bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia
Authors: Becker Kyra J. , McCarron Richard M. , Ruetzler Christl , Laban Olgica , Sternberg Esther , Flanders Kathleen C. , Hallenbeck John M.
Published: July 2002

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmedin vivoby a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterizedin vitroby spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor β1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor β1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.
CooA, a CO-sensing transcription factor from
Rhodospirillum rubrum
, is a CO-binding heme protein
Authors: Shelver Daniel , Kerby Robert L. , He Yiping , Roberts Gary P.
Published: July 2002

Biological sensing of small molecules such as NO, O
2
, and CO is an important area of research; however, little is know about how CO is sensed biologically. The photosynthetic bacterium
Rhodospirillum rubrum
responds to CO by activating transcription of two operons that encode a CO-oxidizing system. A protein, CooA, has been identified as necessary for this response. CooA is a member of a family of transcriptional regulators similar to the cAMP receptor protein and fumavate nitrate reduction from
Escherichia coli
. In this study we report the purification of wild-type CooA from its native organism,
R. rubrum
, to greater than 95% purity. The purified protein is active in sequence-specific DNA binding in the presence of CO, but not in the absence of CO. Gel filtration experiments reveal the protein to be a dimer in the absence of CO. Purified CooA contains 1.6 mol heme per mol of dimer. Upon interacting with CO, the electronic spectrum of CooA is perturbed, indicating the direct binding of CO to the heme of CooA. A hypothesis for the mechanism of the protein’s response to CO is proposed.
The nucleocapsid protein specifically anneals tRNALys-3onto a noncomplementary primer binding site within the HIV-1 RNA genomein vitro
Authors: Chan Barden , Musier-Forsyth Karin
Published: July 2002

HIV type 1 (HIV-1) specifically uses host cell tRNALys-3as a primer for reverse transcription. The 3′ 18 nucleotides of this tRNA are complementary to a region on the HIV RNA genome known as the primer binding site (PBS). HIV-1 has a strong preference for maintaining a lysine-specific PBSin vivo, and viral genomes with mutated PBS sequences quickly revert to be complementary to tRNALys-3. To investigate the mechanism for the observed PBS reversion eventsin vitro, we examined the capability of the nucleocapsid protein (NC) to anneal various tRNA primer sequences onto either complementary or noncomplementary PBSs. We show that NC can anneal different full-length tRNAs onto viral RNA transcripts derived from the HIV-1 MAL or HXB2 isolates, provided that the PBS is complementary to the tRNA used. In contrast, NC promotes specific annealing of only tRNALys-3onto an RNA template (HXB2) whose PBS sequence has been mutated to be complementary to the 3′ 18 nt of human tRNAPro. Moreover, HIV-1 reverse transcriptase extends this binary complex from the proline-specific PBS. The formation of the noncomplementary binary complex does not occur when a chimeric tRNALys/Procontaining proline-specific D and anticodon domains is used as the primer. Thus, elements outside the acceptor-TΨC domains of tRNALys-3play an important role in preferential primer usein vitro. Our results support the hypothesis that mutant PBS reversion is a result of tRNALys-3annealing onto and extension from a PBS that specifies an alternate host cell tRNA.
Adaptive evolution via a major gene effect: Paedomorphosis in the Mexican axolotl
Authors: Voss S. Randal , Shaffer H. Bradley
Published: July 2002

Although adaptive evolution is thought to depend primarily on mutations of small effect, major gene effects may underlie many of the important differences observed among species in nature. The Mexican axolotl (
Ambystoma mexicanum
) has a derived mode of development that is characterized by metamorphic failure (paedomorphosis), an adaptation for an entirely aquatic life cycle. By using an interspecific crossing design and genetic linkage analysis, a major quantitative trait locus for expression of metamorphosis was identified in a local map of amplified fragment length polymorphisms. These data are consistent with a major gene hypothesis for the evolution of paedomorphosis in
A. mexicanum
.
Coexpression of nuclear receptor partners increases their solubility and biological activities
Authors: Li Chuan , Schwabe John W. R. , Banayo Ester , Evans Ronald M.
Published: July 2002

