bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of a class of selective cholecystokinin type B receptor antagonists having potent anxiolytic activity.
Authors: Hughes J , Boden P , Costall B , Domeney A , Kelly E , Horwell D C , Hunter J C , Pinnock R D , Woodruff G N
Published: May 2006

PD134308 and PD135158 are potent and selective antagonists at the cholecystokinin type B (CCK-B) receptors with IC50 values of 1.6 nM and 3.5 nM, respectively, in the radioligand binding assay and Ke values of 7.82 and 12.9 nM, respectively, in their blocking action on CCK responses in the rat lateral hypothalamic slice. PD134308 and PD135158 produced potent anxiolytic effects in the mouse black/white box test after either subcutaneous or oral administration. There was no evidence of the development of tolerance to the anxiolytic action of either PD134308 or PD135158 in mice treated twice daily for 7 days, nor was there any sign of withdrawal anxiogenesis after abrupt termination of this treatment. Both CCK-B antagonists were able to suppress the withdrawal anxiogenesis and produce an anxiolytic effect in mice previously made tolerant to diazepam. PD134308 and PD135158 produced potent anxiolytic effects in the rat elevated plus maze test and the rat social interaction test. The effects were comparable in magnitude to those seen with diazepam. However, unlike diazepam, PD134308 and PD135158 did not produce sedation. The CCK-B antagonists also showed powerful anxiolytic activity in the "marmoset human threat test." These results provide evidence of a selective role for CCK-B receptors in the control of anxiety. PD134308 and PD135158 are members of a class of anxiolytic agents that have a greatly improved profile compared with benzodiazepines or serotonin-related anxiolytics.
Prolactin effects on the dietary regulation of mouse mammary tumor virus proviral DNA expression.
Authors: Hamada N , Engelman R W , Tomita Y , Chen R F , Iwai H , Good R A , Day N K
Published: May 2006

Chronic energy-intake restriction inhibits mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/Ou mice by greater than 90%. We have shown that associated with suppression of mammary tumorigenesis there is a reduction or inhibition of circulating prolactin, MMTV particles expressed, and MMTV mRNA transcription in mammary glands (and in most organs tested). To understand the concerted action of prolactin, energy-consumption level, and MMTV on inducing mammary tumors, experiments were designed to control prolactin and energy levels in order to evaluate their effects on MMTV mRNA expression. Mice on restricted diets were grafted with adenohypophyses, and mice fed ad libitum were treated with the dopaminomimetic agent octahydrobenzo [g]quinoline. Adenohypophyseal grafting significantly increased prolactin in dietary (energy)-restricted mice, and this effect was associated with an increase in MMTV mRNA expression within the mammary gland; a linear correlation between prolactin levels and MMTV mRNA expression in the mammary gland was found. Conversely, elimination of the nocturnal peak of circulating prolactin by i.p. injection of dopaminomimetic octahydrobenzo [g]quinoline to mice fed ad libitum delayed (by 8 weeks) and reduced (even as long as 25 weeks) mammary gland MMTV mRNA expression. These findings associate prolactin influences with MMTV mRNA production in mice and help explain the link between chronic energy-intake restriction and reduced MMTV gene expression.
Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts.
Authors: Li H M , Theg S M , Bauerle C M , Keegstra K
Published: May 2006

Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations. Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain. The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor proteins of ferredoxin and plastocyanin are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are competent to be assembled into holo forms.
An inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in liver nuclei.
Authors: Nicotera P , Orrenius S , Nilsson T , Berggren P O
Published: May 2006

Recent studies in our laboratory have revealed the existence of an ATP- and calmodulin-dependent Ca2+ uptake system in rat liver nuclei that can promote increases in the free Ca2+ concentration in the nuclear matrix. In the present investigation we show that liver nuclei possess a marked ability to sequester and buffer Ca2+, suggesting a potential role for the nucleus in the regulation of the cytosolic free Ca2+ concentration. In addition, we demonstrate that the intracellular messenger, inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3], stimulates the release of a fraction of the nuclear Ca2+ and transiently lowers the intranuclear free Ca2+ concentration. The Ins(1,4,5)P3-stimulated Ca2+ release is followed by Ca2+ reuptake into an inositol phosphate-insensitive nuclear compartment. Together, these results demonstrate that liver nuclei contain, at least, two Ca2+ pools, one of which is releasable by Ins(1,4,5)P3. These findings are consistent with a role for the nucleus in the modulation of the cytosolic free Ca2+ level by agonists and suggest that the control of the nuclear Ca2+ load by second messengers may participate in the regulation of intranuclear Ca2(+)-dependent processes by hormones and other agents.
Isolation of carp genes encoding major histocompatibility complex antigens.
Authors: Hashimoto K , Nakanishi T , Kurosawa Y
Published: May 2006

