bluuebunny/crossref_metadata_2025 huggingface dataset:
Brain drug delivery of small molecules using immunoliposomes
Authors: Huwyler Jörg , Wu Dafang , Pardridge William M.
Published: July 2002

Immunoliposomes (antibody-directed liposomes) were used in the
present study for delivery of the antineoplastic agent daunomycin to
the rat brain. A coupling procedure was introduced, which allows
conjugation of a thiolated antibody to maleimide-grafted 85-nm
liposomes sterically stabilized with PEG. Antibody was thereby coupled
to the terminal end of a PEG-conjugated linker lipid. No brain uptake
of PEG-conjugated liposomes carrying [
3
H]daunomycin was
observed. However, brain targeting of immunoliposomes carrying
[
3
H]daunomycin was mediated by the OX26 monoclonal
antibody to the rat transferrin receptor, which is selectively enriched
at the brain microvascular endothelium that comprises the blood–brain
barrier
in vivo
. Coupling of 30 OX26 antibodies per
liposome resulted in optimal brain delivery. Saturation of delivery was
observed at higher antibody densities. Determination of brain levels of
immunoliposomes over 24 h revealed that immunoliposomes accumulate
in brain tissue. Brain targeting of immunoliposomes was not observed in
immunoliposomes conjugated with a mouse IgG
2a
isotype
control. In addition, coinjection of free OX26 saturated plasma
clearance of immunoliposomes. Since a single liposome may carry
≥10,000 drug molecules, the use of PEG-conjugated immunoliposomes
increases the drug carrying capacity of the monoclonal antibody by up
to 4 logarithmic orders in magnitude. In summary, specific
OX26-mediated targeting of daunomycin to the rat brain was achieved by
the use of an immunoliposome-based drug delivery system.
The chimeric genes AML1/MDS1 and AML1/EAP inhibit AML1B activation at the CSF1R promoter, but only AML1/MDS1 has tumor-promoter properties.
Authors: Zent C S , Mathieu C , Claxton D F , Zhang D E , Tenen D G , Rowley J D , Nucifora G
Published: July 2002

The (3;21)(q26;q22) translocation associated with treatment-related myelodysplastic syndrome, treatment-related acute myeloid leukemia, and blast crisis of chronic myeloid leukemia results in the expression of the chimeric genes AML1/EAP, AML1/MDS1, and AML1/EVI1. AML1 (CBFA2), which codes for the alpha subunit of the heterodimeric transcription factor CBF, is also involved in the t(8;21), and the gene coding for the beta subunit (CBFB) is involved in the inv(16). These are two of the most common recurring chromosomal rearrangements in acute myeloid leukemia. CBF corresponds to the murine Pebp2 factor, and CBF binding sites are found in a number of eukaryotic and viral enhancers and promoters. We studied the effects of AML1/EAP and AML1/MDS1 at the AML1 binding site of the CSF1R (macrophage-colony-stimulating factor receptor gene) promoter by using reporter gene assays, and we analyzed the consequences of the expression of both chimeric proteins in an embryonic rat fibroblast cell line (Rat1A) in culture and after injection into athymic nude mice. Unlike AML1, which is an activator of the CSF1R promoter, the chimeric proteins did not transactivate the CSF1R promoter site but acted as inhibitors of AML1 (CBFA2). AML1/EAP and AML1/MDS1 expressed in adherent Rat1A cells decreased contact inhibition of growth, and expression of AML1/MDS1 was associated with acquisition of the ability to grow in suspension culture. Expression of AML1/MDS1 increased the tumorigenicity of Rat1A cells injected into athymic nude mice, whereas AML1/EAP expression prevented tumor growth. These results suggest that expression of AML1/EAP and AML1/MDS1 can interfere with normal AML1 function, and that AML1/MDS1 has tumor-promoting properties in an embryonic rat fibroblast cell line.
P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.
Authors: de Graaf D , Sharma R C , Mechetner E B , Schimke R T , Roninson I B
Published: July 2002

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.
Poliovirus chimeras replicating under the translational control of genetic elements of hepatitis C virus reveal unusual properties of the internal ribosomal entry site of hepatitis C virus.
Authors: Lu H H , Wimmer E
Published: July 2002

