bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.
Authors: Lin C , Ahmad M , Gordon D , Cashmore A R
Published: May 2006

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.
Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.
Authors: Hur Y , Hwang D J , Zoubenko O , Coetzer C , Uckun F M , Tumer N E
Published: May 2006

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.
Crossover and noncrossover recombination during meiosis: timing and pathway relationships.
Authors: Storlazzi A , Xu L , Cao L , Kleckner N
Published: May 2006

During meiosis, crossovers occur at a high level, but the level of noncrossover recombinants is even higher. The biological rationale for the existence of the latter events is not known. It has been suggested that a noncrossover-specific pathway exists specifically to mediate chromosome pairing. Using a physical assay that monitors both crossovers and noncrossovers in cultures of yeast undergoing synchronous meiosis, we find that both types of products appear at essentially the same time, after chromosomes are fully synapsed at pachytene. We have also analyzed a situation in which commitment to meiotic recombination and formation of the synaptonemal complex are coordinately suppressed (mer1 versus mer1 MER2++). We find that suppression is due primarily to restoration of meiosis-specific double-strand breaks, a characteristic of the major meiotic recombination pathway. Taken together, the observations presented suggest that there probably is no noncrossover-specific pathway and that restoration of intermediate events in a single pairing/recombination pathway promotes synaptonemal complex formation. The biological significant of noncrossover recombination remains to be determined, however.
p53 deficiency does not affect the accumulation of point mutations in a transgene target.
Authors: Sands A T , Suraokar M B , Sanchez A , Marth J E , Donehower L A , Bradley A
Published: May 2006

DNA repair is required by organisms to prevent the accumulation of mutations and to maintain the integrity of genetic information. Mammalian cells that have been treated with agents that damage DNA have an increase in p53 levels, a p53-dependent arrest at G1 in the cell cycle, and a p53-dependent apoptotic response. It has been hypothesized that this block in cell cycle progression is necessary to allow time for DNA repair or to direct the damaged cell to an apoptotic pathway. This hypothesis predicts that p53-deficient cells would have an abnormal apoptotic response and exhibit a "mutator" phenotype. Using a sensitive assay for the accumulation of point mutations, small deletions, and insertions, we have directly tested whether p53-deficient cells exhibit an increased frequency of mutation before and after exposure to DNA-damaging agents. We report that wild-type and p53-deficient fibroblasts, thymocytes, and tumor tissue have indistinguishable rates of point mutation accumulation in a transgenic lacI target gene. These results suggest that the role of p53 in G1 checkpoint control and tumor suppression does not affect the accumulation of point mutations.
Genetic dissection of Alzheimer disease, a heterogeneous disorder.
Authors: Schellenberg G D
Published: May 2006

The genetics of Alzheimer disease (AD) are complex and not completely understood. Mutations in the amyloid precursor protein gene (APP) can cause early-onset autosomal dominant AD. In vitro studies indicate that cells expressing mutant APPs overproduce pathogenic forms of the A beta peptide, the major component of AD amyloid. However, mutations in the APP gene are responsible for 5% or less of all early-onset familial AD. A locus on chromosome 14 is responsible for AD in other early-onset AD families and represents the most severe form of the disease in terms of age of onset and rate of decline. Attempts to identify the AD3 gene by positional cloning methods are underway. At least one additional early-onset AD locus remains to be located. In late-onset AD, the apolipoprotein E gene allele epsilon 4 is a risk factor for AD. This allele appears to act as a dose-dependent age-of-onset modifier. The epsilon 2 allele of this gene may be protective. Other late-onset susceptibility factors remain to be identified.
Cortical activity flips among quasi-stationary states.
Authors: Abeles M , Bergman H , Gat I , Meilijson I , Seidemann E , Tishby N , Vaadia E
Published: May 2006