In the evolution of the adaptive immune system unique to vertebrates, teleost fish occupy the critical position. This is the most primitive class of lower vertebrates in which the capacity for acute allograft rejections can be demonstrated, thus suggesting the presence of major histocompatibility complex (MHC) antigens and, therefore, T cells. Here, we report the identification of two putative MHC-antigen-encoding sequences in the carp Cyprinus carpio. One, identified as TLAI alpha-1, had reasonable homology to MHC class I heavy chains of mammalian and avian species, while the other, identified as TLAII beta-1, was homologous to MHC class II beta chain of the aforementioned higher vertebrates. For these isolations of fish MHC genes, we have identified two highly conserved amino acid sequence blocks surrounding two cysteine residues in the second domain of MHC class II beta chains as well as the third domain of class I heavy chains of humans, mice, and chickens. Two kinds of mixed oligonucleotide probes corresponding to these two regions were synthesized. The carp genomic DNA was subjected to amplification by polymerase chain reaction using the above two synthetic DNA fragments as primers. Subsequently, two different DNA sequences sandwiched by these primers were isolated from the amplified products. Their use as secondary probes led to the identification of TLAI alpha-1 and TLAII beta-1. We also discuss the applicability of the above approach for isolation from lower vertebrates of other genes belonging to the immunoglobulin super-family as well as the evolutionary origin of vertebrate MHC antigens.
2,3,7,8-Tetrachlorodibenzo-p-dioxin causes an extensive alteration of 17 beta-estradiol metabolism in MCF-7 breast tumor cells.
Authors: Spink D C , Lincoln D W , Dickerman H W , Gierthy J F
Published: May 2006

MCF-7 breast tumor cells form multicellular foci in vitro when supplemented with 17 beta-estradiol (E2). In the presence of E2 and the aryl hydrocarbon-receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), MCF-7 cells grow to confluence but do not form foci. To investigate the role of E2 metabolism in this antiestrogenic effect of TCDD, analyses were performed by capillary GC/MS. The results revealed that pretreatment of MCF-7 cultures with TCDD (10 nM) rapidly depletes E2. In untreated cultures supplemented with 10 nM E2, the concentration of free E2 decreased to 4 nM in the first 12 hr, followed by a slower rate of decline. After 3 days most E2 in the medium was in conjugated form(s); 1.7 nM was present as free E2, and 2.9 nM was released by treatment with glucuronidase/sulfatase. In TCDD-treated cultures, E2 declined to 290 pM in 12 hr and after 2 days was not detected (less than 100 pM) either as free steroid or after treatment with glucuronidase/sulfatase. Intracellular E2 and estrone were likewise depleted by pretreatment with TCDD. Microsomes from TCDD-treated cells showed highly elevated aryl hydrocarbon-hydroxylase activity and catalyzed hydroxylations of E2 at C-2, C-4, C-15 alpha, and C-6 alpha with a combined rate of 0.85 nmol/min per nmol of cytochrome P-450 at saturating E2. These results suggest that depletion of E2 by enhanced metabolism accounts for the antiestrogenic activity of TCDD in MCF-7 cells.
The E1 glycoprotein of an avian coronavirus is targeted to the cis Golgi complex.
Authors: Machamer C E , Mentone S A , Rose J K , Farquhar M G
Published: May 2006

It was previously reported that the E1 protein of an avian coronavirus was targeted to the juxtanuclear region in COS cells expressing the protein from cloned cDNA, suggesting that the protein contains information for targeting to the Golgi complex. The first of three membrane-spanning domains was required for intracellular targeting, because a mutant E1 (delta m1,2) lacking this domain was delivered to the plasma membrane. We have used immunoelectron microscopy to localize the wild-type E1 protein within Golgi elements of COS cells and AtT-20 cells expressing these proteins from recombinant vaccinia vectors. By immunoperoxidase and immunogold labeling, the wild-type E1 protein was localized to one or two cisternae located on one side of the Golgi stack that could be identified as the cis side in AtT-20 cells. In contrast, the mutant E1 protein was detected in all cisternae across the stack as well as at the plasma membrane. When the E1 proteins were immunoprecipitated and subjected to digestion with endoglycosidase H, the majority of the wild-type E1 glycoprotein was endoglycosidase H sensitive, whereas the majority of the mutant E1 was processed to an endoglycosidase H-resistant, polylactosaminoglycan-containing form. The findings indicate that the wild-type E1 protein is specifically targeted to cis Golgi cisternae and are consistent with the assumption that the first membrane-spanning domain is required for targeting to the cis Golgi.
Transgenic mouse model of the mild dominant form of osteogenesis imperfecta.
Authors: Bonadio J , Saunders T L , Tsai E , Goldstein S A , Morris-Wiman J , Brinkley L , Dolan D F , Altschuler R A , Hawkins J E , Bateman J F
Published: May 2006

