bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-induced increase in protein kinase C activity in plasma membrane of mast cells.
Authors: White J R , Pluznik D H , Ishizaka K , Ishizaka T
Published: May 2006

Bridging of cell-bound IgE antibody molecules on colony-stimulating factor-dependent mouse mast cell line (PT-18) cells by multivalent antigen induces phospholipid methylation, a transient rise in intracellular cAMP, intracellular mobilization and uptake of Ca2+, and formation of diacylglycerol followed by histamine release. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C activity in the plasma membrane, which is accompanied by a slight decrease in the enzyme in cytosol. Protein kinase C activity in the membrane fraction reached maximum within 30 sec after antigen challenge and then gradually declined. The increase of the enzyme activity in the membrane could not be explained by a shift of the enzyme from cytosol, and it suggested that bridging of IgE-receptor may induce a modulation of existing enzyme to a state of higher catalytic activity. Phorbol 12-myristate 13-acetate also induced a rapid but persistent increase in protein kinase C activity in the membrane fraction of mast cells. However, the increase in the enzyme activity in the membrane was accompanied by a marked decrease in the enzyme in cytosol.
1,25-Dihydroxyvitamin D3 causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow.
Authors: Roodman G D , Ibbotson K J , MacDonald B R , Kuehl T J , Mundy G R
Published: May 2006

1,25-Dihydroxyvitamin D3 induces cells derived from mononuclear phagocytes such as HL-60 and U937 to differentiate to macrophage-like cells and causes alveolar macrophages to form multinucleated cells in vitro. Since the osteoclast is thought to be derived from early marrow cells of the macrophage lineage, we cultured normal primate marrow mononuclear cells in medium containing 1,25-dihydroxy-vitamin D3. We found that large multinucleated cells (3-30 nuclei per cell) formed after 2-3 weeks of culture. Addition of 1,25-dihydroxyvitamin D3 to these cultures markedly stimulated multinucleated cell formation. Other vitamin D analogues, parathyroid hormone, prostaglandin E2, and calcitonin failed to stimulate multinucleated cell formation. However, calcitonin inhibited the stimulation of multinucleated cell formation by 1,25-dihydroxyvitamin D3. The multinucleated cells that formed in these cultures contained a tartrate-resistant acid phosphatase activity, a marker enzyme for osteoclasts. This activity was increased by 1,25-dihydroxyvitamin D3 and parathyroid hormone, and calcitonin inhibited the increase in acid phosphatase activity stimulated by these compounds. Ultrastructurally, the multinucleated cells had several features similar to those of osteoclasts. These data suggest that the multinucleated cells in our culture have several characteristics of osteoclasts and that the potent bone-resorbing activity of 1,25-dihydroxyvitamin D3 in vivo and in vitro may be mediated in part by stimulation of marrow mononuclear cells to form osteoclasts.
Proteins tightly associated with the termini of replicative form DNA of Kilham rat virus, an autonomous parvovirus.
Authors: Wobbe C R , Mitra S
Published: May 2006

Revie et al. [Revie, D., Tseng, B. Y., Grafstrom, R. H. & Goulian, M. (1979) Proc. Natl. Acad. Sci. USA 76, 5539-5543] have proposed that the double-stranded replicative form (RF) DNA of the autonomous rodent parvovirus H-1 has protein of 60 kDa covalently bound at its 5' termini. We present evidence that the RF DNA of a similar rodent parvovirus, Kilham rat virus (KRV), also has covalently bound protein. NaDodSO4/polyacrylamide gel electrophoresis of purified, 125I-labeled RF DNA shows that proteins of 68-72, 66, 64, and 55 kDa copurify with the DNA during velocity and equilibrium sedimentation in the presence of detergents and 4 M guanidine HCl. Phenol extraction in the presence of 2-mercaptoethanol removes the 68- to 72-kDa proteins, but the 66-, 64-, and 55-kDa proteins remain tightly, but noncovalently, bound. The latter polypeptides also appear to associate with protease-treated RF DNA when mixed with uninfected cell extract. Following removal of these proteins by electrophoresis in NaDodSO4/agarose gels, two proteins (called RF TP-90 and RF TP-40), of about 90 and 40 kDa, become evident. These remain bound to the DNA and are released only after nuclease digestion of the DNA. These two proteins, apparently not of viral origin, are associated with terminal restriction fragments of the RF DNA and appear to be covalently bound to the 5' termini of both strands.
Autoimmune anti-androgen-receptor antibodies in human serum.
Authors: Liao S , Witte D
Published: May 2006

Circulating autoantibodies to human and rat androgen receptors are present at high titers in the blood sera of some patients with prostate diseases. The antibodies from some serum samples were associated with a purified IgG fraction and interacted with the 3.8S cytosolic androgen-receptor complexes of rat ventral prostate to form 9- to 12S units. Other serum samples, however, formed 14- to 19S units, suggesting that other immunoglobulins might be involved. In the presence of an anti-human immunoglobulin as a second antibody, the androgen-receptor-antibody complexes could be immunoprecipitated. The antibodies interacted with the nuclear and the cytosolic androgen-receptor complexes, either the DNA-binding or the nonbinding form, but not with receptors for estradiol, progestin, or dexamethasone from a variety of sources. Human testosterone/estradiol-binding globulin, rat epididymal androgen-binding protein, or rat prostate alpha-protein (a nonreceptor steroid-binding protein) also did not interact with the antibodies to form immunoprecipitates. About 37% of male and 3% of female serum samples screened had significant antibody titer. The chance of finding serum with a high titer is much better in males older than 66 years than in the younger males or females at all ages. The presence of the high-titer antibodies may make it possible to prepare monoclonal antibodies to androgen receptors without purification of the receptors for immunization.
Analysis of optical spectra from single crystals of
Rhodopseudomonas viridis
reaction centers
Authors: Knapp E. W. , Fischer S. F. , Zinth W. , Sander M. , Kaiser W. , Deisenhofer J. , Michel H.
Published: May 2006