Parallel recordings of spike trains of several single cortical neurons in behaving monkeys were analyzed as a hidden Markov process. The parallel spike trains were considered as a multivariate Poisson process whose vector firing rates change with time. As a consequence of this approach, the complete recording can be segmented into a sequence of a few statistically discriminated hidden states, whose dynamics are modeled as a first-order Markov chain. The biological validity and benefits of this approach were examined in several independent ways: (i) the statistical consistency of the segmentation and its correspondence to the behavior of the animals; (ii) direct measurement of the collective flips of activity, obtained by the model; and (iii) the relation between the segmentation and the pair-wise short-term cross-correlations between the recorded spike trains. Comparison with surrogate data was also carried out for each of the above examinations to assure their significance. Our results indicated the existence of well-separated states of activity, within which the firing rates were approximately stationary. With our present data we could reliably discriminate six to eight such states. The transitions between states were fast and were associated with concomitant changes of firing rates of several neurons. Different behavioral modes and stimuli were consistently reflected by different states of neural activity. Moreover, the pair-wise correlations between neurons varied considerably between the different states, supporting the hypothesis that these distinct states were brought about by the cooperative action of many neurons.
De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.
Authors: Fra A M , Williamson E , Simons K , Parton R G
Published: May 2006

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.
Human fatty acid synthase: properties and molecular cloning.
Authors: Jayakumar A , Tai M H , Huang W Y , al-Feel W , Hsu M , Abu-Elheiga L , Chirala S S , Wakil S J
Published: May 2006

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.
Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15.
Authors: Johnston J A , Bacon C M , Finbloom D S , Rees R C , Kaplan D , Shibuya K , Ortaldo J R , Gupta S , Chen Y Q , Giri J D
Published: May 2006

The cytokines interleukin 2 (IL-2) and IL-15 have similar biological effects on T cells and bind common hematopoietin receptor subunits. Pathways that involve Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to be important for hematopoietin receptor signaling. In this study we identify the STAT proteins activated by IL-2 and IL-15 in human T cells. IL-2 and IL-15 rapidly induced the tyrosine phosphorylation of STAT3 and STAT5, and DNA-binding complexes containing STAT3 and STAT5 were rapidly activated by these cytokines in T cells. IL-4 induced tyrosine phosphorylation and activation of STAT3 but not STAT5. JAK1 and JAK3 were tyrosine-phosphorylated in response to IL-2 and IL-15. Hence, the JAK and STAT molecules that are activated in response to IL-2 and IL-15 are similar but differ from those induced by IL-4. These observations identify the STAT proteins activated by IL-2 and IL-15 and therefore define signaling pathways by which these T-cell growth factors may regulate gene transcription.
Critical role of the interleukin 2 (IL-2) receptor gamma-chain-associated Jak3 in the IL-2-induced c-fos and c-myc, but not bcl-2, gene induction.
Authors: Kawahara A , Minami Y , Miyazaki T , Ihle J N , Taniguchi T
Published: May 2006

The interleukin 2 receptor (IL-2R) consists of three subunits, the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two Janus family protein tyrosine kinases (PTKs), Jak1 and Jak3, were shown to associate with IL-2R beta c and IL-2R gamma c, respectively, and their PTK activities are increased after IL-2 stimulation. A Jak3 mutant with truncation of the C-terminal PTK domain lacks its intrinsic kinase activity but can still associate with IL-2R gamma c. In a hematopoietic cell line, F7, that responds to either IL-2 or IL-3, overexpression of this Jak3 mutant results in selective inhibition of the IL-2-induced activation of Jak1/Jak3 PTKs and of cell proliferation. Of the three target nuclear protooncogenes of the IL-2 signaling, c-fos and c-myc genes, but not the bcl-2 gene, were found to be impaired. On the other hand, overexpression of the dominant negative form of the IL-2R gamma c chain, which lacks most of its cytoplasmic domain, in F7 cells resulted in the inhibition of all three protooncogenes. These results provide a further molecular basis for the critical role of Jak3 in IL-2 signaling and also suggest a Jak PTK-independent signaling pathway(s) for the bcl-2 gene induction by IL-2R.
Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor.
Authors: Reppert S M , Godson C , Mahle C D , Weaver D R , Slaugenhaupt S A , Gusella J F
Published: May 2006