The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in
Escherichia coli
to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR–RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.
Unwinding of the box I element of a herpes simplex virus type 1 origin by a complex of the viral origin binding protein, single-strand DNA binding protein, and single-stranded DNA
Authors: Lee Sam S.-K. , Lehman I. R.
Published: July 2002

The herpes simplex virus type 1 (HSV-1) genome contains three origins of replication: ori
L
and two copies of ori
S
. These origins contain specific sequences, box I and box II, linked by an AT-rich segment, that are recognized by an HSV-1-encoded origin binding protein (UL9 protein) which also possesses DNA helicase activity. Despite its intrinsic helicase activity, the UL9 protein is unable to unwind ori
S
or the box I element of ori
S
, either in the presence or absence of the HSV-1-encoded single-strand DNA binding protein, ICP8. However, a complex of the UL9 protein and ICP8 can unwind box I if it contains a 3′ single-stranded tail at least 18 nt in length positioned downstream of box I. These findings suggest a model for the initiation of HSV-1 DNA replication in which a complex consisting of the UL9 protein bound to box I, and ICP8 bound to single-stranded DNA generated at the A+T rich linker, perhaps as a consequence of transcription, unwinds an HSV-1 origin of replication to provide access to the replication machinery with the consequent initiation of viral DNA replication. This mode of unwinding is distinct from that observed for other animal viruses—e.g., simian virus 40 or bovine papilloma virus—in which the initiator protein, T antigen, or E1 protein alone, unwinds elements of the origin sequence, and the single-strand DNA binding protein serves only to keep the separated strands apart.
Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of
O
-acetylated ganglioside: A putative acetyl-CoA transporter
Authors: Kanamori Akiko , Nakayama Jun , Fukuda Michiko N. , Stallcup William B. , Sasaki Katsutoshi , Fukuda Minoru , Hirabayashi Yoshio
Published: July 2002

By expression cloning using COS-1 cells stably transfected with GD3-synthase (COS-1/GD3
+
) as a recipient cell line, we have isolated a cDNA, termed AT-1, encoding a novel protein required for the formation of
O
-acetylated (Ac) gangliosides. The cDNA encodes a protein with multitransmembrane spanning domains with a leucine zipper motif. It consists of 549 amino acids and has a molecular mass of 60.9 kDa. Although both
O
-Ac-GD3 and
O
-Ac-GT3 were barely detectable in recipient cells or cells transfected with the vector alone, their amount increased significantly in transfectants containing AT-1. When semi-intact cells prepared by treatment with streptolysin O were incubated with [Ac-
14
C]Ac-CoA, increased incorporation of radioactivity was found in those cells transfected with AT-1 when compared with the mock transfectants. Northern blot analysis showed two major transcripts of 3.3 and 4.3 kb in all tissues examined. Immunohistochemical study with an antibody specific to the AT-1 protein suggested that it is most probably expressed in the endoplasmic reticulum membrane. Based on these results, the protein encoded by AT-1 is suggested to be an Ac-CoA transporter that is involved in the process of
O
-acetylation.
Stage specificity, dose response, and doubling dose for mouse minisatellite germ-line mutation induced by acute radiation
Authors: Dubrova Yuri E. , Plumb Mark , Brown Julia , Fennelly Jan , Bois Philippe , Goodhead Dudley , Jeffreys Alec J.
Published: July 2002

