bluuebunny/crossref_metadata_2025 huggingface dataset:
Short-term exposure to pregnancy levels of estrogen prevents mammary carcinogenesis
Authors: Rajkumar Lakshmanaswamy , Guzman Raphael C. , Yang Jason , Thordarson Gudmundur , Talamantes Frank , Nandi Satyabrata
Published: July 2002

It is well established that pregnancy early in life reduces the
risk of breast cancer in women and that this effect is universal. This
phenomenon of parity protection against mammary cancer is also observed
in rodents. Earlier studies have demonstrated that short-term
administration of estradiol (E) in combination with progesterone mimics
the protective effect of parity in rats. In this study, the lowest
effective E dosage for preventing mammary cancer was determined. Rats
were injected with
N
-methyl-
N
-nitrosourea
at 7 weeks of age; 2 weeks later, the rats were subjected to sustained
treatment with 20 μg, 100 μg, 200 μg, or 30 mg of E in silastic
capsules for 3 weeks. Treatments with 100 μg, 200 μg, and 30 mg of
E resulted in serum levels of E equivalent to those of pregnancy and
were highly effective in preventing mammary cancer. E treatment (20
μg) did not result in pregnancy levels of E and was not effective in
reducing the mammary cancer incidence. In another set of experiments,
we determined the effect of different durations of E with or without
progesterone treatments on mammary carcinogenesis. These experiments
indicate that a period as short as one-third the period of gestation is
sufficient to induce protection against mammary carcinogenesis. The
pioneering aspect of our study in contrast to long-term estrogen
exposure, which is thought to increase the risk of breast cancer, is
that short-term sustained treatments with pregnancy levels of E can
induce protection against frank mammary cancer.
Estrogen receptors activate atrial natriuretic peptide in the rat heart
Authors: Jankowski Marek , Rachelska Grazyna , Donghao Wang , McCann Samuel M. , Gutkowska Jolanta
Published: July 2002

In this study, semiquantitative reverse transcription–PCR analysis
showed that estrogen receptor α (ERα) and β (ERβ) mRNAs are
developmentally regulated in the rat heart. We found that ERα mRNA
was low in all heart chambers of 4-day-old rats, but was elevated in
the atria (6- to 18-fold) and ventricles (3- to 4-fold) of adult rats.
Western blotting analysis confirmed that these differences were
efficiently translated into 67-kDa ERα protein. ERβ mRNA was
expressed at its highest level in the left atrium and was 3- to 4-fold
lower in other heart chambers of 4-day-old animals. In adult rats ERβ
was decreased dramatically in the left atrium (20-fold) and, to a
lesser extent in the other heart chambers (2- to 4-fold). Significant
ER changes occurred already in the first week after birth. Accordingly,
estrogen regulation in cells from neonatal hearts, as reported in
several studies, may not correspond to that occurring in fully
differentiated adult hearts, because of an altered degree of ER
expression. In adult rats, ovariectomy decreases atrial ERα, the
atria/body weight ratio, and atrial natriuretic peptide (ANP)
transcription. Treatment of ovariectomized rats with 17-β-estradiol
(25 μg, 10 days, s.c.) reversed these changes. In addition, there was
no effect of ovariectomy and 17-β-estradiol supplementation on
systolic blood pressure, but in ovariectomized rats a decreased heart
rate followed 17-β-estradiol administration. Similar to the effects
on ERα in the atria, ovariectomy lowered plasma ANP levels, and
17-β-estradiol administration restored ANP in the plasma of
ovariectomized rats. Changes in plasma ANP correlated with changes in
ANP content in the right atrium, as demonstrated by RIA. Increased ANP
expression and secretion in response to ERα activation may be a
protective mechanism in the heart.
Origin of multidrug resistance in cells with and without multidrug resistance genes: Chromosome reassortments catalyzed by aneuploidy
Authors: Duesberg Peter , Stindl Reinhard , Hehlmann Ruediger
Published: July 2002

