bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of cysteines inPlasmodium falciparumcircumsporozoite protein: Interactions with heparin can rejuvenate inactive protein mutants
Authors: Rathore Dharmendar , McCutchan Thomas F.
Published: July 2002

Various pathogenic bacteria, viruses, and protozoan bind to glycosaminoglycan-based receptors on host cells and initiate an infection. Sporozoites ofPlasmodiumpredominantly express circumsporozoite (CS) protein on their surface, which binds to heparan sulfate proteoglycans on liver cell surface that subsequently leads to malaria. Here we show that the interaction of free heparin with this parasite ligand has the potential to be a critical component of invasion. CS protein ofP. falciparumcontains four cysteines at positions 361, 365, 396, and 401. In this study, all four cysteine residues were mutagenized to alanine both individually and in different combinations. Conversion of cysteine 396 to alanine (protein CS3) led to a 10-fold increase in the binding activity of the protein to HepG2 cells. Replacement of cysteines at positions 361, 365, and 401 either alone or in different combinations led to a near total loss of binding. Surprisingly, activity in these inactive mutants could be effectively restored in the presence of submolar concentrations of heparin. Heparin also up-regulated binding of CS3 at submolar concentrations with respect to the protein but down-regulated binding when present in excess. Given the significantly different concentrations of heparin in different organs of the host and thein vitroresults described here one can considerin vivoramifications of this phenomenon for pathogen targeting of specific organs and for the functional effects of antigenic variation on receptor ligand interaction.
A Stat3-interacting protein (StIP1) regulates cytokine signal transduction
Authors: Collum Robert G. , Brutsaert Siska , Lee Gladys , Schindler Christian
Published: July 2002

Genetic and biochemical studies have led to the identification of the
St
at3-
I
nteracting
P
rotein StIP1. The preferential association of StIP1 with inactive (i.e., unphosphorylated) Stat3 suggests that it may contribute to the regulation of Stat3 activation. Consistent with this possibility, StIP1 also exhibits an affinity for members of the Janus kinase family. Overexpression of the Stat3-binding domain of StIP1 blocks Stat3 activation, nuclear translocation, and Stat3-dependent induction of a reporter gene. These studies indicate that StIP1 regulates the ligand-dependent activation of Stat3, potentially by serving as a scaffold protein that promotes the interaction between Janus kinases and their Stat3 substrate. The ability of StIP1 to associate with several additional members of the signal transducer and activator of transcription family suggests that StIP1 may serve a broader role in cytokine-signaling events.
Inhalation delivery of proteins from ethanol suspensions
Authors: Choi Won Seon , Murthy G. G. Krishna , Edwards David A. , Langer Robert , Klibanov Alexander M.
Published: July 2002

To circumvent inherent problems associated with pulmonary
administration of aqueous-solution and dry-powder protein drugs,
inhalation delivery of proteins from their suspensions in absolute
ethanol was explored both
in vitro
and
in
vivo
. Protein suspensions in ethanol of up to 9% (wt/vol)
were readily aerosolized with a commercial compressor nebulizer.
Experiments with enzymic proteins revealed that nebulization caused no
detectable loss of catalytic activity; furthermore, enzyme suspensions
in anhydrous ethanol retained their full catalytic activity for at
least 3 weeks at room temperature. With the use of
Zn
2+
-insulin, conditions were elaborated that produced
submicron protein particles in ethanol suspensions. The latter
(insulin/EtOH) afforded respirable-size aerosol particles after
nebulization. A 40-min exposure of laboratory rats to 10 mg/ml
insulin/EtOH aerosols resulted in a 2-fold drop in the blood glucose
level and a marked rise in the serum insulin level. The bioavailability
based on estimated deposited lung dose of insulin delivered by
inhalation of ethanol suspension aerosols was 33% (relative to an
equivalent s.c. injection), i.e., comparable to those observed in rats
after inhalation administration of dry powder and aqueous solutions of
insulin. Inhalation of ethanol in a relevant amount/time frame
resulted in no detectable acute toxic effects on rat lungs or airways,
as reflected by the absence of statistically significant inflammatory
or allergic responses, damage to the alveolar/capillary barrier, and
lysed and/or damaged cells.
Prostate formation in a marsupial is mediated by the testicular androgen 5α-androstane-3α,17β-diol
Authors: Shaw Geoffrey , Renfree Marilyn B. , Leihy Michael W. , Shackleton Cedric H. L. , Roitman Esther , Wilson Jean D.
Published: July 2002

