bluuebunny/crossref_metadata_2025 huggingface dataset:
The tumor spectrum in
FHIT-
deficient mice
Authors: Zanesi Nicola , Fidanza Vincenzo , Fong Louise Y. , Mancini Rita , Druck Teresa , Valtieri Mauro , Rüdiger Thomas , McCue Peter A. , Croce Carlo M. , Huebner Kay
Published: July 2002

Mice carrying one inactivated
Fhit
allele (
Fhit
+/− mice) are highly susceptible to tumor induction by
N-
nitrosomethylbenzylamine, with 100% of
Fhit
+/− mice exhibiting tumors of the forestomach/squamocolumnar junction vs. 25% of
Fhit
+/+ controls. In the current study a single
N-
nitrosomethylbenzylamine dose was administered to
Fhit
+/+, +/−, and −/− mice to compare carcinogen susceptibility in +/- and −/−
Fhit-
deficient mice. At 29 weeks after treatment, 7.7% of wild-type mice had tumors. Of the
Fhit
−/− mice 89.5% exhibited tumors (average 3.3 tumors/mouse) of the forestomach and squamocolumnar junction; half of the −/− mice had medium (2 mm diameter) to large (>2 mm) tumors. Of the
Fhit
+/− mice 78% exhibited tumors (average 2.4 tumors/mouse) and 22% showed medium to large tumors. Untreated
Fhit-
deficient mice have been observed for up to 2 years for spontaneous tumors.
Fhit
+/− mice (average age 21 mo) exhibit an average of 0.94 tumors of different types;
Fhit
−/− mice (average age 16 mo) also showed an array of tumors (average 0.76 tumor/mouse). The similar spontaneous and induced tumor spectra observed in mice with one or both
Fhit
alleles inactivated suggests that
Fhit
may be a one-hit tumor suppressor gene in some tissues.
High rate of
CAD
gene amplification in human cells deficient in MLH1 or MSH6
Authors: Chen Sihong , Bigner Sandra H. , Modrich Paul
Published: July 2002

MutS and MutL homologs have been implicated in multiple genetic stabilization pathways. The activities participate in the correction of DNA biosynthetic errors, are involved in cellular responses to certain types of DNA damage, and serve to ensure the fidelity of genetic recombination. We show here that the rate of
CAD
(carbamyl-P synthetase/aspartate transcarbamylase/dihydroorotase) gene amplification is elevated 50- to 100-fold in human cell lines deficient in MLH1 or MSH6, as compared with mismatch repair-proficient control cells. Fluorescence
in situ
hybridization indicates that these amplification events are the probable consequence of unequal sister chromatid exchanges involving chromosome 2, as well as translocation events involving other chromosomes. These results implicate MutSα and MutLα in the suppression of gene amplification and suggest that defects in this genetic stabilization function may contribute to the cancer predisposition associated with mismatch repair deficiency.
Histone deacetylase inhibitors reduce polyglutamine toxicity
Authors: McCampbell Alexander , Taye Addis A. , Whitty Leslie , Penney Ellen , Steffan Joan S. , Fischbeck Kenneth H.
Published: July 2002

Polyglutamine diseases include at least nine
neurodegenerative disorders, each caused by a CAG repeat expansion in a
different gene. Accumulation of mutant polyglutamine-containing
proteins occurs in patients, and evidence from cell culture and animal
experiments suggests the nucleus as a site of pathogenesis. To
understand the consequences of nuclear accumulation, we created a cell
culture system with nuclear-targeted polyglutamine. In our system, cell
death can be mitigated by overexpression of full-length cAMP response
element binding protein (CREB)-binding protein (CBP) or its
amino-terminal portion alone. CBP is one of several histone
acetyltransferases sequestered by polyglutamine inclusions. We found
histone acetylation to be reduced in cells expressing mutant
polyglutamine. Reversal of this hypoacetylation, which can be achieved
either by overexpression of CBP or its amino terminus or by treatment
with deacetylase inhibitors, reduced cell loss. These findings suggest
that nuclear accumulation of polyglutamine can lead to altered protein
acetylation in neurons and indicate a novel therapeutic strategy for
polyglutamine disease.
Cell cycle-dependent modulation of telomerase activity in tumor cells.
Authors: Zhu X , Kumar R , Mandal M , Sharma N , Sharma H W , Dhingra U , Sokoloski J A , Hsiao R , Narayanan R
Published: July 2002

Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. We tested this hypothesis by arresting human tumor cell lines at different stages of the cell cycle. Induction of quiescence by serum deprivation did not affect telomerase activity. Cells arrested at the G1/S phase of the cell cycle showed similar levels of telomerase to asynchronous cultures; progression through the S phase was associated with increased telomerase activity. The highest level of telomerase activity was detected in S-phase cells. In contrast, cells arrested at G2/M phase of the cell cycle were almost devoid of telomerase activity. Diverse cell cycle blockers, including transforming growth factor beta1 and cytotoxic agents, also caused inhibition of telomerase activity. These results establish a direct link between telomerase activity and progression through the cell cycle.
Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.
Authors: Härle-Bachor C , Boukamp P
Published: July 2002

Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.
Cloning and characterization of cDNAs for matrix metalloproteinases of regenerating newt limbs.
Authors: Miyazaki K , Uchiyama K , Imokawa Y , Yoshizato K
Published: July 2002

Matrix metalloproteinases (MMPs) of regenerating urodele limbs have been suggested to play crucial roles in the process of the dedifferentiation of cells in the damaged tissues and the ensuing blastema formation because the activation of MMPs is an early and conspicuous event occurring in the amputated limb. MMP cDNAs were cloned as products of the reverse transcription-PCR from cDNA libraries of newt limbs, and their structures were characterized. Three cDNAs encoding newt MMPs (2D-1, 2D-19, and 2D-24) have been cloned from second day postamputation regenerating limbs, and a cDNA (EB-1) was cloned from early bud-stage regenerating limbs. These cDNAs included the full-length coding regions. The deduced amino acid sequences of 2D-1, 2D-19, 2D-24, and EB-1 had a homology with mammalian MMP9, MMP3/10, MMP3/10, and MMP13, respectively. The basic motif of these newt MMP genes was similar to mammalian counterparts and contained regions encoding a putative signal sequence, a propeptide, an active site with three zinc-binding histidine residues, a calcium-binding domain, a hemopexin region, and three key cysteine residues. However, some unique molecular evolutionary features were also found in the newt MMPs. cDNAs of 2D-19 and 2D-24 contained a specific insertion and deletion, respectively. The insertion of 2D-19 is threonine-rich, similar to the threonine cluster found in the collagenase-like sea urchin hatching enzyme. Northern blot analysis showed that the expression levels of the newt MMPs were dramatically increased after amputation, suggesting that they play an important role(s) in tissue remodeling of the regenerating limb.
Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein.
Authors: Williamson R A , Peretz D , Smorodinsky N , Bastidas R , Serban H , Mehlhorn I , DeArmond S J , Prusiner S B , Burton D R
Published: July 2002

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.
Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).
Authors: Joseph K , Ghebrehiwet B , Peerschke E I , Reid K B , Kaplan A P
Published: July 2002

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.
ADrosophilagene structurally and functionally homologous to the mammalian 70-kDa S6 kinase gene
Authors: Watson Kellie L. , Chou Margaret M. , Blenis John , Gelbart William M. , Erikson Raymond L.
Published: July 2002

cDNAs encoding theDrosophila70-kDa S6 kinase (S6K) were isolated by low-stringency hybridization with mammalian p70S6kprobes. Conceptual translation ofS6kcDNA sequences yields a product containing all of the canonical features typical of serine/threonine kinases and has 78% amino acid identity in the catalytic domain with the human p70S6khomologue. TheS6kgene, located at polytene chromosome site 65D, gives rise to two predominant transcripts of 3.0 and 5.0 kb and at least two smaller transcripts (<3.0 kb) that are found in whole-animal RNAs at all stages of development. Blood cells derived from the hematopoietic organs of ribosomal protein S6 (RpS6air8) mutant animals express higher levels of the smallerS6ktranscripts, suggesting tissue- or genotype-specific differences in the regulation of theS6kgene.DrosophilaS6K expressed in COS or NIH 3T3 cells phosphorylates mammalian RPS6 in a mitogen-dependent wortmannin- and rapamycin-sensitive manner, suggesting that its regulation is similiar to mammalian p70S6k.
The evolution of begging: Signaling and sibling competition
Authors: Rodríguez-Gironés Miguel A. , Cotton Peter A. , Kacelnik Alex
Published: July 2002