Cancer cells and aneuploid cell lines can acquire resistance
against multiple unrelated chemotherapeutic drugs that are over
3,000-fold those of normal levels and display spontaneous resistances
up to 20-fold of normal levels. Two different mechanisms were proposed
for this phenotype: (
i
) classical mutation of drug
metabolizing genes or (
ii
) chromosome reassortments,
catalyzed by cancer- and cell line-specific aneuploidy, which generate,
via new gene dosage combinations, a plethora of cancer phenotypes,
including drug resistance. To distinguish between these mechanisms, we
have asked whether three mouse cell lines can become drug resistant,
from which two or three genes have been deleted, and on which multidrug
resistance is thought to depend:
Mdr1a
,
Mdr1b
, and
Mrp1
. Because all three lines
could acquire multidrug resistance and were aneuploid, whereas diploid
mouse cells could not, we conclude that aneuploid cells become drug
resistant via specific chromosome assortments, independent of putative
resistance genes. We have asked further whether aneuploid
drug-resistant Chinese hamster cells revert spontaneously to drug
sensitivity in the absence of cytotoxic drugs at the high rates that
are typical of chromosome reassortments catalyzed by aneuploidy or at
the very low or zero rates (i.e., deletion) of gene mutation. We found
that four drug-resistant hamster cell lines reverted to drug
sensitivity at rates of about 2–3% per generation, whereas two
closely related lines remained resistant under our conditions. Thus,
the karyotypic instability generated by aneuploidy emerges as the
common source of the various levels of drug resistance of cancer cells:
minor spontaneous resistances reflect accidental chromosome
assortments, the high selected resistances reflect complex specific
assortments, and multidrug resistance reflects new combinations of
unselected genes located on the same chromosomes as selected genes.
Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H
Authors: Wisniewski Michele , Balakrishnan Mini , Palaniappan C. , Fay Philip J. , Bambara Robert A.
Published: July 2002

HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA
genome while synthesizing the minus strand of DNA. Many RNA fragments,
including the polypurine tracts, remain annealed to the new DNA.
Several RTs are believed to bind after synthesis to degrade all RNA
fragments except the polypurine tracts by a polymerization-independent
mode of RNase H activity. For this latter process, we found that RT
positions the RNase H active site approximately 18 nt from the 5′ end
of the RNA, making the primary cut. The enzyme rebinds or slides toward
the 5′ end of the RNA to make a secondary cut creating two products
8–9 nt long. RT then binds the new 5′ end of the RNA created by the
first primary or the secondary cuts to make the next primary cut. In
addition, we observed another type of RNase H cleavage specificity. RT
aligns the RNase H active site to the 3′ end of the RNA, cutting 5
residues in. We determined the relative rates of these cuts, defining
their temporal order. Results show that the first primary cut is
fastest, and the secondary and 5-nt cuts occur next at similar rates.
The second primary cuts appear last. Based on these results, we present
a model by which RT progressively cleaves RNA fragments.
Distinctive higher-order chromatin structure at mammalian centromeres
Authors: Gilbert Nick , Allan James
Published: July 2002

The structure of the higher-order chromatin fiber has not been defined in detail. We have used a novel approach based on sucrose gradient centrifugation to compare the conformation of centromeric satellite DNA-containing higher-order chromatin fibers with bulk chromatin fibers obtained from the same mouse fibroblast cells. Our data show that chromatin fibers derived from the centromeric domain of a chromosome exist in a more condensed structure than bulk chromatin whereas pericentromeric chromatin fibers have an intermediate conformation. From the standpoint of current models, our data are interpreted to suggest that satellite chromatin adopts a regular helical conformation compatible with the canonical 30-nm chromatin fiber whereas bulk chromatin fibers appear less regularly folded and are perhaps intermittently interrupted by deformations. This distinctive conformation of the higher-order chromatin fiber in the centromeric domain of the mammalian chromosome could play a role in the formation of heterochromatin and in the determination of centromere identity.
Mutations ofCD40gene cause an autosomal recessive form of immunodeficiency with hyper IgM
Authors: Ferrari Simona , Giliani Silvia , Insalaco Antonella , Al-Ghonaium Abdulaziz , Soresina Anna R. , Loubser Michael , Avanzini Maria A. , Marconi Massimo , Badolato Raffaele , Ugazio Alberto G. , Levy Yves , Catalan Nadia , Durandy Anne , Tbakhi Abdelghani , Notarangelo Luigi D. , Plebani Alessandro
Published: July 2002

CD40 is a member of the tumor necrosis factor receptor superfamily, expressed on a wide range of cell types including B cells, macrophages, and dendritic cells. CD40 is the receptor for CD40 ligand (CD40L), a molecule predominantly expressed by activated CD4+T cells. CD40/CD40L interaction induces the formation of memory B lymphocytes and promotes Ig isotype switching, as demonstrated in mice knocked-out for eitherCD40LorCD40gene, and in patients with X-linked hyper IgM syndrome, a disease caused byCD40L/TNFSF5gene mutations. In the present study, we have identified three patients with an autosomal recessive form of hyper IgM who fail to express CD40 on the cell surface. Sequence analysis ofCD40genomic DNA showed that one patient carried a homozygous silent mutation at the fifth base pair position of exon 5, involving an exonic splicing enhancer and leading to exon skipping and premature termination; the other two patients showed a homozygous point mutation in exon 3, resulting in a cysteine to arginine substitution. These findings show that mutations of theCD40gene cause an autosomal recessive form of hyper IgM, which is immunologically and clinically undistinguishable from the X-linked form.
Femtosecond direct observation of charge transfer between bases in DNA
Authors: Wan Chaozhi , Fiebig Torsten , Schiemann Olav , Barton Jacqueline K. , Zewail Ahmed H.
Published: July 2002

