bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunogene therapy of tumors with vaccine based onXenopushomologous vascular endothelial growth factor as a model antigen
Authors: Wei Yu-quan , Huang Mei-juan , Yang Li , Zhao Xia , Tian Ling , Lu You , Shu Jing-mei , Lu Chong-jiu , Niu Ting , Kang Bin , Mao Yun-qiu , Liu Fen , Wen Yan-jun , Lei Shong , Luo Feng , Zhou Li-qun , Peng Feng , Jiang Yu , Liu Ji-yan , Zhou Hao , Wang Qing-ru , He Qiu-ming , Xiao Fei , Lou Yan-yan , Xie Xing-jiang , Li Qiu , Wu Yang , Ding Zhen-yu , Hu Bin , Hu Min , Zhang Wei
Published: July 2002

Overcoming immune tolerance of the growth factors associated with tumor growth should be a useful approach to cancer therapy by active immunity. We used vascular endothelial growth factor (VEGF) as a model antigen to explore the feasibility of the immunogene tumor therapy with a vaccine based on a single xenogeneic homologous gene, targeting the growth factors associated with angiogenesis. To test this concept, we constructed a plasmid DNA encodingXenopushomologous VEGF (XVEGF-p) and control vectors. We found that immunogene tumor therapy with a vaccine based on XVEGF was effective at both protective and therapeutic antitumor immunity in several tumor models in mice. VEGF-specific autoantibodies in sera of mice immunized with XVEGF-p could be found in Western blotting analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGF-mediated endothelial cell proliferationin vitro, and at antitumor activity and the inhibition of angiogenesis by adoptive transferin vivo. The elevation of VEGF in the sera of the tumor-bearing mice could be abrogated with XVEGF-p immunization. The antitumor activity and production of VEGF-specific autoantibodies, significantly elevated IgG1 and IgG2b, could be abrogated by the depletion of CD4+T lymphocytes. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factors associated with tumor growth in a cross reaction with single xenogeneic homologous gene and may be of importance in the further exploration of the applications of other xenogeneic homologous genes identified in human and other animal genome sequence projects in cancer therapy.
High-affinity ouabain binding by a chimeric gastric H
+
,K
+
-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α
1
-subunit of rat Na
+
,K
+
-ATPase
Authors: Koenderink Jan B. , Hermsen Harm P. H. , Swarts Herman G. P. , Willems Peter H. G. M. , De Pont Jan Joep H. H. M.
Published: July 2002

Na
+
,K
+
-ATPase and gastric
H
+
,K
+
-ATPase are two related enzymes that are
responsible for active cation transport.
Na
+
,K
+
-ATPase activity is inhibited
specifically by ouabain, whereas H
+
,K
+
-ATPase
is insensitive to this drug. Because it is not known which parts of the
catalytic subunit of Na
+
,K
+
-ATPase are
responsible for ouabain binding, we prepared chimeras in which small
parts of the α-subunit of H
+
,K
+
-ATPase
were replaced by their counterparts of the α
1
-subunit of
rat Na
+
,K
+
-ATPase. A chimeric enzyme in which
transmembrane segments 5 and 6 of H
+
,K
+
-ATPase
were replaced by those of Na
+
,K
+
-ATPase could
form a phosphorylated intermediate, but hardly showed a
K
+
-stimulated dephosphorylation reaction. When
transmembrane segments 3 and 4 of Na
+
,K
+
-ATPase
were also included in this chimeric ATPase, K
+
-stimulated
dephosphorylation became apparent. This suggests that there is a direct
interaction between the hairpins M3-M4 and M5-M6. Remarkably, this
chimeric enzyme, HN34/56, had obtained a high-affinity
ouabain-binding site, whereas the rat
Na
+
,K
+
-ATPase, from which the hairpins
originate, has a low affinity for ouabain. The low affinity of the rat
Na
+
,K
+
-ATPase previously had been attributed to
the presence of two charged amino acids in the extracellular domain
between M1 and M2. In the HN34/56 chimera, the M1/M2 loop, however,
originates from H
+
,K
+
-ATPase, which has two
polar uncharged amino acids on this position. Placement of two charged
amino acids in the M1/M2 loop of chimera HN34/56 results in a
decreased ouabain affinity. This indicates that although the M1/M2
loop affects the ouabain affinity, binding occurs when the M3/M4 and
M5/M6 hairpins of Na
+
,K
+
-ATPase are present.
Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione
S
-transferase Pi
Authors: Henderson Colin J. , Wolf C. Roland , Kitteringham Neil , Powell Helen , Otto Diana , Park B. Kevin
Published: July 2002

