bluuebunny/crossref_metadata_2025 huggingface dataset:
Intercellular recognition: quantitation of initial binding events.
Authors: McClay D R , Wessel G M , Marchase R B
Published: May 2006

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena--an initial recognition event and a subsequent stabilization--is supported by the use of a cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 4 degrees C in sealed compartments. The compartments are inverted and a centrifugal force is then applied to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chicken neural retina cells was determined to be on the order of 10(-5) dyne. Topographic specificities of single neural retina cells for retinal monolayers from various regions of the retina were detected with this assay and corresponded to those observed in more traditional assays at 37 degrees C. Also observed were two time- and temperature-dependent stabilization processes in which the force required for dislodgment increased. One of the stabilization processes was sensitive to dinitrophenol and was inactive at 4 degrees C; the second was still active in metabolically blocked cells. The metabolic-dependent process resulted in interactions at least 13 times as strong as the initial binding. The metabolic-independent process resulted in about a 2-fold increase in binding strength and had a temperature dependence similar to that of membrane diffusional phenomena.
Deoxyribonuclease I sensitivity of plasmid genomes in teratocarcinoma-derived stem and differentiated cells.
Authors: Huebner K , Linnenbach A , Weidner S , Glenn G , Croce C M
Published: May 2006

The DNase I (EC 3.1.21.1) sensitivities of the simian virus 40 (SV40) genome, the pBR322 genome, and the herpes simplex virus type 1 thymidine kinase (HSV-1 tk) gene have been compared in teratocarcinoma-derived stem (12-1) and differentiated (12-1a) cell lines established by transfection of thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)-deficient F9 cells with DNA from a tripartite plasmid genome consisting of the pBR322 genome, the SV40 genome, and the HSV-1 tk gene. HSV-1 tk is present in both stem and differentiated cells; SV40 early proteins are present in differentiated cells but not in stem cells; the pBR322 genome is not expressed in either cell type. The SV40 and pBR322 genomes are more sensitive to DNase I digestion in stem cells than in differentiated cells, reflecting the DNase I-hypersensitivity of total stem-cell chromatin. The HSV-1 tk gene is the least sensitive to DNase I digestion in both cell types.
B-lymphocyte responses to trinitrophenyl-conjugated Ficoll: requirement for T lymphocytes and Ia-bearing adherent cells.
Authors: Letvin N L , Benacerraf B , Germain R N
Published: May 2006

These studies were done to characterize the cellular requirements for B-lymphocyte responses to the haptenated polysaccharide trinitrophenyl-conjugated Ficoll. By using an in vitro microculture system, it was demonstrated that hapten-specific anti-trinitrophenyl-conjugated Ficoll plaque-forming cell responses by B lymphocytes require Ia-bearing adherent accessory cells and Thy 1+ Lyt 1+2- nylon wool-nonadherent (T) lymphocytes. Such T cells could be primed in vivo with nonderivatized Ficoll to show carrier-specific helper cell function for derivatized Ficoll responses in vitro. The implications of these findings for our understanding of b-lymphocyte triggering by so-called T-independent antigens are discussed.
Analysis of factor VIII coagulant antigen in normal, thrombin-treated, and hemophilic plasma.
Authors: Weinstein M , Chute L , Deykin D
Published: May 2006

The relationship between Factor VIII coagulant antigen (VIII:CAg) and Factor VIII-associated von Willebrand factor (VIII:vWF), and the effect of thrombin on VIII:CAg have been determined in plasma by using complexes of VIII:CAg and 125I-labeled human anti-VIII:CAg-Fab. Antibody-treated plasma samples were electrophoresed on NaDodSO4/polyacrylamide agarose gels and analyzed by autoradiography. The major VIII:CAg-125I-labeled Fab complex that persisted in NaDodSO4 had Mr 3.2 x 10(5). This Mr value was confirmed by column chromatography and sucrose density centrifugation and is presumed to reflect a free VIII:CAg of Mr 2.7 x 10(5). Minor bands were also present on autoradiograms of normal plasma corresponding to Mr values of 2.5, 1.85, and 1.7 x 10(5) (free VIII:CAg related proteins with Mr values of 2.0, 1.35, and 1.2 x 10(5), respectively). None of the VIII:CAg bands was present in plasma samples from five patients with severe hemophilia A. No radioactivity was associated with VIII:vWF multimers on NaDodSO4 gels. Thrombin treatment of normal plasma eliminated the radioactive band at 3.2 x 10(5) and increased the intensity of a band of Mr 1.7 x 10(5). Generation of this presumed VIII:CAg fragment of Mr is approximately equal to 1.2 x 10(5) coincided with a thrombin-induced increase in Factor VIII coagulant activity. These data demonstrate that the form of VIII:CAg detected in normal plasma is not covalently linked to VIII:vWF multimers and is absent in plasma from five hemophilia A patients. Thrombin-induced proteolysis of VIII:CAg can be detected in microliter quantities of normal plasma.
Molecular lesion affecting the ADP-combining site in a mutant isozyme of erythrocyte pyruvate kinase.
Authors: Paglia D E , Valentine W N
Published: May 2006

