bluuebunny/crossref_metadata_2025 huggingface dataset:
Stochastic pairing of heavy-chain and kappa light-chain variable gene families occurs in polyclonally activated B cells.
Authors: Kaushik A , Schulze D H , Bonilla F A , Bona C , Kelsoe G
Published: May 2006

Frequencies of 25 immunoglobulin heavy-chain and kappa light-chain variable (VH + V kappa) gene-family pairings expressed in splenic B-cell populations were determined by hybridization of VH- and V kappa-family-specific DNA probes to mitogen-induced B-cell colonies from C57BL/6 mice or hybridomas derived from BALB/c and NZB mice. Both analyses support the conclusion that VH and V kappa gene families pair without bias; as would be expected for random association, the frequencies of specific VH + V kappa pairs may be estimated by the product of the independent VH and V kappa frequencies. Based upon the frequencies at which 9 VH and 12 V kappa gene families are expressed, we calculated the expected usage for approximately 100 VH + V kappa family pairings in neonatal and adult C57BL/6 mice. Variability in the expression of such VH + V kappa pairings is considerable; pairs representing greater than 10% to less than 0.01% of the splenic B-cell population occur. This variability is most pronounced in the neonate, where 6 VH + V kappa family pairs account for nearly 40% of all mitogen-reactive B cells. As the neonate matures, the distribution of frequencies for VH + V kappa pairings becomes more nearly uniform. This process may underlie the patterned acquisition of humoral immune responsiveness.
Bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase: complete amino acid sequence and localization of phosphorylation sites.
Authors: Sakata J , Uyeda K
Published: May 2006

We have shown previously that bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (EC 2.7.1.105/3.1.3.46) is phosphorylated by cAMP-dependent protein kinase and protein kinase C; phosphorylation results in activation of kinase. This activation of heart enzyme is in contrast to results with the liver isozyme, in which phosphorylation by cAMP-dependent protein kinase inhibits the kinase activity. As an initial step toward understanding this difference between the isozymes we have determined the DNA sequence of the heart enzyme and analyzed the amino acid sequence with special emphasis on the location of the phosphorylation site. We isolated and sequenced two overlapping cDNA fragments, which together could encode the complete amino acid sequence of bovine heart fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase, a protein of 530 amino acids, with a calculated molecular weight of 60,679. Since the deduced protein contained amino acid sequences identical to the sequences of four known tryptic peptides from this enzyme we concluded that the deduced protein sequence did represent bovine heart enzyme. In addition, a cDNA fragment hybridized to a 4-kilobase mRNA from bovine heart. The phosphorylation sites of the heart enzyme were located near the C terminus, whereas the phosphorylation site of the liver isozyme is known to be located near the N terminus. These opposite locations of the phosphorylation sites may explain the contrasting effect of the covalent modification on the enzymes' activities.
Expansion of the alpha 2-adrenergic receptor family: cloning and characterization of a human alpha 2-adrenergic receptor subtype, the gene for which is located on chromosome 2.
Authors: Lomasney J W , Lorenz W , Allen L F , King K , Regan J W , Yang-Feng T L , Caron M G , Lefkowitz R J
Published: May 2006

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple alpha 2-adrenergic receptor (alpha 2AR) subtypes. We have cloned a human alpha 2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned alpha 2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotide-binding protein-coupled receptors, but whose structure most closely resembles that of the alpha 2ARs. Competition curve analysis of the binding properties of the receptor expressed in COS-7 cells with a variety of adrenergic ligands demonstrates a unique alpha 2AR pharmacology. Hybridization with somatic cell hybrids shows that the gene for this receptor is located on chromosome 2. Northern blot analysis of various rat tissues shows expression in liver and kidney. The unique pharmacology and tissue localization of this receptor suggest that this is an alpha 2AR subtype not previously identified by classical pharmacological or ligand binding approaches.
Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.
Authors: Sherman P A , Fyfe J A
Published: May 2006

The human immunodeficiency virus (HIV) integration protein, a potential target for selective antiviral therapy, was expressed in Escherichia coli. The purified protein, free of detectable contaminating endonucleases, selectively cleaved double-stranded DNA oligonucleotides that mimic the U3 and the U5 termini of linear HIV DNA. Two nucleotides were removed from the 3' ends of both the U5 plus strand and the U3 minus strand; in both cases, cleavage was adjacent to a conserved CA dinucleotide. The reaction was metal-ion dependent, with a preference for Mn2+ over Mg2+. Reaction selectivity was further demonstrated by the lack of cleavage of an HIV U5 substrate on the complementary (minus) strand, an analogous substrate that mimics the U3 terminus of an avian retrovirus, and an HIV U5 substrate in which the conserved CA dinucleotide was replaced with a TA dinucleotide. Such an integration protein-mediated cleavage reaction is expected to occur as part of the integration event in the retroviral life cycle, in which a double-stranded DNA copy of the viral RNA genome is inserted into the host cell DNA.
Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2.
Authors: Sadhu K , Reed S I , Richardson H , Russell P
Published: May 2006