Germ-line mutation induction at mouse minisatellite loci by acute irradiation with x-rays was studied at premeiotic and postmeiotic stages of spermatogenesis. An elevated paternal mutation rate was found after irradiation of premeiotic spermatogonia and stem cells, whereas the frequency of minisatellite mutation after postmeiotic irradiation of spermatids was similar to that in control litters. In contrast, paternal irradiation did not affect the maternal mutation rate. A linear dose–response curve for paternal mutation induced at premeiotic stages was found, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. High frequencies of spontaneous and induced mutations at minisatellite loci allow mutation induction to be evaluated at low doses of exposure in very small population samples, which currently makes minisatellite DNA the most powerful tool for monitoring radiation-induced germ-line mutation.
Broad spectrum chemokine antagonistic activity of a human poxvirus chemokine homolog
Authors: Damon Inger , Murphy Philip M. , Moss Bernard
Published: July 2002

A secreted CC chemokine homolog, encoded by the MC148 gene of molluscum contagiosum virus, potently interfered with the chemotaxis of human monocytes, lymphocytes, and neutrophils in response to a large number of CC and CXC chemokines with diverse receptor specificities. Evidence that the viral protein binds to human chemokine receptors was obtained by competition binding and calcium mobilization experiments. The broad spectrum chemokine antagonistic activity of MC148 can explain the prolonged absence of an inflammatory response in skin tumors that harbor replicating molluscum contagiosum virus.
Deposition-related sites K5/K12 in histone H4 are not required for nucleosome deposition in yeast
Authors: Ma Xiao-Jun , Wu Jiansheng , Altheim Brent A. , Schultz Michael C. , Grunstein Michael
Published: July 2002

Histone H4 can be acetylated at N-terminal lysines K5, K8, K12, and K16, but newly synthesized H4 is diacetylated at K5/K12 in diverse organisms. This pattern is widely thought to be important for histone deposition onto replicating DNA. To investigate the importance of K5/K12 we have mutagenized these lysines in yeast and assayed for nucleosome assembly. Assaying was done in the absence of the histone H3 N terminus, which has functions redundant with those of H4 in histone deposition. Nucleosome assembly was assayed by three methods. Because nucleosome depletion may be lethal, we examined cell viability. We also analyzed nucleosome assembly
in vivo
and
in vitro
by examining plasmid superhelicity density in whole cells and supercoiling in yeast cell extracts. All three approaches demonstrate that mutagenizing K5 and K12 together does not prevent cell growth and histone deposition
in vivo
or
in vitro
. Therefore, K5/K12 cannot be required for nucleosome assembly in yeast. It is only when the first three sites of acetylation—K5, K8, and K12—are mutagenized simultaneously that lethality occurs and assembly is most strongly decreased both
in vivo
and
in vitro
. These data argue for the redundancy of sites K5, K8, and K12 in the deposition of yeast histone H4.
Thermostable archaealO6-alkylguanine-DNA alkyltransferases
Authors: Skorvaga Milan , Raven Neil D. H. , Margison Geoffrey P.
Published: July 2002

Archaea represent some of the most ancient organisms on earth, and they have relatively uncharacterized DNA repair processes. We now show, using anin vitroassay, that extracts of two Crenarchaeota (Sulfolobus acidocaldariusandPyrobaculum islandicum)and two Euryarchaeota (Pyrococcus furiosusandThermococcus litoralis) contain the DNA repair proteinO6-alkylguanine-DNA alkyltransferase (ATase). The ATase activities found in the archaea were extremely thermostable, with half-lives at 80°C ranging from 0.5 hr (S. acidocaldarius) to 13 hr (T. litoralis). The temperature optima of the four proteins ranged from ≈75 to ≈100°C, although activity was seen at 37°C, the temperature optimum of theEscherichia coliand human ATases. In all cases, preincubaton of extracts with a short oligonucleotide containing a singleO6-methylguanine residue caused essentially complete loss of ATase activity, suggesting that the alkylphosphotriester-DNA alkyltransferase activity seen in some prokaryotes is not present in Archaea. The ATase fromPyrobaculum islandicumhad an apparent molecular mass of 15 kDa, making it the smallest of these proteins so far described. In higher organisms, ATase is responsible for the repair of toxic and mutagenicO6-alkylguanine lesions in alkylated DNA. The presence of ATase in these primitive organisms therefore suggests that endogenous or exogenous exposure to agents that generate appropriate substrates in DNA may be an early event in evolution.
Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P
Authors: Plehn-Dujowich Debora , Altman Sidney
Published: July 2002