Development of the male urogenital tract in mammals is mediated by testicular androgens. It has been tacitly assumed that testosterone acts through its intracellular metabolite dihydrotestosterone (DHT) to mediate this process, but levels of these androgens are not sexually dimorphic in plasma at the time of prostate development. Here we show that the 3α-reduced derivative of DHT, 5α-androstane-3α,17β-diol (5α-adiol), is formed in testes of tammar wallaby pouch young and is higher in male than in female plasma in this species during early sexual differentiation. Administration of 5α-adiol caused formation of prostatic buds in female wallaby pouch young, and in tissue minces of urogenital sinus and urogenital tubercle radioactive 5α-adiol was converted to DHT, suggesting that circulating 5α-adiol acts through DHT in target tissues. We conclude that circulating 5α-adiol is a key hormone in male development.
Sequential recruitment of steroid receptor coactivator-1 (SRC-1) and p300 enhances progesterone receptor-dependent initiation and reinitiation of transcription from chromatin
Authors: Liu Zheng , Wong Jiemin , Tsai Sophia Y. , Tsai Ming-Jer , O'Malley Bert W.
Published: July 2002

Employing a cell-free chromatin transcription system that recapitulates progesterone receptor (PR)-mediated transcription
in vivo
, we have investigated further the coactivator functions of steroid receptor coactivator-1 (SRC-1) in terms of its functional domains as well as cooperation with other coactivators in PR transactivation. By analyzing wild-type and mutant SRC-1 with liganded PR in the chromatin transcription system
in vitro
, the basic helix–loop–helix/Per-Arnt-Sim domain, the p300-binding domain, and the carboxyl-terminal region (containing the PR-binding site) of SRC-1 were shown to be important for PR transactivation. Although in context of a synthetic promoter its histone acetyltransferase activity was nonessential for PR-mediated transcription, SRC-1 was observed to act synergistically with p300 to enhance PR transactivation from chromatin. Moreover, SRC-1 and p300 were found to function cooperatively to increase the efficiency of productive transcription initiation and reinitiation. Further analysis of synergism between SRC-1 and p300 revealed an obligatory “sequential” recruitment of SRC-1 and p300 to liganded PR. Efficient recruitment of p300 required the presence of SRC-1. In addition, functional analysis of SRC-2 and SRC-3 coactivators indicated that the SRC family modulated PR transactivation from chromatin by a similar mechanism.
Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.
Authors: Kolluri R , Tolias K F , Carpenter C L , Rosen F S , Kirchhausen T
Published: July 2002

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets. The disease arises from mutations in the gene encoding the WAS protein. T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization. We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42. This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins. A weaker interaction was also detected with Rac1 using WAS protein from cell lysates. It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal. Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.
Similarities and dissimilarities of phage genomes.
Authors: Blaisdell B E , Campbell A M , Karlin S
Published: July 2002

Genomic similarities and contrasts are investigated in a collection of 23 bacteriophages, including phages with temperate, lytic, and parasitic life histories, with varied sequence organizations and with different hosts and with different morphologies. Comparisons use relative abundances of di-, tri-, and tetranucleotides from entire genomes. We highlight several specific findings. (i) As previously shown for cellular genomes, each viral genome has a distinctive signature of short oligonucleotide abundances that pervade the entire genome and distinguish it from other genomes. (ii) The enteric temperate double-stranded (ds) phages, like enterobacteria, exhibit significantly high relative abundances of GpC = GC and significantly low values of TA, but no such extremes exist in ds lytic phages. (iii) The tetranucleotide CTAG is of statistically low relative abundance in most phages. (iv) The DAM methylase site GATC is of statistically low relative abundance in most phages, but not in P1. This difference may relate to controls on replication (e.g., actions of the host SeqA gene product) and to MutH cleavage potential of the Escherichia coli DAM mismatch repair system. (v) The enteric temperate dsDNA phages form a coherent group: they are relatively close to each other and to their bacteria] hosts in average differences of dinucleotide relative abundance values. By contrast, the lytic dsDNA phages do not form a coherent group. This difference may come about because the temperate phages acquire more sequence characteristics of the host because they use the host replication and repair machinery, whereas the analyzed lytic phages are replicated by their own machinery. (vi) The nonenteric temperate phages with mycoplasmal and mycobacterial hosts are relatively close to their respective hosts and relatively distant from any of the enteric hosts and from the other phages. (vii) The single-stranded RNA phages have dinucleotide relative abundance values closest to those for random sequences, presumably attributable to the mutation rates of RNA phages being much greater than those of DNA phages.
Polarity of microtubule assemblies during neuronal cell migration.
Authors: Rakic P , Knyihar-Csillik E , Csillik B
Published: July 2002

