bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased liposome extravasation in selected tissues: effect of substance P.
Authors: Rosenecker J , Zhang W , Hong K , Lausier J , Geppetti P , Yoshihara S , Papahadjopoulos D , Nadel J A
Published: July 2002

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.
Chromosomal double-strand break repair in Ku80-deficient cells.
Authors: Liang F , Romanienko P J , Weaver D T , Jeggo P A , Jasin M
Published: July 2002

The x-ray sensitive hamster cell line xrs-6 is deficient in DNA double-strand break (DSB) repair and exhibits impaired V(D)J recombination. The molecular defect in this line is in the 80-kDa subunit of the Ku autoantigen, a protein that binds to DNA ends and recruits the DNA-dependent protein kinase to DNA. Using an I-SceI endonuclease expression system, chromosomal DSB repair was examined in xrs-6 and parental CHO-K1 cell lines. A DSB in chromosomal DNA increased the yield of recombinants several thousand-fold above background in both the xrs-6 and CHO-K1 cells, with recombinational repair of DSBs occurring in as many as 1 of 100 cells electroporated with the endonuclease expression vector. Thus, recombinational repair of chromosomal DSBs can occur at substantial levels in mammalian cells and it is not grossly affected in our assay by a deficiency of the Ku autoantigen. Rejoining of broken chromosome ends (end-joining) near the site of the DSB was also examined. In contrast to recombinational repair, end-joining was found to be severely impaired in the xrs-6 cells. Thus, the Ku protein appears to play a critical role in only one of the chromosomal DSB repair pathways.
Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.
Authors: Vothknecht U C , Kannangara C G , von Wettstein D
Published: July 2002

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.
Chemical communication in scarab beetles: reciprocal behavioral agonist-antagonist activities of chiral pheromones.
Authors: Leal W S
Published: July 2002

A novel mechanism of reciprocal behavioral agonist-antagonist activities of enantiomeric pheromones plays a pivotal role in overcoming the signal-to-noise problem derived from the use of a single-constituent pheromone system in scarab beetles. Female Anomala osakana produce (S, Z)-5-(+)-(1-decenyl)oxacyclopentan-2-one, which is highly attractive to males; the response is completely inhibited even by 5% of its antipode. These two enantiomers have reverse roles in the Popillia japonica sex pheromone system. Chiral GC-electroantennographic detector experiments suggest that A. osakana and P. japonica have both R and S receptors that are responsible for behavioral agonist and antagonist responses.
HIV replication in CD4+T cells of HIV-infected individuals is regulated by a balance between the viral suppressive effects of endogenous β-chemokines and the viral inductive effects of other endogenous cytokines
Authors: Kinter Audrey L. , Ostrowski Mario , Goletti Delia , Oliva Alessandra , Weissman Drew , Gantt Kira , Hardy Elena , Jackson Robert , Ehler Linda , Fauci Anthony S.
Published: July 2002

This study demonstrates that the β-chemokines macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and, RANTES (regulated on activation, normally T-cell expressed and secreted) inhibit human immunodeficiency virus (HIV) replication in anti-CD3 or recall antigen-stimulated peripheral blood mononuclear cells (PBMCs) of asymptomatic HIV-infected subjects. Significant levels of β-chemokines were produced by both CD4+and CD8+PBMC subsets from HIV-infected individuals. Neutralization of endogenous MIP-1α, MIP-1β, and RANTES did not rescue HIV replication in cultures to which greater than 10% CD8+T cells had been added, indicating that the HIV suppressor activity of CD8+T cells cannot be explained entirely by the β-chemokines. However, significant enhancement of viral replication was observed upon neutralization of endogenous β-chemokines in CD8-depleted or CD4+PBMCs from most donors, particularly in cultures with low inducible levels of HIV production. In contrast, certain endogenous proinflammatory cytokines induced HIV replication in these same cells. These data suggest that the levels of HIV replication in CD4+PBMC reflect the balance of the opposing effects of endogenous suppressive factors, such as the β-chemokines, and HIV-inducing cytokines, such as tumor necrosis factor α and interleukin 1β.
Older males secrete luteinizing hormone and testosterone more irregularly, and jointly more asynchronously, than younger males
Authors: Pincus Steven M. , Mulligan Thomas , Iranmanesh Ali , Gheorghiu Sylvia , Godschalk Michael , Veldhuis Johannes D.
Published: July 2002