Chimeric genomes of poliovirus (PV) have been constructed in which the cognate internal ribosomal entry site (IRES) element was replaced by genetic elements of hepatitis C virus (HCV). Replacement of PV IRES with nt 9-332 of the genotype Ib HCV genome, a sequence comprising all but the first eight residues of the 5' nontranslated region (5'NTR) of HCV, resulted in a lethal phenotype. Addition of 366 nt of the HCV core-encoding sequence downstream of the HCV 5'NTR yielded a viable PV/HCV chimera, which expressed a stable, small-plaque phenotype. This chimeric genome encoded a truncated HCV core protein that was fused to the N terminus of the PV polyprotein via an engineered cleavage site for PV proteinase 3CPpro. Manipulation of the HCV core-encoding sequence of this viable chimera by deletion and frameshift yielded results suggesting that the 5'-proximal sequences of the HCV open reading frame were essential for viability of the chimera and that the N-terminal basic region of the HCV core protein is required for efficient replication of the chimeric virus. These data suggest that the bona fide HCV IRES includes genetic information mapping to the 5'NTR and sequences of the HCV open reading frame. PV chimeras replicating under translational control of genetic elements of HCV can serve to study HCV IRES function in vivo and to search for anti-HCV chemotherapeutic agents.
Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.
Authors: Baruch D I , Gormely J A , Ma C , Howard R J , Pasloske B L
Published: July 2002

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.
Spare the rod, spoil the regulation: Necessity for a myosin rod
Authors: Trybus Kathleen M. , Freyzon Yelena , Faust Lynn Z. , Sweeney H. Lee
Published: July 2002

Regulation of a variety of cellular contractile events requires
that vertebrate smooth and non-muscle myosin II can achieve an
“off” state. To examine the role of the myosin rod in this
process, we determined the minimal size at which a myosin molecule is
capable of regulation via light chain phosphorylation. Expressed smooth
muscle myosin subfragments with as many as 100 amino acids of the
coiled-coil rod sequence did not dimerize and were active independently
of phosphorylation. To test whether dimerization per se restores
regulation of ATPase activity, mutants were expressed with varying
lengths of rod sequence, followed by C-terminal leucine zippers to
stabilize the coiled-coil. Dimerization restored partial regulation,
but the presence of a length of rod approximately equal to the myosin
head was necessary to achieve a completely off state. Partially
regulated short dimers could be converted into fully regulated
molecules by addition of native rod sequence after the zipper. These
results suggest that the myosin rod mediates specific interactions with
the head that are required to obtain the completely inactive state of
vertebrate smooth and non-muscle myosins. If these interactions are
prohibited under cellular conditions, unphosphorylated crossbridges can
slowly cycle.
Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (
Lupinus angustifolius
L.) expressing a sunflower seed albumin gene
Authors: Molvig Lisa , Tabe Linda M. , Eggum Bjorn O. , Moore Andrew E. , Craig Stuart , Spencer Donald , Higgins Thomas J. V.
Published: July 2002

With the aim of improving the nutritive value of an important grain legume crop, a chimeric gene specifying seed-specific expression of a sulfur-rich, sunflower seed albumin was stably transformed into narrow-leafed lupin (
Lupinus angustifolius
L.). Sunflower seed albumin accounted for 5% of extractable seed protein in a line containing a single tandem insertion of the transferred DNA. The transgenic seeds contained less sulfate and more total amino acid sulfur than the nontransgenic parent line. This was associated with a 94% increase in methionine content and a 12% reduction in cysteine content. There was no statistically significant change in other amino acids or in total nitrogen or total sulfur contents of the seeds. In feeding trials with rats, the transgenic seeds gave statistically significant increases in live weight gain, true protein digestibility, biological value, and net protein utilization, compared with wild-type seeds. These findings demonstrate the feasibility of using genetic engineering to improve the nutritive value of grain crops.
Myofibrillogenesis visualized in living embryonic cardiomyocytes
Authors: Dabiri Guissou A. , Turnacioglu Kenan K. , Sanger Jean M. , Sanger Joseph W.
Published: July 2002

Myofibril formation was visualized in cultured live cardiomyocytes that were transfected with plasmids expressing green fluorescent protein (GFP) linked to the Z-band protein, α-actinin. The expression of this fluorescent protein provided an
in vivo
label for structures containing α-actinin. The GFP–α-actinin fusion protein was incorporated into Z-bands, intercalated discs, and attachment plaques, as well as into the punctate aggregates, or Z-bodies, that are thought to be the precursors of Z-bands. Observations of live cells over several days in culture permitted us to test aspects of several theories of myofibril assembly that had been proposed previously based on the study of fixed cells. Fine fibrils, called premyofibrils, that formed
de novo
at the spreading edges of cardiomyocytes, contained punctate concentrations of α-actinin, termed Z-bodies. The punctate Z-bodies grew and aligned with Z-bodies in adjacent fibrils. With increasing time, adjacent fibrils and Z-bodies appeared to fuse and form mature myofibrils and Z-bands in cytoplasmic regions where the linear arrays of Z-bodies had been. These new myofibrils became aligned with existing myofibrils at their Z-bands to form myofibrils that spanned the length of the spread cell. These results are consistent with a model that postulates that the fibrils that form
de novo
near the cell membrane are premyofibrils—i.e., the precursors of mature myofibrils.
Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation
Authors: Kaneko Kiyotoshi , Zulianello Laurence , Scott Michael , Cooper Carol M. , Wallace Andrew C. , James Thomas L. , Cohen Fred E. , Prusiner Stanley B.
Published: July 2002

Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrP
Sc
). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrP
C
) using scrapie-infected mouse (Mo) neuroblastoma cells transfected with chimeric Hu/MoPrP genes even though protein X has not yet been isolated. Substitution of a Hu residue at position 214 or 218 prevented PrP
Sc
formation. The side chains of these residues protrude from the same surface of the C-terminal α-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 also prevented PrP
Sc
formation: at a mechanistic level, these mutant PrPs appear to act as “dominant negatives” by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.
The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism
Authors: Stoffel Markus , Duncan Stephen A.
Published: July 2002

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named
MODY1
). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.
Role of the major histocompatibility complex class IIEagene in lupus susceptibility in mice
Authors: Ibnou-Zekri Nabila , Iwamoto Masahiro , Fossati Liliane , McConahey Patricia J. , Izui Shozo
Published: July 2002

The gene(s) encoded within major histocompatibility complex (MHC) act as one of the major genetic elements contributing to the susceptibility of murine systemic lupus erythematosus (SLE). We have recently demonstrated that lupus susceptibility is more closely linked to the I-E−H-2bhaplotype than to the I-E+H-2dhaplotype in lupus-prone BXSB and (NZB × BXSB)F1hybrid mice. To investigate whether the reduced susceptibility to SLE in H-2dmice is related to the expression of the MHC class IIEagene (absent in H-2bmice), we determined the possible role of theEagene as a lupus protective gene in mice. Our results showed that (i) the development of SLE was almost completely prevented in BXSB (H-2b) mice expressing two copies of theEadtransgene at the homozygous level as well as in BXSB H-2k(I-E+) congenic mice as for H-2dBXSB mice, and (ii) the expression of two functionalEa(transgenic and endogenous) genes in either H-2d/b(NZB × BXSB)F1or H-2k/b(MRL × BXSB)F1mice provided protection from SLE at levels comparable to those conferred by the H-2d/dor H-2k/khaplotype. In addition, the level of theEagene-mediated protection appeared to be dependent on the genetic susceptibility to SLE in individual lupus-prone mice. Our results indicate that the reduced susceptibility associated with the I-E+H-2dand H-2khaplotypes (versus the I-E−H-2bhaplotype) is largely, if not all, contributed by the apparent autoimmune suppressive effect of theEagene, independently of the expression of the I-A or other MHC-linked genes.
Aberrant trafficking of hepatitis B virus glycoproteins in cells in which N-glycan processing is inhibited
Authors: Lu Xuanyong , Mehta Anand , Dadmarz Mitra , Dwek Raymond , Blumberg Baruch S. , Block Timothy M.
Published: July 2002

The role of N-glycan trimming in glycoprotein fate and function is unclear. We have recently shown that hepatitis B virus (HBV) DNA is not efficiently secreted from cells in which α-glucosidase mediated N-glycan trimming is inhibited. Here it is shown that, in cells in glucosidase-inhibited cells, viral DNA, accompanied by envelope and core proteins, most likely accumulate within lysosomal compartments. Pulse–chase experiments show that although the viral glycoproteins (L, M, and S) are dysfunctional, in the sense that they do not mediate virion egress and are not efficiently secreted from the cell, they all still leave the endoplasmic reticulum (ER). Surprisingly, however, the glycoproteins retained within the cell were not rapidly degraded, appearing as aggregates, enriched for L and M, with intracellular half-lives exceeding 20 h. Moreover, by 24 h after synthesis, a substantial fraction of the detained glycoproteins appeared to return to the ER, although a considerable amount was also found in the lysosomes. To our knowledge, this is the first report that shows, as a consequence of inhibiting glycosylation processing, certain glycoproteins (
i
) become dysfunctional and aggregate, yet still depart from the ER, and (
ii
) have extended rather than shortened half-lives. Taken together, these data suggest that proper intracellular routing of HBV glycoproteins requires ER glucosidase function. It is hypothesized that failure to process N-glycan causes HBV glycoproteins to aggregate and that impaired protein–protein interactions and trafficking are the result of misfolding.
Hyperresponsive febrile reactions to interleukin (IL) 1α and IL-1β, and altered brain cytokine mRNA and serum cytokine levels, in IL-1β-deficient mice
Authors: Alheim Katarina , Chai Zhen , Fantuzzi Giamila , Hasanvan Homa , Malinowsky David , Di Santo Elena , Ghezzi Pietro , Dinarello Charles A. , Bartfai Tamas
Published: July 2002

