bluuebunny/crossref_metadata_2025 huggingface dataset:
A yeast cyclophilin gene essential for lactate metabolism at high temperature.
Authors: Davis E S , Becker A , Heitman J , Hall M N , Brennan M B
Published: May 2006

The cyclophilins are a family of ubiquitous eukaryotic proteins first identified by high affinity for cyclosporin A (CsA). The immunosuppressant and cytotoxic effects of CsA are thought to result from formation of a toxic complex between cyclophilin and CsA rather than from inhibition of cyclophilin function. The physiological role(s) of the cyclophilins is unknown. Cyclophilins have in vitro peptidylprolyl cistrans isomerase (PPIase) activity, and thus may be involved in protein folding in vivo. We have isolated a yeast cyclophilin gene, CPR3, which encodes a presumptive mitochondrial isoform. While CPR3 disruption mutants lack any phenotype at 30 degrees C, they are unable to grow on L-lactate at 37 degrees C. Disruptions of two other cyclophilin genes (CPR1, CPR2) and of FPR1, the gene encoding an FK506 binding protein with PPIase activity, do not affect growth on L-lactate at 37 degrees C. L-Lactate metabolism requires transcriptional induction of CYB2, the gene encoding flavocytochrome b2; cpr3 mutants induce transcription of this gene normally. This result demonstrates a conditional lethal phenotype for a cyclophilin mutation and presents a system for genetic and biochemical analysis of cyclophilin function.
Isolation, characterization, and transcription of the gene encoding mouse mast cell protease 7.
Authors: McNeil H P , Reynolds D S , Schiller V , Ghildyal N , Gurley D S , Austen K F , Stevens R L
Published: May 2006

A gene that encodes mouse mast cell protease (mMCP) 7 (also known as mouse mast cell tryptase 2) was isolated by genomic cloning with a cDNA that encodes mMCP-6, a tryptase in serosal mast cells. cDNAs encoding mMCP-7 were isolated from a bone-marrow-derived mast cell cDNA library. The mMCP-7 gene spans 2.3 kilobases and contains five exons rather than six, as found in the mMCP-6 and human mast cell tryptase I genes. Comparison of the 5' end of the transcript with the genomic sequence indicated that the region corresponding to the first intron in the mMCP-6 and human tryptase I genes is not spliced during transcription of mMCP-7 mRNA because of a point mutation at the intron 1 acceptor splice site; this results in a 5' untranslated region of 195 nucleotides, which is longer than that of any other known mast cell-specific transcript. mMCP-7 is 71-76% homologous with mMCP-6 and with dog and human mast cell tryptases, and it is the most acidic mast cell protease, with an overall net charge of -10. RNA blot analyses revealed that the mMCP-7 gene is transcribed in bone-marrow-derived mast cells but is not transcribed in mature serosal mast cells or in mucosal mast cell-enriched intestinal tissue of Trichinella spiralis-infected mice. Transcription of the mMCP-7 gene by differentiating bone-marrow-derived mast cells occurred within 1 week of bone-marrow culture but decreased dramatically after 3 weeks. Thus, the mMCP-7 gene displays a number of unusual structural characteristics and is distinctive in its transient and selective expression in immature mast cells maintained in interleukin 3-enriched medium.
Efficient transfer and sustained high expression of the human glucocerebrosidase gene in mice and their functional macrophages following transplantation of bone marrow transduced by a retroviral vector.
Authors: Ohashi T , Boggs S , Robbins P , Bahnson A , Patrene K , Wei F S , Wei J F , Li J , Lucht L , Fei Y
Published: May 2006

A recombinant retroviral vector (MFG-GC) was used to study the efficiency of transduction of the human gene encoding glucocerebrosidase (GC; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45), in mouse hematopoietic stem cells and expression in their progeny. Transfer of the GC gene to CFU-S (spleen cell colony-forming units) in primary and secondary recipients was virtually 100%. In mice 4-7 months after transplantation, highly efficient transfer of the human gene to bone marrow cells capable of long-term reconstitution was confirmed by detection of one or two copies per mouse genome in hematopoietic tissues and in cultures of pure macrophages. Expression of the human gene exceeded endogenous activity by several fold in primary and secondary CFU-S, tissues from long-term reconstituted mice, and explanted macrophages cultures. These studies are evidence of the feasibility of efficient transfer of the GC gene to hematopoietic stem cells and expression in their progeny for many months after reconstitution. The results of this study strengthen the rationale for gene therapy as a treatment for Gaucher disease.
Tumor necrosis factor alpha response elements in the HLA-DRA promoter: identification of a tumor necrosis factor alpha-induced DNA-protein complex in astrocytes.
Authors: Panek R B , Moses H , Ting J P , Benveniste E N
Published: May 2006

