Conformation of malformin A: a proton magnetic resonance and a circular dichroism study

Malformin A is a cyclic pentapeptide with an intramolecular disulfide bridge. The conformation in solution of this molecule has been studied by NMR and CD. The 270 MHz Proton spectrum in dimethyl sulfoxide is well resolved and the peaks corresponding to the five residues have been assigned. From the temperature dependence of chemical shifts of the peptide protons and from the exchange rate of these protons, it is concluded that the NH proton of one Cys is shielded from the solvent. This observation and H? N? _αC? H angles, estimated from the corresponding coupling constants, a proposed conformation of the peptide backbone. From the H? _βC? _αC? H coupling constants, a P chirality for the disulfide bridge is proposed. Such a conformation is confirmed by the circular dichroism spectrum which shows a negative band at λ > 250 nm. It is concluded that the conformation of malformin A is rigid and that the disulfide bridge is exposed to interact with biological receptors.     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Targeting IL-15 receptor-bearing cells with an antagonist mutant IL-15/Fc protein prevents disease development and progression in murine collagen-induced arthritis

It has been suggested that the inflammatory cytokine IL-15 plays an important role in the development of several autoimmune diseases, including rheumatoid arthritis. We have generated a unique lytic and antagonistic IL-15 mutant/Fcgamma2a fusion protein (CRB-15) that targets the IL-15R. In the present study we examined the effects of targeting the IL-15R on the prevention and treatment of collagen-induced arthritis (CIA) in mice and probed the possible mechanisms of action of this IL-15 mutant/Fcgamma2a protein. Upon immunization with type II collagen, DBA/1 mice develop severe articular inflammation and destruction. Treatment of DBA/1 mice with a brief course of CRB-15 at the time of type II collagen challenge markedly inhibited the incidence and severity of arthritis. Moreover, in animals with ongoing established arthritis, treatment with CRB-15 effectively blocked disease progression compared with that in control-treated animals. The therapeutic effect of CRB-15 on either disease development or disease progression is remarkably stable, because withdrawal of treatment did not lead to disease relapse. A detailed analysis revealed that treatment with CRB-15 decreased synovitis in the joints; reduced bone erosion and cartilage destruction; reduced in situ production of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-17; and decreased the responder frequency of autoreactive T cells. Our study suggests that the effective targeting of IL-15R-triggered events with CRB-15 can be of therapeutic importance in the treatment of rheumatoid arthritis.     

Synthesis of 2,2′-Anhydro-2-Hydroxy- and 6,2′-Anhydro-6-Hydroxy-1-β-D-Arabindfuranosylnicotinamide as Conformationally Restricted Nicotinamide Nucleoside Analogs1

Abstract

1-(β-D-Ribofuranosy1)-2(1H)-pyridone-3-carboxamide (6a) and the 6(1H)-pyridone derivative (6b) were prepared by condensation of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3) with 2- and 6-hydroxynicotinic acid, respectively, to 4a and 4b, followed by conversion of the carboxylic acid function of 4a,b into their corresponding carboxamides 5, and then deprotection of 5. Bath 6a and 6b were then treated with 1,3-dichlom-1,1,3,3-tetraisopropyldisiloxane to give the corresponding 3′,5′-O-TPDS derivatives, 7a and 7b. Mesylation of 7a,b with mesyl chloride in pyridine afforded the stable, protected mesylates 8a,b. Upon de-O-silylation of 8a,b with ET₃NHF gave a mixture of unprotectd mesylates 9a,b and 2,2-anhydro- and 6,2′-anhydronucleosides, 1a and 1b. Upon storage of 9a,b at man temperature, they are quantitatively converted into 1a,b. Mild alkaline hydrolysis of 1a,b afforded their corresponding arabino nucleosides 10a,b.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.     

GENETIC DIFFERENTIATION OF POLISH POPULATIONS OF SOREX ARANEUS L. III. INTERCHROMOSOMAL RECOMBINATION IN A HYBRID ZONE

Two parapatric chromosomal races of the common shrew (Sorex araneus) in Poland differ in their complement of metacentric arm combinations: hk, io, gr, nm (race IV), and hi, ko, gm, np (race II). In hybrids, these eight race-diagnostic metacentrics form two randomly segregating complexes. The first complex (C₁) occurs in the form of a ring configuration ok/kh/hi/io, or a chain o/ok/kh/hi/i (when there is Robertsonian polymorphism of the element io). The second complex (C₂) always takes the form of a six-element chain configuration r/rg/gm/mn/np/p. The C₂ complex may be shortened to five or even four elements, when acrocentrics g, m and n are present. In the contact zone we found shrews of pure races (race II or IV), as well as hybrids with C₁ or C₂ complexes, and recombinants hi, ko, gr, nm. Complex heterozygotes are likely to suffer reduced fertility due to malsegregation at meiosis. However, the C₁ hybrids with ring configurations occur with a high frequency throughout the contact zone. This suggest that their fitness is only slightly lowered relative to pure race individuals, in contrast to the hybrids with C₁ or C₂ chain configurations, which presumably have a more heavily reduced fertility. On the other hand, at the center of the zone there is a high proportion of recombinants, which, being chromosomal homozygotes, should display normal meiotic segregation. Furthermore, the high frequencies of recombinants within the contact zone should facilitate gene flow between the races. The occurrence of recombinants plays a similar role as the appearance of the maximum frequencies of acrocentric homozygotes described in several contact zones of S. araneus.     

Seasonal depth distribution and thermal experience of the non-indigenous round goby Neogobius melanostomus in the Baltic Sea: implications to key trophic relations

Biological Invasions - Native to the Ponto-Caspian region, the benthic round goby (Neogobius melanostomus) has invaded several European inland waterbodies as well as the North American Great Lakes...     

Dynamics of gapped DNA recognition by human polymerase beta

Kinetics of human polymerase beta binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of approximately 600-1200 s(-1). This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5'-terminal PO(4)(-) group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap.     

The role of karyopherins in the regulated sumoylation of septins

In the yeast Saccharomyces cerevisiae, several components of the septin ring are sumoylated during anaphase and then abruptly desumoylated at cytokinesis. We show that septin sumoylation is controlled by the interactions of two enzymes of the sumoylation pathway, Siz1p and Ulp1p, with the nuclear transport machinery. The E3 ligase Siz1p is imported into the nucleus by the karyopherin Kap95p during interphase. In M phase, Siz1p is exported from the nucleus by the karyopherin Kap142p/Msn5p and subsequently targeted to the septin ring, where it participates in septin sumoylation. We also show that the accumulation of sumoylated septins during mitosis is dependent on the interactions of the SUMO isopeptidase Ulp1p with Kap121p and Kap95p-Kap60p and the nuclear pore complex (NPC). In addition to sequestering Ulp1 at the NPC, Kap121p is required for targeting Ulp1p to the septin ring during mitosis. We present a model in which Ulp1p is maintained at the NPC during interphase and transiently interacts with the septin ring during mitosis.     

Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.