Absorption spectra and light-induced absorbance changes of crystals from
Rhodopseudomonas viridis
reaction centers are recorded. A theoretical analysis of the absorption and circular dichroism spectra is presented, yielding a consistent picture of spectroscopic and structural information.
The relationship between the "TATA" sequence and transcription initiation sites at the HIS4 gene of Saccharomyces cerevisiae.
Authors: Nagawa F , Fink G R
Published: May 2006

Transcription of the HIS4 gene begins at a single site (I) at position -60 from the ATG that begins translation. We have made linker insertions/deletions in the 5' noncoding region to identify the elements required for the specificity of transcription initiation. Although there are four sequences that begin TATA and are near the start of transcription (-170, -132, -123, and -102) only the sequence at -123 (TATA-123) is required for transcription initiation. By inserting synthetic oligonucleotides into a mutant from which TATA-123 had been deleted, we found that just TATA or TATAA does not work but that TATAAA functions almost as well as the wild-type sequence. This hexamer does not work in the opposite orientation (TTTATA). When a synthetic TATA sequence is placed upstream from the normal site, the site of initiation also moves upstream in a roughly cometric way even when TATA-123 is present. Analysis of transcripts in strains where the distance between the TATA sequence and the wild-type site of transcription initiation (I site) has been altered shows that in yeast, unlike higher cells, transcription does not initiate at a strictly defined distance from the TATA sequence. Constructions that alter the distance between the TATA and the I site or remove the I site change the pattern of transcription initiation without affecting the level of HIS4 expression. Deletions that eliminate the I site produce heterogeneous transcripts and deletions that substantially shorten the distance between TATA-123 and the I site initiate at multiple sites downstream from the I site. Thus, both the TATA and the sequences downstream from it determine the pattern of transcription initiation.
Homozygosity, effective number of alleles, and interdeme differentiation in subdivided populations.
Authors: Nagylaki T
Published: May 2006

The amount and pattern of genetic variability in a geographically structured population at equilibrium under the joint action of migration, mutation, and random genetic drift is studied. The monoecious, diploid population is subdivided into panmictic colonies that exchange migrants. Self-fertilization does not occur; generations are discrete and nonoverlapping; the analysis is restricted to a single locus in the absence of selection; every allele mutates to new alleles at the same rate. It is shown that if the number of demes is finite and migration does not alter the deme sizes, then population subdivision produces interdeme differentiation and the mean homozygosity and the effective number of alleles exceed their panmictic values. A simple relation between the mean probability of identity and the mean homozygosity is established. The results apply to a dioecious population if the migration pattern and mutation rate are sex independent.
Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor.
Authors: Gamble J R , Harlan J M , Klebanoff S J , Vadas M A
Published: May 2006

Recombinant human tumor necrosis factor (TNF) was found to enhance the adherence of human peripheral blood neutrophils to human umbilical vein endothelial (HUVE) cell monolayers in vitro. The enhancement was due to the effects both on neutrophils and HUVE cells. The effect on neutrophils was maximally induced within 5 min and did not require protein or RNA synthesis. By contrast, maximal effects on HUVE cells took 4 hr to develop and required de novo protein and RNA synthesis; however, exposure of HUVE cells to TNF for as little as 5 min was sufficient to initiate changes leading to maximal adherence of neutrophils at 4 hr. Both the effect on neutrophils and that on HUVE cells were blocked by a monoclonal antibody against TNF. TNF also rapidly induced an increased surface expression of neutrophil antigens recognized by monoclonal antibodies directed against epitopes of a glycoprotein required for optimum adherence and for complement component C3bi receptor (CR3) function. Thus, the mechanism of action of TNF may involve the regulation of expression of cell surface molecules. Our observations show that TNF induces a process central to the development of all inflammatory reactions and that both blood neutrophils and endothelial cells are targets of TNF action. The regulation of inflammatory reactions by TNF or antagonists of TNF has wide-ranging clinical implications.
Cyclical secretion of prorenin during the menstrual cycle: synchronization with luteinizing hormone and progesterone.
Authors: Sealey J E , Atlas S A , Glorioso N , Manapat H , Laragh J H
Published: May 2006

Plasma prorenin, a high molecular weight precursor form of renin, (renin, EC 3.4.23.15; old number, EC 3.4.99.19), was measured three times weekly in normal young women during the menstrual cycle and was related to changes in luteinizing hormone, estradiol, and progesterone. In all subjects a stable baseline level of prorenin occurred during the follicular phase. Then, simultaneously or soon after the luteinizing hormone peak, plasma prorenin consistently increased about 2-fold. Baseline prorenin ranged from 18 to 40 ng per ml per hr, and peak prorenin ranged from 35 to 65 ng per ml per hr. The maximum increase in prorenin averaged 80%. Prorenin remained elevated during the mid-luteal phase of the menstrual cycle and returned to baseline during the late-luteal phase in coordination with the decrease in progesterone. The changes in prorenin were not synchronized with changes in active renin which was significantly increased only during the mid-luteal phase. These findings suggest that prorenin may be involved in reproductive physiology.
Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA.
Authors: Kam W , Clauser E , Kim Y S , Kan Y W , Rutter W J
Published: May 2006

