition is controlled primarily by the kinematics of the collisional interaction. Variations in the intermolecular potential play strictly a secondary role.
Major proteinase movement upon stable serpin–proteinase complex formation
Authors: Stratikos Efstratios , Gettins Peter G. W.
Published: July 2002

To determine whether formation of the stable complex between a serpin and a target proteinase involves a major translocation of the proteinase from its initial position in the noncovalent Michaelis complex, we have used fluorescence resonance energy transfer to measure the separation between fluorescein attached to a single cysteine on the serpin and tetramethylrhodamine conjugated to the proteinase. The interfluorophore separation was determined for the noncovalent Michaelis-like complex formed between α
1
-proteinase inhibitor (Pittsburgh variant) and anhydrotrypsin and for the stable complex between the same serpin and trypsin. A difference in separation between the two fluorophores of ≈21 Å was found for the two types of complex. This demonstrates a major movement of the proteinase in going from the initial noncovalent encounter complex to the kinetically stable complex. The change in interfluorophore separation is most readily understood in terms of movement of the proteinase from the reactive center end of the serpin toward the distal end, as the covalently attached reactive center loop inserts into β-sheet A of the serpin.
Induction of long-term depression and rebound potentiation by inositol trisphosphate in cerebellar Purkinje neurons
Authors: Khodakhah Kamran , Armstrong Clay M.
Published: July 2002

Cerebellar Purkinje neurons receive two major excitatory inputs, the climbing fibers (CFs) and parallel fibers (PFs). Simultaneous, repeated activation of CFs and PFs results in the long-term depression (LTD) of the amplitude of PF-evoked synaptic currents. To induce LTD, activation of CFs may be substituted with depolarization of the Purkinje neuron to turn on voltage-activated calcium channels and increase the intracellular calcium concentration. The role of PFs in the induction of LTD, however, is less clear. PFs activate glutamate metabotropic receptors that increase phosphoinositide turnover and elevate cytosolic inositol 1,4,5-trisphosphate (InsP
3
). It has been proposed that calcium release from intracellular stores via InsP
3
receptors may be important in the induction of LTD. We studied the role of InsP
3
in the induction of LTD by photolytic release of InsP
3
from its biologically inactive “caged” precursor in voltage-clamped Purkinje neurons in acutely prepared cerebellar slices. We find that InsP
3
-evoked calcium release is as effective in LTD induction as activation of PFs. InsP
3
-induced LTD was prevented by calcium chelator 1,2-bis(2-amino phenoxy)ethane-
N
,
N
,
N
′,
N
′-tetraacetic acid. LTD produced either by repeated activation of PFs combined with depolarization (PF+ΔV), or by InsP
3
combined with depolarization (InsP
3
+ΔV) saturated at ≈50%. Maximal LTD induced by PF+ΔV could not be further increased by InsP
3
+ΔV and vice versa, which suggests that both protocols for induction of LTD share a common path. In addition to inducing LTD, photo-release of InsP
3
+ΔV resulted in the rebound potentiation of inhibitory synaptic currents. In the presence of heparin, an InsP
3
receptor antagonist, repeated activation of PF+ΔV failed to induce LTD, suggesting that InsP
3
receptors play an important role in LTD induction under physiological conditions.
Top-down versus bottom-up and the Ruritanian bean bug
Authors: Hassell M. P. , Crawley M. J. , Godfray H. C. J. , Lawton J. H.
Published: July 2002

In a recent article, Hunter uses the late George Varley and George Gradwell’s long-term data on the winter moth (Operophtera brumata) and green tortrix (Tortrix viridana) populations to propose a method of quantifying the relative importance of top-down effects (because of natural enemies) and bottom-up effects (because of resource competition) in influencing population dynamics. We believe this approach is deeply flawed. Using Varley and Gradwell’s winter moth study, we show that the problems with Hunter’s analysis lie in his misinterpretation of the population dynamics and his inappropriate use of statistical techniques. We also emphasize the importance of distinguishing clearly between two quite different things: firstly, top-down and bottom-up regulation of populations and secondly, the much simpler task of categorizing factors affecting changes in population density as either top-down or bottom-up processes.
The influence of depicted illumination on brightness
Authors: Williams S. Mark , McCoy Allison N. , Purves Dale
Published: July 2002