New statistical perspectives on the secretory patterns of both
luteinizing hormone (LH) and testosterone (T) may prove useful in
further understanding the aging process, and possibly ultimately in
improving the diagnosis and treatment of spermatogenetic failure and
loss of sexual interest. We examined serum concentration time-series
for LH and T in 14 young (21–34 years of age) and 11 aged (62–74
years of age) healthy men. For each subject, blood samples were
obtained at 2.5-min intervals during a sleep period, with an average
sampling duration of 7 hr. For each of LH and T, we used the
model-independent statistic approximate entropy (ApEn) to quantify the
irregularity of the serum concentration time-series; to quantify joint
LH–T secretory asynchrony, we employed the recently introduced
cross-ApEn. Although mean (and SD) LH and T concentrations were
indistinguishable in the two age groups (
P
>
0.25), for LH, aged subjects had greater ApEn values (1.525 ±
0.221) than younger individuals (1.207 ± 0.252),
P
< 0.003, indicating more irregular secretion in
the older cohort. For T, aged subjects also had greater ApEn values
(1.622 ± 0.120) than younger counterparts (1.384 ± 0.228),
P
< 0.004. In young, but not older men, ApEn(T)
significantly exceeded ApEn(LH),
P
< 0.02. Aged
subjects had greater cross-ApEn values (1.961 ± 0.121) than
younger subjects (1.574 ± 0.249),
P
<
10
−4
, with nearly 100% sensitivity and specificity,
indicating greater LH–T asynchrony in the older group. In conjunction
with previous findings of greater irregularity of growth hormone
release with increasing age, we propose that increased secretory
irregularity with advancing age may be a widespread hormonal
phenomenon. Finally, theoretically, we clarify the need for
quantifications such as ApEn and cross-ApEn via a study of a
“variable lag” pulsatile process, and empirically note the
potential wide applicability of cross-ApEn to quantify asynchrony in
interconnected (hormonal) networks.
A variant of DNA polymerase β acts as a dominant negative mutant
Authors: Bhattacharyya Nandan , Banerjee Sipra
Published: July 2002

In eukaryotic cells, DNA polymerase β (polβ) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polβ gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208–236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polβ proteins in colorectal tumors. To elucidate the potential functional consequences of polβ truncation, stable HeLa cell lines were established from cloned WT and variant polβ
Δ208–236
. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polβ may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polβ
Δ208–236
cDNA. Reverse transcription–PCR and Western blot analyses showed that the new cell line, 16.3ΔP, expresses the WT and the truncated polβ mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore,
in vivo
the 16.3ΔP cells were more sensitive to
N
-methyl-
N
′-nitro-
N
-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3ΔP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polβ acts as a dominant negative mutant. The defective polβ may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.
Functional cleavage of the common cytokine receptor γ chain (γc) by calpain
Authors: Noguchi Masayuki , Sarin Apurva , Aman M. Javad , Nakajima Hiroshi , Shores Elizabeth W. , Henkart Pierre A. , Leonard Warren J.
Published: July 2002

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γc) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleavein vitro-translated wild-type γc, but not γccontaining a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γcin a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γc. Moreover, in single positive CD4+thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γcblocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γcrepresents a mechanism by which γc-dependent signaling can be controlled.
Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor
Authors: Morinaga Naoko , Moss Joel , Vaughan Martha
Published: July 2002

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from
Saccharomyces cerevisiae
, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5′ and 3′ rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (≈208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a ≈7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.
Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase
Authors: Hampton Randolph Y. , Bhakta Hetal
Published: July 2002

Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the
UBC7
gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on
UBC7
and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.
Plastid-localized acetyl-CoA carboxylase of bread wheat is encoded by a single gene on each of the three ancestral chromosome sets
Authors: Gornicki Piotr , Faris Justin , King Ian , Podkowinski Jan , Gill Bikram , Haselkorn Robert
Published: July 2002