A human term (third trimester) placental alkaline phosphatase (PLAP; EC 3.1.3.1) cDNA was isolated from a human placental lambda gt11 cDNA library. The expression library was screened by using rabbit antibodies against PLAP and oligonucleotide probes. DNA sequence analysis of a positive clone with an insert of 2.7 kilobase pairs allowed us to predict the complete amino acid sequence of PLAP (530 residues), which coincided with the reported 42 N-terminal amino acid sequence of PLAP except at position 3. Contrary to the previous supposition that there was no amino acid sequence homology between PLAP and Escherichia coli alkaline phosphatase (471 residues), we found 30% overall homology, with regions of strong homology including the putative active site and the metal-binding sites. The 44-residue C-terminal extension of PLAP has a stretch of 17 hydrophobic amino acids, which presumably anchors the protein to the plasma membrane, a change perhaps necessary for the transition from a bacterial periplasmic enzyme to a mammalian membrane-associated enzyme. We have also localized PLAP-related DNA sequences mainly on chromosome 2 and to a lesser degree on chromosome 17. It seems likely therefore that the PLAP gene resides on chromosome 2 and other member(s) of the alkaline phosphatase family may exist (on this chromosome and) on chromosome 17.
Noninvasive detection and monitoring of regional myocardial ischemia in situ using depth-resolved 31P NMR spectroscopy.
Authors: Bottomley P A , Herfkens R J , Smith L S , Brazzamano S , Blinder R , Hedlund L W , Swain J L , Redington R W
Published: May 2006

Phosphorus (31P) NMR spectra showing the relative concentrations of phosphocreatine, ATP, and Pi were recorded noninvasively from localized regions in the left ventricles of dog hearts in situ by using depth-resolved surface-coil spectroscopy at 1.5 T. Proton (1H) NMR surface-coil imaging was used to position 31P NMR coils and to determine the location of depth-resolved volumes immediately prior to 31P examination. Occlusion of the left anterior descending coronary artery produced regional ischemia detected as changes in the ratios of phosphocreatine, ATP, and Pi and by changes in the pH measured from the spectra. Spectral changes were not typically observed in regions adjacent to ischemic myocardium. Reperfusion produced some recovery, and ventricular fibrillation resulted in deterioration in high-energy metabolites. The location and size of ischemic tissue was measured by single-photon-emission computed tomography (SPECT) and gamma-ray counting or by staining excised hearts. The technique should permit the long-term noninvasive monitoring of the metabolic response of the heart to pathologic processes and allow assessment of interventions.
Efficient transcription of a protein-coding gene from the RNA polymerase I promoter in transfected cells.
Authors: Grummt I , Skinner J A
Published: May 2006

The activity of the mouse ribosomal promoter was examined after fusion to the gene coding for chloramphenicol acetyltransferase (CAT) and transfection into mouse cells. Very little CAT enzyme but high levels of CAT-specific RNA correctly initiated at the ribosomal DNA start site were synthesized. The amount of specific transcripts was neither influenced by long stretches of upstream spacer sequences nor by the insertion of the Moloney murine sarcoma virus enhancer. The deletion mutant pMr delta-39, which has been shown to be fully active in vitro, exhibited a 90% decrease in template activity in vivo. A mutant in which 22 base pairs of ribosomal DNA (between positions -35 and -14) were substituted by foreign DNA sequences proved transcriptionally inactive. The fusion genes were only transcribed in mouse cells, indicating that species-specific transcription factors are involved in ribosomal promoter recognition.
Expression of the mouse serum albumin gene introduced into differentiated and dedifferentiated rat hepatoma cells.
Authors: Deschatrette J , Fougere-Deschatrette C , Corcos L , Schimke R T
Published: May 2006

A 23-kilobase-pair segment of DNA containing the entire mouse serum albumin gene as well as 2.2 kilobase pairs of 5' and 4.3 kilobase pairs of 3' flanking sequences has been introduced into pSV2dhfr, a plasmid in which expression of the mouse dihydrofolate reductase cDNA is under the control of simian virus 40 sequences. This vector, pSV2dhfr-alb, was used to transfect differentiated and variant dedifferentiated rat hepatoma cells. Nine independent clones of transfected differentiated cells secrete considerable amounts of mouse albumin, while the expression of the normal rat albumin is the same as in nontransfected cells. In contrast, only small amounts of mouse and rat albumin are produced by transfected dedifferentiated cells. The amounts of albumin mRNA present in the cells are consistent with the amounts of albumin produced. These results show that a transfected gene can be regulated in a fashion consistent with the overall differentiation profile of the cell.
Isolation of the non-glycosylated proteins of desmosomes and immunolocalization of a third plaque protein: desmoplakin III.
Authors: Gorbsky G , Cohen S M , Shida H , Giudice G J , Steinberg M S
Published: May 2006

