bluuebunny/crossref_metadata_2025 huggingface dataset:
Encapsulation of doxorubicin in liver-targeted erythrocytes increases the therapeutic index of the drug in a murine metastatic model.
Authors: Zocchi E , Tonetti M , Polvani C , Guida L , Benatti U , De Flora A
Published: May 2006

Doxorubicin-loaded, glutaraldehyde-treated murine erythrocytes, once reinjected into circulation, are rapidly taken up by liver and lungs and behave as an organ-targeted, slow delivery system for the encapsulated drug. The antitumor activity of encapsulated doxorubicin (former generic name, adriamycin) was compared with that of the free drug in a murine hepatic and pulmonary tumor model. This was obtained by intrasplenic injection of L1210 lymphoma cells followed by splenectomy. Different schedules of treatment of tumor-bearing mice with erythrocyte-encapsulated or free doxorubicin were investigated. The optimal schedule of treatment for free doxorubicin proved to be i.v. bolus administration on the day of splenectomy. Under these conditions, the dose producing 50% inhibition of metastatic growth in the liver, as measured by inhibition of 5-[125I]iodo-2'-deoxyuridine uptake 9 days after tumor induction, was 6.3 mg/kg for free doxorubicin and 0.48 mg/kg for the encapsulated drug. In these conditions pulmonary tumor development was even more efficiently prevented by encapsulated doxorubicin as compared with the free drug. The values of the therapeutic index (TI), defined as the ratio between the maximal tolerated dose (LD10) and the minimal effective dose (ED90, producing 90% inhibition of liver metastatic growth), were 4.2 and 1.8 for encapsulated and free doxorubicin, respectively.
Constraints on the interpretation of nonquantal acetylcholine release from frog neuromuscular junctions.
Authors: Meriney S D , Young S H , Grinnell A D
Published: May 2006

In the frog nerve-muscle preparation, there is evidence for nonquantal release of acetylcholine (ACh) at a level 100 times that attributable to spontaneous quantal events (miniature endplate potentials). It is widely assumed that nonquantal release occurs near the sites of quantal release (active zones) in the nerve terminal, close to the postsynaptic muscle membrane. This high level of nonquantal ACh release has led to the suggestion that it may serve a trophic function at the nerve-muscle junction. However, the precise origin and mechanism of nonquantal release have not been determined. We have used outside-out patches of ACh receptor-rich membrane as a sensitive technique for the direct measurement of ACh release from highly localized regions of enzymatically treated nerve terminals and have found little detectable ACh "leakage" from active-zone regions. If all of the nonquantal ACh release were localized to the under surface of the nerve terminal, we estimate that we would have detected more than 10 times the low level detected with our patch probe. Furthermore, although quantal release was easily measurable, vesicular exocytosis (hypothesized to insert ACh transport proteins into the plasma membrane, thereby producing the leak) did not increase single-channel activity in the patch probe above that attributable to quantal release. We conclude that, at rest, the active-zone region of nerve terminals is not a major source of nonquantal ACh release and that vesicular exocytosis does not noticeably increase the level of nonquantal release from the nerve terminal. Thus, with biochemical measurements that indicate that spontaneous ACh release is relatively unchanged by prior denervation, these results question the assumed source and mechanism of nonquantal release and the suggestion that leakage of ACh from active-zone regions plays a trophic role in nerve-muscle interaction.
Phosphorylation and associated translocation of the 87-kDa protein, a major protein kinase C substrate, in isolated nerve terminals.
Authors: Wang J K , Walaas S I , Sihra T S , Aderem A , Greengard P
Published: May 2006

A protein of 87 kilodaltons (87 kDa) was previously identified as a major specific substrate for protein kinase C in neuronal and other tissues. We have now studied the effect of protein kinase C-catalyzed phosphorylation of this protein on its association with membranes in isolated nerve terminals (synaptosomes) from rat cerebral cortex. Incubation of synaptosomal membranes under conditions associated with activation of protein kinase C led to the release of the phosphorylated 87-kDa protein into the incubation medium. In intact synaptosomes, activation of protein kinase C by phorbol esters or by depolarization-induced Ca2+ influx caused an increased phosphorylation of the 87-kDa protein and its translocation from membrane to cytosol. This translocation showed time courses, calcium dependency, and reversibility similar to those observed for the protein kinase C-induced phosphorylation of the protein. These results suggest that protein kinase C-catalyzed phosphorylation of the 87-kDa protein is responsible for its subcellular translocation into the cytosol of nerve terminals.
A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.
Authors: Rosen D R , Martin-Morris L , Luo L Q , White K
Published: May 2006

We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.
Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation.
Authors: Hung M C , Yan D H , Zhao X Y
Published: May 2006

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.
Nitrogen-dependent regulation of photosynthetic gene expression
Authors: Plumley F. Gerald , Schmidt Gregory W.
Published: May 2006