Osteogenesis imperfecta type I is a mild, dominantly inherited, connective tissue disorder characterized by bone fragility. Mutations in type I collagen account for all known cases. In Mov-13 mice, integration of a murine retrovirus within the first intron of the alpha 1(I) collagen gene results in a null allele blocked at the level of transcription. This study demonstrates that mutant mice heterozygous for the null allele are a model of osteogenesis imperfecta type I. A defect in type I collagen production is associated with dominant-acting morphological and functional defects in mineralized and nonmineralized connective tissue and with progressive hearing loss. The model provides an opportunity to investigate the effect of a reduced amount of type I collagen on the structure and integrity of extracellular matrix. It also may represent a system in which therapeutic strategies to strengthen connective tissue can be developed.
Identification of potato nuclear proteins binding to the distal promoter region of the proteinase inhibitor II gene.
Authors: Sánchez-Serrano J J , Peña-Cortés H , Willmitzer L , Prat S
Published: May 2006

Potato nuclear proteins specifically bind to a DNA sequence at the most 5' distal region of the promoter of a potato proteinase inhibitor II gene. Binding studies using the electrophoretic mobility-shift assay showed the appearance of two protein-DNA complexes in the presence of both tuber and leaf nuclear protein extracts. Mechanical wounding of the leaves had no effect on the amount of specific protein-DNA complexes formed. DNase I protection analysis and binding to synthetic oligonucleotides identified the sequence 5'-GAGGGTATTTTCGTAA-3' as the target for the noncooperative binding of two potato nuclear proteins to the upstream element. Methylation interference experiments showed that guanine nucleotides separated by one turn of the DNA helix were in close contact with the proteins. The binding ability of a series of mutated synthetic oligonucleotides further defined the sequence requirements for protein binding, which appeared to contact one side of the DNA helix.
The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.
Authors: Wildgoose P , Kazim A L , Kisiel W
Published: May 2006

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 microM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.
Prenylated proteins: synthesis of geranylgeranylcysteine and identification of this thioether amino acid as a component of proteins in CHO cells.
Authors: Epstein W W , Lever D C , Rilling H C
Published: May 2006

Prenylated proteins, labeled in the isoprenoid residue by growing CHO cells in medium containing [5-3H]mevalonate, were degraded by three different proteolytic procedures, enzymatic or alkaline hydrolysis as well as hydrazinolysis. The products thus obtained were analyzed by HPLC with chemically prepared all-trans-geranylgeranylcysteine as a standard. About 10% of the radioactive products released by each lytic procedure showed the same chromatographic properties as geranylgeranylcysteine. This verifies the earlier conclusion, based on less-direct evidence, that this thioether derivative of cysteine is a component of naturally occurring proteins. The finding of this modified amino acid as a product of hydrazinolysis indicates that it is a carboxyl-terminal amino acid and that it is not carboxyl-methylated.
Cell wall regeneration in Chlamydomonas: accumulation of mRNAs encoding cell wall hydroxyproline-rich glycoproteins.
Authors: Adair W S , Apt K E
Published: May 2006

The unicellular alga Chlamydomonas reinhardtii is surrounded by a cell wall composed entirely of hydroxyproline-rich glycoproteins (HRGPs). When the walls of vegetative cells are removed with the enzyme gamete autolysin (g-lysin), they regenerate a matrix within 3-4 hr. In vitro translation of mRNAs isolated from g-lysin-treated cells showed significant increases and decreases in abundance of several mRNAs encoding proline-rich polypeptides. Because the population of up-regulated mRNAs is likely to include species encoding cell wall components, expression of genes for two outer wall HRGPs (GP1 and GP2) was analyzed during wall regeneration by using cDNAs isolated from a C. reinhardtii lambda gt11 library. Transcripts encoding GP1 and GP2 were elevated severalfold within the first hour or regeneration, suggesting that upregulation of HRGP mRNAs is a primary response to cell wall removal by g-lysin. Cell wall regeneration in Chlamydomonas provides an accessible system to study HRGP gene expression during matrix development.
Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein.
Authors: Gray P W , Barrett K , Chantry D , Turner M , Feldmann M
Published: May 2006

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNF alpha with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNF alpha with an affinity of 2.5 x 10(-9) M. This binding can be competitively inhibited with unlabeled TNF alpha or lymphotoxin (TNF beta).
Resting and activated subsets of mouse multipotent hematopoietic stem cells.
Authors: Spangrude G J , Johnson G R
Published: May 2006

The fluorescent vital dye rhodamine 123 (Rh-123), which preferentially accumulates in mitochondrial membranes, can be used as a probe to indicate mitochondrial and hence cellular activity. In this study, mouse bone marrow hematopoietic stem cells were subdivided into Rh-123lo, Rh-123med, and Rh-123hi populations. The Rh-123lo (resting) population was significantly enriched in cells with a higher proliferative potential compared to the Rh-123hi (activated) population. The resting population exhibited a 20-fold greater ability to differentiate into splenic colony-forming units (CFU-S) relative to the activated population, whereas the activated population contained about 4-fold more day 13 CFU-S on primary transfer relative to the resting population. The two populations produced morphologically distinct splenic colonies; however, the frequency and morphology of in vitro colonies were very similar. Only the resting population provided sufficient stem cells to transfer long-term hematopoietic repopulation to secondary recipient animals after lethal irradiation. On a single cell level, the resting and activated populations exhibited an equivalent ability to differentiate into lymphoid and myeloid progeny. These observations provide further insight into the heterogeneous nature of CFU-S and directly demonstrate that multipotent hematopoietic stem cells are heterogeneous with regard to their clonogenic capacities.
Contraction of myofibrils in the presence of antibodies to myosin subfragment 2.
Authors: Harrington W F , Karr T , Busa W B , Lovell S J
Published: May 2006

