bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence tomographic imaging in turbid media using early-arriving photons and Laplace transforms
Authors: Wu Jun , Perelman Lev , Dasari Ramachandra R. , Feld Michael S.
Published: July 2002

We present a multichannel tomographic technique to detect fluorescent objects embedded in thick (6.4 cm) tissue-like turbid media using early-arriving photons. The experiments use picosecond laser pulses and a streak camera with single photon counting capability to provide short time resolution and high signal-to-noise ratio. The tomographic algorithm is based on the Laplace transform of an analytical diffusion approximation of the photon migration process and provides excellent agreement between the actual positions of the fluorescent objects and the experimental estimates. Submillimeter localization accuracy and 4- to 5-mm resolution are demonstrated. Moreover, objects can be accurately localized when fluorescence background is present. The results show the feasibility of using early-arriving photons to image fluorescent objects embedded in a turbid medium and its potential in clinical applications such as breast tumor detection.
The catalytic subunit of yeast telomerase
Authors: Counter Christopher M. , Meyerson Matthew , Eaton Elinor Ng , Weinberg Robert A.
Published: July 2002

Telomerase is an RNA-directed DNA polymerase, composed of RNA and protein subunits, that replicates the telomere ends of linear eukaryotic chromosomes. Using a genetic strategy described here, we identify the product of the
EST2
gene, Est2p, as a subunit of telomerase in the yeast
Saccharomyces cerevisiae
. Est2p is required for enzyme catalysis, as mutations in
EST2
were found to result in the absence of telomerase activity. Immunochemical experiments show that Est2p is an integral subunit of the telomerase enzyme. Critical catalytic residues present in RNA-directed DNA polymerases are conserved in Est2p; mutation of one such residue abolishes telomerase activity, suggesting a direct catalytic role for Est2p.
CytA enables CryIV endotoxins of
Bacillus thuringiensis
to overcome high levels of CryIV resistance in the mosquito,
Culex quinquefasciatus
Authors: Wirth M. C. , Georghiou G. P. , Federici B. A.
Published: July 2002

Cry proteins produced by
Bacillus thuringiensis
are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito,
Culex quinquefasciatus
, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of
B. thuringiensis
. Resistance at the LC
95
level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC
95
level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.
Ultraspiracle: An invertebrate nuclear receptor for juvenile hormones
Authors: Jones Grace , Sharp Phillip A.
Published: July 2002

Juvenile hormones (JH), a sesquiterpenoid group of ligands that regulate developmental transitions in insects, bind to the nuclear receptor ultraspiracle (USP). In fluorescence-based binding assays, USP protein binds JH III and JH III acid with specificity, adopting for each ligand a different final conformational state. JH III treatment of
Saccharomyces cerevisiae
expressing a LexA-USP fusion protein stabilizes an oligomeric association containing this protein, as detected by formation of a protein–DNA complex, and induces USP-dependent transcription in a reporter assay. We propose that regulation of morphogenetic transitions in invertebrates involves binding of JH or JH-like structures to USP.
Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells
Authors: Massuda Edmond S. , Dunphy Edward J. , Redman Rebecca A. , Schreiber Jennifer J. , Nauta Lauren E. , Barr Frederic G. , Maxwell Ian H. , Cripe Timothy P.
Published: July 2002

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.
Ectopic expression of A-mybin transgenic mice causes follicular hyperplasia and enhanced B lymphocyte proliferation
Authors: DeRocco Susan E. , Iozzo Renato , Ma XiaoPing , Schwarting Roland , Peterson David , Calabretta Bruno
Published: July 2002

The A-mybgene is a transcription factor that shares structural and functional similarities with the v-myboncogene. To date, v-mybis the onlymybgene directly implicated in tumorigenesis, a property attributed to its transactivating ability. Recent studies have demonstrated that A-myb, like v-myb, is a potent transcriptional activator, raising the possibility that A-mybmay also participate in oncogenesis. To test this hypothesis, we generated fusion constructs that contained the human A-mybcDNA under control of the mouse metallothionein promoter and the mouse mammary tumor virus long terminal repeat. These constructs were inserted into the germ line of mice, and the functional consequences of ectopic A-mybexpression were examined. Although transgene expression was detected in a wide range of tissues, abnormalities were confined primarily to hematopoietic tissues. After a 9-month latency, A-mybtransgenic mice developed hyperplasia of the spleen and lymph nodes. Enlarged tissues contained a polyclonally expanded B lymphocyte population that expressed a germinal center-cell phenotype. Transgenic B lymphocytes showed increased DNA synthesis in response to low dose mitogen stimulation, suggesting that A-mybmay contribute to hyperplasia by increasing the rate of B cell proliferation.
A diffusible cytotoxin of
Haemophilus ducreyi
Authors: Cope Leslie D. , Lumbley Sheryl , Latimer Jo L. , Klesney-Tait Julia , Stevens Marla K. , Johnson Linda S. , Purven Maria , Munson Robert S. , Lagergard Teresa , Radolf Justin D. , Hansen Eric J.
Published: July 2002