The cytokine tumor necrosis factor alpha (TNF-alpha) alone does not induce class II major histocompatibility complex (MHC) expression in most primary cells but can regulate ongoing class II expression in either a positive or negative fashion. The mechanism(s) by which TNF-alpha enhances interferon gamma (IFN-gamma)-induced class II expression was examined in a primary cell type, the astrocyte, by transient transfection of the HLA-DRA promoter linked to a chloramphenicol acetyltransferase reporter gene (DRA-CAT). We show that TNF-alpha, while having no effect on its own, can synergize with IFN-gamma to increase the level of promoter activity of a DRA-CAT construct. Three known sequences--W, X, and Y--are required for TNF-alpha enhancement of IFN-gamma-induced promoter activity. The corollary effect of TNF-alpha on DNA-binding proteins specific for these elements was examined. A previous report described a DNA-binding protein, IFN-gamma-enhanced factor X (IFNEX), which is upregulated by IFN-gamma in astrocytes and is specific for the X box of the DRA promoter. In this study, we found that TNF-alpha alone did not induce any nuclear proteins; however, combined treatment of astrocytes with both IFN-gamma and TNF-alpha induced a DNA-protein complex of slower electrophoretic mobility than IFNEX. The TNF-alpha-induced complex (TIC-X) has specificity for the X element of the DRA promoter. These results suggest a mechanism by which TNF-alpha enhances IFN-gamma-induced class II MHC expression via the formation of TIC-X.
Transcription factor IIA is inactivated during terminal differentiation of avian erythroid cells.
Authors: Bungert J , Waldschmidt R , Kober I , Seifart K H
Published: May 2006

Avian histone H5 and alpha A-globin genes are transcribed much more efficiently in whole cell extracts derived from immature polychromatic erythrocytes than in extracts from mature duck erythrocytes. We found that these differential activities are detectable only if assayed with promoters containing a functional TATA box. The addition of either highly purified human or recombinant yeast transcription factor IIA (TFIIA) to extracts from mature erythrocytes resulted in a significant increase in transcription from TATA-containing promoters, whereas transcription from TATA-less promoters remained unaffected. Moreover, the activity of TFIIA was found to be reduced in extracts from mature erythrocytes. These data support the proposition that inactivation of TFIIA may contribute to a general repression of gene activity in avian erythrocytes, and only those genes with alternative mechanisms of initiation complex formation continue to be expressed in these cells. In the case of the histone H5 gene, such an alternative mechanism could be mediated via the interaction between duck erythrocyte upstream stimulating factor and TFIID.
Poly(ADP-ribose) polymerase activity in mononuclear leukocytes of 13 mammalian species correlates with species-specific life span.
Authors: Grube K , Bürkle A
Published: May 2006

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a role in DNA repair and the recovery of cells from DNA damage. We compared poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) activities in Percoll gradient-purified, permeabilized mononuclear leukocytes from mammalian species of different maximal life span. Saturating concentrations of a double-stranded octameric oligonucleotide were applied to provide a direct and maximal stimulation of PARP. Our results on 132 individuals from 13 different species yield a strong positive correlation between PARP activity and life span (r = 0.84; P << 0.001), with human cells displaying approximately 5 times the activity of rat cells. Intraspecies comparisons with both rat and human cells from donors of all age groups revealed some decline of PARP activity with advancing age, but it was only weakly correlated. No significant polymer degradation was detectable under our assay conditions, ruling out any interference by poly(ADP-ribose) glycohydrolase activity. By Western blot analysis of mononuclear leukocytes from 11 species, using a crossreactive antiserum directed against the extremely well-conserved NAD-binding domain, no correlation between the amount of PARP protein and the species' life spans was found, suggesting a greater specific enzyme activity in longer-lived species. We propose that a higher poly(ADP-ribosyl)ation capacity in cells from long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span.
Domain motions in phosphoglycerate kinase: determination of interdomain distance distributions by site-specific labeling and time-resolved fluorescence energy transfer.
Authors: Haran G , Haas E , Szpikowska B K , Mas M T
Published: May 2006

3-Phosphoglycerate kinase is composed of two globular domains separated by a wide cleft. The substrate binding sites are situated on the inner surfaces of the two domains. By analogy to other kinases, it has been postulated that the catalytic mechanism of phosphoglycerate kinase involves a hinge bending domain motion that brings the substrates together to allow phosphoryl transfer. To characterize this large-scale conformational change, as well as the dynamics of the unliganded enzyme in solution, we have applied site-directed mutagenesis and time-resolved nonradiative energy transfer techniques. Two genetically engineered cysteines (Cys-135 and Cys-290), one in each of the two domains, were covalently labeled with a donor and acceptor pair of fluorescent probes. Analysis of subnanosecond fluorescence decay curves yielded the equilibrium distribution of interdomain distances. In the absence of substrates, the distribution of distances between the two labeled sites was very broad, with a full width at half maximum estimated as 20 A or broader, indicative of a large number of conformational substates in solution. The mean distance, 31.5 +/- 1 A, was 8 A smaller than in the crystal structure. Upon addition of ATP alone or of ATP and 3-phosphoglycerate, the average distance increased to 38 +/- 1 A and the width of the distribution decreased. Addition of 3-phosphoglycerate alone induced a similar but smaller change. The rate of conformational state fluctuations (interconversion between states) was found to be slow on the nanosecond time scale, as expected for a protein with a relatively large interdomain contact area.
Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I.
Authors: Yao V J , Spudich J L
Published: May 2006