The cytoplasmic plaque of the spot desmosome or macula adhaerens mediates the attachment of bundles of intermediate filaments to the plasma membrane. We have isolated from a bovine epidermal desmosome preparation a fraction that is highly enriched in the non-glycosylated desmosomal proteins. Plastic-embedded and thin-sectioned high-speed pellets of this fraction reveal closely packed filaments that resemble plaque regions of the low pH whole desmosome preparation from which they are derived. NaDodSO4/polyacrylamide gel electrophoresis reveals four major, non-glycosylated proteins of 240, 210, 81, and 77 kDa. In agreement with a previous study, we find the 240- and 210-kDa proteins (desmoplakins I and II) to be closely related, whereas the 81- and 77-kDa proteins are unique. This is shown both immunologically and by one-dimensional proteolytic peptide mapping. Monospecific, polyclonal rabbit antibodies were prepared against the 81-kDa protein and used, in conjunction with protein A-complexed colloidal gold particles (PAG), to immunolocalize this antigen on ultrathin sections of bovine muzzle epidermis. On antibody-labeled sections, PAG particles were associated principally with the desmosomal cytoplasmic plaque. Sections exposed to preimmune serum showed little or no labeling. We conclude that the 81-kDa protein, like the 240/210-kDa protein family, is one of the major components of the desmosomal plaque. We designate it as "desmoplakin III." The location of the 77-kDa protein remains to be definitively established.
Anti-peripheral myelin antibody in patients with demyelinating neuropathy: quantitative and kinetic determination of serum antibody by complement component 1 fixation.
Authors: Koski C L , Humphrey R , Shin M L
Published: May 2006

The role of anti-peripheral nerve myelin antibody (anti-PNM Ab) in the pathogenesis of acquired demyelination of peripheral nerve is unclear, in part, due to the poor correlation between antibody and disease activity. Previous studies show that only 27-50% of patients with acute demyelinating neuropathy or Guillain-Barré syndrome (GBS) had serum Abs to peripheral nerve or PNM as demonstrated by consumption of hemolytic activity of serum complement 1 (C1) fixation and transfer assay, quantitative determinations of anti-PNM Ab showed significantly high titers in the serum of patients with GBS, chronic and recurrent polyneuritis, and paraproteinemia associated with peripheral neuropathy. All 11 patients with acute-phase GBS had Ab titers 6-56 times higher than controls. In 6 GBS patients, serial Ab determinations showed that titers were highest on admission, fell rapidly the first week, and became undetectable or barely detectable by the third week. Declining Ab titers coincided with cessation of clinical progression. In 3 GBS patients, depletion of serum IgM lowered anti-PNM Ab titers significantly, whereas IgG depletion failed to produce a similar effect. This study shows that the C1 fixation and transfer assay is a sensitive method to detect anti-PNM Ab in the serum of patients with a variety of demyelinating neuropathies and provides good correlation between Ab level and the clinical course of GBS patients. It may provide important information about the pathogenesis of the demyelinating neuropathies.
Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences.
Authors: Reik W , Weiher H , Jaenisch R
Published: May 2006

A bacterial suppressor tRNA gene was introduced into the long terminal repeat of the Moloney murine leukemia virus (Mo-MuLV) proviral genome to construct a retrovirus that allows easy cloning of the provirus with flanking host sequences. A replication competent virus, Mo-MuLV sup containing a tRNA amber suppressor gene, was derived that replicates to high titers in tissue culture cells and stably transduces the bacterial gene. The recombinant virus can efficiently replicate in vivo when microinjected into midgestation embryos or when injected into newborn mice and displays the same tissue tropism as wild-type Mo-MuLV. The suppressor gene in Mo-MuLV sup is functional in bacteria and allows efficient recovery of proviral genomes. This was shown by ligation of DNA from infected cells to phage lambda Charon 4A arms and selective growth of recombinant phages on su- host cells. All recovered phages contained Mo-MuLV proviral sequences and, because of the high cloning capacity of phage lambda, 1-11 kilobases of flanking host DNA. This virus should facilitate studying virus-host interactions in tissue culture cells and in animals.
Identification of a membrane-associated interleukin 1 in macrophages.
Authors: Kurt-Jones E A , Beller D I , Mizel S B , Unanue E R
Published: May 2006

We have found a surface membrane-associated interleukin 1 (IL-1) with potent thymocyte and T-cell stimulatory activity on peptone-elicited peritoneal macrophages. The IL-1 activity was demonstrated on both fixed macrophage monolayers and on isolated membranes from unfixed macrophages. Membrane IL-1 was induced by adherence and/or by adding heat-killed Listeria monocytogenes to macrophage cultures. The macrophage membrane IL-1 was similar functionally and antigenically to soluble IL-1, but its expression could be temporally dissociated from IL-1 secretion; membrane IL-1 was induced earlier and persisted longer than IL-1 secretion during in vitro macrophage culture. Moreover, when cultured macrophages that had ceased both secretion and membrane expression of IL-1 were restimulated by adding heat-killed Listeria, substantial membrane IL-1 was induced in the absence of detectable IL-1 secretion. Membrane IL-1 appears to be an integral membrane protein since it was solubilized by detergent but was not eluted by EDTA, high salt, or low pH treatment of the membranes.
Human leukemia cell maturation induced by a T-cell lymphokine isolated from medium conditioned by normal lymphocytes.
Authors: Leung K , Chiao J W
Published: May 2006