A G protein-coupled receptor for the pineal hormone melatonin was recently cloned from mammals and designated the Mel1a melatonin receptor. We now report the cloning of a second G protein-coupled melatonin receptor from humans and designate it the Mel1b melatonin receptor. The Mel1b receptor cDNA encodes a protein of 362 amino acids that is 60% identical at the amino acid level to the human Mel1a receptor. Transient expression of the Mel1b receptor in COS-1 cells results in high-affinity 2-[125I]iodomelatonin binding (Kd = 160 +/- 30 pM). In addition, the rank order of inhibition of specific 2-[125I]iodomelatonin binding by eight ligands is similar to that exhibited by the Mel1a melatonin receptor. Functional studies of NIH 3T3 cells stably expressing the Mel1b melatonin receptor indicate that it is coupled to inhibition of adenylyl cyclase. Comparative reverse transcription PCR shows that the Mel1b melatonin receptor is expressed in retina and, to a lesser extent, brain. PCR analysis of human-rodent somatic cell hybrids maps the Mel1b receptor gene (MTNR1B) to human chromosome 11q21-22. The Mel1b melatonin receptor may mediate the reported actions of melatonin in retina and participate in some of the neurobiological effects of melatonin in mammals.
Mutant rat phosphatidylinositol/phosphatidylcholine transfer proteins specifically defective in phosphatidylinositol transfer: implications for the regulation of phospholipid transfer activity.
Authors: Alb J G , Gedvilaite A , Cartee R T , Skinner H B , Bankaitis V A
Published: May 2006

The mammalian phosphatidylinositol/phosphatidylcholine transfer proteins (PI-TPs) catalyze exchange of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. We find that Ser-25, Thr-59, Pro-78, and Glu-248 make up a set of rat (r) PI-TP residues, substitution of which effected a dramatic reduction in the relative specific activity for PI transfer activity without significant effect on PC transfer activity. Thr-59 was of particular interest as it is a conserved residue in a highly conserved consensus protein kinase C phosphorylation motif in metazoan PI-TPs. Replacement of Thr-59 with Ser, Gln, Val, Ile, Asn, Asp, or Glu effectively abolished PI transfer capability but was essentially silent with respect to PC transfer activity. These findings identify rPI-TP residues that likely cooperate to form a PI head-group binding/recognition site or that lie adjacent to such a site. Finally, the selective sensitivity of the PI transfer activity of rPI-TP to alteration of Thr-59 suggests a mechanism for in vivo regulation of rPI-TP activity.
Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor.
Authors: Korte M , Carroll P , Wolf E , Brem G , Thoenen H , Bonhoeffer T
Published: May 2006

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) gene family, has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes such as long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF gene. We then used hippocampal slices from these mice to investigate whether LTP was affected by this mutation. Homo- and heterozygous mutant mice showed significantly reduced LTP in the CA1 region of the hippocampus. The magnitude of the potentiation, as well as the percentage of cases in which LTP could be induced successfully, was clearly affected. According to the criteria tested, important pharmacological, anatomical, and morphological parameters in the hippocampus of these animals appear to be normal. These results suggest that BDNF might have a functional role in the expression of LTP in the hippocampus.
Structure of human T-cell receptors specific for an immunodominant myelin basic protein peptide: positioning of T-cell receptors on HLA-DR2/peptide complexes.
Authors: Wucherpfennig K W , Hafler D A , Strominger J L
Published: May 2006

T-cell receptors (TCRs) recognize peptide bound within the relatively conserved structural framework of major histocompatibility complex (MHC) class I or class II molecules but can discriminate between closely related MHC molecules. The structural basis for the specificity of ternary complex formation by the TCR and MHC/peptide complexes was examined for myelin basic protein (MBP)-specific T-cell clones restricted by different DR2 subtypes. Conserved features of this system allowed a model for positioning of the TCR on DR2/peptide complexes to be developed: (i) The DR2 subtypes that presented the immunodominant MBP peptide differed only at a few polymorphic positions of the DR beta chain. (ii) TCR recognition of a polymorphic residue on the helical portion of the DR beta chain (position DR beta 67) was important in determining the MHC restriction. (iii) The TCR variable region (V) alpha 3.1 gene segment was used by all of the T-cell clones. TCR V beta usage was more diverse but correlated with the MHC restriction--i.e., with the polymorphic DR beta chains. (iv) Two clones with conserved TCR alpha chains but different TCR beta chains had a different MHC restriction but a similar peptide specificity. The difference in MHC restriction between these T-cell clones appeared due to recognition of a cluster of polymorphic DR beta-chain residues (DR beta 67-71). MBP-(85-99)-specific TCRs therefore appeared to be positioned on the DR2/peptide complex such that the TCR beta chain contacted the polymorphic DR beta-chain helix while the conserved TCR alpha chain contacted the nonpolymorphic DR alpha chain.
Expression of a plant viral polycistronic mRNA in yeast, Saccharomyces cerevisiae, mediated by a plant virus translational transactivator.
Authors: Sha Y , Broglio E P , Cannon J F , Schoelz J E
Published: May 2006

