bluuebunny/crossref_metadata_2025 huggingface dataset:
Trichostatin A causes selective loss of DNA methylation in
Neurospora
Authors: Selker Eric U.
Published: July 2002

Both DNA methylation and hypoacetylation of core histones are frequently associated with repression of gene expression. Possible connections between these processes were investigated by taking advantage of genes controlled by methylation in
Neurospora crassa
. Trichostatin A (TSA), a potent inhibitor of histone deacetylase, derepressed a copy of
hph
that was repressed by DNA methylation which resulted from repeat-induced point mutation (RIP) acting on sequences flanking
hph
. Derepression by TSA was comparable to derepression by the inhibitor of DNA methylation, 5-azacytidine. TSA treatment also repressed an allele of
am
whose expression depends on methylation of an adjacent transposon,
Tad
. DNA methylation in the
hph
and
Tad
/
am
regions was greatly reduced by TSA treatment. TSA also caused hypomethylation of other methylated alleles of
am
generated by RIP. In contrast, TSA did not affect methylation of several other methylated genomic sequences examined, including the nucleolar rDNA and the inactivated transposon
Punt
RIP1
. Several possible models are discussed for the observed selective demethylation induced by TSA. The implication that acetylation of chromatin proteins can directly or indirectly control DNA methylation raises the possibility that connections between protein acetylation and DNA methylation result in self-reinforcing epigenetic states.
The
Candida albicans KRE9
gene is required for cell wall β-1,6-glucan synthesis and is essential for growth on glucose
Authors: Lussier Marc , Sdicu Anne-Marie , Shahinian Serge , Bussey Howard
Published: July 2002

We have isolated
CaKRE9
, a gene from
Candida albicans
, that is a functional homologue of the
Saccharomyces cerevisiae KRE9
gene involved in β-1,6-glucan synthesis. Disruption of the
CaKRE9
gene in
C. albicans
shows that CaKre9p is required for the synthesis or assembly of this fungal polymer. Homozygous null disruptants of
CaKRE9
grow poorly on galactose and fail to form hyphae in serum, and, in growth medium containing glucose, the gene is essential. Thus, the
CaKRE9
gene product is a potentially useful candidate as a target for fungal-specific drugs.
Characterization of a calmodulin kinase II inhibitor protein in brain
Authors: Chang Bill H. , Mukherji Sucheta , Soderling Thomas R.
Published: July 2002

Ca
2+
/calmodulin-dependent protein kinase II (CaM-KII) regulates numerous physiological functions, including neuronal synaptic plasticity through the phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. To identify proteins that may interact with and modulate CaM-KII function, a yeast two-hybrid screen was performed by using a rat brain cDNA library. This screen identified a unique clone of 1.4 kb, which encoded a 79-aa brain-specific protein that bound the catalytic domain of CaM-KII α and β and potently inhibited kinase activity with an IC
50
of 50 nM. The inhibitory protein (CaM-KIIN), and a 28-residue peptide derived from it (CaM-KIINtide), was highly selective for inhibition of CaM-KII with little effect on CaM-KI, CaM-KIV, CaM-KK, protein kinase A, or protein kinase C. CaM-KIIN interacted only with activated CaM-KII (i.e., in the presence of Ca
2+
/CaM or after autophosphorylation) by using glutathione
S
-transferase/CaM-KIIN precipitations as well as coimmunoprecipitations from rat brain extracts or from HEK293 cells cotransfected with both constructs. Colocalization of CaM-KIIN with activated CaM-KII was demonstrated in COS-7 cells transfected with green fluorescent protein fused to CaM-KIIN. In COS-7 cells phosphorylation of transfected α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors by CaM-KII, but not by protein kinase C, was blocked upon cotransfection with CaM-KIIN. These results characterize a potent and specific cellular inhibitor of CaM-KII that may have an important role in the physiological regulation of this key protein kinase.
Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII
Authors: Frank Peter , Braunshofer-Reiter Christa , Wintersberger Ulrike , Grimm Rudolf , Büsen Werner
Published: July 2002

Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in
Escherichia coli
leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from
E. coli
, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.
An HP1-like protein is missing from transcriptionally silent micronuclei of
Tetrahymena
Authors: Huang Hui , Wiley Emily A. , Lending Craig R. , Allis C. David
Published: July 2002