IL-1β is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1α and IL-1β, and compared these with response to LPS (i.p.) in wild-type and IL-1β-deficient mice. The IL-1β deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1β, IL-1α, or LPS induced hyperresponsive fevers in the IL-1β-deficient mice. We also observed phenotypic differences between wild-type and IL-1β-deficient mice in hypothalamic basal mRNA levels for IL-1α and IL-6, but not for IL-1β-converting enzyme or IL-1 receptor type I or type II. The IL-1α mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1β-deficient mice as compared with wild-type mice. The IL-1β-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type α levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1β plays an important but not obligatory role in fever induction by LPS or IL-1α, as well as in the induction of serum tumor necrosis factor type α and corticosterone responses either by LPS or by IL-1α or IL-1β.
AlF
3
mimics the transition state of protein phosphorylation in the crystal structure of nucleoside diphosphate kinase and MgADP
Authors: Xu Ying-Wu , Moréra Solange , Janin Joël , Cherfils Jacqueline
Published: July 2002

Nucleoside diphosphate kinase reversibly transfers the γ-phosphate of ATP onto its active site histidine. We have investigated the transition state of histidine phosphorylation with the high-resolution crystal structures of the enzyme from
Dictyostelium discoideum
with MgADP and either aluminium or beryllium fluoride. The bound aluminium fluoride species is the neutral species AlF
3
and not the more common AlF
4
−
. AlF
3
forms a trigonal bipyramid that makes it an accurate analog of the transition state of the γ-phosphate of ATP undergoing transfer to the catalytic histidine. Its axial ligands are a histidine nitrogen and a β-phosphate oxygen. Beryllium fluoride also binds at the same position and with the same ligands but in a tetrahedral geometry resembling the Michaelis complex rather than the transition state. The two x-ray structures show explicit enzyme–substrate interactions that discriminate between the ground and the transition states of the reaction. They also illustrate the partially dissociative geometry of the transition state of phosphoryl transfer and demonstrate the potential applications of metallofluorides for the study of kinase mechanisms.
Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation
Authors: Jaaro Hanna , Rubinfeld Hadara , Hanoch Tamar , Seger Rony
Published: July 2002

Mitogen-activated protein kinase kinase (MEK) is a dual-specificity protein kinase that is located primarily in the cellular cytosol, both prior to and upon mitogenic stimulation. The existence of a nuclear export signal in the N-terminal domain of MEK [Fukuda, M., Gotoh, I., Gotoh, Y. & Nishida, E. (1996)
J. Biol. Chem.
271, 20024–20028] suggests that there are circumstances under which MEK enters the nucleus and must be exported. Using mutants of MEK, we show that the deletion of the nuclear export signal sequence from constitutively active MEK caused constitutive localization of MEK in the nucleus of COS7 and HEK-293T cells. However, when the same region was deleted from a catalytically inactive MEK, cytoplasmic localization was observed in resting cells, which turned nuclear upon stimulation. Confocal microscopy of COS7 cells expressing the above mutants showed localization of the active MEK in the nuclear envelope and also in the cell periphery. The differences in cellular localization between the wild-type and mutant MEKs are not due to severe changes in specificity because the recombinant, constitutively active MEK that lacked its N-terminal region exhibited the same substrate specificity as the wild-type MEK, both
in vitro
and in intact cells. Taken together, our results indicate that upon mitogenic stimulation, MEK, like extracellular signal responsive kinase and p90
RSK
, is massively translocated to the nucleus. Rapid export from the nucleus, which is mediated by the nuclear export signal, is probably the cause for the cytoplasmic distribution observed with wild-type MEK.
Insulin-like growth factor I protects and rescues hippocampal neurons against β-amyloid- and human amylin-induced toxicity
Authors: Doré Sylvain , Kar Satyabrata , Quirion Rémi
Published: July 2002