We demonstrate that the cauliflower mosaic virus (CaMV) gene VI product can transactivate the expression of a reporter gene in bakers' yeast, Saccharomyces cerevisiae. The gene VI coding sequence was placed under the control of the galactose-inducible promoter GAL1, which is presented in the yeast shuttle vector pYES2, to create plasmid JS169. We also created a chloramphenicol acetyltransferase (CAT) reporter plasmid, JS161, by inserting the CAT reporter gene in-frame into CaMV gene II and subsequently cloning the entire CaMV genome into the yeast vector pRS314. When JS161 was transformed into yeast and subsequently assayed for CAT activity, only a very low level of CAT activity was detected in cellular extracts. To investigate whether the CaMV gene VI product would mediate an increase in CAT activity, we cotransformed yeast with JS169 and JS161. Upon induction with galactose, we found that CAT activity in yeast transformed with JS161 and JS169 was about 19 times higher than the level in the transformants that contained only JS161. CAT activity was dependent on the presence of the gene VI protein, because essentially no CAT activity was detected in yeast cells grown in the presence of glucose, which represses expression from the GAL1 promoter. RNase protection assays showed that the gene VI product had no effect on transcription from the 35S RNA promoter, demonstrating that regulation was occurring at the translation level. This yeast system will prove useful for understanding how the gene VI product of CaMV mediates the translation of genes present on a eukaryotic polycistronic mRNA.
Sphingosine: a mediator of acute renal tubular injury and subsequent cytoresistance.
Authors: Iwata M , Herrington J , Zager R A
Published: May 2006

The goal of this study was to determine whether sphingosine and ceramide, second messengers derived from sphingolipid breakdown, alter kidney proximal tubular cell viability and their adaptive responses to further damage. Adult human kidney proximal tubular (HK-2) cells were cultured for 0-20 hr in the presence or absence of sphingosine, sphingosine metabolites (sphingosine 1-phosphate, dimethylsphingosine), or C2, C8, or C16 ceramide. Acute cell injury was assessed by vital dye exclusion and tetrazolium dye transport. Their subsequent impact on superimposed ATP depletion/Ca2+ ionophore-induced damage was also assessed. Sphingosine (> or = 10 microM), sphingosine 1-phosphate, dimethylsphingosine, and selected ceramides (C2 and C8, but not C16) each induced rapid, dose-dependent cytotoxicity. This occurred in the absence of DNA laddering or morphologic changes of apoptosis, suggesting a necrotic form of cell death. Prolonged exposure (20 hr) to subtoxic sphingosine doses (< or = 7.5 microM) induced substantial cytoresistance to superimposed ATP depletion/Ca2+ ionophore-mediated damage. Conversely, neither short-term sphingosine treatment (< or = 8.5 hr) nor 20-hr exposures to any of the above sphingosine/ceramide derivatives/metabolites or various free fatty acids reproduced this effect. Sphingosine-induced cytoresistance was dissociated from the extent of cytosolic Ca2+ loading (indo-1 fluorescence), indicating a direct increase in cell resistance to attack. We conclude that sphingosine can exert dual effects on proximal renal tubular viability: in high concentrations it induces cell necrosis, whereas in low doses it initiates a cytoresistant state. These results could be reproduced in human foreskin fibroblasts, suggesting broad-based relevance to the area of acute cell injury and repair.
Targeting cancer micrometastases with monoclonal antibodies: a binding-site barrier.
Authors: Saga T , Neumann R D , Heya T , Sato J , Kinuya S , Le N , Paik C H , Weinstein J N
Published: May 2006

Monoclonal antibodies penetrate bulky tumors poorly after intravenous administration, in part because of specific binding to the target antigen. Experiments presented here demonstrate an analogous phenomenon in micrometastases; poor antibody penetration, attributable to a "binding-site barrier" phenomenon, can be seen in guinea pig micrometastases as small as 300 microns in diameter. Increasing the dose of antibody can partially overcome this limitation, but at a cost in specificity.
Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.
Authors: Sidorenko S P , Law C L , Chandran K A , Clark E A
Published: May 2006

The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway.
Preservation of hemostatic and structural properties of rehydrated lyophilized platelets: potential for long-term storage of dried platelets for transfusion.
Authors: Read M S , Reddick R L , Bode A P , Bellinger D A , Nichols T C , Taylor K , Smith S V , McMahon D K , Griggs T R , Brinkhous K M
Published: May 2006