A methylated membrane protein of 97 kDa was suggested on the basis of mutant analysis to transduce signals from the phototaxis receptor sensory rhodopsin I to the flagellar motor in Halobacterium halobium. Here we report isolation of the proposed transducer protein, cloning of its gene based on partial protein sequences, the complete gene sequence, and analysis of the encoded primary structure. The 1611-base-pair gene termination codon overlaps the initiator ATG of the sopI gene, which encodes the sensory rhodopsin I apoprotein. The predicted size of 57 kDa for the methylated protein indicates an aberrant electrophoretic migration on SDS/polyacrylamide gels, as occurs with other acidic halophilic proteins. Putative promotor elements are located in an A+T-rich region upstream of the gene. Comparison of the translated nucleotide sequence with N-terminal sequence of the purified protein shows the protein is synthesized without a processed leader peptide and the N-terminal methionine is removed in the mature protein. The deduced protein sequence predicts two transmembrane helices near the N terminal that would anchor the protein to the membrane. Beyond this hydrophobic region of 46 residues, the remainder of the protein (536-amino acid residues total) is hydrophilic. The C-terminal 270 residues contain a region homologous to the signaling domains of eubacterial transducers (e.g., Escherichia coli Tsr protein), flanked by two regions homologous to the methylation domains of the transducer family. The protein differs from E. coli Tsr in that it does not have an extramembranous-receptor binding domain but instead has a more extended cytoplasmic region. Coexpression of the methyl-accepting protein gene (designated htrI) and sopI restores sensory rhodopsin I phototaxis to a mutant (Pho81) that contains a deletion in the htrI/sopI region. These results extend the eubacterial transducer family to the archaebacteria and substantiate the proposal that the methylated membrane protein functions as a signal-transducing relay between sensory rhodopsin I and cytoplasmic sensory-pathway components.
Spectral enhancement of proteins: biological incorporation and fluorescence characterization of 5-hydroxytryptophan in bacteriophage lambda cI repressor.
Authors: Ross J B , Senear D F , Waxman E , Kombo B B , Rusinova E , Huang Y T , Laws W R , Hasselbacher C A
Published: May 2006

We have used a tryptophan-requiring Escherichia coli auxotroph to replace the three tryptophan residues of lambda cI repressor with 5-hydroxy-L-tryptophan (5-OHTrp). By using a nonleaky promoter, we have achieved > 95% replacement of tryptophan in the repressor. We show that the absorbance and fluorescence properties of 5-OHTrp-lambda cI are clearly distinct from lambda cI repressor and that the fluorescence of 5-OHTrp-lambda cI repressor can be observed selectively in the presence of exogenous tryptophan. We also show that the 5-OHTrp-lambda cI repressor functional properties, as assessed by measurement of binding constants for self-association and for association to operator DNA, and structural properties, as assessed by fluorescence, are indistinguishable from the native repressor. Based on these results, we anticipate that the availability of spectrally enhanced proteins will significantly enhance the utility of both fluorescence and phosphorescence spectroscopies to study protein structure and function in complex interacting systems.
Expression of yeast hexokinase in pancreatic beta cells of transgenic mice reduces blood glucose, enhances insulin secretion, and decreases diabetes.
Authors: Epstein P N , Boschero A C , Atwater I , Cai X , Overbeek P A
Published: May 2006

It has been proposed that endogenous hexokinases of the pancreatic beta cell control the rate of glucose-stimulated insulin secretion and that genetic defects that reduce beta-cell hexokinase activity may lead to diabetes. To test these hypotheses, we have produced transgenic mice that have a 2-fold increase in hexokinase activity specific to the pancreatic beta cell. This increase was sufficient to significantly augment glucose-stimulated insulin secretion of isolated pancreatic islets, increase serum insulin levels in vivo, and lower the blood glucose levels of transgenic mice by 20-50% below control levels. Elevation of hexokinase activity also significantly reduced blood glucose levels of diabetic mice. These results confirm the role of beta-cell hexokinase activity in the regulation of insulin secretion and glucose homeostasis. They also provide strong support for the proposal that reductions in beta-cell hexokinase activity can produce diabetes.
Ovariectomy selectively reduces the concentration of transforming growth factor beta in rat bone: implications for estrogen deficiency-associated bone loss.
Authors: Finkelman R D , Bell N H , Strong D D , Demers L M , Baylink D J
Published: May 2006

