bluuebunny/crossref_metadata_2025 huggingface dataset:
24- and 26-homo-1,25-dihydroxyvitamin D3: preferential activity in inducing differentiation of human leukemia cells HL-60 in vitro.
Authors: Ostrem V K , Tanaka Y , Prahl J , DeLuca H F , Ikekawa N
Published: May 2006

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D3, promotes the differentiation of HL-60 human promyelocytic leukemia cells into monocytes. Differentiation changes include the induction of phagocytosis, the initiation of nitroblue tetrazolium-reducing activity, and the appearance of nonspecific acid esterase. We have found that the 24-homo- and 26-homo-1,25-dihydroxyvitamin D3 and their delta 22 analogues are 10-fold more potent than 1,25-dihydroxyvitamin D3 in inducing differentiation of HL-60 cells in vitro. In vivo, these analogues show activity similar to 1,25-dihydroxyvitamin D3 in stimulating intestinal calcium transport in vitamin D-deficient rats. The 24-homoanalogues are significantly less active, whereas the 26-homo derivatives are more active than the natural hormone in mobilizing calcium from bone. This unusual activity pattern cannot be explained on the basis of the affinity of these analogues for the 1,25-dihydroxyvitamin D3 intracellular receptor: both 24-homo- and 26-homo-1,25-dihydroxyvitamin D3 have the same effectiveness as 1,25-dihydroxyvitamin D3 in displacing the tritiated hormone from its receptor in rat intestine or HL-60 cells. These analogues of 1,25-dihydroxyvitamin D3 may be of some interest as possible therapeutic substances, or as tools in understanding the action of 1,25-dihydroxyvitamin D3 in inducing differentiation.
Floral determination in internode tissues of day-neutral tobacco first occurs many nodes below the apex
Authors: Singer Susan R. , McDaniel Carl N.
Published: May 2006

The temporal and spatial patterns of floral determination in cultured internode segments of
Nicotiana tabacum
L. cv. Wisconsin 38 were investigated. Segments from internodes, when cultured on hormone-free medium, either produced some
de novo
floral shoots or produced only vegetative shoots. Segments that produced some floral shoots were considered to contain florally determined tissues. The first internode tissues to exhibit floral determination were located 24 nodes (about 15 cm) below the terminal meristem on plants that had 21 leaves of length ≥3 cm (i.e., plants of age 21). Since the terminal meristem was morphologically vegetative at this age, floral determination in internode tissues did not result from a signal emanating from floral structures. As the plant continued growing, internode tissues progressively higher on the main axis gained the capacity to produce
de novo
floral shoots in culture. These data indicate that the inductive signal which elicited the state of floral determination in internode tissues was active in the upper half of the plant beginning at about age 21.
Hereditary thrombophilia: identification of nonsense and missense mutations in the protein C gene.
Authors: Romeo G , Hassan H J , Staempfli S , Roncuzzi L , Cianetti L , Leonardi A , Vicente V , Mannucci P M , Bertina R , Peschle C
Published: May 2006

The structure of the gene for protein C, an anticoagulant serine protease, was analyzed in 29 unrelated patients with hereditary thrombophilia and protein C deficiency. Gene deletion(s) or gross rearrangement(s) was not demonstrable by Southern blot hybridization to cDNA probes. However, two unrelated patients showed a variant restriction pattern after Pvu II or BamHI digestion, due to mutations in the last exon: analysis of their pedigrees, including three or seven heterozygotes, respectively, with approximately 50% reduction of both enzymatic and antigen level, showed the abnormal restriction pattern in all heterozygous individuals, but not in normal relatives. Cloning of protein C gene and sequencing of the last exon allowed us to identify a nonsense and a missense mutation, respectively. In the first case, codon 306 (CGA, arginine) is mutated to an inframe stop codon, thus generating a new Pvu II recognition site. In the second case, a missense mutation in the BamHI palindrome (GGATCC----GCATCC) leads to substitution of a key amino acid (a tryptophan to cysteine substitution at position 402), invariantly conserved in eukaryotic serine proteases. These point mutations may explain the protein C-deficiency phenotype of heterozygotes in the two pedigrees.
Somatic diversification of S107 from an antiphosphocholine to an anti-DNA autoantibody is due to a single base change in its heavy chain variable region.
Authors: Giusti A M , Chien N C , Zack D J , Shin S U , Scharff M D
Published: May 2006

The S107 myeloma cell line expresses the germ-line sequence of the T15 antiphosphocholine (P-Cho) antibody, which is the major antibody made by BALB/c mice in response to P-Cho, either on a variety of bacterial polysaccharides or when attached to a protein carrier. We have previously reported that a somatic mutant of the S107 cell line produces an antibody that has lost the ability to bind P-Cho and has acquired binding for double-stranded DNA. This antibody has a substitution of an alanine for a glutamic acid at residue 35 in the heavy chain variable region. We now show that this amino acid substitution is due to a single A-C transversion, which is the only nucleotide change in the heavy and light chain variable regions. Further, it appears that this change is due to somatic mutation rather than to gene conversion.
Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains.
Authors: Perry G , Friedman R , Shaw G , Chau V
Published: May 2006