Insulin-like growth factors (IGF-I and IGF-II) are well known trophic factors and their specific receptors are uniquely distributed throughout the brain, being especially concentrated in the hippocampal formation. IGFs possess neurotrophic activities in the hippocampus, an area severely affected in Alzheimer disease. These data, together with the evidence that β-amyloid (Aβ)-derived peptides likely play an important role in the neurodegenerative process observed in Alzheimer disease, led us to investigate if IGFs could be neuroprotective to hippocampal neurons against toxicity induced by amyloidogenic derivatives. Exposure of rat primary hippocampal neurons to different concentrations of Aβ25–35, Aβ1–40, Aβ1–42, and human amylin produced marked toxicity, while similar concentrations of two control Aβ peptides—reverse (Aβ40–1) and scrambled sequence (Aβ25–35)—and rat amylin failed to exhibit any significant effect on neuronal survival. IGF-I (10–100 nM) significantly protected hippocampal neurons against neurotoxicity induced by Aβ derivatives and human amylin. The homolog IGF-II was also effective although less potent than IGF-I suggesting the involvement of a typical IGF-I receptor in the observed neuroprotective effect. Most interestingly, IGF-I (10–100 nM) was even able to rescue neurons pre-exposed (up to 4 days) to amyloidogenic peptides. Other neurotrophic factors are reported to lack such rescuing abilities. These results suggest that IGF-I may have unique properties as a potent neuroprotective and neurorescuing agent against amyloid-related neurotoxicity.
Overexpression of transforming growth factor β1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia
Authors: Schulick Andrew H. , Taylor Allen J. , Zuo Wen , Qiu Chang-bin , Dong Gang , Woodward Robert N. , Agah Ramtin , Roberts Anita B. , Virmani Renu , Dichek David A.
Published: July 2002

Uninjured rat arteries transduced with an adenoviral vector expressing an active form of transforming growth factor β1 (TGF-β1) developed a cellular and matrix-rich neointima, with cartilaginous metaplasia of the vascular media. Explant cultures of transduced arteries showed that secretion of active TGF-β1 ceased by 4 weeks, the time of maximal intimal thickening. Between 4 and 8 weeks, the cartilaginous metaplasia resolved and the intimal lesions regressed almost completely, in large part because of massive apoptosis. Thus, locally expressed TGF-β1 promotes intimal growth and appears to cause transdifferentiation of vascular smooth muscle cells into chondrocytes. Moreover, TGF-β1 withdrawal is associated with regression of vascular lesions. These data suggest an unexpected plasticity of the adult vascular smooth muscle cell phenotype and provide an etiology for cartilaginous metaplasia of the arterial wall. Our observations may help to reconcile divergent views of the role of TGF-β1 in vascular disease.
A phenotype-based screen for embryonic lethal mutations in the mouse
Authors: Kasarskis Andrew , Manova Katia , Anderson Kathryn V.
Published: July 2002

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in
Drosophila
,
Caenorhabditis elegans
, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the
open brain
(
opb
) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.
Lipid nanotubes as substrates for helical crystallization of macromolecules
Authors: Wilson-Kubalek Elizabeth M. , Brown Rhoderick E. , Celia Hervé , Milligan Ronald A.
Published: July 2002

A general approach for crystallization of proteins in a fast and simple manner would be of immense interest to biologists studying protein structure–function relationships. Here, we describe a method that we have developed for promoting the formation of helical arrays of proteins and macromolecular assemblies. Electron micrographs of the arrays are suitable for helical image analysis and three-dimensional reconstruction. We show that hydrated mixtures of the glycolipid galactosylceramide (GalCer) and derivatized lipids or charged lipids form unilamellar nanotubules. The tubules bind proteins in a specific manner via high affinity ligands on the polar head groups of the lipid or via electrostatic interactions. By doping the GalCer with a novel nickel-containing lipid, we have been able to form helical arrays of two histidine-tagged proteins. Similarly, doping with a biotinylated lipid allows crystallization of streptavidin. Finally, three proteins with affinity for positively or negatively charged lipid layers formed helical arrays on appropriately charged tubules. The generality of this method may allow a wide variety of proteins to be crystallized on lipid nanotubes under physiological conditions.
In vivo
and
in vitro
complementation of the N-terminal domain of enzyme I of the
Escherichia coli
phosphotransferase system by the cloned C-terminal domain
Authors: Fomenkov Alexey , Valiakhmetov Airat , Brand Ludwig , Roseman Saul
Published: July 2002