The active migration of neurons from their sites of origin to their final destinations requires the unidirectional translocation of the nuclei and somatic cytoplasm within the growing leading processes. To explore the cellular machinery underlying this translocation, we determined the polarity of microtubules situated within the leading and trailing processes of migrating cerebellar granule cells in situ. Our analysis reveals that the newly assembled positive ends of the microtubules in the leading process uniformly face the growing tip, while their disintegrating negative ends face the nucleus. In the trailing process, by contrast, microtubule arrays are of mixed polarity. We suggest that the dynamics of slow polymerization in combination with fast disintegration of oriented microtubules create "push" and "pull" forces that contribute to the piston-like saltatory displacement of the nucleus and cytoplasm within the membrane cylinder of the leading process of the migrating neuron.
Activation of Stat 5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.
Authors: Mellitzer G , Wessely O , Decker T , Meinke A , Hayman M J , Beug H
Published: July 2002

Self renewal of normal erythroid progenitors is induced by the receptor tyrosine kinase c-ErbB, whereas other receptors (c-Kit/Epo-R) regulate erythroid differentiation. To address possible mechanisms that could explain this selective activity of c-ErbB, we analyzed the ability of these receptors to activate the different members of the Stat transcription factor family. Ligand activation of c-ErbB induced the tyrosine phosphorylation, DNA-binding, and reporter gene transcription of Stat 5b in erythroblasts. In contrast, ligand activation of c-Kit was unable to induce any of these effects in the same cells. Activation of the erythropoietin receptor caused specific DNA-binding of Stat 5b, but failed to induce reporter gene transcription. These biochemical findings correlate perfectly with the selective ability of c-ErbB to cause sustained self renewal in erythroid progenitors.
Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.
Authors: Ioffe E , Liu Y , Stanley P
Published: July 2002

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.
Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.
Authors: Matsuoka S , Thompson J S , Edwards M C , Bartletta J M , Grundy P , Kalikin L M , Harper J W , Elledge S J , Feinberg A P
Published: July 2002

Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors.
Cloning of a polycistronic cDNA from tomato encoding γ-glutamyl kinase and γ-glutamyl phosphate reductase
Authors: García-Ríos Mario , Fujita Tomomichi , LaRosa P. Christopher , Locy Robert D. , Clithero Jean M. , Bressan Ray A. , Csonka Laszlo N.
Published: July 2002

We isolated from a tomato cDNA library the tomPRO1locus, which encodes γ-glutamyl kinase (GK) and γ-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. InEscherichia coli, tomPRO1directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified fromE. colirecognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.
Spectrum of 100-kyr glacial cycle: Orbital inclination, not eccentricity
Authors: Muller Richard A. , MacDonald Gordon J.
Published: July 2002

Spectral analysis of climate data shows a strong narrow peak with
period ≈100 kyr, attributed by the Milankovitch theory to changes in
the eccentricity of the earth’s orbit. The narrowness of the peak does
suggest an astronomical origin; however the shape of the peak is
incompatible with both linear and nonlinear models that attribute the
cycle to eccentricity or (equivalently) to the envelope of the
precession. In contrast, the orbital inclination parameter gives a good
match to both the spectrum and bispectrum of the climate data.
Extraterrestrial accretion from meteoroids or interplanetary dust is
proposed as a mechanism that could link inclination to climate, and
experimental tests are described that could prove or disprove this
hypothesis.
A point mutation leads to altered product specificity in β-lactamase catalysis
Authors: Lewis Evan R. , Winterberg Kelly M. , Fink Anthony L.
Published: July 2002

β-Lactamases are the primary cause of β-lactam antibiotic resistance in many pathogenic organisms. The β-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme. Examination of the structure of the class A β-lactamase from
Bacillus licheniformis
suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule. When N170L β-lactamase was reacted with penicillins, a novel product was formed. We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine–oxazolinone as the initial product. The oxazolinone subsequently undergoes rapid breakdown leading to the formation of
N
-phenylacetylglycine and
N
-formylpenicillamine. This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of β-lactamase into that of
d
-Ala-
d
-Ala-carboxypeptidase interacting with benzylpenicillin.
Neural codes: Firing rates and beyond
Authors: Gerstner Wulfram , Kreiter Andreas K. , Markram Henry , Herz Andreas V. M.
Published: July 2002