Erythrocyte pyruvate kinase (PK) from a patient with PK deficiency was characterized according to internationally standardized procedures. In addition to low activity, the mutant isozyme displayed impaired kinetics specifically affecting the ADP-combining site:Km (ADP) was 3-5 times greater than normal when determined at three different concentrations of phosphoenolpyruvate (P-enolpyruvate). Maximum reaction velocities were not achieved until ADP was 10 times the concentration normally required for control PK. Substrate inhibition by high concentrations of ADP and competitive inhibition by ATP were markedly diminished. Other nucleoside diphosphates normally capable of replacing ADP in the PK reaction were less effective with the mutant isozyme than with PK from controls or from subjects with other forms of PK deficiency, and Michaelis--Menten constants for several of these (UDP, GDP, CDP) were significantly elevated. Whereas all previously known PK kinetic defects have involved the substrate P-enolpyruvate, the half-saturation constant K0.5s (P-enolpyruvate) for this mutant isozyme was normal, as was its response to fructose-1,6-bisphosphate activation.
Differential immunologic reactivity and processing of glycoproteins gA and gB of herpes simplex virus types 1 and 2 made in Vero and HEp-2 cells.
Authors: Pereira L , Dondero D , Norrild B , Roizman B
Published: May 2006

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) specify five major glycoproteins designated as gA, gB, gC, gD, and gE. Previous studies have shown that gA and gB differ in electrophoretic mobility but not in reactivity with antisera prepared to each of these glycoproteins. Moreover, gA and gB of HSV-1 crossreact in serologic tests with the corresponding glycoproteins of HSV-2. In this paper, we report on the reactivities of gA and gB of HSV-1 and HSV-2 with 24 independently derived monoclonal antibodies reactive with these antigens. Our results show the following: (i) Electrophoretic mobilities of HSV-1 and HSV-2 glycoproteins gA and gB made in HEp-2 cells are significantly less than those made in Vero cells. (ii) All monoclonal antibodies precipitated both gA and gB made in infected HEp-2 cells. These include 12 monoclonal antibodies that neutralized virus and 12 that did not. (iii) HSV-2 glycoproteins gA and gB made in HEp-2 cells contain type-specific domains. However, monoclonal antibodies produced by one clone directed to these domains did not react with glycoproteins made in Vero cells. (iv) Lysates of infected Vero cells contain three lower molecular weight polypeptides that also reacted with monoclonal antibodies directed to glycoproteins gA and gB. These polypeptides are virus specific inasmuch as those specified by HSV-1 differ in electrophoretic mobilities from those specified by HSV-2. these polypeptides are absent in lysates of HEp-2 cells.
Morphine and opioid peptides reduce inhibitory synaptic potentials in hippocampal pyramidal cells in vitro without alteration of membrane potential.
Authors: Siggins G R , Zieglgänsberger W
Published: May 2006

We used intracellular recording in the hippocampal slice in vitro to characterize further the mechanisms behind the unusual excitatory action of opiates and opioid peptides on hippocampal pyramidal cells in vivo. No significant effect on resting membrane potential, input resistance, or action potential size in cortical area 1 (CA1) pyramidal cells was observed with morphine sulfate, beta-endorphin, [Met5]enkephalin, or [D-Ala2, D-Leu5]enkephalin at 1-50 microM. However, in all cells studied, these agents markedly reduced the size of inhibitory postsynaptic potentials generated by stimulation of the stratum radiatum or alveus. Excitatory postsynaptic potentials were also diminished in many of these cells. The effects of the opioids were antagonized by naloxone. These results are consistent with excitation of pyramidal neurons by a disinhibitory mechanism.
Differentiation of the open and closed states of the ionic channels of nicotinic acetylcholine receptors by tricyclic antidepressants.
Authors: Schofield G G , Witkop B , Warnick J E , Albuquerque E X
Published: May 2006

The actions of two clinically important dibenzocycloheptane antidepressant drugs, amitriptyline and nortriptyline, were studied on ionic channels of nicotinic acetylcholine (AcCho) receptors at the neuromuscular junction of frog skeletal muscle. Amitriptyline (5-10 microM) and nortriptyline (1-2 microM), like imipramine (5-10 microM), did not react with the nicotinic AcCho receptor but caused a voltage- and time-dependent decrease in the peak amplitude of the endplate current (epc). The time constant of epc decay, however, retained its voltage sensitivity. The voltage- and time-dependent effect of amitriptyline was nonlinear with regard to the current/voltage (I/V) relationship. Nortriptyline also had a more pronounced voltage- and time-dependent effect evidenced by a hysteresis loop in the I/V relationship of the epc was eliminated by the use of 50-msec stepwise changes of the membrane potential. The nonlinearity and hysteresis were due to a time-dependent phenomenon and did not involve previous AcCho receptor activation. The rate constant of the voltage- and time-dependent decrease in epc amplitude was sensitive to the membrane electric field and varied linearly with the membrane potential. Iontophoretically elicited epcs were much more depressed by both drugs than were spontaneous miniature epcs. There was no effect on the time constant of miniature epc decay, single-channel lifetime, or conductance. Thus (as we have pointed out in our histrionicotoxin studies) the primary site of action of these agents presumably is the activated but nonconducting species of the ionic channel of the nicotinic AcCho receptor. These agents, particularly nortriptyline, point to several different binding sites of the ionic channel and are suitable tools for the separation of the effects on peak current amplitude from its time constant of decay.
Biosynthesis of membrane-derived oligosaccharides: a periplasmic phosphoglyceroltransferase.
Authors: Goldberg D E , Rumley M K , Kennedy E P
Published: May 2006