Neurofibrillary tangles (NFT) and neurites associated with senile plaques (SP) in Alzheimer disease-affected brain tissues were specifically immunostained with affinity-purified antibody preparations directed against ubiquitin. In addition, a class of neurites seen in brain regions containing NFT and SP were also specifically stained. Cross-reactivity of the ubiquitin antisera for tau protein, neurofilament proteins, and high molecular weight microtubule-associated proteins (MAPs) were ruled out by (i) the inability of the ubiquitin antisera to stain these proteins in immunoblotting experiments and (ii) the inability of tau, neurofilament, and MAP preparations, when preincubated with the ubiquitin antisera, to inhibit the selective neurofibrillar staining observed. Our results are consistent with the suggestion that ubiquitin is covalently associated with the insoluble neurofibrillary material of NFT and SP. We propose that the ubiquitin-mediated degradative pathway may be ineffective in removing these fibrillar structures in Alzheimer disease brain.
Site of anticonvulsant action on sodium channels: autoradiographic and electrophysiological studies in rat brain.
Authors: Worley P F , Baraban J M
Published: May 2006

The anticonvulsants phenytoin and carbamazepine interact allosterically with the batrachotoxin binding site of sodium channels. In the present study, we demonstrate an autoradiographic technique to localize the batrachotoxin binding site on sodium channels in rat brain using [3H]batrachotoxinin-A 20-alpha-benzoate (BTX-B). Binding of [3H]BTX-B to brain sections is dependent on potentiating allosteric interactions with scorpion venom and is displaced by BTX-B (Kd approximately 200 nM), aconitine, veratridine, and phenytoin with the same rank order of potencies as described in brain synaptosomes. The maximum number of [3H]BTX-B binding sites in forebrain sections (approximately 1 pmol/mg of protein) also agrees with biochemical determinations. Autoradiographic localizations indicate that [3H]BTX-B binding sites are not restricted to cell bodies and axons but are present in synaptic zones throughout the brain. For example, a particularly dense concentration of these sites in the substantia nigra is associated with afferent terminals of the striatonigral projection. By contrast, myelinated structures possess much lower densities of binding sites. In addition, we present electrophysiological evidence that synaptic transmission, as opposed to axonal conduction, is preferentially sensitive to the action of aconitine and veratridine. Finally, the synaptic block produced by these sodium channel activators is inhibited by phenytoin and carbamazepine at therapeutic anticonvulsant concentrations. Thus, these anticonvulsants may limit seizure spread not only by affecting all-or-none conduction by axonal sodium channels but also by modulating graded aspects of synaptic transmission.
Structural similarity of bovine lung prostaglandin F synthase to lens epsilon-crystallin of the European common frog.
Authors: Watanabe K , Fujii Y , Nakayama K , Ohkubo H , Kuramitsu S , Kagamiyama H , Nakanishi S , Hayaishi O
Published: May 2006

Cloned cDNA sequences specific for prostaglandin F (PGF) synthase have been isolated from a cDNA library of bovine lung mRNA sequences. Nucleotide-sequence analyses of cloned cDNA inserts have revealed that PGF synthase consists of a 969-base pair open reading frame coding for a 323-amino acid polypeptide with a Mr of 36,666. The sequence analysis indicates that bovine lung PGF synthase shows 62% identical plus conservative substitutions compared with human liver aldehyde reductase [Wermuth, B., Omar, A., Forster, A., Francesco, C., Wolf, M., Wartburg, J.P., Bullock, B. & Gabbay, K.H. (1987) in Enzymology and Molecular Biology of Carbonyl Metabolism: Aldehyde Dehydrogenase, Aldo-Keto Reductase, and Alcohol Dehydrogenase, eds. Weiner, H. & Flynn, T.G. (Liss, New York), pp. 297-307], which is similar to PGF synthase in molecular weight and substrate specificity. However, comparison of the amino acid sequence of PGF synthase with the National Biomedical Research Foundation protein data base reveals that the sequences of 225 amino acids from C termini of epsilon-crystallin of the European common frog (Rana temporaria) [Tomarev, S.I., Zinovieva, R.D., Dolgilevich, S.M., Luchin, S.V., Krayev, A.S., Skryabin, K.G. & Gause, G.G. (1984) FEBS Lett. 171, 297-302] and of PGF synthase show 77% identical and conservative substitutions without deletions/additions. The result suggests that European common frog lens epsilon-crystallin is identical to bovine lung PGF synthase.
X-ray Laue diffraction from crystals of xylose isomerase.
Authors: Farber G K , Machin P , Almo S C , Petsko G A , Hajdu J
Published: May 2006