Computational neuroscience has contributed significantly to
our understanding of higher brain function by combining experimental
neurobiology, psychophysics, modeling, and mathematical analysis. This
article reviews recent advances in a key area: neural coding and
information processing. It is shown that synapses are capable of
supporting computations based on highly structured temporal codes. Such
codes could provide a substrate for unambiguous representations of
complex stimuli and be used to solve difficult cognitive tasks, such as
the binding problem. Unsupervised learning rules could generate the
circuitry required for precise temporal codes. Together, these results
indicate that neural systems perform a rich repertoire of computations
based on action potential timing.
A bacterial basic region leucine zipper histidine kinase regulating toluene degradation
Authors: Lau Peter C. K. , Wang Ying , Patel Ashok , Labbé Diane , Bergeron Hélène , Brousseau Roland , Konishi Yasuo , Rawlings Merriann
Published: July 2002

The two-component signal transduction pathways in bacteria use a histidine–aspartate phosphorelay circuit to mediate cellular changes in response to environmental stimuli. Here we describe a novel two-component
todST
system, which activates expression of the toluene degradation (
tod
) pathway in
Pseudomonas putida
F1. The
todS
gene is predicted to encode a sensory hybrid kinase with two unique properties—a basic region leucine zipper dimerization motif at the N terminus and a duplicated histidine kinase motif. Evidence from a synthetic peptide model suggests that TodS binds as a dimer to a pseudopalindromic sequence (5′-TGACTCA), which resembles the recognition sequence of the eukaryotic transcription factors Fos and Jun. These results provide additional evidence that bacteria and eukaryotes share common regulatory motifs. The
todT
gene product, a response regulator, was overproduced as a fusion protein in
Escherichia coli
, and the purified protein was found to bind specifically to a 6-bp palindromic DNA structure in the
tod
control region. The phosphorylated form of TodT appears to be the activator of
tod
structural genes. This is the first report of a two-component system that regulates aromatic metabolism in bacteria.
Mammalian Golgi apparatus UDP-
N-
acetylglucosamine transporter: Molecular cloning by phenotypic correction of a yeast mutant
Authors: Guillen Eduardo , Abeijon Claudia , Hirschberg Carlos B.
Published: July 2002

Transporters in the Golgi apparatus membrane translocate nucleotide sugars from the cytosol into the Golgi lumen before these can be substrates for the glycosylation of proteins, lipids, and proteoglycans. We have cloned the mammalian Golgi membrane transporter for uridine diphosphate-
N-
acetylglucosamine by phenotypic correction with cDNA from MDCK cells of a recently characterized
Kluyveromyces lactis
mutant deficient in Golgi transport of the above nucleotide sugar. Phenotypically corrected transformants were separated from mutants in a fluorescent-activated cell sorter after labeling of
K. lactis
cells with fluorescein isothiocyanate (FITC) conjugated to
Griffonia simplicifolia
II lectin, which binds terminal
N-
acetylglucosamine. A 2-kb DNA fragment was found to restore the wild-type cell lectin binding phenotype, which reverted to the mutant one upon loss of the plasmid. The DNA fragment contained an ORF encoding a hydrophobic, multitransmembrane spanning protein of 326 aa that had only 22% amino acid sequence identity with the corresponding transporter from
K. lactis
but showed 53% amino acid sequence identity to the mammalian UDP-galactose transporters and 40% to the CMP-sialic acid transporter. Golgi vesicles from the transformant regained their ability to transport UDP-GlcNAc in an assay
in vitro
. The above results demonstrate that the mammalian Golgi UDP-GlcNAc transporter gene has all of the necessary information for the protein to be expressed and targeted functionally to the Golgi apparatus of yeast and that two proteins with very different amino acid sequences may transport the same solute within the same Golgi membrane.
Unequal fidelity of leading strand and lagging strand DNA replication on the
Escherichia
coli
chromosome
Authors: Fijalkowska Iwona J. , Jonczyk Piotr , Tkaczyk Magdalena Maliszewska , Bialoskorska Malgorzata , Schaaper Roel M.
Published: July 2002

We have investigated the question whether during chromosomal DNA replication in
Escherichia coli
the two DNA strands may be replicated with differential accuracy. This possibility of differential replication fidelity arises from the distinct modes of replication in the two strands, one strand (the leading strand) being synthesized continuously, the other (the lagging strand) discontinuously in the form of short Okazaki fragments. We have constructed a series of
lacZ
strains in which the
lac
operon is inserted into the bacterial chromosome in the two possible orientations with regard to the chromosomal replication origin
oriC
. Measurement of
lac
reversion frequencies for the two orientations, under conditions in which mutations reflect replication errors, revealed distinct differences in mutability between the two orientations. As gene inversion causes a switching of leading and lagging strands, these findings indicate that leading and lagging strand replication have differential fidelity. Analysis of the possible mispairs underlying each specific base pair substitution suggests that the lagging strand replication on the
E. coli
chromosome may be more accurate than leading strand replication.
Normal hepatic glucose production in the absence of GLUT2 reveals an alternative pathway for glucose release from hepatocytes
Authors: Guillam Marie-Thérèse , Burcelin Rémy , Thorens Bernard
Published: July 2002

