bluuebunny/crossref_metadata_2025 huggingface dataset:
EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract
Authors: Dottori Mirella , Hartley Lynne , Galea Mary , Paxinos George , Polizzotto Mark , Kilpatrick Trevor , Bartlett Perry F. , Murphy Mark , Köntgen Frank , Boyd Andrew W.
Published: July 2002

Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.
A novel structure involved in the formation of liver endothelial cell fenestrae revealed by using the actin inhibitor misakinolide
Authors: Braet Filip , Spector Ilan , De Zanger Ronald , Wisse Eddie
Published: July 2002

Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.
Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA
Authors: Waterhouse Peter M. , Graham Michael W. , Wang Ming-Bo
Published: July 2002

Many examples of extreme virus resistance and posttranscriptional gene silencing of endogenous or reporter genes have been described in transgenic plants containing sense or antisense transgenes. In these cases of either cosuppression or antisense suppression, there appears to be induction of a surveillance system within the plant that specifically degrades both the transgene and target RNAs. We show that transforming plants with virus or reporter gene constructs that produce RNAs capable of duplex formation confer virus immunity or gene silencing on the plants. This was accomplished by using transcripts from one sense gene and one antisense gene colocated in the plant genome, a single transcript that has self-complementarity, or sense and antisense transcripts from genes brought together by crossing. A model is presented that is consistent with our data and those of other workers, describing the processes of induction and execution of posttranscriptional gene silencing.
Transgenic cattle produced by reverse-transcribed gene transfer in oocytes
Authors: Chan Anthony W. S. , Homan E. Jane , Ballou Linda U. , Burns Jane C. , Bremel Robert D.
Published: July 2002

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.
Suppression of protein synthesis in brain during hibernation involves inhibition of protein initiation and elongation
Authors: Frerichs Kai U. , Smith Carolyn B. , Brenner Michael , DeGracia Donald J. , Krause Gary S. , Marrone Laura , Dever Thomas E. , Hallenbeck John M.
Published: July 2002

Protein synthesis (PS) has been considered essential to sustain mammalian life, yet was found to be virtually arrested for weeks in brain and other organs of the hibernating ground squirrel,
Spermophilus tridecemlineatus
. PS,
in vivo
, was below the limit of autoradiographic detection in brain sections and, in brain extracts, was determined to be 0.04% of the average rate from active squirrels. Further, it was reduced 3-fold in cell-free extracts from hibernating brain at 37°C, eliminating hypothermia as the only cause for protein synthesis inhibition (active, 0.47 ± 0.08 pmol/mg protein per min; hibernator, 0.16 ± 0.05 pmol/mg protein per min,
P
< 0.001). PS suppression involved blocks of initiation and elongation, and its onset coincided with the early transition phase into hibernation. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2α are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a 3-fold increase in ribosomal mean transit times in cell-free extracts from hibernators (active, 2.4 ± 0.7 min; hibernator, 7.1 ± 1.4 min,
P
< 0.001). No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate “trickle” blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state.
The neuropeptide Y/agouti gene-related protein (AGRP) brain circuitry in normal, anorectic, and monosodium glutamate-treated mice
Authors: Broberger Christian , Johansen Jeanette , Johansson Carolina , Schalling Martin , Hökfelt Tomas
Published: July 2002

Neuropeptide Y (NPY) and the endogenous melanocortin receptor antagonist, agouti gene-related protein (AGRP), coexist in the arcuate nucleus, and both exert orexigenic effects. The present study aimed primarily at determining the brain distribution of AGRP. AGRP mRNA-expressing cells were limited to the arcuate nucleus, representing a major subpopulation (95%) of the NPY neurons, which also was confirmed with immunohistochemistry. AGRP-immunoreactive (-ir) terminals all contained NPY and were observed in many brain regions extending from the rostral telencephalon to the pons, including the parabrachial nucleus. NPY-positive, AGRP-negative terminals were observed in many areas. AGRP-ir terminals were reduced dramatically in all brain regions of mice treated neonatally with monosodium glutamate as well as of mice homozygous for the anorexia mutation. Terminals immunoreactive for the melanocortin peptide α-melanocyte-stimulating hormone formed a population separate from, but parallel to, the AGRP-ir terminals. Our results show that arcuate NPY neurons, identified by the presence of AGRP, project more extensively in the brain than previously known and indicate that the feeding regulatory actions of NPY may extend beyond the hypothalamus.
Induction of antigen-specific T cell anergy: An early event in the course of tumor progression
Authors: Staveley-O’Carroll Kevin , Sotomayor Eduardo , Montgomery Jami , Borrello Ivan , Hwang Leon , Fein Steve , Pardoll Drew , Levitsky Hyam
Published: July 2002