Nitrogen-limited
Chlamydomonas reinhardtii
is chlorotic and very deficient in chlorophyll a/b light-harvesting complexes (LHC). Rates of synthesis of photosynthetic proteins, but especially the LHC apoproteins, are reduced 10- to 40-fold. Moderately high levels of chloroplast transcripts accumulate in nitrogen-limited cells, and there is a correlation between chloroplast DNA levels and chloroplast mRNA abundance. In contrast, nuclear transcripts encoding LHCII and ribulose 1,5-bisphosphate carboxylase small subunits are markedly reduced. Thus, nitrogen availability affects chloroplast protein synthesis by inhibition of translation and, to a lesser extent, chloroplast DNA amplification. Regulation of nuclear-encoded photosynthetic proteins by nitrogen is achieved through mechanisms affecting transcription and/or mRNA stability.
Mutations in RNA polymerase II enhance or suppress mutations in GAL4.
Authors: Allison L A , Ingles C J
Published: May 2006

The activation domains of eukaryotic DNA-binding transcription factors, such as GAL4, may regulate transcription by contacting RNA polymerase II. One potential site on RNA polymerase II for such interactions is the C-terminal tandemly repeated heptapeptide domain in the largest subunit (RPO21). We have changed the number of heptapeptide repeats in this yeast RPO21 C-terminal domain and have expressed these mutant RNA polymerase II polypeptides in yeast cells containing either wild-type or defective GAL4 proteins. Although the number of RPO21 heptapeptide repeats had no effect on the activity of wild-type GAL4, changing the length of the C-terminal domain modified the ability of mutant GAL4 proteins to activate transcription. Shorter or longer RPO21 C-terminal domains enhanced or partially suppressed, respectively, the effects of deletions in the transcriptional-activation domains of GAL4. The same RPO21 mutations also affected transcriptional activation by a GAL4-GCN4 chimera. These data suggest that the activation domains of DNA-binding transcription factors could interact, either directly or indirectly, with the heptapeptide repeats of RNA polymerase II.
Water intake in rats subjected to hypothalamic immunoneutralization of angiotensin II, atrial natriuretic peptide, vasopressin, or oxytocin.
Authors: Franci C R , Kozlowski G P , McCann S M
Published: May 2006

To investigate the influence of various peptides on control of dehydration-induced drinking, water intake elicited by overnight water deprivation was analyzed in groups of male rats after intracerebroventricular (third ventricle, icv) injection of 2 microliters of normal rabbit serum or an equal volume of antiserum directed against angiotensin II (Ab-AII), atrial natriuretic peptide, vasopressin, or oxytocin. There was no difference in water intake after normal rabbit serum and antiserum injections when water was offered immediately after icv injections. Water intake was greatly reduced by Ab-AII when water was offered 1 hr and 3 hr after icv injection. The other antisera were partially effective only when water was offered 3 hr after icv injection. The dipsogenic effect of icv injection of AII in normally hydrated rats was reduced only by icv injection of Ab-AII 3 hr before and not by the other antisera. Ab-AII injected icv had no effect on the drinking that occurred just before and after the onset of darkness and that was associated with eating (prandial drinking). The results indicate that AII is primarily responsible for dehydration-induced drinking, and the other peptides may play a permissive role since their antisera were partially effective, with longer latencies after antiserum injection, which is perhaps the result of gradual diffusion to effective sites within the hypothalamus. In contrast, endogenous AII appears to play little, if any, role in prandial drinking.
Biregular classification of Fano 3-folds and Fano manifolds of coindex 3
Authors: Mukai Shigeru
Published: May 2006

The Fano 3-folds and their higher dimensional analogues are classified over an arbitrary field
k
[unk] C by applying the theory of vector bundles (in the case
B
2
= 1) and the theory of extremal rays (in the case
B
2
≥ 2). An
n
-dimensional smooth projective variety
X
over
k
is a Fano manifold if its first Chern class
c
1
(
X
) ε
H
2
(
X, Z
) is positive in the sense of Kodaira [Kodaira, K. (1954)
Ann. Math.
60, 28-48] (or ample). If
n
= 3 and
c
1
(
X
) generates
H
2
(
X, Z
), then either (
i
)
X
is a complete intersection in a Grassmann variety
G
with respect to a homogeneous vector bundle
E
on
G
: the rank of
E
is equal to codim

G
X

and
X
is isomorphic to the zero locus of a global section of
E
, (
ii
)
X
is a linear section of a 10-dimensional spinor variety
X
12
10
[unk] P

k

15
, or (
iii
)
X
is isomorphic to a double cover of P

k

3
, a 3-dimensional quadric

Q
k

3
, or a quintic del Pezzo 3-fold
V
5
[unk] P

k

6
. If
n
= 4 and
c
1
(
X
) is divisible by 2, then
X
[unk] C is isomorphic to (
a
) a complete intersection in a homogeneous space or its double cover, (
b
) a product of P
1
and a Fano 3-fold, (
c
) the blow-up of
Q
4
[unk]
P
5
along a line or along a conic, or (
d
) a P
1
-bundle compactifying a line bundle on P
3
or on
Q
3
[unk] P
4
.
Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties.
Authors: Ono Y , Fujii T , Ogita K , Kikkawa U , Igarashi K , Nishizuka Y
Published: May 2006