The polymerase (
PB2
) and nucleocapsid (
NP
) genes encoded by the genome of influenza virus are essential for replication of the virus. When synthetic genes that express RNAs for external guide sequences targeted to the mRNAs of the
PB2
and
NP
genes are stably incorporated into mouse cells in tissue culture, infection of these cells with influenza virus is nonproductive. Endogenous RNase P cleaves the targeted influenza virus mRNAs when they are in a complex with the external guide sequences. Targeting two different mRNAs simultaneously inhibits viral particle production more efficiently than does targeting only one mRNA.
Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway
Authors: Gross-Mesilaty Shlomit , Reinstein Eyal , Bercovich Beatrice , Tobias Karin E. , Schwartz Alan L. , Kahana Chaim , Ciechanover Aaron
Published: July 2002

We have previously shown that the degradation of c-myc and N-
myc in vitro
is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins
in vivo
and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins
in vitro
. Expression of E6-16 in cells results in significant shortening of the
t
1/2
of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s—E2-14 kDa and E3α involved in the “N-end rule” pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc⋅E6⋅E6–AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.
Lithium acutely inhibits and chronically up-regulates and stabilizes glutamate uptake by presynaptic nerve endings in mouse cerebral cortex
Authors: Dixon John F. , Hokin Lowell E.
Published: July 2002

We previously reported that lithium stimulated extracellular glutamate accumulation in monkey and mouse cerebrocortical slices. We report here that this is caused by lithium-induced inhibition of glutamate uptake into the slice. Glutamate release was amplified 5-fold over inhibition of uptake. When the effects of lithium and the specific glutamate transporter inhibitors,
l
-trans-pyrrolidine-2,4-dicarboxylic acid and dihydrokainic acid, were plotted as glutamate accumulation vs. inhibition of glutamate uptake, the plots were superimposable. This finding strongly indicates that lithium-induced glutamate accumulation is caused entirely by inhibition of uptake. With cerebrocortical synaptosomes, inhibition of glutamate uptake was greater than in slices, suggesting that presynaptic nerve endings are the primary site of inhibition of uptake by lithium. Inhibition of uptake was caused by a progressive lowering of
V
max
, as the lithium concentration was increased, whereas the
K
m
remained constant, indicating that lithium inhibited the capacity of the transporter but not its affinity. Chronic treatment of mice with lithium, achieving a blood level of 0.7 mM, which is on the low side of therapeutic, up-regulated synaptosomal uptake of glutamate. This would be expected to exert an antimanic effect. Lithium is a mood stabilizer, dampening both the manic and depressive phases of bipolar disorder. Interestingly, although the uptake of glutamate varied widely in individual control mice, uptake in lithium-treated mice was stabilized over a narrow range (variance in controls, 0.423; in lithium treated, 0.184).
Molecular cloning and characterization of human caspase-activated DNase
Authors: Mukae Naomi , Enari Masato , Sakahira Hideki , Fukuda Yoji , Inazawa Johji , Toh Hiroyuki , Nagata Shigekazu
Published: July 2002

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart.
In vitro
transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional
CAD
gene was localized to human chromosome 1p36.3 by fluorescent
in situ
hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.
The ATP–metallothionein complex
Authors: Jiang Li-Juan , Maret Wolfgang , Vallee Bert L.
Published: July 2002

We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (
K
d
= 176 ± 33 μM, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol–disulfide interchange with Ellman’s reagent [5,5′-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di- or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two critical cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding constant and the cellular concentration of ATP indicate that in the cell MT could be essentially saturated with ATP at low concentrations of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.
Expression profiles of multiple genes in single neurons of Alzheimer’s disease
Authors: Chow Nienwen , Cox Chris , Callahan Linda M. , Weimer Jill M. , Guo LiRong , Coleman Paul D.
Published: July 2002