Little is known about the virulence mechanisms employed by
Haemophilus ducreyi
in the production of genital ulcers. This Gram-negative bacterium previously has been shown to produce a soluble cytotoxic activity that kills HeLa and HEp-2 cells. We have now identified a cluster of three
H. ducreyi
genes that encode this cytotoxic activity. The predicted proteins encoded by these genes are most similar to the products of the
Escherichia coli cdtABC
genes that comprise the cytolethal distending toxin (CDT) of this enteric pathogen. Eleven of 12
H. ducreyi
strains were shown to possess this gene cluster and culture supernatants from these strains readily killed HeLa cells. The culture supernatant from a single strain of
H. ducreyi
that lacked these genes was unable to kill HeLa cells. When the
H. ducreyi cdtABC
gene cluster was cloned into
E. coli
, culture supernatant from the recombinant
E. coli
clone killed HeLa cells. A monoclonal antibody that neutralized this soluble cytotoxic activity of
H. ducreyi
was shown to bind to the
H. ducreyi cdtC
gene product. This soluble
H. ducreyi
cytotoxin may play a role in the development or persistence of the ulcerative lesions characteristic of chancroid.
In situactivation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation
Authors: Islam Md. Shahidul , Leibiger Ingo , Leibiger Barbara , Rossi Daniela , Sorrentino Vincenzo , Ekström Tomas J. , Westerblad Håkan , Andrade Francisco H. , Berggren Per-Olof
Published: July 2002

Molecular mechanisms that regulatein situactivation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells fromob/obmice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+from ER, giving rise to a slow and small increase in [Ca2+]iin beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+release by 4-CEP, giving rise to Ca2+spikes. In glucose-stimulated beta cells forskolin induced Ca2+spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude thatin situactivation of RY2in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.
HCC-2, a human chemokine: Gene structure, expression pattern, and biological activity
Authors: Pardigol Andreas , Forssmann Ulf , Zucht Hans-Dieter , Loetscher Pius , Schulz-Knappe Peter , Baggiolini Marco , Forssmann Wolf-Georg , Mägert Hans-Jürgen
Published: July 2002

Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named “HCC-2.” The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKβ8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1γ, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1α. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.
SIRE-1
, a
copia/Ty1
-like retroelement from soybean, encodes a retroviral envelope-like protein
Authors: Laten Howard M. , Majumdar Arpita , Gaucher Eric A.
Published: July 2002

The soybean genome hosts a family of several hundred, relatively homogeneous copies of a large,
copia/Ty1
-like retroelement designated
SIRE-1
. A copy of this element has been recovered from a
Glycine max
genomic library. DNA sequence analysis of two
SIRE-1
subclones revealed that
SIRE-1
contains a long, uninterrupted, ORF between the 3′ end of the
pol
ORF and the 3′ long terminal repeat (LTR), a region that harbors the
env
gene in retroviral genomes. Conceptual translation of this second ORF produces a 70-kDa protein. Computer analyses of the amino acid sequence predicted patterns of transmembrane domains, α-helices, and coiled coils strikingly similar to those found in mammalian retroviral envelope proteins. In addition, a 65-residue, proline-rich domain is characterized by a strong amino acid compositional bias virtually identical to that of the 60-amino acid, proline-rich neutralization domain of the feline leukemia virus surface protein. The assignment of
SIRE-1
to the
copia/Ty1
family was confirmed by comparison of the conceptual translation of its reverse transcriptase-like domain with those of other retroelements. This finding suggests the presence of a proretrovirus in a plant genome and is the strongest evidence to date for the existence of a retrovirus-like genome closely related to
copia/Ty1
retrotransposons.
13
C NMR study of the effects of leptin treatment on kinetics of hepatic intermediary metabolism
Authors: Cohen Sheila M. , Werrmann Jeffrey G. , Tota Michael R.
Published: July 2002

