Responses of transformed Catharanthus roseus roots to femtomolar concentrations of salicylic acid.

Catharanthus roseus transformed roots were cultured in the presence of salicylic acid (SA) at concentrations between 0.1 fM and 100 pM and the effect on root growth was evaluated. Significant morphological changes in the lateral roots were recorded on day two in the SA treatment. Presence of SA increased root cap size and caused the appearance of lateral roots closer to the root tip. The bioassay was sensitive enough to allow testing of low concentrations of other growth regulators that may affect root morphology and physiology.     

Rab1b interacts with GBF1 and modulates both ARF1 dynamics and COPI association

Assembly of the cytosolic coat protein I (COPI) complex at the ER-Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t(1/2) 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.     

Membrane-associated phosphoinositides-specific phospholipase C forms from <Emphasis Type="Italic">Catharanthus roseus</Emphasis> transformed roots

We have previously reported that Catharanthus roseus transformed roots contain at least two phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC) activities, one soluble and the other membrane associated. Detergent, divalent cations, and neomycin differentially regulate these activities and pure protein is required for a greater understanding of the function and regulation of this enzyme. In this article we report a partia purification of membrane-associated PLC. We found that there are at least two forms of membrane-associated PLC in transformed roots of C. roseus. These forms were separated on the basis of their affinity for heparin. One form shows an affinity for heparin and elutes at approx 600 mM KCl. This form has a molecular mass of 67 kDa by size exclusion chromatography and Western blot analysis, whereas the other form does not bind to heparin and has a molecular mass of 57 kDa. Possible differential regulation of these forms during transformed root growth is discussed.     

Modulation of oxidative damage by nitroxide free radicals

Piperidine nitroxides like 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) are persistent free radicals in non-acidic aqueous solutions and organic solvents that may have value as therapeutic agents in medicine. In biological environments, they undergo mostly reduction to stable hydroxylamines but can also undergo oxidation to reactive oxoammonium compounds. Reactions of the oxoammonium derivatives could have adverse consequences including chemical modification of vital macromolecules and deleterious effects on cell signaling. An examination of their reactivity in aqueous solution has shown that oxoammonium compounds can oxidize almost any organic as well as many inorganic molecules found in biological systems. Many of these reactions appear to be one-electron transfers that reduce the oxoammonium to the corresponding nitroxide species, in contrast to a prevalence of two-electron reductions of oxoammonium in organic solvents. Amino acids, alcohols, aldehydes, phospholipids, hydrogen peroxide, other nitroxides, hydroxylamines, phenols and certain transition metal ions and their complexes are among reductants of oxoammonium, causing conversion of this species to the paramagnetic nitroxide. On the other hand, thiols and oxoammonium yield products that cannot be detected by ESR even under conditions that would oxidize hydroxylamines to nitroxides. These products may include hindered secondary amines, sulfoxamides and sulfonamides. Thiol oxidation products other than disulfides cannot be restored to thiols by common enzymatic reduction pathways. Such products may also play a role in cell signaling events related to oxidative stress. Adverse consequences of the reactions of oxoammonium compounds may partially offset the putative beneficial effects of nitroxides in some therapeutic settings.     

Bulky melamine-based Zn-porphyrin tweezer as a CD probe of molecular chirality

The transfer of chirality from a guest molecule to an achiral host is the subject of significant interest especially when, upon chiral induction, the chiroptical response of the host/guest complex can effectively report the absolute configuration (AC) of the guest. For more than a decade, dimeric metalloporphyrin hosts (tweezers) have been successfully applied as chirality probes for determination of the AC for a wide variety of chiral synthetic compounds and natural products. The objective of this study is to investigate the utility of a new class of melamine-bridged Zn-porphyrin tweezers as sensitive AC reporters. A combined approach based on an experimental CD analysis and a theoretical prediction of the prevailing interporphyrin helicity demonstrates that these tweezers display favorable properties for chiral recognition. Herein, we discuss the application of the melamine-bridged tweezer to the chiral recognition of a diverse set of chiral guests, such as 1,2-diamines, α-amino-esters and amides, secondary alcohols, and 1,2-amino-alcohols. The bulky periphery and the presence of a rigid porphyrin linkage lead, in some cases, to a more enhanced CD sensitivity than that reported earlier with other tweezers.     