The priming of tumor-antigen-specific T cells is critical for the initiation of successful anti-tumor immune responses, yet the fate of such cells during tumor progression is unknown. Naive CD4+T cells specific for an antigen expressed by tumor cells were transferred into tumor-bearing mice. Transient clonal expansion occurred early after transfer, accompanied by phenotypic changes associated with antigen recognition. Nevertheless, these cells had a diminished response to peptide antigenin vitroand were unable to be primedin vivo. The development of antigen-specific T cell anergy is an early event in the tumor-bearing host, and it suggests that tolerance to tumor antigens may impose a significant barrier to therapeutic vaccination.
RNA-binding protein TIAR is essential for primordial germ cell development
Authors: Beck Andreas R. P. , Miller Ira J. , Anderson Paul , Streuli Michel
Published: July 2002

Primordial germ cells (PGCs) give rise to both eggs and sperm via complex maturational processes that require both cell migration and proliferation. However, little is known about the genes controlling gamete formation during the early stages of PGC development. Although several mutations are known to severely reduce the number of PGCs reaching and populating the genital ridges, the molecular identity of only two of these genes is known: the c-
kit
receptor protein tyrosine kinase and the c-
kit
ligand (the steel factor). Herein, we report that mutant mice lacking TIAR, an RNA recognition motif/ribonucleoprotein-type RNA-binding protein highly expressed in PGCs, fail to develop spermatogonia or oogonia. This developmental defect is a consequence of reduced survival of PGCs that migrate to the genital ridge around embryonic day 11.5 (E11.5). The numbers of PGCs populating the genital ridge in TIAR-deficient embryos are severely reduced compared to wild-type embryos by E11.5 and in the mutants PGCs are completely absent at E13.5. Furthermore, TIAR-deficient embryonic stem cells do not proliferate in the absence of exogenous leukemia inhibitory factor in an
in vitro
methylcellulose culture assay, supporting a role for TIAR in regulating cell proliferation. Because the development of PGCs relies on the action of several growth factors, these results are consistent with a role for TIAR in the expression of a survival factor or survival factor receptor that is essential for PGC development. TIAR-deficient mice thus provide a model system to study molecular mechanisms of PGC development and possibly the basis for some forms of idiopathic infertility.
The chemotaxis system, but not chemotaxis, is essential for swarming motility in
Escherichia coli
Authors: Burkart Mark , Toguchi Adam , Harshey Rasika M.
Published: July 2002

The chemotaxis system plays an essential role in swarm cell differentiation and motility. We show in this study that two (Tsr and Tar) of the four chemoreceptors in
Escherichia coli
can support swarming individually, but sensing their most powerful chemoattractants is not necessary. Conditions that abolish chemotaxis toward serine (presence of serine concentrations that saturate Tsr, or mutations in Tsr that destroy serine binding) have no effect on swarming. Similar results were obtained for the aspartate and maltose chemoreceptor Tar. We also show that although a mutation in the signaling domain of Tsr that inhibits CheA kinase abolishes swarming, nonchemotactic flagellar switch mutants can swarm. Our results suggest that during swarming, the chemoreceptors signal through the chemotaxis pathway and induce swarmer cell differentiation in response to signals other than their known chemoeffectors.
Diffusion of peroxynitrite across erythrocyte membranes
Authors: Denicola Ana , Souza José M. , Radi Rafael
Published: July 2002