The recent discovery of leptin receptors in peripheral tissue raises questions about which of leptin’s biological actions arise from direct effects of the hormone on extraneural tissues and what intracellular mechanisms are responsible for leptin’s effects on carbohydrate and lipid metabolism. The present study is focused on the action of leptin on hepatic metabolism. Nondestructive
13
C NMR methodology was used to follow the kinetics of intermediary metabolism by monitoring flux of
13
C-labeled substrate through several multistep pathways. In perfused liver from either
ob/ob
or lean mice, we found that acute treatment with leptin
in vitro
modulates pathways controlling carbohydrate flux into
13
C-labeled glycogen, thereby rapidly enhancing synthesis by an insulin-independent mechanism. Acute treatment of
ob/ob
liver also caused a rapid stimulation of long-chain fatty acid synthesis from
13
C-labeled acetyl-CoA by the
de novo
synthesis route. Chronic leptin treatment
in vivo
induced homeostatic changes that resulted in a tripling of the rate of glycogen synthesis via the gluconeogenic pathway from [2-
13
C]pyruvate in
ob/ob
mouse liver perfused in the absence of the hormone. Consistent with the
13
C NMR results, leptin treatment of the
ob/ob
mouse
in vivo
resulted in significantly increased hepatic glycogen synthase activity. Chronic treatment with leptin
in vivo
exerted the opposite effect of acute treatment
in vitro
and markedly decreased hepatic
de novo
synthesis of fatty acids in
ob/ob
mouse liver. In agreement with the
13
C NMR findings, activities of hepatic acetyl-CoA carboxylase and fatty acid synthase were significantly reduced by chronic treatment of the
ob/ob
mouse with leptin. Our data represent a demonstration of direct effects of leptin in the regulation of metabolism in the intact functioning liver.
A genetic defect resulting in mild low-renin hypertension
Authors: Wilson Robert C. , Dave-Sharma Swati , Wei Ji-Qing , Obeyesekere Varuni R. , Li Kevin , Ferrari Paolo , Krozowski Zygmunt S. , Shackleton Cedric H. L. , Bradlow Leon , Wiens Timothy , New Maria I.
Published: July 2002

Severe low-renin hypertension has few known causes. Apparent mineralocorticoid excess (AME) is a genetic disorder that results in severe juvenile low-renin hypertension, hyporeninemia, hypoaldosteronemia, hypokalemic alkalosis, low birth weight, failure to thrive, poor growth, and in many cases nephrocalcinosis. In 1995, it was shown that mutations in the gene (
HSD11B2
) encoding the 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) cause AME. Typical patients with AME have defective 11β-HSD2 activity, as evidenced by an abnormal ratio of cortisol to cortisone metabolites and by an exceedingly diminished ability to convert [11-
3
H]cortisol to cortisone. Recently, we have studied an unusual patient with mild low-renin hypertension and a homozygous mutation in the
HSD11B2
gene. The patient came from an inbred Mennonite family, and though the mutation identified her as a patient with AME, she did not demonstrate the typical features of AME. Biochemical analysis in this patient revealed a moderately elevated cortisol to cortisone metabolite ratio. The conversion of cortisol to cortisone was 58% compared with 0–6% in typical patients with AME whereas the normal conversion is 90–95%. Molecular analysis of the
HSD11B2
gene of this patient showed a homozygous C→T transition in the second nucleotide of codon 227, resulting in a substitution of proline with leucine (P227L). The parents and sibs were heterozygous for this mutation.
In vitro
expression studies showed an increase in the
K
m
(300 nM) over normal (54 nM). Because ≈40% of patients with essential hypertension demonstrate low renin, we suggest that such patients should undergo genetic analysis of the
HSD11B2
gene.
Epidermaltrans-urocanic acid and the UV-A-induced photoaging of the skin
Authors: Hanson Kerry M. , Simon John D.
Published: July 2002