Overdose of acetaminophen, a widely used analgesic drug, can result
in severe hepatotoxicity and is often fatal. This toxic reaction is
associated with metabolic activation by the P450 system to form a
quinoneimine metabolite,
N
-acetyl-
p
-benzoquinoneimine (NAPQI),
which covalently binds to proteins and other macromolecules to cause
cellular damage. At low doses, NAPQI is efficiently detoxified,
principally by conjugation with glutathione, a reaction catalyzed in
part by the glutathione
S
-transferases (GST), such as
GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we
examined acetaminophen metabolism and liver damage in mice nulled for
GstP (
GstP1/P2
(−/−)
). Contrary to our
expectations, instead of being more sensitive, GstP null mice were
highly resistant to the hepatotoxic effects of this compound. No
significant differences between wild-type
(
GstP1/P2
(+/+)
) mice and
GstP1/P2
(−/−)
nulls in either the rate
or route of metabolism, particularly to glutathione conjugates, or in
the levels of covalent binding of acetaminophen-reactive metabolites to
cellular protein were observed. However, although a similar rapid
depletion of hepatic reduced glutathione (GSH) was found in both
GstP1/P2
(+/+)
and
GstP1/P2
(−/−)
mice, GSH levels only
recovered in the
GstP1/P2
(−/−)
mice.
These data demonstrate that GstP does not contribute
in
vivo
to the formation of glutathione conjugates of
acetaminophen but plays a novel and unexpected role in the toxicity of
this compound. This study identifies new ways in which GST can modulate
cellular sensitivity to toxic effects and suggests that the level of
GST Pi may be an important and contributing factor in the sensitivity
of patients with acetaminophen-induced hepatotoxicity.
Probing the open state of cytochrome P450cam with ruthenium-linker substrates
Authors: Dunn Alexander R. , Dmochowski Ivan J. , Bilwes Alexandrine M. , Gray Harry B. , Crane Brian R.
Published: July 2002

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-Å deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.
Activation of the Akt-related cytokine-independent survival kinase requires interaction of its phox domain with endosomal phosphatidylinositol 3-phosphate
Authors: Virbasius Joseph V. , Song Xi , Pomerleau Darcy P. , Zhan Yong , Zhou G. Wayne , Czech Michael P.
Published: July 2002

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3′-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.
Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees
Authors: Forns Xavier , Thimme Robert , Govindarajan Sugantha , Emerson Suzanne U. , Purcell Robert H. , Chisari Francis V. , Bukh Jens
Published: July 2002

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the ΔHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with ΔHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The ΔHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.
Attractive and defensive functions of the ultraviolet pigments of a flower (
Hypericum calycinum
)
Authors: Gronquist Matthew , Bezzerides Alexander , Attygalle Athula , Meinwald Jerrold , Eisner Maria , Eisner Thomas
Published: July 2002

The flower of
Hypericum calycinum
, which appears uniformly yellow to humans, bears a UV pattern, presumably visible to insects. Two categories of pigments, flavonoids and dearomatized isoprenylated phloroglucinols (DIPs), are responsible for the UV demarcations of this flower. Flavonoids had been shown previously to function as floral UV pigments, but DIPs had not been demonstrated to serve in that capacity. We found the DIPs to be present in high concentration in the anthers and ovarian wall of the flower, suggesting that the compounds also serve in defense. Indeed, feeding tests done with one of the DIPs (hypercalin A) showed the compound to be deterrent and toxic to a caterpillar (
Utetheisa ornatrix
). The possibility that floral UV pigments fulfill both a visual and a defensive function had not previously been contemplated. DIPs may also serve for protection of female reproductive structures in other plants, for example in hops (
Humulus lupulus
). The DIPs of hops are put to human use as bitter flavoring agents and preservatives in beer.
Expanded polyglutamines in
Caenorhabditis elegans
cause axonal abnormalities and severe dysfunction of PLM mechanosensory neurons without cell death
Authors: Parker J. Alex , Connolly John B. , Wellington Cheryll , Hayden Michael , Dausset Jean , Neri Christian
Published: July 2002