We report the identification and cloning of a 28-kDa polypeptide (p28) in
Tetrahymena
macronuclei that shares several features with the well studied heterochromatin-associated protein HP1 from
Drosophila
. Notably, like HP1, p28 contains both a chromodomain and a chromoshadow domain. p28 also shares features with linker histone H1, and like H1, p28 is multiply phosphorylated, at least in part, by a proline-directed, Cdc2-type kinase. As such, p28 is referred to as Hhp1p (for H1/HP1-like protein). Hhp1p is missing from transcriptionally silent micronuclei but is enriched in heterochromatin-like chromatin bodies that presumably comprise repressed chromatin in macronuclei. These findings shed light on the evolutionary conserved nature of heterochromatin in organisms ranging from ciliates to humans and provide further evidence that HP1-like proteins are not exclusively associated with permanently silent chromosomal domains. Our data support a view that members of this family also associate with repressed states of euchromatin.
Efficient translesion replication in the absence of
Escherichia coli
Umu proteins and 3′–5′ exonuclease proofreading function
Authors: Vandewiele Dominique , Borden Angela , O’Grady Paul I. , Woodgate Roger , Lawrence Christopher W.
Published: July 2002

Translesion replication (TR) past a cyclobutane pyrimidine dimer in
Escherichia coli
normally requires the UmuD′
2
C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3′–5′ exonuclease proofreading activity of the pol III ɛ-subunit also is disabled. TR was measured in isogenic
uvrA6
Δ
umuDC
strains carrying the dominant negative
dnaQ
allele,
mutD5
, or Δ
dnaQ spq
-
2
mutations by transfecting them with single-stranded M13-based vectors containing a specifically located
cis
-
syn
T–T dimer. As expected, little TR was observed in the Δ
umuDC dnaQ
+
strain. Surprisingly, 26% TR occurred in UV-irradiated Δ
umuDC mutD5
cells, one-half the frequency found in a

uvrA6 umuDC
+

mutD5 strain.
lexA3
(Ind
−
) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for ≈70% of the TR, and another LexA-independent, responsible for the remaining ≈30%. Curiously, the Δ
umuDC
Δ
dnaQ spq
-
2
strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the
spq
-
2
allele, a
dnaE
mutation required for viability in Δ
dnaQ
strains, since introduction of
spq
-
2
into the Δ
umuDC mutD5
strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995)
J. Bacteriol.
177, 6041–6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the Δ
umuDC mutD5
strain is unchanged by introduction of a Δ
polB
mutation.
Identification of the
Meg1
/
Grb10
imprinted gene on mouse proximal chromosome 11, a candidate for the Silver–Russell syndrome gene
Authors: Miyoshi Naoki , Kuroiwa Yoshimi , Kohda Takashi , Shitara Hiroshi , Yonekawa Hiromichi , Kawabe Tohru , Hasegawa Hideaki , Barton Sheilla C. , Surani M. Azim , Kaneko-Ishino Tomoko , Ishino Fumitoshi
Published: July 2002

In a systematic screen for maternally expressed imprinted genes using subtraction hybridization with androgenetic and normal fertilized mouse embryos, seven candidate maternally expressed genes (
Megs
) have been isolated, including the
H19
and
p57

Kip2

genes that are known to be maternally expressed. Herein, we demonstrate that an imprinted gene,
Meg1
, is apparently identical to
Grb10
(growth factor receptor-bound protein 10), which is located on mouse proximal chromosome 11. Grb10 protein was reported to bind to the insulin receptor and/or the insulin-like growth factor (IGF) I receptor via its src homology 2 domain and to inhibit the associated tyrosine kinase activity that is involved in the growth promoting activities of insulin and IGFs (IGF-I and -II). Thus, it is probable that
Meg1
/
Grb10
is responsible for the imprinted effects of prenatal growth retardation or growth promotion caused by maternal or paternal duplication of proximal chromosome 11 with reciprocal deficiencies (MatDp.prox11 or PatDp.prox11), respectively. In the human, it has been reported that the maternal uniparental disomy 7 is responsible for the Silver–Russell syndrome (SRS) whose effects include pre- and postnatal growth retardation and other dysmorphologies. The human homologue
GRB10
on chromosome 7q11.2–12 is a candidate gene for Silver–Russell syndrome.
Basic fibroblast growth factor induces cell migration and proliferation after glia-specific gene transfer in mice
Authors: Holland Eric C. , Varmus Harold E.
Published: July 2002

Basic fibroblast growth factor (bFGF) is overexpressed in most high-grade human gliomas, implying that it is involved in the pathogenesis of these tumors. To assess the biological effect of inappropriate production of bFGF in normal astrocytes, we developed a system for glia-specific gene transfer in transgenic mice. A transgene encoding the receptor for subgroup A avian leukosis virus and controlled by the astrocyte-specific glial fibrillary acidic protein promoter permits efficient glia-specific transfer of genes carried by subgroup A avian leukosis virus vectors. With this system, we have demonstrated that bFGF induces proliferation and migration of glial cells
in vivo,
without the induction of tumors.
Both allelic variation and expression of nuclear and cytoplasmic transcripts of
Hsr-omega
are closely associated with thermal phenotype in
Drosophila
Authors: McKechnie Stephen W. , Halford Michael M. , McColl Gawain , Hoffmann Ary A.
Published: July 2002