5′-End fragments of two genes encoding plastid-localized acetyl-CoA carboxylase (ACCase; EC
6.4.1.2
) of wheat (
Triticum aestivum
) were cloned and sequenced. The sequences of the two genes,
Acc-1,1
and
Acc-1,2
, are 89% identical. Their exon sequences are 98% identical. The amino acid sequence of the biotin carboxylase domain encoded by
Acc-1,1
and
Acc-1,2
is 93% identical with the maize plastid ACCase but only 80–84% identical with the cytosolic ACCases from other plants and from wheat. Four overlapping fragments of cDNA covering the entire coding region were cloned by PCR and sequenced. The wheat plastid ACCase ORF contains 2,311 amino acids with a predicted molecular mass of 255 kDa. A putative transit peptide is present at the N terminus. Comparison of the genomic and cDNA sequences revealed introns at conserved sites found in the genes of other plant multifunctional ACCases, including two introns absent from the wheat cytosolic ACCase genes. Transcription start sites of the plastid ACCase genes were estimated from the longest cDNA clones obtained by 5′-RACE (rapid amplification of cDNA ends). The untranslated leader sequence encoded by the
Acc-1
genes is at least 130–170 nucleotides long and is interrupted by an intron. Southern analysis indicates the presence of only one copy of the gene in each ancestral chromosome set. The gene maps near the telomere on the short arm of chromosomes 2A, 2B, and 2D. Identification of three different cDNAs, two corresponding to genes
Acc-1,1
and
Acc-1,2
, indicates that all three genes are transcriptionally active.
The core domain of a new retina specific RGS protein stimulates the GTPase activity of transducin
in vitro
Authors: Faurobert Eva , Hurley James B.
Published: July 2002

GTP hydrolysis by the transducin α subunit is stimulated by a membrane-bound protein. The identity of this GTPase-activating protein (GAP) is not yet known, but the recent identification of a new gene family encoding regulator of G protein signaling (RGS) proteins raises the possibility that the transducin GAP is an RGS protein. Biochemical evidence shows that RGS proteins act as GAPs for α subunits of the G
i
subfamily of G proteins. To identify an RGS protein that could be a GAP for the transducin α subunit, we investigated the expression of RGS proteins in the retina and identified a new RGS domain, RET-RGS-d, which is specifically expressed in the retina.
In situ
RNA hybridization analyses revealed that RET-RGS-d is expressed in photoreceptor cells as well as in other cells of the retina. Recombinant RET-RGS-d accelerates single turnover hydrolysis of GTP by transducin. We used RET-RGS-d to isolate a full-length cDNA, RET-RGS1, encoding a new RGS protein with a C terminus that corresponds to RET-RGS-d. The N-terminal half of RET-RGS1 contains a putative transmembrane domain and a string of nine cysteines that are potential substrates for multiple palmitoylation. These findings suggest that RET-RGS1 is an integral membrane protein and that it is a candidate for the membrane-associated protein responsible for the GAP activity detected in photoreceptor membranes.
Correlation of germ-line mutations and two-hit inactivation of the
WT1
gene with Wilms tumors of stromal–predominant histology
Authors: Schumacher V. , Schneider S. , Figge A. , Wildhardt G. , Harms D. , Schmidt D. , Weirich A. , Ludwig R. , Royer-Pokora B.
Published: July 2002

The
WT1
gene, located on chromosome 11p13, is mutated in a low number of Wilms tumors (WTs). Germ-line mutations in the
WT1
gene are found in patients with bilateral WT and/or associated genital tract malformations (GU). We have identified 19 hemizygous
WT1
gene mutations/deletions in 64 patient samples. The histology of the tumors with mutations was stromal–predominant in 13, triphasic in 3, blastemal–predominant in 1, and unknown in 2 cases. Thirteen of 21 patients with stromal–predominant tumors had
WT1
mutations and 10 of these were present in the germ line. Of the patients with germ-line alterations, six had GU and a unilateral tumor, two had a bilateral tumor and normal GU tracts, and two had a unilateral tumor and normal GU. Three mutations were tumor-specific and were found in patients with unilateral tumors without GU. These data demonstrate a correlation of
WT1
mutations with stromal–predominant histology, suggesting that a germ-line mutation in
WT1
predisposes to the development of tumors with this histology. Twelve mutations are nonsense mutations resulting in truncations at different positions in the WT1 protein and only two are missense mutations. Of the stromal–predominant tumors, 67% showed loss of heterozygosity, and in one tumor a different somatic mutation in addition to the germ-line mutation was identified. These data show that in a large proportion of a histopathologically distinct subset of WTs the classical two-hit inactivation model, with loss of a functional WT1 protein, is the underlying cause of tumor development.
His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity
Authors: Lu Jade M.-Y. , Bush Daniel R.
Published: July 2002