Membrane-derived oligosaccharides (MDO) are glucose-containing constituents of the periplasmic space of Escherichia coli whose biosynthesis is closely linked to the metabolism of membrane phospholipids. A periplasmic enzyme has now been discovered that catalyzes the transfer of phosphoglycerol residues between species of MDO or to certain glucose-containing model substrates such as gentiobiose (6-O-beta-D-glucopyranosyl-D-glucose). The partially purified enzyme has an apparent molecular weight of about 56,000 in gel permeation chromatography, and has an absolute requirement for divalent cations, of which Mn2+ is most active. Although the transferase activity appears to be the physiological function of the enzyme, at low concentrations of acceptor, the enzyme (Enz) acts as a cyclase, with the liberation of cyclic 1(3),2-phosphoglycerol, suggesting the following mechanism: (formula, see text).
In vitro synthesis, glycosylation, and membrane insertion of the four subunits of Torpedo acetylcholine receptor.
Authors: Anderson D J , Blobel G
Published: May 2006

We have characterized the early biosynthetic forms of the Torpedo electroplax acetylcholine receptor by using a cell-free protein synthesizing system. We obtained primary translation products of approximately 38, 50, 49, and 60 kilodaltons for the alpha, beta, gamma, and delta polypeptides, respectively, by using immunoprecipitation with subunit-specific antisera. These chains could each be labeled by the formylated initiator [35S]Met-tRNA. On cotranslational incubation with pancreatic rough microsomes, glycosylated forms of each subunit were obtained that had molecular weights close to those of their mature authentic counterparts. Extensive trypsinization reduced the glycosylated forms of the receptor subunits to glycosylated membrane-protected fragments of approximately 35 (alpha), 37 (beta), 45 (gamma), and 44 (delta) kilodaltons. In this system, then, each receptor chain spans the membrane at least once. This in vitro-synthesized material apparently exhibited neither oligomeric assembly nor alpha-bungarotoxin binding.
Microfilament or microtubule assembly or disassembly against a force.
Authors: Hill T L
Published: May 2006

Microtubules (tubulin) or bundles of microfilaments (actin) are thought to cause movement, in some instances, by disassembly or assembly of subunits. Possible examples are the pulling of a chromosome toward a pole in mitosis (anaphase) or the deformation of a cell membrane to change the shape of a cell. This paper examines the relevant elementary bioenergetic considerations when assembly or disassembly of an aggregate occurs against a resisting force. The problem is considered, in the first section, without NTPase activity. Sickle cell hemoglobin aggregation in vivo is an example. In the second section, the tubulin GTPase and actin ATPase activities are included in the analysis.
Frequency of thermostability variants: estimation of total "rare" variant frequency in human populations.
Authors: Mohrenweiser H W , Neel J V
Published: May 2006

Eight erythrocyte enzymes were examined for thermostability in an unselected sample of 100 newborn infants. Three thermolabile variants, one each of lactate dehydrogenase, glucosephosphate isomerase, and glucose-6-phosphate dehydrogenase, were identified, none of which was detectable as a variant by standard electrophoretic techniques. All were inherited. This frequency of 3.8 heritable thermostabiliy variants per 1000 determinations is to be compared with a frequency of electrophoretically detectable variants of 1.1 per 1000 determinations, a frequency of 2.4 enzyme-deficiency variants per 1000 determinations, and a frequency of 1.1 hypo/hyperactive enzyme-activity variants per 1000 determinations in this human newborn population. The total measured frequency of individuals with rare enzyme deficiency or electrophoretic or thermostability (or both) variants at these loci is 8.3 per 1000 determinations. A similar distribution and frequency is seen when the comparison is limited to the seven loci studied by all techniques. It is clear that not all of the electrophoretic and thermostability variants present in the population are detected by the techniques used in this study. Accordingly, it is estimated that the true frequency of carriers of a rare variant for each of these enzyme-coding loci averages greater than 10/1000. Some implications of these frequencies for human disease are discussed.
Human nucleolus organizers on nonhomologous chromosomes can share the same ribosomal gene variants.
Authors: Krystal M , D'Eustachio P , Ruddle F H , Arnheim N
Published: May 2006

The distributions of three human ribosomal gene polymorphisms among individual chromosomes containing nucleolus organizers were analyzed by using mouse--human hybrid cells. Different nucleolus organizers can contain the same variant, suggesting the occurrence of genetic exchanges among ribosomal gene clusters on nonhomologous chromosomes. Such exchanges appear to occur less frequently in mice. This difference is discussed in terms of the nucleolar organization and chromosomal location of ribosomal gene clusters in humans and mice.
Isolation of enterotoxin structural gene deletion mutations in Vibrio cholerae induced by two mutagenic vibriophages.
Authors: Mekalanos J J , Moseley S L , Murphy J R , Falkow S
Published: May 2006