Inducible heat shock genes are considered a major component of the molecular mechanisms that confer cellular protection against a variety of environmental stresses, in particular high temperature extremes. We have tested the association between expression of the heat shock RNA gene
hsr-omega
and thermoresistance by generating thermoresistant lines of
Drosophila melanogaster
after application of two distinct regimes of laboratory selection. One set of lines was selected for resistance to knockdown by heat stress and the other was similarly selected but before selection a mild heat exposure known to increase resistance (heat hardening) was applied. A cross between resistant and susceptible lines confirmed our earlier observation that increased thermal tolerance cosegregates with allelic variation in the
hsr-omega
gene. This cosegregating variation is attributed largely to two haplotype groups. Using quantitative reverse transcription–PCR, we find evidence for divergent phenotypic responses in the two selection regimes, involving both structural and regulatory changes in
hsr-omega
. Lines selected after hardening showed increased levels of the cytoplasmic transcript but decreased levels of the nuclear transcript. Lines selected without hardening showed decreased levels of the cytoplasmic transcript. The allelic frequency changes at
hsr-omega
could not by themselves account for the altered transcription patterns. Our results support the idea that the functional RNA molecules transcribed from
hsr-omega
are an important and polymorphic regulatory component of an insect thermoresistance phenotype.
Characterization of a human
RPD3
ortholog, HDAC3
Authors: Emiliani Stephane , Fischle Wolfgang , Van Lint Carine , Al-Abed Yousef , Verdin Eric
Published: July 2002

Histone acetylation levels in cells result from a dynamic equilibrium between competing histone acetylases and deacetylases. Changes in histone acetylation levels occur during both transcriptional activation and silencing. Cloning of the cDNA for a human histone deacetylase (HDAC1) has shown that it represents a human ortholog of the yeast transcriptional regulator
RPD3
. We have screened the expressed sequence tag database (National Center for Biotechnology Information) with the yeast RPD3 sequence and identified a human ortholog of
RPD3
, HDAC3. This cDNA encodes a protein of 428 amino acids with 58% sequence identity with HDAC1p. By using a specific polyclonal antiserum recognizing the C-terminal domain of HDAC3p and Western blotting, we detected a single ∼49-kDa band in several tumor cell lines. HDAC3p is expressed predominantly in the nuclear compartment. Immunoprecipitation experiments with either an antiserum against HDAC3p or an anti-FLAG antiserum and a flagged HDAC3 cDNA showed that HDAc3p exhibits deacetylase activity both on free histones and on purified nucleosomes. This deacetylase activity is inhibited by trichostatin, trapoxin, and butyrate
in vitro
to the same degree as the deacetylase activity associated to HDAC1p. These observations identify another member of a growing family of human HDAC genes.
Place cell discharge is extremely variable during individual passes of the rat through the firing field
Authors: Fenton André A. , Muller Robert U.
Published: July 2002

The idea that the rat hippocampus stores a map of space is based on
the existence of “place cells” that show
“location-specific” firing. The discharge of place cells is
confined with remarkable precision to a cell-specific part of the
environment called the cell’s “firing field.” We demonstrate
here that firing is not nearly as reliable in the time domain as in the
positional domain. Discharge during passes through the firing field was
compared with a model with Poisson variance of the location-specific
firing determined by the time-averaged positional firing rate
distribution. Place cells characteristically fire too little or too
much compared with expectations from the random model. This fundamental
property of place cells is referred to as “excess firing
variance” and has three main implications: (
i
) Place
cell discharge is not only driven by the summation of many small,
asynchronous excitatory synaptic inputs. (
ii
) Place cell
discharge may encode a signal in addition to the current head location.
(
iii
) The excess firing variance helps explain why the
errors in computing the rat’s position from the simultaneous activity
of many place cells are large.
Transcriptional map of 170-kb region at chromosome 11p15.5: Identification and mutational analysis of the
BWR1A
gene reveals the presence of mutations in tumor samples
Authors: Schwienbacher Christine , Sabbioni Silvia , Campi Marco , Veronese Angelo , Bernardi Guido , Menegatti Agnese , Hatada Izuho , Mukai Tsunehiro , Ohashi Hirofumi , Barbanti-Brodano Giuseppe , Croce Carlo M. , Negrini Massimo
Published: July 2002

