Two Arabidopsis threonine aldolases are nonredundant and compete with threonine deaminase for a common substrate pool

Amino acids are not only fundamental protein constituents but also serve as precursors for many essential plant metabolites. Although amino acid biosynthetic pathways in plants have been identified, pathway regulation, catabolism, and downstream metabolite partitioning remain relatively uninvestigated. Conversion of Thr to Gly and acetaldehyde by Thr aldolase (EC 4.1.2.5) was only recently shown to play a role in plant amino acid metabolism. Whereas one Arabidopsis thaliana Thr aldolase (THA1) is expressed primarily in seeds and seedlings, the other (THA2) is expressed in vascular tissue throughout the plant. Metabolite profiling of tha1 mutants identified a >50-fold increase in the seed Thr content, a 50% decrease in seedling Gly content, and few other significant metabolic changes. By contrast, homozygous tha2 mutations cause a lethal albino phenotype. Rescue of tha2 mutants and tha1 tha2 double mutants by overproduction of feedback-insensitive Thr deaminase (OMR1) shows that Gly formation by THA1 and THA2 is not essential in Arabidopsis. Seed-specific expression of feedback-insensitive Thr deaminase in both tha1 and tha2 Thr aldolase mutants greatly increases seed Ile content, suggesting that these two Thr catabolic enzymes compete for a common substrate pool.     

Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

Therapeutic induction of regulatory, cytotoxic CD8+ T cells in multiple sclerosis

In the setting of autoimmunity, one of the goals of successful therapeutic immune modulation is the induction of peripheral tolerance, a large part of which is mediated by regulatory/suppressor T cells. In this report, we demonstrate a novel immunomodulatory mechanism by an FDA-approved, exogenous peptide-based therapy that incites an HLA class I-restricted, cytotoxic suppressor CD8+ T cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA; Copaxone) induces differential up-regulation of GA-reactive CD8+ T cell responses. We now show that these GA-induced CD8+ T cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8+ T cell-mediated suppression, compared with healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8+ T cells from GA-treated patients and healthy subjects, but not those from untreated patients with MS, exhibit potent, HLA class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced cytotoxic CD8+ T cells can directly kill CD4+ T cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4+ T cells and may depend on presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8+ T cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease.     

A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps

Vacuolar-type H⁺-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists. Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum. PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H⁺-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases. Heterologous expression of PVP in Saccharomyces cerevisiae yields a M_r 64 000 membrane polypeptide that specifically catalyzes Mg²⁺-dependent PPi hydrolysis. The existence of PVP implies that PPi-energized H⁺-translocation is phylogenetically more deeply rooted than previously thought.     

Design,synthesis, and characterization of 2,2-bis(2,4-dinitrophenyl)-2-(phosphonatomethylamino)acetate as a herbicidal and biological active agent

The present study was designed to synthesize the bioactive molecule 2,2-bis(2,4-dinitrophenyl)-2-(phosphonatomethylamino)acetate (1), having excellent applications in the field of plant protection as a herbicide. Structure of newly synthesized molecule 1 was confirmed by using the elemental analysis, mass spectrometric, NMR, UV-visible, and FTIR spectroscopic techniques. To obtain better structural insights of molecule 1, 3D molecular modeling was performed using the GAMESS programme. Microbial activities of 1 were checked against the pathogenic strains Aspergillus fumigatus (NCIM 902) and Salmonella typhimurium (NCIM 2501). Molecule 1 has shown excellent activities against fungal strain A. fumigates (35 μg/l) and bacterial strain S. typhimurium (25 μg/l). To check the medicinal significance of molecule 1, interactions with bovine serum albumin (BSA) protein were checked. The calculated value of binding constant of molecule 1–BSA complex was 1.4 × 10⁶ M^?1, which were similar to most effective drugs like salicylic acid. More significantly, as compared to herbicide glyphosate, molecule 1 has exhibited excellent herbicidal activities, in pre- and post-experiments on three weeds; barnyard grass (Echinochloa Crus), red spranglitop (Leptochloa filiformis), and yellow nuts (Cyperus Esculenfus). Further, effects of molecule 1 on plant growth-promoting rhizobacterial (PGPR) strains were checked. More interestingly, as compared to glyphosate, molecule 1 has shown least adverse effects on soil PGPR strains including the Rhizobium leguminosarum (NCIM 2749), Pseudomonas fluorescens (NCIM 5096), and Pseudomonas putida (NCIM 2847).     

Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance because of frequent natural exposure and their use in medical applications. Depending on the preparation studied and the end point measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. We prepared highly purified chitosan and chitin and examined the capacity of these glycans to stimulate murine macrophages to release the inflammasome-associated cytokine IL-1β. We found that although chitosan was a potent NLRP3 inflammasome activator, acetylation of the chitosan to chitin resulted in a near total loss of activity. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL-1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. In contrast, chitin is relatively inert.     