Peroxynitrite anion (ONOO
−
) is a reactive species of increasingly recognized biological relevance that contributes to oxidative tissue damage. At present, however, there is limited knowledge about the mechanisms of peroxynitrite diffusion through biological compartments. In this work we have studied the diffusion of peroxynitrite across erythrocyte membranes. In solution, peroxynitrite rapidly reacts with oxyhemoglobin to yield methemoglobin, with
k
2
= (10.4 ± 0.3) × 10
3
M
−1
⋅s
−1
at pH 7.4 and 25°C. Addition of peroxynitrite to intact erythrocytes caused oxidation of intracellular oxyhemoglobin to methemoglobin. Oxidation yields in red blood cells at pH 7.0 were approximately 40% of those obtained in solution, which results mostly from competition of other cytosolic components for peroxynitrite. Indeed, rather small differences were observed between oxidation yields in lysates compared with intact erythrocytes, in particular at acidic and neutral pH values, indicating that membrane was not precluding peroxynitrite diffusion. Incubation of erythrocytes at pH 7.0 with 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), a specific inhibitor of anion exchange, resulted in up to 50% inhibition of oxyhemoglobin oxidation by peroxynitrite. More protection by DIDS was achieved at alkaline pH, while no effect was observed at pH 5.5, where 95% of peroxynitrite is in the acidic form, ONOOH (pK
a
= 6.8). In addition, peroxynitrite caused nitration of intracellular hemoglobin, in a process that was enhanced in thiol-depleted erythrocytes. Our results indicate that peroxynitrite is able to cross the erythrocyte membrane by two different mechanisms: in the anionic form through the DIDS-inhibitable anion channel, and in the protonated form by passive diffusion.
Parallel analysis of genetic selections using whole genome oligonucleotide arrays
Authors: Cho Raymond J. , Fromont-Racine Micheline , Wodicka Lisa , Feierbach Becket , Stearns Timothy , Legrain Pierre , Lockhart David J. , Davis Ronald W.
Published: July 2002

Thousands of genes have recently been sequenced in organisms ranging from
Escherichia coli
to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of
Saccharomyces cerevisiae
, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein–protein interaction screens were carried out for
S. cerevisiae
genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.
The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu
Authors: Ogawa Akira , Murata Koichi , Mizuno Shigeki
Published: July 2002

Perhaps the most striking fact about early Cenozoic avian history some 70 million years ago was the rapid radiation of large, flightless, ground-living birds. It has been suggested that, for a time, there was active competition between these large terrestrial birds and the early mammals. Probably reflecting the above noted early start of
Ratitae
of the infraclass
Eoaves
, the presumptive sex chromosomes of their present day survivors, such as the emu and the ostrich, largely remained homomorphic. The signs of genetic differentiation between their still-homomorphic Z and W chromosomes were tested by using two marker genes (Z-linked
ZOV3
and the gene for the iron-responsive element-binding protein) and one marker sequence of a part of a presumptive pseudogene (W-linked EE0.6 of the chicken). Their homologues, maintaining 71–92% identities to the chicken counterparts, were found in both the emu (
Dromaius novaehollandiae
) and the ostrich (
Struthio camelus
). Their locations were visualized on chromosome preparations by fluorescence
in situ
hybridization. In the case of the emu, these three marker sequences were localized on both members of the fifth pair of a female, thus revealing no sign yet of genetic differentiation between the Z and the W. The finding was the same with regard to both members of the fourth pair of male ostriches. In the female ostrich, however, the sequence of the gene for the iron-responsive element-binding protein was missing from one of the pairs, thus revealing the differentiation by a small deletion of the W from the Z.
Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence
Authors: Kouprina Natalay , Annab Lois , Graves Joan , Afshari Cynthia , Barrett J. Carl , Resnick Michael A. , Larionov Vladimir
Published: July 2002

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (
HPRT
) sequence at one end and an 189-bp
Alu
repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire
HPRT
gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various
Alu
positions as much as 600 kb upstream. These YACs were retrofitted with a Neo
R
and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the
HPRT
isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.
Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body
Authors: LaMorte Vickie J. , Dyck Jacqueline A. , Ochs Robert L. , Evans Ronald M.
Published: July 2002