Charge transfer in supramolecular assemblies of DNA is unique
because of the notion that the π-stacked bases within the duplex may
mediate the transport, possibly leading to damage and/or repair. The
phenomenon of transport through π-stacked arrays over a long distance
has an analogy to conduction in molecular electronics, but the
mechanism still needs to be determined. To decipher the elementary
steps and the mechanism, one has to directly measure the dynamics in
real time and in suitably designed, structurally well characterized DNA
assemblies. Here, we report our first observation of the femtosecond
dynamics of charge transport processes occurring between bases within
duplex DNA. By monitoring the population of an initially excited
2-aminopurine, an isomer of adenine, we can follow the charge transfer
process and measure its rate. We then study the effect of different
bases next to the donor (acceptor), the base sequence, and the distance
dependence between the donor and acceptor. We find that the charge
injection to a nearest neighbor base is crucial and the time scale is
vastly different: 10 ps for guanine and up to 512 ps for inosine.
Depending on the base sequence the transfer can be slowed down or
inhibited, and the distance dependence is dramatic over the range of 14
Å. These observations provide the time scale, and the range and
efficiency of the transfer. The results suggest the invalidity of an
efficient wire-type behavior and indicate that long-range transport is
a slow process of a different mechanism.
Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.
Authors: Mösch H U , Roberts R L , Fink G R
Published: July 2002

RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.
A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function.
Authors: Kumar V , Jayasuryan N , Kumar R
Published: July 2002

The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses. Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137). To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis. A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot. Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays. We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation. Deletion of the nonconserved regions B or F also had no deleterious effects. Deletions of regions C and D resulted in a significant loss of function. Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid. The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity. Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.
Second generation hybrid polar compounds are potent inducers of transformed cell differentiation.
Authors: Richon V M , Webb Y , Merger R , Sheppard T , Jursic B , Ngo L , Civoli F , Breslow R , Rifkind R A , Marks P A
Published: July 2002

Hybrid polar compounds, of which hexamethylenebisacetamide (HMBA) is the prototype, are potent inducers of differentiation of murine erythroleukemia (MEL) cells and a wide variety of other transformed cells. HMBA has been shown to induce differentiation of neoplastic cells in patients, but is not an adequate therapeutic agent because of dose-limiting toxicity. We report on a group of three potent second generation hybrid polar compounds, diethyl bis-(pentamethylene-N,N-dimethylcarboxamide) malonate (EMBA), suberoylanilide hydroxamic acid (SAHA), and m-carboxycinnamic acid bis-hydroxamide (CBHA) with optimal concentrations for inducing MEL cells of 0.4 mM, 2 microM, and 4 microM, respectively, compared to 5 mM for HMBA. All three agents induce accumulation of underphosphorylated pRB; increased levels of p2l protein, a prolongation of the initial G1 phase of the cell cycle; and accumulation of hemoglobin. However, based upon their effective concentrations, the cross-resistance or sensitivity of an HMBA-resistant MEL cell variant, and differences in c-myb expression during induction, these differentiation-inducing hybrid polar compounds can be grouped into two subsets, HMBA/EMBA and SAHA/CBHA. This classification may prove of value in selecting and planning prospective preclinical and clinical studies toward the treatment of cancer by differentiation therapy.
Urokinase-type plasminogen activator is effective in fibrin clearance in the absence of its receptor or tissue-type plasminogen activator.
Authors: Bugge T H , Flick M J , Danton M J , Daugherty C C , Romer J , Dano K , Carmeliet P , Collen D , Degen J L
Published: July 2002

The availability of gene-targeted mice deficient in the urokinase-type plasminogen activator (uPA), urokinase receptor (uPAR), tissue-type plasminogen activator (tPA), and plasminogen permits a critical, genetic-based analysis of the physiological and pathological roles of the two mammalian plasminogen activators. We report a comparative study of animals with individual and combined deficits in uPAR and tPA and show that these proteins are complementary fibrinolytic factors in mice. Sinusoidal fibrin deposits are found within the livers of nearly all adult mice examined with a dual deficiency in uPAR and tPA, whereas fibrin deposits are never found in livers collected from animals lacking uPAR and rarely detected in animals lacking tPA alone. This is the first demonstration that uPAR has a physiological role in fibrinolysis. However, uPAR-/-/tPA-/- mice do not develop the pervasive, multi-organ fibrin deposits, severe tissue damage, reduced fertility, and high morbidity and mortality observed in mice with a combined deficiency in tPA and the uPAR ligand, uPA. Furthermore, uPAR-/-/tPA-/- mice do not exhibit the profound impairment in wound repair seen in uPA-/-/tPA-/- mice when they are challenged with a full-thickness skin incision. These results indicate that plasminogen activation focused at the cell surface by uPAR is important in fibrin surveillance in the liver, but that uPA supplies sufficient fibrinolytic potential to clear fibrin deposits from most tissues and support wound healing without the benefit of either uPAR or tPA.
Inhibitors of HIV-1 replication that inhibit HIV integrase.
Authors: Robinson W E , Reinecke M G , Abdel-Malek S , Jia Q , Chow S A
Published: July 2002