Huntington's disease (HD) is a dominant neurodegenerative disease caused by polyglutamine (polyQ) expansion in the protein huntingtin (htt). HD pathogenesis appears to involve the production of mutated N-terminal htt, cytoplasmic and nuclear aggregation of htt, and abnormal activity of htt interactor proteins essential to neuronal survival. Before cell death, neuronal dysfunction may be an important step of HD pathogenesis. To explore polyQ-mediated neuronal toxicity, we expressed the first 57 amino acids of human htt containing normal [19 Gln residues (Glns)] and expanded (88 or 128 Glns) polyQ fused to fluorescent marker proteins in the six touch receptor neurons of
Caenorhabditis elegans
. Expanded polyQ produced touch insensitivity in young adults. Noticeably, only 28 ± 6% of animals with 128 Glns were touch sensitive in the tail, as mediated by the PLM neurons. Similar perinuclear deposits and faint nuclear accumulation of fusion proteins with 19, 88, and 128 Glns were observed. In contrast, significant deposits and morphological abnormalities in PLM cell axons were observed with expanded polyQ (128 Glns) and partially correlated with touch insensitivity. PLM cell death was not detected in young or old adults. These animals indicate that significant neuronal dysfunction without cell death may be induced by expanded polyQ and may correlate with axonal insults, and not cell body aggregates. These animals also provide a suitable model to perform
in vivo
suppression of polyQ-mediated neuronal dysfunction.
Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis
Authors: Fiorucci Stefano , Mencarelli Andrea , Palazzetti Barbara , Distrutti Eleonora , Vergnolle Nathalie , Hollenberg Morley D. , Wallace John L. , Morelli Antonio , Cirino Giuseppe
Published: July 2002

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH2, a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH2, whereas the scramble control peptide, LSIGRL-NH2, was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH2, but not the scramble peptide, directly inhibited IFN-γ secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.
The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes
Authors: Zak Elena , Norling Birgitta , Maitra Radhashree , Huang Fang , Andersson Bertil , Pakrasi Himadri B.
Published: July 2002

During oxygenic photosynthesis in cyanobacteria and chloroplasts of plants and eukaryotic algae, conversion of light energy to biologically useful chemical energy occurs in the specialized thylakoid membranes. Light-induced charge separation at the reaction centers of photosystems I and II, two multisubunit pigment-protein complexes in the thylakoid membranes, energetically drive sequential photosynthetic electron transfer reactions in this membrane system. In general, in the prokaryotic cyanobacterial cells, the thylakoid membrane is distinctly different from the plasma membrane. We have recently developed a two-dimensional separation procedure to purify thylakoid and plasma membranes from the genetically widely studied cyanobacterium
Synechocystis
sp. PCC 6803. Immunoblotting analysis demonstrated that the purified plasma membrane contained a number of protein components closely associated with the reaction centers of both photosystems. Moreover, these proteins were assembled in the plasma membrane as chlorophyll-containing multiprotein complexes, as evidenced from nondenaturing green gel and low-temperature fluorescence spectroscopy data. Furthermore, electron paramagnetic resonance spectroscopic analysis showed that in the partially assembled photosystem I core complex in the plasma membrane, the P700 reaction center was capable of undergoing light-induced charge separation. Based on these data, we propose that the plasma membrane, and not the thylakoid membrane, is the site for a number of the early steps of biogenesis of the photosynthetic reaction center complexes in these cyanobacterial cells.
Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa
Authors: Kusakabe Hirokazu , Szczygiel Monika A. , Whittingham David G. , Yanagimachi Ryuzo
Published: July 2002