The proton–sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton–sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from
Arabidopsis thaliana
is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased
V
max
) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction.
Reconstitution of HIV-1 Rev nuclear export: Independent requirements for nuclear import and export
Authors: Love Dona C. , Sweitzer Thomas D. , Hanover John A.
Published: July 2002

The Rev protein of HIV-1 actively shuttles between nucleus and cytoplasm and mediates the export of unspliced retroviral RNAs. The localization of shuttling proteins such as Rev is controlled by the relative rates of nuclear import and export. To study nuclear export in isolation, we generated cell lines expressing a green fluorescent protein-labeled chimeric protein consisting of HIV-1 Rev and a hormone-inducible nuclear localization sequence. Steroid removal switches off import thus allowing direct visualization of the Rev export pathway in living cells. After digitonin permeabilization of these cells, we found that a functional nuclear export sequence (NES), ATP, and fractionated cytosol were sufficient for nuclear export
in vitro.
Nuclear pore-specific lectins and leptomycin B were potent export inhibitors. Nuclear export was not inhibited by antagonists of calcium metabolism that block nuclear import. These data further suggest that nuclear pores do not functionally close when luminal calcium stores are depleted. The distinct requirements for nuclear import and export argue that these competing processes may be regulated independently. This system should have wide applicability for the analysis of nuclear import and export.
Time course of reactions controlled and gated by intramolecular dynamics of proteins: Predictions of the model of random walk on fractal lattices
Authors: Kurzynski M. , Palacz K. , Chelminiak P.
Published: July 2002

Computer simulations of random walk on the Sierpiński gasket and percolation clusters demonstrate that the short, initial condition-dependent stage of protein involving reactions can dominate the progress of the reaction over the main stage described by the standard kinetics. This phenomenon takes place if the intramolecular conformational transition dynamics modeled by the stochastic process is slow enough and the initial conformational substate of the protein already belongs to the transition state of the reaction. Both conditions are realized in two kinds of experiments: small ligand rebinding to protein after laser flash photolysis and direct recording of single protein channel activity. The model considered suggests simple analytical formulae that can explain the time behavior of the processes observed and its variation with temperature. The initial condition-dependent stage, and not the stage described by the standard kinetics, is expected as responsible for the coupling of component reactions in the complete catalytic cycles and more complex processes of biological free energy transduction.
Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin
Authors: Lin Jing , Westler William M. , Cleland W. Wallace , Markley John L. , Frey Perry A.
Published: July 2002

NMR investigations have been carried out of complexes between bovine chymotrypsin A
α
and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin:
N
-Acetyl-
l
-Phe-CF
3
,
N
-Acetyl-Gly-
l
-Phe-CF
3
,
N
-Acetyl-
l
-Val-
l
-Phe-CF
3
, and
N
-Acetyl-
l
-Leu-
l
-Phe-CF
3
. The D/H fractionation factors (
φ
) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-H
δ1
) in these four complexes at 5°C were in the range
φ
= 0.32–0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-H
ɛ1
(δ
2,2-dimethylsilapentane-5-sulfonic acid
8.97–9.18), the exchange rate of the His 57-H
δ1
proton with bulk water protons (284–12.4 s
−1
), and the activation enthalpies for this hydrogen exchange (14.7–19.4 kcal⋅mol
−1
). It was found that the previously noted correlations between the inhibition constants (
K
i
170–1.2 μM) and the chemical shifts of His 57-H
δ1
(δ
2,2-dimethylsilapentane-5-sulfonic acid
18.61–18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998)
Biochemistry
37, 11940–11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-H
δ1
, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.
Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors
Authors: Sata Makoto , Donaldson Julie G. , Moss Joel , Vaughan Martha
Published: July 2002

The
Saccharomyces cerevisiae
Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of ≈200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are ≈20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His
6
-tagged Sec7 domain from ySec7p (rySec7d) synthesized in
Escherichia coli
enhanced binding of guanosine 5′-[γ-[
35
S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5′-[γ-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.
A new structure for the murine
Xist
gene and its relationship to chromosome choice/counting during X-chromosome inactivation
Authors: Hong Young-Kwon , Ontiveros Sara D. , Chen Caifu , Strauss William M.
Published: July 2002