The cellular role of the PML-containing nuclear bodies also known as ND10 or PODs remains elusive despite links to oncogenesis and viral replication. Although a potential role in transcription has been considered, direct evidence has been lacking. By developing a novel
in vivo
nucleic acid labeling approach, we demonstrate the existence of nascent RNA polymerase II transcripts within this nuclear body. In addition, PML and the transactivation cofactor, CREB binding protein (CBP), colocalize within the nucleus. Furthermore, we show that CBP in contrast to PML is distributed throughout the internal core of the structure. Collectively, these findings support a role for this nuclear body in transcriptional regulation.
E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium
Authors: Auersperg Nelly , Pan Jie , Grove Bryon D. , Peterson Todd , Fisher Janet , Maines-Bandiera Sarah , Somasiri Aruna , Roskelley Calvin D.
Published: July 2002

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.
Interfacial membrane docking of cytosolic phospholipase A
2
C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance
Authors: Ball Andy , Nielsen Robert , Gelb Michael H. , Robinson Bruce H.
Published: July 2002

The C2 domain of cytosolic phospholipase A
2
(C2
cPLA2
) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids. By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2
cPLA2
s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2
cPLA2
with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-
sn
-glycero-3-phosphomethanol. The structure reveals that the two calcium-binding regions on C2
cPLA2
that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane. CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted. The long axis of the cylindrical C2
cPLA2
molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups. Such an orientation places the two bound calcium ions close to the membrane surface. All together, the results provide structural support for previous proposals that binding of C2
cPLA2
to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core. The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface.
Guanosine triphosphatase stimulation of oncogenic Ras mutants
Authors: Ahmadian Mohammad Reza , Zor Tsaffrir , Vogt Dorothee , Kabsch Wolfgang , Selinger Zvi , Wittinghofer Alfred , Scheffzek Klaus
Published: July 2002

Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13. Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off. Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells. Here, we show that the guanosine 5′-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5′-diphosphate-bound inactive Ras and DABP-P
i
. The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants. Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged. An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction. The crystal structures of Ras-bound DABP-β,γ-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety. The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction.
Heat-inactivated proteins are rescued by the DnaK⋅J-GrpE set and ClpB chaperones
Authors: Motohashi Ken , Watanabe Yohei , Yohda Masafumi , Yoshida Masasuke
Published: July 2002

Functional chaperone cooperation between Hsp70 (DnaK) and Hsp104 (ClpB) was demonstrated
in vitro
. In a eubacterium
Thermus thermophilus
, DnaK and DnaJ exist as a stable trigonal ring complex (
T
DnaK⋅J complex) and the
dnaK
gene cluster contains a
clpB
gene. When substrate proteins were heated at high temperature, none of the chaperones protected them from heat inactivation, but the
T
DnaK⋅J complex could suppress the aggregation of proteins in an ATP- and
T
GrpE-dependent manner. Subsequent incubation of these heated preparations at moderate temperature after addition of
T
ClpB resulted in the efficient reactivation of the proteins. Reactivation was also observed, even though the yield was low, if the substrate protein alone was heated and incubated at moderate temperature with the
T
DnaK⋅J complex,
T
GrpE,
T
ClpB, and ATP. Thus, all these components were necessary for the reactivation. Further, we found that
T
GroEL/ES could not substitute
T
ClpB.
Impairment of spermatogenesis in mice lacking a functional aromatase (cyp 19) gene
Authors: Robertson Kirsten M. , O’Donnell Liza , Jones Margaret E. E. , Meachem Sarah J. , Boon Wah Chin , Fisher Carolyn R. , Graves Kathy H. , McLachlan Robert I. , Simpson Evan R.
Published: July 2002