Entry into mitosis during the somatic cell cycle is regulated in response to signals that monitor the completion of DNA replication, the integrity of the nuclear genome, and, possibly, the increase in cellular mass during the cell cycle. It has been postulated that the operation of this cell cycle control involves the gradual accumulation of rate-limiting mitotic inducers, which trigger nuclear division when their cellular concentration reaches a critical level. We have cloned a human gene, which we call CDC25, whose product may function as a mitotic inducer. This human gene encodes a protein with a predicted molecular mass of 53,000 daltons whose C-terminal domain shares about 37% sequence identity with the fission yeast cdc25+ mitotic inducer. The human CDC25 gene rescues the defect of a fission yeast temperature-sensitive (ts) cdc25ts mutant that is unable to initiate mitosis. In HeLa cells CDC25 mRNA levels are very low in G1 and increase at least 4-fold as cells progress towards M phase. These data suggest that in human cells, as in fission yeast, the accumulation of CDC25 mitotic inducer during G2 may play a key role in regulating the timing of mitosis.
Brain concentrations of benzodiazepines are elevated in an animal model of hepatic encephalopathy.
Authors: Basile A S , Pannell L , Jaouni T , Gammal S H , Fales H M , Jones E A , Skolnick P
Published: May 2006

Brain extracts from rats with hepatic encephalopathy due to thioacetamide-induced fulminant hepatic failure contained 4- to 6-fold higher concentrations of substances that inhibit radioligand binding to benzodiazepine receptors than corresponding control rat extracts. Both isocratic and gradient-elution HPLC indicated that this inhibitory activity was localized in 3-8 peaks with retention times corresponding to deschlorodiazepam, deschlorolorazepam, lorazepam, oxazepam, diazepam, and N-desmethyldiazepam. The presence of diazepam and N-desmethyldiazepam was confirmed by mass spectroscopy. Both mass spectroscopic and radiometric techniques indicated that the concentrations of N-desmethyldiazepam and diazepam in brain extracts from encephalopathic rats were 2-9 and 5-7 times higher, respectively, than in control brain extracts. While benzodiazepines have been identified previously in mammalian and plant tissues, this report demonstrates that concentrations of these substances are increased in a pathophysiological condition. These findings provide a rational basis for the use of benzodiazepine receptor antagonists in the management of hepatic encephalopathy in humans.
The B subunit of a rat heteromeric CCAAT-binding transcription factor shows a striking sequence identity with the yeast Hap2 transcription factor.
Authors: Maity S N , Vuorio T , de Crombrugghe B
Published: May 2006

CBF is a heteromeric mammalian transcription factor that binds to CCAAT sequences in a number of promoters such as the two type I collagen promoters, the albumin promoter, the major histocompatibility complex class II promoter, and others. It is composed of two components, A and B, that are both needed for DNA binding. We have isolated a rat cDNA containing the complete 341-amino acid coding sequence of the B component of CBF. Expression of this cDNA in vitro generates a polypeptide that shows the same dependency on the A component as the native B component in the formation of a complex with a CCAAT-containing DNA. The C-terminal portion of the B component from residue 260 to residue 312 shows a 75% sequence identity with a portion of the Hap2 protein, a component of a heteromeric CCAAT-binding protein in yeast. In contrast, the rest of the protein shows little sequence homology with Hap2, although both proteins contain glutamine-rich domains. In the B component of CBF this domain spans the amino-terminal 60% of the protein, whereas in Hap2 this domain is much smaller. Hence, only a few changes in one domain of this protein were tolerated during evolution between yeast and mammals, whereas the rest of the protein diverged much more extensively.
Distribution and self-organization of photosynthetic pigments in micelles: implication for the assembly of light-harvesting complexes and reaction centers in the photosynthetic membrane.
Authors: Scherz A , Rosenbach-Belkin V , Fisher J R
Published: May 2006

The addition of bacteriochlorophylls and bacteriopheophytins to formamide/water, 3:1 (vol/vol), (or water) containing small spherical micelles of Triton X-100 leads to the reorganization of the detergent into micelles that consist of 5000-40,000 amphiphilic molecules. The pigment distribution within the micelles was determined by modified Poisson statistics taking into consideration the various sizes of micelles. Pigment dimerization occurred in micelles with more than a single occupant and was driven by a free-energy change of -4.5 kcal/mol (1 cal = 4.184 J) for bacteriochlorophyll a in formamide/water, -7.6 kcal/mol for bacteriopheophytin a in formamide/water, and -6.6 kcal/mol for bacteriopheophytin a in water. These values correspond to the room temperature equilibrium constants 2.2 x 10(3) M-1, 3.9 x 10(5) M-1, and 7.5 x 10(4) M-1, respectively. The incorporation of bacteriochlorophylls with attached small formamide polymers and the subsequent dimerization of these pigments in the lipid phase provide a model for studying the synergetic organization of polypeptides and bacteriochlorophyll clusters in the photosynthetic membrane.
Complementary DNA encoding the human T-cell FK506-binding protein, a peptidylprolyl cis-trans isomerase distinct from cyclophilin.
Authors: Maki N , Sekiguchi F , Nishimaki J , Miwa K , Hayano T , Takahashi N , Suzuki M
Published: May 2006