In this report, we present structural data for the murine
Xist
gene. The data presented in this paper demonstrate that the murine
Xist
transcript is at least 17.4 kb, not 14.3 kb as previously reported. The new structure of the murine
Xist
gene described herein has seven exons, not six. Exon VII encodes an additional 3.1 kb of information at the 3′ end. Exon VII contains seven possible sites for polyadenylation; four of these sites are located in the newly discovered 3′ end. Consequently, it is possible that several distinct transcripts may be produced through differential polyadenylation of a primary transcript. Alternative use of polyadenylation signals could result in size changes for exon VII. Two major species of
Xist
are detectable by Northern analysis, consistent with differential polyadenylation. In this paper, we propose a model for the role of the
Xist
3′ end in the process of X-chromosome counting and choice during embryonic development.
Immunization against genital herpes with a vaccine virus that has defects in productive and latent infection
Authors: Da Costa Xavier J. , Jones Cheryl A. , Knipe David M.
Published: July 2002

An effective vaccine for genital herpes has been difficult to achieve because of the limited efficacy of subunit vaccines and the safety concerns about live viruses. As an alternative approach, mutant herpes simplex virus strains that are replication-defective can induce protective immunity. To increase the level of safety and to prove that replication was not needed for immunization, we constructed a mutant herpes simplex virus 2 strain containing two deletion mutations, each of which eliminated viral replication. The double-mutant virus induces protective immunity that can reduce acute viral shedding and latent infection in a mouse genital model, but importantly, the double-mutant virus shows a phenotypic defect in latent infection. This herpes vaccine strain, which is immunogenic but has defects in both productive and latent infection, provides a paradigm for the design of vaccines and vaccine vectors for other sexually transmitted diseases, such as AIDS.
TID1, a human homolog of theDrosophilatumor suppressorl(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions
Authors: Syken Josh , De-Medina Tali , Münger Karl
Published: July 2002

Mitochondria have emerged as central regulators of apoptosis. Here, we show thatTID1, a human homolog of theDrosophilatumor suppressorlethal (2) tumorous imaginal discs, l(2)tid, encodes two mitochondrial matrix proteins, designated hTid-1Land hTid-1S. These splice variants are both highly conserved members of the DnaJ family of proteins, which regulate the activity of and confer substrate specificity to Hsp70 proteins. Both hTid-1Land hTid-1Scoimmunoprecipitate with mitochondrial Hsp70. Expression of hTid-1Lor hTid-1Shave no apparent capacity to induce apoptosis but have opposing effects on apoptosis induced by exogenous stimuli. Expression of hTid-1Lincreases apoptosis induced by both the DNA-damaging agent mitomycin c and tumor necrosis factor α. This activity is J domain-dependent, because a J domain mutant of hTid-1Lcan dominantly suppress apoptosis. In sharp contrast, expression of hTid-1Ssuppresses apoptosis, whereas expression of a J domain mutant of hTid-1Sincreases apoptosis. Hence, we propose thatTID1gene products act to positively and negatively modulate apoptotic signal transduction or effector structures within the mitochondrial matrix.
A model-based approach for assessingin vivocombination therapy interactions
Authors: Lopez Angela M. , Pegram Mark D. , Slamon Dennis J. , Landaw Elliot M.
Published: July 2002

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach comparesin vivotumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.
tRNA
Val
-heterodimeric maxizymes with high potential as geneinactivating agents: Simultaneous cleavage at two sites in HIV-1 tat mRNA in cultured cells
Authors: Kuwabara Tomoko , Warashina Masaki , Nakayama Aya , Ohkawa Jun , Taira Kazunari
Published: July 2002

It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNA
Val
-promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNA
Val
-driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNA
Val
-driven maxizymes tested to date have been more effective than tRNA
Val
-driven “standard” hammerhead ribozymes, the tRNA
Val
-driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.
A different approach to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase
Authors: Sarkissian Christineh N. , Shao Zhongqi , Blain Françoise , Peevers Rosalie , Su Hongsheng , Heft Robert , Chang Thomas M. S. , Scriver Charles R.
Published: July 2002