It is well established that spermatogenesis is controlled by gonadotrophins and testosterone. However, a role for estrogens in male reproduction recently was suggested in adult mice deficient in estrogen receptor α. These mice became infertile primarily because of an interruption of fluid reabsorption by the efferent ductules of the epididymis, thus leading to a disruption of the seminiferous epithelium [Hess, R. A., Bunick, D., Lee, K. H., Bahr, J., Taylor, J. A., Korach, K. S., and Lubahn, D. B. (1997)Nature (London)390, 509–512]. Despite the demonstration of the aromatase enzyme, which converts androgens to estrogens, and estrogen receptors within the rodent seminiferous epithelium, the role of aromatase and estrogen in germ cell development is unknown. We have investigated spermatogenesis in mice that lack aromatase because of the targeted disruption of thecyp19gene (ArKO). Male mice deficient in aromatase were initially fertile but developed progressive infertility, until their ability to sire pups was severely impaired. The mice deficient in aromatase developed disruptions to spermatogenesis between 4.5 months and 1 year, despite no decreases in gonadotrophins or androgens. Spermatogenesis primarily was arrested at early spermiogenic stages, as characterized by an increase in apoptosis and the appearance of multinucleated cells, and there was a significant reduction in round and elongated spermatids, but no changes in Sertoli cells and earlier germ cells. In addition, Leydig cell hyperplasia/hypertrophy was evident, presumably as a consequence of increased circulating luteinizing hormone. Our findings indicate that local expression of aromatase is essential for spermatogenesis and provide evidence for a direct action of estrogen on male germ cell development and thus fertility.
Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitmentin vivo
Authors: Clish Clary B. , O’Brien Jennifer A. , Gronert Karsten , Stahl Gregory L. , Petasis Nicos A. , Serhan Charles N.
Published: July 2002

Aspirin (ASA) triggers a switch in the biosynthesis of lipid mediators, inhibiting prostanoid production and initiating 15-epi-lipoxin generation through the acetylation of cyclooxygenase II. These aspirin-triggered lipoxins (ATL) may mediate some of ASA’s beneficial actions and therefore are of interest in the search for novel antiinflammatories that could manifest fewer unwanted side effects. Here, we report that design modifications to native ATL structure prolong its biostabilityin vivo. In mouse whole blood, ATL analogs protected at carbon 15 [15(R/S)-methyl-lipoxin A4(ATLa1)] and the omega end [15-epi-16-(para-fluoro)-phenoxy-LXA4(ATLa2)] were recoverable to ≈90 and 100% at 3 hr, respectively, compared with a ≈40% loss of native lipoxin A4. ATLa2retains bioactivity and, at levels as low as ≈24 nmol/mouse, potently inhibited tumor necrosis factor-α-induced leukocyte recruitment into the dorsal air pouch. Inhibition was evident by either local intra-air pouch delivery (≈77% inhibition) or systemic delivery by intravenous injection (≈85% inhibition) and proved more potent than local delivery of ASA. Rank order for inhibiting polymorphonuclear leukocyte infiltration was: ATLa2(10 μg, i.v.) ≈ATLa2(10 μg, local) ≈dexamethasone (10 μg, local) >ASA (1.0 mg, local). Applied topically to mouse ear skin, ATLa2also inhibited polymorphonuclear leukocyte infiltration induced by leukotriene B4(≈78% inhibition) or phorbol ester (≈49% inhibition), which initiates endogenous chemokine production. These results indicate that this fluorinated analog of natural aspirin-triggered lipoxin A4is bioavailable by either local or systemic delivery routes and is a more potent and precise inhibitor of neutrophil accumulation than is ASA.
A novel function for transglutaminase 1: Attachment of long-chain ω-hydroxyceramides to involucrin by ester bond formation
Authors: Nemes Zoltán , Marekov Lyuben N. , Fésüs Lázló , Steinert Peter M.
Published: July 2002

Transglutaminases (TGases) are defined as enzymes capable of forming isopeptide bonds by transfer of an amine onto glutaminyl residues of a protein. Here we show that the membrane-bound form of the TGase 1 enzyme can also form ester bonds between specific glutaminyl residues of human involucrin and a synthetic analog of epidermal specific ω-hydroxyceramides. The formation of a ≈5-nm-thick lipid envelope on the surface of epidermal keratinocytes is an important component of normal barrier function. The lipid envelope consists of ω-hydroxyceramides covalently linked by ester bonds to cornified envelope proteins, most abundantly to involucrin. We synthesized an analog of natural ω-hydroxyceramides
N-
[16-(16-hydroxyhexadecyl)oxypalmitoyl]sphingosine (lipid Z). When recombinant human TGase 1 and involucrin were reacted on the surface of synthetic lipid vesicles containing lipid Z, lipid Z was attached to involucrin and formed saponifiable protein–lipid adducts. By mass spectroscopy and sequencing of tryptic lipopeptides, the ester linkage formation used involucrin glutamine residues 107, 118, 122, 133, and 496 by converting the γ-carboxamido groups to lipid esters. Several of these residues have been found previously to be attached to ceramides
in vivo
. Mass spectrometric analysis after acetonide derivatization also revealed that ester formation involved primarily the ω-hydroxyl group of lipid Z. Our data reveal a dual role for TGase 1 in epidermal barrier formation and provide insights into the pathophysiology of lamellar ichthyosis resulting from defects of TGase 1 enzyme.
Thirty-seven candidate genes for polycystic ovary syndrome: Strongest evidence for linkage is with follistatin
Authors: Urbanek Margrit , Legro Richard S. , Driscoll Deborah A. , Azziz Ricardo , Ehrmann David A. , Norman Robert J. , Strauss Jerome F. , Spielman Richard S. , Dunaif Andrea
Published: July 2002