Phenotypically nontoxinogenic mutants of Vibrio cholerae were isolated after infection with either of two mutagenic vibriophages, VcAI and VcA2ctsl. DNA isolated from these mutants was analyzed for toxin gene sequences by the Southern blotting method with 32P-labeled probes derived from the cloned A and B subunit genes for the heat-labile enterotoxin of Escherichia coli, designated LT. Several of the mutant isolates were shown by this method to have lost all sequences hybridizing to the LT probes, indicating that these clones contain deletion mutations that removed the structural gene(s) for cholera toxin. The mutants were prototrophic and grew normally, in vitro, demonstrating that the toxin is not essential for the growth and viability of V. cholerae. Moreover, the toxin gene deletion mutants multiplied well in vivo in ligated rabbit intestine. Because of these growth properties and the stability of deletion mutations, these strains are promising candidates for testing as live oral vaccine strains for protection against cholera.
Monoclonal antibodies against human factor VIII molecular neutralize antihemophilic factor and ristocetin cofactor activities.
Authors: Sola B , Avner P , Sultan Y , Jeanneau C , Maisonneuve P
Published: May 2006

A series of monoclonal antibodies have been raised against a preparation of the factor VIII/von Willebrand factor molecule. Of the seven hybridomas showing specific activity against the factor VIII molecule in a solid-phase radioimmunoassay, three (F4.55, F4.77, and F4.264) have been shown to partially inhibit ristocetin-induced platelet aggregation and two (F4.115 and F4.415) inhibit the antihemophilic activity of the factor VIII molecule. An additional monoclonal antibody was directed against a contaminant of the factor VIII preparation and is an antifibrinogen antibody.
Mapping of transcription initiation and termination signals on Xenopus laevis ribosomal DNA.
Authors: Bakken A , Morgan G , Sollner-Webb B , Roan J , Busby S , Reeder R H
Published: May 2006

We have injected cloned derivatives of Xenopus laevis ribosomal genes into X. laevis oocyte nuclei and examined the resulting transcription complexes in the electron microscope. From this work we conclude that the promoter lies somewhere within a region between -320 nucleotides upstream and +113 nucleotides downstream from the site of transcription initiation. This assignment agrees with inferences based on sequence conservation. It further suggests that the duplicated initiation region sequences located further out in the spacer ("Bam islands") are not required for the normal high densities of RNA polymerase loading seen on ribosomal genes. Concerning termination, the cluster of four Ts that forms part of the HindIII restriction site at the 3' end of the gene appears to be part of the normal termination signal. Termination still occurs when only three Ts are present, but reduction to two Ts damages termination. Because clusters of three Ts appear at several sites within the gene, it is likely that sequences adjacent to the T cluster also are required for normal termination. In addition, we present evidence for a fail-safe termination site just upstream from the site of transcription initiation.
Structure of fibroblastic intermediate filaments: analysis of scanning transmission electron microscopy.
Authors: Steven A C , Wall J , Hainfeld J , Steinert P M
Published: May 2006

The structure of fibroblastic intermediate filaments from Chinese hamster ovary cells has been investigated by scanning transmission electron microscopy. Freshly extracted (native) filaments were compared with filaments reassembled in vitro from purified decamin. From digital micrographs of unstained specimens, direct measurements of linear mass density were performed on many individual filaments. Native filaments beyond a certain minimal length constitute a homogeneous population, averaging 38 +/- 4 kilodaltons (kDal)/nm. A minor but distinct polymorphic variant (23 +/- 4 kDal/nm) was also present as very short filaments or end-segments; these may represent breakdown products or assembly intermediates. Analysis of reassembled filaments demonstrates that the in vitro assembly reaction is--in the main--faithful, although the distribution of their mass measurements is appreciably broader than that of the native data. In addition to the predominant type at 37 +/- 4 kDal/nm and a minor component at 26 +/- 4 kDal/nm, small amounts of a third, more massive, polymorphic variant at 52 +/- 5 kDal/nm were also present. Micrographs of negatively stained specimens clearly demonstrate that the filaments are composed of bundles of protofilaments--each 2-3 nm in diameter--and also reveal an axial periodicity of about 46 nm. The implications of these findings are discussed for three classes of model previously proposed for the structure of intermediate filaments.
Nucleotide sequence of the transforming gene of simian sarcoma virus.
Authors: Devare S G , Reddy E P , Robbins K C , Andersen P R , Tronick S R , Aaronson S A
Published: May 2006

The sequence of the transforming region of simian sarcoma virus (SSV) has been determined by using molecularly cloned viral DNA. This region encompassed the 1.0-kilobase pair woolly monkey cell-derived insertion sequence, v-sis, and flanking simian sarcoma-associated viral (SSAV) sequences. A 675-nucleotide-long open reading frame commenced 19 nucleotides within the SSAV sequences to the left of the v-sis helper viral junction and terminated within v-sis itself. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame, and potential polyadenylylation sites were identified downstream both within v-sis and in helper viral sequences beyond v-sis. The recombinational event that led to the generation of SSV occurred in the middle of two functional codons, indicating that SSAV provided the regulatory elements for transcription as well as the initiation codon for translation of SSV cell-derived transforming sequences.
Extracellular matrix promotes mammary epithelial growth and differentiation in vitro.
Authors: Wicha M S , Lowrie G , Kohn E , Bagavandoss P , Mahn T
Published: May 2006