Phosphoproteome Analysis of Functional Mitochondria Isolated from Resting Human Muscle Reveals Extensive Phosphorylation of Inner Membrane Protein Complexes and Enzymes

Mitochondria play a central role in energy metabolism and cellular survival, and consequently mitochondrial dysfunction is associated with a number of human pathologies. Reversible protein phosphorylation emerges as a central mechanism in the regulation of several mitochondrial processes. In skeletal muscle, mitochondrial dysfunction is linked to insulin resistance in humans with obesity and type 2 diabetes. We performed a phosphoproteomics study of functional mitochondria isolated from human muscle biopsies with the aim to obtain a comprehensive overview of mitochondrial phosphoproteins. Combining an efficient mitochondrial isolation protocol with several different phosphopeptide enrichment techniques and LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, including 116 phosphoserine, 23 phosphothreonine, and 16 phosphotyrosine residues. The relatively high number of phosphotyrosine residues suggests an important role for tyrosine phosphorylation in mitochondrial signaling. Many of the mitochondrial phosphoproteins are involved in oxidative phosphorylation, tricarboxylic acid cycle, and lipid metabolism, i.e. processes proposed to be involved in insulin resistance. We also assigned phosphorylation sites in mitochondrial proteins involved in amino acid degradation, importers and transporters, calcium homeostasis, and apoptosis. Bioinformatics analysis of kinase motifs revealed that many of these mitochondrial phosphoproteins are substrates for protein kinase A, protein kinase C, casein kinase II, and DNA-dependent protein kinase. Our results demonstrate the feasibility of performing phosphoproteome analysis of organelles isolated from human tissue and provide novel targets for functional studies of reversible phosphorylation in mitochondria. Future comparative phosphoproteome analysis of mitochondria from healthy and diseased individuals will provide insights into the role of abnormal phosphorylation in pathologies, such as type 2 diabetes.Mitochondria are the primary energy-generating systems in eukaryotes. They play a crucial role in oxidative metabolism, including carbohydrate metabolism, fatty acid oxidation, and urea cycle, as well as in calcium signaling and apoptosis (1, 2). Mitochondrial dysfunction is centrally involved in a number of human pathologies, such as type 2 diabetes, Parkinson disease, and cancer (3). The most prevalent form of cellular protein post-translational modifications (PTMs),1 reversible phosphorylation (4–6), is emerging as a central mechanism in the regulation of mitochondrial functions (7, 8). The steadily increasing numbers of reported mitochondrial kinases, phosphatases, and phosphoproteins imply an important role of protein phosphorylation in different mitochondrial processes (9–11).Mass spectrometry (MS)-based proteome analysis is a powerful tool for global profiling of proteins and their PTMs, including protein phosphorylation (12, 13). A variety of proteomics techniques have been developed for specific enrichment of phosphorylated proteins and peptides and for phosphopeptide-specific data acquisition techniques at the MS level (14). Enrichment methods based on affinity chromatography, such as titanium dioxide (TiO₂) (15–17), zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) (18), immobilized metal affinity chromatography (IMAC) (19, 20), and ion exchange chromatography (strong anion exchange and strong cation exchange) (21, 22), have shown high efficiencies for enrichment of phosphopeptides (14). Recently, we demonstrated that calcium phosphate precipitation (CPP) is highly effective for enriching phosphopeptides (23). It is now generally accepted that no single method is comprehensive, but combinations of different enrichment methods produce distinct overlapping phosphopeptide data sets to enhance the overall results in phosphoproteome analysis (24, 25). Phosphopeptide sequencing by mass spectrometry has seen tremendous advances during the last decade (26). For example, MS/MS product ion scanning, multistage activation, and precursor ion scanning are effective methods for identifying serine (Ser), threonine (Thr), and tyrosine (Tyr) phosphorylated peptides (14, 26).A “complete” mammalian mitochondrial proteome was reported by Mootha and co-workers (27) and included 1098 proteins. The mitochondrial phosphoproteome has been characterized in a series of studies, including yeast, mouse and rat liver, porcine heart, and plants (19, 28–31). To date, the largest data set by Deng et al. (30) identified 228 different phosphoproteins and 447 phosphorylation sites in rat liver mitochondria. However, the in vivo phosphoproteome of human mitochondria has not been determined. A comprehensive mitochondrial phosphoproteome is warranted for further elucidation of the largely unknown mechanisms by which protein phosphorylation modulates diverse mitochondrial functions.The percutaneous muscle biopsy technique is an important tool in the diagnosis and management of human muscle disorders and has been widely used to investigate metabolism and various cellular and molecular processes in normal and abnormal human muscle, in particular the molecular mechanism underlying insulin resistance in obesity and type 2 diabetes (32). Skeletal muscle is rich in mitochondria and hence a good source for a comprehensive proteomics and functional analysis of mitochondria (32, 33).The major aim of the present study was to obtain a comprehensive overview of site-specific phosphorylation of mitochondrial proteins in functionally intact mitochondria isolated from human skeletal muscle. Combining an efficient protocol for isolation of skeletal muscle mitochondria with several different state-of-the-art phosphopeptide enrichment methods and high performance LC-MS/MS, we identified 155 distinct phosphorylation sites in 77 mitochondrial phosphoproteins, many of which have not been reported before. We characterized this mitochondrial phosphoproteome by using bioinformatics tools to classify functional groups and functions, including kinase substrate motifs.     