HIV-1 replication depends on the viral enzyme integrase that mediates integration of a DNA copy of the virus into the host cell genome. This enzyme represents a novel target to which antiviral agents might be directed. Three compounds, 3,5-dicaffeoylquinic acid, 1-methoxyoxalyl-3,5-dicaffeoylquinic acid, and L-chicoric acid, inhibit HIV-1 integrase in biochemical assays at concentrations ranging from 0.06-0.66 microgram/ml; furthermore, these compounds inhibit HIV-1 replication in tissue culture at 1-4 microgram/ml. The toxic concentrations of these compounds are fully 100-fold greater than their antiviral concentrations. These compounds represent a potentially important new class of antiviral agents that may contribute to our understanding of the molecular mechanisms of viral integration. Thus, the dicaffeoylquinic acids are promising leads to new anti-HIV therapeutics and offer a significant advance in the search for new HIV enzyme targets as they are both specific for HIV-1 integrase and active against HIV-1 in tissue culture.
Construction of an improved mycoinsecticide overexpressing a toxic protease.
Authors: St Leger R , Joshi L , Bidochka M J , Roberts D W
Published: July 2002

Mycoinsecticides are being used for the control of many insect pests as an environmentally acceptable alternative to chemical insecticides. A key aim of much recent work has been to increase the speed of kill and so improve commercial efficacy of these biocontrol agents. This might he achieved by adding insecticidal genes to the fungus, an approach considered to have enormous potential for the improvement of biological pesticides. We report here the development of a genetically improved entomopathogenic fungus. Additional copies of the gene encoding a regulated cuticle-degrading protease (Pr1) from Metarhizium anisopliae were inserted into the genome of M. anisopliae such that Pr1 was constitutively overproduced in the hemolymph of Manduca sexta, activating the prophenoloxidase system. The combined toxic effects of Pr1 and the reaction products of phenoloxidase caused larvae challenged with the engineered fungus to exhibit a 25% reduction in time of death and reduced food consumption by 40% compared to infections by the wild-type fungus. In addition, infected insects were rapidly melanized, and the resulting cadavers were poor substrates for fungal sporulation. Thus, environmental persistence of the genetically engineered fungus is reduced, thereby providing biological containment.
Purification, molecular cloning, and expression of the mammalian sigma1-binding site.
Authors: Hanner M , Moebius F F , Flandorfer A , Knaus H G , Striessnig J , Kempner E , Glossmann H
Published: July 2002

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.
A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice.
Authors: Olee T , Pierangeli S S , Handley H H , Le D T , Wei X , Lai C J , En J , Novotny W , Harris E N , Woods V L , Chen P P
Published: July 2002

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.
Mice lacking the gene encoding tissue-type plasminogen activator show a selective interference with late-phase long-term potentiation in both Schaffer collateral and mossy fiber pathways.
Authors: Huang Y Y , Bach M E , Lipp H P , Zhuo M , Wolfer D P , Hawkins R D , Schoonjans L , Kandel E R , Godfraind J M , Mulligan R , Collen D , Carmeliet P
Published: July 2002

The gene encoding tissue-type plasminogen activator (t-PA) is an immediate response gene, downstream from CREB-1 and other constitutively expressed transcription factors, which is induced in the hippocampus during the late phase of long-term potentiation (L-LTP). Mice in which the t-PA gene has been ablated (t-PA-/-) showed no gross anatomical, electrophysiological, sensory, or motor abnormalities but manifest a selective reduction in L-LTP in hippocampal slices in both the Schaffer collateral-CA1 and mossy fiber-CA3 pathways. t-PA-/- mice also exhibit reduced potentiation by cAMP analogs and D1/D5 agonists. By contrast, hippocampal-dependent learning and memory were not affected in these mice, whereas performance was impaired on two-way active avoidance, a striatum-dependent task. These results provide genetic evidence that t-PA is a downstream effector gene important for L-LTP and show that modest impairment of L-LTP in CA1 and CA3 does not result in hippocampus-dependent behavioral phenotypes.
Functional analysis of retinoid Z receptor beta, a brain-specific nuclear orphan receptor.
Authors: Greiner E F , Kirfel J , Greschik H , Dörflinger U , Becker P , Mercep A , Schüle R
Published: July 2002