Glucose production by liver is a major physiological function, which is required to prevent development of hypoglycemia in the postprandial and fasted states. The mechanism of glucose release from hepatocytes has not been studied in detail but was assumed instead to depend on facilitated diffusion through the glucose transporter GLUT2. Here, we demonstrate that in the absence of GLUT2 no other transporter isoforms were overexpressed in liver and only marginally significant facilitated diffusion across the hepatocyte plasma membrane was detectable. However, the rate of hepatic glucose output was normal. This was evidenced by (
i
) the hyperglycemic response to i.p. glucagon injection; (
ii
) the
in vivo
measurement of glucose turnover rate; and (
iii
) the rate of release of neosynthesized glucose from isolated hepatocytes. These observations therefore indicated the existence of an alternative pathway for hepatic glucose output. Using a [
14
C]-pyruvate pulse-labeling protocol to quantitate neosynthesis and release of [
14
C]glucose, we demonstrated that this pathway was sensitive to low temperature (12°C). It was not inhibited by cytochalasin B nor by the intracellular traffic inhibitors brefeldin A and monensin but was blocked by progesterone, an inhibitor of cholesterol and caveolae traffic from the endoplasmic reticulum to the plasma membrane. Our observations thus demonstrate that hepatic glucose release does not require the presence of GLUT2 nor of any plasma membrane glucose facilitative diffusion mechanism. This implies the existence of an as yet unsuspected pathway for glucose release that may be based on a membrane traffic mechanism.
Abrogation of the G
2
cell cycle checkpoint associated with overexpression of HSIX1: A possible mechanism of breast carcinogenesis
Authors: Ford Heide L. , Kabingu Edith N. , Bump Edward A. , Mutter George L. , Pardee Arthur B.
Published: July 2002

While conducting a search for cell cycle-regulated genes in human mammary carcinoma cells, we identified HSIX1, a recently discovered member of a new homeobox gene subfamily. HSIX1 expression was absent at the onset of and increased toward the end of S phase. Since its expression pattern is suggestive of a role after S phase, we investigated the effect of HSIX1 in the G
2
cell cycle checkpoint. Overexpression of HSIX1 in MCF7 cells abrogated the G
2
cell cycle checkpoint in response to x-ray irradiation. HSIX1 expression was absent or very low in normal mammary tissue, but was high in 44% of primary breast cancers and 90% of metastatic lesions. In addition, HSIX1 was expressed in a variety of cancer cell lines, suggesting an important function in multiple tumor types. These data support the role for homeobox genes in tumorigenesis/tumor progression, possibly through a cell cycle function.
Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single G
βγ
subunit
Authors: Zamponi Gerald W. , Snutch Terry P.
Published: July 2002

We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (α
1B
+ β
1b
+ α
2
δ subunits) by exogenously applied purified G protein βγ subunits. In the absence of G
βγ
, barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified G
βγ
in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of G
βγ
-dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant τ for the recovery from facilitation was sensitive to exogenous G
βγ
, so that the inverse of τ linearly depended on the G
βγ
concentration. Overall, the data are consistent with a model whereby a single G
βγ
molecule dissociates from the channel during the prepulse, and that reassociation of G
βγ
with the channel after the prepulse occurs as a bimolecular reaction.
Rescue of
Caenorhabditis elegans
pharyngeal development by a vertebrate heart specification gene
Authors: Haun Christina , Alexander Jonathan , Stainier Didier Y. , Okkema Peter G.
Published: July 2002

Development of pharyngeal muscle in nematodes and cardiac muscle in vertebrates and insects involves the related homeobox genes
ceh-22
,
nkx2.5
, and
tinman
, respectively. To determine whether the nematode and vertebrate genes perform similar functions, we examined activity of the zebrafish
nkx2.5
gene in transgenic
Caenorhabditis elegans
. Here, we report that ectopic expression of
nkx2.5
in
C. elegans
body wall muscle can directly activate expression of both the endogenous
myo-2
gene, a
ceh-22
target normally expressed only in pharyngeal muscle, and a synthetic reporter construct controlled by a multimerized CEH-22 binding site.
nkx2.5
also efficiently rescues a
ceh-22
mutant when expressed in pharyngeal muscle. Together, these results indicate that
nkx2.5
and
ceh-22
provide a single conserved molecular function. Further, they suggest that an evolutionarily conserved mechanism underlies heart development in vertebrates and insects and pharyngeal development in nematodes.
Reinnervation of cross-regenerated gustatory nerve fibers into amiloride-sensitive and amiloride-insensitive taste receptor cells
Authors: Ninomiya Yuzo
Published: July 2002