The recently discovered macrolide FK506 has been demonstrated to have potent immunosuppressive activity at concentrations 100-fold lower than cyclosporin A, a cyclic undecapeptide that is used to prevent rejection after transplantation of bone marrow and organs, such as kidney, heart, and liver. After the recent discovery that the cyclosporin A-binding protein cyclophilin is identical to peptidylprolyl cis-trans isomerase, a cellular binding protein for FK506 was found to be distinct from cyclophilin but to have the same enzymatic activity. In this study, we isolated a cDNA coding for FK506-binding protein (FKBP) from human peripheral blood T cells by using mixed 20-mer oligonucleotide probes synthesized on the basis of the sequence, Glu-Asp-Gly-Lys-Lys-Phe-Asp, reported for bovine FKBP. The DNA isolated contained an open reading frame encoding 108 amino acid residues. The first 40 residues of the deduced amino acid sequence were identical to those of the reported amino-terminal sequence of bovine FKBP, indicating that the DNA sequence isolated represents the gene coding for FKBP. Computer-assisted analysis of the deduced amino acid sequence indicates that FKBP exhibits no internal homology and does not have significant sequence similarity to any other amino acid sequences of known proteins, including cyclophilin. This result suggests that two catalytically similar proteins, cyclophilin and FKBP, evolved independently. In Northern blot analysis, mRNA species of approximately 1.8 kilobases that hybridized with human FKBP cDNA were detected in poly(A)+ RNAs from brain, lung, liver, and placental cells and leukocytes. Induction of Jurkat leukemic T cells with phorbol 12-myristate 13-acetate and ionomycin did not affect the level of FKBP mRNA. Southern blot analysis of human genomic DNA digested with different restriction enzymes suggests the existence of only a few copies of the DNA sequence encoding FKBP. This is in contrast to the result that as many as 20 copies of the cyclophilin gene and possible pseudogenes may be present in the mammalian genome.
High-resolution two-dimensional polyacrylamide gel electrophoresis reveals a glucose-response protein of 65 kDa in pancreatic islet cells.
Authors: Collins H W , Buettger C , Matschinsky F
Published: May 2006

High-resolution two-dimensional PAGE was used to search for glucose-response proteins in isolated pancreatic islets that were labeled with [35S]methionine at ambient glucose concentrations of 0-18 mM. A 65-kDa protein, isoelectric focusing point of approximately 6.6-7.0, was discovered that showed at least a 20-fold stimulation of radiolabeling when glucose in the labeling medium was increased from 3 to 18 mM, in contrast to a 2.5-fold enhancement of label incorporation into total islet proteins. This 65-kDa protein is evident after 30 min of labeling with 18 mM glucose and is preferentially synthesized compared to its nearest neighbors after both 30 and 60 min of labeling. Glucose induction of the 65-kDa protein was virtually blocked by D-mannoheptulose. Glucose induction of this 65-kDa protein is in practically all aspects comparable to glucose induction of insulin and glucokinase in pancreatic beta cells. A working hypothesis is developed proposing that glucose-response proteins or "glucospondins" are pivotal constituents of pancreatic islet cells and that their discovery and exploration promise new insights into normal and pathological islet cell function.
Microinjection of a protein-tyrosine-phosphatase inhibits insulin action in Xenopus oocytes.
Authors: Cicirelli M F , Tonks N K , Diltz C D , Weiel J E , Fischer E H , Krebs E G
Published: May 2006

A protein-tyrosine-phosphatase (PTPase 1B; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), specific for phosphotyrosyl residues, was microinjected into Xenopus oocytes. This resulted in a 3- to 5-fold increase in PTPase activity over endogenous levels. The PTPase blocked the insulin-stimulated phosphorylation of tyrosyl residues on endogenous proteins, including a protein having a molecular mass in the same range as the beta subunit of the insulin or insulin-like growth factor I receptor. PTPase 1B also blocked the activation of an S6 peptide kinase--i.e., an enzyme recognizing a peptide having the sequence RRLSSLRA found in a segment of ribosomal protein S6 and known to be activated early in response to insulin. On the other hand, the insulin stimulation of an S6 kinase, detected by using 40S ribosomes as substrate, was unaffected even though PTPase 1B partially prevented the phosphorylation of ribosomal protein S6 in vivo. Mono Q chromatography of insulin-treated oocyte extracts revealed two main peaks of S6 kinase activity. Fractions from the first peak displayed S6 peptide kinase activity that was essentially abolished in profiles from PTPase 1B-injected oocytes. Material from the second peak, which was best revealed by using 40S ribosomes as substrate and had comparatively little S6 peptide kinase activity, was minimally affected by PTPase 1B. These observations suggest that at least two distinct "S6 kinases" are involved in ribosomal protein S6 phosphorylation in vivo and that the activation pathways for these enzymes differ in their sensitivity to PTPase 1B.
Guanosine 3'-diphosphate 5'-diphosphate is not required for growth rate-dependent control of rRNA synthesis in Escherichia coli.
Authors: Gaal T , Gourse R L
Published: May 2006