In many species, young solicit food from their parents, which
respond by feeding them. Because of the difference in genetic make-up
between parents and their offspring and the consequent conflict, this
interaction is often studied as a paradigm for the evolution of
communication. Existent theoretical models demonstrate that chick
signaling and parent responding can be stable if solicitation is a
costly signal. The marginal cost of producing stronger signals allows
the system to converge to an equilibrium: young beg with intensity that
reflects their need, and parents use this information to maximize their
own inclusive fitness. However, we show that there is another
equilibrium where chicks do not beg and parents’ provisioning effort
is optimal with respect to the statistically probable distribution of
chicks’ states. Expected fitness for parents and offspring at the
nonsignaling equilibrium is higher than at the signaling equilibrium.
Because nonsignaling is stable and it is likely to be the ancestral
condition, we would like to know how natural systems evolved from
nonsignaling to signaling. We suggest that begging may have evolved
through direct sibling fighting before the establishment of a parental
response, that is, that nonsignaling squabbling leads to signaling. In
multiple-offspring broods, young following a condition-dependent
strategy in the contest for resources provide information about their
condition. Parents can use this information even though it is not an
adaptation for communication, and evolution will lead the system to the
signaling equilibrium. This interpretation implies that signaling
evolved in multiple-offspring broods, but given that signaling is
evolutionarily stable, it would also be favored in species which
secondarily evolved single-chick broods.
Evolutionary theory predicts late-life mortality plateaus
Authors: Mueller Laurence D. , Rose Michael R.
Published: July 2002

Most demographic data indicate a roughly exponential increase in
adult mortality with age, a phenomenon that has been explained in terms
of a decline in the force of natural selection acting on age-specific
mortality. Scattered demographic findings suggest the existence of a
late-life mortality plateau in both humans and dipteran insects,
seemingly at odds with both prior data and evolutionary theory.
Extensions to the evolutionary theory of aging are developed which
indicate that such late-life mortality plateaus are to be expected when
enough late-life data are collected. This expanded theory predicts
late-life mortality plateaus, with both antagonistic pleiotropy and
mutation accumulation as driving population genetic mechanisms.
Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.
Authors: Magnani M , Casabianca A , Fraternale A , Brandi G , Gessani S , Williams R , Giovine M , Damonte G , De Flora A , Benatti U
Published: July 2002

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.
PU.1 can participate in an active enhancer complex without its transcriptional activation domain
Authors: Pongubala Jagan M. R. , Atchison Michael L.
Published: July 2002

The transcription factor PU.1 is necessary for the development of multiple hematopoietic lineages and contributes to the activity of the immunoglobulin κ 3′ enhancer. A variety of proteins bind to the 3′ enhancer (PU.1, PIP, ATF1, CREM, c-Fos, c-Jun, and E2A), but the mechanism of 3′-enhancer activity and the proteins necessary for its activity are presently unclear. We show here that PU.1 participates with other transcription factors in forming a higher-order complex with 3′-enhancer DNA sequences. Each protein is necessary for formation of this complex. Individually, transcription factors that bind to the 3′ enhancer do not appreciably stimulate transcription in a cell type in which the 3′ enhancer is normally silent (NIH 3T3). However, mixture of multiple transcription factors (PU.1, PIP, c-Fos, and c-Jun) can greatly activate the enhancer. PU.1 is necessary for maximal enhancer activity, but mutants of PU.1 that lack the transcriptional activation domain are nearly as efficient at stimulating enhancer activity as the wild-type PU.1 protein. PU.1 apparently can activate transcription by playing an architectural role in interactions with other transcription factors.
Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GH in early embryogenesis?
Authors: Pantaleon M. , Whiteside E. J. , Harvey M. B. , Barnard R. T. , Waters M. J. , Kaye P. L.
Published: July 2002

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages. In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose–response curves for GH stimulation of both 3-
O
-methyl glucose transport and protein synthesis. Maximal stimulation of 40–50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GH. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription–PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.
F-actin and G-actin binding are uncoupled by mutation of conserved tyrosine residues in maize actin depolymerizing factor (ZmADF)
Authors: Jiang Chang-Jie , Weeds Alan G. , Khan Safina , Hussey Patrick J.
Published: July 2002