The striking illusions produced by simultaneous brightness contrast generally are attributed to the center-surround receptive field organization of lower order neurons in the primary visual pathway. Here we show that the apparent brightness of test objects can be either increased or decreased in a predictable manner depending on how light and shadow are portrayed in the scene. This evidence suggests that perceptions of brightness are generated empirically by experience with luminance relationships, an idea whose implications we pursue in the accompanying paper.
Rendering the inedible edible: Circumvention of a millipede’s chemical defense by a predaceous beetle larva (Phengodidae)
Authors: Eisner Thomas , Eisner Maria , Attygalle Athula B. , Deyrup Mark , Meinwald Jerrold
Published: July 2002

The larva of the phengodid beetle,
Phengodes laticollis
, feeds on the millipede,
Floridobolus penneri
, without risking exposure to the repellent benzoquinones ordinarily ejected by the millipede from its defensive glands when attacked. The phengodid subdues the millipede by piercing the millipede’s integument with its hollow sickle-shaped mandibles and apparently injecting gastric fluid. The infusion abruptly paralyzes the millipede, which thereby is prevented from discharging its glands. As the phengodid then imbibes the liquefied systemic contents of the dead millipede, the millipede’s benzoquinones remain harmlessly confined to the glands, prevented from diffusing into the millipede’s body cavity by the glands’ impervious cuticular lining. At the end of the meal only the millipede’s skeletal armor and glandular sacs remain uneaten. Analysis of such discarded sacs showed these to contain benzoquinones in amounts commensurate with those present in replete glands of living millipedes.
Solution structure and peptide binding studies of the C-terminal Src homology 3-like domain of the diphtheria toxin repressor protein
Authors: Wang Guangshun , Wylie Gregory P. , Twigg Pamela D. , Caspar Donald L. D. , Murphy John R. , Logan Timothy M.
Published: July 2002

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe
2+
binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five β-strands and three helices arranged into a partially orthogonal, two-sheet β-barrel, similar to the structure observed in the crystalline Co
2+
complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in β-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.
Mapping the sensitivity of T cells with an optical trap: Polarity and minimal number of receptors for Ca2+signaling
Authors: Wei Xunbin , Tromberg Bruce J. , Cahalan Michael D.
Published: July 2002

Contact with antigen-presenting cells (APCs) initiates an activation cascade within T lymphocytes, including a rise in cytosolic calcium, lymphokine production, and cell division. Although T cell–APC physical contact is required for an immune response, little is known about the patterns of cellular interactions and their relation to activation. Calcium imaging combined with an optical trap enabled the T cell contact requirements and polarity to be investigated at the single-cell level. APCs or anti-CD3 mAb-coated beads were trapped with a laser and placed at different locations along the T cell, which has a polarized appearance defined by the shape and direction of crawling. T cells were 3-fold more sensitive to APC contact made at the leading edge of the T cell than with contact made at the tail. Anti-CD3 mAb-coated 6-μm beads induced calcium signaling with ≈10-fold higher frequency and ≈4-fold shorter latency on contact with the leading edge of the T cell than on contact with the trailing edge. Alterations in antibody density (2 to 500 per μm2) and bead size (1 to 6 μm in diameter) were used to determine the spatial requirements and the minimal number of receptors which must be engaged to transmit a positive signal. T cell response percentage, latency, and calcium-signaling pattern (transient vs. sustained or oscillatory) depended on antibody density on the bead. The presence of ≈170 anti-CD3 mAb within the contact area elicited a detectable T cell calcium response. We propose here that engagement of no more than 340 T cell receptors (≈1% of the total on the cell) is sufficient to initiate Ca2+signaling. The minimal contact area was ≈3 μm2.
Messenger RNA translation state: The second dimension of high-throughput expression screening
Authors: Zong Qin , Schummer Michèl , Hood Leroy , Morris David R.
Published: July 2002