Sex differences in kidney gene expression during the life cycle of F344 rats

Background

The kidney functions in key physiological processes to filter blood and regulate blood pressure via key molecular transporters and ion channels. Sex-specific differences have been observed in renal disease incidence and progression, as well as acute kidney injury in response to certain drugs. Although advances have been made in characterizing the molecular components involved in various kidney functions, the molecular mechanisms responsible for sex differences are not well understood. We hypothesized that the basal expression levels of genes involved in various kidney functions throughout the life cycle will influence sex-specific susceptibilities to adverse renal events.

Methods

Whole genome microarray gene expression analysis was performed on kidney samples collected from untreated male and female Fischer 344 (F344) rats at eight age groups between 2 and 104 weeks of age.

Results

A combined filtering approach using statistical (ANOVA or pairwise t test, FDR 0.05) and fold-change criteria (>1.5 relative fold change) was used to identify 7,447 unique differentially expressed genes (DEGs). Principal component analysis (PCA) of the 7,447 DEGs revealed sex-related differences in mRNA expression at early (2 weeks), middle (8, 15, and 21 weeks), and late (104 weeks) ages in the rat life cycle. Functional analysis (Ingenuity Pathway Analysis) of these sex-different genes indicated over-representation of specific pathways and networks including renal tubule injury, drug metabolism, and immune cell and inflammatory responses. The mRNAs that code for the qualified urinary protein kidney biomarkers KIM-1, Clu, Tff3, and Lcn2 were also observed to show sex differences.

Conclusions

These data represent one of the most comprehensive in-life time course studies to be published, assessing sex differences in global gene expression in the F344 rat kidney. PCA and Venn analyses reveal specific periods of sexually dimorphic gene expression which are associated with functional categories (xenobiotic metabolism and immune cell and inflammatory responses) of key relevance to acute kidney injury and chronic kidney disease, which may underlie sex-specific susceptibility. Analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

     

Synthesis,angiopreventive activity,and in vivo tumor inhibition of novel benzophenone–benzimidazole analogs

Aim

The development of anticancer drugs with specific targets is of prime importance in modern biology. This study investigates the angiopreventive and in vivo tumor inhibition activities of novel synthetic benzophenone–benzimidazole analogs.

Main methods

The multistep synthesis of novel benzophenone–benzimidazole analogs (8a–n) allowing substitution with methoxy, methyl and halogen groups at different positions on the identical chemical backbone and the variations in the number of substituents were synthesized and characterized. The newly synthesized compounds were further evaluated for cytotoxic and antiproliferative effects against Ehrlich ascites carcinoma (EAC) cells. The potent lead compounds were further assessed for antiangiogenic effects in a CAM model and a tumor-induced vasculature in vivo model. The effect of angioprevention on tumor growth was verified in a mouse model.

Key findings

The cytotoxicity studies revealed that compounds 8f and 8n are strongly cytotoxic. Analyzing the structure–activity relationship, we found that an increase in the number of methyl groups in addition to methoxy substitution at the para position of the benzoyl ring in compound 8n resulted in higher potency compared to 8f. Furthermore, neovessel formation in in vivo systems, such as the chorioallantoic membrane (CAM) and tumor-induced mice peritoneum models, was significantly suppressed and reflected the tumor inhibition observed in mice.

Significance

These results suggest the potential clinical application of compound 8n as an antiangiogenic drug for cancer therapy.     

Pilot and feasibility study: comparative proteomic analysis by 2-DE MALDI TOF/TOF MS reveals 14-3-3 proteins as putative biomarkers of response to neoadjuvant chemotherapy in ER-positive breast cancer

Neoadjuvant chemotherapy is used to treat oestrogen receptor-positive breast cancer however chemo-resistance is a major obstacle in this molecular subtype. The ability to predict tumour response would allow chemotherapy administration to be directed towards patients who would most benefit, thus maximising treatment efficacy. We aimed to identify protein biomarkers associated with response to neoadjuvant chemotherapy, in a pilot study using comparative 2-DE MALDI TOF/TOF MS proteomic analysis of breast tumour samples. A total of 3 comparative proteomic experiments were performed, comparing protein expression between chemotherapy-sensitive and chemotherapy-resistant oestrogen receptor-positive invasive ductal carcinoma tissue samples. This identified a list of 132 unique proteins that were significantly differentially expressed (≥ 2 fold) in chemotherapy resistant samples, 57 of which were identified in at least two experiments. Ingenuity? Pathway Analysis was used to map the 57 DEPs onto canonical signalling pathways. We implicate several isoforms of 14-3-3 family proteins (theta/tau, gamma, epsilon, beta/alpha and zeta/delta), which have previously been associated with chemotherapy resistance in breast cancer. Extensive clinical validation is now required to fully assess the role of these proteins as putative markers of chemotherapy response in luminal breast cancer subtypes.