The primary structure of the zeta subspecies of rat brain protein kinase C was deduced from its overlapping cDNAs. The zeta subspecies of protein kinase C consists of 592 amino acid residues with the calculated molecular mass of 67,740 Da and has regulatory and protein kinase domains in its amino- and carboxyl-terminal halves, respectively. Although all members of the protein kinase C family so far identified have a tandem repeat of the characteristic cysteine-rich zinc-finger-like sequence in the regulatory domain, the zeta subspecies contains only one set of this sequence. Northern (RNA)-blot hybridization analysis indicated that two major RNA transcripts of the zeta subspecies with different lengths may be generated by the use of different polyadenylylational signals. The enzyme was expressed in COS-7 cells by transfection with the cDNA construct encoding its whole sequence. It showed an approximate molecular mass of 64,000 Da upon SDS/PAGE. The enzyme activity was significantly dependent on phospholipid but was independent of the presence of Ca2+ or diacylglycerol, when assayed with calf thymus H1 histone as a phosphate acceptor protein. The zeta subspecies expressed in COS-7 cells did not appear to show binding activity of phorbol ester. The structural and biochemical properties indicate that the zeta subspecies is related to, but distinct from, other subspecies of protein kinase C. Perhaps, this subspecies belongs to another entity of the enzyme family.
Gestational changes in calbindin-D9k in rat uterus, yolk sac, and placenta: implications for maternal-fetal calcium transport and uterine muscle function.
Authors: Mathieu C L , Burnett S H , Mills S E , Overpeck J G , Bruns D E , Bruns M E
Published: May 2006

Calbindin-D9k was quantified and its cellular location was defined in uterus, yolk sac, and placenta. In late gestation (days 17 to term) coordinated induction of calbindin-D9k was seen in uterine epithelial lining cells and juxtaposed yolk sac visceral epithelium as well as the intraplacental yolk sac epithelium. The induction of calbindin-D9k in these cells coincided with the time of exponential fetal bone growth and maximal fetal accumulation of calcium, suggesting a role of the protein in these epithelial layers in maternal-fetal calcium transport. Dynamic changes also occurred in the calbindin-D9k contents of the two layers of uterine smooth muscle (outer longitudinal and inner circular) during mid- and late gestation. During early pregnancy (days 0-4), calbindin-D9k was present in the two smooth muscle layers. By midgestation (day 10), calbindin-D9k had decreased by a factor of 10 in these tissue layers. During late gestation calbindin-D9k rebounded in the inner circular smooth muscle layer. These uterine changes of early and midgestation were reproduced by the endocrine changes of pseudopregnancy. Progesterone appeared to be a good candidate for controlling the midgestational decrease of uterine muscle calbindin-D9k, as it blunted estrogen's induction of the protein in the muscle layers and stroma in a dose-dependent manner. Changes in myometrial calbindin-D9k may reflect variations in muscular calcium storage, thereby representing alterations in potential for contraction.
Evolution of the first metabolic cycles.
Authors: Wächtershäuser G
Published: May 2006

There are two alternatives concerning the origin of life: the origin may be heterotrophic or autotrophic. The central problem within the theory of an autotrophic origin is the first process of carbon fixation. I here propose the hypothesis that this process is an autocatalytic cycle that can be retrodictively constructed from the extant reductive citric acid cycle by replacing thioesters by thioacids and by assuming that the required reducing power is obtained from the oxidative formation of pyrite (FeS2). This archaic cycle is strictly chemoautotrophic: photoautotrophy is not required. The cycle is catalytic for pyrite formation and autocatalytic for its own multiplication. It is a consequence of this hypothesis that the postulated cycle cannot exist as a single isolated cycle but must be a member of a network of concatenated homologous cycles, from which all anabolic pathways appear to have sprung.
Identification of a viral gene encoding a ubiquitin-like protein.
Authors: Guarino L A
Published: May 2006

The baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV, which is representative of the MNPV subtype in which the virions may contain many nucleocapsids within a single viral envelope) encodes a protein, v-ubi, that has 76% identity with the eukaryotic protein ubiquitin. Transcriptional mapping indicated that the gene for v-ubi was transcribed during the late phase of viral infection. Two transcriptional start sites potentially encoding v-ubi were identified. Both sites were contained within a sequence motif common to baculovirus late genes. A recombinant virus, AcUbi-beta Gal, encoding a ubiquitin-beta-galactosidase fusion protein was constructed to monitor the temporal regulation of v-ubi gene during viral infection. The fusion protein was expressed maximally at 14-18 hr postinfection, consistent with its classification as a late protein. The amount of ubiquitin-beta-galactosidase fusion protein that accumulated in AcUbi-beta Gal-infected cells by 48 hr postinfection was approximately 14% of the level of beta-galactosidase that was synthesized under control of the polyhedrin promoter. Transcriptional analysis confirmed that synthesis of the fusion protein was directed by the v-ubi gene promoter. AcUbi-beta Gal also produced normal levels of authentic viral ubiquitin message. Southern blot analysis of AcUbi-beta Gal and 15 additional isolates revealed that the fusion sequences had not recombined at the ubiquitin locus. A polyubiquitin gene was isolated and sequenced from Spodoptera frugiperda, a lepidopteran host cell line for AcMNPV. The predicted amino acid sequence of the product of the host gene is identical to animal ubiquitin.
Fibroblast-mediated acceleration of human epithelial tumor growth in vivo.
Authors: Camps J L , Chang S M , Hsu T C , Freeman M R , Hong S J , Zhau H E , von Eschenbach A C , Chung L W
Published: May 2006

Transformed fibroblasts coinoculated with epithelial cells accelerated the growth and shortened the latency period of human epithelial tumors in athymic mice. Addition of NbF-1 fibroblasts caused epithelial tumors to grow from five marginally tumorigenic or "nontumorigenic" (nontumor-forming) human tumor cell lines or strains: PC-3 (prostate), WH (bladder), MDA-436 (breast), and cells derived from the ascites fluids of patients with metastatic renal pelvic or prostate cancers. Evidence for the human and epithelial nature of these experimental tumors was provided by histologic, immunohistochemical, Southern and dot-blot hybridization, and cytogenetic analyses. Transformed fibroblasts induced predominantly carcinosarcomas, whereas nontumorigenic fibroblasts (NIH 3T3) and lethally irradiated transformed fibroblasts induced exclusively carcinomas. The fibroblast-epithelial interaction appears to occur bidirectionally and does not result from cell fusion. Because coculture experiments in vitro did not demonstrate an increased cell proliferation, it appears that undefined host factors can influence tumor growth. This tumor model may be useful in drug-screening programs and in mechanistic studies of factors regulating human tumor growth and progression.
Asynchronous replication of homologous loci on human active and inactive X chromosomes.
Authors: Schmidt M , Migeon B R
Published: May 2006

The two X chromosomes in mammalian females replicate asynchronously, the inactive later than the active one. Using BrdUrd-sensitive restriction and UV irradiation to identify newly synthesized DNA directly on Southern blots, and restriction fragment length differences to discriminate alleles on active and inactive human X chromosomes, we examined the replication of hypoxanthine phosphoribosyltransferase (HPRT) and clotting factor IX (F9) loci in clonal populations of mouse-human hybrids. We find that HPRT replicates at different times during the period of DNA synthesis (S phase), depending on its activity: It replicates in early S phase, when expressed (on the active X chromosome), and in late S phase when silent (on the inactive X chromosome). Furthermore, when reactivated, the derepressed locus is earlier replicating, supporting a relationship between replication and transcription. Neither F9 allele is expressed in these cells, and both replicate in the second half of S phase, (slightly earlier on active than on inactive X chromosome).
Pigeons with ablated pyriform cortex home from familiar but not from unfamiliar sites.
Authors: Papi F , Casini G
Published: May 2006

Homing behavior was tested in pigeons (Columba livia) after removing a portion of the ventrolateral telencephalon, which receives extensive projections from the olfactory bulb and is comparable with the mammalian pyriform cortex. Ablated pigeons show unaffected cardiac responses to odorous stimuli but altered homing behavior. After the operation, the birds were trained by repeated flock releases along with control birds from a site 40 km from the loft. After being released singly from this familiar site, the ablated birds turned out to be unaffected by the operation. In releases from two unfamiliar sites, ablated birds, unlike control birds, were not homeward oriented and were mostly lost. The ablation of the pyriform cortex has the same effect on homing behavior as olfactory deprivation. It can be concluded that the pyriform cortex plays an important role in the specific mechanisms linking olfactory inputs with the navigational response.
Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana.
Authors: Ma H , Yanofsky M F , Meyerowitz E M
Published: May 2006

We have isolated a gene coding for a G protein alpha subunit from the flowering plant Arabidopsis thaliana. This gene, named GPA1, was isolated by using a DNA probe generated by polymerase chain reaction based on protein sequences from mammalian and yeast G protein alpha subunits. The sequences of genomic and cDNA clones indicate that GPA1 has 14 exons, and the deduced amino acid sequence shows that the GPA1 gene product (GP alpha 1) has 383 amino acid residues (44,582 Da). The GP alpha 1 protein exhibits similarity to all known G protein alpha subunits--36% of its amino acids are identical and 73% are similar (identical and conservative changes) to mammalian inhibitory guanine nucleotide-binding regulatory factor alpha subunits and transducins. Furthermore, the GP alpha 1 protein has all of the consensus regions for a GTP-binding protein. The GPA1-encoded mRNA of 1.55 kilobases is most abundant in vegetative plant tissues, as determined by RNA blot analysis. Restriction fragment length polymorphism mapping experiments show that GPA1 is approximately 1.2 centimorgans from the visible marker er on chromosome 2.
Protein kinase C activity is not responsible for the expression of long-term potentiation in hippocampus.
Authors: Muller D , Buchs P A , Dunant Y , Lynch G
Published: May 2006