Actin depolymerizing factors (ADF) are stimulus responsive actin cytoskeleton modulating proteins. They bind both monomeric actin (G-actin) and filamentous actin (F-actin) and, under certain conditions, F-actin binding is followed by filament severing. In this paper, using mutant maize ADF3 proteins, we demonstrate that the maize ADF3 binding of F-actin can be spatially distinguished from that of G-actin. One mutant, zmadf3–1, in which Tyr-103 and Ala-104 (equivalent to destrin Tyr-117 and Ala-118) have been replaced by phenylalanine and glycine, respectively, binds more weakly to both G-actin and F-actin compared with maize ADF3. A second mutant, zmadf3–2, in which both Tyr-67 and Tyr-70 are replaced by phenylalanine, shows an affinity for G-actin similar to maize ADF3, but F-actin binding is abolished. The two tyrosines, Tyr-67 and Tyr-70, are in the equivalent position to Tyr-82 and Tyr-85 of destrin, respectively. Using the tertiary structure of destrin, yeast cofilin, and
Acanthamoeba actophorin
, we discuss the implications of removing the aromatic hydroxyls of Tyr-82 and Tyr-85 (i.e., the effect of substituting phenylalanine for tyrosine) and conclude that Tyr-82 plays a critical role in stabilizing the tertiary structure that is essential for F-actin binding. We propose that this tertiary structure is maintained as a result of a hydrogen bond between the hydroxyl of Tyr-82 and the carbonyl of Tyr-117, which is located in the long α-helix; amino acid components of this helix (Leu-111 to Phe-128) have been implicated in G-actin and F-actin binding. The structures of human destrin and yeast cofilin indicate a hydrogen distance of 2.61 and 2.77 Å, respectively, with corresponding bond angles of 99.5° and 113°, close to the optimum for a strong hydrogen bond.
Lack of cell cycle regulation of telomerase activity in human cells
Authors: Holt Shawn E. , Aisner Dara L. , Shay Jerry W. , Wright Woodring E.
Published: July 2002

Conflicting reports have appeared concerning the cell cycle regulation of telomerase activity and its possible repression during quiescence and cell differentiation. We have reexamined these issues in an attempt to uncover the basis for the discrepancies. Variations in extracted telomerase activity during the cell cycle are not observed in cells sorted on the basis of DNA content. Variations are observed in cells synchronized using some biochemical cell cycle inhibitors, but only with those agents where cellular toxicity is evident. A progressive decline in telomerase activity is observed in cells whose growth rate is reduced from seven to eight population doublings per week to one to two doublings per week. Telomerase is largely absent in cells that truly exit the cell cycle and do not divide over the 7-day period. Although it is not necessary for all cell types to regulate telomerase in the same way, we conclude that in the immortal cultured cell lines examined, extracted telomerase activity does not change significantly during progression through the stages of the cell cycle. Telomerase activity generally correlates with growth rate and is repressed in cells that exit the cell cycle and become quiescent.
Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone
Authors: Armitage Bruce , Ly Danith , Koch Troels , Frydenlund Henrik , Ørum Henrik , Batz Hans G. , Schuster Gary B.
Published: July 2002

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson–Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA–DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA–DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.
Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid
Authors: Kang Jing X. , Li Yunyuan , Leaf Alexander
Published: July 2002

Retinoic acid (RA) exerts diverse biological effects in the control of cell growth in embryogenesis and oncogenesis. These effects of RA are thought to be mediated by the nuclear retinoid receptors. Mannose-6-phosphate (M6P)/insulin-like growth factor-II (IGF-II) receptor is a multifunctional membrane glycoprotein that is known to bind both M6P and IGF-II and function primarily in the binding and trafficking of lysosomal enzymes, the activation of transforming growth factor-β, and the degradation of IGF-II. M6P/IGF-II receptor has recently been implicated in fetal development and carcinogenesis. Despite the functional similarities between RA and the M6P/IGF-II receptor, no direct biochemical link has been established. Here, we show that the M6P/IGF-II receptor also binds RA with high affinity at a site that is distinct from those for M6P and IGF-II, as identified by a photoaffinity labeling technique. We also show that the binding of RA to the M6P/IGF-II receptor enhances the primary functions of this receptor. The biological consequence of the interaction appears to be the suppression of cell proliferation and/or induction of apoptosis. These findings suggest that the M6P/IGF-II receptor mediates a RA response pathway that is important in cell growth regulation. This discovery of the interaction of RA with the M6P/IGF-II receptor may have important implications for our understanding of the roles of RA and the M6P/IGF-II receptor in development, carcinogenesis, and lysosomal enzyme-related diseases.
Pore size of the malaria parasite’s nutrient channel
Authors: Desai Sanjay A. , Rosenberg Robert L.
Published: July 2002