In a muscle-based version of in vitro motility assays, the unloaded shortening velocity of rabbit skeletal myofibrils has been determined in the presence and absence of affinity-column-purified polyclonal antibodies directed against the subfragment-2 region of myosin. Contraction was initiated by photohydrolysis of caged ATP and the time dependence of shortening was monitored by an inverted microscope equipped with a video camera. Antibody-treated myofibrils undergo unloaded shortening in a fast phase with initial rates and half-times comparable to control (untreated) myofibrils, despite a marked reduction in the isometric force of skinned muscle fibers in the presence of the antibodies. In antibody-treated myofibrils, this process is followed by a much slower phase of contraction, terminating in elongated structures with well-defined sarcomere spacings (approximately 1 micron) in contrast to the supercontracted globular state of control myofibrils. These results suggest that although the unloaded shortening of myofibrils (and in vitro motility of actin filaments over immobilized myosin heads) can be powered by myosin heads, the subfragment-2 region as well as the myosin head contributes to force production in actively contracting muscle.
Growth hormone and adipose differentiation: growth hormone-induced antimitogenic state in 3T3-F442A preadipose cells.
Authors: Corin R E , Guller S , Wu K Y , Sonenberg M
Published: May 2006

An additional activity for pituitary growth hormone is described--i.e., the in vitro induction of an antimitogenic state in murine 3T3-F442A preadipocyte fibroblasts. We previously developed a serum-free, hormonally defined medium permissive for the adipose differentiation of 3T3-F442A cells. When 3T3-F442A fibroblasts were maintained in serum-free medium without insulin but with growth hormone (2 nM), typical adipose differentiation did not occur. However, we found that growth hormone induced a state of cellular refractoriness to the mitogenic stimulus of fetal bovine serum as assayed by de novo DNA synthesis. The mitogen refractory condition (i.e., the antimitogenic state) was time-dependent (half maximal at approximately 2.5 days) and growth hormone concentration-dependent (half maximal and maximal at approximately 0.05 and 2.0 nM, respectively). The antimitogenic state was specifically induced by growth hormone and was not mediated by insulin-like growth factor I or prolactin. The growth hormone-induced antimitogenic state was completely reversible. The antimitogenic state was not induced by growth hormone in 3T3-C2 cells, a sister clone of 3T3 cells that exhibits essentially no adipose conversion. The kinetics for growth hormone-dependent commitment to adipose differentiation and induction of the antimitogenic state were similar. We suggest a relationship of growth hormone-induced antimitogenic state and the growth hormone-induced adipose differentiation of 3T3-F442A cells.
Sterol phylogenesis and algal evolution.
Authors: Nes W D , Norton R A , Crumley F G , Madigan S J , Katz E R
Published: May 2006

The stereochemistry of several sterol precursors and end products synthesized by two fungal-like micro-organisms Prototheca wickerhamii (I) and Dictyostelium discoideum (II) have been determined by chromatographic (TLC, GLC, and HPLC) and spectral (UV, MS, and 1H NMR) methods. From I and II the following sterols were isolated from the cells: cycloartenol, cyclolaudenol, 24(28)-methylenecycloartanol, ergosterol, protothecasterol, 4alpha-methylergostanol, 4alpha-methylclionastanol, clionastanol, 24beta-ethylcholesta-8,22-enol, and dictyosterol. In addition, the mechanism of C-24 methylation was investigated in both organisms by feeding to I [2-3H]lanosterol, [2-3H]cycloartenol, [24-3H]lanosterol, and [methyl-2H3]methionine and by feeding to II [methyl-2H3]methionine. The results demonstrate that the 24beta configuration is formed by different alkylation routes in I and II. The Delta25(27) route operates in I while the Delta24(28) route operates in II. Based on what is known in the literature regarding sterol distribution and phylogenesis together with our findings that the stereochemical outcome of squalene oxide cyclization leads to the production of cycloartenol rather than lanosterol (characteristic of the fungal genealogy) and the chirality of the C-24 alkyl group is similar in the two nonphotosynthetic microbes (beta oriented), we conclude that Prototheca is an apoplastic Chlorella (i.e., an alga) and that Dictyostelium as well as the other soil amoebae that synthesize cycloartenol evolved from algal rather than fungal ancestors.
Polymerase chain reaction evidence for human immunodeficiency virus 1 neutralization by passive immunization in patients with AIDS and AIDS-related complex.
Authors: Karpas A , Hewlett I K , Hill F , Gray J , Byron N , Gilgen D , Bally V , Oates J K , Gazzard B , Epstein J E
Published: May 2006