rRNA synthesis in Escherichia coli is subject to at least two regulation systems, growth rate-dependent control and stringent control. The inverse correlation between rRNA synthesis rates and guanosine 3'-diphosphate 5'-diphosphate (ppGpp) levels under various physiological conditions has led to the supposition that ppGpp is the mediator of both control mechanisms by inhibiting transcription from rrn P1 promoters. Recently, relA- spoT- strains have been constructed in which both ppGpp synthesis pathways most likely have been removed (M. Cashel, personal communication). We have confirmed that such strains produce no detectable ppGpp and therefore offer a direct means for testing the involvement of ppGpp in the regulation of rRNA synthesis in vivo. Stringent control was determined by measurement of rRNA synthesis after amino acid starvation, while growth rate control was determined by measurement of rRNA synthesis under different nutritional conditions. As expected, the relA- spoT- strain is relaxed for stringent control. However, growth rate-dependent regulation is unimpaired. These results indicate that growth rate regulation can occur in the absence of ppGpp and imply that ppGpp is not the mediator, or at least is not the sole mediator, of growth rate-dependent control. Therefore, growth rate-dependent control and stringent control may utilize different mechanisms for regulating stable RNA synthesis.
Inhibition of carcinoma and melanoma cell growth by type 1 transforming growth factor beta is dependent on the presence of polyunsaturated fatty acids.
Authors: Newman M J
Published: May 2006

Improved serum-free media were developed for the anchorage-dependent growth of A549 human lung carcinoma and B16 mouse melanoma cell lines in vitro. Type 1 transforming growth factor beta (TGF-beta 1) inhibited the growth of A549 or B16 cells under serum-free conditions or in the presence of 10% serum by 15-33%. In contrast, in the presence of micrograms/ml concentrations of polyunsaturated fatty acids (PUFAs), picomolar concentrations of TGF-beta 1 irreversibly inhibited the serum-free growth of A549 or B16 cells by 90-100%. The PUFAs alone had little effect on cell growth. Cell growth inhibition by TGF-beta 1 was not potentiated by saturated fatty acids, monounsaturated fatty acids, or prostaglandins. Inhibition of A549 or B16 cell growth by TGF-beta 1 in the presence of PUFAs was almost completely reversed by the antioxidant vitamin E, suggesting a role for lipid peroxidation in this process. Inhibition of A549 or B16 cell growth by TGF-beta 1 in the presence of 5% fetal calf serum was also potentiated by PUFAs and partially reversed by antioxidants. The presence of retinoic acid was required for maximal PUFA-dependent growth inhibition of A549 or B16 cells by TGF-beta 1 under some, but not all, conditions. These results suggest that inhibition of carcinoma and melanoma cell growth by TGF-beta 1 is mediated, in large part, by PUFAs.
Female copying increases the variance in male mating success.
Authors: Wade M J , Pruett-Jones S G
Published: May 2006

Theoretical models of sexual selection assume that females choose males independently of the actions and choice of other individual females. Variance in male mating success in promiscuous species is thus interpreted as a result of phenotypic differences among males which females perceive and to which they respond. Here we show that, if some females copy the behavior of other females in choosing mates, the variance in male mating success and therefore the opportunity for sexual selection is greatly increased. Copying behavior is most likely in non-resource-based harem and lek mating systems but may occur in polygynous, territorial systems as well. It can be shown that copying behavior by females is an adaptive alternative to random choice whenever there is a cost to mate choice. We develop a statistical means of estimating the degree of female copying in natural populations where it occurs.
Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell lines.
Authors: Holmes J , Clark S , Modrich P
Published: May 2006

Nuclear extracts derived from HeLa and Drosophila melanogaster KC cell lines have been found to correct single base-base mispairs within open circular DNA heteroduplexes containing a strand-specific, site-specific incision located 808 base pairs from the mismatch. Correction in both extract systems is strand specific, being highly biased to the incised DNA strand. Different mispairs within a homologous set of heteroduplexes were processed with different efficiencies (G.T greater than G.G approximately equal to A.C greater than C.C), and correction was accompanied by mismatch-dependent DNA synthesis localized to the region spanning the mispair and the strand break, thus demonstrating that mismatch recognition is associated with the repair reaction. Correction of each of these heteroduplexes was abolished by aphidicolin but was relatively insensitive to the presence of high concentrations of ddTTP, indicating probable involvement of alpha and/or delta class DNA polymerase(s). These findings suggest that higher eukaryotic cells possess a general, strand-specific mismatch repair system analogous to the Escherichia coli mutHLS and the Streptococcus pneumoniae hexAB pathways, systems that contribute in a major way to the genetic stability of these bacterial species.
In vitro transcription of two rRNA genes of the archaebacterium Sulfolobus sp. B12 indicates a factor requirement for specific initiation.
Authors: Hüdepohl U , Reiter W D , Zillig W
Published: May 2006

We describe a cell-free transcription system for the archaebacterium Sulfolobus sp. B12 that specifically initiates transcription at the 5S rRNA-encoding DNA and the 16S/23S rRNA-encoding DNA promoters of the same species. With this crude extract system, specific initiation was absolutely dependent on the box A motif, a highly conserved promoter element in archaebacteria located approximately 25 base pairs upstream of transcription initiation sites. In vitro transcription of the rRNA genes by purified RNA polymerase, however, resulted in semi-specific, box A-independent initiation, indicating that factor(s) in the crude extract were necessary for the highly specific box A-dependent transcription. Fractionation of the cell-free extract by sucrose-gradient centrifugation resulted in the identification of a low molecular weight fraction complementing purified RNA polymerase to an extract-like specificity.
Photoreduction of NADP+ by isolated reaction centers of photosystem II: requirement for plastocyanin.
Authors: Arnon D I , Barber J
Published: May 2006