The malaria parasite,
Plasmodium falciparum
, requires large amounts of nutrients to sustain its rapid growth within the human red blood cell. A recently identified ion channel on the surface of the intraerythrocytic parasite may provide direct access to these nutrients in the red blood cell cytosol. Evidence supporting this role was obtained by incorporating this channel into planar lipid bilayers. In bilayers, this channel has conductance and gating properties identical to the
in situ
channel, passes soluble macromolecules of up to 1400 Da, and functions as a high capacity, low affinity molecular sieve. These properties, remarkably similar to those of a pore on
Toxoplasma gondii
(another protozoan parasite causing human disease), suggest a novel class of channels used by these intracellular parasites to acquire nutrients from host cytosol.
A substance P antagonist, RP-67,580, ameliorates a mouse meningoencephalitic response toTrypanosoma brucei brucei
Authors: Kennedy Peter G. E. , Rodgers Jean , Jennings Francis W. , Murray Max , Leeman Susan E. , Burke Joanne M.
Published: July 2002

Mice infected with the protozoan parasiteTrypanosoma brucei bruceiand treated subcuratively with the trypanocidal drug diminazene aceturate develop an acute inflammatory meningoencephalitis with associated astrocytic proliferation. This reaction is very similar to that seen in the fatal posttreatment reactive encephalopathies that can occur in human African trypanosomiasis. The 11-amino acid neuropeptide substance P (SP) has recently been identified as a mediator in many inflammatory responses, and the development of potent, highly specific, nonpeptide SP antagonists has provided a new opportunity to investigate the possible involvement of SP in a variety of pathological conditions. We therefore postulated that SP may play a role in the development of the posttreatment inflammatory encephalopathy found in this experimental mouse model of African trypanosomiasis. In the present study RP-67,580, a SP antagonist that binds specifically to NK-1 receptors, was given intraperitoneally at a dose of 2 mg/kg twice daily to mice in which a severe meningoencephalitis had been produced. A significant reduction in both the severity of the inflammatory response (P= 0.0001) as well as the degree of astrocyte activation (P< 0.001) was found in the brains of these animals as compared with control mice that had not received RP-67,580. An inactive enantiomer of this SP antagonist, RP-68,651, had no effect on the central nervous system inflammatory reaction. We conclude from these findings that the neuropeptide SP plays a key role in the development of the severe central nervous system inflammatory response associated with African trypanosomiasis.
Transient and selectable transformation of the parasitic protist
Trichomonas vaginalis
Authors: Delgadillo Maria G. , Liston David R. , Niazi Kayvan , Johnson Patricia J.
Published: July 2002

We have developed methods to transiently and selectably transform the human-infective protist
Trichomonas vaginalis
. This parasite, a common cause of vaginitis worldwide, is one of the earlier branching eukaryotes studied to date. We have introduced three heterologous genes into
T. vaginalis
by electroporation and have used the 5′ and 3′ untranslated regions of the endogenous gene α-succinyl CoA synthetase B (
α-SCSB
) to drive transcription of these genes. Transient expression of two reporter proteins, chloramphenicol acetyltransferase (CAT) or luciferase, was detected when electroporating in the presence of 50 μg closed-circular construct. Optimal levels of expression were observed using ≈2.5 × 10
8
T. vaginalis
cells and 350 volts, 960 μFd for electroporation; however, other conditions also led to significant reporter gene expression. A time course following the expression of CAT in
T. vaginalis
transient transformants revealed the highest level of expression 8–21 hr postelectroporation and showed that CAT activity is undetectable using TLC by 99 hr postelectroporation. The system we established to obtain selectable transformants uses the neomycin phosphotransferase (
neo
) gene as the selectable marker. Cells electroporated with 20 μg of the NEO construct were plated in the presence of 50 μg/ml paromomycin and incubated in an anaerobic chamber. The paromomycin-resistant colonies that formed within 3–5 days were cultivated in the presence of drug and DNA was isolated for analyses. The NEO construct was shown to be maintained episomally, as a closed-circle, at between 10–30 copies per cell. The ability to transiently and selectably transform
T. vaginalis
should greatly enhance research on this important human parasite.
Human cytolytic and interferon γ-secreting CD8+T lymphocytes specific forMycobacterium tuberculosis
Authors: Lalvani Ajit , Brookes Roger , Wilkinson Robert J. , Malin Adam S. , Pathan Ansar A. , Andersen Peter , Dockrell Hazel , Pasvol Geoffrey , Hill Adrian V. S.
Published: July 2002

