Sugar conformation of a stereospecific 2′-R or 2′-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants

The sugar conformation of a DNA decamer was studied with proton-proton ³J coupling constants. Two samples, one comprising stereospecifically labeled 2-R-²H for all residues and the other 2-S-²H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980–6986; Nucleosides Nucleotides (1995) 14, 333–336], the deuterium labeling being highly stereospecific 99% for all 2-²H, 98% for 2-²H of A, C, and T, and 93% for 2-²H of G). The ³J values of all H1-H2 and H1-H2 pairs, and several H2-H3 and H2-H3 pairs were determined by line fitting of 1D spectra with 0.1–0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3-exo and C2-endo with _m values of 26° to 44°, except for the second and 3 terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from J_H1H2 and J_H1H2 values. For C10, the N–S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1-exo conformation with 27° distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.     

Activation of immunoglobulin control elements in trasgenic mice

To assess the role interleukins and mitogens play in regulating immunoglobulin (Ig) gene expression via the Ig enhancer and promoter, transgenic mice carrying two different Ig gene regulatory regions were generated. One, EkCAT, contains the Ig heavy chain enhancer (E) and the light chain promoter driving the chloramphenicol acetyltransferase (CAT) gene. In the other, EkCAT, CAT is under the control of the promoter alone. E and relative activity were assessed by CAT assay. In EkCAT mice, low CAT expression was consistently found in spleen, bone marrow, mesenteric lymph node, and thymus but not in brain, lung, or kidney. In EkCAT mice, CAT expression was detectable just above background in lymphoid tissues, suggesting a basic level of tissue specificity in the absence of the enhancer. Whole spleen cell cultures prepared from the mice were treated with lymphokines and mitogens. Lipopolysaccharide (LPS), concanavilin A (Con A), interleukin 6 (IL-6), and interferon- (IFN-) increased CAT expression to varying extents in cells derived from EkCAT mice but not in spleen cells prepared from EkCAT mice. Thus, the presence of E, in addition to the promoter, is essential for the stimulation of CAT expression mediated by these factors. B cells from EkCAT mice were separated by density into populations of small and large cells. In untreated small B cells, no CAT expression was detected and only addition of LPS resulted in an increase in CAT expression. In large B cells, CAT was expressed at a low level without addition of exogenous factors. Incubation with LPS, IL-6, Con A and IFN- caused CAT expression to increase several-fold. This transgenic system provides a means to identify exogenous factors that activate Ig enhancers and promoters.This work has been submitted in partial fulfillment of the requirements for the doctoral degree from the George Washington University.     

The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

Cytokinesis in the green alga Eremosphaera viridis: Plasmalemma formation from “open membranes”

Summary Cytokinesis in the unicellular chlorococcalean alga Eremosphaera viridis de Bary has been investigated by electron microscopy of thin sections. The new plasmalemmata of the daughter cells in this organism form centrifugally within a phycoplast. Unlike other cell division systems each new plasmalemma is formed, not by the fusion of vesicles, but rather by the fusion of open membranes which are characteristically heavily stained. Measurements of these open membranes reveal that they are 11 nm thick with a central 4,5 nm unstained portion. The possible origin of these open membranes as burst-open vesicles has been suggested from the presence of intensely straining vesicles in the vicinity of the cell equator. Calculations of vesicle and open membrane surface areas support this contention.     

DNA Replication and Cell Cycle Progression Regulatedby Long Range Interaction between Protein Complexes bound to DNA

A nonstationary interaction that controlsDNA replication and the cell cycle isderived from many-body physics in achemically open T cell. The model predictsa long range force F() =– (/2) (1 – )(2 – )between thepre-replication complexes (pre-RCs) boundby the origins in DNA, = /N being the relativedisplacement of pre-RCs, the number of pre-RCs, Nthe number of replicons to be replicated,and the compressibilitymodulus in the lattice of pre-RCs whichbehaves dynamically like an elasticallybraced string. Initiation of DNAreplication is induced at the threshold = N by a switch ofsign of F'(), fromattraction (–) and assembly in the G ₁ phase (0<<N), to repulsion (+) and partialdisassembly in the S phase (N< < 2N), withrelease of licensing factors from pre-RCs,thus explaining prevention ofre-replication. Replication is terminatedby a switch of sign of force at = 2N, from repulsion inS phase back to attraction in G ₂, when all primed replicons havebeen duplicated once. F(0) = 0corresponds to a resting cell in theabsence of driving force at = 0. The model thus ensures that the DNAcontent in G ₂ cells is exactlytwice that of G ₁ cells. The switch of interaction at the R-point, at which N pre-RCs have been assembled, starts the release of Rb protein thus also explaining the shift in the Rb phosphorylation from mitogen-dependent cyclinD to mitogen-independent cyclin E.Shape,slope and scale of the response curvesderived agree well with experimental datafrom dividing T cells and polymerising MTs,the variable length of which is due to anonlinear dependence of the growthamplitude on the initial concentrations oftubulin dimers and guanosine-tri-phosphate(GTP). The model also explains the dynamic instabilityin growing MTs.     