The carrier of photosynthetically generated reducing power is the iron-sulfur protein ferredoxin, which provides directly, or via NADP+, reducing equivalents needed for CO2 assimilation and other metabolic reactions in the cell. It is now widely held that, in oxygenic photosynthesis, the generation of reduced ferredoxin-NADP+ requires the collaboration in series of two photosystems: photosystem II (PSII), which energizes electrons to an intermediate reducing potential and transfers them to photosystem I (PSI), which in turn is solely competent to energize electrons to the strong reducing potential required for the reduction of ferredoxin-NADP+ (the Z scheme). This investigation tested the premise of an alternative scheme, which envisions that PSII, without the involvement of PSI, is also capable of photoreducing ferredoxin-NADP+. We report here unexpected findings consistent with the alternative scheme. Isolated PSII reaction centers (completely free of PSI components), when supplemented with ferredoxin, ferredoxin-NADP+ oxidoreductase, and a PSII electron donor,1,5-diphenylcarbazide, gave a significant photoreduction of NADP+. A striking feature of this electron transfer from a PSII donor to the perceived terminal acceptor of PSI was its total dependence on catalytic quantities of plastocyanin, a copper-containing electron-transport protein hitherto known only as an electron donor to PSI.
Enhancement of RNA polymerase binding to promoters by a transcriptional activator, OmpR, in Escherichia coli: its positive and negative effects on transcription.
Authors: Tsung K , Brissette R E , Inouye M
Published: May 2006

The OmpR binding sequence (OBS) in the upstream region of the ompF promoter of Escherichia coli was fused to 27 synthetic promoters. Transcription from a number of weak promoters, regardless of their sequences, was dramatically activated in the presence of OmpR, a transcriptional activator. In vivo DNA footprinting revealed that OmpR enhanced the binding of RNA polymerase to the promoters. This enhancement was essential for transcription of weak promoters, while OmpR binding to the OBS fused to a strong promoter was inhibitory for transcription. These results indicate that OmpR stabilizes the formation of an RNA polymerase-promoter complex, possibly a closed promoter complex, and that a transcription activator can serve not only as a positive but also as a negative regulator for gene expression.
Microtubules in the metaphase-arrested mouse oocyte turn over rapidly.
Authors: Gorbsky G J , Simerly C , Schatten G , Borisy G G
Published: May 2006

After ovulation mammalian oocytes arrest in second meiotic metaphase. We asked whether the microtubules that comprise the meiotic spindle of mouse oocytes were stable or were undergoing rapid cycles of assembly and disassembly. Porcine brain tubulin, derivatized with biotin or x-rhodamine [5- (and -6)-carboxy-x-rhodamine], was microinjected into living oocytes. Biotinylated tubulin incorporated into the meiotic spindle to apparent equilibrium within 15 min. To assess quantitatively the rates of disassembly and assembly of the microtubules, small domains within the spindles of oocytes injected with x-rhodamine-tubulin were photobleached and their recovery was analyzed by digital imaging microscopy. Fluorescence recovery in the spindles was rapid and extensive, plateauing to an average of 83% at 4 min. The calculated half-time for turnover of the spindle microtubules was 77 sec. In contrast, fluorescence recovery of the spindle midbodies in telophase oocytes was much more limited, averaging approximately 22% at 4 min. These data indicate that most microtubules within the arrested metaphase spindle of the mouse oocyte undergo rapid cycles of assembly and disassembly. Microtubules of the telophase midbody are more stable.
The epidermal growth factor receptor and the product of the neu protooncogene are members of a receptor tyrosine phosphorylation cascade.
Authors: Connelly P A , Stern D F
Published: May 2006

The protein product of the neu protooncogene, p185, is a tyrosine kinase with a high degree of sequence homology to the epidermal growth factor (EGF) receptor. Although p185 does not bind EGF, EGF stimulates tyrosine phosphorylation of p185. To determine the mechanism of this interaction we have used a vaccinia virus/bacteriophage T7-based transient gene expression system to induce production of normal and kinase-deficient forms of p185 in the absence and presence of EGF receptors. Tyrosine phosphorylation of kinase-deficient p185 was observed, but only in the presence of the EGF receptor. These findings strongly support the hypothesis that p185 is a substrate for the EGF receptor tyrosine kinase in a tyrosine kinase cascade.
Molecular analysis of the hydrogenosomal ferredoxin of the anaerobic protist Trichomonas vaginalis.
Authors: Johnson P J , d'Oliveira C E , Gorrell T E , Müller M
Published: May 2006