Human myelogeneous leukemia cells in liquid culture can be induced to mature along the monocyte/macrophage pathway by a maturation inducer derived from the conditioned medium of activated human T lymphocytes. Serum-free conditioned medium was used for the isolation of the T-cell lymphokine. The maturation inducer was purified approximately equal to 6000-fold by ammonium sulfate precipitation, low-salt elution from DEAE-Sepharose CL-6B, gel filtration on Bio-Gel A-0.5m, and NaDodSO4/PAGE under nonreducing conditions. The molecular weight of the maturation inducer was 36,000-58,000 on NaDodSO4/PAGE. Terminal differentiation associated with inhibition of leukemia cell proliferation and expression of mature cell properties was observed with the isolated maturation inducer, identical to the activity observed with the unfractionated conditioned medium. Cell-cycle analysis revealed that the proportion of replicating S-phase cells was reduced from 40% to 7% after initial interaction of the maturation inducer with cells. The differentiating cells simultaneously acquired monocyte antigen, membrane complement receptors, phagocytic function, and monocyte/macrophage morphology. The maturation-inducing activity is dose-dependent, with more inducer causing the development of more mature cells in a shorter time period. The maturation inducer was shown to be stable after pH 2 treatment, independent of interleukin 2 and colony-stimulating factor, devoid of alpha-, beta-, and gamma-interferon, and not affected by antibody to interferon. The maturation inducer may play a role as a physiological regulator of monocytic and leukemia cell development.
Evidence for a role of endogenous corticotropin-releasing factor in cold, ether, immobilization, and traumatic stress.
Authors: Nakane T , Audhya T , Kanie N , Hollander C S
Published: May 2006

The role of corticotropin-releasing factor (CRF) in four model stresses (cold, ether, immobilization, and trauma) was examined in the guinea pig by using passive immunoneutralization with anti-CRF antiserum. Plasma corticotropin levels were measured at various times after exposure to stress, and groups treated with CRF antiserum were compared with those treated with normal rabbit serum. Of the four stresses tested, ether had the most pronounced effect on corticotropin secretion. Treatment with anti-CRF inhibited most of the ether-induced corticotropin secretory response, the difference between the normal serum- and the anti-CRF antiserum-treated groups being significant at 5 and 10 min (P less than 0.01). Corticotropin responses to cold stress in the two groups differed at the 0.05 level of significance at 10 and 20 min. After administration of trauma (leg fracture), a statistically significant difference (P less than 0.01) between the two groups also was evident, albeit only at 20 min. During immobilization, corticotropin levels differed significantly from control only in the normal serum-treated group but not in the anti-CRF-treated group. These findings show that CRF antiserum was effective in reducing corticotropin levels, indicating that CRF has an important role in mediating corticotropin response to stress. The fact that neutralization was incomplete might be due to an inability of the antiserum to sufficiently neutralize the endogenous CRF or, more likely, reflects the contribution of additional mediators, notably catecholamines and vasopressin, of corticotropin release upon stress.
Lithium induces corticotropin secretion and desensitization in cultured anterior pituitary cells.
Authors: Zatz M , Reisine T D
Published: May 2006

Lithium stimulated corticotropin (ACTH) secretion by mouse pituitary tumor cells (AtT-20/D16-16) and by normal rat anterior pituitary cells in primary culture. Effects were observed at less than 2 mM LiCl. ACTH secretion was comparable in magnitude to that induced by other secretagogues, was calcium dependent, and was inhibited by somatostatin. Lithium also induced changes in [3H]inositide metabolism; these changes accompanied and were correlated with changes in ACTH secretion. The most prominent and reliable effect was to increase [3H]inositol monophosphate. Other secretagogues had no effect on [3H]inositides in the presence or absence of lithium. Pretreatment with lithium for 3 hr desensitized the cells to the effects of subsequent exposure to lithium. The cells were not desensitized to lithium by pretreatment with other secretagogues, nor were they desensitized by lithium to the effects of corticotropin-releasing factor, high potassium, or forskolin. However, pretreatment with lithium did desensitize the cells to stimulation by phorbol esters. The interaction between lithium and phorbol esters suggests the involvement of inositide metabolism and protein kinase C in the regulation of ACTH secretion and possibly of other hormones or neurotransmitters. It also suggests new avenues of research into the basis of lithium's psychopharmacological effects.
Purified hydrophobic proteins, chargerins, are essential for energy transduction in oxidative phosphorylation.
Authors: Higuti T , Takigawa M , Kotera Y , Oka H , Uchida J , Arakaki R , Fujita T , Ogawa T
Published: May 2006

Studies on anisotropic inhibitors, a unique type of inhibitor of energy transduction in oxidative phosphorylation, suggested that redox reactions generate two kinds of negative charges on the outer surface of mitochondrial inner membranes, on redox complexes and on F0, and that the inhibitors inhibit energy transduction by binding to these negative charges. Recent experiments on photoaffinity labeling of mitochondria with monoazide ethidium, which is an anisotropic inhibitor, showed that the inhibitor specifically binds to a hydrophobic protein of the membranes. In the present work the mitochondrial components labeled with monoazide ethidium were further purified and two kinds of hydrophobic proteins (apparent molecular masses, 8 and 13 kDa) were found to be specifically labeled with the inhibitor. These proteins were named chargerin I and II, respectively. Redox reactions greatly increased the molar ratio of ethidium bound to chargerin I and II in mitochondria, reflecting a conformational change of the chargerins coupled with the redox reactions. It was also shown that antibody against chargerin II specifically inhibited ATP synthesis in mitoplasts (inner membranes plus matrix) prepared from rat liver mitochondria. Thus, the present findings show that chargerins have an essential role in energy transduction in oxidative phosphorylation in rat liver mitochondria, in good accord with the conformational coupling model of the H+ pumps and ATP synthesis.
Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport.
Authors: Grinstein S , Cohen S , Goetz J D , Rothstein A , Gelfand E W
Published: May 2006