Protective immunity toMycobacterium tuberculosisis poorly understood, but mounting evidence, at least in animal models, implicates major histocompatibility complex class I-restricted CD8+T cells as an essential component. By using a highly sensitive assay for single cell interferon γ release, we screened an array ofM. tuberculosisantigen-derived peptides congruent with HLA class I allele-specific motifs. We identified CD8+T cells specific for epitopes in the early secretory antigenic target 6 during active tuberculosis, after clinical recovery and in healthy contacts. Unrestimulated cells exhibited peptide-specific interferon γ secretion, whereas lines or clones recognized endogenously processed antigen and showed cytolytic activity. These results provide direct evidence for the involvement of CD8+cytotoxic T lymphocytes in host defense againstM. tuberculosisin humans and support current attempts to generate protective cytotoxic T lymphocyte responses againstM. tuberculosisby vaccination.
Optimizing the stability of single-chain proteins by linker length and composition mutagenesis
Authors: Robinson Clifford R. , Sauer Robert T.
Published: July 2002

Linker length and composition were varied in libraries of
single-chain Arc repressor, resulting in proteins with effective
concentrations ranging over six orders of magnitude (10 μM–10 M).
Linkers of 11 residues or more were required for biological activity.
Equilibrium stability varied substantially with linker length, reaching
a maximum for glycine-rich linkers containing 19 residues. The effects
of linker length on equilibrium stability arise from significant and
sometimes opposing changes in folding and unfolding kinetics. By fixing
the linker length at 19 residues and varying the ratio of Ala/Gly or
Ser/Gly in a 16-residue-randomized region, the effects of linker
flexibility were examined. In these libraries, composition rather than
sequence appears to determine stability. Maximum stability in the
Ala/Gly library was observed for a protein containing 11 alanines and
five glycines in the randomized region of the linker. In the Ser/Gly
library, the most stable protein had seven serines and nine glycines in
this region. Analysis of folding and unfolding rates suggests that
alanine acts largely by accelerating folding, whereas serine acts
predominantly to slow unfolding. These results demonstrate an important
role for linker design in determining the stability and folding
kinetics of single-chain proteins and suggest strategies for optimizing
these parameters.
Implications of macromolecular crowding for signal transduction and metabolite channeling
Authors: Rohwer Johann M. , Postma Pieter W. , Kholodenko Boris N. , Westerhoff Hans V.
Published: July 2002

The effect of different total enzyme concentrations on the flux through the bacterial phospho
enol
pyruvate:carbohydrate phosphotransferase system (PTS)
in vitro
was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of
Escherichia coli
. The dependence of the flux on the protein concentration was more than linear but less than quadratic. The combined flux–response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of ≈1.8 with increasing protein concentrations in the assay. Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux–response coefficient, in one case to values of ≈1. PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration. This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes. High concentrations of the microsolute glycerol did not affect the combined flux–response coefficient. The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway. Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover. The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed.
WW domain-mediated interactions reveal a spliceosome-associated protein that binds a third class of proline-rich motif: The proline glycine and methionine-rich motif
Authors: Bedford Mark T. , Reed Robin , Leder Phili
Published: July 2002

Pre-mRNA splicing requires the bridging of the 5′ and 3′ ends of the intron. In yeast, this bridging involves interactions between the WW domains in the splicing factor PRP40 and a proline-rich domain in the branchpoint binding protein, BBP. Using a proline-rich domain derived from formin (a product of the murine limb deformity locus), we have identified a family of murine formin binding proteins (FBP’s), each of which contains one or more of a special class of tyrosine-rich WW domains. Two of these WW domains, in the proteins FBP11 and FBP21, are strikingly similar to those found in the yeast splicing factor PRP40. We show that FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains. Moreover, FBP21 interacts directly with the U1 snRNP protein U1C, the core snRNP proteins SmB and SmB′, and the branchpoint binding protein SF1/mBBP. Thus, FBP21 may play a role in cross-intron bridging of U1 and U2 snRNPs in the mammalian A complex.
A family of phase-variable restriction enzymes with differing specificities generated by high-frequency gene rearrangements
Authors: Dybvig Kevin , Sitaraman Ramakrishnan , French C. Todd
Published: July 2002

The
hsd
genes of
Mycoplasma pulmonis
encode restriction and modification enzymes exhibiting a high degree of sequence similarity to the type I enzymes of enteric bacteria. The S subunits of type I systems dictate the DNA sequence specificity of the holoenzyme and are required for both the restriction and the modification reactions. The
M. pulmonis
chromosome has two
hsd
loci, both of which contain two
hsdS
genes each and are complex, site-specific DNA inversion systems. Embedded within the coding region of each
hsdS
gene are a minimum of three sites at which DNA inversions occur to generate extensive amino acid sequence variations in the predicted S subunits. We show that the polymorphic
hsdS
genes produced by gene rearrangement encode a family of functional S subunits with differing DNA sequence specificities. In addition to creating polymorphisms in
hsdS
sequences, DNA inversions regulate the phase-variable production of restriction activity because the other genes required for restriction activity (
hsdR
and
hsdM
) are expressed only from loci that are oriented appropriately in the chromosome relative to the
hsd
promoter. These data cast doubt on the prevailing paradigms that restriction systems are either selfish or function to confer protection from invasion by foreign DNA.
The 30-kDa C-terminal domain of the RecB protein is critical for the nuclease activity, but not the helicase activity, of the RecBCD enzyme from
Escherichia coli
Authors: Yu Misook , Souaya Jehanne , Julin Douglas A.
Published: July 2002