Enzyme I (EI) is the first protein in the phosphoryl transfer sequence from phospho
enol
pyruvate (PEP) to sugar in carbohydrate uptake via the bacterial PEP:glycose phosphotransferase system. The EI monomer/dimer transition may regulate the phosphotransferase system because only the EI dimer is autophosphorylated by PEP. We previously showed that the EI monomer comprises two major domains: (
i
) a compact, protease-resistant N-terminal domain (EI-N), containing the active site His, and (
ii
) a flexible, protease-sensitive C-terminal domain (EI-C), which is required for EI dimerization. EI-N interacts with the second protein, HPr, and phospho-HPr, but EI-N neither dimerizes nor is phosphorylated by PEP. We report here the molecular cloning and some properties of EI-C. EI-C is rapidly proteolyzed
in vivo
. Therefore, two different overexpression vectors encoding fusion proteins were constructed. Fusion Xa contains MalE (the maltose-binding protein), the four-amino acid sequence required by protease factor Xa, followed by EI-C. Fusion G contains His-Tyr between MalE and EI-C and is cleaved by the protease genenase. Homogenous EI-C was isolated from fusion G. [
32
P]PEP phosphorylated EI-N when supplemented with EI-C, fusion Xa, or fusion G. EI-C may act catalytically. Complementation was also demonstrated
in vivo
. An
Escherichia coli ptsI
deletion grew on mannitol as the sole source of carbon after it was transformed with two compatible vectors; one vector encoded EI-N and the other encoded fusion Xa or fusion G. The molecular details underlying important properties of EI can now be studied.
A catalytic antibody against cocaine prevents cocaine’s reinforcing and toxic effects in rats
Authors: Mets Berend , Winger Gail , Cabrera Camilo , Seo Susan , Jamdar Subhash , Yang Ginger , Zhao Kang , Briscoe Richard J. , Almonte Rowena , Woods James H. , Landry Donald W.
Published: July 2002

Cocaine addiction and overdose have long defied specific treatment. To provide a new approach, the high-activity catalytic antibody mAb 15A10 was elicited using a transition-state analog for the hydrolysis of cocaine to nontoxic, nonaddictive products. In a model of cocaine overdose, mAb 15A10 protected rats from cocaine-induced seizures and sudden death in a dose-dependent fashion; a noncatalytic anticocaine antibody did not reduce toxicity. Consistent with accelerated catalysis, the hydrolysis product ecgonine methyl ester was increased >10-fold in plasma of rats receiving mAb 15A10 and lethal amounts of cocaine. In a model of cocaine addiction, mAb 15A10 blocked completely the reinforcing effect of cocaine in rats. mAb 15A10 blocked cocaine specifically and did not affect behavior maintained by milk or by the dopamine reuptake inhibitor bupropion. This artificial cocaine esterase is a rationally designed cocaine antagonist and a catalytic antibody with potential for medicinal use.
Cloning and characterization of a third human lysyl hydroxylase isoform
Authors: Passoja Kaisa , Rautavuoma Kati , Ala-Kokko Leena , Kosonen Taru , Kivirikko Kari I.
Published: July 2002

Lysyl hydroxylase (EC
1.14.11.4
), a homodimer, catalyzes the formation of hydroxylysine in collagens. Recently, an isoenzyme termed lysyl hydroxylase 2 has been cloned from human sources [M. Valtavaara, H. Papponen, A.-M. Pirttilä, K. Hiltunen, H. Helander and R. Myllylä (1997)
J. Biol. Chem.
272, 6831–6834]. We report here on the cloning of a third human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3. The cDNA clones encode a 738 amino acid polypeptide, including a signal peptide of 24 residues. The overall amino acid sequence identity between the processed human lysyl hydroxylase 3 and 1 polypeptides is 59%, and that between the processed lysyl hydroxylase 3 and 2 polypeptides is 57%, whereas the identity to the processed
Caenorhabditis elegans
polypeptide is only 45%. All four recently identified critical residues at the catalytic site, two histidines, one aspartate, and one arginine, are conserved in all these polypeptides. The mRNA for lysyl hydroxylase 3 was found to be expressed in a variety of tissues, but distinct differences appear to exist in the expression patterns of the three isoenzyme mRNAs. Recombinant lysyl hydroxylase 3 expressed in insect cells by means of a baculovirus vector was found to be more soluble than lysyl hydroxylase 1 expressed in the same cell type. No differences in catalytic properties were found between the recombinant lysyl hydroxylase 3 and 1 isoenzymes. Deficiency in lysyl hydroxylase 1 activity is known to cause the type VI variant of the Ehlers–Danlos syndrome, and it is therefore possible that deficiency in lysyl hydroxylase 3 activity may lead to some other variant of this syndrome or to some other heritable connective tissue disorder.
A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate
Authors: Lott William B. , Pontius Brian W. , von Hippel Peter H.
Published: July 2002