The effect of intracapillary media feed protocols on hollow fiber cell culture

Summary Two intracapillary (IC) media feed protocols termed media rich and media lean were examined in an effort to understand the effect of this variable on hollow fiber cell cultures. The media rich protocol emphasized a high volume IC media per day (5 liters) containing no serum and a normal amount of extracapillary (EC) media serum (10% v/v). Alternatively, the media lean protocol used up to 1.0 liter of IC media per day containing 5% v/v serum and increased EC media serum (20% v/v). Both protocols produced substantial amounts of antibody in 25 days using HFN7.1 hybridoma cells (ATCC CRL 1606), however the media rich protocol produced twice as much antibody as the media lean protocol. The metabolism of the cells was dramatically different as measured by glucose uptake rate (GUR) with media lean cells having a six-fold lower GUR. Our results indicate that the media rich protocol is useful for producing larger amounts of antibody in a short time frame. The media lean protocol may be considered when the production costs of antibody, particularly media and serum, is the overriding concern.     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

Moral commitment without objectivity or illusion: Comments on Ruse and Woolcock

Peter Woolcock, in Ruse's Darwinian Meta-Ethics: A Critique, argues that the subjectivist (nonobjectivist) Darwinian metaethics proposed by Michael Ruse (in Taking Darwin Seriously) cannot work, because the illusion of objectivity that Ruse claims is essential to morality breaks down when it is recognized as illusion, and there then remain no good reasons for acknowledging or following moral obligations. Woolcock, however, is mistaken in supposing that moral behaviour requires rational motivation. Ruse's Darwinian metaethical analysis shows why such objective support for morality is neither plausible nor necessary; and when that is recognized, it can also be seen that Ruse's proposed illusion of moral objectivity is superfluous.     

Beitr?ge zum Verhalten des Krabbentauchers(Plautus alle alle)

Summary The behavior of the Little Auk(Plautus alle alle) has been studied in June/ July 1968 in West-Spitzbergen.In the shelf-area we counted 32–75 Little Auks per km² flying or swimming in the sea. Flight behavior is described. On small rocky ledges before the entrances of the breeding caves, singing, display, sleeping and preening take place. Mass singing (Massengesang), display flight and parade (Imponierflug und Imponiergehen) and billing (Schnäbeln) are described. The calls of the Little Auk consist of 5 variable elements.     

HbF-Lesvos: An HbF variant due to a novel Gγ mutation (:Gγ 75 ATA→ACA) detected in a Greek family

A TC substitution at position 402 of the G globin gene results in an isoleucine to threonine substitution at codon 75 of the G globin chain and the formation of HbF-Lesvos [₂G₂ 75 (E19) IleThr].     

Assignment of human ferritin genes to chromosomes 11 and 19q13.3→19qter

Summary Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.319qter produced human L type ferritin. This confirms the previous assignment of the ferritin gene to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human H type ferritin suggesting that the gene for the H subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either H or L type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.     

Extent and high frequency of a short conversion between the human Aγ and Gγ fetal globin genes

Summary Southern blotting and DNA sequencing after polymerase chain reaction (PCR) amplification provide evidence for the frequent occurrence (in 7 out of 24 chromosomes) of a short conversion GA in the 3 end of the human fetal A globin gene. This short conversion is characterized by the presence, 3 nucleotides downstream from the termination codon of the A gene, of the TCAC sequence that is normally present at the equivalent position at the 3 end of the G gene; it is therefore identical to a conversion already described. Interestingly, we have found that this conversion is associated with the presence of theHindIII polymorphic restriction site in the A IVS2, occuppying an equivalent position in both the G and A genes. Our observations strengthen the hypothesis that the presence of the HindIII polymorphic restriction site in A IVS2 and the presence of the sequence TCAC at the 3 end of the A gene might be the result of a single conversion event.     