Polycystic ovary syndrome (PCOS) is a common endocrine disorder of women, characterized by hyperandrogenism and chronic anovulation. It is a leading cause of female infertility and is associated with polycystic ovaries, hirsutism, obesity, and insulin resistance. We tested a carefully chosen collection of 37 candidate genes for linkage and association with PCOS or hyperandrogenemia in data from 150 families. The strongest evidence for linkage was with the follistatin gene, for which affected sisters showed increased identity by descent (72%; χ
2
= 12.97; nominal
P
= 3.2 × 10
−4
). After correction for multiple testing (33 tests), the follistatin findings were still highly significant (
P
c
= 0.01). Although the linkage results for
CYP11A
were also nominally significant (
P
= 0.02), they were no longer significant after correction. In 11 candidate gene regions, at least one allele showed nominally significant evidence for population association with PCOS in the transmission/disequilibrium test (χ
2
≥ 3.84; nominal
P
< 0.05). The strongest effect in the transmission/disequilibrium test was observed in the
INSR
region (
D19S884
; allele 5; χ
2
= 8.53) but was not significant after correction. Our study shows how a systematic screen of candidate genes can provide strong evidence for genetic linkage in complex diseases and can identify those genes that should have high (or low) priority for further study.
Domain structure and lipid interaction of recombinant yeast Tim44
Authors: Weiss Celeste , Oppliger Wolfgang , Vergères Guy , Demel Rudy , Jenö Paul , Horst Martin , de Kruijff Ben , Schatz Gottfried , Azem Abdussalam
Published: July 2002

Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE. To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in
Escherichia coli
and studied the properties of the pure protein and its interaction with model lipid membranes. Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain. The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone. Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region.
A tubular endosomal fraction from rat liver: Biochemical evidence of receptor sorting by default
Authors: Vergés Marcel , Havel Richard J. , Mostov Keith E.
Published: July 2002

We previously have isolated an endosomal fraction from rat liver, termed receptor-recycling compartment (RRC), which is highly enriched in recycling receptors and in the transcytotic polymeric Ig receptor (pIgR). We now have analyzed the RRC fraction by immunoisolation and found that no uniquely transcytotic elements were present, because recycling receptors and the pIgR were coisolated on the same elements. In addition, RRC was very rich in proteins previously shown to be associated with recycling endosomes, such as rab 11, cellubrevin, and endobrevin, but relatively poor in early endosome antigen 1. As RRC contains mainly tubules and small vesicles, our results indicate that it is enriched in elements of a tubular endosomal compartment involved in receptor sorting. Biochemical analysis showed that the density of recycling receptors and transcytotic pIgR in RRC membranes was similar to that in early endosome membranes. This observation supports the idea that increasing membrane surface area by endosome tubulation is the main mechanism to ensure efficient receptor sorting and, at the same time, locates RRC in a common step of the endocytotic system before final receptor segregation into distinct recycling and transcytotic pathways.
Constructing primate phylogenies from ancient retrovirus sequences
Authors: Johnson Welkin E. , Coffin John M.
Published: July 2002