Chromosome stability was maintained in mouse spermatozoa after freeze-drying or freezing without cryoprotection in a simple Tris⋅HCl buffer containing EGTA (50 mM) and NaCl (50 mM). The ability of spermatozoa to activate oocytes spontaneously was not destroyed by freeze-drying or freezing without cryoprotection in this solution. Embryos derived after injecting oocytes with sperm heads from rehydrated freeze-dried and from thawed spermatozoa developed normally. Provided the DNA integrity of the sperm nucleus is maintained, embryos can be generated by the intracytoplasmic sperm injection technique (ICSI) from severely damaged spermatozoa that are no longer capable of normal physiological activity. This procedure was effective for preserving spermatozoa from strains (C57BL/6J, 129/SvJ, and BALB/c) in which the fertility of spermatozoa frozen conventionally is extremely poor. The technique provides an effective means of storing mouse spermatozoa from many different inbred, mutant, and transgenic strains for biomedical research.
Responses of python gastrointestinal regulatory peptides to feeding
Authors: Secor Stephen M. , Fehsenfeld Drew , Diamond Jared , Adrian Thomas E.
Published: July 2002

In the Burmese python (
Python molurus
), the rapid up-regulation of gastrointestinal (GI) function and morphology after feeding, and subsequent down-regulation on completing digestion, are expected to be mediated by GI hormones and neuropeptides. Hence, we examined postfeeding changes in plasma and tissue concentrations of 11 GI hormones and neuropeptides in the python. Circulating levels of cholecystokinin (CCK), glucose-dependent insulinotropic peptide (GIP), glucagon, and neurotensin increase by respective factors of 25-, 6-, 6-, and 3.3-fold within 24 h after feeding. In digesting pythons, the regulatory peptides neurotensin, somatostatin, motilin, and vasoactive intestinal peptide occur largely in the stomach, GIP and glucagon in the pancreas, and CCK and substance P in the small intestine. Tissue concentrations of CCK, GIP, and neurotensin decline with feeding. Tissue distributions and molecular forms (as determined by gel-permeation chromatography) of many python GI peptides are similar or identical to those of their mammalian counterparts. The postfeeding release of GI peptides from tissues, and their concurrent rise in plasma concentrations, suggests that they play a role in regulating python-digestive responses. These large postfeeding responses, and similarities of peptide structure with mammals, make pythons an attractive model for studying GI peptides.
Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea
Authors: Fabarius Alice , Willer Andreas , Yerganian George , Hehlmann Ruediger , Duesberg Peter
Published: July 2002

Aneuploidy is ubiquitous in cancer, and its phenotypes are inevitably dominant and abnormal. In view of these facts we recently proposed that aneuploidy is sufficient for carcinogenesis generating cancer-specific aneusomies via a chain reaction of autocatalytic aneuploidizations. According to this hypothesis a carcinogen initiates carcinogenesis via a random aneuploidy. Aneuploidy then generates transformation stage-specific aneusomies and further random aneusomies autocatalytically, because it renders chromosome segregation and repair mechanisms error-prone. The hypothesis predicts that several specific aneusomies can cause the same cancers, because several chromosomes also cooperate in normal differentiation. Here we describe experiments on the Chinese hamster (CH) that confirm this hypothesis. (
i
) Random aneuploidy was detected before transformation in up to 90% of CH embryo cells treated with the carcinogen nitrosomethylurea (NMU). (
ii
) Several specific aneusomies were found in 70–100% of the aneuploid cells from colonies transformed with NMU
in vitro
and from tumors generated by NMU-transformed cells in syngeneic animals. Among the aneuploid
in vitro
transformed cells, 79% were trisomic for chromosome 3, and 59% were monosomic for chromosome 10, compared with 8% expected for random distribution of any aneusomy among the 12 CH chromosomes. Moreover, 52% shared both trisomy 3 and monosomy 10 compared with 0.6% expected for random distribution of any two aneusomies. Among the tumor cells, 65% were trisomic for chromosome 3, 51% were trisomic for chromosome 5, and 30% shared both trisomies. Aneuploid cells without these specific aneusomies may contain minor transformation-specific aneusomies or may be untransformed. (
iii
) Random aneusomies and structurally altered chromosomes increased with the generations of transformed cells to the point where their origins became unidentifiable in tumors. We conclude that specific aneusomies are necessary for carcinogenesis.
Recombinational repair of gaps in DNA is asymmetric in Ustilago maydis and can be explained by a migrating D-loop model.
Authors: Ferguson D O , Holloman W K
Published: July 2002