The retinoid Z receptor beta (RZR beta), an orphan receptor, is a member of the retinoic acid receptor (RAR)/thyroid hormone receptor (TR) subfamily of nuclear receptors. RZR beta exhibits a highly restricted brain-specific expression pattern. So far, no natural RZR beta target gene has been identified and the physiological role of the receptor in transcriptional regulation remains to be elucidated. Electrophoretic mobility shift assays reveal binding of RZR beta to monomeric response elements containing the sequence AnnTAGGTCA, but RZR beta-mediated transactivation of reporter genes is only achieved with two property spaced binding sites. We present evidence that RZR beta can function as a cell-type-specific transactivator. In neuronal cells, GaI-RZR beta fusion proteins function as potent transcriptional activators, whereas no transactivation can be observed in nonneuronal cells. Mutational analyses demonstrate that the activation domain (AF-2) of RZR beta and RAR alpha are functionally interchangeable. However, in contrast to RAR and TR, the RZR beta AF-2 cannot function autonomously as a transactivation domain. Furthermore, our data define a novel repressor function for the C-terminal part of the putative ligand binding domain. We propose that the transcriptional activity of RZR beta is regulated by an interplay of different receptor domains with coactivators and corepressors.
Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.
Authors: Rostas J A , Brent V A , Voss K , Errington M L , Bliss T V , Gurd J W
Published: July 2002

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.
Molecular basis for the exquisite sensitivity of
Mycobacterium tuberculosis
to isoniazid
Authors: Zhang Y. , Dhandayuthapani S. , Deretic V.
Published: July 2002

The exceptional sensitivity of
Mycobacterium
tuberculosis
to isonicotinic acid hydrazide (INH) lacks
satisfactory definition.
M. tuberculosis
is a natural
mutant in
oxyR
, a central regulator of peroxide stress
response. The
ahpC
gene, which encodes a critical
subunit of alkyl hydroperoxide reductase, is one of the targets usually
controlled by
oxyR
in bacteria. Unlike in mycobacterial
species less susceptible to INH, the expression of
ahpC
was below detection limits at the protein level in INH-sensitive
M. tuberculosis
and
Mycobacterium bovis
strains. In contrast, AhpC was detected in several series of isogenic
INH-resistant (INH
r
) derivatives. In a demonstration of the
critical role of
ahpC
in sensitivity to INH, insertional
inactivation of
ahpC
on the chromosome of
Mycobacterium smegmatis
, a species naturally insensitive
to INH, dramatically increased its susceptibility to this compound.
These findings suggest that AhpC counteracts the action of INH and that
the levels of its expression may govern the intrinsic susceptibility of
mycobacteria to this front-line antituberculosis drug.
Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase
Authors: Setya Amit , Murillo Michael , Leustek Thomas
Published: July 2002

Sulfate-assimilating organisms reduce inorganic sulfate for
Cys biosynthesis. There are two leading hypotheses for the mechanism of
sulfate reduction in higher plants. In one, adenosine 5′-phosphosulfate
(APS) (5′-adenylylsulfate) sulfotransferase carries out reductive
transfer of sulfate from APS to reduced glutathione. Alternatively, the
mechanism may be similar to that in bacteria in which the enzyme,
3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase, catalyzes
thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA
were cloned from
Arabidopsis thaliana
termed
APR1
, -
2
, and -
3
, that
functionally complement a
cysH
, PAPS reductase mutant
strain of
Escherichia coli
. The coding sequence of the
APR
clones is homologous with PAPS reductases from
microorganisms. In addition, a carboxyl-terminal domain is homologous
with members of the Trx superfamily. Further genetic analysis showed
that the
APR
clones can functionally complement a mutant
strain of
E. coli
lacking Trx, and an APS kinase,
cysC
. mutant. These results suggest that the
APR
enzyme may be a Trx-independent APS reductase. Cell
extracts of
E. coli
expressing
APR
showed
Trx-independent sulfonucleotide reductase activity with a preference
for APS over PAPS as a substrate.
APR
-mediated APS
reduction is dependent on dithiothreitol, has a pH optimum of
8.5, is stimulated by high ionic strength, and is sensitive to
inactivation by 5′-adenosinemonophosphate (5′-AMP). 2′-AMP, or
3′-phosphoadenosine-5′-phosphate (PAP), a competitive inhibitor of PAPS
reductase, do not affect activity. The
APR
enzymes may
be localized in different cellular compartments as evidenced by the
presence of an amino-terminal transit peptide for plastid localization
in
APR1
and
APR3
but not
APR2
. Southern blot analysis confirmed that the
APR
clones are members of a small gene family, possibly
consisting of three members.
Chemical nature of the light emitter of the
Aequorea
green fluorescent protein
Authors: Niwa Haruki , Inouye Satoshi , Hirano Takashi , Matsuno Tatsuki , Kojima Satoshi , Kubota Masayuki , Ohashi Mamoru , Tsuji Frederick I.
Published: July 2002