Currently, therapeutic platelet concentrates can be stored for only 5 days. We have developed a procedure that permits long-term storage of fixed and lyophilized platelets that retain hemostatic properties after rehydration. These rehydrated lyophilized platelets (RL platelets) restore hemostasis in thrombocytopenic rats and become incorporated in the hemostatic plug of bleeding time wounds of normal dogs as well as von Willebrand disease dogs with partially replenished plasma von Willebrand factor. Ultrastructurally, these platelets are well preserved and are comparable to control normal washed platelets. Flow cytometry analysis shows that RL platelets react with antibodies to the major surface receptors, glycoprotein (GP)Ib and GPIIb/IIIa. These receptors are involved in platelet agglutination, aggregation, and adhesion. In vitro functional tests document the ability of RL platelets to adhere to denuded subendothelium and to spread on a foreign surface. Circulating RL platelets participated in carotid arterial thrombus formation induced in normal canine subjects. The participation of RL platelets in these vital hemostatic properties suggests that with further development they could become a stable platelet product for transfusion.
Systemin activates synthesis of wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway.
Authors: Constabel C P , Bergey D R , Ryan C A
Published: May 2006

Tomato plants overexpressing a prosystemin gene that encodes the precursor of a mobile wound signal called systemin have been shown previously to constitutively synthesize extraordinarily high levels of two defensive proteinase inhibitor proteins in leaves in the absence of wounding. We herein report that leaves of these transgenic plants possess enhanced levels of another defensive protein, polyphenol oxidase (PPO) at levels that are up to 70-fold higher than levels found in leaves of wild-type plants. Supplying young wild-type tomato plants with systemin through cut stems induced PPO activity in leaves, and wounding lower leaves of young tomato plants induced PPO activity in both wounded and unwounded leaves to levels equal to those induced by systemin. Exposing young tomato plants to methyl jasmonate vapor caused an increase in PPO activity equivalent to levels found in plants overexpressing the prosystemin gene. The data indicate that PPO and proteinase inhibitor genes are coactivated systemically by wounding via the octadecanoid signal transduction pathway and that systemin has a much broader role in signaling plant defensive genes than was previously known.
Functional interaction of yeast and human TATA-binding proteins with an archaeal RNA polymerase and promoter.
Authors: Wettach J , Gohl H P , Tschochner H , Thomm M
Published: May 2006

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.
p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Uso1p.
Authors: Sapperstein S K , Walter D M , Grosvenor A R , Heuser J E , Waters M G
Published: May 2006

A recently discovered vesicular transport factor, termed p115, is required along with N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins for in vitro Golgi transport. p115 is a peripheral membrane protein found predominantly on the Golgi. Biochemical and electron microscopic analyses indicate that p115 is an elongated homodimer with two globular "heads" and an extended "tail" reminiscent of myosin II. We have cloned and sequenced cDNAs for bovine and rat p115. The predicted translation products are 90% identical, and each can be divided into three domains. The predicted 108-kDa bovine protein consists of an N-terminal 73-kDa globular domain followed by a 29-kDa coiled-coil dimerization domain, a linker segment of 4 kDa, and a highly acidic domain of 3 kDa. p115 is related to Uso1p, a protein required for endoplasmic reticulum to Golgi vesicular transport in Saccharomyces cerevisiae, which has a similar "head-coil-acid" domain structure. The p115 and Uso1p heads are similar in size, have approximately 25% sequence identity, and possess two highly homologous regions (62% and 60% identity over 34 and 53 residues, respectively). There is a third region of homology (50% identity over 28 residues) between the coiled-coil and acidic domains. Although the acidic nature of the p115 and Uso1p C termini is conserved, the primary sequence is not. We discuss these results in light of the proposed function of p115 in membrane targeting and/or fusion.
Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10(-7).
Authors: Gross H J , Verwer B , Houck D , Hoffman R A , Recktenwald D
Published: May 2006