Recombinational repair of double-stranded DNA gaps was investigated in Ustilago maydis. The experimental system was designed for analysis of repair of an autonomously replicating plasmid containing a cloned gene disabled by an internal deletion. It was discovered that crossing over rarely accompanied gap repair. The strong bias against crossing over was observed in three different genes regardless of gap size. These results indicate that gap repair in U. maydis is unlikely to proceed by the mechanism envisioned in the double-stranded break repair model of recombination, which was developed to account for recombination in Saccharomyces cerevisiae. Experiments aimed at exploring processing of DNA ends were performed to gain understanding of the mechanism responsible for the observed bias. A heterologous insert placed within a gap in the coding sequence of two different marker genes strongly inhibited repair if the DNA was cleaved at the promoter-proximal junction joining the insert and coding sequence but had little effect on repair if the DNA was cleaved at the promoter-distal junction. Gene conversion of plasmid restriction fragment length polymorphism markers engineered in sequences flanking both sides of a gap accompanied repair but was directionally biased. These results are interpreted to mean that the DNA ends flanking a gap are subject to different types of processing. A model featuring a single migrating D-loop is proposed to explain the bias in gap repair outcome based on the observed asymmetry in processing the DNA ends.
Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.
Authors: Dauter Z , Wilson K S , Sieker L C , Moulis J M , Meyer J
Published: July 2002

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.
Modular cis-regulatory organization of developmentally expressed genes: two genes transcribed territorially in the sea urchin embryo, and additional examples.
Authors: Kirchhamer C V , Yuh C H , Davidson E H
Published: July 2002

The cis-regulatory systems that control developmental expression of two sea urchin genes have been subjected to detailed functional analysis. Both systems are modular in organization: specific, separable fragments of the cis-regulatory DNA each containing multiple transcription factor target sites execute particular regulatory subfunctions when associated with reporter genes and introduced into the embryo. The studies summarized here were carried out on the CyIIIa gene, expressed in the embryonic aboral ectoderm and on the Endo16 gene, expressed in the embryonic vegetal plate, archenteron, and then midgut. The regulatory systems of both genes include modules that control particular aspects of temporal and spatial expression, and in both the territorial boundaries of expression depend on a combination of negative and positive functions. In both genes different regulatory modules control early and late embryonic expression. Modular cis-regulatory organization is widespread in developmentally regulated genes, and we present a tabular summary that includes many examples from mouse and Drosophila. We regard cis-regulatory modules as units of developmental transcription control, and also of evolution, in the assembly of transcription control systems.
The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.
Authors: Sakurai H , Kim Y J , Ohishi T , Kornberg R D , Fukasawa T
Published: July 2002

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.
In vitro activation of human herpesviruses 6 and 7 from latency.
Authors: Katsafanas G C , Schirmer E C , Wyatt L S , Frenkel N
Published: July 2002

Human herpesviruses 6 and 7 (HHV-6 and HHV-7) are prevalent lymphotropic viruses that infect more than 80% of children at infancy or during early childhood. Infection ranges from asymptomatic to severe disease. HHV-6B causes exanthem subitum. The virus can be recovered from peripheral blood mononuclear cells during the acute phase of exanthem subitum, but the host remains latently infected throughout life. In immunocompromised patients undergoing kidney, liver, or bone marrow transplantation latent HHV-6B is reactivated, at times causing severe or fatal disease. Here, we describe the establishment of an in vitro system for reactivation of HHV-6B and HHV-7 from latency. HHV-7 is reactivated from latently infected peripheral blood mononuclear cells by T-cell activation. HHV-6B could not be reactivated under similar conditions; however, the latent HHV-6B could be recovered after the cells were infected with HHV-7. Once reactivated, the HHV-6B genomes became prominent and the HHV-7 disappeared. We conclude that HHV-7 can provide a transacting function(s) mediating HHV-6 reactivating from latency. Understanding the activation process is critical for the development of treatments to control the activation of latent viruses so as to avoid these sometimes life threatening infections in transplant recipients.
The biology of vision of Drosophila.
Authors: Zuker C S
Published: July 2002