We have determined the primary structure of the [2Fe-2S]ferredoxin of the anaerobic protist Trichomonas vaginalis. This protein, situated in the hydrogenosome, is composed of 93 amino acids. A comparison of T. vaginalis ferredoxin with greater than 80 other ferredoxins shows the closest similarity to [2Fe-2S]putidaredoxin of the aerobic bacterium Pseudomonas putida and a lesser one to mitochondrial [2Fe-2S]ferredoxins of vertebrates. This similarity is reflected in the overall primary structure and in the spacing of cysteine residues coordinating the iron-sulfur center. The primary structure, but not the environment of the iron-sulfur center, also shows similarity with [2Fe-2S]ferredoxins of photosynthetic organisms and halobacteria. We have cloned and analyzed the T. vaginalis ferredoxin gene. The gene is present in a single copy and devoid of introns. It gives rise to a transcript with unusually short 5' and 3' untranslated regions of 16 and 18 nucleotides, respectively. DNA sequence analysis of the gene predicts an additional 8 amino acids at the amino terminus which are absent from the purified protein. This amino-terminal region of the protein is characterized by properties typical of mitochondrial presequences.
Complementary DNA cloning of a receptor for tumor necrosis factor and demonstration of a shed form of the receptor.
Authors: Heller R A , Song K , Onasch M A , Fischer W H , Chang D , Ringold G M
Published: May 2006

Tumor necrosis factor (TNF) receptor (TNFR) was isolated as a 68-kDa glycoprotein from UC/HeLa 2-5 cells developed from a parental B-cell line (UC cells) to overexpress the receptor. Tryptic digests of two separate TNFR preparations provided amino acid sequences of four different peptides. Amino-terminal analysis indicated the presence of the amino-acid sequence Val-Ala-Phe-Thr-Pro, reported to be the amino-terminal sequence of a 30-kDa urinary TNF-binding protein II. Examination of the cultured medium of UC/HeLa 2-5 cells showed an abundance of a 40-kDa TNF-binding protein, indicating that the previously cited 30-kDa TNF-binding protein II is likely to be a shed form of the TNFR. Based on the peptide sequences, oligonucleotides were synthesized, and two of these were used as primers in the polymerase chain reaction to amplify cDNA sequences from poly(A)+ RNA of UC/HeLa 2-5 cells. These PCR fragments were radiolabeled and used to screen a cDNA library made from UC/HeLa 2-5 mRNA. Further analysis identified cDNA sequences that encoded the amino acid sequences of all four TNFR peptides. RNA blot-hybridization analysis of UC/HeLa 2-5 mRNA revealed a 3.8-kilobase transcript of the same size as the mRNA in the parental UC cells. Genomic Southern blots indicated the presence of a single gene in parental cells and a second, amplified gene in TNFR-overexpressing cells, suggesting amplification of the transfected gene as a possible mechanism for the increase in TNFR numbers in UC/HeLa 2-5 cells.
Molecular cloning of cDNA encoding a second cellular retinoic acid-binding protein.
Authors: Giguère V , Lyn S , Yip P , Siu C H , Amin S
Published: May 2006

The vitamin A derivative retinoic acid is known to be a potent agent for control of differentiation and proliferation of epithelial cells and to exert profound effects on pattern formation during embryogenesis. Its action at the molecular level appears to be mediated by two distinct classes of proteins: a family of nuclear receptors that regulates gene transcription in a ligand-dependent fashion and a small cellular retinoic acid-binding protein (CRABP) for which a precise function remains to be elucidated. In this report, we describe the identification (by molecular cloning of its cDNA) of an isoform of CRABP, referred to as CRABP-II, expressed at high levels during mouse embryogenesis and in adult skin. We also show that CRABP-II mRNA levels are induced by at least 50-fold upon treatment of F9 teratocarcinoma cells with retinoic acid. The up-regulation of the gene encoding CRABP-II by its ligand suggests that CRABP-II might be involved in a feedback regulatory role in the mechanism of action of retinoic acid on cellular differentiation.
Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses.
Authors: Wistow G , Anderson A , Piatigorsky J
Published: May 2006

In apparent contrast to most other tissues, the ocular lenses in vertebrates show striking differences in protein composition between taxa, most notably in the recruitment of different enzymes as major structural proteins. This variability appears to be the result of at least partially neutral evolutionary processes, although there is also evidence for selective modification in molecular structure. Here we describe a bird, the chimney swift (Chaetura pelagica), that lacks delta-crystallin/argininosuccinate lyase, usually the major crystallin of avian lenses. Clearly, delta-crystallin is not specifically required for a functionally effective avian lens. Furthermore the lens composition of the swift is more similar to that of the related hummingbirds than to that of the barn swallow (Hirundo rustica), suggesting that phylogeny is more important than environmental selection in the recruitment of crystallins. However differences in epsilon-crystallin/lactate dehydrogenase-B sequence between swift and hummingbird and other avian and reptilian species suggest that selective pressures may also be working at the molecular level. These differences also confirm the close relationship between swifts and hummingbirds.
Patterns of expression of position-dependent integrated transgenes in mouse embryo.
Authors: Bonnerot C , Grimber G , Briand P , Nicolas J F
Published: May 2006

The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program. We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues. Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter. Unexpectedly, however, the expression is temporally and spatially regulated. Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression. These results show that, for expression, the integrated construct must be complemented by elements of the genome. These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter. The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography. These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm. This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments. Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution.
Genes for Plasmodium falciparum surface antigens cloned by expression in COS cells.
Authors: Elliott J F , Albrecht G R , Gilladoga A , Handunnetti S M , Neequaye J , Lallinger G , Minjas J N , Howard R J
Published: May 2006