The genomes of modern humans are riddled with thousands of endogenous retroviruses (HERVs), the proviral remnants of ancient viral infections of the primate lineage. Most HERVs are nonfunctional, selectively neutral loci. This fact, coupled with their sheer abundance in primate genomes, makes HERVs ideal for exploitation as phylogenetic markers. Endogenous retroviruses (ERVs) provide phylogenetic information in two ways: (i) by comparison of integration site polymorphism and (ii) by orthologous comparison of evolving, proviral, nucleotide sequence. In this study, trees are constructed with the noncoding long terminal repeats (LTRs) of several ERV loci. Because the two LTRs of an ERV are identical at the time of integration but evolve independently, each ERV locus can provide two estimates of species phylogeny based on molecular evolution of the same ancestral sequence. Moreover, tree topology is highly sensitive to conversion events, allowing for easy detection of sequences involved in recombination as well as correction for such events. Although other animal species are rich in ERV sequences, the specific use of HERVs in this study allows comparison of trees to a well established phylogenetic standard, that of the Old World primates. HERVs, and by extension the ERVs of other species, constitute a unique and plentiful resource for studying the evolutionary history of the Retroviridae and their animal hosts.
Late changes in spliceosomal introns define clades in vertebrate evolution
Authors: Venkatesh Byrappa , Ning Yana , Brenner Sydney
Published: July 2002

The evolutionary origin of spliceosomal introns has been the subject of much controversy. Introns are proposed to have been both lost and gained during evolution. If the gain or loss of introns are unique events in evolution, they can serve as markers for phylogenetic analysis. We have made an extensive survey of the phylogenetic distribution of seven spliceosomal introns that are present in
Fugu
genes, but not in their mammalian homologues; we show that these introns were acquired by actinopterygian (ray-finned) fishes at various stages of evolution. We have also investigated the intron pattern of the rhodopsin gene in fishes, and show that the four introns found in the ancestral chordate rhodopsin gene were simultaneously lost in a common ancestor of ray-finned fishes. These changes in introns serve as excellent markers for phylogenetic analysis because they reliably define clades. Our intron-based cladogram establishes the difficult-to-ascertain phylogenetic relationships of some ray-finned fishes. For example, it shows that bichirs (
Polypterus
) are the sister group of all other extant ray-finned fishes.
A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA
Authors: Waisfisz Quinten , de Winter Johan P. , Kruyt Frank A. E. , de Groot Jan , van der Weel Laura , Dijkmans Lonneke M. , Zhi Yu , Arwert Fré , Scheper Rik J. , Youssoufian Hagop , Hoatlin Maureen E. , Joenje Hans
Published: July 2002

Fanconi anemia (FA) is a recessively inherited disease characterized at the cellular level by spontaneous chromosomal instability and specific hypersensitivity to cross-linking agents. FA is genetically heterogeneous, comprising at least eight complementation groups (A-H). We report that the protein encoded by the gene mutated in complementation group G (FANCG) localizes to the cytoplasm and nucleus of the cell and assembles in a molecular complex with the FANCA protein, both
in vivo
and
in vitro
. Endogenous FANCA/FANCG complex was detected in both non-FA cells and in FA cells from groups D and E. By contrast, no complex was detected in specific cell lines belonging to groups A and G, whereas reduced levels were found in cells from groups B, C, F, and H. Wild-type levels of FANCA/FANCG complex were restored upon correction of the cellular phenotype by transfection or cell fusion experiments, suggesting that this complex is of functional significance in the FA pathway. These results indicate that the cellular FA phenotype can be connected to three biochemical subtypes based on the levels of FANCA/FANCG complex. Disruption of the complex may provide an experimental strategy for chemosensitization of neoplastic cells.
BRCA1-associated growth arrest is RB-dependent
Authors: Aprelikova Olga N. , Fang Bruno S. , Meissner Eric G. , Cotter Shane , Campbell Mel , Kuthiala Aalok , Bessho Mika , Jensen Roy A. , Liu Edison T.
Published: July 2002