Many changes have been described in the brains of Alzheimer’s disease (AD) patients, including loss of neurons and formation of senile plaques and neurofibrillary tangles. The molecular mechanisms underlying these pathologies are unclear. Northern blot, dot-blot, and reverse transcription-coupled PCR analyses have demonstrated altered expression levels of multiple messages in AD brain. Because not all cells are equally affected by the disease, these methods obviously cannot study the changes in relation to disease states of individual cells. We address this problem by using antisense RNA profiling of single cells. We present expression profiles of single neurons at early and late stages of AD and describe statistical tools for data analysis. With multivariate canonical analysis, we were able to distinguish the disease state on the basis of altered expression of multiple messages. To validate this approach, we compared results obtained by this approach with results obtained by
in situ
hybridization analysis. When the neurofilament medium subunit was used as a marker, our results from an antisense RNA profiling revealed no change in neurofilament medium subunit expression between early- and late-stage AD, consistent with findings obtained with
in situ
hybridization. However, our results obtained by either analysis at the single-cell level differed from the reported decrease in AD neocortex obtained by Northern blot analysis [Kittur, S., Hoh, J., Endo, H., Tourtellotte, W., Weeks, B. S., Markesbery, W. & Adler, W. (1994)
J. Geriatr. Psychiatry Neurol.
7, 153–158]. Thus, the strategy of using the single-cell antisense RNA approach to identify altered gene expression in postmortem AD brain, followed by detailed
in situ
hybridization studies for genes of interest, is valuable in the study of the molecular mechanisms underlying AD neuropathology.
Molecular evidence for multiple origins of Insectivora and for a new order of endemic African insectivore mammals
Authors: Stanhope Michael J. , Waddell Victor G. , Madsen Ole , de Jong Wilfried , Hedges S. Blair , Cleven Gregory C. , Kao Diana , Springer Mark S.
Published: July 2002

The traditional views regarding the mammalian order Insectivora are that the group descended from a single common ancestor and that it is comprised of the following families: Soricidae (shrews), Tenrecidae (tenrecs), Solenodontidae (solenodons), Talpidae (moles), Erinaceidae (hedgehogs and gymnures), and Chrysochloridae (golden moles). Here we present a molecular analysis that includes representatives of all six families of insectivores, as well as 37 other taxa representing marsupials, monotremes, and all but two orders of placental mammals. These data come from complete sequences of the mitochondrial 12S rRNA, tRNA-Valine, and 16S rRNA genes (2.6 kb). A wide range of different methods of phylogenetic analysis groups the tenrecs and golden moles (both endemic to Africa) in an all-African superordinal clade comprised of elephants, sirenians, hyracoids, aardvark, and elephant shrews, to the exclusion of the other four remaining families of insectivores. Statistical analyses reject the idea of a monophyletic Insectivora as well as traditional concepts of the insectivore suborder Soricomorpha. These findings are supported by sequence analyses of several nuclear genes presented here: vWF, A2AB, and α-β hemoglobin. These results require that the order Insectivora be partitioned and that the two African families (golden moles and tenrecs) be placed in a new order. The African superordinal clade now includes six orders of placental mammals.
Relationship between donor age and the replicative lifespan of human cells in culture: A reevaluation
Authors: Cristofalo Vincent J. , Allen Robert G. , Pignolo Robert J. , Martin Bernard G. , Beck Jeanne C.
Published: July 2002

Normal human diploid fibroblasts have a finite replicative lifespanin vitro,which has been postulated to be a cellular manifestation of agingin vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared “healthy,” according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.
Multiple independent origins of mitochondrial gene order in birds
Authors: Mindell David P. , Sorenson Michael D. , Dimcheff Derek E.
Published: July 2002