The Laue method (stationary crystal, polychromatic x-rays) was used to collect native and heavy-atom-derivative data on crystals of xylose isomerase (EC 5.3.1.5). These data were used to find the heavy-atom positions. The positions found by use of Laue data are the same as those found by use of monochromatic data collected on a diffractometer. These results confirm that Laue diffraction data sets, which can be obtained on a millisecond time scale, can be used to locate small molecules bound to protein active sites. The successful determination of heavy-atom positions also indicates that x-ray crystallographic data collected by the Laue method can be used to solve protein structures.
Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells.
Authors: Grosovsky A J , de Boer J G , de Jong P J , Drobetsky E A , Glickman B W
Published: May 2006

The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage.
Somatic and germ-line reverse mutation rates of the retrovirus-induced dilute coat-color mutation of DBA mice.
Authors: Seperack P K , Strobel M C , Corrow D J , Jenkins N A , Copeland N G
Published: May 2006

At present, the dilute (dv) coat-color mutation of DBA mice provides the only simple means for measuring the relative somatic and germ-line reverse mutation rates of retrovirus-induced mutations in mammals. The dv mutation was generated by the spontaneous integration of an ecotropic murine leukemia virus into noncoding sequences of the dilute locus. Reversion of the dv mutation occurs by provirus excision and is mediated by homologous recombination events involving the viral long terminal repeat sequences. Although numerous independent germ-line d+ revertants have been identified, somatic d+ revertants have not been reported previously. During the past 5 years, we have screened more than one million mice homozygous for the dv mutation to determine whether we could identify somatic d+ revertants. This survey has resulted in the identification of a somatic d+ revertant and has provided a data base from which we can estimate the relative somatic and germ-line excision frequencies of retroviruses in mice and speculate about the nature of homologous recombination events producing d+ revertant alleles.
Olfaction by melanophores: what does it mean?
Authors: Lerner M R , Reagan J , Gyorgyi T , Roby A
Published: May 2006

Hypotheses on general olfaction can be divided into two broad groups: those that predict the existence of olfactory-specific olfactory receptor proteins and those that do not. Recently, much attention has been paid to the discovery of an odorant-stimulated adenylate cyclase in purified olfactory cilia. This finding has, for the most part, been accepted as evidence that the former hypotheses are correct. Here we report that frog melanophores, which are nonolfactory in nature, disperse their melanosomes in response to the same types and concentrations of odorants used in the investigations of olfactory cilia and that pigment dispersion is accompanied by rises in intracellular cAMP levels. The effects show that the existence of a cAMP-based second messenger system in olfactory cilia is not in itself proof of the existence of olfactory-specific olfactory receptor proteins. Also they explain the basis of Ottoson's pioneering work of 30 years ago on the electrical responses of frog olfactory epithelium to stimulation with alcohols. The results suggest that there could be two mechanisms that are important for the detection of odorants: one based on specific receptors, the other nonspecific, but both working through activation of cAMP.
Identification of a truncated form of the nerve growth factor receptor.
Authors: DiStefano P S , Johnson E M
Published: May 2006

Schwann cells express growth factor (NGF) receptors on their cell surface in response to axotomy, a phenomenon that can be demonstrated both in vivo and in vitro. The predominant form of the NGF receptor on Schwann cells exists as an approximately equal to 80-kDa band, as determined by NaDodSO4/PAGE. We demonstrate that cultured Schwann cells shed a truncated (50-kDa) form of the NGF receptor (NGF-Rt) into their medium. Other cell types that shed the NGF-Rt into medium include a rat schwannoma and, to a lesser extent, PC12 cells and superior cervical ganglion neurons. NGF-Rt was not found in media conditioned by mixed neuron/glia cultures from various brain regions, or anterior pituitary cells derived from rat. In vivo, NGF-Rt was present in neonatal rat urine, and its presence was developmentally regulated: levels were high in postnatal day-1 rat urine and declined to low, but detectable, levels by weeks 4 and 8. NGF-Rt was also found in amniotic fluid and in the stomach contents of fetal rats. Maternal urine (pre- and postnatal) had slightly elevated NGF-Rt levels over normal adult urine. NGF-Rt was detected in rat plasma and showed developmental regulation similar to that found for urine. In addition, a 77-kDa receptor species was detected in plasma during early development. Finally, NGF-Rt was significantly elevated in the urine of adult rats with bilateral sciatic nerve lesions. These findings suggest that the developmentally regulated release of NGF-Rt, present in plasma and other body fluids, plays a regulatory role in nervous system development.
Electrical coupling between rods and cones in the tiger salamander retina.
Authors: Wu S M , Yang X L
Published: May 2006