Phototransduction systems in vertebrates and invertebrates share a great deal of similarity in overall strategy but differ significantly in the underlying molecular machinery. Both are rhodopsin-based G protein-coupled signaling cascades displaying exquisite sensitivity and broad dynamic range. However, light activation of vertebrate photoreceptors leads to activation of a cGMP-phosphodiesterase effector and the generation of a hyperpolarizing response. In contrast, activation of invertebrate photoreceptors, like Drosophila, leads to stimulation of phospholipase C and the generation of a depolarizing receptor potential. The comparative study of these two systems of phototransduction offers the opportunity to understand how similar biological problems may be solved by different molecular mechanisms of signal transduction. The study of this process in Drosophila, a system ideally suited to genetic and molecular manipulation, allows us to dissect the function and regulation of such a complex signaling cascade in its normal cellular environment. In this manuscript I review some of our recent findings and the strategies used to dissect this process.
The 3′-untranslated region of CaMKIIα is a cis-acting signal for the localization and translation of mRNA in dendrites
Authors: Mayford Mark , Baranes Danny , Podsypanina Katrina , Kandel Eric R.
Published: July 2002

Neuronal signaling requires that synaptic proteins be appropriately
localized within the cell and regulated there. In mammalian neurons,
polyribosomes are found not just in the cell body, but also in
dendrites where they are concentrated within or beneath the dendritic
spine. The α subunit of
Ca
2+
-calmodulin-dependent protein kinase II
(CaMKIIα) is one of only five mRNAs known to be present within the
dendrites, as well as in the soma of neurons. This targeted subcellular
localization of the mRNA for CaMKIIα provides a possible cell
biological mechanism both for controlling the distribution of the
cognate protein and for regulating independently the level of protein
expression in individual dendritic spines. To characterize the
cis-acting elements involved in the localization of dendritic mRNA we
have produced two lines of transgenic mice in which the CaMKIIα
promoter is used to drive the expression of a
lacZ
transcript, which either contains or lacks the 3′-untranslated region
of the CaMKIIα gene. Although both lines of mice show expression in
forebrain neurons that parallels the expression of the
endogenous CaMKIIα gene, only the
lacZ
transcripts bearing the 3′-untranslated region are localized to
dendrites. The β-galactosidase protein shows a variable level of
expression along the dendritic shaft and within dendritic spines, which
suggests that neurons can control the local biochemistry of the
dendrite either through differential localization of the mRNA or
variations in the translational efficiency at different sites along the
dendrite.
Two functionally distinct subsites for the binding of internal blockers to the pore of voltage-activated K
+
channels
Authors: Baukrowitz Thomas , Yellen Gary
Published: July 2002

Many blockers of Na
+
and K
+
channels act by
blocking the pore from the intracellular side. For Shaker
K
+
channels, such intracellular blockers vary in their
functional effect on slow (C-type) inactivation: Some blockers
interfere with C-type inactivation, whereas others do not. These
functional differences can be explained by supposing that there are two
overlapping “subsites” for blocker binding, only one of which
inhibits C-type inactivation through an allosteric effect. We find that
the ability to bind to these subsites depends on specific structural
characteristics of the blockers, and correlates with the effect of
mutations in two distinct regions of the channel protein. These
interactions are important because they affect the ability of blockers
to produce use-dependent inhibition.
Energy considerations show that low-barrier hydrogen bonds do not offer a catalytic advantage over ordinary hydrogen bonds
Authors: Warshel Arieh , Papazyan Arno
Published: July 2002