Two genes encoding membrane antigens of Plasmodium falciparum were isolated by transient expression in mammalian cells and selection with human immune sera from African adults exposed to P. falciparum malaria. COS-7 cells were transfected with a plasmid expression library constructed from P. falciparum genomic DNA, and cells expressing reactive malaria antigens on their surface were enriched by adherence to antibody-coated dishes. One of the genes isolated is distinctive in that it does not contain repeat sequences typical of many malarial genes cloned by immunoscreening of bacterial expression libraries. The second gene apparently encodes a polymorphic version of the P. falciparum merozoite surface antigen Ag513, since the two sequences are identical in the 5' and 3' coding regions but diverge completely in the center. The COS-7 expression system provides an alternate means for cloning genes encoding malarial membrane antigens by using those antibodies in complex immune sera that bind membrane-associated, nondenatured molecules.
Simulation of shape changes and adhesion phenomena in an elastic model of erythrocytes.
Authors: Leibler S , Maggs A C
Published: May 2006

We present simulations of a model of a closed membrane that shares important features with erythrocytes: resistance to bending and shear, membrane asymmetry, and an osmotic pressure difference between the interior and exterior. By varying a few parameters we obtain several realistic (e.g., biconcave and cup-like) shapes whose fluctuations, analogous to flickering of erythrocytes, and mutual transformations are studied in thermal equilibrium. Our simulations form a basis for quantitative analysis of recent experiments done on erythrocytes and artificial bilayer vesicles. They also predict effects that could be observed in experiments such as an "'unbinding" phenomenon, i.e., a separation of adhering cells induced by thermal fluctuations.
Modulation of the N-methyl-D-aspartate channel by extracellular H+.
Authors: Tang C M , Dichter M , Morad M
Published: May 2006

The influence of external [H+] on whole-cell and single-channel currents activated by glutamate agonists was studied in rat hippocampal neurons. In the pH range between 6.6 and 8.0, changes in external [H+] had negligible influence on the amplitude and kinetics of the monovalent ion-carrying currents activated by the agonists quisqualate and kainate. The divalent ion-carrying N-methyl-D-aspartate (NMDA)-activated current, on the other hand, was strongly modulated by extracellular [H+]. Increased external [H+] suppressed, whereas decreased external [H+] enhanced, the NMDA-activated current. Changes in internal [H+] had little or no effect on the NMDA-activated current. Modulation of the NMDA-activated current resulted primarily from changes in the number of channel openings. Neither the unitary conductance nor the individual open dwell-times were significantly affected. These results suggest that the protonation site is on the external aspect of the channel and is far removed from the channel permeation pathway. Because interactions between H+, NMDA, and glycine in activating the current were predominantly noncompetitive, our results suggest that the modulatory effect of H+ was not associated with changes in receptor-agonist affinities. These results suggest that modulation of the NMDA-receptor channel by [H+] may be an intrinsic protective mechanism by which calcium influx into neurons is regulated, particularly in hypoxic/ischemic conditions.
Molecular genetics of X chromosome-linked color vision among populations of African and Japanese ancestry: high frequency of a shortened red pigment gene among Afro-Americans.
Authors: Jørgensen A L , Deeb S S , Motulsky A G
Published: May 2006

Red-green color vision in humans is mediated by the X chromosome-linked highly homologous red and green pigment genes. Color vision defects are caused by deletions and fusions involving these genes. However, we found the frequency of molecular abnormalities among Caucasians to be twice as high as that of phenotypic color vision defects. Among Japanese the frequency of phenotypic and molecular color vision defects was similar (approximately 5%). Among Afro-Americans, molecular defects (largely green-red fusion genes) were at least five times more frequent (21%) than phenotypic color vision defects (approximately 4%). In addition, 35% of Afro-Americans, 2% of Japanese, and less than 1% of Caucasians had a shortened red pigment gene not associated with phenotypic color vision defects. This gene lacked 1.9 kilobases in its first intron and had the identical size as the green pigment gene from which it presumably originated by gene conversion in an ancestral African population. This gene and the closely linked glucose-6-phosphate dehydrogenase A+ variant were in linkage equilibrium. A model for the evolutionary origin of the color vision pigment genes in higher primates is portrayed.
Selective osmotic effect on diffusion of plasma membrane lipids in maize protoplasts.
Authors: Furtula V , Khan I A , Nothnagel E A
Published: May 2006

Osmotic levels in the range typically used during plant protoplast isolation and incubation were investigated with regard to effects on the lateral diffusion of lipid probes in the plasma membrane. The lateral diffusion coefficient of a fluorescent sterol probe in the plasma membrane of maize (Zea mays L.) root protoplasts in a medium containing 0.45 M mannitol was 4 times faster than when the medium contained 0.9 M mannitol. The lateral diffusion coefficient of a fluorescent phospholipid probe, however, did not change over this range of mannitol concentrations. Similar diffusion characteristics were observed when the medium contained trehalose instead of mannitol. Slower lateral diffusion of the sterol probe at higher osmolality was also observed when KCl/CaCl2-based osmotic media were used with protoplasts isolated by a mechanical, rather than by an enzymic, method. Extraction and quantitation of total lipids from protoplasts showed that both the phospholipid and sterol contents per protoplast decreased with increasing osmolality, while the sterol/phospholipid ratio increased. These results demonstrate that osmotic stress induces selective changes in both the composition and biophysical properties of plant membranes.
Structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease.
Authors: DesJarlais R L , Seibel G L , Kuntz I D , Furth P S , Alvarez J C , Ortiz de Montellano P R , DeCamp D L , Babé L M , Craik C S
Published: May 2006