Chromosome region 11p15.5 harbors unidentified genes involved in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome. The genetic analysis of a 170-kb region at 11p15.5 between loci
D11S601
and
D11S679
resulted in the identification of six transcriptional units. Three genes,
hNAP2
,
CDKN1C
, and
KVLQT1
, are well characterized, whereas three genes are novel. The three additional genes were designated
BWR1A
,
BWR1B
, and
BWR1C
. Full-length cDNAs for these three genes were cloned and nucleotide sequences were determined. While our work was in progress,
BWR1C
cDNA was described as
IPL
[Qian, N., Franck, D., O’Keefe, D., Dao, D., Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. & Tycko, B. (1997)
Hum. Mol. Genet.
6, 2021–2029]. The cloning and mapping of these genes together with the fine mapping of the three known genes indicates that the transcriptional map of this region is likely to be complete. Because this region frequently is altered in neoplasms and in the genetic disease Beckwith–Wiedemann syndrome, we carried out a mutational analysis in tumor cell lines and Beckwith–Wiedemann syndrome samples that resulted in the identification of genetic alterations in the
BWR1A
gene: an insertion that introduced a stop codon in the breast cancer cell line BT549 and a point mutation in the rhabdomyosarcoma cell line TE125-T. These results indicate that
BWR1A
may play a role in tumorigenesis.
A tripotential glial precursor cell is present in the developing spinal cord
Authors: Rao Mahendra S. , Noble Mark , Mayer-Pröschel Margot
Published: July 2002

We have isolated a tripotential glial precursor cell population from spinal cords of E13.5 rats.In vitro,these A2B5+E-NCAM−glial-restricted precursor (GRP) cells can undergo extensive self-renewal, and can differentiate into oligodendrocytes and two distinct astrocyte populations, but do not differentiate into neurons. The differentiation potential of GRP cells is retained through at least three cycles of expansion and recloning. Unlike oligodendrocyte-type 2 astrocyte progenitor cells, freshly isolated GRP cells do not respond to platelet-derived growth factor as a mitogen or survival factor, nor do GRP cells differentiate into oligodendrocytes—or even survive—when plated in mitogen-free chemically defined medium. Exposure to fetal calf serum induces GRP cells to differentiate into A2B5−fibroblast-like astrocytes, whereas growth in the presence of basic fibroblast growth factor and ciliary neurotrophic factor induces the generation of A2B5+process-bearing astrocytes. The early appearance of GRP cells during spinal cord development suggests that they may represent the earliest GRP cell population.
Recessive and dominant mutations in the ethylene biosynthetic gene
ACS
5
of
Arabidopsis
confer cytokinin insensitivity and ethylene overproduction, respectively
Authors: Vogel John P. , Woeste Keith E. , Theologis Athanasios , Kieber Joseph J.
Published: July 2002

We identified a set of cytokinin-insensitive mutants by using a
screen based on the ethylene-mediated triple response observed after
treatment with low levels of cytokinins. One group of these mutants
disrupts
ACS5
, a member of the
Arabidopsis
gene family that encodes
1-aminocyclopropane-1-carboxylate synthase, the first enzyme in
ethylene biosynthesis. The ACS5 isoform is mainly responsible for the
sustained rise in ethylene biosynthesis observed in response to low
levels of cytokinin and appears to be regulated primarily by a
posttranscriptional mechanism. Furthermore, the dominant
ethylene-overproducing mutant
eto2
was found to be the
result of an alteration of the carboxy terminus of
ACS5
,
suggesting that this domain acts as a negative regulator of ACS5
function.
Membrane voltage initiates Ca
2+
waves and potentiates Ca
2+
increases with abscisic acid in stomatal guard cells
Authors: Grabov Alexander , Blatt Michael R.
Published: July 2002

In higher plants changes and oscillations in cytosolic free Ca
2+
concentration ([Ca
2+
]
i
) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca
2+
]
i
are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca
2+
]
i
to voltage and its interaction with ABA. We recorded [Ca
2+
]
i
by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact
Vicia
guard cells. Free-running oscillations between voltages near −50 mV and −200 mV were associated with oscillations in [Ca
2+
]
i
, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca
2+
]
i
to increase, often in excess of 1 μM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca
2+
]
i
that spread centripetally from the peripheral cytoplasm within 5–10 s and relaxed over 40–60 s thereafter. The [Ca
2+
]
i
increases showed a voltage threshold near −120 mV and were sensitive to external Ca
2+
concentration. Substituting Mn
2+
for Ca
2+
to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca
2+
]
i
rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca
2+
]
i
. These results demonstrate a capacity for voltage to evoke [Ca
2+
]
i
increases, they point to a dual interaction with ABA in triggering and propagating [Ca
2+
]
i
increases, and they implicate a role for voltage in “conditioning” [Ca
2+
]
i
signals that regulate ion channels for stomatal function.
Pneumocysterol [(24
Z
)-ethylidenelanost-8-en-3β-ol], a rare sterol detected in the opportunistic pathogen
Pneumocystis carinii hominis
: Structural identity and chemical synthesis
Authors: Kaneshiro Edna S. , Amit Zunika , Swonger Mardie M. , Kreishman George P. , Brooks Elwood E. , Kreishman Mara , Jayasimhulu Koka , Parish Edward J. , Sun Hang , Kizito Stephen A. , Beach David H.
Published: July 2002