Electrical coupling between rods and cones was studied in the salamander (Ambystoma tigrinum) retina by measuring the light responses and spectral sensitivities of rods and cones and by measuring the voltage responses from a rod to current pulses injected into a cone. A population of 10-20% of the photoreceptors exhibited a mixed-response waveform of the rod and the cone under dark-adapted conditions, and a response waveform closely resembled that of a cone in the presence of background illumination. Lucifer yellow injection revealed that these cells are morphologically identical to rods, and thus they are named rodcs. Dark-adapted rodcs exhibited a rod-like spectral sensitivity with a peak at approximately 520 nm that shifted to a cone-like spectral sensitivity with a peak at approximately 620 nm in response to background light (Purkinje shift). The voltage response of a rodc to a -1-nA current step injected into an adjacent cone is approximately 3.6 times larger than that of a rod to the same current step. These results indicate that there is a population of rods (rodcs) in the tiger salamander retina that is strongly coupled to the cones and that these cells allow significant mixture of rod and cone signals at the photoreceptor level.
Immunodetection of estrogen receptor in epithelial and stromal tissues of neonatal mouse uterus.
Authors: Korach K S , Horigome T , Tomooka Y , Yamashita S , Newbold R R , McLachlan J A
Published: May 2006

The tissue distribution and levels of estrogen receptor in neonatal mouse uterine tissue were determined in epithelial and stromal fractions separated by mild enzymatic treatment. Proteins of the isolated fractions were separated by gel electrophoresis and receptor was detected on immunoblots with monoclonal antibody H-222. Estrogen receptor protein was detectable in samples of reproductive tract tissue from 5- and 10-day-old mice. The level of receptor in 5-day-old animals was lower per unit DNA in epithelial cells than in stroma. Receptor levels were increased in both tissue types after treatment with diethylstilbestrol, but not with progesterone. Receptor protein present in these neonatal tissues was able to bind steroid as evidenced by affinity labeling with tamoxifen aziridine. Immunohistochemistry on sections of uteri from 4- and 10-day-old mice confirmed the biochemical results and indicated lower nuclear straining in epithelial cells than in stromal cells of uteri of 4-day-old mice. These results demonstrated that estrogen receptor protein is present in both epithelium and stroma of the neonatal mouse uterus, but at a higher level in stromal cells.
Hairy roots are more sensitive to auxin than normal roots
Authors: Shen Wen Hui , Petit Annik , Guern Jean , Tempé Jacques
Published: May 2006

Responses to auxin of
Lotus corniculatus
root tips or protoplasts transformed by
Agrobacterium rhizogenes
strains 15834 and 8196 were compared to those of their normal counterparts. Three different types of experiments were performed, involving long-term, medium-term, or short-term responses to a synthetic auxin, 1-naphthaleneacetic acid. Root tip elongation, proton excretion by root tips, and transmembrane electrical potential difference of root protoplasts were measured as a function of exogenous auxin concentration. The sensitivity of hairy root tips or protoplasts to exogenous auxin was found to be 100-1000 times higher than that of untransformed material.
Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.
Authors: Gronwald R G , Grant F J , Haldeman B A , Hart C E , O'Hara P J , Hagen F S , Ross R , Bowen-Pope D F , Murray M J
Published: May 2006

The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately 5.7 kb) and a minor (approximately 4.8 kb) transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an approximately equal to 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF (i.e., PDGF dimers composed of two B chains or an A and a B chain). Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.
ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells.
Authors: Horio M , Gottesman M M , Pastan I
Published: May 2006

Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the "MDR1" gene, whose product is the plasma membrane P-glycoprotein. Plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulate [3H]vinblastine in an ATP-dependent manner. This transport is osmotically sensitive, with an apparent Km of 38 microM for ATP and of approximately equal to 2 microM for vinblastine. The nonhydrolyzable analog adenosine 5'-[beta, gamma-imido]triphosphate does not substitute for ATP but is a competitive inhibitor of ATP for the transport process. Vanadate, an ATPase inhibitor, is a potent noncompetitive inhibitor of transport. These results indicate that hydrolysis of ATP is probably required for active transport of vinblastine. Several other drugs to which multidrug-resistant cell lines are resistant inhibit transport, with relative potencies as follows: vincristine greater than actinomycin D greater than daunomycin greater than colchicine = puromycin. Verapamil and quinidine, which reverse the multidrug-resistance phenotype, are good inhibitors of the transport process. These results confirm that multidrug-resistant cells express an energy-dependent plasma membrane transporter for hydrophobic drugs, and establish a system for the detailed biochemical analysis of this transport process.
Suppression of the Escherichia coli ssb-1 mutation by an allele of groEL.
Authors: Ruben S M , VanDenBrink-Webb S E , Rein D C , Meyer R R
Published: May 2006

A series of spontaneous suppressors to the temperature-sensitive phenotype of the single-stranded DNA-binding protein mutation ssb-1 were isolated. A genomic library of EcoRI fragments from one of these suppressor strains was prepared by using pBR325 as the cloning vector. A 10.0-kilobase class of inserts was identified as carrying the ssb-1 gene itself. A second class of 8.3-kilobase inserts was shown to contain the groE region by (i) restriction analysis, (ii) Southern hybridization of the 8.3-kilobase insert to groE+ DNA, and (iii) identification of the gene products by similar migration on polyacrylamide gels. Subcloning demonstrated that an intact mutant groEL gene was necessary for suppression and that plasmids carrying the 8.3-kilobase insert could suppress mutants carrying groES- but not groEL- genes for phage lambda growth. The suppressor, designated as groEL411, was specific for the ssb-1 allele. In ssb-1 groEL411 cells, DNA synthesis stopped after a shift to 42.5 degrees C but rapidly recovered within minutes. The data suggest a direct interaction between the single-stranded DNA-binding protein and GroEL proteins in DNA replication.
Dynamics of trimethoprim bound to dihydrofolate reductase.
Authors: Searle M S , Forster M J , Birdsall B , Roberts G C , Feeney J , Cheung H T , Kompis I , Geddes A J
Published: May 2006

The conformation of a small molecule in its binding site on a protein is a major factor in the specificity of the interaction between them. In this paper, we report the use of 1H and 13C NMR spectroscopy to study the fluctuations in conformation of the anti-bacterial drug trimethoprim when it is bound to its "target," dihydrofolate reductase. 13C relaxation measurements reveal dihedral angle changes of +/- 25 degrees to +/- 35 degrees on the subnanosecond time scale, while 13C line-shape analysis demonstrates dihedral angle changes of at least +/- 65 degrees on the millisecond time scale. 1H NMR shows that a specific hydrogen bond between the inhibitor and enzyme, which is believed to make an important contribution to binding, makes and breaks rapidly at room temperature.
Positive regulation of pertussis toxin expression.
Authors: Gross R , Rappuoli R
Published: May 2006

Although the genus Bordetella contains several closely related species, pertussis toxin (PT) is produced only by phase I Bordetella pertussis. In this work we have studied the regulation of expression of the PT operon and investigated why PT is produced by phase I and not by phase III B. pertussis despite the presence of the PT genes. We have constructed a vector for Bordetella species that contains the PT promoter fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene, and we have used it to identify the regulatory elements involved in the transcription of the PT operon. Efficient transcription of these genes requires at least two features: (i) the 170-base-pair DNA sequence upstream from the start site of transcription and (ii) a trans-activating factor encoded by the vir locus. Bordetella parapertussis and Bordetella bronchiseptica, although endowed with a functional trans-activating system, do not produce PT because of mutations within their PT promoter regions. In contrast, phase III Bordetella species do not show any trans activity.
Release of vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin-(1-7) heptapeptide.
Authors: Schiavone M T , Santos R A , Brosnihan K B , Khosla M C , Ferrario C M
Published: May 2006

We have recently shown that hydrolysis of labeled angiotensin I in canine brainstem homogenate causes a rapid accumulation of the heptapeptide angiotensin-(1-7) [Ang-(1-7)]. Although this angiotensin fragment has no vasopressor activity, its consistent generation in brain homogenate led us to study its potential neurosecretory effects in the rat hypothalamo-neurohypophysial system (HNS) in vitro. Ang-(1-7) or angiotensin II (Ang II) was added to HNS perifusate in concentrations of 0.04, 0.4, and 4 microM, and release of arginine vasopressin (AVP) during each treatment was quantified as a percentage of the AVP release detected in the preceding collection period. Base-line release of AVP averaged 281 +/- 47 pg per 15 min (mean +/- SEM) in HNS explants (five experiments, five explants per chamber) perifused in Krebs solution at 37 degrees C, after a 1-hr equilibration period. At 0.04 microM, Ang II or Ang-(1-7) did not stimulate AVP release. Ang II increased AVP release over the control value by 172% +/- 44% and 268% +/- 66% at 0.4 and 4 microM, respectively; the same concentrations of Ang-(1-7) increased AVP release by 134% +/- 12% and 216% +/- 45%. The responses to Ang II and Ang-(1-7) at the highest concentration were both significant (P less than 0.05), and comparison by two-way analysis of variance indicated that Ang II and Ang-(1-7) were equipotent in stimulating AVP release over the range of concentrations studied. In the presence of the competitive Ang II antagonist [Sar1,Thr8]Ang II (20 microM), the release of AVP increased approximately equal to 2-fold. Neither Ang II nor Ang-(1-7) (4 microM) caused a further enhancement of AVP release in the presence of [Sar1,Thr8]Ang II. These data suggest that a hydrophobic residue in position 8 of the angiotensin peptide is not essential for activation of angiotensin receptors in the rat HNS. Moreover, the equipotence of Ang II and Ang-(1-7) indicates that Ang-(1-7) may participate in the control of AVP release.
Reduced CO2/O2 specificity of ribulose-bisphosphate carboxylase/oxygenase in a temperature-sensitive chloroplast mutant of Chlamydomonas.
Authors: Chen Z X , Chastain C J , Al-Abed S R , Chollet R , Spreitzer R J
Published: May 2006