Phorbol 12-myristate 13-acetate and other phorbol esters induce an intracellular alkalinization in rat thymic lymphocytes. An extracellular acidification can be recorded concomitantly. This transmembrane H+ (equivalent) flux is dependent on external Na+ and is amiloride sensitive. Phorbol esters also stimulate an amiloride-sensitive uptake of 22Na+, suggesting activation of Na+o/H+i exchange. Only those phorbol derivatives that are tumor promoters and activators of protein kinase C stimulate the antiport. Activation of the Na+/H+ exchange is brought about by a change in the cytoplasmic pH sensitivity of the antiport. Activation of the Na+/H+ exchanger by phorbol esters results in membrane hyperpolarization, due to indirect stimulation of the electrogenic Na+/K+ pump by the increased intracellular Na+ concentration. Increased Na+/H+ exchange also produces cell swelling, which may be one of the earliest manifestations of the growth-promoting properties of the phorbol esters.
Oxygen chemoreception by carotid body cells in culture.
Authors: Fishman M C , Greene W L , Platika D
Published: May 2006

Chemoreceptors for oxygen reside within the carotid body, but it is not known which cells actually sense hypoxia and by what mechanisms they transduce this information into afferent signals in the carotid sinus nerve. We have developed systems for the growth of glomus cells of the carotid body in dissociated cell culture. Here we demonstrate that, as in vivo, these cells contain the putative neurotransmitters dopamine, serotonin, and norepinephrine. Oxygen tension regulates the rate of dopamine secretion from the glomus cells. Similar to chemically stimulated catecholamine secretion from other adrenergic cells this hypoxia-stimulated release requires extracellular calcium. These results are compatible with the suggestion that the glomus cells of the carotid body are chemoreceptor cells and that they signal hypoxia by regulated secretion of dopamine.
Unusual proton exchange properties of Z-form poly[d(G-C)]
Authors: Mirau Peter A. , Kearns David R.
Published: May 2006

Real-time solvent-exchange NMR has been used to study the proton exchange in Z-form poly[d(G-C)]. In 4.5 M NaCl the most slowly exchanging protons (about two orders of magnitude slower than in B-DNA) are identified as the guanine imino proton and the cytosine amino proton hydrogen bonded to the guanine carbonyl. Both protons exchange at the same rate and the exchange follows single-exponential kinetics and cannot be catalyzed, implying that the exchanges of the two protons both occur from the same transient solvent-exposed state. The exchange depends strongly on temperature and the activation energy for exchange (≈22 kcal/mol) is the same as the activation energy for the B → Z transition. The rate of proton exchange is identical with the B → Z transition rate, both measured in 4.5 M NaCl. The correlation between the B → Z kinetics and the proton exchange also extends to 3.25 M NaClO
4
solutions, in which both rates are 5 times faster. This unexpected parallelism between the B → Z transition kinetics and the Z exchange kinetics indicates that the rate-limiting steps in the two processes are similar.
Extensive but reversible depletion of ATP via adenylate cyclase in rat adipocytes.
Authors: Chung F Z , Weber H W , Appleman M M
Published: May 2006

In adipocytes, adenylate cyclase is positively regulated by beta-adrenergic agents and negatively regulated by adenosine. Incubation of adipocytes with adenosine deaminase relieves the inhibition of adenylate cyclase by destroying the adenosine that the cells release into the medium. When adipocytes are incubated with adenosine deaminase and the beta-adrenergic agent isoproterenol, most of their ATP is converted to AMP in 5 min. Either isoproterenol or adenosine deaminase alone has little or no effect. In the additional presence of the phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one (Ro 20-1724) cAMP accumulates instead of AMP. Under these conditions, cAMP represents 40-50% of the total intracellular adenine nucleotides, and ATP only 5%. N6-(L-2-phenylisopropyl)adenosine, a deaminase-resistant adenosine agonist, prevents beta-adrenergic stimulation. 8-(p-Sulfophenyl)theophylline and 3-isobutyl-1-methylxanthine are both adenosine antagonists that can replace the deaminase in permitting beta-adrenergic stimulation of adenylate cyclase, but only the latter also inhibits the phosphodiesterase and causes accumulation of cAMP. When the ATP-depleted adipocytes are washed with fresh medium, the nucleoside triphosphate level can be restored within 5 min. The ATP-restored adipocytes can respond rapidly to a second dose of isoproterenol and adenosine antagonist. These findings point out the important role of adenosine in controlling adenylate cyclase activity and the possible involvement of adenylate cyclase in the control of energy flow in rat adipocytes.
Evidence for higher rates of nucleotide substitution in rodents than in man.
Authors: Wu C I , Li W H
Published: May 2006