Previous work showed that production of transforming growth factor beta (TGF-beta) by osteoblast-like rat UMR 106 cells was increased by 17 beta-estradiol at physiological concentrations. To determine whether ovariectomy alters the concentration of TGF-beta in rat long bones, female Sprague-Dawley rats were either sham-operated (n = 19) or ovariectomized (n = 19), pair-fed a semisynthetic diet for 6 weeks, and sacrificed. Tibial and femoral diaphyses were removed and extracted by demineralization. Ovariectomy lowered serum estrogen; did not alter body weight, serum magnesium, or serum 1,25-dihydroxyvitamin D; and produced only modest differences in serum calcium and phosphate concentrations. Hydroxyproline was higher and extractable protein was lower in bones from ovariectomized rats than in bones from sham-operated rats; calcium content did not differ between the two groups of animals. Ovariectomy lowered the concentration of TGF-beta in bone but did not change the concentration of insulin-like growth factors I or II compared with values in bone from control animals. The reduction of bone TGF-beta was evident 6 weeks after surgery but not at 3 weeks. Treatment of ovariectomized rats with estrogen eliminated the TGF-beta deficit. To determine whether 17 beta-estradiol increased TGF-beta production by normal bone cells, mouse osteoblasts were treated for 2 days with 17 beta-estradiol. The production of TGF-beta was increased almost 2-fold by 1 nM 17 beta-estradiol, and short-term treatment stimulated the intracellular accumulation of TGF-beta 1 mRNA. We conclude that ovariectomy reduces deposition of TGF-beta in rat bone and that diminished skeletal TGF-beta could play a role in the pathogenesis of bone loss, fractures, and microfractures that occur in estrogen-deficient states. Our results support the possibility that estrogen and bone TGF-beta may be necessary for normal maintenance of the skeleton in female rats.
Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy.
Authors: Lynch C M , Clowes M M , Osborne W R , Clowes A W , Miller A D
Published: May 2006

Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli beta-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.
Environmental issues and the law.
Authors: Henrichs R
Published: May 2006

This report first briefly reviews the history and lessons learned from our legislation regarding protection of our environment. Then, these lessons are combined with principles of general jurisprudence to suggest avenues for regulation of industrial ecology. Even though the law has important limitations, it can be used as a tool to identify national goals and to promote their achievement by means of incentives or sanctions.
Educating and training industrial ecologists for the 1990s.
Authors: Troxell D E
Published: May 2006

For industrial ecology to become a reality and flourish, many talents must be integrated into a single system. Basically, there are four types of technologists who must be brought together to create a system: (i) technical specialists-those that can take the state of knowledge to the state of the art and eventually to the state of practice; (ii) technical "generalists"-those that can integrate diverse technologies into a comprehensive solution; (iii) technical managers-those that can lead technical efforts to an optimal solution (these people must have management skills in their area); and (iv) program managers-those that can understand and merge the overall efforts of technology, business, schedule, and finance. The initial questions for industrial ecology in the 1990s are: Do these people presently exist in industry? What will be needed to bring new people (recent graduates) into the field of industrial ecology? For this decade, the people who will develop and lead the concept of industrial ecology are presently in industry. However, certainly few, if any, today understand the concept of industrial ecology as a system issue. Training will be needed for these practitioners of the 1990s so that the overall goal and the mechanism for achieving industrial ecology are understood. A three-tiered approach is presented to define the education/training needed, to conduct training on an industry basis, and then to initiate the training to be done within a corporation. The three tiers propose the use of universities, professional societies, and industry groups. Having universities participate in the training of the 1990s industrial ecologists will give universities insight to the development of industrial ecology, and access to industry needs so that curriculum can be initiated to educate the industrial ecologists of the future.
Solid-liquid phase boundaries of lens protein solutions.
Authors: Berland C R , Thurston G M , Kondo M , Broide M L , Pande J , Ogun O , Benedek G B
Published: May 2006

We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf gamma-crystallin proteins: gamma II, gamma IIIa, and gamma IIIb. We also studied the liquidus line for solutions of native gamma IV-crystallin calf lens protein, which consists of 85% gamma IVa/15% gamma IVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-liquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location of the liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures greater than 70 degrees C: gamma IIIa and gamma IV, and those in which liquidus lines flatten at temperatures less than 50 degrees C: gamma II and gamma IIIb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.
Restricted heterogeneity in T-cell antigen receptor V beta gene usage in the lymph nodes and arthritic joints of mice.
Authors: Haqqi T M , Anderson G D , Banerjee S , David C S
Published: May 2006