BRCA1
is a susceptibility gene for breast and ovarian cancer with growth-inhibitory activity for which the mechanism of action remains unclear. When introduced into cells,
BRCA1
inhibits growth of some but not all cell lines. In an attempt to uncover the mechanism of growth suppression by
BRCA1
, we examined a panel of cell lines for their ability to reduce colony outgrowth in response to
BRCA1
overexpression. Of all variables tested, only those cells with wild-type pRb were sensitive to
BRCA1
-induced growth suppression. In cells with an intact
rb
gene, inactivation of pRb by HPV E7 abrogates the growth arrest imposed by
BRCA1
. In accordance with these observations, we found that
BRCA1
could not suppress BrdUrd uptake in primary fibroblasts from
rb
−/− mice and exhibited an intermediate ability to inhibit DNA synthesis in
rb
+/− as compared with
rb
+/+ cells. We further found that the BRCA1 protein complexes with the hypophosphorylated form of pRb. This binding is localized to amino acids 304–394 of BRCA1 protein and requires the ABC domain of pRb. In-frame deletion of BRCA1 fragment involved in interaction with pRb completely abolished the growth-suppressive property of
BRCA1
. Although it has been reported that
BRCA1
interacts with p53, we find the p53 status did not affect the ability of
BRCA1
to suppress colony formation. Our data suggest that the growth suppressor function of
BRCA1
depends, at least in part, on Rb.
Evidence for a protective role of metallothionein-1 in focal cerebral ischemia
Authors: van Lookeren Campagne Menno , Thibodeaux Harold , van Bruggen Nick , Cairns Belinda , Gerlai Robert , Palmer James T. , Williams Simon P. , Lowe David G.
Published: July 2002

Metallothioneins (MTs) are a family of metal binding proteins that have been proposed to participate in a cellular defense against zinc toxicity and free radicals. In the present study, we investigated whether increased expression of MT in MT-1 isoform-overexpressing transgenic mice (MT-TG) affords protection against mild focal cerebral ischemia and reperfusion. Transient focal ischemia was induced in control (wild type) and MT-TG mice by occluding the right middle cerebral artery for 45 min. Upon reperfusion, cerebral edema slowly developed and peaked at 24 hr as shown by T2-weighted MRI. The volume of affected tissue was on the average 42% smaller in MT-TG mice compared with control mice at 6, 9, 24, and 72 hr and 14 days postreperfusion (
P
< 0.01). In addition, functional studies showed that 3 weeks after reperfusion MT-TG mice showed a significantly better motor performance compared with control mice (
P
= 0.011). Although cortical baseline levels of MT-1 mRNA were similar in control and MT-TG mice, there was an increase in MT-1 mRNA levels in the ischemic cortex of MT-TG mice to 7.5 times baseline levels compared with an increase to 2.3 times baseline levels in control mice 24 hr after reperfusion. In addition, MT-TG mice showed an increased MT immunoreactivity in astrocytes, vascular endothelial cells, and neurons 24 hr after reperfusion whereas in control mice MT immunoreactivity was restricted mainly to astrocytes and decreased in the infarcted tissue. These results provide evidence that increased expression of MT-1 protects against focal cerebral ischemia and reperfusion.
Efficient folding of proteins with multiple disulfide bonds in the
Escherichia coli
cytoplasm
Authors: Bessette Paul H. , Åslund Fredrik , Beckwith Jon , Georgiou George
Published: July 2002

Under physiological conditions, the
Escherichia coli
cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (
trxB gor
mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or
trxB
mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by co-expression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of
E. coli
FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.
Defects in inositol 1,4,5-trisphosphate receptor expression, Ca
2+
signaling, and insulin secretion in the
anx
7(+/−) knockout mouse
Authors: Srivastava Meera , Atwater Illani , Glasman Mirta , Leighton Ximena , Goping Gertrude , Caohuy Hung , Miller Georgina , Pichel José , Westphal Heiner , Mears David , Rojas Eduardo , Pollard Harvey B.
Published: July 2002

The mammalian
anx7
gene codes for a Ca
2+
-activated GTPase, which supports Ca
2+
/GTP-dependent secretion events and Ca
2+
channel activities
in vitro
and
in vivo
. To test whether
anx7
might be involved in Ca
2+
signaling in secreting pancreatic β cells, we knocked out the
anx7
gene in the mouse and tested the insulin-secretory properties of the β cells. The nullizygous anx7 (−/−) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/−) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/−) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca
2+
channel functions are normal. However, electrooptical recordings indicate that the (+/−) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP
3
)-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP
3
receptor expression and function in pancreatic islets. The profound increase in islets, β cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic β cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca
2+
signaling through IP
3
-sensitive Ca
2+
stores.