A flow cytometric assay was developed to detect rare cancer cells in blood and bone marrow. Multiple markers; each identified by a separate color of immunofluorescence (yellow and two shades of red), are used to reliably identify the cancer cells. Blood or bone marrow cells, which are not of interest but interfere in detecting the cancer cells, are identified by a panel of immunofluorescence markers, each of which has the same color (green). Thus, the rare cancer cells of interest are yellow and two different shades of red but not green. The requirement that the rare cancer cell be simultaneously positive for three separate colors (the specific markers) and negative for a fourth color (the exclusion color) allowed detection of as few as one cancer cell in 10(7) nucleated blood cells (a frequency of 10(-7). To test this rare-event assay prior to clinical studies, a model study was performed in which the clinical sample was simulated by mixing small numbers of cells from the breast carcinoma line BT-20 with peripheral blood mononuclear cells. We detected statistically significant numbers of BT-20 cells at mixing frequencies of 10(-5), 10(-6), and 10(-7). In control samples, no target events (BT-20) were observed when more than 10(8) cells were analyzed. For additional confirmation that the BT-20 cells in the model study were correctly identified and counted, the BT-20 cells (and only BT-20 cells) were covalently stained with a fifth fluorescence dye, 7-amino-4-chloromethylcoumarin (CMAC). CMAC fluorescence data were not used in the assay for detecting BT-20 cells. Only after the analysis using data from the specific and exclusion colors had been completed were the events identified as BT-20 cells checked for CMAC fluorescence. The putative BT-20 events were always found to be positive for CMAC fluorescence, which further increases confidence in the assay. Manual data analysis and an automated computer program were compared. Results were comparable with the manual and automated methods, but the automated "genetic algorithm" always found more BT-20 events. Cell sorting of BT-20 cells from samples that contained BT-20 at frequencies of 10(-5), 10(-6), and 10(-7) provided further evidence that these rare cells could be reliably detected. The good performance of the assay with the model system will encourage further studies on clinical samples.
Effect of erythrocyte membrane on extracellular development of the erythrocytic cycle of Plasmodium falciparum.
Authors: Williams J H , Gill G S , Trager W
Published: May 2006

We have earlier reported conditions that support the axenic development in vitro of a complete asexual erythrocytic cycle of Plasmodium falciparum. Up to 30% of merozoites showed initial differentiation into trophic forms (rings) viable at 14 hr. However, only approximately 1% of the merozoites would develop further into trophozoites and early schizonts viable at 36 hr. In efforts to increase the number of late stage parasites, we have now found a significant favorable effect of the addition of erythrocyte ghosts. Doubling the quantity of erythrocyte membrane in the erythrocyte sonicate medium resulted in approximate doubling of the number of trophozoites and early schizonts. These results indicate that components of the erythrocyte membrane are essential for the complete development of the erythrocytic cycle.
The period clock gene is expressed in central nervous system neurons which also produce a neuropeptide that reveals the projections of circadian pacemaker cells within the brain of Drosophila melanogaster.
Authors: Helfrich-Förster C
Published: May 2006

The period protein (PER) is a essential component of the circadian clock in Drosophila melanogaster. Although PER-containing pacemaker cells have been previously identified in the brain, the neuronal network that comprises the circadian clock remained unknown. Here it is shown that some PER neurons are also immunostained with an antiserum against the crustacean pigment-dispersing hormone (PDH). This antiserum reveals the entire arborization pattern of these pacemaker cells. The arborizations of these neurons are appropriate for modulation of the activity of many neurons and they might interact with PER-containing glial cells. A putative physiological role of PDH in the circadian system is discussed.
An aphid-resistance locus is tightly linked to the nematode-resistance gene, Mi, in tomato.
Authors: Kaloshian I , Lange W H , Williamson V M
Published: May 2006

Tomato lines from diverse breeding programs were evaluated in the field for resistance to a natural infestation of the potato aphid, Macrosiphum euphorbiae, in Davis, CA. It was noted that all lines that carried the nematode-resistance gene, Mi, displayed aphid resistance. A greenhouse assay for aphid resistance was developed to investigate this relationship. Association of nematode and aphid resistances in near-isogenic lines suggested that these traits are tightly linked. Analysis of an F2 population segregating for nematode resistance indicated that aphid resistance segregated as a single major locus genetically linked to Mi. The name Meu1 is proposed for this locus. It is likely that Meu1 was introduced into tomato along with Mi from the wild species Lycopersicon peruvianum. The presence of aphid resistance in the line Motelle, which contains a very small region of introgressed DNA, and the lack of recombinants suggest that Meu1 is tightly linked to Mi or possibly is the same gene. The map-based strategy currently being used to clone Mi should be applicable to cloning Meu1.
Chemoattraction of male gametes by a pheromone produced by female gametes of Chlamydomonas.
Authors: Starr R C , Marner F J , Jaenicke L
Published: May 2006