We tried to assess the long-term safety and potential efficacy of passive immunization in AIDS-related-complex (ARC) and AIDS patients. We also wanted to establish whether hyperimmune plasma from healthy human immunodeficiency virus 1 (HIV-1)-infected individuals clears the cell-free virus from circulation. Using the polymerase chain reaction (PCR), we were able to provide conclusive evidence that hyperimmune plasma is effective and maintains long-term neutralization of viremia. Using the cell test, we found that in most patients the total antibody level was maintained; in one of the ARC patients, it actually increased 8-fold and has remained at that level for nearly 2 years. The CD4+ cell count decreased in the AIDS patients but was stable in the ARC patient. Clinically, there was an initial improvement in all patients, but five of six of the advanced/terminal AIDS patients had died by month 17. Our studies suggest that passive immunization may be safe in ARC and AIDS patients. It reduces HIV-1 viremia to levels undetectable even by PCR. To advanced/terminal patients, the benefit is of limited duration, while to ARC patients it may be long-term. Therefore, passive immunization should start early in the disease.
Adoptive transfer of autoimmune diabetes and thyroiditis to athymic rats.
Authors: McKeever U , Mordes J P , Greiner D L , Appel M C , Rozing J , Handler E S , Rossini A A
Published: May 2006

We describe the induction of autoimmune diabetes, insulitis, and thyroiditis in athymic rats following injections of major histocompatibility complex compatible spleen cells. Lymphocytes with these capabilities were found in normal rats of the YOS, WAG, PVG, and diabetes-resistant BB strains, and in diabetes-prone BB rats. Adoptive transfer was facilitated by prior in vivo depletion of RT6.1+ regulatory T cells and in vitro mitogen activation of donor spleen cells. By RT6 depleting diabetes-resistant donors and using nude recipients, transfer of diabetes and thyroiditis was accomplished by using fresh, unstimulated spleen cells. The data suggest that organ-specific autoreactive cells may be present to various degrees but suppressed to a variable extent in many rat strains. The equilibrium between autoreactive and regulatory cells appears to determine the expression of autoimmunity.
Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction.
Authors: June C H , Fletcher M C , Ledbetter J A , Schieven G L , Siegel J N , Phillips A F , Samelson L E
Published: May 2006

The binding of antigen to the multicomponent T-cell receptor (TCR) activates several signal transduction pathways via coupling mechanisms that are poorly understood. One event that follows antigen receptor engagement is the activation of inositol phospholipid-specific phospholipase C (PLC). TCR activation by antigen, lectins, or anti-TCR monoclonal antibody has also been shown to cause increases in tyrosine phosphorylation of TCR-zeta and other substrates, suggesting stimulation of protein tyrosine kinase (PTK) activity. A critical question is whether these two pathways, PLC and PTK, are independently activated or whether one initiates and/or regulates the other. In the former case, PLC activation could be coupled to the TCR via a GTP-binding protein (G protein). We have reported, however, that tyrosine phosphorylation of intracellular substrates precedes detection of PLC activation and intracellular calcium elevation, suggesting that inositol phospholipid turnover in T cells is initiated by a PTK pathway. In this study, we test this hypothesis by treating T cells with the drug herbimycin A. We demonstrate that this agent inhibits substrate tyrosine phosphorylation, TCR-mediated inositol phospholipid hydrolysis, and calcium elevation. In contrast, under these conditions G-protein-mediated PLC activity, as tested by addition of aluminum fluoride, remains intact. Furthermore, whereas herbimycin treatment prevents TCR-mediated interleukin 2 production and interleukin 2 receptor expression, phorbol ester-induced effects are substantially resistant to herbimycin. The drug thus appears to abrogate TCR-mediated signaling without affecting distal signaling mechanisms.
Evidence for susceptibility of intrathymic T-cell precursors and their progeny carrying T-cell antigen receptor phenotypes TCR alpha beta + and TCR gamma delta + to human immunodeficiency virus infection: a mechanism for CD4+ (T4) lymphocyte depletion.
Authors: Schnittman S M , Denning S M , Greenhouse J J , Justement J S , Baseler M , Kurtzberg J , Haynes B F , Fauci A S
Published: May 2006