Pneumocystis carinii
pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus,
P. carinii
lacks ergosterol. Instead, the pathogen synthesizes a number of distinct Δ
7
, 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C
32
sterol were found in human-derived
P. carinii hominis
. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived
P. carinii carinii
, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C
31
and C
32
sterols were identified as euphorbol (24-methylenelanost-8-en-3β-ol) and pneumocysterol [(24
Z
)-ethylidenelanost-8-en-3β-ol], respectively. The identification of these and other 24-alkylsterols in
P. carinii hominis
suggests that (
i
) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (
ii
) 24-alkylsterols are important components of the pathogen’s membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (
iii
) the inefficacy of azole drugs against
P. carinii
can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in
P. carinii
are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.
p27 and Rb are on overlapping pathways suppressing tumorigenesis in mice
Authors: Park Michele S. , Rosai Juan , Nguyen Hai T. , Capodieci Paola , Cordon-Cardo Carlos , Koff Andrew
Published: July 2002

The commitment of cells to replicate and divide correlates with the activation of cyclin-dependent kinases and the inactivation of Rb, the product of the retinoblastoma tumor suppressor gene. Rb is a target of the cyclin-dependent kinases and, when phosphorylated, is inactivated. Biochemical studies exploring the nature of the relationship between cyclin-dependent kinase inhibitors and Rb have supported the hypothesis that these proteins are on a linear pathway regulating commitment. We have been able to study this relationship by genetic means by examining the phenotype of
Rb
+/−
p27
−/− mice. Tumors arise from the intermediate lobe cells of the pituitary gland in
p27
−/− mice, as well as in
Rb
+/− mice after loss of the remaining wild-type allele of Rb. Using these mouse models, we examined the genetic interaction between Rb and p27. We found that the development of pituitary tumors in
Rb
+/− mice correlated with a reduction in p27 mRNA and protein expression. To determine whether the loss of p27 was an indirect consequence of tumor formation or a contributing factor to the development of this tumor, we analyzed the phenotype of
Rb
+/−
p27
−/− mice. We found that these mice developed pituitary adenocarcinoma with loss of the remaining wild-type allele of Rb and a high-grade thyroid C cell carcinoma that was more aggressive than the disease in either
Rb
+/− or
p27
−/− mice. Importantly, we detected both pituitary and thyroid tumors earlier in the
Rb
+/−
p27
−/− mice. We therefore propose that Rb and p27 cooperate to suppress tumor development by integrating different regulatory signals.
Broad tumor-associated expression and recognition by tumor-derived γδ T cells of MICA and MICB
Authors: Groh Veronika , Rhinehart Rebecca , Secrist Heather , Bauer Stefan , Grabstein Kenneth H. , Spies Thomas
Published: July 2002

Human MHC class I-related molecules, MICA and MICB, are stress-induced antigens that are recognized by a subset of γδ T cells expressing the variable region Vδ1. This functional association has been found to be limited to intestinal epithelium, where these T cells are prevalent and where MICA and, presumably, MICB are mainly expressed. However, increased frequencies of Vδ1 γδ T cells have been observed in various epithelial tumors; moreover, MICA/B are expressed on diverse cultured epithelial tumor cells. With freshly isolated tumor specimens, expression of MICA/B was documented in many, but not all, carcinomas of the lung, breast, kidney, ovary, prostate, and colon. In tumors that were positive for MICA/B, the frequencies of Vδ1 γδ T cells were significantly higher than in those that were negative. Vδ1 γδ T cell lines and clones derived from different tumors recognized MICA/B on autologous and heterologous tumor cells. In accord with previous evidence, no constraints were observed in these interactions, such as those imposed by specific peptide ligands. Thus, MICA/B are tumor-associated antigens that can be recognized, in an apparently unconditional manner, by a subset of tumor-infiltrating γδ T cells. These results raise the possibility that an induced expression of MICA/B, by conditions that may be related to tumor homeostasis and growth, could play a role in immune responses against tumors.
A structural snapshot of base-pair opening in DNA
Authors: van Aalten Daan M. F. , Erlanson Daniel A. , Verdine Gregory L. , Joshua-Tor Leemor
Published: July 2002

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-Å crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)
2
with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the
Hae
III methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine.
Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin α6β1: Implications for murine fertilization
Authors: Chen Michellee S. , Tung Kenneth S. K. , Coonrod Scott A. , Takahashi Yuji , Bigler Dora , Chang Alice , Yamashita Yoshio , Kincade Paul W. , Herr John C. , White Judith M.
Published: July 2002

CD9 is a tetraspan protein that associates with several β1 integrins, including α6β1. Because α6β1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin β), we first asked whether CD9 is present on murine eggs and whether it functions in sperm–egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm–egg binding and fusionin vitroin a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin β disintegrin domain. (Both ligands bind to the egg via α6β1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin α6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, α6A and CD9 both are expressed on the apical epithelial surface at the uterine–oviduct junction. These findings correlate with the observation that fertilin β “knockout” sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin β−/−male mice. Our results suggest that high-avidity binding between fertilin β (ADAM 2) and α6β1 requires cooperation between α6β1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm–egg interactions.
Drosophila
proteins related to vertebrate DNA (5-cytosine) methyltransferases
Authors: Hung Ming-Shiu , Karthikeyan Narayanan , Huang Bauling , Koo Hshi-Chi , Kiger John , Shen C.-K. James
Published: July 2002