The RecBCD enzyme from
Escherichia coli
is an ATP-dependent helicase and an ATP-stimulated nuclease. The 3′ → 5′ exonuclease activity on double-stranded DNA is suppressed when the enzyme encounters a recombinational hot spot, called chi (χ). We have prepared a RecB deletion mutant (RecB
1–929
) by using results of limited proteolysis experiments that indicated that the RecB subunit consists of two main domains. The RecB
1–929
protein, comprising the 100-kDa N-terminal domain of RecB, is an ATP-dependent helicase and a single-stranded DNA-dependent ATPase. Reconstitution of RecB
1–929
with RecC and RecD leads to processive unwinding of a linearized plasmid. However, the reconstituted RecB
1–929
CD enzyme has lost the single-strand endo- and exonuclease and the double-strand exonuclease activities of the RecBCD enzyme. These results show that the 30-kDa C-terminal domain of RecB has an important role in the nuclease activity of RecBCD. On the basis of these findings, we propose the RecB C-terminal domain swing model to explain RecBCD’s transformation from a 3′ → 5′ exonuclease to a helicase when it meets a χ site.
Alterations in NF-κB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-κB
Authors: Seitz Cornelia S. , Lin Qun , Deng Helen , Khavari Paul A.
Published: July 2002

Stratified epithelium contains a mitotically active basal layer of cells that cease proliferating, then migrate outwards and undergo terminal differentiation. The control of this process, which is abnormal in cutaneous neoplasia and inflammation, is not well understood. In normal epidermis, NF-κB proteins were found to exist in the cytoplasm of basal cells and then to localize in the nuclei of suprabasal cells, suggesting a role for NF-κB in the switch from proliferation to growth arrest and differentiation. Functional blockade of NF-κB by expressing dominant-negative NF-κB inhibitory proteins in transgenic murine and human epidermis produced hyperplastic epitheliumin vivo. Consistent with this, application of a pharmacologic inhibitor of NF-κB to intact skin induced epidermal hyperplasia. In contrast, overexpression of active p50 and p65 NF-κB subunits in transgenic epithelium produced hypoplasia and growth inhibition. These data suggest that spatially restricted NF-κB activation occurs in stratified epithelium and indicate that NF-κB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition.
Highly water-permeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung
Authors: Dobbs Leland G. , Gonzalez Robert , Matthay Michael A. , Carter Ethan P. , Allen Lennell , Verkman A. S.
Published: July 2002

Water permeability measured between the airspace and vasculature in intact sheep and mouse lungs is high. More than 95% of the internal surface area of the lung is lined by alveolar epithelial type I cells. The purpose of this study was to test whether osmotic water permeability (
P
f
) in type I alveolar epithelial cells is high enough to account for the high
P
f
of the intact lung.
P
f
measured between the airspace and vasculature in the perfused fluid-filled rat lung by the pleural surface fluorescence method was high (0.019 ± 0.004 cm/s at 12°C) and weakly temperature-dependent (activation energy 3.7 kcal/mol). To resolve the contributions of type I and type II alveolar epithelial cells to lung water permeability,
P
f
was measured by stopped-flow light scattering in suspensions of purified type I or type II cells obtained by immunoaffinity procedures. In response to a sudden change in external solution osmolality from 300 to 600 mOsm, the volume of type I cells decreased rapidly with a half-time (
t
1/2
) of 60–80 ms at 10°C, giving a plasma membrane
P
f
of 0.06–0.08 cm/s.
P
f
in type I cells was independent of osmotic gradient size and was weakly temperature-dependent (activation energy 3.4 kcal/mol). In contrast,
t
1/2
for type II cells in suspension was much slower, ≈1 s;
P
f
for type II cells was 0.013 cm/s. Vesicles derived from type I cells also had a very high
P
f
of 0.06–0.08 cm/s at 10°C that was inhibited 95% by HgCl
2
. The
P
f
in type I cells is the highest measured for any mammalian cell membrane and would account for the high water permeability of the lung.
A peroxisome proliferator-activated receptor γ ligand inhibits adipocyte differentiation
Authors: Oberfield Jennifer L. , Collins Jon L. , Holmes Christopher P. , Goreham Donna M. , Cooper Joel P. , Cobb Jeffery E. , Lenhard James M. , Hull-Ryde Emily A. , Mohr Christopher P. , Blanchard Steven G. , Parks Derek J. , Moore Linda B. , Lehmann Jürgen M. , Plunket Kelli , Miller Ann B. , Milburn Michael V. , Kliewer Steven A. , Willson Timothy M.
Published: July 2002