We have used PCR to study the expression of T-cell antigen receptor beta RNA containing particular variable region (V) elements from transcripts directly in the cells isolated from joints and lymph nodes of B10.Q mice (H-2q) immunized with chicken type II collagen. Our data show that the T cells present in arthritic joints expressed only a few V beta transcripts--V beta 2, -6, -7, -8.2, -9, -10, and -15. V beta 6 and -8.2 were expressed predominantly (six out of seven animals) while others were expressed at a relatively low level in different animals. In lymph node cells, transcripts for V beta 6, -8.2, and -9 were detected in four out of seven animals. The data indicate that in collagen-induced arthritis there is a restrictive usage of TCR V beta elements and that V beta 6 and -8.2 are probably used preferentially.
Carcinogen-induced frameshift mutagenesis in repetitive sequences.
Authors: Lambert I B , Napolitano R L , Fuchs R P
Published: May 2006

We have constructed plasmids pS3G-1 and pSG4 that contain single acetylaminofluorene adducts within contiguous runs of three (5'-CCCG1G2G3-3') and four (5'-CG1GGG4T-3') guanine residues, respectively. In Escherichia coli, the frequency of induced -1 frameshift mutations was strongly dependent on the position of modification: pS3G-G3 was approximately 100-fold and 10-fold more mutagenic than pS3G-G1 and pS3G-G2, respectively; pSG4-G4 was approximately 600-fold more mutagenic than pSG4-G1. Mutagenesis was SOS-dependent and was markedly reduced in bacteria that were proficient in nucleotide excision repair as compared to a repair-deficient uvrA6 mutant. DNA sequencing showed that -1 frameshift events in pS3G-1 consisted of either targeted mutations (greater than 90% of induced mutations) within the guanine sequence or semitargeted mutations (greater than 10%) in the 5' flanking repetitive cytosine sequence. Semitargeted events, which were observed when acetylaminofluorene modification was at G1 and G2, show that a lesion can reduce the fidelity of replication at positions 5' to its location on the template strand. No semitargeted frameshifts were observed in plasmid pSG4, which lacks a repetitive sequence 5' to the adduct. Our results are consistent with a model for frameshift mutagenesis in which the acetylaminofluorene adduct (i) allows accurate incorporation of cytosine opposite the bulky lesion during DNA synthesis and (ii) impedes elongation of primer/template termini formed opposite the adduct or 5' to the adduct on the template strand, providing increased opportunity for the formation of slipped frameshift intermediates.
Hemispheric differences in avian song discrimination.
Authors: Cynx J , Williams H , Nottebohm F
Published: May 2006

Auditory input to the right or left forebrain of adult male zebra finches (Taeniopygia guttata) was disrupted by lesioning the ipsilateral auditory relay nucleus of the thalamus. These birds were then presented with two kinds of auditory discriminations: (i) between their own song and the song of a cage mate; (ii) between two versions of an unfamiliar zebra finch song that differed only in the harmonic profile of one of the syllables. Right-side lesion birds did better than left-side lesion ones at discriminating between their own song and the song of a cage mate; left-side lesion birds did better on the harmonic profile task. We suggest that the two halves of the zebra finch brain process conspecific sounds differently, as seems to be the case for humans.
A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.
Authors: Singer-Sam J , Goldstein L , Dai A , Gartler S M , Riggs A D
Published: May 2006

Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.
Ontogeny of a biological clock in Drosophila melanogaster.
Authors: Sehgal A , Price J , Young M W
Published: May 2006

Drosophila melanogaster born and reared in constant darkness exhibit circadian locomotor activity rhythms as adults. However, the rhythms of the individual flies composing these populations are not synchronized with one another. This lack of synchrony is evident in populations of flies commencing development at the same time, indicating that a biological clock controlling circadian rhythmicity in Drosophila begins to function without a requirement for light and without a developmentally imparted phase. It is possible to synchronize the phases of rhythms produced by dark-reared flies with light treatments ending as early as the developmental transition from embryo to first-instar larva: Light treatments occurring at developmental times preceding hatching of the first-instar larva fail to synchronize adult locomotor activity rhythms, while treatments ending at completion of larval hatching entrain these rhythms. The synchronized rhythmic behavior of adult flies receiving such light treatments suggests that a clock controlling circadian rhythms may function continuously from the time of larval hatching to adulthood.
A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini.
Authors: Padfield P J , Balch W E , Jamieson J D
Published: May 2006

In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.
Phase-separation inhibitors and prevention of selenite cataract.
Authors: Clark J I , Steele J E
Published: May 2006