Single nerve fiber responses to NaCl and their inhibition by amiloride were compared among the chorda tympani (CT) and glossopharyngeal (IXth), and their cross-regenerated nerves in the C57BL/KsJ mice. The CT nerve innervating the anterior part of the tongue contained approximately equal numbers of two types of NaCl-responsive neurons; one type showed strong suppression of NaCl responses by amiloride [amiloride-sensitive (AS) type], and the other type showed only weak or no suppression of NaCl responses by amiloride [amiloride-insensitive (AI) type]. In contrast, the IXth nerve innervating the posterior part of the tongue has almost exclusively the AI type. This relative abundance of the AS and AI types of fibers was not altered by cross-regeneration of the two gustatory nerves into the reverse tongue regions. This suggests that regenerated taste axons selectively recouple with the appropriate type of receptor cell whether they innervate the front or the back of the tongue. Such selective synapse reformation may help explain the stability of response profiles of taste neurons during continual receptor cell turnover.
Catalytic properties of selenophosphate synthetases: Comparison of the selenocysteine-containing enzyme from
Haemophilus influenzae
with the corresponding cysteine-containing enzyme from
Escherichia coli
Authors: Lacourciere Gerard M. , Stadtman Thressa C.
Published: July 2002

The
selD
gene from
Haemophilus influenzae
has been overexpressed in
Escherichia coli
. The expressed protein was purified to homogeneity in a four-step procedure and then carboxymethylated by reaction with chloroacetate. N-terminal sequencing by Edman degradation identified residue 16 as carboxymethyl selenocysteine, which corresponded to the essential cysteine residue in the glycine-rich sequence of the
E. coli
selenophosphate synthetase. It would be expected that an ionized selenol of a selenocysteine in place of a catalytically essential cysteine residue would result in an enzyme with increased catalytic activity. To test this hypothesis we kinetically characterized the selenocysteine containing selenophosphate synthetase from
H. influenzae
and compared its catalytic activity to that of the cysteine containing selenophosphate synthetase from
E. coli
. Our characterization revealed the
K
m
values for the two substrates, selenide and ATP, were similar for both enzymes. However, the selenocysteine-containing enzyme did not exhibit the expected higher catalytic activity. Based on these results we suggest a role of selenocysteine in
H. influenzae
that is not catalytic.
Chromosome healing in mouse embryonic stem cells
Authors: Sprung Carl N. , Reynolds Gloria E. , Jasin Maria , Murnane John P.
Published: July 2002

The addition of new telomeres to the ends of broken chromosomes, termed chromosome healing, has been extensively studied in unicellular organisms; however, its role in the mammalian cell response to double-strand breaks is unknown. A system for analysis of chromosome healing, which involves the integration of plasmid sequences immediately adjacent to a telomere, has been established in mouse embryonic stem cells. This “marked” telomere contains a
neo
gene for positive selection in G418, an I-
Sce
I endonuclease recognition sequence for introducing double-strand breaks, and a herpes simplex virus thymidine kinase gene for negative selection with ganciclovir for cells that have lost the telomere. Transient expression of the I-
Sce
I endonuclease results in terminal deletions involving telomeric repeat sequences added directly onto the end of the broken chromosome. The sites of addition of the new telomeres contain short regions of complementarity to telomeric repeat sequences. The most common site of addition is the last A of the ATAA 3′ overhang generated by the I-
Sce
I endonuclease, without the loss of a single nucleotide from the end of the chromosome. The next most frequent site involved 5 bp of complementarity, which occurred after the loss of four nucleotides from the end of the chromosome. The new telomeres are generally much shorter than in the parental cell line, and most increase in size with time in culture. These results demonstrate that chromosome healing is a mechanism for repair of chromosome breaks in mammalian cells.
Sequence-tagged connectors: A sequence approach to mapping and scanning the human genome
Authors: Mahairas Gregory G. , Wallace James C. , Smith Kim , Swartzell Steven , Holzman Ted , Keller Andrew , Shaker Ron , Furlong Jeff , Young Janet , Zhao Shaying , Adams Mark D. , Hood Leroy
Published: July 2002