The jellyfish
Aequorea victoria
possesses in the
margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that
serves as the ultimate light emitter in the bioluminescence reaction of
the animal. The protein is made up of 238 amino acid residues in a
single polypeptide chain and produces a greenish fluorescence
(λ
max
= 508 nm) when irradiated with long ultraviolet
light. The fluorescence is due to the presence of a chromophore
consisting of an imidazolone ring, formed by a post-translational
modification of the tripeptide
-Ser
65
-Tyr
66
-Gly
67
-. GFP has been
used extensively as a reporter protein for monitoring gene expression
in eukaryotic and prokaryotic cells, but relatively little is known
about the chemical mechanism by which fluorescence is produced. To
obtain a better understanding of this problem, we studied a peptide
fragment of GFP bearing the chromophore and a synthetic model compound
of the chromophore. The results indicate that the GFP chromophore
consists of an imidazolone ring structure and that the light emitter is
the singlet excited state of the phenolate anion of the chromophore.
Further, the light emission is highly dependent on the microenvironment
around the chromophore and that inhibition of isomerization of the
exo
-methylene double bond of the chromophore accounts
for its efficient light emission.
Highly selective isolation of human DNAs from rodent–human hybrid cells as circular yeast artificial chromosomes by transformation-associated recombination cloning
Authors: Larionov Vladimir , Kouprina Natalya , Graves Joan , Resnick Michael A.
Published: July 2002

T
ransformation-
a
ssociated
r
ecombination (TAR) can be exploited in yeast to clone
human DNAs. TAR cloning was previously accomplished using one or two
telomere-containing vectors with a common human repeat(s) that could
recombine with human DNA during transformation to generate yeast
artificial chromosomes (YACs). On basis of the proposal that broken DNA
ends are more recombinogenic than internal sequences, we have
investigated if TAR cloning could be applied to the generation of
circular YACs by using a single centromere vector containing various
human repeats at opposite ends. Transformation with these vectors along
with human DNA led to the efficient isolation of circular YACs with a
mean size of ≈150 kb. The circular YACs are stable and they can be
easily separated from yeast chromosomes or moved into bacterial cells
if the TAR vector contains an
Escherichia coli
F-factor
cassette. More importantly, circular TAR cloning enabled the selective
isolation of human DNAs from monochromosomal human–rodent hybrid cell
lines. Although <2% of the DNA in the hybrid cells was human, as much
as 80% of transformants had human DNA YACs when a TAR cloning vector
contained
Alu
repeats. The level of enrichment of human
DNA was nearly 3000-fold. A comparable level of enrichment was
demonstrated with DNA isolated from a radiation hybrid cell line
containing only 5 Mb of human DNA. A high selectivity of human DNA
cloning was also observed for linear TAR cloning with two telomere
vectors. No human–rodent chimeras were detected among YACs generated
by TAR cloning. The results with a circular TAR cloning vector or two
vectors differed from results with a single-telomere vector in that the
latter often resulted in a series of terminal deletions in linear YACs.
This could provide a means for physical mapping of cloned material.
The brain-specific activator p35 allows Cdk5 to escape inhibition by p27Kip1 in neurons.
Authors: Lee M H , Nikolic M , Baptista C A , Lai E , Tsai L H , Massagué J
Published: July 2002

Cell cycle withdrawal in postmitotic cells involves cyclin-dependent kinase (Cdk) inhibitors that repress cell cycle Cdk activity. During mouse neurogenesis, cortical postmitotic neurons are shown here to accumulate high levels of the p27 Cdk inhibitor compared with their progenitor neuroblasts. Elevated p27 levels in staged embryo brain extracts correlate with p27 binding to Cdk2, and Cdk inactivation. Yet, Cdk5, which is associated with the noncyclin activator p35 in neurons, remains active in the presence of high p27 levels. Both in vitro and in vivo, p27 and related inhibitors can recognize a cyclin D-Cdk5 complex but not a p35-Cdk5 complex. The results indicate that the choice of activator determines the susceptibility of Cdk5 to p27 and related Cdk inhibitors, and thus its ability to act in postmitotic cells.
Association of glycolate oxidation with photosynthetic electron transport in plant and algal chloroplasts.
Authors: Goyal A , Tolbert N E
Published: July 2002

