Reversal of alpha,alpha'-Dipyridyl-induced Porphyrin Synthesis in Etiolated and Greening Red Kidney Bean Leaves

The chemical induction of porphyrin synthesis has been investigated in etiolated and greening leaves of Phaseolus vulgaris L. var. Red Kidney. When these leaves are incubated in darkness with solutions of transition metal ion chelators such as α,α′-dipyridyl, 1,10-phenanthroline, pyridine-2-aldoxime, or other related aromatic heterocyclic nitrogenous bases, they synthesize large amounts of protochlorophyllide and Mg protoporphyrins. Greening leaves produce more porphyrin than do etiolated leaves under such conditions. If the leaves are then transferred to 1 millimolar solutions of various transition metal salts such as Fe²⁺, Zn²⁺, or Co²⁺ (but not Mn²⁺ or Mg²⁺), Mg protoporphyrin (monomethyl ester) synthesis immediately ceases and the pigment(s) rapidly disappear(s); protochlorophyllide synthesis gradually diminishes during 4 to 8 hours of treatment. The loss in Mg protoporphyrin(s) can be accounted for by a simultaneous increase in protochlorophyllide in partially greened leaves but not in etiolated leaves. In the latter, the decline in Mg protoporphyrin(s) initiated by the application of Zn²⁺ is retarded by low temperature and anaerobiosis but not by respiratory inhibitors. Cycloheximide inhibits the loss of Mg protoporphyrin(s) but does not affect their conversion to protochlorophyllide.     

A Combination of CD28 (rs1980422) and IRF5 (rs10488631) Polymorphisms Is Associated with Seropositivity in Rheumatoid Arthritis: A Case Control Study

IntroductionThe aim of the study was to analyse genetic architecture of RA by utilizing multiparametric statistical methods such as linear discriminant analysis (LDA) and redundancy analysis (RDA).MethodsA total of 1393 volunteers, 499 patients with RA and 894 healthy controls were included in the study. The presence of shared epitope (SE) in HLA-DRB1 and 11 SNPs (PTPN22 C/T (rs2476601), STAT4 G/T (rs7574865), CTLA4 A/G (rs3087243), TRAF1/C5 A/G (rs3761847), IRF5 T/C (rs10488631), TNFAIP3 C/T (rs5029937), AFF3 A/T (rs11676922), PADI4 C/T (rs2240340), CD28 T/C (rs1980422), CSK G/A (rs34933034) and FCGR3A A/C (rs396991), rheumatoid factor (RF), anti–citrullinated protein antibodies (ACPA) and clinical status was analysed using the LDA and RDA.ResultsHLA-DRB1, PTPN22, STAT4, IRF5 and PADI4 significantly discriminated between RA patients and healthy controls in LDA. The correlation between RA diagnosis and the explanatory variables in the model was 0.328 (Trace = 0.107; F = 13.715; P = 0.0002). The risk variants of IRF5 and CD28 genes were found to be common determinants for seropositivity in RDA, while positivity of RF alone was associated with the CTLA4 risk variant in heterozygous form. The correlation between serologic status and genetic determinants on the 1st ordinal axis was 0.468, and 0.145 on the 2nd one (Trace = 0.179; F = 6.135; P = 0.001). The risk alleles in AFF3 gene together with the presence of ACPA were associated with higher clinical severity of RA.ConclusionsThe association among multiple risk variants related to T cell receptor signalling with seropositivity may play an important role in distinct clinical phenotypes of RA. Our study demonstrates that multiparametric analyses represent a powerful tool for investigation of mutual relationships of potential risk factors in complex diseases such as RA.     

Functional diversity of LAP2alpha and LAP2beta in postmitotic chromosome association is caused by an alpha-specific nuclear targeting domain

Lamina-associated polypeptide 2alpha (LAP2alpha) is a non-membrane-bound isoform of the LAP2 family implicated in nuclear structure organization. We show that during postmitotic nuclear assembly LAP2alpha associates with chromosomes prior to accumulation of the membrane-bound isoform LAP2beta, although both proteins contain the same putative chromatin interaction domains located in their common N-terminal regions. By transient and stable expression of various N- and C-terminal LAP2alpha deletion mutants in HeLa cells, we identified an approximately 350-amino-acid-long region in the C-terminal alpha-specific domain of the protein that is required for retention of LAP2alpha in interphase nuclei and for association with mitotic chromosomes, while the N-terminal domain seemed to be dispensable for these interactions. In vitro chromosome binding studies using recombinant LAP2alpha mutants revealed that this LAP2alpha-specific 'nuclear targeting domain' was essential and sufficient for association with chromosomes. These data suggested a functional diversity of chromosome binding properties of LAP2 isoforms.     

Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly

The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.     

Lamins and lamin-associated proteins in aging and disease

Lamins, together with the lamin-associated proteins of the inner nuclear membrane, are structural proteins in the nucleus that mediate mechanical stress resistance. Novel findings show that lamin complexes also have scaffolding functions in the formation and regulation of higher order chromatin and in epigenetic regulatory pathways. Furthermore, lamins serve as scavenging complexes and regulators of signaling molecules in diverse pathways. Lamin complexes in the nuclear interior contribute to retinoblastoma-mediated cell cycle regulation. Because of their multiple and diverse roles, lamins are linked to an increasing number of human diseases. The molecular mechanisms of these diseases, which are just beginning to emerge, may involve cell cycle and differentiation defects in adult stem cells and genomic instability.     

Complementation of the Chlamydomonas reinhardtii arg7-8 (arg2) point mutation by recombination with a truncated nonfunctional ARG7 gene

Chlamydomonas reinhardtii arg7-8 (arg2) mutant strains carrying a hitherto undescribed mutation in their argininosuccinate lyase gene (ARG7) that leads to arginine auxotrophy have been used together with the corresponding wild-type gene as a very reliable transformation system since 1989. In this study, we finally identify the molecular nature of the arg7-8 mutation as a (6073)G to A transition in exon 9 of ARG7 leading to a (288)Gly to Ser exchange near the active site of the protein. The same mutation was found in the ARG7 genes of three commonly used C. reinhardtii laboratory strains, namely cw15-302 arg2, CC-48, and CC-1618. We did not observe exact spontaneous reversion of the arg7-8 allele in our study, but did identify two different and rare intragenic suppressor mutations, (27)Leu to Phe and (285)Tyr to Phe. In our hands, only transformation of the arg7-8 strain with a truncated nonfunctional wild-type ARG7 gene lacking 124 codons at its 5' end led to exact reversion of the mutant base (6073)A to the wild-type (6073)G, presumably by recombination. This system offers a positive selection scheme for homologous recombination (HR) and may, therefore, be useful to the methodical improvement of recombination in Chlamydomonas.