A small variance in the antigenicity but not function of recombinant β-lactoglobulin purified from the culture supernatant of transformed yeast cells

We purified recombinant bovine -lactoglobulin (r-LG) from the culture supernatant of transformed yeast and investigated whether r-LG maintained the functional ability and antigenicity of native -LG. Immunostaining following gel electrophoresis and reversed-phase high-performance liquid chromatography confirmed that r-LG was purified homogeneously. r-LG showed almost the same retinol-binding ability as native -LG purified from bovine milk. However, affinities of two anti--LG monoclonal antibodies (mAbs) to r-LG were different from those to native -LG, although three other mAbs bound these two proteins equally. Since our panel of five mAbs has been previously shown to be able to detect structural changes occurring in -LG, this variance in antigenicity can be attributed to conformational differences between r-LG and native -LG. Then, we studied which step in the production and purification procedure was responsible for altering the antigenicity of r-LG. Bovine milk native -LG was added to several steps in this procedure and purified in the same manner as r-LG. The results suggested that incubation in the yeast culture had adverse effects on maintaining the antigenicity of this recombinant protein. We conclude from these results that even if no difference between the native and recombinant proteins can be detected by functional analysis, some subtle conformational change which can be distinguished by mAbs may be incorporated into the recombinant protein during its production and ultimately cause a different immune reaction in vivo.Abbreviations -LG, -lactoglobulin; r-LG, recombinant -LG; PBS, phosphate-buffered saline; PBS-Tween, PBS containing 0.05% Tween 20; ELISA, enzyme-linked immunosorbent assay.     

Evolution of the interleukin-1 gene family in mammals

The phylogeny of interleukin-1 family genes shows that human interleukin-1 (IL-1) is more closely related to IL-1 of the bovine than to IL-1 of the mouse, whereas human interleukin-1 (IL-1) is more closely related to IL-1 of the mouse than to IL-1 of the bovine. The IL-1 receptor antagonist (IL-1) shows homology to the C-terminal region of both IL-1 and IL-1. In the C-terminal region, the IL-1 genes of human and mouse have diverged more from each other at nonsynonymous sites than have either IL-1 or IL-1; because the same pattern is not seen at synonymous sites, it must be due not to a difference in mutation rate but rather to a greater degree of functional constraint on this region in the IL-1 and IL-1 proteins than in the IL-1 protein. But synonymous sites in IL-1 of mouse have evolved more rapidly than in IL-1 of human, indicating a higher rate of mutation in the former gene. In the N-terminal region of the protein, nonsynonymous sites have evolved at similar rates in IL-1 and IL-1. The first exon of the IL-1 gene, which encodes the leader peptide, shows evidence of homology with the first exon of IL-1, which is not translated. Thus, it seems likely that IL-1 evolved by duplication of an IL-1 gene and loss of expression of exons 2–4. Correspondence to: A.L. Hughes     

Electrophoretic variation between class II molecules expressed on HLA-DRw8 homozygous typing cells reveals multiple distinct haplotypes

Two-dimensional (2D) gel electrophoresis of immunoprecipitated HLA-DR antigens from eight homozygous typing cells (HTC) expressing the HLA-DRw8 specificity revealed a clustering of polymorphic chain patterns into distinct electrophoretic variants. The variant patterns correlate with three discrete HLA-D clusters that are defined in the mixed leukocyte culture reaction (MLR) using DRw8-positive HTC. These HLA-D clusters have been provisionally designated Dw8.1, detected primarily in Caucasoids, Dw8.2, detected primarily in American Indians, and Dw8.3, detected predominantly in Orientals. All three HLA-Dw8.1 cell lines express a single DR-locus product as defined by immunoprecipitation with a DR-specific monoclonal antibody, P4.1. This DR chain is identical among the Dw8.1 cell lines and different from the DR chains of the Dw8.2 and Dw8.3 cell lines. Two separate Dw8.2 HTC express a shared DR chain that is slightly more basic than the 8.1 DR molecule; interestingly, one of these lines also expresses an additional DR-like chain not found in the other cells. Thus, the two lines defining the Dw8.2 cluster share one distinct class 11 molecule, but differ in another and therefore are not biochemically HLA-identical. Cells from the Dw8.3 cluster are likewise distinct from all other Dw8 clusters. One additional DRw8-positive HTC has been analyzed and found to be distinct from the Dw8.1, 8.2 and 8.3 clusters by both MLR and 2D gels. lmmunoprecipitates using monoclonal antibody 1B5 [anti-DR and anti-DQ(DS)] identify additional polymorphic class II variants among the cell lines tested. These data indicate that HLA-DRw8 is a public serologic specificity present on class II molecules expressed on multiple distinct haplotypes. These haplotypes differ from each other in expression of polymorphic class II molecules encoded by at least two HLA loci. They also differ in HLA-D, even though they all type as HLA-DRw8 homozygous. In Dw8.2, variation in expressed chains is not reflected in variation in HLA-D, indicating that MLR, as well as serologic typing, does not detect the full degree of allelic polymorphism within HLA.     