Individuals infected by the human immunodeficiency virus type 1 (HIV-1) demonstrate progressive depletion and qualitative dysfunction of the helper T4 (CD4+) cell population. Mechanisms proposed for attrition of CD4+ T cells include direct cytopathicity of these mature cells following infection as well as infection of early T-lymphocyte progenitors. The latter mechanism could lead to failure to regenerate mature functioning CD4+ T cells. The present study determines the susceptibility of thymocytes at various stages of maturity to infection with HIV-1. Various normal thymocyte populations were inoculated with HIV-1, including unfractionated (UF), CD3- CD4- CD8- ["triple negative" (TN)], CD4+ CD8+ ["double positive" (DP)] thymocytes, and thymocyte populations obtained by limited dilution cloning. Cultures were studied for the presence of HIV-1 DNA by polymerase chain reaction in addition to examination for reverse transcriptase activity. We determined that transformed T-cell and thymocyte cell lines completely lacking CD4 were not susceptible to infection by HIV-1, whereas all of the following lines were: UF thymocytes (70-90% CD4hi+); DP thymocytes (99% CD4hi+); TN thymocytes (0% CD4hi+); and TCR alpha beta +, TCR gamma delta +, or CD16+ CD3- (natural killer) thymocyte clones expressing variable levels of CD4 and representing the progeny of TN thymocytes. [TCR alpha beta + and TCR gamma delta + refer to the chains of the T-cell antigen receptor (TCR), and CD4hi refers to a strong rightward shift (greater than 30 linear channels) of the CD4 curve on flow cytometric analysis compared with control.] Monoclonal antibodies (mAbs) to CD4 (T4a epitope) but not to CD3 (T3) were capable of blocking infection of mature and immature CD4hi+ thymocytes. Moreover, anti-CD4(T4a) mAbs also inhibited infection of CD4hi- TN thymocytes, indicating that these T-cell precursors--despite their apparent "triple negativity" (CD3- CD4hi- CD8-)--expressed sufficient CD4 molecules to become infected. Cell sorter analysis with a panel of CD4 mAbs demonstrated a mean shift of the mean fluorescence channel (MFC) with CD4 mAbs on TN thymocytes of 6 +/- 4 MFC units. Thus, intrathymic T-cell precursors and their progeny representing many stages of T-cell ontogeny are susceptible to infection by HIV-1, including early TN thymocytes, which express very low levels of CD4. Infection of multiple stages and multiple subsets of the T-cell lineage in man, mediated via the CD4 molecule, may explain the inability of the T-cell pool to regenerate in the setting of progressive HIV infection.
Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes.
Authors: Tigyi G , Matute C , Miledi R
Published: May 2006

A method was developed to produce monoclonal antibodies to brain cell antigens by using Xenopus oocytes as immunological vectors. The method consists in injecting Xenopus oocytes with rat brain mRNA to express foreign proteins and using the oocytes for immunization. Immunizations were preceded by immunotolerization of mice to antigens of native oocyte membranes. With this approach we generated a set of monoclonal antibodies that are specific markers for the cerebellar "pinceau"--a unique complex synapse formed between basket cell terminals and the initial segment of the Purkinje cell axon. Our findings reveal an immunoreactivity highly localized at the pinceau and its late expression beginning at postnatal day 19 during cerebellar development.
In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product.
Authors: Leinfelder W , Forchhammer K , Veprek B , Zehelein E , Böck A
Published: May 2006

The selD gene from Escherichia coli, whose product is involved in selenium metabolism, has been cloned and sequenced. selD codes for a protein of 347 amino acids with a calculated molecular weight of 36,687. Analysis of the selD gene product through expression of the gene in the phage T7 promoter/polymerase system confirmed the predicted molecular weight of the protein. Gene disruption experiments demonstrated that the SelD protein is required both for the incorporation of selenium into the modified nucleoside 5-methylaminomethyl-2-selenouridine of tRNA and for the biosynthesis of selenocysteine from an L-serine residue esterbonded to tRNA(Ser)(UCA). tRNA(Ser)(UCA) has been purified, aminoacylated with L-serine, and used as a substrate for the development of an in vitro system for selenocysteine biosynthesis. Efficient formation of selenocysteinyl-tRNA(Ser)(UCA) was achieved by using extracts in which both the selD and the selA gene products were overproduced. The results demonstrate that selenocysteine is synthesized from L-serine bound to tRNA(UCA) and they are in accord with SelD functioning as a donor of reduced selenium.
Two oncogenes, v-fos and v-ras, cooperate to convert normal keratinocytes to squamous cell carcinoma.
Authors: Greenhalgh D A , Welty D J , Player A , Yuspa S H
Published: May 2006

Previous studies have implicated the rasHa oncogene in the initiation of skin carcinogenesis and the fos oncogene in malignant progression of premalignant skin cell lines. To determine if these two oncogenes are sufficient to convert normal keratinocytes to cancer cells, freshly isolated mouse keratinocytes were coinfected with replication-defective (psi-2) v-rasHa and v-fos viruses in culture. When tested in nude mice within several days of infection, v-fos/v-rasHa-coinfected keratinocytes produced squamous cell carcinomas. Introduction of v-fos alone resulted in normal or hyperplastic skin, whereas v-rasHa alone produced squamous papillomas. These results indicate that two oncogenes are sufficient to produce the malignant phenotype in epidermal cells. Furthermore, they clearly link the fos oncogene with malignant conversion. Since fos acts as a transcriptional regulator of other genes, malignant conversion may be an indirect consequence of the overexpression of the fos-encoded protein leading to a change in the expression of fos-controlled cellular genes.
Beta-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes.
Authors: Yue D T , Herzig S , Marban E
Published: May 2006

cAMP-dependent phosphorylation clearly increases current through cardiac L-type Ca channels, but the molecular manifestation of this effect remains controversial. Previous work implicates either an increase in the number of functional channels or graded changes in the gating of individual channels. We now find that single cardiac Ca channels display three patterns of activity ("modes") and that isoproterenol or 8-bromoadenosine 3',5'-cyclic monophosphate redistributes the relative proportions of modes such that the two most active (mode 1, bursts of brief openings; mode 2, very long-lasting openings) are favored (P less than 0.05; n = 7). Conversely, a pattern of sparse brief openings (mode 0a) is selectively inhibited (P less than 0.01). Despite differences in the relative frequencies of the various modes before and during drug exposure, the gating within each mode is not detectably changed. We conclude that potentiation of highly active modes of Ca channel gating underlies the enhancement of calcium influx by beta-adrenergic stimulation.
Tumor necrosis factor alpha functions in an autocrine manner in the induction of human immunodeficiency virus expression.
Authors: Poli G , Kinter A , Justement J S , Kehrl J H , Bressler P , Stanley S , Fauci A S
Published: May 2006