Low-barrier hydrogen bonds have recently been proposed as a major
factor in enzyme catalysis. Here we evaluate the feasibility of
transition state (TS) stabilization by low-barrier hydrogen bonds in
enzymes. Our analysis focuses on the facts that (
i
) a
low-barrier hydrogen bond is less stable than a regular hydrogen bond
in water, (
ii
) TSs are more stable in the enzyme active
sites than in water, and (
iii
) a nonpolar active site
would destabilize the TS relative to its energy in water. Combining
these points and other experimental and theoretical facts in a
physically consistent framework shows that a low-barrier hydrogen bond
cannot stabilize the TS more than an ordinary hydrogen bond. The reason
for the large catalytic effect of active site hydrogen bonds is that
their formation entails a lower reorganization energy than their
solution counterparts, due to the preorganized enzyme environment.
Multiple potential germ-line helicases are components of the germ-line-specific P granules of
Caenorhabditis elegans
Authors: Gruidl M. E. , Smith P. A. , Kuznicki K. A. , McCrone J. S. , Kirchner J. , Roussell D. L. , Strome S. , Bennett K. L.
Published: July 2002

Two components of the germ-line-specific P granules of the nematode
Caenorhabditis elegans
have been
identified using polyclonal antibodies specific for each. Both
components are putative germ-line RNA helicases (GLHs) that contain
CCHC zinc fingers of the type found in the RNA-binding nucleocapsid
proteins of retroviruses. The predicted GLH-1 protein has four CCHC
fingers; GLH-2 has six. Both GLH proteins localize in the P granules at
all stages of germ-line development. However, the two
glh
genes display different patterns of RNA and protein
accumulation in the germ lines of hermaphrodites and males. Injection
of antisense
glh-1
or
glh-2
RNA into
wild-type worms causes some offspring to develop into sterile adults,
suggesting that either or both genes are required for normal germ-line
development. As these very similar
glh
genes physically
map within several hundred kilobases of one another, it seems likely
that they represent a fairly recent gene duplication event.
Phospholipase C β4 is involved in modulating the visual response in mice
Authors: Jiang Huiping , Lyubarsky Arkady , Dodd Robert , Vardi Noga , Pugh Edward , Baylor Denis , Simon Melvin I. , Wu Dianqing
Published: July 2002

Expression of G protein-regulated phospholipase C (PLC) β4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC β4 may play a role in the mammalian visual process. A mouse line that lacks PLC β4 was generated and the physiological significance of PLC β4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC β4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC β4-null mice showed 4-fold reduction in the maximal amplitude of the rod a- and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC β4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC β4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC β4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell–cell interactions between rods and other retinal cells.
A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins
Authors: Kim Sung-Su , Chen Yung-Ming , O’Leary Eileen , Witzgall Ralph , Vidal Marc , Bonventre Joseph V.
Published: July 2002

The Krüppel-associated box A (KRAB-A) domain is an
evolutionarily conserved transcriptional repressor domain present in
approximately one-third of zinc finger proteins of the
Cys
2
-His
2
type. Using the yeast two-hybrid
system, we report the isolation of a cDNA encoding a novel murine
protein, KRAB-A interacting protein 1 (KRIP-1) that physically
interacts with the KRAB-A region. KRIP-1 is a member of the RBCC
subfamily of the RING finger, or Cys
3
HisCys
4
,
family of zinc binding proteins whose other members are known to play
important roles in differentiation, oncogenesis, and signal
transduction. The KRIP-1 protein has high homology to TIF1, a putative
modulator of ligand-dependent activation function of nuclear receptors.
A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult
mouse tissues studied. When a GAL4–KRIP-1 fusion protein is expressed
in COS cells with a chloramphenicol acetyltransferase reporter
construct with five GAL4 binding sites, there is dose-dependent
repression of transcription. Thus, KRIP-1 interacts with the KRAB-A
region of C
2
H
2
zinc finger proteins and may
mediate or modulate KRAB-A transcriptional repressor activity.
CD8+T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages
Authors: Moriuchi Hiroyuki , Moriuchi Masako , Combadiere Christophe , Murphy Philip M. , Fauci Anthony S.
Published: July 2002

It has been demonstrated that CD8+T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4+T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8+T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8+T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8+T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8+T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8+T-cell supernatants; and (iii) the CD8+T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8+T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8+T-cell-derived HIV-suppressor factors.
The cutoff phenomenon in finite Markov chains.
Authors: Diaconis P
Published: July 2002