The variation of the phase-separation temperature (Tc) was studied in lenses during formation of cataracts induced by a subcutaneous injection of sodium selenite. In normal control animals, the Tc decreased monotonically with increasing age. Approximately 2 days after administration of the selenite the Tc decreased sharply to a minimum, and then at day 4 the Tc increased dramatically toward body temperature. Mature irreversible cataracts formed approximately 6 days after injection of the selenite. Intraperitoneal administration of WR-77913, a phase-separation inhibitor, prevented the abnormal variation of Tc in vivo. When injected into control animals without selenite, WR-77913 produced no abnormal variation in Tc. The results confirm that Tc is a sensitive measure of early changes in the lens and that opacification associated with abnormal variation in Tc can be prevented in vivo by using a phase-separation inhibitor.
Axial distances in discrete Möbius groups.
Authors: Gehring F W , Martin G J
Published: May 2006

We describe here new lower bounds for the trace of the commutator of a discrete two-generator group of Möbius transformations. These results lead to sharp estimates for the distance between the axes of elliptics in a discrete group and hence to new bounds for the volume of hyperbolic manifolds and orbifolds that will be discussed elsewhere.
Primary structure of Euplotes raikovi pheromones: comparison of five sequences of pheromones from cells with variable mating interactions.
Authors: Raffioni S , Miceli C , Vallesi A , Chowdhury S K , Chait B T , Luporini P , Bradshaw R A
Published: May 2006

The amino acid sequences of five pheromones, Er-2, Er-3, Er-9, Er-11, and Er-20, secreted by cells of different mating types of the ciliated protozoa Euplotes raikovi, have been determined by automated Edman analyses of the whole proteins and germane fragments. In each case, the molecular mass was determined by plasma desorption or laser desorption mass spectrometry and was in excellent agreement with the calculated values. Where available, the determined sequences were also in accord with the corresponding segments of the precursor molecules predicted from relevant nucleic acid sequences. Of the five, two were found to be identical (Er-2 and Er-9) and one (Er-3) was identical to a pheromone previously sequenced (Er-1), even though mating pair formation was found to take place (although to a limited extent) when cells secreting those pheromones were combined in a mixture. Comparison of the five unique sequences suggested a closer relationship between Er-1 (Er-3) and Er-10 and between Er-11 and Er-20 (44% and 56% identity, respectively) than was generally observed among the other members. This pairing was also supported by hydrophobicity analyses. Interestingly, Er-20 cannot, as a rule, induce cell union in any of the other cell types, including cells secreting Er-11, despite the fact that Er-20 and Er-11 are the most similar of the five unique sequences. Thus sequence identity and secondary structure profiles are not a good indicator of biological relatedness as manifested in heterologous receptor interaction.
Identification of the promoter of the myelomonocytic leukocyte integrin CD11b.
Authors: Hickstein D D , Baker D M , Gollahon K A , Back A L
Published: May 2006

The CD11b (or macrophage-1 antigen; MAC-1) subunit of the leukocyte integrin family forms a noncovalently associated heterodimeric structure with the CD18 (beta) subunit on the surface of human granulocytes and monocyte/macrophages, where it enables these myeloid cells to participate in a variety of adherence-related activities. Expression of the CD11b subunit is restricted to cells of the myelomonocytic lineage and depends upon the stage of differentiation with the most mature myeloid cells expressing the highest levels of CD11b. To study the regulation of CD11b expression, a genomic clone corresponding to the 5' region of the CD11b gene was isolated from a human chromosome 16 library. Primer extension and RNase protection assays identified two major transcriptional start sites, located 90 base pairs and 54 base pairs upstream from the initiation methionine. DNA sequence analysis of 1.7 kilobases of the 5' flanking sequence of the CD11b gene indicated the absence of a "CAAT" or "TATA" box; however, potential binding sites for the transcription activators Sp1, PU.1, ets, and AP-2 are present, as well as retinoic acid response elements. The 1.7-kilobase CD11b promoter sequence displayed functional activity in transient transfection assays in the monocytic cell line THP-1 and the myeloid cell line HL-60. In contrast, this 1.7-kilobase promoter sequence did not display functional activity in the Jurkat T-lymphoid cell line. Detailed characterization of the CD11b promoter sequence should provide insight into the molecular events regulating the tissue-specific and developmental stage-specific expression of the CD11b molecule in myelomonocytic cells.
Telomere-related markers for the pseudoautosomal region of the mouse genome.
Authors: Eicher E M , Lee B K , Washburn L L , Hale D W , King T R
Published: May 2006