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate glucose and lipid homeostasis. The PPARγ subtype plays a central role in the regulation of adipogenesis and is the molecular target for the 2,4-thiazolidinedione class of antidiabetic drugs. Structural studies have revealed that agonist ligands activate the PPARs through direct interactions with the C-terminal region of the ligand-binding domain, which includes the activation function 2 helix. GW0072 was identified as a high-affinity PPARγ ligand that was a weak partial agonist of PPARγ transactivation. X-ray crystallography revealed that GW0072 occupied the ligand-binding pocket by using different epitopes than the known PPAR agonists and did not interact with the activation function 2 helix. In cell culture, GW0072 was a potent antagonist of adipocyte differentiation. These results establish an approach to the design of PPAR ligands with modified biological activities.
Differentiation between vasculoprotective and uterotrophic effects of ligands with different binding affinities to estrogen receptors α and β
Authors: Mäkelä Sari , Savolainen Hanna , Aavik Einari , Myllärniemi Marjukka , Strauss Leena , Taskinen Eero , Gustafsson Jan-Åke , Häyry Pekka
Published: July 2002

Estrogen-based drug therapy in cardiovascular diseases has been difficult because it has not been possible to separate the wanted vasculoprotective effect from the unwanted effects of the hormone to the reproductive system. Here, we demonstrate that, after endothelial denudation of rat carotid artery, the mRNA of the classical estrogen receptor (ERα) is constitutively expressed at a low level whereas the expression of the novel ERβ mRNA increases >40-fold. Under
in situ
hybridization and immunohistochemistry, ERβ mRNA and protein colocalize with the smooth muscle cells in the media and neointima. Treatment of ovariectomized female rats with the isoflavone phytoestrogen genistein, which shows 20-fold higher binding affinity to ERβ than to ERα, or with 17β-estradiol, which does not differentiate between the two receptors, provides similar dose-dependent vasculoprotective effect in rat carotid injury model. In addition in concentrations <10 μM, both ligands are equally inhibitory to the replication and migration of smooth muscle cells
in vitro
. However, only treatment with 17β-estradiol, but not with genistein, is accompanied with a dose-dependent uterotrophic effect. The results suggest that preferential targeting to ERβ will provide vasculoprotective estrogen analogs devoid of effects to the reproductive system.
Cholesterol efflux to apolipoprotein AI involves endocytosis and resecretion in a calcium-dependent pathway
Authors: Takahashi Yukihiro , Smith Jonathan D.
Published: July 2002

We previously have described the cAMP-mediated induction of cholesterol and phospholipid efflux from the murine macrophage RAW264 cell line to lipid-free apolipoprotein acceptors. This induction of cholesterol efflux is associated with increased binding and association of apolipoprotein to the cells. In the present study, using primarily apolipoprotein AI (apoAI) as the acceptor, cAMP-dependent cholesterol efflux to apolipoprotein acceptors was associated with apoAI binding to coated pits, cellular uptake, and resecretion. After cell association and washing, 58% of the apoAI was resecreted during a 90-min chase period. In addition, after apoAI uptake and washing, cholesterol efflux was observed during a chase period without additional acceptors. Cholesterol efflux was partially blocked by chlorpromazine and hypertonic media, two inhibitors of coated pit endocytosis. Cholesterol efflux to apoAI was found to depend on extracellular calcium. By temporally separating the cAMP induction phase from the apoAI chase phase, calcium was found to be required during the apoAI chase phase rather than during the cAMP induction period. In the absence of calcium the 8-Br-cAMP-mediated induction of apoAI binding was maintained, but the specific apoAI cellular association was inhibited. The data are consistent with a model for cholesterol efflux to apolipoproteins that involves a calcium-dependent endocytic pathway, followed by recycling and the subsequent release of the nascent lipoprotein particle from the cell.