Phenylketonuria (PKU), with its associated hyperphenylalaninemia (HPA) and mental retardation, is a classic genetic disease and the first to have an identified chemical cause of impaired cognitive development. Treatment from birth with a low phenylalanine diet largely prevents the deviant cognitive phenotype by ameliorating HPA and is recognized as one of the first effective treatments of a genetic disease. However, compliance with dietary treatment is difficult and when it is for life, as now recommended by an internationally used set of guidelines, is probably unrealistic. Herein we describe experiments on a mouse model using another modality for treatment of PKU compatible with better compliance using ancillary phenylalanine ammonia lyase (PAL, EC
4.3.1.5
) to degrade phenylalanine, the harmful nutrient in PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase (EC
1.14.16.1
), which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. We describe (
i
) an efficient recombinant approach to produce PAL enzyme, (
ii
) testing of PAL in orthologous
N
-ethyl-
N′
-nitrosourea (ENU) mutant mouse strains with HPA, and (
iii
) proofs of principle (PAL reduces HPA)—both pharmacologic (with a clear dose–response effect vs. HPA after PAL injection) and physiologic (protected enteral PAL is significantly effective vs. HPA). These findings open another way to facilitate treatment of this classic genetic disease.
Telomerase in kinetoplastid parasitic protozoa
Authors: Cano M. I. N. , Dungan J. M. , Agabian N. , Blackburn E. H.
Published: July 2002

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species:
Trypanosoma brucei, Leishmania major
, and
Leishmania tarentolae
. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N.,
et al.
(1994)
Science
266, 2011–2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996)
EMBO J.
15, 5928–5935]. The activity in
T. brucei
extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The
in vitro
properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of
T. brucei
. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.
Molecular modeling andin vitroactivity of an HIV-1-encoded glutathione peroxidase
Authors: Zhao Lijun , Cox Arthur G. , Ruzicka Jan A. , Bhat Ajita A. , Zhang Weiqing , Taylor Ethan Will
Published: July 2002

Based on theoretical evidence, it has been proposed that HIV-1 may encode several selenoprotein modules, one of which (overlapping theenvgp41-coding region) has highly significant sequence similarity to the mammalian selenoprotein glutathione peroxidase (GPx; EC1.11.1.9). The similarity score of the putative HIV-1 viral GPx homolog relative to an aligned set of known GPx is 6.3 SD higher than expected for random sequences of similar composition. Based on that alignment, a molecular model of the HIV-1 GPx was constructed by homology modeling from the bovine GPx crystal structure. Despite extensive truncation relative to the cellular GPx gene, the structural core and the geometry of the catalytic triad of selenocysteine, glutamine, and tryptophan are well conserved in the viral GPx. All of the insertions and deletions predicted by the alignment proved to be structurally feasible. The model is energetically favorable, with a computed molecular mechanics strain energy close to that of the bovine GPx structure, when normalized on a per-residue basis. However, considering the remote homology, this model is intended only to provide a working hypothesis allowing for a similar active site and structural core. To validate the theoretical predictions, we cloned the hypothetical HIV-1 gene and found it to encode functional GPx activity when expressed as a selenoprotein in mammalian cells. In transfected canine kidney cells, the increase in GPx activity ranged from 21% to 43% relative to controls (average 30%,n= 9,P< 0.0001), whereas, in transfected MCF7 cells, which have low endogenous GPx activity, a near 100% increase was observed (average 99%,n= 3,P< 0.05).
Cognitive skills among Nepalese child labourers
Authors: Alaraudanjoki Esa , Regmi Murari P. , Ahonen Timo , Nurmi Jari‐Erik , Ruoppila Isto
Published: July 2002

The cognitive skills of 61 Nepalese 10–14‐year‐old working children with at least 2 years working experience (WE) were compared to two groups of children, beginners (N=29) with less than 1 year of WE and a school group (N = 104) matched for age and ethnic background. All the children (N=194) were tested by the Bender test, WISC‐R for Arithmetic, Digit Span, and the Word Fluency test. The main results showed that the school group was better in all the cognitive tests, except for Digit Span Backwards, where the working group had the highest average score. The second main finding shows no major differences in cognitive skills between the beginner and working groups. However, the work experience as such was related to decreasing visuoconstructive skills and improving Digit Span Forward scores. This suggests that work in the carpet factories seem to develop the verbal short‐term memory functions of the children, while their visuoconstructive skills decrease by work experience. The third main finding showed that the boys were better in arithmetic skills than the girls when they had long working experience.
Influence of a macrolide antibiotic, roxithromycin, on mast cell growth and activation in vitro
Authors: Shimane Toshikazu , Asano Kazuhito , Suzuki Mayumi , Hisamitsu Tadashi , Suzaki Harumi
Published: July 2002

Background: Long‐term administration of macrolide antibiotics is recognized to be able to favorably modify the clinical condition of inflammatory diseases, such as diffuse panbronchiolitis and cystic fibrosis. However, the precise mechanisms by which macrolide antibiotics could improve clinical conditions of the patients
are not well understood.