Long-term potentiation (LTP) in hippocampus has been proposed to result from a tonic activation of protein kinase C. This hypothesis predicts that stimulation of the kinase would produce a smaller change in response size on potentiated versus control pathways and, conversely, that inhibition of the kinase would reduce potentiated inputs to a greater degree than control responses. We tested these predictions using phorbol esters to activate and using the antagonist H-7 to inhibit protein kinase C; we found that the actions of these drugs on synaptic transmission were not affected by prior induction of LTP. Both compounds, however, significantly decreased the contribution of N-methyl-D-aspartate receptors to synaptic potentials, a result that accounts for the suppressive effects of these compounds on LTP formation. Thus protein kinase C is probably not involved in the expression of LTP but may play a role in the receptor-mediated events participating in its induction.
cAMP-dependent phosphorylation and inactivation of yeast transcription factor ADR1 does not affect DNA binding.
Authors: Taylor W E , Young E T
Published: May 2006

Transcription factor ADR1 increases the level of ADH2 gene expression 200-fold by binding to a palindromic upstream activation sequence (UAS1) in the glucose-repressible ADH2 promoter in Saccharomyces cerevisiae. cAMP-dependent protein kinase (cAPK) phosphorylates ADR1 in vitro and a yeast strain with elevated cAPK activity inhibits the ability of ADR1 to activate ADH2 transcription in vivo [Cherry, J. R., Johnson, T. R., Dollard, C., Schuster, J. R. & Denis, C. L. (1988) Cell 56, 409-419]. Intact ADR1 protein was detected at comparable levels in extracts made from repressed or derepressed yeast cells, indicating that glucose repression is not due to absence of ADR1. ADR1 in extracts made from glucose-repressed and -derepressed cells bound UAS1 DNA with similar affinities despite having greatly different abilities to activate ADH2 gene expression in vivo. A mutant form of ADR1 encoded by ADR1-5c, which has an altered consensus sequence for phosphorylation by cAPK conferred constitutive expression on ADH2 but bound DNA to the same extent as wild-type ADR1 protein. Similarly, normal DNA binding was seen for ADR1 produced in mutants with altered levels of cAPK activity. Because inactivation of ADR1 by phosphorylation has no detectable effect on either DNA binding or ADR1 levels, ADR1 probably binds to UAS1 constitutively and phosphorylation prevents it from promoting transcription.
Integration of human immunodeficiency virus type 1 DNA in vitro.
Authors: Farnet C M , Haseltine W A
Published: May 2006

A highly efficient cell-free system for the integration of human immunodeficiency virus type 1 DNA is described. Linear viral DNA synthesis occurs in the cytoplasm of newly infected cells, reaching peak levels 4 hr after infection. The linear viral DNA molecules present in cytoplasmic extracts are capable of integrating into heterologous DNA targets in vitro. The viral DNA resides in a high molecular weight nucleoprotein structure that can be separated from the bulk of cellular protein and nucleic acid without a detectable decrease in the ability to integrate in vitro.
Funnel-web spider venom and a toxin fraction block calcium current expressed from rat brain mRNA in Xenopus oocytes.
Authors: Lin J W , Rudy B , Llinás R
Published: May 2006

Injection of rat brain mRNA into Xenopus oocytes has been shown to induce a calcium current (ICa) that is insensitive to dihydropyridine and omega-conotoxin. We examined the effect of funnel-web spider venom on two aspects of this expressed ICa: (i) the calcium-activated chloride current [ICl(Ca)] and (ii) the currents carried by barium ions through calcium channels (IBa). In the presence of 1.8 mM extracellular calcium, ICl(Ca) tail current became detectable between -30 and -40 mV from a holding potential of -80 mV and reached a maximal amplitude between 0 and +10 mV. Total spider venom partially (83%) and reversibly blocked the calcium-activated chloride current without changing its voltage sensitivity. A chromatographic toxin fraction from the venom also blocked this current (64%). The venom had a minimal effect on INa and IK. Direct investigation of inward current mediated by calcium channels was carried out in high-barium solution. IBa had a higher threshold of activation (-30 to -20 mV) and reached its maximal amplitude at about +20 mV. Total venom or a partly purified chromatographic toxic fraction blocked IBa partially and reversibly without changing its current-voltage characteristics. Furthermore, the extent of the total venom block depended on the concentration of extracellular barium. Only 35% of the IBa was blocked in 60 mM Ba2+, whereas the block increased to 65% and 71%, respectively, for 40 and 20 mM Ba2+. On the basis of these results, we propose that the calcium channels expressed from rat brain mRNA in Xenopus oocytes is similar to the recently discovered P-type channels.
Progressive changes in the protein composition of the nuclear matrix during rat osteoblast differentiation.
Authors: Dworetzky S I , Fey E G , Penman S , Lian J B , Stein J L , Stein G S
Published: May 2006