The study of growth and differentiation of mammary epithelium has been hampered by the difficulty of maintaining these functions in vitro. We describe a system for the primary culture of rat mammary epithelium on an acellular matrix derived from whole rat mammary glands that maintains growth and differentiation for months. Cultures plated on this complex substratum produce 50 times the alpha-lactalbumin of those on tissue culture dishes and 5 times the alpha-lactalbumin of those on floating collagen gels as determined by radioimmunoassay. Unlike cultures grown on floating collagen gels, which rapidly lose the ability to secrete the milk sugar lactose, mammary cells on this matrix retain this ability for over 30 days in culture. The organ specificity of this mammary extracellular material is shown by the failure of extracellular matrix prepared from rat liver to support mammary differentiation. Within a given culture dish, cells on the surface of mammary extracellular matrix are more differentiated than those on the adjacent plastic. This is demonstrated by their increased alpha-lactalbumin content as shown by indirect immunofluorescence, and by their increased ability to bind fluorescein-conjugated peanut lectin. Cells on the surface of the matrix continue to synthesize DNA as determined by [3H]thymidine incorporation and autoradiography. Even when mammary epithelial cells are plated at low density, cell division continues until the matrix is covered with a confluent layer. We propose that the limited growth, differentiation, and survival of mammary cells in previously described in vitro systems may have been due to substrate that were inadequate to support these functions.
Isolation of a cDNA clone for the murine transplantation antigen H-2Kb.
Authors: Reyes A A , Schöld M , Itakura K , Wallace R B
Published: May 2006

A library of cloned cDNAs constructed from the poly(A)+RNA of the murine thymoma cell line EL4 (b haplotype) was screened with a probe encoding a short region of the H-2Kb transplantation antigen. One of the clones isolated, pH202, contains a region that can code for a transplantation antigen with an amino acid sequence 98% homologous to that previously published for H-2Kb. Based on this high degree of homology, pH202 appears to encode the H-2Kb antigen from amino acid 66 through the carboxy terminus, including 386 nucleotides of 3'-untranslated sequence. The amino acid sequence deduced from pH202 suggests that the H-2Kb antigen is actually 2 amino acids longer than previously reported (a total of 348 residues). Four other differences in amino acid assignments are seen. Analysis of the DNA sequences of pH202 and other H-2 clones previously described in the literature suggests that alternative routes of splicing at the 3' end of the coding region are involved in the production of different transplantation antigen mRNAs.
Regression of left ventricular hypertrophy and prevention of left ventricular dysfunction by captopril in the spontaneously hypertensive rat.
Authors: Pfeffer J M , Pfeffer M A , Mirsky I , Braunwald E
Published: May 2006

To determine whether chronic antihypertensive therapy prevents the progression of cardiac hypertrophy and the deterioration in cardiac performance observed in spontaneously hypertensive rats (SHR) with long-term hypertension, 14-month-old female SHR and normotensive American Wistar rats (NWR) were treated for 10 months with an inhibitor of angiotensin I-converting enzyme, captopril (2 g/liter of drinking water). Captopril reduced the marked left ventricular hypertrophy of 24-month-old SHR (untreated, 4.37 +/- 0.2 mg/g of body weight; treated, 3.01 +/- 0.1 mg/g; P less than 0.02) to levels observed in 6-month-old SHR. Treatment prevented the reductions in baseline and maximal aortic blood flows that occurred in SHR between ages 12 and 24 months yet had no effect on the blood flows of NWR. The diminished maximal stroke volume of untreated SHR was ejected from a significantly increased left ventricular end-diastolic volume, so that the ejection-fraction index was markedly reduced (24-month-old untreated NWR, 84 +/- 3%; untreated SHR, 56 +/- 5%; P less than 0.001). Therapy restores this index in SHR to normal (77 +/- 4%). The relationship between ejection-fraction index, and afterload was also normal in treated SHR. Thus, chronic therapy with captopril produced a marked regression of cardiac hypertrophy and prevented the deterioration of cardiac performance in SHR with long-standing hypertension.
Trimeric intermediate in the in vivo folding and subunit assembly of the tail spike endorhamnosidase of bacteriophage P22.
Authors: Goldenberg D , King J
Published: May 2006

Newly synthesized tail spike polypeptide chains mature from trypsin- and NaDodSO4-sensitive unfolded chains to trypsin- and NaDodSO4-resistant native trimers with a t1/2 of 5 min at 30 degrees C. A metastable intermediate in subunit folding and assembly was trapped by chilling and isolated by electrophoresis through nondenaturing gels in the cold. A fraction of the intermediate could be matured into native trimers in vitro by incubating at physiological temperature. Mixing experiments with electrophoretically distinct mutant proteins showed that the precursor that matured in vitro represented three tail spike polypeptide chains already associated with each other but not fully folded. Identification of this intermediate reveals that the processes of polypeptide chain folding and subunit assembly are coupled in this large structural protein.
Cycloartenol-derived sterol biosynthesis in the Peronosporales
Authors: Warner Steven A. , Eierman David F. , Sovocool G. Wayne , Domnas Aristotle J.
Published: May 2006