Photosynthetic carbon metabolism is initiated by ribulose-bisphosphate carboxylase/oxygenase (Rubisco), which uses both CO2 and O2 as substrates. One 2-phosphoglycolate (P-glycolate) molecule is produced for each O2 molecule fixed. P-glycolate has been considered to be metabolized exclusively via the oxidative photosynthetic carbon cycle. This paper reports an additional pathway for P-glycolate and glycolate metabolism in the chloroplasts. Light-dependent glycolate or P-glycolate oxidation by osmotically shocked chloroplasts from the algae Dunaliella or spinach leaves was measured by three electron acceptors, methyl viologen (MV), potassium ferricyanide, or dichloroindophenol. Glycolate oxidation was assayed with 3-(3,4)-dichlorophenyl)-1,1-dimethylurea (DCMU) as oxygen uptake in the presence of MV at a rate of 9 mol per mg of chlorophyll per h. Washed thylakoids from spinach leaves oxidized glycolate at a rate of 22 mol per mg of chlorophyll per h. This light-dependent oxidation was inhibited completely by SHAM, an inhibitor of quinone oxidoreductase, and 75% by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), which inhibits electron transfer from plastoquinone to the cytochrome b6f complex. SHAM stimulated severalfold glycolate excretion by algal cells, Dunaliella or Chlamydomonas, and by isolated Dunaliella chloroplasts. Glycolate and P-glycolate were oxidized about equally well to glyoxylate and phosphate. On the basis of results of inhibitor action, the possible site which accepts electrons from glycolate or P-glycolate is a quinone after the DCMU site but before the DBMIB site. This glycolate oxidation is a light-dependent, SHAM-sensitive, glycolate-quinone oxidoreductase system that is associated with photosynthetic electron transport in the chloroplasts.
Frequency-domain techniques enhance optical mammography: Initial clinical results
Authors: Franceschini Maria Angela , Moesta K. Thomas , Fantini Sergio , Gaida Gerhard , Gratton Enrico , Jess Helge , Mantulin William W. , Seeber Marcel , Schlag Peter M. , Kaschke Michael
Published: July 2002

We present a novel approach to optical mammography and initial clinical results. We have designed and developed a frequency-domain (110-MHz) optical scanner that performs a transillumination raster scan of the female breast in approximately 3 min. The probing light is a dual-wavelength (690 and 810 nm, 10-mW average power), 2-mm-diameter laser beam, and the detection optical fiber is 5 mm in diameter. The ac amplitude and phase data are processed with use of an algorithm that performs edge effect corrections, thereby enhancing image contrast. This contrast enhancement results in a greater tumor detectability compared with simple light intensity images. The optical mammograms are displayed on a computer screen in real time. We present x-ray and optical mammograms from two patients with breast tumors. Our initial clinical results show that the frequency-domain scanner, even at the present stage of development, has the potential to be a useful tool in mammography.
Role of the casein kinase I isoform, Hrr25, and the cell cycle-regulatory transcription factor, SBF, in the transcriptional response to DNA damage in
Saccharomyces cerevisiae
Authors: Ho Yuen , Mason Stephen , Kobayashi Ryuji , Hoekstra Merl , Andrews Brenda
Published: July 2002

In the budding yeast,
Saccharomyces cerevisiae
, DNA damage or ribonucleotide depletion causes the transcriptional induction of an array of genes with known or putative roles in DNA repair. The ATM-like kinase, Mec1, and the serine/threonine protein kinases, Rad53 and Dun1, are required for this transcriptional response. In this paper, we provide evidence suggesting that another kinase, Hrr25, is also involved in the transcriptional response to DNA damage through its interaction with the transcription factor, Swi6. The Swi6 protein interacts with Swi4 to form the SBF complex and with Mbp1 to form the MBF complex. SBF and MBF are required for the G
1
-specific expression of G
1
cyclins and genes required for S-phase. We show that Swi6 associates with and is phosphorylated by Hrr25
in vitro
. We find that
swi4
,
swi6
, and
hrr25
mutants, but not
mbp1
mutants, are sensitive to hydroxyurea and the DNA-damaging agent methyl methanesulfonate and are defective in the transcriptional induction of a subset of DNA damage-inducible genes. Both the sensitivity of
swi6
mutants to methyl methanesulfonate and hydroxyurea and the transcriptional defect of
hrr25
mutants are rescued by overexpression of
SWI4
, implicating the SBF complex in the Hrr25/Swi6-dependent response to DNA damage.
Cell–cell interaction in prostate gene regulation and cytodifferentiation
Authors: Liu Alvin Y. , True Lawrence D. , LaTray Leah , Nelson Peter S. , Ellis William J. , Vessella Robert L. , Lange Paul H. , Hood Leroy , van den Engh Ger
Published: July 2002