When the coding regions of 11 genes from rodents (mouse or rat) and man are compared with those from another mammalian species (usually bovine), it is found that rodents evolve significantly faster than man. The ratio of the number of nucleotide substitutions in the rodent lineage to that in the human lineage since their divergence is 2.0 for synonymous substitutions and 1.3 for nonsynonymous substitutions. Rodents also evolve faster in the 5' and 3' untranslated regions of five different mRNAs; the ratios are 2.6 and 3.1, respectively. The numbers of nucleotide substitutions between members of the beta-globin gene family that were duplicated before the man-mouse split are also higher in mouse than in man. The difference is, again, greater for synonymous substitutions than for nonsynonymous substitutions. This tendency is more consistent with the neutralist view of molecular evolution than with the selectionist view. A simple explanation for the higher rates in rodents is that rodents have shorter generation times and, thus, higher mutation rates. The implication of our findings for the study of molecular phylogeny is discussed.
Characterization of a temperature-sensitive mutant of mouse FM3A cells defective in DNA replication.
Authors: Murakami Y , Yasuda H , Miyazawa H , Hanaoka F , Yamada M
Published: May 2006

The characterization of a temperature-sensitive mutant (tsFT20 strain, dnats) of mouse FM3A cells is reported. After incubation of tsFT20 cells at the nonpermissive temperature (39 degrees C), DNA synthesis ceased with little change in either RNA or protein synthesis. Flow-microfluorometric analysis revealed that the cell cycle of tsFT20 cells grown at 39 degrees C for 16 hr was similar to that of wild-type cells that were synchronized at the G1/S boundary and at S phase by treatment with aphidicolin, a specific inhibitor of DNA polymerase alpha. The DNA polymerase alpha activity of tsFT20 cells measured in crude cell extracts or in purified preparations was inactivated more rapidly at 39 degrees C than the activity of wild-type cells. In the growth revertants of the tsFT20 cell strain, the heat lability of DNA polymerase alpha decreased. These data suggest that tsFT20 is a temperature-sensitive mutant of DNA polymerase alpha or of a factor associated with DNA polymerase alpha that is essential for its activity.
Translational control by adenovirus: lack of virus-associated RNAI during adenovirus infection results in phosphorylation of initiation factor eIF-2 and inhibition of protein synthesis.
Authors: Siekierka J , Mariano T M , Reichel P A , Mathews M B
Published: May 2006

The dl331 mutant of adenovirus serotype 5 fails to produce virus-associated (VA) RNAI, and cells infected with this mutant do not synthesize proteins efficiently at late times in infection. The translational defect occurs at the level of polypeptide chain initiation, and cell-free extracts prepared from dl331-infected cells exhibit the defect observed in vivo. Addition of either eukaryotic initiation factor 2 (eIF-2) or guanine nucleotide exchange factor (GEF) to these cell-free extracts restores translational activity, with GEF functioning more efficiently in this regard. These results suggest that cells infected with the dl331 mutant develop a translational block at the level of GEF-catalyzed guanine nucleotide exchange and that this block is most likely established through phosphorylation of the alpha subunit of eIF-2. In the present investigation we show that endogenous HeLa cell GEF activity is significantly reduced in cells infected with the dl331 mutant. Further, in contrast to cells infected with wild-type serotype 2 adenovirus, dl331-infected cells contain increased eIF-2 alpha kinase activity. These results indicate that VA RNAI plays a role in suppressing eIF-2 alpha kinase activity during adenovirus infection of HeLa cells.
A 20-kDa protein associated with the murine T-cell antigen receptor is phosphorylated in response to activation by antigen or concanavalin A.
Authors: Samelson L E , Harford J , Schwartz R H , Klausner R D
Published: May 2006

Antigen or concanavalin A activation of a murine T-cell hybrid specific for pigeon cytochrome c and restricted to the Ek alpha:Ek beta immune response-associated (Ia) molecule resulted in phosphorylation of a 20-kDa protein that was specifically coprecipitated by a monoclonal antibody binding the T-cell antigen receptor. There was no evidence for phosphorylation of the antigen receptor itself. The phosphorylation of the 20-kDa polypeptide was dependent on the concentration of antigen or lectin used to activate the T-cell hybrid and reached a maximum 40 min after the addition of antigen. Moreover, the phosphorylation induced by antigen in the presence of Ia molecule-bearing B cells was specifically blocked by the addition of appropriate anti-Ia molecule monoclonal antibodies. The 20-kDa protein was also radioiodinated with a hydrophobic photoactivatable labeling reagent. The amount of iodinated 20-kDa protein immunoprecipitable with the anti-receptor antibody did not increase with T-cell activation, indicating that the phosphorylation occurred on a molecule that was constitutively associated with the antigen receptor. Concanavalin A also induced phosphorylation of a 20-kDa polypeptide in a second antigen-specific major histocompatibility complex-restricted T-cell hybrid. Again, the phosphorylated polypeptide was precipitated only by a monoclonal antibody specific for the antigen receptor on this hybrid. Thus, the antigen or concanavalin A-induced activation of T-cell hybrids results in the rapid phosphorylation of a 20-kDa protein that is associated with the T-cell antigen receptor.
Molecular dynamics simulation of photodissociation of carbon monoxide from hemoglobin.
Authors: Henry E R , Levitt M , Eaton W A
Published: May 2006