Selected species of the order Peronosporales, which are unable to epoxidize squalene and thus synthesize sterols, are able to metabolize exogenous cycloartenol to lanosterol and in some organisms to fucosterol, ergosterol, and cholesterol. Lanosterol was less effectively utilized but some ergosterol and cholesterol were yielded. Fucosterol was very efficiently metabolized by most species to ergosterol, Δ
7
-ergostenol, Δ
5
-ergostenol, cholestanol, and cholesterol. Several unknown sterols were observed in most trials. These data suggest a vestigial sterol synthetic pathway derived from cycloartenol, followed by possible isomerization to lanosterol and then to other sterols.
Immunoregulation in senescence: increased inducibility of antigen-specific suppressor T cells and loss of cell sensitivity to immunosuppression in aging mice.
Authors: Doria G , Mancini C , Adorini L
Published: May 2006

Azobenzenearsonate (ABA)-specific T cell-mediated suppression has been studied in aging mice. ABA-specific suppressor T cells were induced in young and old mice by injection of ABA conjugated to syngeneic spleen cells (ABA-SC). These suppressor cells were tested for their ability to suppress the in vitro anti-trinitrophenyl (TNP) antibody response of lymph node cells obtained from ABA-keyhole limpet hemocyanin (KLH)-primed young or old mice and cultured with TNP-ABA-KLH. Suppressor T cells were found to be more easily induced in old than in young mice but to suppress less efficiently the antibody response of cells from old than from young mice. The increased inducibility of antigen-specific suppressor T cells in old mice is compatible with the age-dependent decline of immune responsiveness to exogenous antigens. The loss of cell sensitivity to antigen-specific immunosuppression as well as the lack of evidence for increased nonspecific suppression in old mice is consistent with the age-related increase in autoimmune disorders. These findings provide a unifying explanation for the most relevant immunological phenomena of senescence.
Cloning of human immunoglobulin epsilon chain genes: evidence for multiple C epsilon genes.
Authors: Nishida Y , Miki T , Hisajima H , Honjo T
Published: May 2006

An active human epsilon chain gene was cloned from a phage library containing partial EcoRI digests of IgE-producing myeloma DNA, using the human JH (joining) gene fragment as a probe. The epsilon chain gene clone was identified by partial nucleotide sequence determination. The germ-line constant region gene of the epsilon chain (C epsilon gene) was cloned from a human fetal liver DNA library, using the cloned epsilon chain gene as a probe. Comparative studies on the human and mouse germ-line epsilon chain genes revealed that the switch (S) sequence is more conserved than the coding sequence. Restriction endonuclease BamHI digestion of human DNA produced three C epsilon fragments of 3.0, 6.5, and 9.2 kilobases, which were named C epsilon 1, C epsilon 2, and C epsilon 3 genes, respectively. We found the three C epsilon gene fragments in all of the human DNA preparations from eleven individuals. The C epsilon gene expressed in the myeloma was identified as the C epsilon 1 gene. Because the C epsilon 2 gene is deleted from the myeloma DNA, the order of the C epsilon genes is likely to be 5'-C epsilon 2-C epsilon 1-C epsilon 3-3', assuming that all the C epsilon genes are on chromosome 14. The germ-line C epsilon 3 gene was also cloned from the myeloma DNA. Characterization of the C epsilon 3 gene revealed that it does not have the S region, suggesting that it might be a pseudogene.
Vascular smooth muscle cell growth kinetics in vivo in aged rats.
Authors: Stemerman M B , Weinstein R , Rowe J W , Maciag T , Fuhro R , Gardner R
Published: May 2006

Age is a risk factor in the development of atherosclerosis. In this study we investigated the hypothesis that proliferation of vascular smooth muscle cells (SMCs), an integral part of atherosclerotic plaque formation, changes with age. SMC growth kinetics of old rats (21-24 months) were compared to those of young adult rats (3-4 months). Rat aortas were denuded of their endothelium and the animals were killed after [3H]thymidine and Evans blue injections at 0-28 days after denudation. Incorporation of [3H]thymidine into SMC peaked in the young animals by day 2, whereas the older animals responded to endothelial removal with greater incorporation at day 2 and a more sustained rate of incorporation peaking at day 4. The [3H]thymidine incorporation curves decreased sharply from their peaks at 2 and 4 days, respectively, and paralleled each other after day 7. [3H]Thymidine uptake reflected the subsequent SMC intimal growth as measured morphometrically, with old animals showing greater numbers of intimal SMC than did the younger animals. The difference in response of SMC to injury with age suggests that aging produces a change in the vascular SMC that enhances proliferation. This change in response implies that the more pronounced atherosclerotic plaque growth seen with aging may be a result of an age-related increase in response to injury rather than merely the accumulation of time-related intimal change.
Centruroides toxin, a selective blocker of surface Na+ channels in skeletal muscle: voltage-clamp analysis and biochemical characterization of the receptor.
Authors: Jaimovich E , Ildefonse M , Barhanin J , Rougier O , Lazdunski M
Published: May 2006