DNA methylation at CpG residues is closely associated with a number of biological processes during vertebrate development. Unlike the vertebrates, however, several invertebrate species, including the
Drosophila
, do not have apparent DNA methylation in their genomes. Nor have there been reports on a DNA (5-cytosine) methyltransferase (CpG MTase) found in these invertebrates. We now present evidence for two CpG MTase-like proteins expressed in
Drosophila
cells. One of these, DmMTR1, is a protein containing peptide epitopes immunologically related to the conserved motifs I and IV in the catalytic domain of the mammalian dnmt1. DmMTR1 has an apparent molecular mass of 220 kDa and, similar to mammalian dnmt1, it also interacts
in vivo
with the proliferating cell nuclear antigen. During interphase of the syncytial
Drosophila
embryos, the DmMTR1 molecules are located outside the nuclei, as is dnmt1 in the mouse blastocyst. However, DmMTR1 appears to be rapidly transported into, and then out of the nuclei again, as the embryos undergo mitotic waves. Immunofluorescent data indicate that DmMTR1 molecules “paint” the whole set of condensed
Drosophila
chromosomes throughout the mitotic phase, suggesting they may play an essential function in the cell-cycle regulated condensation of the
Drosophila
chromosomes. Through search in the genomic database, we also have identified a
Drosophila
polypeptide, DmMT2, that exhibits high sequence homology to the mammalian dnmt2 and the yeast CpG MTase homolog pmt1. The expression of DmMT2 appears to be developmentally regulated. We discuss the evolutionary and functional implications of the discovery of these two
Drosophila
proteins related to mammalian CpG MTases.
Identification of naturally processed and HLA-presented Epstein–Barr virus peptides recognized by CD4+or CD8+T lymphocytes from human blood
Authors: Herr Wolfgang , Ranieri Elena , Gambotto Andrea , Kierstead Lisa Salvucci , Amoscato Andrew A. , Gesualdo Loreto , Storkus Walter J.
Published: July 2002

The broad clinical implementation of cancer vaccines targeting the induction of specific T cell-mediated immunity is hampered because T cell defined tumor-associated peptides are currently available for only a restricted range of tumor types. Current epitope identification strategies requirea priorithe generation of T “indicator” cell lines that specifically recognize the tumor antigenic epitope inin vitroassay systems. An alternative to this strategy is the use of “memory” T cells freshly isolated from the peripheral blood of patients with cancer in concert with sensitive effector cell readout assays (such as the cytokine enzyme-linked immunospot assay) and MS to identify relevant peptide epitopes. In a model system, we have evaluated the capacity of natural Epstein–Barr virus (EBV)-transformed B-lymphoblastoid cell line-extracted peptides to activate “memory” viral-specific CD4+or CD8+T cells freshly isolated from the blood of an EBV-seropositive individual using the IFN-γ enzyme-linked immunospot assay. After HPLC fractionation and loading onto autologous dendritic cells, multiple naturally processed HLA class I and II-associated lymphoblastoid cell line-derived peptides were isolated that were capable of inducing IFN-γ spot production by “memory” T lymphocytes. Using MS analysis on a HPLC fraction recognized by CD8+T cells, we were able to sequence natural 9-, 10-, and 11-mer peptides naturally processed from the latent EBV antigen LMP-2 (latentmembraneprotein-2) and presented in the context of HLA-A2. This approach provides a useful methodology for the future identification of MHC-presented viral and tumor epitopes using freshly isolated patient materials.
Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains
Authors: Chihade Joseph W. , Schimmel Paul
Published: July 2002

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of
Escherichia coli
alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1–2 kcal⋅mol
−1
of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.
Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1
Authors: Kim Young Ho , Choi Cheol Yong , Kim Yongsok
Published: July 2002

Posttranslational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the recently identified family of nuclear protein kinases that act as corepressors for homeodomain transcription factors. Here, we show that HIPK2 is regulated by a ubiquitin-like protein, SUMO-1. We demonstrate that HIPK2 localizes to nuclear speckles (dots) by means of a speckle-retention signal. This speckle-retention signal contains a domain that interacts with a mouse ubiquitin-like protein conjugating (E2) enzyme, mUBC9. In cultured cells, HIPK2 is covalently modified by SUMO-1, and the SUMO-1 modification of HIPK2 correlates with its localization to nuclear speckles (dots). Thus, our results provide firm evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots).
Axon pathology in Parkinson’s disease and Lewy body dementia hippocampus contains α-, β-, and γ-synuclein
Authors: Galvin James E. , Uryu Kunihiro , Lee Virginia M.-Y. , Trojanowski John Q.
Published: July 2002