Quantitation of RNA polymerase subunits in Escherichia coli during exponential growth and after bacteriophage T4 infection

Summary A method was developed to measure the amounts of RNA polymerase subunits, , , and in crude extracts of Escherichia coli. The proteins were labelled by growing the cells in ³⁵S-sulphate containing media. For measuring and , the cell lysate was electrophoresed on 6% polyacrylamide gels containing SDS and the and bands cut out and counted. For measuring and , the cell lysate was co-electrophoresed with dansylated RNA polymerase on 8% polyacrylamide gels containing SDS. The fluorescent bands were cut out, the proteins eluted, and the and subunits further purified on polyacrylamide gels containing 8 molar urea.The results are: (1) is the subunit of the core RNA polymerase which is present in limiting amount. (2) The core enzyme, as measured by , constitutes a constant fraction of total cellular protein (0.9%), independent of the bacterial growth rate. (3) The subunit is made in excess and is probably regulated independently. (4) The subunit is present in 0.3–0.4 times the amount of the core enzyme. (5) All four subunits are fully conserved after bacteriophage T4 infection.     

RNA polymerase subunit biosynthesis in Bacillus subtilis

Summary The relative rates of RNA polymerase biosynthesis in Bacillus subtilis has been examined under steady-state growth conditions. The synthesis of RNA polymerase subunits (, , , ) has been followed by subunit fractionation of immunoprecipitated [³H]-labelled samples on SDS-polyacrylamide gels. The stoichiometries of ::: subunits have been determined from cultures pulse-labelled during steady-state growth. The results suggest that an unassembled pool of the -subunit exists from which the holoenzyme is formed.Upon shift-up from acetate to glycerol containing medium, a rapid rise in the differential rate of core enzyme synthesis was observed, while the rate of synthesis of the -subunit was not stimulated. During shift-down, a concomitant reduction in the rate of synthesis of all subunits occurred for the first 20 min after the shift; thereafter, a rate of synthesis characteristic of the new growth rate was established.As cultures enter sporulation, an immediate reduction in the rate of -subunit synthesis was demonstrated.     

Die Zeteraktivit?t der Amsel (Turdus merula)

Zusammenfassung Das Zetern der Amseln wurde von 1969 bis 1972 in einem sehr regelmäßig besetzten Revier automatisch registriert. Sowohl das Zetern sämtlicher registrierter Amseln, als auch das Zetern bestimmter Individuen gehorchen den meisten Aschoff'schen Regeln. Zwischen Individuen sind die Zeteranfänge äußerst unterschiedlich, die Zeterenden dagegen nicht. Die individuellen Zeteranfänge reichen in der Vergleichszeit von ca. 50 min bis 20 min vor Sonnenaufgang (Mittelwerte für 1/3 Monat).

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The alarm-call activity of the Blackbird

Summary In a very regulary occupied district the alarm-call (Zetern) of blackbirds was automatically recorded from 1969 till 1972. The Zetern of all recorded blackbirds as well as the Zetern of single individuals obey to most of Aschoff's rules. Among the individuals the beginnings of the Zetern are extremly different, but not the endings of the Zetern.The interindividual beginnings of Zetern in the comparable time reach from about 50 min. to about 20 min. before sunrise (mean values for 1/3 of a month).

     

Glycosphingolipids of leukocytes are unbranched at the galactopyranosyl residue and contain fucosylα-1-3-N-acetylglucosamine structures

Glycolipids of peripheral leukocytes which had been used for the production of interferon were separated into oligoglycosylceramides, polyglycosylceramides and polyglycosylpeptides (erythroglycan). Neutral oligoglycosylceramides comprised glucosylceramide, galactosylceramide, lactosylceramide, lactotriaosylceramide, globotriaosylceramide andneolactotetraosylceramide. Globotetraosylceramide was not detected. Glycolipids which were more complex thanneolactotetraosylceramide belonged exclusively to theneolacto series of compounds and were essentially unbranched at galactopyranosyl residues. The polyglycosylceramide fraction contained a glycolipid with a probable structure Gal1-4(Fuc1-3) GlcNAc1-3Gal1-4GlcNAc1-3 Gal1-4GlcNAc1-3Gal1-4Glc1-1ceramide. Polyglycosylpeptides were found only in trace amounts and were also unbranched at galactopyranosyl residues. All glycoconjugates studies did not contain significant amounts of carbohydrate structures derived from ABH immunodominant groups.Nomenclature Gal1-4Gal1-4GlcCer Lactotrioasylcermide (LcOse₃Cer) - Gal1-4Gal1-4GlcCer globotriaosylceramide, (GbOse₄Cer) - GalNAc1-3Gal1-4 Gal1-4GlcCer globoside (globotetraosylceramide, GbOse₄Cer) - Gal1-4GlcNAc1-3Gal1-4GlcCer paragloboside (lacto-N-neo tetraosylceramide,nLcOse₄Cer)