The pseudoautosomal (PA) region of the mammalian genome is the region of the X and Y chromosomes that shares extensive DNA sequence homology and is of special interest because it may play an essential role during male meiosis. We have identified three telomere-related restriction fragments from the PA region of the mouse genome, using an oligonucleotide probe composed of the mammalian telomere consensus sequence TTAGGG. PA assignment of two C57BL/6J-derived fragments was initially suggested by analysis of DNAs from progeny sired by C57BL/6J males carrying the rearranged Y chromosome, Y*: the hybridization intensity of both fragments was concordant with the sex-chromosome complement of the offspring. Further analysis indicated that both fragments were present in female and male F1, mice regardless of the sex of their C57BL/6J parent--a criterion for autosomal or PA linkage. Both fragments were closely linked to each other and located on the X chromosome distal to amelogenin (Amg)--in agreement with X or PA linkage. Confirmation of the PA derivation of these fragments was accomplished by following their segregation in a cross involving XY* males mated to DBA/2J females. A similar experiment identified a third PA-derived restriction fragment of LT/SvEi origin. Identification of PA-derived telomere-related restriction fragments will enable further genetic analysis of this region of the mouse genome.
ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ.
Authors: Haycock J W , Ahn N G , Cobb M H , Krebs E G
Published: May 2006

Tyrosine hydroxylase (TH) is phosphorylated at four sites in situ and in vivo, and the protein kinases that phosphorylate three of these sites (Ser8,Ser19,Ser40) have been identified. In intact cells, the phosphorylation of the fourth site (Ser31) is increased in response to phorbol esters or nerve growth factor (NGF). Here, we show that Ser31 is phosphorylated by ERK1 and ERK2, two myelin basic protein and microtubule-associated protein kinases. Extracts of NGF- or bradykinin-treated PC12 rat pheochromocytoma cells were fractionated on Mono Q columns. Protein kinase activity toward Ser31 in TH was present in two peaks corresponding to myelin basic protein kinase activities previously identified as ERK1 and ERK2. Phosphorylation of purified TH in vitro by both kinases was selective for Ser31 up to at least 0.6 mol of phosphate per mol of TH subunit. Treatment of intact PC12 cells with bradykinin or NGF increased both the phosphorylation of TH-Ser31 in situ and the catalytic activity of ERKs (measured subsequently in vitro with myelin basic protein as substrate). Pretreatment of the cells with genistein (a protein-tyrosine kinase inhibitor) decreased the bradykinin- but not the NGF-induced changes in both TH-Ser31 phosphorylation and ERK activity. Genistein also inhibited the increases in Ser31 phosphorylation produced by phorbol dibutyrate, muscarine, and Ba2+. The data indicate that ERK activity is responsible for phosphorylating TH at Ser31 in intact cells and suggest that TH-Ser31 phosphorylation may be regulated by multiple signaling pathways that converge at or prior to the activation of the ERKs.
Serotype conversion in Vibrio cholerae O1.
Authors: Stroeher U H , Karageorgos L E , Morona R , Manning P A
Published: May 2006

Vibrio cholerae O1 exists as two major serotypes, Inaba and Ogawa, which are associated with the O antigen of the lipopolysaccharide and are capable of unequal reciprocal interconversion. The 20-kilobase rfb regions encoding O-antigen biosynthesis in strains 569B (Inaba) and O17 (Ogawa) have been cloned in Escherichia coli K-12 and the nucleotide sequences have been determined. Besides several base substitutions and a small deletion in the 569B sequence relative to O17, there is a single nucleotide change resulting in a TGA stop codon within the gene for the 32-kDa RfbT protein. We have demonstrated that rfbT is responsible for serotype conversion (Inaba to Ogawa). The construction of a specific rfbT mutation in the Ogawa strain O17, and the ability of the gene from O17 to complement Inaba strains to Ogawa, confirmed rfbT as the gene required for the Ogawa serotype. By Southern hybridization and sequencing of PCR products of a number of strains, we have shown that the changes observed in one Inaba strain (569B) are not conserved in other Inaba strains. This may explain why some Inaba strains are able to convert to Ogawa whereas others are not. The protein encoded by rfbT has been identified and expressed in E. coli K-12 using a phage T7 expression system. Amino-terminal analysis of partially purified protein has identified the translational start of the protein. Primer extension studies have enabled the 5' end of the mRNA to be defined. It exists as a separate transcript from the rest of the rfb region, and the distinctive G + C content of rfbT suggests that it has been acquired from a non-Vibrio source.
Chronic deafferentation in monkeys differentially affects nociceptive and nonnociceptive pathways distinguished by specific calcium-binding proteins and down-regulates gamma-aminobutyric acid type A receptors at thalamic levels.
Authors: Rausell E , Cusick C G , Taub E , Jones E G
Published: May 2006