Aim: The present study was designed to examine the influence of macrolide antibiotics on effector cell functions responsible for inflammation through the choice of roxithromycin (RXM) and mast cell.

Methods: Mast cells were induced by long‐term culture of splenocytes from BALB/c mice. RXM was added to the cultures at seeding and then every 4‐5 days, when the culture medium was replaced with a fresh one. The influence of RXM on mast cell growth was evaluated by counting the number of cells grown on the 16th day. We also examined the influence of RXM on mast cell activation by examining histamine release and inflammatory cytokine secretion.

Results and conclusion: RXM could not inhibit mast cell growth, even when splenocytes were exposed to 100μg/ml of RXM throughout the entire culture periods. RXM also could not suppress histamine release from cultured mast cells in response to nonimmunological and immunological stimulations. However, RXM could suppress inflammatory cytokine, interleukin‐1β, interleukin‐6, granulocyte macrophage‐colony stimulating factor and tumor necrosis factor‐α, secretions induced by concanavalin A stimulation at a concentration of as little as 0.5μg/ml. These results may suggest that RXM modulated the ability of mast cells to secrete inflammatory cytokines and results in improvement of clinical condition of chronic inflammatory diseases.
Cyclosporine and inflammatory bowel disease: buying time
Authors: Meijssen M. A. C.
Published: July 2002

Cyclosporine is an effective drug in acute exacerbations of corticosteroid resistant ulcerative colitis, but its efficacy to maintain disease remission is not clear. Cyclosporine may not be as effective in Crohn′s disease. However, being a rapidly acting immunosuppressant, cyclosporine may be a valuable therapeutic option in the short‐term to treat corticosteroid resistant Crohn′s disease and ulcerative colitis.
Antioxidants and mucosa protectives: realistic therapeutic options in inflammatory bowel disease?
Authors: Kruidenier L. , Verspaget H. W.
Published: July 2002

Oxidative damage is involved in the pathogenic process of idiopathic chronic inflammatory bowel disease. Although specific intervention in the oxidative cascade showed promising results in animal models and preliminary patient trials, the clinical efficacy of antioxidants still has to be established. Mucosa protection, for example by dietary fatty acids, seems to attenuate the intestinal inflammatory process as well but awaits definite clinical proof for the treatment of inflammatory bowel disease
Pouchitis
Authors: Schouten W. R.
Published: July 2002

Pouchitis is a major long‐term complication of the continent ileostomy as well as the ileoanal pouch anastomosis. When diagnosed on the basis of clinical, endoscopic and histologic features, this syndrome has been demonstrated almost exclusively in patients with ulcerative colitis. The clinical course, the endoscopic findings and the histologic abnormalities resemble those of ulcerative colitis. The association with extra‐intestinal manifestations further supports the hypothesis that pouchitis represents ulcerative colitis in the small bowel. All ileal reservoirs show bacterial overgrowth, especially of anaerobes. As a response to this altered intraluminal environment chronic inflammation and incomplete colonic metaplasia occur. The efficiency of metronidazole does suggest that bacteriological factors play an important role in the pathogenesis of pouchitis.
Airway Responsiveness: Role of Inflammation, Epithelium Damage and Smooth Muscle Tension
Authors: Gourgoulianis K. I. , Domali A. , Molyvdas P. A.
Published: July 2002

The purpose of this study was the effect of epithelium damage on mechanical responses of airway smooth muscles under different resting tension. We performed acetylcholine (ACh) (10-5M)‐induced contraction on tracheal strips from 30 rabbits in five groups (0.5, 1, 1.5, 2 and 2.5 g) before and after epithelium removal. At low resting tension (0.5‐1.5g), the epithelium removal decreased the ACh‐induced contractions. At 2g resting tension, the epithelium removal increased the ACh‐induced contractions of airways with intact epithelium about 20%. At 2.5 g resting tension, the elevation of contraction is about 25% (p < 0.01). Consequently, after epithelium loss, the resting tension determines the airway smooth muscles responsiveness. In asthma, mediators such as ACh act on already contracted inflammatory airways, which results in additional increase of contraction. In contrast, low resting tension, a condition that simulates normal tidal breathing, protects from bronchoconstriction even when the epithelium is damaged.