The sequence-tagged connector (STC) strategy proposes to generate sequence tags densely scattered (every 3.3 kilobases) across the human genome by arraying 450,000 bacterial artificial chromosomes (BACs) with randomly cleaved inserts, sequencing both ends of each, and preparing a restriction enzyme fingerprint of each. The STC resource, containing end sequences, fingerprints, and arrayed BACs, creates a map where the interrelationships of the individual BAC clones are resolved through their STCs as overlapping BAC clones are sequenced. Once a seed or initiation BAC clone is sequenced, the minimum overlapping 5′ and 3′ BAC clones can be identified computationally and sequenced. By reiterating this “sequence-then-map by computer analysis against the STC database” strategy, a minimum tiling path of clones can be sequenced at a rate that is primarily limited by the sequencing throughput of individual genome centers. As of February 1999, we had deposited, together with The Institute for Genomic Research (TIGR), into GenBank 314,000 STCs (≈135 megabases), or 4.5% of human genomic DNA. This genome survey reveals numerous genes, genome-wide repeats, simple sequence repeats (potential genetic markers), and CpG islands (potential gene initiation sites). It also illustrates the power of the STC strategy for creating minimum tiling paths of BAC clones for large-scale genomic sequencing. Because the STC resource permits the easy integration of genetic, physical, gene, and sequence maps for chromosomes, it will be a powerful tool for the initial analysis of the human genome and other complex genomes.
Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca
2+
channels by using knockout mice
Authors: Shapiro Mark S. , Loose Michael D. , Hamilton Susan E. , Nathanson Neil M. , Gomeza Jesus , Wess Jürgen , Hille Bertil
Published: July 2002

There are five known subtypes of muscarinic receptors (M
1
–M
5
). We have used knockout mice lacking the M
1
, M
2
, or M
4
receptors to determine which subtypes mediate modulation of voltage-gated Ca
2+
channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca
2+
channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca
2+
channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M
2
receptor knockout mice; the slow and voltage-independent pathway is absent from the M
1
receptor knockout mice; and neither pathway is affected in the M
4
receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.
NMR characterization of lignins in
Arabidopsis
altered in the activity of ferulate 5-hydroxylase
Authors: Marita Jane M. , Ralph John , Hatfield Ronald D. , Chapple Clint
Published: July 2002

Nuclear magnetic resonance (NMR) of isolated lignins from an
Arabidopsis
mutant deficient in ferulate 5-hydroxylase (F5H) and transgenic plants derived from the mutant by overexpressing the
F5H
gene has provided detailed insight into the compositional and structural differences between these lignins. Wild-type
Arabidopsis
has a guaiacyl-rich, syringyl-guaiacyl lignin typical of other dicots, with prominent β-aryl ether (β–O–4), phenylcoumaran (β–5), resinol (β–β), biphenyl/dibenzodioxocin (5–5), and cinnamyl alcohol end-group structures. The lignin isolated from the
F5H
-deficient
fah1–2
mutant contained only traces of syringyl units and consequently enhanced phenylcoumaran and dibenzodioxocin levels. In
fah1–2
transgenics in which the
F5H
gene was overexpressed under the control of the cauliflower mosaic virus 35S promoter, a guaiacyl-rich, syringyl/guaiacyl lignin similar to the wild type was produced. In contrast, the isolated lignin from the
fah1–2
transgenics in which
F5H
expression was driven by the cinnamate 4-hydroxylase promoter was almost entirely syringyl in nature. This simple lignin contained predominantly β-aryl ether units, mainly with
erythro
-stereochemistry, with some resinol structures. No phenylcoumaran or dibenzodioxocin structures (which require guaiacyl units) were detectable. The overexpression of syringyl units in this transgenic resulted in a lignin with a higher syringyl content than that in any other plant we have seen reported.
Survival of reproductive behaviors in estrogen receptor β gene-deficient (βERKO) male and female mice
Authors: Ogawa Sonoko , Chan Johnny , Chester April E. , Gustafsson Jan-Åke , Korach Kenneth S. , Pfaff Donald W.
Published: July 2002