In isogamous species of Chlamydomonas, such as Chlamydomonas reinhardtii and Chlamydomonas eugametos, the sexual process involves the use of flagella agglutinins by which the gametes of compatible strains adhere through chance encounter and ultimately pair and fuse to form zygotes. In a newly described heterogamous species, Chlamydomonas allensworthii, the sexual process is initiated by the chemoattraction of small sperm to a sexually competent female gamete, which continues to secrete the pheromone until it has fused with one of the sperm so attracted. From bacteria-free female strains of C. allensworthii, the chemoat-tractant has been isolated and identified as a pentosylated hydroquinone (Mr = 532) whose spectral, chemical, and physical properties are in accord with the structure of a 2,3-dimethyl-5-(triprenylcarboxymethyl)-1,4-benzohydroquinone-1-(beta-xyloside). A rapid bioassay of the pheromone uses DEAE-Toyopearl 650M beads to which the pheromone adsorbs. When such activated beads are placed in a suspension of sperm, they act as surrogate females and attract the small motile sperm. The purified pheromone shows activity at a concentration as low as 1 pM.
Efficient screening of retroviral cDNA expression libraries.
Authors: Kitamura T , Onishi M , Kinoshita S , Shibuya A , Miyajima A , Nolan G P
Published: May 2006

Expression cloning of cDNAs was first described a decade ago and was based on transient expression of cDNA libraries in COS cells. In contrast to transient transfection of plasmids, retroviral gene transfer delivers genes stably into a wide range of target cells. We utilize a simple packaging system for production of high-titer retrovirus stock from cDNA libraries to establish a cDNA expression cloning system. In two model experiments, murine interleukin (IL)-3-dependent Ba/F3 cells were infected with libraries of retrovirally expressed cDNA derived from human T-cell mRNA or human IL-3-dependent TF-1 cell line mRNA. These infected Ba/F3 cells were selected for the expression of CD2 by flow cytometry or for the alpha subunit of the human IL-3 receptor (hIL-3R alpha) by factor-dependent growth. CD2 (frequency, 1 in 10(4)) and hIL-3R alpha (frequency, 1 in 1.5 x 10(5)) cDNAs were readily detected in small-scale experiments, indicating this retroviral expression cloning system is efficient enough to clone low-abundance cDNAs by their expression or function.
Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.
Authors: Wu J , Perrin D M , Sigman D S , Kaback H R
Published: May 2006

Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.
Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.
Authors: Shcherban T Y , Shi J , Durachko D M , Guiltinan M J , McQueen-Mason S J , Shieh M , Cosgrove D J
Published: May 2006

Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.
Molecular cloning, characterization, and functional expression of rat oxidosqualene cyclase cDNA.
Authors: Abe I , Prestwich G D
Published: May 2006

A cDNA encoding rat oxidosqualene lanosterol-cyclase [lanosterol synthase; (S)-2,3-epoxysqualene mutase (cyclizing, lanosterol-forming), EC 5.4.99.7] was cloned and sequenced by a combination of PCR amplification, using primers based on internal amino acid sequence of the purified enzyme, and cDNA library screening by oligonucleotide hybridization. An open reading frame of 2199 bp encodes a M(r) 83,321 protein with 733 amino acids. The deduced amino acid sequence of the rat enzyme showed significant homology to the known oxidosqualene cyclases (OSCs) from yeast and plant (39-44% identity) and still retained 17-26% identity to two bacterial squalene cyclases (EC 5.4.99.-). Like other cyclases, the rat enzyme is rich in aromatic amino acids and contains five so-called QW motifs, highly conserved regions with a repetitive beta-strand turn motif. The binding site sequence for the 29-methylidene-2,3-oxidosqualene (29-MOS), a mechanism-based irreversible inhibitor specific for the vertebrate cyclase, is well-conserved in all known OSCs. The hydropathy plot revealed a rather hydrophilic N-terminal region and the absence of a hydrophobic signal peptide. Unexpectedly, this microsomal membrane-associated enzyme showed no clearly delineated transmembrane domain. A full-length cDNA was constructed and subcloned into a pYEUra3 plasmid, selected in Escherichia coli cells, and used to transform the OSC-deficient uracil-auxotrophic SGL9 strain of Saccharomyces cerevisiae. The recombinant rat OSC expressed was efficiently labeled by the mechanism-based inhibitor [3H]29-MOS.
Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.
Authors: Donevan S D , Rogawski M A
Published: May 2006

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.