Cognitive landscape and information: new perspectives to investigate the ecological complexity

Landscape ecology deals with ecological processes in their spatial context. It shares with ecosystem ecology the primate of emergent ecological disciplines. The aim of this contribution is to approach the definition of landscapes using cognitive paradigms. Neutral-based landscape (NbL), individual-based landscape (IbL) and observed-based landscape (ObL) are defined to explore the cognitive mechanisms. NbL represents the undecoded component of the cognitive matrix. The IbL is the portion of landscape perceived by the biological sensors. ObL is the part of the cognitive matrix perceived using the cultural background of the observer. The perceived landscape (PL) is composed by the sum of these three approaches of landscape perception. Two further types of information (sensu Stonier) are recognized in this process of perception: the compressed information, as it is present inside the cognitive matrix, and the decompressed information that will structure the PL when a semiotic relationship operates between the organisms and the cognitive matrix. Scaling properties of these three PL components are recognized in space and time. In NbL scale seems irrelevant, in IbL the perception is filtered by organismic scaling and in ObL the spatio-temporal scale seems of major importance. Definitively, perception is scale-dependent. A combination of the cognitive approach with information paradigms to study landscapes opens new perspectives in the interpretation of ecological complexity.     

A simple and efficient method for isolation of DNA in high mucilaginous plant tissues

A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants.     

Tuberculosis masquerading as malignancy: a multimodality approach to the correct diagnosis – a case report

BACKGROUND: Extrapulmonary tuberculosis is one of the great mimickers of medicine, and often masquerades as malignancy. As a result, patients may be referred to oncologists and surgeons for further evaluation and management, delaying the institution of appropriate anti-tuberculous drug therapy. CASE PRESENTATION: We present the case of a 21 year old man with tuberculous osteomyelitis, who was referred to the Bone and Soft Tissue Sarcoma Service at our institution with a provisional diagnosis of malignancy. Further investigation revealed extensive retroperitoneal abdominal and pelvic lymphadenopathy. The recognition of certain patterns on imaging, and finally the isolation of Mycobacterium tuberculosis from tissue samples obtained under image guidance, enabled the correct diagnosis to be made. CONCLUSION: This case highlights the importance of remaining cognisant of the protean manifestations of extrapulmonary tuberculosis, and illustrates the advantage of a clinically directed multi-modality imaging approach to diagnosis.