Primary cultures of fetal rat calvarial osteoblasts undergo a developmental sequence with respect to the temporal expression of genes encoding osteoblast phenotypic markers. Based on previous suggestions that gene-nuclear matrix associations are involved in regulating cell- and tissue-specific gene expression, we investigated the protein composition of the nuclear matrix during this developmental sequence by using high-resolution two-dimensional gel electrophoresis. The nuclear matrix was isolated at times during a 4-week culture period that represent the three principal osteoblast phenotypic stages: proliferation, extracellular matrix (ECM) maturation, and mineralization. The most dramatic changes in the nuclear matrix protein patterns occurred during transitions from the proliferation to the ECM maturation stage and from ECM maturation to the mineralization period, with only minor variations in the profiles within each period. These stage-specific changes, corresponding to the major transition points in gene expression, indicate that the nuclear matrix proteins reflect the progressive differentiation of the bone cell phenotype. Subcultivation of primary cells delays mineralization, and a corresponding delay was observed for the nuclear matrix protein patterns. Thus, the sequential changes in protein composition of the nuclear matrix that occur during osteoblast differentiation represent distinct stage-specific markers for maturation of the osteoblast to an osteocytic cell in a bone-like mineralized ECM. These changes are consistent with a functional involvement of the nuclear matrix in mediating modifications of developmental gene expression.
Identification of a contact between arginine-180 of the catabolite gene activator protein (CAP) and base pair 5 of the DNA site in the CAP-DNA complex.
Authors: Zhang X P , Ebright R H
Published: May 2006

We have used site-directed mutagenesis to replace amino acid 1 of the recognition alpha-helix of the catabolite gene activator protein (CAP), Arg-180, with glycine and with alanine. Substitution of Arg-180 of CAP eliminated specificity between G.C, A.T, C.G, and T.A at base pair 5 of the DNA half-site. The effect was position-specific: substitution of Arg-180 did not eliminate specificity between G.C, A.T, C.G, and T.A at base pair 7 of the DNA half-site. We conclude, in agreement with the model for the structure of the CAP-DNA complex [Weber, I. & Steitz, T. (1984) Proc. Natl. Acad. Sci. USA 81, 3973-3977; and Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Proc. Natl. Acad. Sci. USA 81, 7274-7278], that Arg-180 of CAP makes a specificity-determining contact with base pair 5 of the DNA half-site in the CAP-DNA complex. The identification of the contact by Arg-180 in this report, in conjunction with the identification of the contact by Glu-181 in a previous report [Ebright, R., Cossart, P., Gicquel-Sanzey, B. & Beckwith, J. (1984) Nature (London) 311, 232-235], provides information sufficient to define the orientation of the helix-turn-helix motif of CAP with respect to DNA in the CAP-DNA complex.
Neurospora crassa A mating-type region.
Authors: Glass N L , Grotelueschen J , Metzenberg R L
Published: May 2006

The mating-type locus of the haploid filamentous fungus Neurospora crassa is a regulatory region that controls entry into the sexual cycle and prevents formation of mixed mating-type heterokaryons in the vegetative phase. The locus consists of alternative sequences called A and a. The A mating-type DNA sequence of Neurospora crassa is composed of a region of 5301 base pairs that has little similarity to the sequence present at the mating-type locus in an a mating-type strain. However, the sequences flanking the mating-type locus in the A haploid and a haploid genome are essentially identical. The region of the A mating-type sequence required for expression of the heterokaryon incompatibility and sexual functions has been localized to a single open reading frame (ORF) encoding a polypeptide of 288 amino acids. Sequence analysis of sterile, heterokaryon-compatible mutants reveals frameshift mutations in this same ORF. The putative 288-amino acid product has a region of similarity to the MAT alpha 1 polypeptide of Saccharomyces cerevisiae.
Protein kinase C in Saccharomyces cerevisiae: comparison with the mammalian enzyme.
Authors: Ogita K , Miyamoto S , Koide H , Iwai T , Oka M , Ando K , Kishimoto A , Ikeda K , Fukami Y , Nishizuka Y
Published: May 2006