This paper describes the effects of a toxin from the scorpion Centruroides suffusus suffusus on frog skeletal muscle. The main findings are the following, (i) Centruroides toxin (CssII) blocks the early phase of the inward sodium current in the muscle that arises from influx via Na+ channels in the surface membrane, but it does not affect the late phase of the inward current that represents flux through Na+ channels in the T-tubule membranes, (ii) CssII, in marked contrast to tetrodotoxin, does not affect contraction of the muscle, (iii) Measurements of the binding of 125I-labeled CssII to a partially purified membrane preparation from the muscle indicate that the Kd of the CssII--receptor complex is approximately 0.4 nM. The half-life for the dissociation of this complex is 3 min at 22 degrees C and 16 min at 2 degrees C. Binding of the radiolabeled toxin varies markedly with pH and becomes insignificant at pH greater than 8.5. Proteolytic digestion of the membrane preparation decreases its ability to bind CssII, suggesting that the receptor is a protein. (iv) The number of binding sites for a radiolabeled derivative of tetrodotoxin on the membrane preparation was similar to that for CssII. However, neither tetrodotoxin nor any of seven other neurotoxins and some local anesthetics that alter the functioning of the Na+ channel have any effect on the binding of CssII to the muscle membrane. These results therefore indicate that CssII belongs to a different class of neurotoxins that has a different receptor on the Na+ channel.
Molecular structure of the octamer d(G-G-C-C-G-G-C-C): modified A-DNA.
Authors: Wang A H , Fujii S , van Boom J H , Rich A
Published: May 2006

The deoxynucleotide fragment d(GpGpCpCpGpGpCpC) was synthesized and crystallized, and its three-dimensional structure was determined by x-ray diffraction techniques to a resolution of 2.25 A. The molecule forms a right-handed double helix in which the two base pairs at either end of the molecule are in the conventional A-DNA conformation, while the central four base pairs have a modified form in which alternate residues have sugar conformations that are closer to those in B-DNA than in A-DNA. The molecules have an intermolecular contact in which the planar terminal guanine . cytosine base pair lies on the flat minor groove surface of the A-DNA helix.
Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.
Authors: Dandekar A M , Robinson E A , Appella E , Qasba P K
Published: May 2006

Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert of p-Wp 52 and shows that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content of half-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.
Structure of a B-DNA dodecamer at 16 K.
Authors: Drew H R , Samson S , Dickerson R E
Published: May 2006

The crystal structure of the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, previously solved and refined at room temperature (290 K), has been analyzed at 16 K (-257 degrees C). The end-to-end winding of the helix does not vary with temperature but remains constant at 10.1 base pairs per turn. Negatively charged phosphate groups throughout the structure do move closer together on cooling, however, probably because of increase in the dielectric constant of water as the temperature is lowered. This has the two-fold effect of reducing the spacing between neighboring double helices from 24.0 to 22.9 A and of narrowing the helix grooves within any isolated molecule. Overall lattice displacements as deduced from crystallographic temperature factors are very much decreased in the 16 K structure, yet displacements at phosphates continue to exceed those of deoxyribose sugars by B = 9 A2 and those of base pairs by B = 22 A2, even at this very low temperature at which practically all thermal motion has been eliminated. These differences, formerly interpreted as evidence for thermal vibration, must now be attributed to static disorder.
Fluvial processes and local lithology controlling abundance, structure, and composition of mussel beds
Authors: Vannote Robin L. , Minshall G. Wayne
Published: May 2006

In the Salmon River Canyon, Idaho, the fresh-water pearl mussel,
Margaritifera falcata
, attains maximum density and age in river reaches where large block-boulders structurally stabilize cobbles and interstitial gravels. We hypothesize that block-boulders prevent significant bed scour during major floods, and these boulder-sheltered mussel beds, although rare, may be critical for population recruitment elsewhere within the river, especially after periodic flood scour of less protected mussel habitat. Mussel shells in Indian middens adjacent to these boulder-stabilized areas suggest that prehistoric tribes selectively exploited the high-density old-aged mussel beds. Locally, canyon reaches are aggrading with sand and gravel, and
M. falcata
is being replaced by
Gonidea angulata
.
Oxidative inactivation of leukotriene C
4
by stimulated human polymorphonuclear leukocytes
Authors: Lee Chong W. , Lewis Robert A. , Corey E. J. , Barton Alan , Oh Hunseung , Tauber Alfred I. , Austen K. Frank
Published: May 2006

