Mathematical model of fructan biosynthesis and polymer length distribution in plants

Background and Aims

There are many unresolved issues concerning the biochemistry of fructan biosynthesis. The aim of this paper is to address some of these by means of modelling mathematically the biochemical processes.

Methods

A model has been constructed for the step-by-step synthesis of fructan polymers. This is run until a steady state is achieved for which a polymer distribution is predicted. It is shown how qualitatively different distributions can be obtained.

Key Results

It is demonstrated how a set of experimental results on polymer distribution can by simulated by a simple parameter adjustments.

Conclusions

Mathematical modelling of fructan biosynthesis can provide a useful tool for helping elucidate the details of the biosynthetic processes.     

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.     

Zur Heredität des Strabismus concomitans

697 children with concomitant strabism, all patients who were seen in the Ophthalmalogical university hospital and outpatient service of the Charité, Berlin, during a certain period, have been examined together with their parents and siblings. In a second series, 3398 12-years old school children (born 1953) from three urban districts of Berlin were examined at the occasion of a vaccination term, and 179 children with strabism were ascertained. Probands as well as their parents and siblings were examined thoroughly according to the criteria of strabism diagnostics. Incidence of squinting among siblings was increased markedly when compared with the population frequency, but did not reach the expectations under rthe assumption of a single, monomeric mode of inheritance. Admixture of phenocopies or new mutants could be excluded. Twin investigations (12 monocygotic and 27 dicygotic pairs) showed a manifestation rate of 94,1% in monocygotic pairs, corresponding to a 3 1/2 times higher concordance in monocygotic as compared with dicygotic twins. From the results discussed, a multifactorial genetic system in combination with a threshold effect seems to be the most likely genetic interpretation. An analysis of pedigrees shows that slight sensoric as well as motoric anomalies might combine in different and continuously varying quantities, contributing to the syndrom of concomitant strabism.     

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor. This designation, however, is controversial as a result of recent studies in which lysophosphatidylinositol (LPI) was identified as a GPR55 agonist. Defining a biological role for GPR55 requires GPR55 selective ligands that have been unavailable. From a β-arrestin, high-throughput, high-content screen of 300000 compounds run in collaboration with the Molecular Libraries Probe Production Centers Network initiative (PubChem AID1965), we identified potent GPR55 selective agonists. By modeling of the GPR55 activated state, we compared the GPR55 binding conformations of three of the novel agonists obtained from the screen, CID1792197, CID1172084, and CID2440433 (PubChem Compound IDs), with that of LPI. Our modeling indicates the molecular shapes and electrostatic potential distributions of these agonists mimic those of LPI; the GPR55 binding site accommodates ligands that have inverted-L or T shapes with long, thin profiles that can fit vertically deep in the receptor binding pocket while their broad head regions occupy a horizontal binding pocket near the GPR55 extracellular loops. Our results will allow the optimization and design of second-generation GPR55 ligands and provide a means for distinguishing GPR55 selective ligands from those interacting with cannabinoid receptors.     

Kinetics control preferential heterodimer formation of platelet-derived growth factor from unfolded A- and B-chains

The folding and assembly of platelet-derived growth factor (PDGF), a potent mitogen involved in wound-healing processes and member of the cystine knot growth factor family, was studied. The kinetics of the formation of disulfide-bonded dimers were investigated under redox reshuffling conditions starting either from unfolded and reduced PDGF-A- or B-chains or an equimolar mixture of both chains. It is shown that in all cases the formation of disulfide-bonded dimers is a very slow process occurring in the time scale of hours with a first-order rate-determining step. The formation of disulfide-bonded PDGF-AA or PDGF-BB homodimers displayed identical kinetics, indicating that both monomeric forms as well as the dimerized homodimer have similar folding and assembly pathways. In contrast, the formation of the heterodimer occurred three times more rapidly compared with the formation of the homodimers. As both monomeric forms revealed similar renaturation kinetics, it can be concluded that the first-order rate-determining folding step does not occur during monomer folding but must be attributed to conformational rearrangements of the dimerized, not yet disulfide-bonded protein. These structural rearrangements allow a more rapid formation of intermolecular disulfide bonds between the two different monomers of a heterodimer compared with the formation of the disulfide bonds between two identical monomers. The preferential formation of disulfide-bonded heterodimers from an equimolar mixture of unfolded A- and B-chains is thus a kinetically controlled process. Moreover, similar activation enthalpies for the formation of all different isoforms suggest that faster heterodimerization is controlled by entropic factors.     

Development of both colorimetric and fluorescence heparinase activity assays using fondaparinux as substrate

Because tumors and other diseases are characterized by increased heparanase levels, human heparanase is a promising drug target and diagnostic marker. Therefore, methods are needed to determine heparanase activity and to examine potential inhibitors. Because of substrate comparability, we used the bacterial enzyme heparinase II (heparinase) for the assay development. Usually the substrate of heparanase assays is heparan sulfate, which has several disadvantages. Because of that, we used fondaparinux, which is being cleaved by both heparanase and heparinase. Two concepts to detect its degradation were examined: measurement of anti-factor Xa activity of fondaparinux and its direct quantification with the fluorescent sensor polymer-H. Using fondaparinux as substrate, the anti-factor Xa assay was shsown to be appropriate to determine heparinase activity. The detection with polymer-H was easier and even faster to perform. Linearity was given with fondaparinux as well as heparan sulfate, and heparin as substrates, but fondaparinux turned out to be most suitable. By modifications (incubation time, fondaparinux concentration, and polymer-H concentration), the limit of quantification and the linear range can be adapted to the respective requirements. In conclusion, a simple, accurate, and robust heparinase assay was developed. It is suitable for heparinase quality control and testing heparinase inhibitors and could be adapted to heparanase.     

Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

MicroRNA-196a and -196b as Potential Biomarkers for the Early Detection of Familial Pancreatic Cancer

Screening programs are recommended for individuals at risk (IAR) from families with familial pancreatic cancer (FPC). However, reliable imaging methods or biomarkers for early diagnosis of pancreatic ductal adenocarcinoma (PC) or its precursor lesions are still lacking. The ability of circulating microRNAs (miRNAs) to discriminate multifocal high-grade precursor lesions or PC from normal was examined. The presence of miRNA-21, -155, -196a, -196b and -210 was analyzed in the serum of transgenic KPC mice to test their ability to distinguish mice with different grades of pancreatic intraepithelial neoplasia (mPanIN1–3) or PC from control mice. Serum levels of miR-196a and -196b were significantly higher in mice with PanIN2/3 lesions (n = 10) or PC (n = 8) as compared to control mice (n = 10) or mice with PanIN1 lesions (n = 10; P = .01). In humans, miR-196a and -196b were also diagnostic. Patients with PC, sporadic (n = 9) or hereditary (n = 10), and IAR with multifocal PanIN2/3 lesions (n = 5) had significantly higher serum levels than patients with neuroendocrine pancreatic tumors (n = 10) or chronic pancreatitis (n = 10), IAR with PanIN1 or no PanIN lesions (n = 5), and healthy controls (n = 10). The combination of both miR-196a and -196b reached a sensitivity of 1 and specificity of 0.9 (area under the curve = 0.99) to diagnose PC or high-grade PanIN lesions. In addition, preoperative elevated serum levels of miR-196a and -196b in patients with PC or multifocal PanIN2/3 lesions dropped to normal after potential curative resection. The combination of miR-196a and -196b may be a promising biomarker test for the screening of IAR for FPC.