Protein kinase C (PKC) was detected in the yeast Saccharomyces cerevisiae with bovine myelin basic protein as the phosphate acceptor. The enzyme was purified at least 500-fold by a four-step column chromatographic procedure (phenyl-Sepharose CL-4B, Mono Q, Heparin-5PW, and hydroxyapatite). The molecular mass was approximately 90 kDa, as estimated by gel-filtration analysis. Yeast PKC was activated by the simultaneous addition of Ca2+, diacylglycerol, and phosphatidylserine. Free arachidonic acid alone could activate the enzyme to some extent. However, yeast PKC did not respond significantly to tumor-promoting phorbol esters. GTP did not serve as phosphate donor. The yeast enzyme showed substrate specificity distinctly different from that of mammalian PKCs. H1 histone and protamine were poor substrates. With myelin basic protein as a model substrate, yeast PKC phosphorylated threonyl residues preferentially, whereas rat brain PKCs phosphorylated seryl residues preferentially. Further studies should elucidate the role of yeast PKC in cellular regulation and cell cycle control.
Two distinct forms of active transcription factor CREB (cAMP response element binding protein).
Authors: Berkowitz L A , Gilman M Z
Published: May 2006

Mammalian cells express two distinct forms of transcription factor CREB (cAMP response element binding protein) that are apparently the products of alternative splicing of the CREB gene transcript. The two proteins differ by a 14-amino acid serine-rich insertion present in one of the CREB isoforms. We show that both CREB isoforms are expressed in many cell types and mammalian species. Both encode proteins that bind specifically to a cAMP response element in vitro. As expected for proteins of this class, the CREB proteins bind DNA as dimers. Both proteins impart cAMP-regulated transcriptional activity to a heterologous DNA-binding domain, showing that cAMP directly modulates the transcriptional stimulatory activity of CREB. The presence of multiple CREB isoforms with identical DNA-binding specificities but differences in the presumed regulatory domain raises the possibility that CREB proteins may be able to integrate distinct regulatory signals at the level of gene transcription.
An insulin response element in the glyceraldehyde-3-phosphate dehydrogenase gene binds a nuclear protein induced by insulin in cultured cells and by nutritional manipulations in vivo.
Authors: Nasrin N , Ercolani L , Denaro M , Kong X F , Kang I , Alexander M
Published: May 2006

Two independent cis-acting insulin response elements (IREs) in the gene encoding glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], designated IRE-A and IRE-B, are sufficient to direct insulin-inducible gene expression. Using the electrophoretic mobility shift assay, a 4-fold increase in the amount of IRE-A DNA bound to nuclear proteins was detected when extracts isolated from insulin-stimulated differentiated 3T3-L1 cells or from the liver of rats refed a high-carbohydrate/low-fat diet after a 72-hr fast were compared to control nuclear extracts. The points of contact between protein and IRE-A DNA may represent a sequence recognized by at least one class of insulin-sensitive transcription factor(s).
nit-2, the major positive-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequence-specific DNA-binding protein.
Authors: Fu Y H , Marzluf G A
Published: May 2006

The nit-2 major nitrogen regulatory gene of Neurospora crassa turns on the expression of various unlinked structural genes that specify nitrogen-catabolic enzymes under nitrogen-limitation conditions. The nit-2 gene encodes a protein of 1036 amino acid residues with a single zinc finger and a downstream basic region that may make up a DNA-binding domain. The zinc-finger domain of the NIT2 protein was synthesized in two ways to examine its DNA-binding activity with gel-band-mobility shift and DNA-footprint experiments. The NIT2 protein binds to specific DNA recognition elements that are located upstream of nitrogen-regulated structural genes. Each recognition element contains at least two copies of a core sequence whose consensus is TATCTA.
Developmental repression of growth and gene expression in Aspergillus.
Authors: Adams T H , Timberlake W E
Published: May 2006

Asexual reproductive development can be initiated in Aspergillus nidulans in the presence of excess nutrients through artificial induction of the developmental regulatory genes brlA or abaA by fusing the genes to the promoter from the alcohol dehydrogenase I gene (alcA) and culturing cells in the presence of an inducing alcohol. Artificially induced development completely inhibits growth and represses expression of the endogenous alcA gene and the coordinately controlled aldehyde dehydrogenase gene (aldA). Repression of alcA and aldA expression probably occurs at both the transcriptional and posttranslational levels. We propose that developmental induction results in a generalized metabolic shutdown, leading to an inability of cells to acquire nutrients from the growth medium. Self-imposed nutrient limitation could reinforce the primary developmental stimulus and ensure progression through the asexual reproductive pathway.
Trans-activation function of a 3' truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues.
Authors: Takada S , Koike K
Published: May 2006

To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3' end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.
Genetic linkage map of 46 DNA markers on human chromosome 16.
Authors: Keith T P , Green P , Reeders S T , Brown V A , Phipps P , Bricker A , Falls K , Rediker K S , Powers J A , Hogan C
Published: May 2006