By using a structure-based computer-assisted search, we have found a butyrophenone derivative that is a selective inhibitor of the human immunodeficiency virus 1 (HIV-1) protease. The computer program creates a negative image of the active site cavity using the crystal structure of the HIV-1 protease. This image was compared for steric complementarity with 10,000 molecules of the Cambridge Crystallographic Database. One of the most interesting candidates identified was bromperidol. Haloperidol, a closely related compound and known antipsychotic agent, was chosen for testing. Haloperidol inhibits the HIV-1 and HIV-2 proteases in a concentration-dependent fashion with a Ki of approximately 100 microM. It is highly selective, having little inhibitory effect on pepsin activity and no effect on renin at concentrations as high as 5 mM. The hydroxy derivative of haloperidol has a similar effect on HIV-1 protease but a lower potency against the HIV-2 enzyme. Both haloperidol and its hydroxy derivative showed activity against maturation of viral polypeptides in a cell assay system. Although this discovery holds promise for the generation of nonpeptide protease inhibitors, we caution that the serum concentrations of haloperidol in normal use as an antipsychotic agent are less than 10 ng/ml (0.03 microM). Thus, concentrations required to inhibit the HIV-1 protease are greater than 1000 times higher than the concentrations normally used. Haloperidol is highly toxic at elevated doses and can be life-threatening. Haloperidol is not useful as a treatment for AIDS but may be a useful lead compound for the development of an antiviral pharmaceutical.
Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides.
Authors: Shimoda N , Toyoda-Yamamoto A , Nagamine J , Usami S , Katayama M , Sakagami Y , Machida Y
Published: May 2006

Most virulence (vir) genes of Agrobacterium tumefaciens that are required for the formation of crown gall tumors are expressed in response to such plant signal molecules as acetosyringone and lignin precursors. The phenolic signals are transduced through a receptor VirA protein in the inner membrane of the bacterial cell. The expression of these genes triggers the transfer of a specific DNA segment, called transferred DNA (T-DNA), from the Ti plasmid to plant cells, and its integration into their nuclear DNA. We show here that a group of aldoses (L-arabinose, D-xylose, D-lyxose, D-glucose, D-mannose, D-idose, D-galactose, and D-talose) can markedly enhance acetosyringone-dependent expression of vir genes when the concentration of acetosyringone is limited (10 microM) but does not enhance the expression of noninducible genes. Likewise, a 2-deoxy-D-glucose, a nonmetabolized sugar, is also effective. When a deletion was introduced into the virA gene in the region encoding the periplasmic portion of the VirA protein, enhancement by glucose disappeared, but vir expression was induced by acetosyringone in this mutant. These results suggest that these sugars directly enhance a signaling process initiated by phenolic inducers that results in an increase in expression of the vir genes.
Molecular phylogeny of Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora and molecular clocks.
Authors: Li W H , Gouy M , Sharp P M , O'hUigin C , Yang Y W
Published: May 2006

Phylogenetic analysis of DNA sequences from primates, rodents, lagomorphs, artiodactyls, carnivores, and birds strongly suggests that the order Rodentia is an outgroup to the other four mammalian orders and that Artiodactyla and Carnivora belong to a superordinal clade. Further, there is strong evidence against the Glires concept, which unites Lagomorpha and Rodentia. The radiation among Lagomorpha, Primates, and Artiodactyla--Carnivora is very bush-like, but there is some evidence that Lagomorpha has branched off first. Thus, the branching sequence for these five orders of mammals seems to be Rodentia, Lagomorpha, Primates, Artiodactyla, and Carnivora. The branching date for Rodentia could be as early as 100 million years ago. The rate of nucleotide substitution in the rodent lineage is shown to be at least 1.5 times higher than those in the other four mammalian lineages.
A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes.
Authors: Roussel M F , Shurtleff S A , Downing J R , Sherr C J
Published: May 2006

Substitution of phenylalanine for tyrosine-809 in the human colony-stimulating factor 1 receptor (CSF-1R) inhibited its ability to transduce ligand-dependent mitogenic signals in mouse NIH 3T3 cells. When combined with an "activating" mutation at codon 301 that induces constitutive CSF-1R tyrosine kinase activity, the codon 809 mutation suppressed ligand-independent cell transformation. Comparative mapping of tryptic phosphopeptides from mutant and wild-type CSF-1R indicated that tyrosine-809 is a site of ligand-dependent receptor phosphorylation in vivo. The mutant receptor was active as a tyrosine kinase in vitro and in vivo, underwent CSF-1-dependent association with a phosphatidylinositol 3-kinase, and induced expression of the protooncogenes c-fos and junB, underscoring its ability to trigger some of the known cellular responses to CSF-1. The mutant receptor is likely to be impaired in its ability to interact with critical cellular effectors whose activity is required for mitogenesis.