Previously, it was shown that the lack of a functional estrogen receptor (ER) α gene (ERα) greatly affects reproduction-related behaviors in both female and male mice. However, widespread expression of a novel second ER gene, ERβ, demanded that we examine the possible participation of ERβ in regulation of these behaviors. In dramatic contrast to our results with ERα knockout (αERKO) males, βERKO males performed at least as well as wild-type controls in sexual behavior tests. Moreover, not only did βERKO males exhibit normal male-typical aggressive behavior, including offensive attacks, but they also showed higher levels of aggression than wild-type mice under certain conditions of social experience. These data revealed a significant interaction between genotype and social experience with respect to aggressive behavior. Finally, females lacking a functional β isoform of the ER gene showed normal lordosis and courtship behaviors, extending in some cases beyond the day of behavioral estrus. These results highlight the importance of ERα for the normal expression of natural reproductive behaviors in both sexes and also provide a background for future studies evaluating ERβ gene contributions to other, nonreproductive behaviors.
“Coarse” stability and bifurcation analysis using time-steppers: A reaction-diffusion example
Authors: Theodoropoulos Constantinos , Qian Yue-Hong , Kevrekidis Ioannis G.
Published: July 2002

Evolutionary, pattern forming partial differential equations (PDEs) are often derived as limiting descriptions of microscopic, kinetic theory-based models of molecular processes (e.g., reaction and diffusion). The PDE dynamic behavior can be probed through direct simulation (time integration) or, more systematically, through stability/bifurcation calculations; time-stepper-based approaches, like the Recursive Projection Method [Shroff, G. M. & Keller, H. B. (1993)
SIAM J. Numer. Anal.
30, 1099–1120] provide an attractive framework for the latter. We demonstrate an adaptation of this approach that allows for a direct, effective (“coarse”) bifurcation analysis of microscopic, kinetic-based models; this is illustrated through a comparative study of the FitzHugh-Nagumo PDE and of a corresponding Lattice–Boltzmann model.
On a Recent “Review Essay” in JHET
Authors: Kurz Heinz D. , Salvadori Neri
Published: July 2002

On September 22, 1994 we signed a contract with Edward Elgar Publishing Limited to edit The Elgar Companion to Classical Economics (ECCE) (Kurz and Salvadori 1998). Dr. Terry Peach accepted our invitation (dated January 10, 1995) to contribute an entry to ECCE by July 15, 1995. He was reminded of the deadline in a letter dated June 19 and again in a letter dated August 28, when we suggested a new deadline of September 30. Our only response from Peach was a letter dated September 5, also to the publisher, informing us that he had “to rescind [his] offer to contribute an entry.” This unexpected withdrawal was justified on the following grounds:

My critical position on the Sraffa-inspired history of economic thought, and particularly on the “Sraffian” interpretation of Ricardo, is probably well known to you. After long deliberation I have decided that I cannot in all conscience make even a minor contribution to a project which, to a pronounced degree, apparently seeks to promote a particular version of the history of economic thought to which I am profoundly opposed.
Making Economic Knowledge: Review of Jan Golinski'sMaking Natural Knowledge
Authors: Sent Esther-Mirjam
Published: July 2002

When I went through my graduate training in economics at Stanford University, I learned that economics consists of ideas. These are often expressed in mathematical terms and can be found in books and articles. To become a successful economist, you have to understand these ideas and come up with variations on them. So, during my first year of graduate studies, I spent the majority of my time working my way through the many books and articles assigned for my classes in microeconomics, macroeconomics, and econometrics. During their lectures, our instructors would walk us through any difficult mathematical manipulations that we encountered in our readings. Surely, I thought, I was on my way to becoming a respected economist. Still, I needed to specialize in a few fields, which I did in my second year.
Peroxisome Biogenesis: Involvement of ARF and Coatomer
Authors: Passreiter Michael , Anton Markus , Lay Dorothee , Frank Rainer , Harter Cordula , Wieland Felix T. , Gorgas Karin , Just Wilhelm W.
Published: July 2002

Peroxisomal membrane protein (Pmp)26p (RnPex11p), a major constituent of induced rat liver peroxisomal membrane, was found to contain a COOH-terminal, cytoplasmically exposed consensus dilysine motif with the potential to bind coatomer. Biochemical as well as immunocytochemical evidence is presented showing that peroxisomes incubated with preparations of bovine brain or rat liver cytosol recruit ADP-ribosylation factor (ARF) and coatomer in a strictly guanosine 5′-O-(3-thiotriphosphate)–dependent manner. Consistent with this observation, ldlF cells expressing a temperature-sensitive mutant version of the ε-subunit of coatomer exhibit elongated tubular peroxisomes possibly due to impaired vesiculation at the nonpermissive temperature. Since overexpression of Pex11p in Chinese hamster ovary wild-type cells causes proliferation of peroxisomes, these data suggest that Pex11p plays an important role in peroxisome biogenesis by supporting ARF- and coatomer-dependent vesiculation of the organelles.