We have constructed a genetic linkage map of human chromosome 16 based on 46 DNA markers that detect restriction fragment length polymorphisms. Segregation data were collected on a set of multigenerational families provided by the Centre d'Etude du Polymorphisme Humain, and maps were constructed using recently developed multipoint analysis techniques. The map spans 115 centimorgans (cM) in males and 193 cM in females. Over much of the chromosome there is a significantly higher frequency of recombination in females than males. Near the alpha-globin locus on the distal part of the short arm, however, there is a significant excess of male recombination. Twenty-seven (59%) of the markers on the map have heterozygosities greater than or equal to 0.50. The largest interval between loci on the sex-average map is 14 cM and the average marker spacing is 3 cM. Using loci on this map, one could detect linkage to a dominant disease on chromosome 16 with as few as 10-15 phase-known meioses.
The complete coding sequence of arg defines the Abelson subfamily of cytoplasmic tyrosine kinases.
Authors: Kruh G D , Perego R , Miki T , Aaronson S A
Published: May 2006

We have previously described partial genomic sequences of arg, a human gene related to c-abl, and shown that it is expressed as a 12-kilobase transcript and is located at chromosome position 1q24-25. In this study we elucidate the complete coding sequence of arg by characterization of cDNA clones. Analysis of the predicted amino acid sequence of arg revealed that it is indeed closely related to that of c-abl. The two proteins are strikingly similar with regard to overall structural architecture as well as the amino acid sequences of their tyrosine kinase and src homologous 2 and 3 domains. In addition, arg, like c-abl, is expressed as two transcripts that result from a process of alternative splicing and encode alternative protein forms that differ only in their amino termini. The two genes define the Abelson subfamily of cytoplasmic tyrosine kinases and share a common homolog in Drosophila.
A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis.
Authors: Greenberg S G , Davies P
Published: May 2006

Paired helical filaments (PHFs) are prominent components of Alzheimer disease (AD) neurofibrillary tangles (NFTs). Rather than isolating NFTs, we selected for PHF populations that can be extracted from AD brain homogenates. About 50% of PHF immunoreactivity can be obtained in 27,200 x g supernatants following homogenization in buffers containing 0.8 M NaCl. We further enriched for PHFs by taking advantage of their insolubility in the presence of zwitterionic detergents and 2-mercaptoethanol, removal of aggregates by filtration through 0.45-microns filters, and sucrose density centrifugation. PHF-enriched fractions contained two to five proteins of 57-68 kDa that displayed the same antigenic properties as PHFs. Since the 57- to 68-kDa PHF proteins are antigenically related to tau proteins, they are similar to the tau proteins previously observed in NFTs. However, further analysis revealed that PHF-associated tau can be distinguished from normal, soluble tau by PHF antibodies that do not recognize human adult tau and by one- and two-dimensional PAGE.
Opioids from immunocytes interact with receptors on sensory nerves to inhibit nociception in inflammation.
Authors: Stein C , Hassan A H , Przewłocki R , Gramsch C , Peter K , Herz A
Published: May 2006

Exogenous opioids can produce localized opioid receptor-mediated antinociception in peripheral inflamed tissue. Previous studies show that activation of endogenous opioids by a cold water swim in rats with hind paw inflammation results in a similar local antinociceptive effect but suggest that pituitary-adrenal opioid pools are not directly involved in producing this effect. Here we show increased amounts of opioid peptides in immune cells infiltrating the inflamed tissue. Furthermore, we demonstrate immunoreactive opioid receptors on peripheral terminals of sensory neurons. The local administration of antibodies against opioid peptides or receptors or systemic pretreatment with the immunosuppressant cyclosporine blocks cold water swim-induced antinociception. These findings suggest that antinociception in inflammation can be brought about by endogenous opioids from immune cells interacting with opioid receptors on peripheral sensory nerves.
Human homolog of the mouse sperm receptor.
Authors: Chamberlin M E , Dean J
Published: May 2006

The human zona pellucida, composed of three glycoproteins (ZP1, ZP2, and ZP3), forms an extracellular matrix that surrounds ovulated eggs and mediates species-specific fertilization. The genes that code for at least two of the zona proteins (ZP2 and ZP3) cross-hybridize with other mammalian DNA. The recently characterized mouse sperm receptor gene (Zp-3) was used to isolate its human homolog. The human homolog spans approximately 18.3 kilobase pairs (kbp) (compared to 8.6 kbp for the mouse gene) and contains eight exons, the sizes of which are strictly conserved between the two species. Four short (8-15 bp) sequences within the first 250 bp of the 5' flanking region in the human Zp-3 homolog are also present upstream of mouse Zp-3. These elements may modulate oocyte-specific gene expression. By using the polymerase chain reaction, a full-length cDNA of human ZP3 was isolated from human ovarian poly(A)+ RNA and used to deduce the structure of human ZP3 mRNA. Certain features of the human and mouse ZP3 transcripts are conserved. Both have unusually short 5' and 3' untranslated regions, both contain a single open reading frame that is 74% identical, and both code for 424 amino acid polypeptides that are 67% the same. The similarity between the two proteins may define domains that are important in maintaining the structural integrity of the zona pellucida, while the differences may play a role in mediating the species-specific events of mammalian fertilization.