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La
3+
concentration in the presence of a constant concentration of Mg
2+
. We show that a bell-shaped curve of cleavage activation is obtained as La
3+
is added in micromolar concentrations in the presence of 8 mM Mg
2+
, with a maximal rate of cleavage being attained in the presence of 3 μM La
3+
. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the
K
d
values for Mg
2+
of 3.5 mM and >50 mM, that these sites bind La
3+
with estimated
K
d
values of 0.9 and >37.5 μM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg
2+
by La
3+
at the stronger (relative to Mg
2+
) binding site activating catalysis and displacement of Mg
2+
by La
3+
at the weaker (relative to Mg
2+
) (relative to Mg
2+
) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2′-oxygen species in the reaction and lowers the pK
a
of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5′-oxygen in the transition state. We suggest structural reasons why the Mg
2+
–La
3+
ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.
Transgenic mice carrying an imbalance in the native ratio of A to B forms of progesterone receptor exhibit developmental abnormalities in mammary glands
Authors: Shyamala G. , Yang X. , Silberstein G. , Barcellos-Hoff M. H. , Dale E.
Published: July 2002

In this report we document the creation of transgenic mice in which the native ratio of A and B forms of progesterone receptor (PR) has been altered by the introduction of additional A form as transgene. We also show that in these mice there is an aberration in mammary development. In ovariectomized prepubertal PR-A transgenic mice, end buds with unusual morphology persist after ovariectomy, and in young adult nonovariectomized mice, mammary glands have extensive lateral branching. The glands of adult mice also exhibit ductal hyperplasia with a disorganized basement membrane and decreased cell–cell adhesion, features commonly associated with neoplasia. Because progesterone is a mitogenic hormone in mammary glands and PR is required for mammary development, these data provide direct evidence that
in vivo
a regulated expression of the two isoforms of PR is critical for appropriate cellular response to progesterone and that for mammary glands this may have major implications to carcinogenesis.
Independent deletions of a pathogen-resistance gene in
Brassica
and
Arabidopsis
Authors: Grant Murray R. , McDowell John M. , Sharpe Andrew G. , de Torres Zabala Marta , Lydiate Derek J. , Dangl Jeffery L.
Published: July 2002

Plant disease resistance (
R
) genes confer race-specific resistance to pathogens and are genetically defined on the basis of intra-specific functional polymorphism. Little is known about the evolutionary mechanisms that generate this polymorphism. Most
R
loci examined to date contain alternate alleles and/or linked homologs even in disease-susceptible plant genotypes. In contrast, the resistance to
Pseudomonas syringae
pathovar
maculicola
(
RPM1
) bacterial resistance gene is completely absent (
rpm1-null
) in 5/5
Arabidopsis thaliana
accessions that lack
RPM1
function. The
rpm1-null
locus contains a 98-bp segment of unknown origin in place of the
RPM1
gene. We undertook comparative mapping of
RPM1
and flanking genes in
Brassica napus
to determine the ancestral state of the
RPM1
locus. We cloned two
B. napus RPM1
homologs encoding hypothetical proteins with ≈81% amino acid identity to
Arabidopsis RPM1
. Collinearity of genes flanking
RPM1
is conserved between
B. napus
and
Arabidopsis
. Surprisingly, we found four additional
B. napus
loci in which the flanking marker synteny is maintained but
RPM1
is absent. These
B. napus rpm1-null
loci have no detectable nucleotide similarity to the
Arabidopsis rpm1-null
allele. We conclude that
RPM1
evolved before the divergence of the Brassicaceae and has been deleted independently in the
Brassica
and
Arabidopsis
lineages. These results suggest that functional polymorphism at
R
gene loci can arise from gene deletions.
Fetal origins of the
TEL-AML1
fusion gene in identical twins with leukemia
Authors: Ford Anthony M. , Bennett Caroline A. , Price Cathy M. , Bruin M. C. A. , Van Wering Elisabeth R. , Greaves Mel
Published: July 2002

The
TEL
(
ETV6
)−
AML1
(
CBFA2
) gene fusion is the most common reciprocal chromosomal rearrangement in childhood cancer occurring in ≈25% of the most predominant subtype of leukemia— common acute lymphoblastic leukemia. The
TEL-AML1
genomic sequence has been characterized in a pair of monozygotic twins diagnosed at ages 3 years, 6 months and 4 years, 10 months with common acute lymphoblastic leukemia. The twin leukemic DNA shared the same unique (or clonotypic) but nonconstitutive
TEL-AML1
fusion sequence. The most plausible explanation for this finding is a single cell origin of the
TEL-AML
fusion in one fetus
in utero
, probably as a leukemia-initiating mutation, followed by intraplacental metastasis of clonal progeny to the other twin. Clonal identity is further supported by the finding that the leukemic cells in the two twins shared an identical rearranged
IGH
allele. These data have implications for the etiology and natural history of childhood leukemia.
Social stress results in altered glucocorticoid regulation and shorter survival in simian acquired immune deficiency syndrome
Authors: Capitanio John P. , Mendoza Sally P. , Lerche Nicholas W. , Mason William A.
Published: July 2002