The premature photoaging of the skin is mediated by the sensitization of reactive oxygen species after absorption of ultraviolet radiation by endogenous chromophores. Yet identification of UV-A-absorbing chromophores in the skin that quantitatively account for the action spectra of the physiological responses of photoaging has remained elusive. This paper reports that thein vitroaction spectrum for singlet oxygen generation after excitation oftrans-urocanic acid mimics thein vivoUV-A action spectrum for the photosagging of mouse skin. The data presented provide evidence suggesting that the UV-A excitation oftrans-urocanic acid initiates chemical processes that result in the photoaging of skin.
Crystal structure of the ribosomal RNA domain essential for binding elongation factors
Authors: Correll Carl C. , Munishkin Alexander , Chan Yuen-Ling , Ren Zhong , Wool Ira G. , Steitz Thomas A.
Published: July 2002

The structure of a 29-nucleotide RNA containing the sarcin/ricin loop (SRL) of rat 28 S rRNA has been determined at 2.1 Å resolution. Recognition of the SRL by elongation factors and by the ribotoxins, sarcin and ricin, requires a nearly universal dodecamer sequence that folds into a G-bulged cross-strand A stack and a GAGA tetraloop. The juxtaposition of these two motifs forms a distorted hairpin structure that allows direct recognition of bases in both grooves as well as recognition of nonhelical backbone geometry and two 5′-unstacked purines. Comparisons with other RNA crystal structures establish the cross-strand A stack and the GNRA tetraloop as defined and modular RNA structural elements. The conserved region at the top is connected to the base of the domain by a region presumed to be flexible because of the sparsity of stabilizing contacts. Although the conformation of the SRL RNA previously determined by NMR spectroscopy is similar to the structure determined by x-ray crystallography, significant differences are observed in the “flexible” region and to a lesser extent in the G-bulged cross-strand A stack.
In vitro
selection of RNA molecules that displace cocaine from the membrane-bound nicotinic acetylcholine receptor
Authors: Ulrich Henning , Ippolito Joseph E. , Pagán Oné R. , Eterovic Vesna A. , Hann Richard M. , Shi Hua , Lis John T. , Eldefrawi Mohyee E. , Hess George P.
Published: July 2002

The nicotinic acetylcholine receptor (AChR) controls signal transmission between cells in the nervous system. Abused drugs such as cocaine inhibit this receptor. Transient kinetic investigations indicate that inhibitors decrease the channel-opening equilibrium constant [Hess, G. P. & Grewer, C. (1998)
Methods Enzymol.
291, 443–473]. Can compounds be found that compete with inhibitors for their binding site but do not change the channel-opening equilibrium? The systematic evolution of RNA ligands by exponential enrichment methodology and the AChR in
Torpedo californica
electroplax membranes were used to find RNAs that can displace inhibitors from the receptor. The selection of RNA ligands was carried out in two consecutive steps: (
i
) a gel-shift selection of high-affinity ligands bound to the AChR in the electroplax membrane, and (
ii
) subsequent use of nitrocellulose filters to which both the membrane-bound receptor and RNAs bind strongly, but from which the desired RNA can be displaced from the receptor by a high-affinity AChR inhibitor, phencyclidine. After nine selection rounds, two classes of RNA molecules that bind to the AChR with nanomolar affinities were isolated and sequenced. Both classes of RNA molecules are displaced by phencyclidine and cocaine from their binding site on the AChR. Class I molecules are potent inhibitors of AChR activity in BC
3
H1 muscle cells, as determined by using the whole-cell current-recording technique. Class II molecules, although competing with AChR inhibitors, do not affect receptor activity in this assay; such compounds or derivatives may be useful for alleviating the toxicity experienced by millions of addicts.
Protein tyrosine phosphatase PTP1B suppresses p210 bcr-abl-induced transformation of Rat-1 fibroblasts and promotes differentiation of K562 cells
Authors: LaMontagne Kenneth R. , Hannon Greg , Tonks Nicholas K.
Published: July 2002

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-abl-expressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl/Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcr-abl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.
Suppression of an absolute defect in Type IV pilus biogenesis by loss-of-function mutations in
pilT
, a twitching motility gene in
Neisseria gonorrhoeae
Authors: Wolfgang Matthew , Park Hae-Sun , Hayes Stanley F. , van Putten Jos P. M. , Koomey Michael
Published: July 2002

Type IV pili of
Neisseria gonorrhoeae
, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the
pilC
gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.
In vitro
assembly of a ribonucleoprotein particle corresponding to the platform domain of the 30S ribosomal subunit
Authors: Agalarov S. C. , Zheleznyakova E. N. , Selivanova O. M. , Zheleznaya L. A. , Matvienko N. I. , Vasiliev V. D. , Spirin A. S.
Published: July 2002

A fragment of the 16S RNA of
Thermus thermophilus
corresponding to the central domain (nucleotides 547–895) has been prepared by transcription
in vitro
. Incubation of this fragment with the total 30S ribosomal proteins has resulted in the formation of a compact 12S ribonucleoprotein particle. This particle contained five
T. thermophilus
proteins corresponding to
Escherichia coli
ribosomal proteins S6, S8, S11, S15, and possibly S18, all of which were previously shown to interact with the central domain of the 16S RNA and to be localized in the platform (side bulge) of the 30S ribosomal subunit. When examined by electron microscopy, isolated particles have an appearance that is similar in size and shape to the corresponding morphological features of the 30S subunit. We conclude that the central domain of the 16S RNA can independently and specifically assemble with a defined subset of ribosomal proteins into a compact ribonucleoprotein particle corresponding to the platform (side bulge) of the 30S subunit.
Toward a plant genomics initiative: Thoughts on the value of cross-species and cross-genera comparisons in the grasses
Authors: McCouch Susan
Published: July 2002

Comparative genomics offers unparalleled opportunities to integrate
historically distinct disciplines, to link disparate biological
kingdoms, and to bridge basic and applied science. Cross-species,
cross-genera, and cross-kingdom comparisons are proving key to
understanding how genes are structured, how gene structure relates to
gene function, and how changes in DNA have given rise to the biological
diversity on the planet. The application of genomics to the study of
crop species offers special opportunities for innovative approaches for
combining sequence information with the vast reservoirs of historical
information associated with crops and their evolution. The grasses
provide a particularly well developed system for the development of
tools to facilitate comparative genetic interpretation among members of
a diverse and evolutionarily successful family. Rice provides
advantages for genomic sequencing because of its small genome and its
diploid nature, whereas each of the other grasses provides
complementary genetic information that will help extract meaning from
the sequence data. Because of the importance of the cereals to the
human food chain, developments in this area can lead directly to
opportunities for improving the health and productivity of our food
systems and for promoting the sustainable use of natural resources.
Unusual features of theDrosophila melanogastertelomere transposable elementHeT-Aare conserved inDrosophila yakubatelomere elements
Authors: Danilevskaya Olga N. , Tan Chen , Wong Jimmy , Alibhai Minna , Pardue Mary-Lou
Published: July 2002

HeT-Awas the first transposable element shown to have a bona fide role in chromosome structure, maintenance of telomeres inDrosophila melanogaster. HeT-Ahas hallmarks of non-long-terminal-repeat (non-LTR) retrotransposable elements but also has several unique features. We have now isolatedHeT-Aelements fromDrosophila yakuba,showing that the retrotransposon mechanism of telomere maintenance predates the separation ofD. melanogasterandD. yakuba(5–15 million years ago).HeT-Aelements from the two species show significant sequence divergence, yet unusual features seen inHeT-Amelare conserved inHeT-Ayak. In both species,HeT-Aelements are found in head-to-tail tandem arrays in telomeric heterochromatin. In both species, nearly half of theHeT-Asequence is noncoding and shows a distinctive imperfect repeat pattern of A-rich segments. Neither element encodes reverse transcriptase. TheHeT-Amelpromoter appears to be intermediate between the promoters of non-LTR and of LTR retrotransposons. TheHeT-Ayakpromoter shows similar features.HeT-Amelhas a frameshift within the coding region.HeT-Ayakdoes not require a frameshift but shows conservation of the polypeptide sequence of the frameshifted product ofD. melanogaster.
Modification of bacterial artificial chromosomes through Chi-stimulated homologous recombination and its application in zebrafish transgenesis
Authors: Jessen Jason R. , Meng Anming , McFarlane Ramsay J. , Paw Barry H. , Zon Leonard I. , Smith Gerald R. , Lin Shuo
Published: July 2002

The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish
GATA-2
genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that
GATA-2
expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.
On defining the rules for interactions between the T cell receptor and its ligand: A critical role for a specific amino acid residue of the T cell receptor β chain
Authors: Wang Fuming , Ono Toshiro , Kalergis Alexis M. , Zhang Weijia , DiLorenzo Teresa P. , Lim Kap , Nathenson Stanley G.
Published: July 2002

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR α chain transgenic mouse in a TCR α-deficient background to define specific structural features in the TCR β chain that are important for the recognition of the VSV/H-2Kbcomplex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR β chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3β loop. We conclude that the amino acid residue at position 98 of the CDR3β loop is a key residue that plays a critical role in determining the specificity of TCR–VSV/H-2Kbinteractions and that a specific length of the CDR3β loop is required to facilitate such interactions. Further, these findings suggest that the α and β chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.
Emergence of a dual-catalytic RNA with metal-specific cleavage and ligase activities: The spandrels of RNA evolution
Authors: Landweber Laura F. , Pokrovskaya Irina D.
Published: July 2002

In vitro
selection, or directed molecular evolution, allows the isolation and amplification of rare sequences that satisfy a functional-selection criterion. This technique can be used to isolate novel ribozymes (RNA enzymes) from large pools of random sequences. We used
in vitro
evolution to select a ribozyme that catalyzes a novel template-directed RNA ligation that requires surprisingly few nucleotides for catalytic activity. With the exception of two nucleotides, most of the ribozyme contributes to a template, suggesting that it is a general prebiotic ligase. More surprisingly, the catalytic core built from randomized sequences actually contains a 7-nt manganese-dependent self-cleavage motif originally discovered in the
Tetrahymena
group I intron. Further experiments revealed that we have selected a dual-catalytic RNA from random sequences: the RNA promotes both cleavage at one site and ligation at another site, suggesting two conformations surrounding at least one divalent metal ion-binding site. Together, these results imply that similar catalytic RNA motifs can arise under fairly simple conditions and that multiple catalytic structures, including bifunctional ligases, can evolve from very small preexisting parts. By breaking apart and joining different RNA strands, such ribozymes could have led to the production of longer and more complex RNA polymers in prebiotic evolution.
Metalloprotease-mediated ligand release regulates autocrine signaling through the epidermal growth factor receptor
Authors: Dong Jianying , Opresko Lee K. , Dempsey Peter J. , Lauffenburger Douglas A. , Coffey Robert J. , Wiley H. Steven
Published: July 2002

Ligands that activate the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors that appear to be proteolytically released by members of the ADAM family of metalloproteases. Because membrane-anchored EGFR ligands are thought to be biologically active, the role of ligand release in the regulation of EGFR signaling is unclear. To investigate this question, we used metalloprotease inhibitors to block EGFR ligand release from human mammary epithelial cells. These cells express both transforming growth factor α and amphiregulin and require autocrine signaling through the EGFR for proliferation and migration. We found that metalloprotease inhibitors reduced cell proliferation in direct proportion to their effect on transforming growth factor α release. Metalloprotease inhibitors also reduced growth of EGF-responsive tumorigenic cell lines and were synergistic with the inhibitory effects of antagonistic EGFR antibodies. Blocking release of EGFR ligands also strongly inhibited autocrine activation of the EGFR and reduced both the rate and persistence of cell migration. The effects of metalloprotease inhibitors could be reversed by either adding exogenous EGF or by expressing an artificial gene for EGF that lacked a membrane-anchoring domain. Our results indicate that soluble rather than membrane-anchored forms of the ligands mediate most of the biological effects of EGFR ligands. Metalloprotease inhibitors have shown promise in preventing spread of metastatic disease. Many of their antimetastatic effects could be the result of their ability to inhibit autocrine signaling through the EGFR.
Conformational variants of class II MHC/peptide complexes induced by N- and C-terminal extensions of minimal peptide epitopes
Authors: Rötzschke O. , Falk K. , Mack J. , Lau J. M. , Jung G. , Strominger J. L.
Published: July 2002

Class II MHC molecules are known to exist in conformational variants. “Floppy” and “compact” forms of murine MHC molecules, for example, are discriminated by their migration behavior on SDS/PAGE and represent empty and ligand-loaded forms. Here we show that formation of distinctly faster-migrating ligand complexes (F-forms) rather than the normal compact (C-) forms of HLA-DR1 or -DR4 results from extensions of minimal peptide epitopes (such as HA306-318 or IC106-120) by ≈10 amino acids at either the N or the C terminus. Two similar but distinct F-forms (FIand FII) were detected, depending on the site of the extension. Both F-forms were characterized by increased surface hydrophobicity and reduced SDS-stability. Native gel separations and size exclusion chromatography indicated that the F-forms had increased hydrodynamic radii compared with the C-form and an apparent size similar to that of empty MHC molecules. The regions on the ligand overhangs responsible for the effect began at a distance of ≈5 amino acids on either side of the epitopes, comprised 4–8 amino acids (i.e., a total overhang of 9–14), and did not have a particular sequence preference. The possible functional significance of these forms is discussed.
Transient expression of DNA and RNA in parasitic helminths by using particle bombardment
Authors: Davis Richard E. , Parra Angelica , LoVerde Philip T. , Ribeiro Eugenia , Glorioso Gina , Hodgson Scott
Published: July 2002

Parasitic helminths (worms belonging to several metazoan phyla) cause considerable morbidity and mortality in humans. They are an important veterinary problem, and they result in significant economic losses in animal grazing and agriculture. Experimental studies on parasitic helminths have been limited by a lack of parasite cell lines and methods for molecular genetic analyses. We evaluated particle bombardment (biolistics) as a strategy to introduce and express nucleic acids in these multicellular parasites. By using embryos of the parasitic nematodeAscarisas a model, we developed methods to introduce and express both DNA and RNA during several stages ofAscarisembryogenesis. Biolistic transfection will facilitate experimental strategies inAscarisembryos complementing other biochemical tools available (e.g.,in vitrowhole-cell embryo extracts for transcription, RNA processing, and translation). Transfection experiments with adult schistosomes further suggest that the biolistic strategy should be applicable to a variety of other parasitic helminths. The development of these methods provides molecular genetic tools to study gene expression and the biology of a variety of types and developmental stages of important helminth parasites.
Synthesis and biological evaluation of antagonists of growth hormone-releasing hormone with high and protracted
in vivo
activities
Authors: Varga József L. , Schally Andrew V. , Csernus Valér J. , Zarándi Márta , Halmos Gábor , Groot Kate , Rékási Zoltán
Published: July 2002

Some antagonists of human growth hormone-releasing hormone (hGH-RH) synthesized previously were shown to inhibit
in vivo
proliferation of various human cancers in nude mice. However, the activity of these analogs requires an increase to assure clinical efficacy. In an attempt to prepare hGH-RH antagonists with a high and protracted activity, we synthesized and biologically tested 22 antagonistic analogs of hGH-RH(1–29)NH
2
. The ability of the antagonists to inhibit hGH-RH-induced GH release was evaluated
in vitro
in a superfused rat pituitary system, as well as
in vivo
after i.v. injection into rats. The binding affinity of the peptides to GH-RH receptors also was determined. All antagonistic analogs had the common core sequence [PhAc-Tyr
1
,
d
-Arg
2
, Phe(4-Cl)
6
(
para
-chlorophenylalanine), Abu
15
(α-aminobutyric acid),Nle
27
]hGH-RH(1–29)NH
2
and contained Arg,
d
-Arg, homoarginine (Har), norleucine (Nle), and other substitutions. The following analogs were determined to have a high and/or protracted antagonistic activity: [PhAc-Tyr
1
,
d
-Arg
2
,Phe(4-Cl)
6
,Arg
9
,Abu
15
,Nle
27
,
d
-Arg
29
]hGH-RH(1–29)NH
2
(JV-1–10), [PhAc-Tyr
1
,
d
-Arg
2
,Phe(4-Cl)
6
,Abu
15
,Nle
27
,
d
-Arg
28
,Har
29
]hGH-RH(1–29)NH
2
(MZ-6–55), [PhAc-Tyr
1
,
d
-Arg
2
,Phe(4-Cl)
6
,Arg
9
,Abu
15
,Nle
27
,
d
-Arg
28
,Har
29
]hGH-RH(1–29)NH
2
(JV-1–36), and [PhAc-Tyr
1
,
d
-Arg
2
,Phe(4-Cl)
6
,Har
9
,Tyr(Me)
10
,Abu
15
,Nle
27
,
d
-Arg
28
,Har
29
]hGH-RH(1–29)NH
2
(JV-1–38). Among the peptides tested, analog JV-1–36 showed the highest GH-RH antagonistic activity
in vitro
and also induced a strong and prolonged inhibition of GH release
in vivo
for at least 30 min. The antagonist JV-1–38 was slightly less potent than JV-1–36 both
in vitro
and
in vivo
but proved to be very long-acting
in vivo
, suppressing the GH-RH-induced GH release even after 60 min. High and protracted
in vivo
activities of these antagonists indicate an improvement over earlier GH-RH analogs. Some of these hGH-RH antagonists could find clinical applications in the treatment of cancers dependent on insulin-like growth factors I and II.
Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat
Authors: Champion H. C. , Bivalacqua T. J. , Hyman A. L. , Ignarro L. J. , Hellstrom W. J. G. , Kadowitz P. J.
Published: July 2002

Nitric oxide (NO), a mediator involved in penile erection, is synthesized by the nitric oxide synthase (NOS) family of enzymes. It has been shown that NOS activity decreases with age. To determine whether adenoviral-mediated overexpression of endothelial NOS (eNOS) could enhance erectile responses, we administered a recombinant adenovirus containing the eNOS gene (AdCMVeNOS) into the corpora cavernosum of the aged rat. Adenoviral expression of the β-galactosidase reporter gene was observed in cavernosal tissue 1 day after intracavernosal administration of AdCMVβgal; 1 day after administration of AdCMVeNOS, transgene expression was confirmed by immunoblot staining of eNOS protein, and cGMP levels were increased. The increase in cavernosal pressure in response to cavernosal nerve stimulation was enhanced in animals transfected with eNOS, and erectile responses to acetylcholine and zaprinast were enhanced at a time when the erectile response to the NO donor sodium 1-(
N
,
N
-diethylamino)diazen-1-ium-1,2-diolate was not altered. These results suggest that
in vivo
gene transfer of eNOS, alone or in combination with a type V phosphodiesterase inhibitor, may constitute a new therapeutic intervention for the treatment of erectile dysfunction.
Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F
2
-isoprostanes in human urine
Authors: Li Hongwei , Lawson John A. , Reilly Muredach , Adiyaman Mustafa , Hwang Seong-Woo , Rokach Joshua , FitzGerald Garret A.
Published: July 2002

Isoprostanes (iPs) are free radical catalyzed prostaglandin isomers. Analysis of individual isomers of PGF
2α
—F
2
-iPs—in urine has reflected lipid peroxidation in humans. However, up to 64 F
2
-iPs may be formed, and it is unknown whether coordinate generation, disposition, and excretion of F
2
-iPs occurs in humans. To address this issue, we developed methods to measure individual members of the four structural classes of F
2
-iPs, using liquid chromatography/tandem mass spectrometry (LC/MS/MS), in which sample preparation is minimized. Authentic standards of F
2
-iPs of classes III, IV, V, and VI were used to identify class-specific ions for multiple reaction monitoring. Using iPF
2α
-VI as a model compound, we demonstrated the reproducibility of the assay in human urine. Urinary levels of all F
2
-iPs measured were elevated in patients with familial hypercholesterolemia. However, only three of eight F
2
-iPs were elevated in patients with congestive heart failure, compared with controls. Paired analyses by GC/MS and LC/MS/MS of iPF
2α
-VI in hypercholesterolemia and of 8,12-
iso
-iPF
2α
-VI in congestive heart failure were highly correlated. This approach will permit high throughput analysis of multiple iPs in human disease.
Design of highly specific cytotoxins by using trans-splicing ribozymes
Authors: Ayre Brian G. , Köhler Uwe , Goodman Howard M. , Haseloff Jim
Published: July 2002

We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus. The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a
Saccharomyces cerevisiae
model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity
in vivo.
Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.
Universal conservation in translation initiation revealed by human and archaeal homologs of bacterial translation initiation factor IF2
Authors: Lee Joon H. , Choi Sang Ki , Roll-Mecak Antonina , Burley Stephen K. , Dever Thomas E.
Published: July 2002

Binding of initiator methionyl-tRNA to ribosomes is catalyzed in prokaryotes by initiation factor (IF) IF2 and in eukaryotes by eIF2. The discovery of both IF2 and eIF2 homologs in yeast and archaea suggested that these microbes possess an evolutionarily intermediate protein synthesis apparatus. We describe the identification of a human IF2 homolog, and we demonstrate by using
in vivo
and
in vitro
assays that human IF2 functions as a translation factor. In addition, we show that archaea IF2 can substitute for its yeast homolog both
in vivo
and
in vitro
. We propose a universally conserved function for IF2 in facilitating the proper binding of initiator methionyl-tRNA to the ribosomal P site.