Leukotriene C
4
(LTC
4
) was metabolized by human polymorphonuclear leukocytes (PMNs) stimulated with phorbol myristate acetate (PMA) into three sets of products. These products differed in mobility on reverse-phase high-performance liquid chromatography (RP HPLC) from LTC
4
and also from leukotriene D
4
(LTD
4
) and leukotriene E
4
(LTE
4
), the sequential products of peptide cleavage of LTC
4
. Products I, II, and III were eluted as doublets with an average retention time for each doublet of 7.5 ± 0.3, 10.5 ± 0.6, and 16.3 ± 1.1 min (mean ± SD), respectively, as compared with 13.8 min for LTC
4
. Doublet I material was biologically inactive and showed <5% of the immunoreactivity of LTC
4
, doublet II material had 1% of the spasmogenic activity of LTC
4
on the guinea pig ileum and was equally immunoreactive, and doublet III material was neither biologically active nor immunoreactive. When [14,15-
3
H]LTC
4
and [
35
S]LTC
4
were metabolized, all three doublet products retained the
3
H label, whereas only the doublet I and doublet II products retained the
35
S label. The UV absorbance spectra of the three sets of metabolites were as follows: doublet I, maximum at 280 nm with shoulders at about 270 and 290 nm; doublet II, maximum at 284.5 nm with shoulders at about 275 and 295 nm; and doublet III, maximum at 269 nm with shoulders at about 259 and 279 nm. The metabolism of LTC
4
to the three classes of functionally inactive products by stimulated PMNs was completely blocked by catalase and azide, indicating a requirement for H
2
O
2
and myeloperoxidase. When hypochlorous acid (HOCl)—considered to be a natural product of the interaction of myeloperoxidase, H
2
O
2
, and chloride ion—was formed chemically and allowed to react with LTC
4
, the resulting products were indistinguishable by UV and HPLC analyses from the doublet II and doublet III metabolites of LTC
4
. The doublet II products were identified as the two diastereoisomeric sulfoxides of LTC
4
by comparison with synthetic reference compounds. The doublet III products were shown to be identical with synthetic samples of (5
S
, 12
S
)- and (5
S
, 12
R
)-6-
trans
-LTB
4
. The formation of two diastereoisomeric LTC
4
sulfoxides and 6-
trans
-LTB
4
can be explained in terms of an
S
-chlorosulfonium ion as the initial reactive intermediate, which subsequently undergoes conversion to product II by hydrolysis and product III by carbocation formation.
Expression of a chemically synthesized human alpha 1 interferon gene.
Authors: De Maeyer E , Skup D , Prasad K S , De Maeyer-Guignard J , Williams B , Meacock P , Sharpe G , Pioli D , Hennam J , Schuch W , Atherton K
Published: May 2006

Cells of Escherichia coli containing a chemically synthesized human alpha 1 interferon (IFN-alpha 1) gene, under control of the lac promoter, make a product with biological properties indistinguishable from those of the natural IFN-alpha 1 [antiviral activity, acid stability, species crossreactivity, inactivation by antisera directed against leukocyte or Namalwa cell interferon, and stimulation of (2'-5')oligoadenylate synthetase activity]. Similar levels of IFN synthesis were obtained when the expression unit (lac promoter plus synthetic IFN-alpha 1 gene) was transplanted into the obligate methylotroph Methylophilus methylotrophus.
Dodecahedrane—The chemical transliteration of Plato's universe (A Review)
Authors: Paquette Leo A.
Published: May 2006

The development of chemical interest in three of Plato's five convex polyhedra is described from an historical perspective. The successful synthesis of 1,16-dimethyldodecahedrane and its structural characteristics are outlined. Finally, an account of recent work leading to the still more aesthetically appealing and ultrasymmetric parent dodecahedrane is given.
Treatment of disordered and ordered systems of polymer chains by lattice methods
Authors: Flory Paul J.
Published: May 2006

Classical lattice theories of systems of long-chain molecules provide estimates of the number Z of
random
configurations to the exclusion of
ordered
ones. The decrease of Z thus estimated to values [unk]1 with decrease in chain flexibility at high densities is genuine, but it does not take account of eligible ordered configurations; the latter are not a subset of the configurations whose numbers are estimated by classical lattice methods. Failure to recognize this fact and the fundamental distinction between disordered and ordered states has engendered misinterpretations and has cast doubt on the validity of lattice-statistical methods. In a system at equilibrium, the decline of Z (disordered) with decrease in chain flexibility must be arrested by a first order transition to an ordered state. The inference that approach of Z (disordered) to values <1 presages a thermodynamic transition of second order is tenable only if the array of ordered configurations, not comprehended by theories in which the mean field of unoccupied lattice sites is random, can be ignored.
Distribution of ribosomal gene length variants among mouse chromosomes.
Authors: Arnheim N , Treco D , Taylor B , Eicher E M
Published: May 2006

The ribosomal genes (rDNA) in mouse inbred strains have a multichromosomal distribution. Using a structural feature of rDNA [variable length rDNA segment (VrDNA)] that shows length polymorphism within and among inbred strains, we studied the chromosomal distribution of the variant ribosomal gene type through genetic analysis. Our results show that five of the length variant classes can be divided into three discrete linkage groups. The variants present on a particular chromosome pair appear to be unique to that pair and absent from nonhomologous chromosomes. The chromosomal location of particular variants appears to be the same in two unrelated inbred strains suggesting that the observed linkage patterns predate the origin of inbred mice. The nonrandom chromosomal distribution of these rDNA classes suggests that only a limited degree of genetic exchange occurs among nucleolus organizer regions on nonhomologous chromosomes. We have localized one particular VrDNA linkage group to chromosome 12. These and other restriction fragment polymorphisms can be used in the construction of detailed mouse linkage maps.
Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.
Authors: Robson K J , Chandra T , MacGillivray R T , Woo S L
Published: May 2006

The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans.