bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mapping of the human major histocompatibility complex by pulsed-field gel electrophoresis.
Authors: Dunham I , Sargent C A , Trowsdale J , Campbell R D
Published: May 2006

Pulsed-field gel electrophoresis and "cosmid walking" have been used to establish a molecular map of the human major histocompatibility complex (MHC). We have isolated approximately equal to 230 kilobases (kb) of genomic DNA in overlapping cosmid clones covering the genes for the second and fourth components of complement (C2 and C4, respectively), factor B, and steroid 21-hydroxylase, and approximately equal to 82 kb of genomic DNA surrounding the genes for the tumor necrosis factors alpha and beta. Single-copy hybridization probes isolated from these cosmid clusters and probes for the known MHC gene loci were hybridized to Southern blots of genomic DNA that had been digested with infrequently cutting restriction endonucleases and separated on pulsed-field gels. The data obtained allowed the construction of a long-range genomic restriction map and indicated that the MHC spans 3800 kb. This map orients the MHC class III gene cluster with respect to the DR subregion; the C2 gene is on the telomeric side of the 21-hydroxylase B gene. In addition we have defined the positions of the genes for the tumor necrosis factors alpha and beta in the human MHC. Genes for the alpha chain of DR and 21-hydroxylase B are separated by at least 300 kb, while the distance between the genes for C2 and tumor necrosis factor alpha is 390 kb. The HLA-B locus lies approximately equal to 250 kb on the telomeric side of the tumor necrosis factor genes.
Subpopulations of MCF7 cells separated by Percoll gradient centrifugation: a model to analyze the heterogeneity of human breast cancer.
Authors: Resnicoff M , Medrano E E , Podhajcer O L , Bravo A I , Bover L , Mordoh J
Published: May 2006

Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.
Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes.
Authors: Lutz H U , Bussolino F , Flepp R , Fasler S , Stammler P , Kazatchkine M D , Arese P
Published: May 2006

Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3 antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which stimulate senescent erythrocytes with respect to bound antibody and complement.
Peroxisomes in wild-type and rosy mutant Drosophila melanogaster.
Authors: Beard M E , Holtzman E
Published: May 2006

This study shows that peroxisomes are abundant in the Malpighian tubule and gut of wild-type Oregon R Drosophila melanogaster and that the peroxisomal population of the rosy-506 eye-color mutant differs from that of the wild type. Catalase activity in wild-type flies is demonstrable in bodies of appearance and centrifugal behavior comparable to the peroxisomes of vertebrate tissues. Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.1.3.22) activity of the Malpighian tubule of wild-type flies is demonstrable cytochemically in bodies like those containing catalase. The rosy-506 mutant flies, with a deletion in the structural gene for xanthine dehydrogenase (xanthine:NAD+ oxidoreductase, EC 1.1.1.204), lack cytochemically demonstrable peroxisomal xanthine oxidase activity. In addition, peroxisomes in the rosy-506 mutants show less intense cytochemical staining for catalase than those in wild-type flies, and biochemical assays indicate that catalase in the rosy mutant is much more accessible to substrate in the absence of detergent than in the wild type. Thus, the rosy-506 mutation appears to affect peroxisomes and may mimic aspects of the defects of peroxisomes in some human metabolic disorders.
Myoglobin-mediated oxygen delivery to mitochondria of isolated cardiac myocytes.
Authors: Wittenberg B A , Wittenberg J B
Published: May 2006

Myoglobin-mediated oxygen delivery to intracellular mitochondria is demonstrated in cardiac myocytes isolated from the hearts of mature rats. Myocytes are held at high ambient oxygen pressure, 40-340 torr (5-45 kPa); sarcoplasmic myoglobin is fully oxygenated. In this condition oxygen availability does not limit respiratory rate; myoglobin-facilitated diffusion contributes no additional oxygen flux and, since oxygen consumption is measured in steady states, the storage function of myoglobin vanishes. Carbon monoxide, introduced stepwise, displaces oxygen from intracellular oxymyoglobin without altering the optical spectrum of the largely oxidized intracellular mitochondria. A large part, about one-third, of the steady-state oxygen uptake is abolished by carbon monoxide blockade of myoglobin oxygenation. The myoglobin-dependent component of the oxygen uptake decreases linearly with decreasing fraction of intracellular oxymyoglobin, with a slope near unity. Studies using inhibitors of mitochondrial electron transport indicate that myoglobin-delivered oxygen uptake depends on electron flow through the mitochondrial electron transport chain. We conclude that cardiac mitochondria accept two additive simultaneous flows of oxygen: a flow of dissolved oxygen to cytochrome oxidase and a flow of myoglobin-bound oxygen to a mitochondrial terminus. Myoglobin-mediated oxygen delivery supports ATP generation by heart cells at physiological ambient oxygen pressure.
Primate involucrins: antigenic relatedness and detection of multiple forms.
Authors: Parenteau N L , Eckert R L , Rice R H
Published: May 2006

Hominoid apes (gorilla, chimpanzee, orangutan, gibbon), Old World monkeys (rhesus, cynomolgus), New World monkeys (owl, cebus), and a prosimian (lemur) express involucrin-like proteins in cultured keratinocytes. Primate involucrins can be precipitated with trichloroacetic acid, resolubilized at pH 8, and subsequently retain aqueous solubility in 67% ethanol. Polyacrylamide gel electrophoresis of keratinocyte extracts after this rapid partial purification has revealed in each species tested one (chimpanzee, orangutan, gibbon) or two (gorilla, rhesus, owl, cebus) antigenically crossreactive proteins that migrate in the vicinity of human involucrin. In the species examined further (gorilla, chimpanzee, rhesus), poly(A)+ mRNA isolated from the cultures directed the cell-free translation of polypeptides with mobilities similar to those extracted from the cells. From five cynomolgus monkeys, three different electrophoretic profiles were obtained, suggesting the existence of different alleles. Quantitative comparisons by a sensitive enzyme-linked immunosorbent assay indicated that certain primate involucrins have a higher density of antigenic determinants than the human protein, whereas others lack some determinant(s). In contrast to those from other species, all of which showed substantial crossreactivity, the lemur protein was minimally immunoreactive by immunoblotting and not clearly detected by solid-phase assay. The electrophoretic and antigenic differences displayed throughout the primate order suggest that this protein has been subject to relatively rapid evolution.
cDNA sequence of a human skeletal muscle ADP/ATP translocator: lack of a leader peptide, divergence from a fibroblast translocator cDNA, and coevolution with mitochondrial DNA genes.
Authors: Neckelmann N , Li K , Wade R P , Shuster R , Wallace D C
Published: May 2006

We have characterized a 1400-nucleotide cDNA for the human skeletal muscle ADP/ATP translocator. The deduced amino acid sequence is 94% homologous to the beef heart ADP/ATP translocator protein and contains only a single additional amino-terminal methionine. This implies that the human translocator lacks an amino-terminal targeting peptide, a conclusion substantiated by measuring the molecular weight of the protein synthesized in vitro. A 1400-nucleotide transcript encoding the skeletal muscle translocator was detected on blots of total RNA from human heart, kidney, skeletal muscle, and HeLa cells by hybridization with oligonucleotide probes homologous to the coding region and 3' noncoding region of the cDNA. However, the level of this mRNA varied substantially among tissues. Comparison of our skeletal muscle translocator sequence with that of a recently published human fibroblast translocator cognate revealed that the two proteins are 88% identical and diverged about 275 million years ago. Hence, tissues vary both in the level of expression of individual translocator genes and in differential expression of cognate translocator genes. Comparison of the base substitution rates of the ADP/ATP translocator and the oxidative phosphorylation genes encoded by mitochondrial DNA revealed that the mitochondrial DNA genes fix 10 times more synonymous substitutions and 12 times more replacement substitutions; yet, these nuclear and cytoplasmic respiration genes experience comparable evolutionary constraints. This suggests that the mitochondrial DNA genes are highly prone to deleterious mutations.
Transformation of Paramecium by microinjection of a cloned serotype gene.
Authors: Godiska R , Aufderheide K J , Gilley D , Hendrie P , Fitzwater T , Preer L B , Polisky B , Preer J R
Published: May 2006

Paramecia of a given serotype express only one of several possible surface proteins called immobilization antigens (i-antigens). A 16-kilobase plasmid containing the gene for immobilization antigen A from Paramecium tetraurelia, stock 51, was injected into the macronucleus of deletion mutant d12, which lacks that gene. Approximately 40% of the injected cells acquired the ability to express serotype A at 34 degrees C. Expression appeared to be regulated normally. The transformed cells, like wild type, could be switched to serotype B by antiserum treatment and culture at 19 degrees C; on transfer to 34 degrees C, they switched back to serotype A expression. Many of the lines retained the ability to express serotype A until autogamy, when the old macronucleus is replaced by a new one derived from the micronucleus. DNA from transformants contained the injected plasmid sequences, which were replicated within the paramecia. No evidence for integration was obtained. The majority of replicated plasmid DNA comigrated with a linearized form of the input plasmid. Nonetheless, the pattern of restriction fragments generated by transformant DNA and that generated by input plasmid DNA are identical and consistent with a circular rather than a linear map. These conflicting observations can be reconciled by assuming that a mixture of different linear fragments is present in the transformants, each derived from the circular plasmid by breakage at a different point. Copy-number determinations suggest the presence of 45,000-135,000 copies of the injected plasmid per transformed cell. These results suggest that the injected DNA contains information sufficient for both controlled expression and autonomous replication in Paramecium.
Repressor of
P
elements in
Drosophila melanogaster
: Cytotype determination by a defective
P
element carrying only open reading frames 0 through 2
Authors: Nitasaka Eiji , Mukai Terumi , Yamazaki Tsuneyuki
Published: May 2006

The
P
element is a type of transposable element in
Drosophila melanogaster
. Characteristics of the syndrome of “hybrid dysgenesis” are due to transposition of
P
elements, and the molecular mechanism for regulation of this transposition has been unknown. In this study a Q strain (which carries only defective
P
elements in its genome but still is able to repress the transposition of complete
P
elements although defective in transposase activity) was used to determine the structure of the
P
element with this repressor (or P cytotype-determining) domain. Examination of the cytotype and structure of the
P
elements of particular strains with reduced copy number of
P
elements showed that the
P
element with a repressor domain was defective, being deleted between bases 1991 and 2448. This region corresponds to most of the third intron [between open reading frame (ORF) 2 and ORF 3] as well as half the ORF 3 of an intact
P
element. Therefore ORF 3 was deemed to be unnecessary for repressor production.
Molecular karyotype of Plasmodium falciparum: conserved linkage groups and expendable histidine-rich protein genes.
Authors: Kemp D J , Thompson J K , Walliker D , Corcoran L M
Published: May 2006

We describe fractionation of the Plasmodium falciparum genome into 14 chromosomal DNA molecules by pulsed-field gel electrophoresis. This number agrees with the number of chromosomes observed by electron microscopic visualization of kinetochores. The assignment of 25 markers to 12 of the 14 chromosomes in three cloned parasite lines demonstrates that chromosomal size variation can greatly change the relative migration of genetically equivalent chromosomes. Deletions that include genes for three different histidine-rich proteins, located on chromosomes 2, 8, and 13, contribute to size differences in some clones. Other karyotypic differences result from chromosome segregation and/or recombination during meiosis.
Inhibition of pluripotent hematopoietic stem cells of bone marrow by large granular lymphocytes.
Authors: Barlozzari T , Herberman R B , Reynolds C W
Published: May 2006

Previous studies suggested that natural killer (NK) cells are involved in the regulation of the growth and differentiation of pluripotent hematopoietic stem cells. To establish whether the effector cells responsible for the in vivo resistance to bone marrow (BM) transplants and the in vitro inhibition of colony-forming units (CFU) may represent identical or overlapping populations, we used a rat system for syngeneic BM transplantation, with and without the transfer of large numbers of peripheral blood large granular lymphocytes (LGLs). BM reconstitution was measured by the in vivo formation of syngeneic CFU in the spleen (CFU-s). Because of the very low frequency of CFU-s in normal rat BM, we fractionated BM cells in Percoll density gradients, which provided a 2- to 5-fold enrichment in CFU-s in the lower-density fractions. Although these fractions contained less than 10% of the total cells, they contained greater than 75% of the CFU-s and allowed for the transfer of significantly fewer donor cells. At the time of BM transplantation, radiation-resistant asialoganglioside GM1-positive LGLs, with high NK activity, accounted for a significant percentage of the lymphoid cells in the irradiated recipient. The in vivo regulatory role of these cells on engraftment was demonstrated by their depletion (by i.v. injection of small amounts of anti-asialo-GM1 antiserum before BM transplantation), which resulted in a significant increase in the number of CFU-s. Conversely, a 50% inhibition in CFU-s was found when CFU-s-enriched BM fractions were preincubated in vitro with LGLs. Additional experiments, involving selective in vivo depletion of NK cells followed by LGL repopulation, directly demonstrated the involvement of LGLs in the regulation and growth of syngeneic pluripotent hematopoietic stem cells. Our results further support the hypothesis that LGLs are involved directly or via humoral factors in the homeostasis and regulation of hematopoietic stem cell growth and differentiation.
Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.
Authors: Thiebaut F , Tsuruo T , Hamada H , Gottesman M M , Pastan I , Willingham M C
Published: May 2006

Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDR1), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little P-glycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels of P-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface of cells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.
A yeast mutant conditionally defective only for reentry into the mitotic cell cycle from stationary phase.
Authors: Drebot M A , Johnston G C , Singer R A
Published: May 2006

We report the isolation of a cold-sensitive mutant of the yeast Saccharomyces cerevisiae that is conditionally defective only for reentry into the mitotic cell cycle from stationary phase. Although actively dividing mutant cells shifted to the restrictive temperature continued to divide, stationary-phase mutant cells placed in fresh medium at the restrictive temperature failed to divide or even perform the cell cycle regulatory step "start" but did lose the characteristic stationary-phase properties of thermotolerance, accumulation of storage carbohydrates, and resistance to cell-wall-lytic enzymes. Order-of-function analysis indicated that the cold-sensitive defect blocked cells during reentry before start of the first mitotic cell cycle. Genetic analysis showed that the mutant phenotype is due to the interaction between two mutations, a cold-sensitive mutation gcs1 and a suppressor mutation sed1. These mutations thus provide the genetic basis for further analysis of stationary phase and the G0 state.
A series of six ligands for the human formyl peptide receptor: tetrapeptides with high chemotactic potency and efficacy.
Authors: Rot A , Henderson L E , Copeland T D , Leonard E J
Published: May 2006

We recently isolated, from culture fluids of Staphylococcus aureus, a chemotactic peptide that comprised equimolar quantities of methionine, leucine, phenylalanine, and isoleucine. It interacted with the formylmethionyl peptide receptor of human leukocytes and had considerably higher potency and efficacy than the widely studied tripeptide agonist fMet-Leu-Phe. On the assumption that the attractant was a formylmethionyl tetrapeptide, we synthesized the six possible sequences and tested the products for chemotactic potency and efficacy, as well as their capacity to inhibit binding of fluorescein isothiocyanate-labeled fMet-Leu-Phe-Lys to human monocytes. The concentrations required for inhibition of fluorescein-labeled fMet-Leu-Phe-Lys binding by the six peptides covered three orders of magnitude. Chemotactic potency (concentration that caused 50% of the maximum chemotactic response) ranged from 3.1 X 10(-11) M to 6.4 X 10(-10) M; efficacy (percentage of monocytes migrating at optimal attractant concentration) ranged from 41% to 66%. When the six synthetic tetrapeptides were ranked for chemotactic efficacy, they paired according to the position of phenylalanine. The average percentage migration was 66% for the two peptides with phenylalanine in position 3, 51% for phenylalanine in position 4, and 41% for phenylalanine in position 2. Since the published value for the percentage of human monocytes with detectable formyl peptide receptors is 60%, it is apparent that the two tetrapeptides with phenylalanine in position 3 (fMet-Ile-Phe-Leu and fMet-Leu-Phe-Ile) are full chemotactic agonists, which are capable of inducing migration of all the receptor-bearing cells. This is in contrast to the tripeptide fMet-Leu-Phe, which induces migration of only 50% of monocytes with receptors (efficacy of 33%). Since the chemotactic efficacy of the six tetrapeptides covers a wide range, the series may be useful to investigate signals that lead to directed movement after occupancy of receptors by chemoattractants.
Asymmetric expression in somites of cytotactin and its proteoglycan ligand is correlated with neural crest cell distribution.
Authors: Tan S S , Crossin K L , Hoffman S , Edelman G M
Published: May 2006

The development of the vertebrate neural crest presents a particularly challenging problem in pattern formation. Several studies have revealed that a population of neural crest cells penetrates the sclerotomal mesenchyme of the somite only in its rostral half. In a search for molecular correlates of this pattern, we have observed that cytotactin and a chondroitin sulfate proteoglycan, two interactive extracellular matrix molecules, show a specialized distribution within the sclerotome. Cytotactin was localized in the rostral half of the sclerotome at about the time of neural crest cell invasion. The proteoglycan was initially diffuse throughout the sclerotome but became restricted to the caudal half after the appearance of cytotactin and invasion of neural crest cells in the rostral half. These distributions were crest cell-independent; they occurred on the same schedule even when all crest cells were removed by surgical extirpation of the neural tube. Furthermore, in tissue culture, somite cells synthesized high levels of both molecules. In vitro, crest cells rounded up in the presence of these molecules and cell migration assays revealed that neither cytotactin nor proteoglycan alone was as good a substratum for crest cell migration as fibronectin. In combination with fibronectin, however, cytotactin or proteoglycan only restricted cell movement but did not prevent it. Taken together, these observations support the hypothesis that cytotactin and the chondroitin sulfate proteoglycan may contribute to pattern formation during embryogenesis by means of their site-restricted distribution, their ability to alter migration on other substrates such as fibronectin, and their ability to induce cell-surface modulation.
Leaf pubescence in buttonwood: Community variation in a putative defense against defoliation
Authors: Schoener Thomas W.
Published: May 2006

Plants have a variety of putative defenses against defoliation by herbivores, among which are pubescent leaves. Buttonwood (
Conocarpus erectus
), a Caribbean tree, shows considerable between-individual variation in this trait, and pubescent leaves have less herbivore damage. Surveying 97 island communities, I documented three patterns expected were pubescent individuals more frequent where herbivory is great. (
i
) Larger islands have a higher percentage of pubescence (larger islands have more herbivores). (
ii
) Islands nearer to a mainland have a higher percentage of pubescence (nearer islands receive more herbivore immigrants). (
iii
) Islands having an extremely abundant predator on foliage arthropods, arboreal lizards, have a smaller percentage of pubescence than no-lizard islands. The third effect, though statistically significant, is weak relative to the direct effects of lizards on one category of their prey (spiders) measured in the same system.
Gene conversion-like events cause steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia.
Authors: Harada F , Kimura A , Iwanaga T , Shimozawa K , Yata J , Sasazuki T
Published: May 2006

Genomic DNAs from twelve Japanese patients with steroid 21-hydroxylase [21-OHase; steroid 21-monooxygenase; steroid, hydrogen-donor:oxygen oxidoreductase (21-hydroxylating); EC 1.14.99.10] deficiency were analyzed by Southern blot hybridization. A 3.7-kilobase (kb) Taq I and a 1.7-kb Pvu II restriction endonuclease fragment that correspond to a 21-OHase B gene were absent from the DNA of two unrelated patients with the salt-wasting form of the disease. However, a 10.5-kb Bgl II fragment corresponding to the region encompassing the 21-OHase B gene was still present in these two patients. The genes encoding 21-OHase were cloned from one of these two patients, who was homozygous by descent for HLA-A26;B39;C4A3;C4B1;DR4. Restriction endonuclease mapping as well as partial nucleotide sequencing analysis revealed that the 21-OHase B gene of the patient has been converted to the pseudogene, 21-OHase A, as far as the critical 0.5-kb sequence was concerned. Thus, the defect was due to both chromosomes each carrying two copies of 21-OHase A pseudogene and lacking functional 21-OHase B gene.
Blood-brain barrier protein recognized by monoclonal antibody.
Authors: Sternberger N H , Sternberger L A
Published: May 2006

An IgG1 mouse monoclonal antibody produced in response to immunization with rat brain homogenate reacted with endothelial cells in the central and peripheral nervous system. Because antibody reactivity was associated with endothelia that have a selective permeability barrier, the antibody was called anti-endothelial-barrier antigen (anti-EBA). Paraffin sections of Bouins'-fixed rat tissue were used for initial screening and subsequent characterization of antibody reactivity. The antibody was generally unreactive with endothelial cells in other organs and with nonendothelial cells in or outside of the nervous system. Antibody binding was greatly reduced or absent in endothelia of the area postrema and choroid plexus, sites known to possess fenestrated blood vessels. In developing rat brain, anti-EBA binding to some microvessels was seen at 3 days postnatally. Anti-EBA reactivity outside the nervous system occurred in spleen and skin. Patchy reaction with portions of some spleen blood vessels and binding to some cells in the spleen were observed. In the skin, small cells, tentatively identified as Langerhans cells, which participate in Ia presentation, were stained. On immunoblots of rat brain microvessel preparations electrophoresed in Na-DodSO4/polyacrylamide gels, anti-EBA reacted with a protein triplet of Mr 30,000, 25,000, and 23,500 components.
Mutants of the catabolite activator protein of Escherichia coli that are specifically deficient in the gene-activation function.
Authors: Irwin N , Ptashne M
Published: May 2006

In the presence of cyclic AMP, the catabolite activator protein (CAP) of Escherichia coli binds DNA and stimulates transcription at a number of promoters. We have examined a model of CAP bound at the gal promoter and, using directed mutagenesis, have isolated CAP mutants that are analogous to the lambda repressor positive control (pc) mutants. These CAP mutants bind DNA but are defective in stimulating transcription at the gal P1 promoter. These mutants are also altered in positive control at the lac and malT promoters, where CAP binds to sites further upstream from the transcription start site.
Picosecond kinetics of fluorescence and absorbance changes in photosystem II particles excited at low photon density
Authors: Schatz Günther H. , Brock Helmut , Holzwarth Alfred R.
Published: May 2006

Oxygen-evolving photosystem II particles (from
Synechococcus
) with about 80 chlorophyll molecules per primary electron donor (P
680
) were used for a correlated study of picosecond kinetics of fluorescence and absorbance changes, detected by the single-photon-timing technique and by a pump-probe apparatus, respectively. Chlorophyll fluorescence decays were biexponential with lifetimes τ
1
= 80 ± 20 ps and τ
2
= 520 ± 120 ps in open reaction centers and τ
1
= 220 ± 30 ps and τ
2
= 1.3 ± 0.15 ns in closed reaction centers. The corresponding fluorescence yield ratio
F
max
/
F
o
was 3-4. Absorbance changes were monitored in the spectral range of 620-700 nm after excitation at 675 nm with 10-ps pulses sufficiently weak (<7 × 10
12
photons/cm
2
per pulse) to avoid singlet-singlet annihilation. With open reaction centers, the absorbance changes could be fit to the sum of three exponentials. The associated absorbance difference spectra were attributed to (
i
) exciton trapping and charge separation (τ = 100 ± 20 ps), (
ii
) the electron-transfer step P
680
+
I
-
Q
A
→ P
680
+
I Q
A
-
(where I is the primary electron acceptor and Q
A
is the first quinone acceptor) (τ = 510 ± 50 ps), and (
iii
) the reduction of P
680
+
by the intact donor side (τ > 10 ns). With closed reaction centers, the absorbance changes were biexponential with lifetimes τ
1
= 170-260 ps and τ
2
= 1.6-1.75 ns. The results are explained in terms of a kinetic model that assumes P
680
to constitute a shallow trap. The results show that Q
A
reduction in these photosystem II particles decreases both the apparent rate and the yield of the primary charge separation by a factor of 2-3 and increases the mean lifetime of excitons in the antenna by a factor of 3-4. Thus, we conclude that the long-lived, nanosecond chlorophyll fluorescence is not charge-recombination luminescence but rather emission from equilibrated excited states of antenna chlorophylls.
Linkage map of the human major histocompatibility complex including the tumor necrosis factor genes.
Authors: Carroll M C , Katzman P , Alicot E M , Koller B H , Geraghty D E , Orr H T , Strominger J L , Spies T
Published: May 2006

The tumor necrosis factor (TNF) alpha and beta gene pair has been linked in the human major histocompatibility complex to HLA-B, HLA-C, and, tentatively, HLA-E and HLA-A on one side and to the class III complement/steroid 21-hydroxylase gene cluster on the other by pulsed-field gel electrophoresis. The TNF genes are located 200 kilobases (kb) centromeric of HLA-B and about 350 kb telomeric of the class III cluster. Together with previous data on the linkage and structures of the class II and class III regions, a restriction map of the entire human major histocompatibility complex of about 3500 kb has been prepared.
Human heavy-chain variable region gene family nonrandomly rearranged in familial chronic lymphocytic leukemia.
Authors: Shen A , Humphries C , Tucker P , Blattner F
Published: May 2006

We have identified a family of human immunoglobulin heavy-chain variable-region (VH) genes, one member of which is rearranged in two affected members of a family in which the father and four of five siblings developed chronic lymphocytic leukemia. Cloning and sequencing of the rearranged VH genes from leukemic lymphocytes of three affected siblings showed that two siblings had rearranged VH genes (VHTS1 and VHWS1) that were 90% homologous. The corresponding germ-line gene, VH251, was found to be part of a small (four gene) VH gene family, which we term VHV. The DNA sequence homology to VHWS1 (95%) and VHTS1 (88%) and identical restriction sites on the 5' side of VH confirm that rearrangement of VH251 followed by somatic mutation produced the identical VH gene rearrangements in the two siblings. VHTS1 is not a functional VH gene; a functional VH rearrangement was found on the other chromosome of this patient. The other two siblings had different VH gene rearrangements. All used different diversity genes. Mechanisms proposed for non-random selection of a single VH gene include developmental regulation of this VH gene rearrangement or selection of a subpopulation of B cells in which this VH has been rearranged.
Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients.
Authors: Cooper G J , Willis A C , Clark A , Turner R C , Sim R B , Reid K B
Published: May 2006

Deposition of amyloid in pancreatic islets is a common feature in human type 2 diabetic subjects but because of its insolubility and low tissue concentrations, the structure of its monomer has not been determined. We describe a peptide, of calculated molecular mass 3905 Da, that was a major protein component of amyloid-rich pancreatic extracts of three type 2 diabetic patients. After collagenase treatment, an extract containing 20-50% amyloid was solubilized by sonication into 70% formic acid and the peptide was purified by gel filtration followed by reverse-phase high-performance liquid chromatography. We term this peptide diabetes-associated peptide, as it was not detected in extracts of pancreas from any of six normal subjects. Diabetes-associated peptide contains 37 amino acids and is 46% identical to the sequences of rat and human calcitonin gene-related peptide, indicating that these peptides are related in evolution. Sequence identities with conserved residues of the insulin A chain were also seen in a 16-residue segment. On extraction, the islet amyloid is particulate and insoluble like the core particles of Alzheimer disease. Their monomers have similar molecular masses, each having a hydropathic region that can probably form beta-pleated sheets. The accumulation of amyloid, including diabetes-associated peptide, in islets may impair islet function in type 2 diabetes mellitus.
Regulation of metallothionein gene expression by 1 alpha,25-dihydroxyvitamin D3 in cultured cells and in mice.
Authors: Karasawa M , Hosoi J , Hashiba H , Nose K , Tohyama C , Abe E , Suda T , Kuroki T
Published: May 2006

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25(OH)2D3], a hormonally active form of vitamin D3, has been shown to modulate cell differentiation and tumor promotion. This report demonstrates that mRNA of the metallothionein (MT) gene was induced by 1 alpha,25(OH)2D3 in cultured epidermal keratinocytes and also in liver, kidney, and skin tissues when 1 alpha-hydroxyvitamin D3, a synthetic precursor of 1 alpha,25(OH)2D3, was applied in vivo. Exposure of FRSK cells, a cell line derived from fetal rat skin keratinocytes, to 1 alpha,25(OH)2D3 at 5 ng/ml (12 nM) increased MT mRNA to almost the same extent as that induced by 10 microM dexamethasone or 1 microM CdCl2. This increase in the level of MT mRNA was evident within 2 hr and was maximal 12-24 hr after the addition of 1 alpha,25(OH)2D3. The induction was dose-dependent with concentrations of 1 alpha,25(OH)2D3 from 0.05 to 5.0 ng/ml. Amounts of MT increased with the increase of MT mRNA induced by 1 alpha,25(OH)2D3. Of the derivatives of vitamin D3 tested, only 1 alpha,25(OH)2D3 caused marked induction. Treatment with cycloheximide did not inhibit MT mRNA induction by 1 alpha,25(OH)2D3. 1 alpha,25(OH)2D3 induced MT mRNA in primary cultures of mouse epidermal keratinocytes but not in IAR-20, a liver cell line. 1 alpha,25(OH)2D3 had a similar effect in vivo: oral administration of 1 alpha-hydroxyvitamin D3 to mice resulted in increased levels of MT mRNA in the liver, kidney, and skin 24 hr later. Increase in the level of MT mRNA may be relevant to some biological actions of 1 alpha,25(OH)2D3.
Host control of plasmid replication: requirement for the sigma factor sigma 32 in transcription of mini-F replication initiator gene.
Authors: Wada C , Imai M , Yura T
Published: May 2006

Replication of F factor or mini-F plasmid is strongly inhibited in the rpoH (htpR) mutants of Escherichia coli deficient in the sigma factor (sigma 32) known to be required for heat shock gene expression. Transcription of the mini-F repE gene encoding a replication initiator protein (E protein) was examined by operon fusion and by direct determination of repE mRNA. The synthesis rate and the level of repE mRNA were found to increase transiently upon temperature upshift (30 degrees C to 42 degrees C) in wild-type cells but to decrease rapidly in the rpoH mutants. Thus sigma 32 appeared to be directly involved in transcription of repE whose product, E protein, in turn activates DNA replication from the mini-F ori2 region. This scheme of host-controlled plasmid replication is further supported by the analysis of transcription in vitro: RNA synthesis can be initiated from the repE promoter by a minor form of RNA polymerase containing sigma 32 but not by the major polymerase containing the normal sigma factor sigma 70. The sigma 32-mediated transcription from the repE promoter is strongly inhibited by the E protein. We conclude that transcription of the mini-F repE gene is mediated by the host transcription factor sigma 32 and is negatively controlled by its own product.
Expression of a synthetic bovine rhodopsin gene in monkey kidney cells.
Authors: Oprian D D , Molday R S , Kaufman R J , Khorana H G
Published: May 2006

We report here the high-level expression of a synthetic gene for bovine rhodopsin in transfected monkey kidney COS-1 cells. Rhodopsin is produced in these cells to a level of 0.3% of the cell protein, and it binds exogenously added 11-cis-retinal to generate the characteristic rhodopsin absorption spectrum. We describe a one-step immunoaffinity procedure for purification of the rhodopsin essentially to homogeneity. The COS-1 cell rhodopsin activates the GTPase activity of bovine transducin in a light-dependent manner with the same specific activity as that of purified bovine rhodopsin. Electron microscopy of immunogold-stained cells indicates that rhodopsin is located in the plasma membrane of the transfected cells and is oriented with the amino terminus on the extracellular side of the membrane. This orientation is analogous to that of rhodopsin in the disk membranes of photoreceptor cells in the bovine retina.
Changes in translational yield regulate tissue-specific expression of beta-glucuronidase.
Authors: Bracey L T , Paigen K
Published: May 2006

The number of beta-glucuronidase (GUS; beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) molecules per cell varies as much as 12-fold among mouse tissues. To identify the regulatory mechanisms responsible, estimates of the rates of GUS protein synthesis (ks) and degradation (kd) were obtained for six tissues in the B6.PAC-Gusn mouse strain, which carries the N haplotype of the GUS gene. Differences in enzyme levels among tissues were predominantly due to differences in rates of enzyme synthesis; only brain differed significantly in the rate of protein degradation. Typically, tissues contain about 2 molecules of GUS mRNA per cell. Differences in GUS mRNA levels were found among tissues, but these were not sufficient to account for observed differences in ks. This suggests that tissues differ in translational yield, which is defined as the product of the efficiency with which the GUS message is translated and the fraction of newly made polypeptides that are successfully matured into GUS tetramers. Experimental estimates of translational yield confirmed that this is indeed a source of tissue differences in GUS gene regulation. This finding also proved to be true of the B haplotype of the GUS gene. The differential regulation of special-function genes is, in general, effected transcriptionally. In contrast, the differential regulation of several "housekeeping" genes has been reported to arise from changes in mRNA maturation and/or stability. It is now apparent that translational yield, which is an aspect of protein synthesis, can also serve as a differential regulatory mechanism.
Structure and function of anti-DNA autoantibodies derived from a single autoimmune mouse.
Authors: Shlomchik M J , Aucoin A H , Pisetsky D S , Weigert M G
Published: May 2006

Four monoclonal anti-DNA antibodies derived from a single autoimmune MRL/lpr mouse were studied. Three of these antibodies showed similarities in DNA binding; the fourth had a much higher specific activity for single-stranded DNA and, in addition, was unique in binding double-stranded DNA and cardiolipin. Complete nucleotide sequences of heavy- and light-chain variable regions demonstrated that all four antibodies are clonally related. The sequences also showed numerous somatic mutations, the distribution of which suggests that positive selection by antigen operated on these clonally related autoantibodies.
Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site.
Authors: Lemaitre M , Bayard B , Lebleu B
Published: May 2006

Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression, but their efficient introduction within intact cells proved to be difficult to realize. As a step toward this goal, small (13- or 15-mer) synthetic oligodeoxyribonucleotides have been coupled at their 3' ends to epsilon-amino groups of lysine residues of poly(L-lysine) (Mr, 14,000). A 15-mer oligonucleotide-poly(L-lysine) conjugate complementary to the initiation region of vesicular stomatitis virus (VSV) N-protein mRNA specifically inhibits the synthesis of VSV proteins and exerts an antiviral activity against VSV when added in the cell culture medium at doses as low as 100 nM. Neither synthesis of cellular proteins nor multiplication of encephalomyocarditis virus was affected significantly by this oligonucleotide conjugate. The data suggest that oligonucleotide-poly(L-lysine) conjugates might become effective for studies on gene expression regulation and for antiviral chemotherapy.
Thermal and pH stability of "beta-benzyme".
Authors: D'Souza V T , Lu X L , Ginger R D , Bender M L
Published: May 2006

The thermal and pH stability of "beta-benzyme", an artificial chymotrypsin based on beta-cyclodextrin, has been studied and compared with the stability of real chymotrypsin. Artificial chymotrypsin is vastly superior to real chymotrypsin with regard to both temperature and pH stability. The reasons for this increased stability are discussed.
Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A.
Authors: Krause J E , Chirgwin J M , Carter M S , Xu Z S , Hershey A D
Published: May 2006

Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and defined by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene.
Allelic inclusion in a pre-B-cell line that generates immunoglobulin heavy chain genes in vitro.
Authors: Beck-Engeser G , Jäck H M , Wabl M
Published: May 2006

In a pre-B-cell line that rearranges its heavy chain gene segments in vitro, we found that the rate of productive rearrangement on one allele was not influenced by the presence of heavy chain protein encoded by the other allele. This shows that allelic exclusion of heavy chain genes is not regulated at the genetic level.
sis (platelet-derived growth factor B chain) gene transcript levels are elevated in human atherosclerotic lesions compared to normal artery.
Authors: Barrett T B , Benditt E P
Published: May 2006

Arterial smooth muscle cell (SMC) proliferation is thought to be an essential aspect of the development of human atherosclerotic lesions. In this study we posed the question, could a growth factor gene be transcriptionally active in atherosclerotic tissue? We found that transcripts from the sis gene, which encodes one of the two chains of platelet-derived growth factor, were present in surgically removed human carotid artery lesions at levels 5-fold greater than the low level of constitutive expression detected in normal artery. This demonstrates that a growth factor could be synthesized endogenously within human atherosclerotic lesions. Although atherosclerotic lesions are composed predominantly of SMC, large numbers of infiltrating macrophages, T cells, and endothelial cells can also be present, raising the possibility that one of these secondary cell types, rather than SMC, could be responsible for the sis transcripts. Human macrophages activated in culture contained 2- to 4-fold more sis RNA, per micrograms of total cellular RNA, than lesions, whereas T cells activated in culture did not contain significant levels. Cultured human endothelial cells expressed sis transcripts at higher levels than macrophages. Since human arterial SMC in culture express receptors for and are mitogenically responsive to platelet-derived growth factor, transcription of the sis gene by cells within lesions, whether these cells are SMC themselves, macrophages, endothelial cells, or another cell type, suggests that an autocrine and/or paracrine proliferative mechanism is important in the pathogenesis of atherosclerosis.
Force generation by muscle fibers in rigor: a laser temperature-jump study.
Authors: Davis J S , Harrington W F
Published: May 2006

A clear prediction of the helix-coil model for force generation in muscle is that force should be produced when the equilibrium (helix-coil) of a rigor (or activated) fiber is perturbed by a temperature jump near the melting temperature of the light meromyosin/heavy meromyosin hinge. An infrared, iodine-photodissociation laser was used to heat the fibers by approximately equal to 5 degrees C in under 1 mus. Under ionic conditions where rigor bridges are predominantly associated with the thick filament backbone, an abrupt drop in tension typical of normal thermoelastic expansion was seen. A similar response was observed below 41 degrees C for thick filament-released rigor bridges. Above this temperature, a rubber-like thermoelastic response was obtained typical of a helix-coil transition. At temperatures near 50 degrees C, the amount of force generated by a rigor fiber was large and comparable to that seen for an activated fiber at 5 degrees C. The relaxation spectra of force generation obtained for both systems (rigor and activated) show a step change followed by a biexponential kinetic process. The reciprocal relaxation times and amplitudes for these individual processes in activated and rigor fibers differ only by factors of 2-4. Force generation in the rigor muscle appears to arise from melting in the subfragment 2 hinge region of the myosin molecule since binding of subfragment 2 to the thick filament backbone inhibits force production. No significant force generation was observed following temperature jumps of relaxed fibers.