A molecular dynamics simulation of the photodissociation of carbon monoxide from the alpha subunit of hemoglobin is described. To initiate photodissociation, trajectories of the liganded molecule were interrupted, the iron-carbon monoxide bond was broken, and the parameters of the iron-nitrogen bonds were simultaneously altered to produce a deoxyheme conformation. Heme potential functions were used that reproduce the energies and forces for the iron out-of-plane motion obtained from quantum mechanical calculations. The effect of the protein on the rate and extent of the displacement of the iron from the porphyrin plane was assessed by comparing the results with those obtained for an isolated complex of heme with imidazole and carbon monoxide. The half-time for the displacement of the iron from the porphyrin plane was found to be 50-150 fs for both the protein and the isolated complex. These results support the interpretation of optical absorption studies using 250-fs laser pulses that the iron is displaced from the porphyrin plane within 350 fs in both hemoglobin and a free heme complex in solution.
Rat Sertoli cells acquire a beta-adrenergic response during primary culture.
Authors: Kierszenbaum A L , Spruill W A , White M G , Tres L L , Perkins J P
Published: May 2006

Two-dimensional polyacrylamide gel electrophoresis and the radioligand (-)-[125I]iodopindolol (125I-Pin) have been used to study isoproterenol-dependent protein phosphorylation and beta-adrenergic receptor availability, respectively, in cultured Sertoli cells and freshly isolated seminiferous tubular segments of sexually immature and mature rats. Sertoli cells prepared from sexually immature rats show progressive 125I-Pin binding in primary cultures that correlates with isoproterenol-induced cell shape changes, redistribution of immunoreactive vimentin, and phosphorylation of this intermediate filament protein. The development of 125I-Pin binding to Sertoli cell lysates is blocked by cycloheximide. Seminiferous tubules do not show significant isoproterenol-dependent vimentin phosphorylation nor 125I-Pin binding. However, vimentin phosphorylation can be induced by follicle-stimulating hormone or a cyclic nucleotide analog. This study stresses the need for correlating pharmacological-induced responses observed in Sertoli cell primary cultures with those in the intact seminiferous tubule.
Spatial distribution of transcripts of the long repeated ETn sequence during early mouse embryogenesis.
Authors: Brûlet P , Condamine H , Jacob F
Published: May 2006

RNA X DNA in situ hybridization revealed a high level of ETn ("early transposon") transcripts in the pluripotent cell lineage of the 3.5- to 7.5-day mouse embryo. Some extra-embryonic ectoderm derivatives also show a high level of ETn transcripts at these stages. Older embryos (8.5 days and later) have a uniform low level of ETn transcripts.
Transposon-like sequences in extrachromosomal circular DNA from mouse thymocytes.
Authors: Fujimoto S , Tsuda T , Toda M , Yamagishi H
Published: May 2006

Small polydisperse circular (spc) DNA was isolated from mouse thymocytes and cloned into the HindIII site of lambda vector Charon 7. Fifty-six recombinants from this spc DNA library were analyzed. R repeats, which were originally found near immunoglobulin genes, were enriched in spc DNA clones relative to their representation in the chromosome. In one clone, the R sequence was linked to Bam and MIF sequences and the contiguous arrangement was truncated from both ends. In another clone, composite Bam/R and R repeats existed as a pair in inverted repeat orientation. Truncation occurred from the 5' side without affecting the 3' ends. In both clones, short direct repeats flanked the repeated sequences. The possible role of R sequences in transposition and circular formation is discussed.
Ionic responses rapidly elicited by activation of protein kinase C in quiescent Swiss 3T3 cells.
Authors: Vara F , Schneider J A , Rozengurt E
Published: May 2006

Diacylglycerol and phorbol esters activate protein kinase C in intact cells. We report here that addition of the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) to quiescent cultures of Swiss 3T3 cells caused a marked increase in the rate of ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na+/K+ pump. The effect was dose-dependent and could be detected after 1 min of exposure to the diacylglycerol. OAG stimulated Na+ influx via an amiloride-sensitive pathway and increased intracellular pH by 0.15 pH unit. Phorbol 12,13-dibutyrate (PBt2) also enhanced ouabain-sensitive 86Rb+ uptake and amiloride-sensitive 22Na+ influx. Prolonged treatment (40 hr) of 3T3 cells with PBt2 at a saturating dose, which reduces the number of PBt2 binding sites and protein kinase C activity, abolished the ionic response of the cells to a subsequent addition of either OAG or PBt2. Appropriate controls using acid "loads" and the Na+ ionophore monensin showed that the function of the Na+/H+ antiport system and of the Na+/K+ pump was not impaired in the PBt2-desensitized cells. We suggest that activation of protein kinase C elicits, either directly or indirectly, enhanced Na+/H+ antiport activity, which, in turn, leads to Na+ influx, intracellular pH modulation, and stimulation of the Na+/K+ pump.
An unusual genetic code in nuclear genes of Tetrahymena.
Authors: Horowitz S , Gorovsky M A
Published: May 2006

We have cloned and partially sequenced two histone H3 genes of Tetrahymena thermophila. The DNA sequences strongly suggest that both genes are active in the vegetatively growing cell. Comparison of the derived amino acid sequences of these two genes with the actual sequence of Tetrahymena histone H3 results in the surprising conclusion that TAA codes for glutamine. This represents the first demonstration of a coding function for this termination codon of the "universal" code. This observation has important implications for the evolution of ciliates and of the genetic code.