Technological advances over the past 10 years have generated powerful tools for parallel analysis of complex biological problems. Among these new technologies, DNA arrays have provided an important experimental approach for identifying changes in the levels of individual mRNA molecules during important cellular transitions. However, cellular behavior is dictated not by mRNA levels, but by the proteins translated from the individual mRNA species. We report a high-throughput method for simultaneously monitoring the translation state and level of individual mRNA species. Messenger RNAs from resting and mitogenically activated fibroblasts were separated, according to degree of ribosome loading, into well-translated and under-translated pools. cDNA probes generated from these fractions were used to interrogate cDNA arrays. Among approximately 1,200 genes analyzed, less than 1% were found to be translationally regulated in response to mitogenic activation, demonstrating the strong selectivity of this regulatory mechanism. This high-throughput approach is shown to be an effective tool for superimposing translation profile on mRNA level for large numbers of genes, as well as for identifying translationally regulated genes for further study.
Endogenous activation of metabotropic glutamate receptors in neocortical development causes neuronal calcium oscillations
Authors: Flint Alexander C. , Dammerman Ryan S. , Kriegstein Arnold R.
Published: July 2002

Oscillations in intracellular free calcium concentration ([Ca
2+
]
i
) occur spontaneously in immature neurons of the developing cerebral cortex. Here, we show that developing murine cortical neurons exhibit calcium oscillations in response to direct activation of the mGluR5 subtype of the group I metabotropic glutamate receptor (mGluR). In contrast, other manipulations that elicit [Ca
2+
]
i
increases produce simple, nonoscillatory changes. Furthermore, we find that spontaneous oscillatory [Ca
2+
]
i
activity is blocked by antagonists of group I mGluRs, suggesting a specific role for mGluR activation in the promotion of oscillatory [Ca
2+
]
i
dynamics in immature cortical neurons. The oscillatory pattern of [Ca
2+
]
i
increases produced by mGluR activation might play a role in the regulation of gene expression and the control of developmental events.
Kinetic equilibrium of forces and molecular events in muscle contraction
Authors: Becker Erwin W.
Published: July 2002

In biomolecular systems, the mechanical transfer of free energy occurs with both high efficiency and high speed. It is shown here that such a transfer can be achieved only if the participating free-energy-storing elements exhibit opposing relationships between their content of free energy and the force they exert in the transfer direction. A kinetic equilibrium of forces (KEF) results, in which the transfer of free energy is mediated essentially by thermal molecular motion. On the basis of present evidence, KEF is used as a guiding principle in developing a mechanical model of the crossbridge cycle in muscle contraction. The model allows the basic features of molecular events to be visualized in terms of plausible structures. Real understanding of the process will require identification of the elements that perform the functions described here. Besides chemomechanical energy transduction, KEF may have a role in other biomolecular processes in which free energy is transferred mechanically over large distances.
Selective disruption of protein aggregation by cyclodextrin dimers
Authors: Leung David K. , Yang Zhiwei , Breslow Ronald
Published: July 2002

β-Cyclodextrin (CD) dimers (
n
= 11) were synthesized and tested against eight enzymes, seven of which were dimeric or tetrameric, for inhibitor activity. Initial screening showed that only
l
-lactate dehydrogenase and citrate synthase were inhibited but only by two specific CD dimers in which two β-CDs were linked on the secondary face by a pyridine-2,6-dicarboxylic group. Further investigation suggested that these CD dimers inhibit the activity of
l
-lactate dehydrogenase and citrate synthase at least in part by disruption of protein–protein aggregation.
Localization of α1,3-fucosyltransferase VI in Weibel–Palade bodies of human endothelial cells
Authors: Schnyder-Candrian Silvia , Borsig Lubor , Moser René , Berger Eric G.
Published: July 2002

Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked α1→3 to
N-
acetylglucosamine. A family of α1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription–PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with β4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel–Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel–Palade bodies suggests a function independent of Golgi-associated glycosylation.