N-caffeoyl-4-amino-n-butyric acid, a new flower-specific metabolite in cultured tobacco cells and tobacco plants

PUT cells were selected from the XD line of cultured tobacco cells (Nicotiana tabacum L. cv. Xanthi-nc) for the ability to utilize putrescine as sole nitrogen source. Previous work had indicated that hydroxycinnamoylputrescines (principally caffeoylputrescine) and 4-amino-n-butyric acid (GABA) are obligatory intermediates in the assimilation of putrescine by PUT cells. The apparent absence in these cells of diamine or polyamine oxidase and pyrroline dehydrogenase, enzymes which catalyze putrescine oxidation in some plant species, led us to propose the following pathway for putrescine oxidation in PUT cells: putrescine----hydroxycinnamoylputrescine----hydroxycinnamoyl - 4-aminobutyraldehyde----hydroxycinnamoyl-GABA----GABA. We tested the hypothesis by looking for the predicted compound, caffeoyl-GABA. A chemical synthesis was developed, and chromatographic and mass spectroscopic procedures were devised for identifying the compound in extracts of cells and plant tissues. Caffeoyl-GABA was found in extracts of PUT cells in micromolar concentrations but was not present in XD cells. Thus, its occurrence in PUT cells appears to be a direct result of selection for the ability to catabolize putrescine. Caffeoyl-GABA has the same distribution in tobacco plants as caffeoylputrescine, i.e. flower buds greater than open flowers greater than floral leaves, green fruit; absent in vegetative tissues.     

Alpha interferon accelerates lateral diffusion of Daudi cell surface differentiation antigens: measurement by fluorescence redistribution after photobleaching

Lateral diffusion coefficients (D) of two surface differentiation antigens (sIgM and Bp35) were determined on interferon-sensitive (-IFs) or resistant (-IFr) Daudi cells by fluorescence photobleaching, using monospecific FITC-anti-IgM or PE-anti-Leu 16 probes. For untreated Daudi -IFs, mean (D) were 5.8 and 5.3 (x10(-10) cm2/sec). These increased, to 11 and 7.9 x 10(-10) cm2/sec (p less than 0.001) within 30 min after binding of recombinant IFN-a (80 to 800 U/10(6) cells), but decreased by up to 4-fold after Con A Mean (D) of identical surface antigens on Daudi-IFr were 8.2 and 9.4 x 10(-10) cm2/sec; and were not altered by IFN-a. Mean (D) of a lipid analog was up to 40-fold higher than for surface proteins and statistically identical in Daudi-IFs and Daudi-IFr. Rapid acceleration by IFN-a of surface protein lateral diffusion in Daudi-IFs obviously could facilitate anti-proliferative signal transduction; by contrast, a baseline increase of (D) in Daudi-IFr was evidently associated with their refractory state.     

Cellular systems for the study of the biosynthesis of polyamines and ethylene,as well as of virus multiplication

Leaves of Chinese cabbage from healthy plants or from those infected with turnip yellow mosaic virus yield protoplasts which convert methionine to protein, S-adenosylmethionine, decarboxylated S-adenosylmethionine, spermidine, spermine and 1-aminocyclopropane-1-carboxylate. The enzyme spermidine synthase is entirely cytosolic and has been purified extensively. An inhibitor of this enzyme, dicyclohexylamine, blocks spermidine synthesis in intact protoplasts, and in so doing stimulates spermine synthesis. Aminoethoxyvinylglycine blocks the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylate, the precursor to ethylene, in protoplasts. This inhibitor markedly stimulates the synthesis of both spermidine and spermine. Essentially all the protoplasts obtained from new leaves of plants infected 7 days earlier are infected. On incubation, such protoplasts convert exogenous methionine to viral protein and viral spermidine whose specific radioactivity is twice that of total cell spermidine. Exogeneous spermidine is also converted to cell putrescine and viral spermidine and spermine. Normal and virus-infected cells are being studied for their content of phenolic acid amides of the polyamines.     

PACAP and PAC1 Receptor Expression in Human Insulinomas

International Journal of Peptide Research and Therapeutics - Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. PACAP...     

Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance

Objectives

The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods.

Methods

Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied.

Results

Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly—p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively.

Conclusions

Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.     

MicroDSC study of Staphylococcus epidermidis growth

Background  

A microcalorimetric study was carried out using a Staphylococcus epidermidis population to determine the reproducibility of bacterial growth and the variability of the results within certain experimental parameters (temperature, bacterial concentration, sample thermal history). Reproducibility tests were performed as series of experiments within the same conditions using either freshly prepared populations or samples kept in cold storage. In both cases, the samples were obtained by serial dilution from a concentrated TSB bacterial inoculum incubated overnight.     

Chronic infections and genetic factors in the development of ischemic stroke

The aim of this study was to examine whether chronic infections and genetic factors of the host play roles in the pathophysiology of acute noncardioembolic ischemic stroke. Blood samples from 59 subjects with ischemic stroke and 52 control patients were investigated by nested PCR for the presence of C. pneumoniae DNA, HCMV DNA and enterovirus RNA, by ELISA for the levels of antibodies to C. pneumoniae, HCMV, HSV, HHV-6, EBV and the inflammatory chemokine IL-8, and by PCR for promoter polymorphism of the IL-8 and CD14 host genes. Associations of stroke with the HCMV IgG and HSV-1 IgA antibody levels were observed. No association of stroke was detected with the presence of C. pneumoniae, HCMV or enterovirus nucleic acids in the peripheral blood, C. pneumoniae IgM, IgG and IgA, the HSV IgG, the EBV IgG, or HHV-6 IgG antibody levels, the pathogen burden, the IL-8 or CD14 promoter polymorphisms, or with the serum levels of IL-8 in the overall study population. These results are consistent with the hypothesis that certain pathogens are involved in the development of ischemic stroke.     

Localization and organization of protein factors involved in chromosome inheritance in Dictyostelium discoideum

Heterochromatin protein 1 (HP1) proteins are highly conserved heterochromatin components required for genomic integrity. We have previously shown that the two HP1 isoforms expressed in Dictyostelium, HcpA and HcpB, are mainly localized to (peri-)centromeric heterochromatin and have largely overlapping functions. However, they cause distinct phenotypes when overexpressed. We show here that these isoforms display quantitative differences in dimerization behavior. Dimerization preference, as well as the mutant phenotype in overexpression strains, depends on the C-terminus containing the hinge and chromo shadow domains. Both Hcp proteins are targeted to distinct subnuclear regions by different chromo shadow domain-dependent and -independent mechanisms. In addition, both proteins bind to DNA and RNA in vitro and binding is independent of the chromo shadow domain. Thus, this DNA and/or RNA binding activity may contribute to protein targeting. To further characterize heterochromatin, we cloned the Dictyostelium homolog of the origin recognition complex subunit 2 (OrcB). OrcB localizes to distinct subnuclear foci that were also targeted by HcpA. In addition, it is associated with the centrosome throughout the cell cycle. The results indicate that, similar to Orc2 homologs from other organisms, it is required for different processes in chromosome inheritance.     

Assembly of Slx4 signaling complexes behind DNA replication forks

Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.