The Chlamydomonas reinhardtii chloroplast mutant 68-4PP is phenotypically indistinguishable from wild type at 25 degrees C but fails to grow photosynthetically at 35 degrees C. It had about 30% of the wild-type level of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) holoenzyme and carboxylase activity when grown at 25 degrees C, but less than 15% when grown at 35 degrees C. Pulse-labeling with 35S showed that the decrease in enzyme level at the restrictive temperature was not a result of reduced synthesis of enzyme subunits. The CO2/O2 specificity factor (VCKO/VOKC, where VC and VO are Vmax values for carboxylation and oxygenation and KC and KO are Km values for CO2 and O2) of the mutant enzyme was found to be significantly less than that of the wild-type enzyme (54 +/- 2 and 62 +/- 1, respectively), and this alteration was accompanied by increases in KO and KC and a decrease in VC/VO. DNA sequencing revealed a single missense mutation in the 68-4PP chloroplast large-subunit gene. This mutation causes leucine to be replaced by phenylalanine at position 290 in the large-subunit polypeptide sequence. These results (i) support previous studies that implicated this region of the large subunit as an important structural component of the enzyme's function and (ii) demonstrate that chloroplast genetic modification of the CO2/O2 specificity factor of a plant-type carboxylase/oxygenase is feasible.
Nuclear posttranscriptional processing of thymidine kinase mRNA at the onset of DNA synthesis.
Authors: Gudas J M , Knight G B , Pardee A B
Published: May 2006

The posttranscriptional regulatory mechanism(s) underlying thymidine kinase (TK) mRNA accumulation was investigated in BALB/c 3T3 cells during their progression from G0 into S phase of the cell cycle. Very little TK mRNA could be detected in either the nuclear or the cytoplasmic compartment from cells harvested in G0 or G1. At the onset of S phase, however, the level of nuclear TK mRNA precursors and mature TK mRNAs increased dramatically. The high molecular weight TK heterogeneous nuclear RNA species detected in the nuclei of S-phase cells were polyadenylylated and hybridized to intron sequences derived from the TK gene. A series of high molecular weight precursors could be chased to lower molecular weight species in the presence of actinomycin D, suggesting an ordered removal of intron sequences with the kinetics of a precursor-product relationship. These results demonstrate a striking change in the nuclear posttranscriptional processing of TK heterogeneous nuclear RNA at the G1-S boundary and, furthermore, define a model system for the examination of RNA-processing events in vivo.
Hox-5.1 defines a homeobox-containing gene locus on mouse chromosome 2.
Authors: Featherstone M S , Baron A , Gaunt S J , Mattei M G , Duboule D
Published: May 2006

We have isolated a murine homeobox-containing gene, Hox-5.1, by virtue of its relatedness to the Hox-1.4 gene. In situ hybridization to metaphase spreads mapped Hox-5.1 to band D of mouse chromosome 2. Sequence comparisons indicate that Hox-5.1 is the murine homolog of the human C13 homeobox-containing gene. Hox-5.1 also bears significant similarity to the Xenopus Xhox-1A homeobox-containing gene and the Drosophila deformed homeotic gene at N-terminal and homeobox regions. Hox-5.1 transcripts were detected in mouse embryos, in adult mouse testis, kidney, heart, and intestine, and in mouse embryonal carcinoma cells treated with retinoic acid. In situ hybridization to sections from whole mouse embryos revealed Hox-5.1 expression in spinal cord and prevertebrae.
Wheat germ agglutinin-positive cells in a stem cell-enriched fraction of mouse bone marrow have potent natural suppressor activity.
Authors: Sugiura K , Inaba M , Ogata H , Yasumizu R , Inaba K , Good R A , Ikehara S
Published: May 2006

In the present study we have characterized natural suppressor (NS) cells, which nonspecifically suppress mitogen responses and mixed-lymphocyte reaction. The strongest NS activity was found in a fraction of relatively low-density cells (1.063 less than p less than 1.075) obtained by equilibrium density centrifugation. Further purification and characterization of these NS cells by using a fluorescence-activated cell sorter indicated that wheat germ agglutinin (WGA)-positive cells have potent NS activity, whereas WGA-negative cells have no NS activity. Spleen colony-forming unit (CFU-S) assays demonstrated a significant correlation between the number of CFU-S cells and the NS activity in the bone marrow. However, WGA-positive cells obtained from the bone marrow of animals treated with 5-fluorouracil, which cells in turn were exposed to 5-fluorouracil, showed a marked reduction in NS activity. These results suggest that the pluripotent stem cells have NS activity when the cells are in the cycling phase but not when the cells are in the G0 phase. It seems possible that apparently primitive cells play an important role in down-regulation of immune responses.
Orientation-dependent function of a short CYC1 DNA fragment in directing mRNA 3' end formation in yeast.
Authors: Ruohola H , Baker S M , Parker R , Platt T
Published: May 2006

We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYC1 mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYC1 fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation of this fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate less than 40% of the control level of full-length mRNA and reveal the presence of a short, stable (approximately equal to 300 nucleotides) poly(A) RNA that represents 60-70% of the transcripts originating from the same promoter. The reverse orientation of the insert allows near-normal levels of full-length mRNA. Mapping of the 3' end of the truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYC1 transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.
Molecular and biological interaction between major histocompatibility complex class I antigens and luteinizing hormone receptors or beta-adrenergic receptors triggers cellular response in mice.
Authors: Solano A R , Cremaschi G , Sánchez M L , Borda E , Sterin-Borda L , Podestá E J
Published: May 2006