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced O
L
(origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.
The expression of the KAI1 gene, a tumor metastasis suppressor, is directly activated by p53
Authors: Mashimo Tomoyuki , Watabe Misako , Hirota Shigeru , Hosobe Sadahiro , Miura Kunio , Tegtmeyer Peter J. , Rinker-Shaeffer Carrie W. , Watabe Kounosuke
Published: July 2002

KAI1 is a tumor metastasis suppressor gene that is capable of inhibiting the metastatic process in animals. The expression of the KAI1 gene also is found to be down-regulated during the tumor progression of prostate, breast, lung, bladder, and pancreatic cancers in humans, and this down-regulation appears to be at or posttranscription level. We have found that the tumor suppressor gene p53 can directly activate the KAI1 gene by interacting with the 5′ upstream region. The p53 responding region is located at approximately 860 bases upstream of the transcriptional initiation site, and it contains a typical tandem repeat of the p53 consensus-binding sequence. A gel-shift mobility analysis showed that this sequence indeed had the ability to bind to the purified p53 protein. Mutations of this sequence abolished the responsiveness to p53 and also the binding ability to the p53 protein. Furthermore, immunohistochemical analysis of 177 samples of human prostate tumors revealed that the expression of the KAI1 gene was correlated strongly to that of the p53 gene and that the loss of these two markers resulted in poor survivals of patients. Our data indicate a direct relationship between p53 and KAI1 genes and suggest that the loss of p53 function, which is commonly observed in many types of cancer, leads to the down-regulation of the KAI1 gene, which may result in the progression of metastasis.
A mutation in human CMP-sialic acid hydroxylase occurred after theHomo-Pandivergence
Authors: Chou Hsun-Hua , Takematsu Hiromu , Diaz Sandra , Iber Jane , Nickerson Elizabeth , Wright Kerry L. , Muchmore Elaine A. , Nelson David L. , Warren Stephen T. , Varki Ajit
Published: July 2002

Sialic acids are important cell-surface molecules of animals in the deuterostome lineage. Although humans do not express easily detectable amounts ofN-glycolylneuraminic acid (Neu5Gc, a hydroxylated form of the common sialic acidN-acetylneuraminic acid, Neu5Ac), it is a major component in great ape tissues, except in the brain. This difference correlates with lack of the hydroxylase activity that converts CMP-Neu5Ac to CMP-Neu5Gc. Here we report cloning of human and chimpanzee hydroxylase cDNAs. Although this chimpanzee cDNA is similar to the murine homologue, the human cDNA contains a 92-bp deletion resulting in a frameshift mutation. The isolated human gene also shows evidence for this deletion. Genomic PCR analysis indicates that this deletion does not occur in any of the African great apes. The gene is localized to 6p22–p23 in both humans and great apes, which does not correspond to known chromosomal rearrangements that occurred during hominoid evolution. Thus, the lineage leading to modern humans suffered a mutation sometime after the common ancestor with the chimpanzee and bonobo, potentially affecting recognition by a variety of endogenous and exogenous sialic acid-binding lectins. Also, the expression of Neu5Gc previously reported in human fetuses and tumors as well as the traces detected in some normal adult humans must be mediated by an alternate pathway.
Expression of the
Hox
gene complex in the indirect development of a sea urchin
Authors: Arenas-Mena César , Martinez Pedro , Cameron R. Andrew , Davidson Eric H.
Published: July 2002

