Comparison of folylpolyglutamate hydrolases of mouse liver,kidney, muscle and brain

Summary The folylpolyglutamate hydrolase activities of mouse liver, kidney, muscle and brain were examined by incorporation of methylenetetrahydrofolate polyglutamate reaction products into a stable ternary complex with tritiated fluorodeoxyuridylate and L. casei thymidylate synthetase. Complexes were separated electrophoretically on the basis of charge associated with the polyglutamyl moieties to determine distribution of chain lengths throughout the time course of the reaction. Tissue folylpolyglutamate hydrolase activities were allowed to utilize endogenous folylpolyglutamate as substrates by incubating crude tissue extracts at pH 7.4 and pH 4.5. Kidney and muscle contained relatively reactive hydrolases which were capable of generating intermediates of essentially all chain lengths from folylpentaglutamate, the predominant endogenous species. The relatively low activity in brain also gave rise to all possible intermediates. Liver contained a high concentration of methylenetetrahydrofolate but little hydrolase activity. The activity present in liver gave rise to essentially no intermediates but yielded only the monoglutamate form of the cofactor. When purified lysosomal preparations from liver and kidney were allowed to react with synthetic folylpolyglutamates, the same specificity with regard to reaction products was observed as with endogenous substrates.     

Combined effect of X‐ray radiation and static magnetic fields on reactive oxygen species in rat lymphocytes in vitro

The aim of this study was to investigate the effect of static magnetic fields (SMF) on reactive oxygen species induced by X‐ray radiation. The experiments were performed on lymphocytes from male albino Wistar rats. After exposure to 3 Gy X‐ray radiation (with a dose rate of 560 mGy/min) the measurement of intracellular reactive oxygen species in lymphocytes, using a fluorescent probe, was done before exposure to the SMF, and after 15 min, 1 and 2 h of exposure to the SMF or a corresponding incubation time. For SMF exposure, 0 mT (50 µT magnetic field induction opposite to the geomagnetic field) and 5 mT fields were chosen. The trend of SMF effects for 0 mT was always opposite that of 5 mT. The first one decreased the rate of fluorescence change, while the latter one increased it. Bioelectromagnetics 34:333–336, 2013. © 2012 Wiley Periodicals, Inc.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet receptor recognition domain on the gamma chain of human fibrinogen and its synthetic peptide analogues

We have shown previously that the domain recognizing receptors on activated human platelets is located on the human fibrinogen gamma chain between residues 400 and 411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767]. To study the correlation between the structure of this segment of the gamma chain and its reactivity toward receptors on ADP-activated human platelets, we designed a series of analogues containing replacements at 9 out of 12 positions. A double substitution of the normal His400-His401 sequence by Ala-Ala reduced the inhibitory potency of the dodecapeptide 3-fold. When Lys406 was replaced by Arg, the inhibitory potency of the dodecapeptide decreased 15 times. On the other hand, substitution of Ala408 with Arg increased the inhibitory potency of the dodecapeptide 6-fold. A drastic decrease in the reactivity of the dodecapeptide toward platelet receptors was observed when Val411 was replaced by leucine or cysteine or tyrosine. A 3-fold decrease in reactivity was noted when Val411 was substituted with phenylalanine. Amidation of the carboxy-terminal Val411 also produced a significant decrease in dodecapeptide reactivity. With seven residues (His400, His401, Leu402, Lys406, Gln407, Asp410, and Val411) preserved, substitution of the intervening five amino acids with nonpolar leucine or polar serine, increasing or decreasing the hydrophobicity of the dodecapeptide, reduced more than 16-fold its inhibitory potency. Rabbit antibody Fab fragments directed against the human fibrinogen gamma-chain peptide encompassing residues 385-411 inhibited 50% of 125I-fibrinogen binding at a 2:1 stoichiometry with regard to 125I-fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Traffic COPs and the formation of vesicle coats

Forward and retrograde trafficking of secretory proteins between the endoplasmic reticulum and the Golgi apparatus is driven by two biochemically distinct vesicle coats, COPI and COPII. Assembly of the coats on their target membranes is thought to provide the driving force for membrane deformation and the selective packaging of cargo and targeting molecules into nascent transport vesicles. This review describes our current knowledge on these issues and discusses how the two coats may be differentially targeted and assembled to achieve protein sorting and transport within the early secretory pathway.     

9-Fluorenylmethyl esters

N alpha-Protected amino acid 9-fluorenylmethyl esters (Fm esters) were prepared by imidazole-catalyzed transesterification of active esters with 9-fluorenylmethanol (9-hydroxymethylfluorene). The new carboxyl protection is unaffected by acids, but is efficiently removed by beta-elimination under the influence of secondary and tertiary amines. Primary amines and ammonia can cause slight amide formation. Deblocking was achieved also by catalytic hydrogenation.     

Ptr1 evolved convergently with RPS2 and Mr5 to mediate recognition of AvrRpt2 in diverse solanaceous species

The Ptr1 (Pseudomonas tomato race 1) locus in Solanum lycopersicoides confers resistance to strains of Pseudomonas syringae pv. tomato expressing AvrRpt2 and Ralstonia pseudosolanacearum expressing RipBN. Here we describe the identification and phylogenetic analysis of the Ptr1 gene. A single recombinant among 585 F2 plants segregating for the Ptr1 locus was discovered that narrowed the Ptr1 candidates to eight nucleotide‐binding leucine‐rich repeat protein (NLR)‐encoding genes. From analysis of the gene models in the S. lycopersicoides genome sequence and RNA‐Seq data, two of the eight genes emerged as the strongest candidates for Ptr1. One of these two candidates was found to encode Ptr1 based on its ability to mediate recognition of AvrRpt2 and RipBN when it was transiently expressed with these effectors in leaves of Nicotiana glutinosa. The ortholog of Ptr1 in tomato and in Solanum pennellii is a pseudogene. However, a functional Ptr1 ortholog exists in Nicotiana benthamiana and potato, and both mediate recognition of AvrRpt2 and RipBN. In apple and Arabidopsis, recognition of AvrRpt2 is mediated by the Mr5 and RPS2 proteins, respectively. Phylogenetic analysis places Ptr1 in a distinct clade compared with Mr5 and RPS2, and it therefore appears to have arisen by convergent evolution for recognition of AvrRpt2.     

Mediation of Clathrin-Dependent Trafficking during Cytokinesis and Cell Expansion by Arabidopsis STOMATAL CYTOKINESIS DEFECTIVE Proteins

STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.     

Membrane trafficking in plants: new discoveries and approaches

The general organization and function of the endomembrane system is highly conserved in eukaryotic cells. In addition, increasing numbers of studies demonstrate that normal plant growth and development are dependent on specialized tissue and subcellular-specific components of the plant membrane trafficking machinery. New approaches, including chemical genomics and proteomics, will likely accelerate our understanding of the diverse functions of the plant endomembrane system.