Tumor necrosis factor alpha (TNF-alpha) is an immunoregulatory cytokine capable of inducing viral expression in cells chronically infected with the human immunodeficiency virus (HIV), such as the promonocytic line U1 and the T-lymphocytic line ACH-2. In the present study, we demonstrate an autocrine mechanism of TNF-alpha-mediated HIV induction. Stimulation of U1 and ACH-2 cells with phorbol 12-myristate 13-acetate (PMA) resulted in the induction of TNF-alpha mRNA and the secretion of TNF-alpha. Of note is the fact that anti-TNF-alpha antibodies significantly suppressed the expression of HIV in PMA-stimulated U1 and ACH-2 cells. Furthermore, anti-TNF-alpha antibodies also suppressed both the constitutive and inducible levels of viral expression in the chronically infected promonocytic clone U33.3. This study illustrates the interrelationship between the regulation of HIV expression and normal immunoregulatory mechanisms in that virus expression, both constitutive and induced, can be modulated by an autocrine pathway involving TNF-alpha, a cytokine involved in the complex network of regulation of the normal human immune response.
Random field and neural information.
Authors: Hervé T , Dolmazon J M , Demongeot J
Published: May 2006

A representation--the Ear-Th representation--of the activity of an assembly of neurons is proposed that allows us to describe simultaneous recorded spike trains through the concept of the random field. This representation intrinsically takes into account the fundamental properties of the neuronal signal: its temporal, stochastic, and spatial nature. As a consequence, a neural network, considered as a kind of parallel random automata, delivers an output random field in response to the excitation provided by a random field that represents the activity of some input fibers. Each random field is represented by its associated Gibbs measure, whose potential is plotted in our representation. This approach is applied to the modeling of an intermediary neural network, which receives its input excitation from the auditory nerve fibers and delivers its response to the next auditory neuronal layer.
Superconnections and internal supersymmetry dynamics.
Authors: Ne'eman Y , Sternberg S
Published: May 2006

In previous papers we proposed a theory of internal supersymmetry using the superalgebra su(n/1) to give rise to a unified structure that included quarks and leptons in 2n-5 generations. In the present paper we suggest that the notion of superconnections as introduced by Quillen provides a natural setting for the dynamics of an internally supersymmetric theory with the Higgs field occurring as the "zero-th order part" of the superconnection. The Higgs mechanism enters quadratically into the curvature of the superconnection and hence quartically into the Lagrangian. The supercovariant derivative gives a coupling of the Higgs field to the matter field similar to that put in "by hand" in the Lagrangian of the Weinberg-Salam theory.
Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants.
Authors: Talon M , Koornneef M , Zeevaart J A
Published: May 2006

Twenty gibberellins (GAs) have been identified in extracts from shoots of the Landsberg erecta line of Arabidopsis thaliana by full-scan gas chromatography-mass spectrometry and Kovats retention indices. Eight of them are members of the early-13-hydroxylation pathway (GA53, GA44, GA19, GA17, GA20, GA1, GA29, and GA8), six are members of the early-3-hydroxylation pathway (GA37, GA27, GA36, GA13, GA4, and GA34), and the remaining six are members of the non-3,13-hydroxylation pathway (GA12, GA15, GA24, GA25, GA9, and GA51). Seven of these GAs were quantified in the Landsberg erecta line of Arabidopsis and in the semidwarf ga4 and ga5 mutants by gas chromatography-selected ion monitoring (SIM) using internal standards. The relative levels of the remaining 13 GAs were compared by the use of ion intensities only. In comparison with the Landsberg erecta line, the ga4 mutant had reduced levels of the 3-hydroxy- and 3,13-dihydroxy-GAs, and it accumulated the 13-hydroxy-GAs, except GA53, and the non-3,13-hydroxy-GAs, except GA12. The GA4 gene encodes, therefore, a protein with 3 beta-hydroxylation activity. The ga5 mutant had reduced levels of the C19-GAs, which indicates that the product of the GA5 gene catalyzes the elimination of C-20 at the aldehyde level. The ga5 mutant also had increased levels of certain C20-GAs, which indicates existence of an additional control, possibly hydroxylation of C-20. The growth-response data, as well as the accumulation of GA9 in the ga4 mutant, indicate that GA9 is not active in Arabidopsis, but it must be 3 beta-hydroxylated to GA4 to become bioactive. It is concluded that the reduced levels of the 3 beta-hydroxy-GAs, GA1 and GA4, are the cause of the semidwarf growth habit of both mutants.
Structural requirements for recognition of the HLA-Dw14 class II epitope: a key HLA determinant associated with rheumatoid arthritis.
Authors: Hiraiwa A , Yamanaka K , Kwok W W , Mickelson E M , Masewicz S , Hansen J A , Radka S F , Nepom G T
Published: May 2006