Hox
complex genes control spatial patterning mechanisms in the development of arthropod and vertebrate body plans.
Hox
genes are all expressed during embryogenesis in these groups, which are all directly developing organisms in that embryogenesis leads at once to formation of major elements of the respective adult body plans. In the maximally indirect development of a large variety of invertebrates, the process of embryogenesis leads only to a free-living, bilaterally organized feeding larva. Maximal indirect development is exemplified in sea urchins. The 5-fold radially symmetric adult body plan of the sea urchin is generated long after embryogenesis is complete, by a separate process occurring within imaginal tissues set aside in the larva. The single
Hox
gene complex of
Strongylocentrotus purpuratus
contains 10 genes, and expression of eight of these genes was measured by quantitative methods during both embryonic and larval developmental stages and also in adult tissues. Only two of these genes are used significantly during the entire process of embryogenesis
per se
, although all are copiously expressed during the stages when the adult body plan is forming in the imaginal rudiment. They are also all expressed in various combinations in adult tissues. Thus, development of a microscopic, free-living organism of bilaterian grade, the larva, does not appear to require expression of the
Hox
gene cluster as such, whereas development of the adult body plan does. These observations reflect on mechanisms by which bilaterian metazoans might have arisen in Precambrian evolution.
Amyloid β peptide alters intracellular vesicle trafficking and cholesterol homeostasis
Authors: Liu Yuanbin , Peterson Daniel A. , Schubert David
Published: July 2002

Amyloid β peptide (Aβ) is thought to play a central role in the pathogenesis of Alzheimer disease (AD). How Aβ induces neurodegeneration in AD is not known. A connection between AD and cholesterol metabolism is suggested by the finding that people with the apolipoprotein E4 allele, a locus coding for a cholesterol-transporting lipoprotein, have a modified risk for both late-onset AD and cardiovascular disease. In the present study we show that both Aβ and submicromolar concentrations of free cholesterol alter the trafficking of a population of intracellular vesicles that are involved in the transport of the reduced form of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT formazan), the formation of which is a widely used cell viability assay. Treatments that change cellular free cholesterol levels also modulate the trafficking of the MTT formazan-containing vesicles, suggesting that the trafficking of these vesicles may be regulated by free cholesterol under physiological conditions. In addition, Aβ decreases cholesterol esterification and changes the distribution of free cholesterol in neurons. These results suggest that the MTT formazan-transporting vesicles may be involved in cellular cholesterol homeostasis and that the alteration of vesicle transport by Aβ may be relevant to the chronic neurodegeneration observed in AD.
A streptavidin mutant with altered ligand-binding specificity
Authors: Reznik Gabriel O. , Vajda Sandor , Sano Takeshi , Cantor Charles R.
Published: July 2002

The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (
K
a
) of 1 × 10
6
M
−1
, two orders of magnitude higher than that for biotin (1 × 10
4
M
−1
). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 × 10
4
M
−1
) was lower than predicted (2.9 × 10
5
M
−1
), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.
Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif
Authors: Potash Mary Jane , Bentsman Galina , Muir Tom , Krachmarov Chavdar , Sova Pavel , Volsky David J.
Published: July 2002

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease
in vitro
and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21–65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif
21–65
blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif
41–65
, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif
21–65
was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif
21–65
inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31–8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.
Modeling stochastic gene expression: Implications for haploinsufficiency
Authors: Cook Daniel L. , Gerber Anthony N. , Tapscott Stephen J.
Published: July 2002

There is increasing recognition that stochastic processes regulate highly predictable patterns of gene expression in developing organisms, but the implications of stochastic gene expression for understanding haploinsufficiency remain largely unexplored. We have used simulations of stochastic gene expression to illustrate that gene copy number and expression deactivation rates are important variables in achieving predictable outcomes. In gene expression systems with non-zero expression deactivation rates, diploid systems had a higher probability of uninterrupted gene expression than haploid systems and were more successful at maintaining gene product above a very low threshold. Systems with relatively rapid expression deactivation rates (unstable gene expression) had more predictable responses to a gradient of inducer than systems with slow or zero expression deactivation rates (stable gene expression), and diploid systems were more predictable than haploid, with or without dosage compensation. We suggest that null mutations of a single allele in a diploid organism could decrease the probability of gene expression and present the hypothesis that some haploinsufficiency syndromes might result from an increased susceptibility to stochastic delays of gene initiation or interruptions of gene expression.
Origin of a chloroplast protein importer
Authors: Bölter Bettina , Soll Jürgen , Schulz Alexander , Hinnah Silke , Wagner Richard
Published: July 2002