Pathogenic α-synuclein (αS) gene mutations occur in rare familial Parkinson’s disease (PD) kindreds, and wild-type αS is a major component of Lewy bodies (LBs) in sporadic PD, dementia with LBs (DLB), and the LB variant of Alzheimer’s disease, but β-synuclein (βS) and γ-synuclein (γS) have not yet been implicated in neurological disorders. Here we show that in PD and DLB, but not normal brains, antibodies to αS and βS reveal novel presynaptic axon terminal pathology in the hippocampal dentate, hilar, and CA2/3 regions, whereas antibodies to γS detect previously unrecognized axonal spheroid-like lesions in the hippocampal dentate molecular layer. The aggregation of other synaptic proteins and synaptic vesicle-like structures in the αS- and βS-labeled hilar dystrophic neurites suggests that synaptic dysfunction may result from these lesions. Our findings broaden the concept of neurodegenerative “synucleinopathies” by implicating βS and γS, in addition to αS, in the onset/progression of PD and DLB.
Long-term exposure to high corticosterone levels attenuates serotonin responses in rat hippocampal CA1 neurons
Authors: Karten Y. J. G. , Nair S. M. , van Essen L. , Sibug R. , Joëls M.
Published: July 2002

Recent studies indicated that hyperactivity of the hypothalamo-pituitary-adrenal system is a considerable risk factor for the precipitation of affective disorders, most notably of major depression. The mechanism by which this hyperactivity eventually leads to clinical symptoms of depression is unknown. In the present animal study, we tested one possible mechanism, i.e., that long-term exposure to high corticosterone levels alters functional responses to serotonin in the hippocampus, an important area in the etiology of depression. Rats were injected daily for 3 weeks with a high dose of corticosterone; electrophysiological responses to serotonin were recorded intracellularly from CA1 pyramidal neurons
in vitro
. We observed that daily injections with corticosterone gradually attenuate the membrane hyperpolarization and resistance decrease mediated by serotonin-1A receptors. We next used single-cell antisense RNA amplification from identified CA1 pyramidal neurons to resolve whether the functional deficits in serotonin responsiveness are accompanied by decreased expression levels of the serotonin-1A receptor. It appeared that expression of serotonin-1A receptors in CA1 pyramidal cells is not altered; this result was supported by
in situ
hybridization. Expression of corticosteroid receptors in the same cells, particularly of the high-affinity mineralocorticoid receptor, was significantly reduced after long-term corticosterone treatment. The present findings indicate that prolonged elevation of the corticosteroid concentration, a possible causal factor for major depression in humans, gradually attenuates responsiveness to serotonin without necessarily decreasing serotonin-1A receptor mRNA levels in pyramidal neurons. These functional changes may occur by a posttranscriptional mechanism or by transcriptional regulation of genes other than the serotonin-1A receptor gene itself.
Calcium electrogenesis in distal apical dendrites of layer 5 pyramidal cells at a critical frequency of back-propagating action potentials
Authors: Larkum M. E. , Kaiser K. M. M. , Sakmann B.
Published: July 2002

Action potentials in juvenile and adult rat layer-5 neocortical pyramidal neurons can be initiated at both axonal and distal sites of the apical dendrite. However, little is known about the interaction between these two initiation sites. Here, we report that layer 5 pyramidal neurons are very sensitive to a critical frequency of back-propagating action potentials varying between 60 and 200 Hz in different neurons. Bursts of four to five back-propagating action potentials above the critical frequency elicited large regenerative potentials in the distal dendritic initiation zone. The critical frequency had a very narrow range (10–20 Hz), and the dendritic regenerative activity led to further depolarization at the soma. The dendritic frequency sensitivity was suppressed by blockers of voltage-gated calcium channels, and also by synaptically mediated inhibition. Calcium-fluorescence imaging revealed that the site of largest transient increase in intracellular calcium above the critical frequency was located 400–700 μm from the soma at the site for initiation of calcium action potentials. Thus, the distal dendritic initiation zone can interact with the axonal initiation zone, even when inputs to the neuron are restricted to regions close to the soma, if the output of the neuron exceeds a critical frequency.
Posttranslational modification of Gα
o1
generates Gα
o3
, an abundant G protein in brain
Authors: Exner Torsten , Jensen Ole N. , Mann Matthias , Kleuss Christiane , Nürnberg Bernd
Published: July 2002