Purified IgG from BALB/c mouse anti-C3H serum exerts positive inotropic and chronotropic effects in C3H mouse atria and induces testosterone synthesis in C3H mouse Leydig cells. The effect depends on IgG concentration and can be abolished by beta-adrenergic-receptor and luteinizing hormone-receptor antagonists. IgG interferes with the binding of dihydroalprenolol and luteinizing hormone. Monoclonal antibodies against major histocompatibility complex class I antigens were active on the Leydig cells of C3H and BALB/c mice. There was a parallelism between the effect of each individual monoclonal antibody with specificity for a particular haplotype and the response of the target cell from the strains carrying such haplotypes. These antibodies could precipitate the soluble luteinizing hormone-receptor complex. The results suggested that bound hormone triggers the association of major histocompatibility class I antigen with the receptor, thereby activating the respective target cells.
Infusions of recombinant human interleukins 1 alpha and 1 beta cause hypercalcemia in normal mice.
Authors: Sabatini M , Boyce B , Aufdemorte T , Bonewald L , Mundy G R
Published: May 2006

The immune cell products interleukins 1 alpha and -beta, which stimulate osteoclast activity in vitro, are among the most potent bone resorbing factors so far described. Although it appears likely that these cytokines are involved in regulation of trabecular bone turnover, nothing is known of their effects on extracellular fluid calcium concentration. In this report, we show the effects of 72-hr subcutaneous infusions of interleukins 1 alpha and -beta on plasma calcium and bone morphology in mice. Both interleukins 1 caused a marked dose-dependent increase in the plasma calcium. In higher doses, which cause the animals to die, the plasma calcium fell preterminally. Quantitative histomorphometry of bone sections showed evidence of increased numbers of osteoclasts and bone resorption surfaces. The effects were similar to those obtained with infusions of parathyroid hormone. These data suggest that in addition to its potential influence on trabecular bone volume, interleukin 1 may also modulate extracellular fluid calcium homeostasis under conditions in which it is produced excessively.
Two mutant alleles of the human cytochrome P-450db1 gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs.
Authors: Skoda R C , Gonzalez F J , Demierre A , Meyer U A
Published: May 2006

The "debrisoquine polymorphism" is a clinically important genetic defect of drug metabolism affecting 5-10% of individuals in Caucasian populations. It is inherited as an autosomal recessive trait. A full-length cDNA for human cytochrome P-450db1, the deficient enzyme (also designated P450IID1 for P450 family II subfamily D isozyme 1), has recently been cloned. Leukocyte DNA from "extensive metabolizers" (EMs) or "poor metabolizers" (PMs) of debrisoquine was examined by Southern analysis. Two polymorphic restriction fragments were associated with the PM phenotype when DNAs from 24 unrelated PM and 29 unrelated EM individuals were probed with P-450db1 cDNA after digestion with Xba I restriction endonuclease and Southern blotting: a polymorphic 44-kilobase (kb) fragment was found in 58% of PMs but only in 3.4% of EMs, and a polymorphic 11.5-kb fragment was present in 33% of PMs but in none of the EMs. Seventy-five percent of PMs had either the 44-kb or the 11.5-kb fragment or both. Segregation of these restriction fragment length polymorphisms in the families of six PM probands demonstrated that each of the two fragments is allelic with the 29-kb fragment present in all EM individuals and suggests that they identify two independent mutated allels of the P-450db1 gene (designated P450C2D1). At least a third mutated allele not detected by these restriction fragment length polymorphisms must be present in the population. The Xba I 44-kb fragment and 11.5-kb fragment were in linkage disequilibrium with restriction fragment length polymorphisms generated by four and five additional restriction endonucleases, respectively, which can be used to identify the same mutant alleles for the P-450db1 gene.
Multiple repeated units in Drosophila melanogaster ribosomal DNA spacer stimulate rRNA precursor transcription.
Authors: Grimaldi G , Di Nocera P P
Published: May 2006

Drosophila melanogaster ribosomal DNA (rDNA) transcriptional units are separated by nontranscribed spacer (NTS) segments consisting of tandemly arranged repeats 95, 330, and 240 base pairs long. NTS sequences stimulate transcription from the rRNA precursor (pre-rRNA) promoter. Primer extension analysis of RNA from cells cotransfected with plasmids carrying NTS sequences of various lengths shows that the activity of the pre-rRNA promoter is directly correlated with the number of 240-base-pair repeats; NTS sequences upstream of these units also stimulate pre-rRNA transcription. The NTS effect might depend upon transcription from duplicated promoters present within the 240- and 330-base-pair repeats. The strength of the pre-RNA promoter correlates in each construct with the level of spacer transcription. The action of spacer sequences, although able to take place over a large distance, is not independent of orientation: stimulation of pre-rRNA transcription is abolished in plasmids carrying inverted NTS segments. Removal of a putative transcription termination site located upstream of the pre-rRNA promoter has no effect on pre-rRNA initiation nor does it substantially alter spacer enhancement.
Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides.
Authors: Goodchild J , Agrawal S , Civeira M P , Sarin P S , Sun D , Zamecnik P C
Published: May 2006

Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.
Transcription complexes that program Xenopus 5S RNA genes are stable in vivo.
Authors: Darby M K , Andrews M T , Brown D D
Published: May 2006

The long-term stability of transcription complexes on 5S RNA genes has been demonstrated in vivo. Complexes on oocyte and somatic-type 5S RNA genes injected into Xenopus laevis oocyte nuclei are stable for at least 4 days. Tissue culture cells and mature erythrocytes have equivalent numbers of somatic 5S RNA genes programmed into transcription complexes, yet the former cell type has a greater than 50-fold higher cellular content of transcription factor IIIA (TFIIIA). Functional transcription complexes on somatic 5S RNA genes in nucleated erythrocytes of Xenopus are stable for weeks, perhaps months, even though a mature erythrocyte has less than two molecules of TFIIIA for each somatic 5S RNA gene. These findings strengthen our proposal that stable transcription complexes are a means of maintaining the differentiated state.
Direct action of the parathyroid hormone-like human hypercalcemic factor on bone.
Authors: Thompson D D , Seedor J G , Fisher J E , Rosenblatt M , Rodan G A
Published: May 2006

Peptides containing residues 1-34 of the human parathyroid hormone (PTH)-like hypercalcemic factor (hHCF), termed hHCF-(1-34)-NH2, produce effects similar to those of PTH in several biological systems in vitro and in vivo. However, there is conflicting evidence regarding the potency of hHCF on bone and, by implication, its role in calcium mobilization and the skeletal contribution to tumor-associated hypercalcemia. To resolve this conflict, the effects of infusing either hHCF-(1-34)-NH2 or a peptide containing residues 1-34 of bovine PTH [bPTH-(1-34)] into unrestrained thyroparathyroidectomized rats on a low calcium diet were compared. Direct effects on bone histology and serum calcium levels, which are totally dependent on calcium mobilization from bone in these animals, were examined. bPTH-(1-34) and hHCF-(1-34)-NH2 were equipotent in producing dose-dependent calcium mobilization from bone. At an infusion rate of 0.1 nmol/hr, both peptides produced hypercalcemia and extensive nephrocalcinosis. Histomorphometric analysis of tibiae from these animals after 48 hr of peptide infusion showed a dose-related increase in osteoclast number from 3-5 cells per mm2 at 0.01 nmol/hr to approximately equal to 32 cells per mm2 at 0.1 nmol/hr of hHCF or bovine PTH. These findings indicate that hHCF has a direct PTH-like effect on bone and, in this model system, the hHCF-(1-34)-NH2 is equipotent to bPTH-(1-34).
Formation of stable transcription complexes as assayed by analysis of individual templates.
Authors: Weintraub H
Published: May 2006

Conditions were established where transient transfection of two marker genes resulted in the expression of one or the other, but not both, in individual cells as assayed by immunofluorescence. Thus, the expression from a single cell reflects the activity of single active transcription templates. Under these conditions, a vector encoding the simian virus 40 large tumor antigen (SV40 T-Ag) driven by the SV40 enhancer and early promoter was transfected into CV-1, L, or HeLa cells yielding, for all three cell types, about 10-30% T-Ag-positive cells as assayed by immunofluorescence. Similar vectors containing either mutated or deleted SV40 enhancers also gave T-Ag-positive cells, but at about 1/100 the frequency. Quantitative analysis showed that T-Ag-positive cells produced about the same amount of T-Ag whether or not an active enhancer was present. Chloramphenicol acetyltransferase-encoding vectors gave the same result. The data are consistent with the hypothesis that at a low, but finite, probability, fully functional transcription complexes can form on a given active template in the absence of enhancer DNA. Enhancers seem to increase the number of active templates. Subcloning experiments suggest that these transcription complexes can be surprisingly stable.
Parathyroid hormone modulates transforming growth factor beta activity and binding in osteoblast-enriched cell cultures from fetal rat parietal bone.
Authors: Centrella M , McCarthy T L , Canalis E
Published: May 2006

Transforming growth factor beta (TGF-beta) is produced by bone cells, is abundant in bone matrix, and regulates bone cell biochemical processes. In osteoblast-enriched fetal rat parietal bone cell cultures, low TGF-beta doses increase DNA synthesis, whereas higher levels are less mitogenic, stimulate collagen production, and decrease alkaline phosphatase activity. Parathyroid hormone by itself has minimal effects on these processes, but it opposes the effects of TGF-beta and alters TGF-beta binding to its receptors in osteoblast-enriched cultures. Some functions ascribed to parathyroid hormone in bone may therefore result from alterations in TGF-beta activity, suggesting that the local effects of TGF-beta in bone are under systemic hormonal control.