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57
+
luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and
PSA
mRNA is one of the abundant messages in these cells. We show that
PSA
expression by the CD57
+
cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57
+
cells are reconstituted with stromal cells. The CD44
+
basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44
+
cells are cocultured with stromal cells. Our studies show that cell–cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.
Neurotrophin release by neurotrophins: Implications for activity-dependent neuronal plasticity
Authors: Canossa Marco , Griesbeck Oliver , Berninger Benedikt , Campana Gabriele , Kolbeck Roland , Thoenen Hans
Published: July 2002

Neurotrophins, secreted in an activity-dependent manner, are thought to be involved in the activity-dependent refinement of synaptic connections. Here we demonstrate that in hippocampal neurons and the rat pheochromocytoma cell line PC12 application of exogenous neurotrophins induces secretion of neurotrophins, an effect that is mediated by the activation of tyrosine kinase neurotrophin receptors (Trks). Like activity-dependent secretion of neurotrophins, neurotrophin-induced neurotrophin secretion requires mobilization of calcium from intracellular stores. Because neurotrophins are likely to be released from both dendrites and axons, neurotrophin-induced neurotrophin release represents a potential positive feedback mechanism, contributing to the reinforcement and stabilization of synaptic connections.
Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of
Saccharomyces cerevisiae
Authors: Yano Ken-ichi , Fukasawa Toshio
Published: July 2002

When galactose is added to logarithmically growing culture of the yeast
Saccharomyces cerevisiae
, a set of genes encoding galactose-metabolizing enzymes (
GAL
genes) starts to be transcribed within a few minutes. This rapid induction involves a serial interplay of Gal3p, Gal80p, and Gal4p. Recent experiments have indicated that a direct interaction between Gal3p and Gal80p plays a pivotal role in an early step of
GAL
induction. Here we demonstrate that complex of Gal3p and Gal80p, otherwise unstable, is stabilized in the presence of 0.1 mM galactose and 0.5 mM ATP. The requirement for galactose and ATP for stable complex formation is also observed by using highly purified Gal3p and Gal80p from yeast. We further show that thus formed Gal3p/Gal80p complex can easily be dissociated when it is washed with buffer lacking galactose. Finally, we show that mutant proteins encoded by

GAL80
S

or
GAL80DE21
, which confer galactose-uninducible phenotype, fail to interact with Gal3p. These results strongly suggest that Gal3p functions as the sensor and transducer of galactose signal in the induction pathway of Gal4p-activated genes.
Barx2
, a new homeobox gene of the
Bar
class, is expressed in neural and craniofacial structures during development
Authors: Jones Frederick S. , Kioussi Chrissa , Copertino Donald W. , Kallunki Pekka , Holst Brent D. , Edelman Gerald M.
Published: July 2002

Homeobox genes are regulators of place-dependent morphogenesis and play important roles in controlling the expression patterns of cell adhesion molecules (CAMs). To identify proteins that bind to a regulatory element common to the genes for two neural CAMs, Ng–CAM and L1, we screened a mouse cDNA expression library with a concatamer of the sequence CCATTAGPyGA and found a new homeobox gene, which we have called
Barx2
. The homeodomain encoded by
Barx2
is 87% identical to that of
Barx1
, and both genes are related to genes at the
Bar
locus of
Drosophila melanogaster
.
Barx1
and
Barx2
also encode an identical stretch of 17 residues downstream of the homeobox; otherwise, they share no appreciable homology.
In vitro
,
Barx2
stimulated activity of an L1 promoter construct containing the CCATTAGPyGA motif but repressed activity when this sequence was deleted. Localization studies showed that expression of
Barx1
and
Barx2
overlap in the nervous system, particularly in the telencephalon, spinal cord, and dorsal root ganglia.
Barx2
was also prominently expressed in the floor plate and in Rathke’s pouch. During craniofacial development,
Barx1
and
Barx2
showed complementary patterns of expression: whereas
Barx1
appeared in the mesenchyme of the mandibular and maxillary processes,
Barx2
was observed in the ectodermal lining of these tissues. Intense expression of
Barx2
was observed in small groups of cells undergoing tissue remodeling, such as ectodermal cells within indentations surrounding the eye and maxillo-nasal groove and in the first branchial pouch, lung buds, precartilagenous condensations, and mesenchyme of the limb. The localization data, combined with
Barx2
’s dual function as activator and repressor, suggest that
Barx2
may differentially control the expression of L1 and other target genes during embryonic development.