Gα
o
, the most abundant G protein in mammalian brain,
occurs at least in two subforms, i.e., Gα
o1
and
Gα
o2
, derived by alternative splicing of the mRNA. A
third Gα
o1
-related isoform, Gα
o3
, has been
purified, representing about 30% of total G
o
in brain.
Initial studies revealed distinct biochemical properties of
Gα
o3
as compared with other Gα
o
isoforms.
In matrix-assisted laser desorption/ionization peptide mass mapping
of gel-isolated Gα
o1
and Gα
o3
, C-terminal
peptides showed a difference of +1 Da for Gα
o3
.
Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp
instead of an Asn at position 346 of Gα
o3
. Gel
electrophoretic analysis of recombinant Gα
o3
showed the
same mobility as native Gα
o3
but distinct to
Gα
o1
. The conversion of
346
Asn→Asp changed
the signaling properties, including the velocity of the basal guanine
nucleotide-exchange reaction, which points to the involvement of the C
terminus in basal guanosine 5′-[γ-thio]triphosphate binding. No
cDNA coding for Gα
o3
was detected, suggesting an
enzymatic deamidation of Gα
o1
by a yet-unidentified
activity. Therefore, Gα heterogeneity is generated not only at the
DNA or RNA levels, but also at the protein level. The relative amount
of Gα
o1
and Gα
o3
differed from cell type to
cell type, indicating an additional principle of G protein regulation.
Histone modification governs the cell cycle regulation of a replication-independent chromatin assembly pathway in
Saccharomyces cerevisiae
Authors: Altheim Brent A. , Schultz Michael C.
Published: July 2002

We describe a replication-independent, cell cycle-regulated
chromatin assembly pathway in budding yeast. The activity of this
pathway is low in S phase extracts but is very high in G
2
,
M, and G
1
cell extracts, with peak activity in late
M/early G
1
. The cell cycle regulation of this pathway
requires a specific pattern of posttranslational modification of
histones H3 and/or H4, which is distinct for H3/H4 present in S
phase versus M and G
1
phase cell extracts. Histone H3/H4
modification is therefore important for the reciprocal control of
replication-dependent and -independent chromatin assembly pathways
during the cell cycle.
Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice
Authors: Gomeza Jesus , Shannon Harlan , Kostenis Evi , Felder Christian , Zhang Lu , Brodkin Jesse , Grinberg Alexander , Sheng Hui , Wess Jürgen
Published: July 2002

Members of the muscarinic acetylcholine receptor family (M1–M5)
are known to be involved in a great number of important central and
peripheral physiological and pathophysiological processes. Because of
the overlapping expression patterns of the M1–M5 muscarinic receptor
subtypes and the lack of ligands endowed with sufficient subtype
selectivity, the precise physiological functions of the individual
receptor subtypes remain to be elucidated. To explore the physiological
roles of the M2 muscarinic receptor, we have generated mice lacking
functional M2 receptors by using targeted mutagenesis in mouse
embryonic stem cells. The resulting mutant mice were analyzed in
several behavioral and pharmacologic tests. These studies showed that
the M2 muscarinic receptor subtype, besides its well documented
involvement in the regulation of heart rate, plays a key role in
mediating muscarinic receptor-dependent movement and temperature
control as well as antinociceptive responses, three of the most
prominent central muscarinic effects. These results offer a rational
basis for the development of novel muscarinic drugs.
Molecular basis for semidominance of missense mutations in the XANTHA-H (42-kDa) subunit of magnesium chelatase
Authors: Hansson Andreas , Kannangara C. Gamini , von Wettstein Diter , Hansson Mats
Published: July 2002

During biosynthesis of bacteriochlorophyll or chlorophyll, three
protein subunits of 140, 70, and 42 kDa interact to insert
Mg
2+
into protoporphyrin IX. The semidominant
Chlorina-125
,
-157
, and
-161
mutants in barley are deficient in this step and
accumulate protoporphyrin IX after feeding on 5-aminolevulinate.
Chlorina-125
,
-157
, and
-161
are allelic to the recessive
xantha-h
mutants and contain G559A, G806A, and C271T
mutations, respectively. These mutations cause single amino acid
substitutions in residues that are conserved in all known primary
structures of the 42-kDa subunit.
In vitro
complementation and reconstitution of Mg-chelatase activity show that
the 42-kDa subunits are defective in the semidominant
Chlorina
mutants. A mutated protein is maintained in the
Chlorina
plastids, unlike in the
xantha–h
plastids. Heterozygous
Chlorina
seedlings have 25–50% of the Mg-chelatase activity of wild-type
seedlings. Codominant expression of active and inactive 42-kDa subunits
in heterozygous
Chlorina
seedlings is likely to produce
two types of heterodimers between the strongly interacting 42-kDa and
70-kDa subunits. Reduced Mg-chelatase activity is explained by the
capacity of heterodimers consisting of mutated 42-kDa and wild-type
70-kDa protein to bind to the 140-kDa subunit. The 42-kDa subunit is
similar to chaperones that refold denatured polypeptides with respect
to its ATP-to-ADP exchange activity and its ability to generate ATPase
activity with the 70-kDa subunit. We hypothesize that the association
of the 42-kDa subunit with the 70-kDa subunit allows them to form a
specific complex with the 140-kDa subunit and that this complex inserts
Mg
2+
into protoporphyrin IX.