From early in the AIDS epidemic, psychosocial stressors have been proposed as contributors to the variation in disease course. To test this hypothesis, rhesus macaques were assigned to stable or unstable social conditions and were inoculated with the simian immunodeficiency virus. Animals in the unstable condition displayed more agonism and less affiliation, shorter survival, and lower basal concentrations of plasma cortisol compared with stable animals. Early after inoculation, but before the emergence of group differences in cortisol levels, animals receiving social threats had higher concentrations of simian immunodeficiency virus RNA in plasma, and those engaging in affiliation had lower concentrations. The results indicate that social factors can have a significant impact on the course of immunodeficiency disease. Socially induced changes in pituitary–adrenal hormones may be one mechanism mediating this relationship.
Diversity and molecular evolution of the
RPS2
resistance gene in
Arabidopsis thaliana
Authors: Caicedo Ana Lucia , Schaal Barbara A. , Kunkel Barbara N.
Published: July 2002

The
RPS2
gene in
Arabidopsis thaliana
governs resistance to strains of the bacterial pathogen,
Pseudomonas syringae
pv.
tomato
, that express the
avrRpt2
gene. The two loci are involved in a gene-for-gene interaction. Seventeen accessions of
A. thaliana
were sequenced to explore the diversity present in the coding region of the
RPS2
locus. An unusually high level of nucleotide polymorphisms was found (1.26%), with nearly half of the observed polymorphisms resulting in amino acid changes in the
RPS2
protein. Seven haplotypes (alleles) were identified and their evolutionary relationships deduced. Several of the alleles conferring resistance were found to be closely related, whereas susceptibility to disease was conferred by widely divergent alleles. The possibility of selection at the
RPS2
locus is discussed.
A protein–protein interaction map of yeast RNA polymerase III
Authors: Flores A. , Briand J.-F. , Gadal O. , Andrau J.-C. , Rubbi L. , Van Mullem V. , Boschiero C. , Goussot M. , Marck C. , Carles C. , Thuriaux P. , Sentenac A. , Werner M.
Published: July 2002

The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein–protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interactin vivowith one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10α, and ABC10β), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.
Tumor-targeted IL-2 amplifies T cell-mediated immune response induced by gene therapy with single-chain IL-12
Authors: Lode Holger N. , Xiang Rong , Duncan Steven R. , Theofilopoulos Argyrios N. , Gillies Stephen D. , Reisfeld Ralph A.
Published: July 2002

Induction, maintenance, and amplification of tumor-protective immunity after cytokine gene therapy is essential for the clinical success of immunotherapeutic approaches. We investigated whether this could be achieved by single-chain IL-12 (scIL-12) gene therapy followed by tumor-targeted IL-2 using a fusion protein containing a tumor-specific recombinant anti-ganglioside GD
2
antibody and IL-2 (ch14.18-IL-2) in a poorly immunogenic murine neuroblastoma model. Herein, we demonstrate the absence of liver and bone marrow metastases after a lethal challenge with NXS2 wild-type cells only in mice (five of six animals) vaccinated with scIL-12-producing NXS2 cells and given a booster injection of low-dose ch14.18-IL-2 fusion protein. This tumor-protective immunity was effective 3 months after initial vaccination, in contrast to control animals treated with a nonspecific fusion protein or an equivalent mixture of antibody and IL-2. Only vaccinated mice receiving the tumor-specific ch14.18-IL-2 fusion protein revealed a reactivation of CD8
+
T cells and subsequent MHC class I-restricted tumor target cell lysis
in vitro
. The sequential increase in the usage of TCR chains Vβ11 and -13 in mouse CD8
+
T cells after vaccination and amplification with ch14.18-IL-2 suggests that the initial polyclonal CD8
+
T cell response is effectively boosted by targeted IL-2. In conclusion, we demonstrate that a successful boost of a partially protective memory T cell immune response that is induced by scIL-12 gene therapy could be generated by tumor-specific targeting of IL-2 with a ch14.18-IL-2 fusion protein. This approach could increase success rates of clinical cancer vaccine trials.