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares ≈22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the
Synechocystis
PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of
Synechocystis
PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.
Menin, the product of the
MEN1
gene, is a nuclear protein
Authors: Guru Siradanahalli C. , Goldsmith Paul K. , Burns A. Lee , Marx Stephen J. , Spiegel Allen M. , Collins Francis S. , Chandrasekharappa Settara C.
Published: July 2002

The
MEN1
gene, mutations in which are responsible for multiple endocrine neoplasia type 1 (MEN1), encodes a 610-amino acid protein, denoted menin. The amino acid sequence of this putative tumor suppressor offers no clue to the function or subcellular location of the protein. We report herein, based on immunofluorescence, Western blotting of subcellular fractions, and epitope tagging with enhanced green fluorescent protein, that menin is located primarily in the nucleus. Enhanced green fluorescent protein-tagged menin deletion constructs identify at least two independent nuclear localization signals (NLS), both located in the C-terminal fourth of the protein. Among the 68 known independent disease-associated mutations, none of the 22 missense and 3 in-frame deletions affect either of the putative NLS sequences. However, if expressed, none of the truncated menin proteins resulting from the 43 known frameshift/nonsense mutations would retain both the NLSs. The precise role(s) of menin in the nucleus remain to be understood.
Role of cyclophilin A in the uptake of HIV-1 by macrophages and T lymphocytes
Authors: Sherry Barbara , Zybarth Gabriele , Alfano Massimo , Dubrovsky Larisa , Mitchell Robert , Rich Daniel , Ulrich Peter , Bucala Richard , Cerami Anthony , Bukrinsky Michael
Published: July 2002

Cyclophilins are a family of proteins that bind cyclosporin A (CsA) and possess peptidyl-prolyl cis-trans isomerase activity. In addition, they are secreted by activated cells and act in a cytokine-like manner, presumably via signaling through a cell surface cyclophilin receptor. More recently, host-derived cyclophilin A (CyPA) has been shown to be incorporated into HIV-1 virions and its incorporation essential for viral infectivity. Here we present evidence supporting a role for viral-associated CyPA in the early events of HIV-1 infection. We report that HIV-1 infection of primary peripheral blood mononuclear cells can be inhibited by: (i) an excess of exogenously added CyPA; (ii) a CsA analogue unable to enter the cells; (iii) neutralizing antibodies to CyPA. Taken together with our observations that recombinant CyPA-induced mobilization of calcium in immortalized, as well as primary, CD4+T lymphocytes, and that incubation of T cells with iodinated CyPA, followed by chemical cross-linking, resulted in the formation of a high molecular mass complex on the cell surface, these results suggest that HIV-1-associated CyPA mediates an early event in viral infection via interaction with a cellular receptor. This interaction may present a target for anti-HIV therapies and vaccines.
“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of
Shigella
spp. and enteroinvasive
Escherichia coli
Authors: Maurelli Anthony T. , Fernández Reinaldo E. , Bloch Craig A. , Rode Christopher K. , Fasano Alessio
Published: July 2002

Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example,
Shigella
spp., the causative agents of bacillary dysentery, differ from the closely related commensal
Escherichia coli
in the presence of a plasmid in
Shigella
that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in ≈90% of
E. coli
strains but is uniformly absent in
Shigella
strains. When the gene for LDC,
cadA
, was introduced into
Shigella flexneri
2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the
S. flexneri
2a and laboratory
E. coli
K-12 genomes in the region of
cadA
revealed a large deletion in
Shigella
. Representative strains of
Shigella
spp. and enteroinvasive
E. coli
displayed similar deletions of
cadA
. Our results suggest that, as
Shigella
spp. evolved from
E. coli
to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these “black holes,” deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.