Chronic deafferentation of skin and peripheral tissues is associated with plasticity of representational maps in cerebral cortex and with perturbations of sensory experience that include severe "central" pain. This study shows that in normal monkeys the nonnociceptive, lemniscal component of the somatosensory pathways at spinal, brainstem, and thalamic levels is distinguished by cells and fibers immunoreactive for the calcium-binding protein parvalbumin, whereas cells of the nociceptive component at these levels are distinguished by immunoreactivity for 28-kDa calbindin. Long-term dorsal rhizotomies in monkeys lead to transneuronal degeneration of parvalbumin cells at brainstem and thalamic sites accompanied in the thalamus by a down-regulation of gamma-aminobutyric acid type A receptors and an apparent increase in activity of calbindin cells preferentially innervated by central pain pathways. Release from inhibition and imbalance in patterns of somatosensory inputs from thalamus to cerebral cortex may constitute subcortical mechanisms for inducing changes in representational maps and perturbations of sensory perception, including central pain.
Synthesis of the early light-inducible protein is controlled by blue light and related to light stress.
Authors: Adamska I , Ohad I , Kloppstech K
Published: May 2006

The early light-inducible proteins (ELIPs) are expressed in developing plants in the first hours of the greening process. Here we report that strong light causing photoinhibition of photosynthesis also induces ELIP transcription and accumulation of the protein in mature green pea plants. Accumulation of ELIP transcript is induced in plants exposed to light intensities above 500 E/m2.s (E, einstein) and is maximal at approximately 1500 E/m2.s. The ELIP mRNA level increases in correlation with the degree of photoinhibition. The increase in ELIP level in the thylakoid membranes parallels the decrease in the amount of D1 protein of the photosystem II reaction center. Examination of ELIP induction as a function of light quality demonstrates that ELIP transcription is specifically induced by blue (410-480 nm) but not by red or far-red light. The level of blue light-induced ELIP transcript is significantly repressed by low-intensity red light. However, the accumulation of ELIP translation product is related to the total amount of blue and red light energy absorbed.
Specific binding of arginine to TAR RNA.
Authors: Tao J , Frankel A D
Published: May 2006

A single arginine residue within the basic region of the human immunodeficiency virus Tat protein mediates specific binding of Tat peptides to a three-nucleotide bulge in TAR RNA. It has been proposed that arginine recognizes TAR by forming a network of hydrogen bonds with two structurally distinct phosphates, an interaction termed the "arginine fork." Here it is shown that L-arginine blocks the Tat peptide/TAR interaction, whereas L-lysine and analogs of arginine that remove specific hydrogen bond donors do not. Experiments using an L-arginine affinity column demonstrate that arginine and the Tat peptides bind to the same site in TAR. Modification of two phosphates located at the junction of the double-stranded stem and bulge and modification of two adenine N7 groups in base-paired regions of TAR interfere with specific arginine binding. The results emphasize the importance of RNA structure in RNA-protein recognition and provide methods to identify arginine-binding sites in RNAs.
Pituitary hyperplasia induced by ectopic expression of nerve growth factor.
Authors: Borrelli E , Sawchenko P E , Evans R M
Published: May 2006

Nerve growth factor (NGF) cDNA was fused to the rat prolactin promoter to induce its ectopic expression in pituitary lactotrophs of transgenic mice. High-level expression of both RNA and functional protein was achieved in two pedigrees. Pituitary cells from these animals secreted biologically active NGF that was capable of inducing rapid differentiation of cocultured PC12 pheochromocytoma cells. Despite this robust expression, transgenic pituitaries failed to show any detectable increase in neuronal innervation. Unexpectedly, we observed a dramatic hyperplasia of lactotrophs resulting in pituitaries 10-100 times larger than normal. These results suggest that NGF, in addition to its previously described effects, may act as a new class of mitogen, with a potential role in oncogenesis.
A 36-base-pair core sequence of locus control region enhances retrovirally transferred human beta-globin gene expression.
Authors: Chang J C , Liu D , Kan Y W
Published: May 2006

The locus control region of the human beta-globin cluster consists of four major DNase I hypersensitive sites (HS). When linked to globin genes, the locus control region confers a high level of erythroid-specific expression of these genes in transgenic mice or transfected erythroid cell lines. We have examined the effect of one of these sites, HS2, on human beta-globin gene expression in a murine erythroleukemia cell line (MEL) after retrovirus-mediated gene transfer. We incorporated a 732- or 412-base-pair (bp) segment of HS2 in the retroviral construct carrying the human beta-globin gene. These fragments rendered the viruses unstable as the human beta-globin gene was rearranged or deleted in all the packaging cell lines examined. On the other hand, when a 36-bp fragment containing the NFE-2/AP-1 binding consensus in this region was inserted into the retroviral construct, we recovered 6 stable packaging cell lines of 12 examined, similar in percentage to the construct with the beta-globin gene alone. The virus titers of the packaging cell lines from these two constructs were similar. We infected MEL cells with viruses produced from three packaging cell lines of each of the two constructs and measured the ratio of human beta-globin to mouse alpha-globin mRNA after hexamethylenebisacetamide induction. The overall level of expression increased 2-fold from 6.0% to 12.7% with the addition of this 36-bp enhancer.