Natural mixing processes modeled by Markov chains often show a sharp cutoff in their convergence to long-time behavior. This paper presents problems where the cutoff can be proved (card shuffling, the Ehrenfests' urn). It shows that chains with polynomial growth (drunkard's walk) do not show cutoffs. The best general understanding of such cutoffs (high multiplicity of second eigenvalues due to symmetry) is explored. Examples are given where the symmetry is broken but the cutoff phenomenon persists.
The resource consumption principle: attention and memory in volumes of neural tissue.
Authors: Montague P R
Published: July 2002

In the cerebral cortex, the small volume of the extracellular space in relation to the volume enclosed by synapses suggests an important functional role for this relationship. It is well known that there are atoms and molecules in the extracellular space that are absolutely necessary for synapses to function (e.g., calcium). I propose here the hypothesis that the rapid shift of these atoms and molecules from extracellular to intrasynaptic compartments represents the consumption of a shared, limited resource available to local volumes of neural tissue. Such consumption results in a dramatic competition among synapses for resources necessary for their function. In this paper, I explore a theory in which this resource consumption plays a critical role in the way local volumes of neural tissue operate. On short time scales, this principle of resource consumption permits a tissue volume to choose those synapses that function in a particular context and thereby helps to integrate the many neural signals that impinge on a tissue volume at any given moment. On longer time scales, the same principle aids in the stable storage and recall of information. The theory provides one framework for understanding how cerebral cortical tissue volumes integrate, attend to, store, and recall information. In this account, the capacity of neural tissue to attend to stimuli is intimately tied to the way tissue volumes are organized at fine spatial scales.
Disruption of lysosomal targeting is associated with insecticidal potency of juvenile hormone esterase
Authors: Bonning Bryony C. , Ward Vernon K. , van Meer Marnix M. M. , Booth Tim F. , Hammock Bruce D.
Published: July 2002

Juvenile hormone esterase (JHE; EC
3.1.1.1
), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein.
Peptidyl-transferase inhibitors have antiviral properties by altering programmed −1 ribosomal frameshifting efficiencies: Development of model systems
Authors: Dinman Jonathan D. , Ruiz-Echevarria Maria J. , Czaplinski Kevin , Peltz Stuart W.
Published: July 2002

The effects of two peptidyl-transferase inhibitors, anisomycin and sparsomycin, on ribosomal frameshifting efficiencies and the propagation of yeast double-stranded RNA viruses were examined. At sublethal doses in yeast cells these drugs specifically alter the efficiency of −1, but not of +1, ribosomal frameshifting. These compounds promote loss of the yeast L-A double-stranded RNA virus, which uses a programmed −1 ribosomal frameshift to produce its Gag-Pol fusion protein. Both of these drugs also change the efficiency of −1 ribosomal frameshifting in yeast and mammalian
in vitro
translation systems, suggesting that they may have applications to control the propagation of viruses of higher eukaryotes, which also use this translational regulatory mechanism. Our results offer a new set of antiviral agents that may potentially have a broad range of applications in the clinical, veterinary, and agricultural fields.
The abalone egg vitelline envelope receptor for sperm lysin is a giant multivalent molecule
Authors: Swanson Willie J. , Vacquier Victor D.
Published: July 2002

Abalone sperm lysin is a 16-kDa acrosomal protein, which nonenzymatically and species selectively creates a hole in the egg vitelline envelope (VE) through which the sperm passes to reach the egg cell membrane. The crystal structures of both monomeric and dimeric lysins have been solved and the sequences of lysins from 20 abalone species have been determined. As a first step in understanding the molecular mechanism by which lysin creates a hole in the VE, its VE receptor was isolated. The VE receptor for lysin (VERL) is an unbranched, rod-like molecule with an approximate relative molecular mass of 2 million; half the mass being carbohydrate. Fluorescence polarization studies showed positive cooperativity in the binding of lysin to VERL (EC
50
≈9 nM) and were consistent with the species selectivity of lysin in dissolving VEs. Each molecule of VERL bound between 126 and 142 molecules of monomeric lysin (two independent assays), showing that VERL possesses repetitive lysin-binding motifs.