Although HLA genes have been shown to be associated with certain diseases, the basis for this association is unknown. Recent studies, however, have documented patterns of nucleotide sequence variation among some HLA genes associated with a particular disease. For rheumatoid arthritis, HLA genes in most patients have a shared nucleotide sequence encoding a key structural element of an HLA class II polypeptide; this sequence element is critical for the interaction of the HLA molecule with antigenic peptides and with responding T cells, suggestive of a direct role for this sequence element in disease susceptibility. We describe the serological and cellular immunologic characteristics encoded by this rheumatoid arthritis-associated sequence element. Site-directed mutagenesis of the DRB1 gene was used to define amino acids critical for antibody and T-cell recognition of this structural element, focusing on residues that distinguish the rheumatoid arthritis-associated alleles Dw4 and Dw14 from a closely related allele, Dw10, not associated with disease. Both the gain and loss of rheumatoid arthritis-associated epitopes were highly dependent on three residues within a discrete domain of the HLA-DR molecule. Recognition was most strongly influenced by the following amino acids (in order): 70 greater than 71 greater than 67. Some alloreactive T-cell clones were also influenced by amino acid variation in portions of the DR molecule lying outside the shared sequence element.
Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.
Authors: Mullinax R L , Gross E A , Amberg J R , Hay B N , Hogrefe H H , Kubitz M M , Greener A , Alting-Mees M , Ardourel D , Short J M
Published: May 2006

We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.
Genetically engineered truncated myosin in Dictyostelium: the carboxyl-terminal regulatory domain is not required for the developmental cycle.
Authors: O'Halloran T J , Spudich J A
Published: May 2006

The study of engineered Dictyostelium mutants with altered or missing myosin has revealed the molecule to be essential both for cytokinesis and for completion of the complex Dictyostelium developmental cycle. To explore the biological role of the carboxyl-terminal portion of the myosin tail, we have created a Dictyostelium cell line bearing a mutation designated my delta C34 in the myosin (mhcA) locus. This cell line produces a truncated myosin protein lacking the 34-kDa carboxyl terminus of the wild-type tail. Southern blots of the mutant cells show that the myosin gene was disrupted by homologous recombination of the transforming plasmid into the myosin locus. Based on in vitro studies of myosin functional domains, the 200-kDa truncated myosin was designed to include a domain important for assembly but to eliminate a domain important for threonine phosphorylation. The mutant cells are defective in cytokinesis, similar to those mutants that are either devoid of myosin (null cells) or contain a truncated 140-kDa myosin (hmm cells). However, unlike previous mutants, the cells carrying the my delta C34 mutation are able to complete the Dictyostelium developmental cycle to form fruiting bodies. Thus a truncated 200-kDa myosin can substitute for native myosin to function in developing cells. These results demonstrate that the 34-kDa carboxyl terminus of myosin, which contributes regulated phosphorylation sites and 20% of the total length of the rod, is not required for the developmental cycle of Dictyostelium.
Opal phytoliths found on the teeth of the extinct ape Gigantopithecus blacki: implications for paleodietary studies.
Authors: Ciochon R L , Piperno D R , Thompson R G
Published: May 2006

Identification of opal phytoliths bonded to the enamel surface of the teeth of Gigantopithecus blacki indicates that this extinct ape had a varied diet of grasses and fruits. By using the scanning electron microscope at magnifications of 2000-6000x specific opal phytoliths were observed and photographed on the fossilized teeth of an extinct species. Since opal phytoliths represent the inorganic remains of once-living plant cells, their documentation on the teeth of Gigantopithecus introduces a promising technique for the determination of diet in extinct mammalian species which should find numerous applications in the field of paleoanthropology as well as vertebrate paleontology.
Beta-globin locus activation regions: conservation of organization, structure, and function.
Authors: Li Q L , Zhou B , Powers P , Enver T , Stamatoyannopoulos G
Published: May 2006

The human beta-globin locus activation region (LAR) comprises four erythroid-specific DNase I hypersensitive sites (I-IV) thought to be largely responsible for activating the beta-globin domain and facilitating high-level erythroid-specific globin gene expression. We identified the goat beta-globin LAR, determined 10.2 kilobases of its sequence, and demonstrated its function in transgenic mice. The human and goat LARs share 6.5 kilobases of homologous sequences that are as highly conserved as the epsilon-globin gene promoters. Furthermore, the overall spatial organization of the two LARs has been conserved. These results suggest that the functionally relevant regions of the LAR are large and that in addition to their primary structure, the spatial relationship of the conserved elements is important for LAR function.