Heterogeneity in photosystem I. Evidence from cation-induced decrease in Photosystem I electron transport

The rate of electron transfer through Photosystem I (reduced 2,6-dichlorophenol indophenol (DCIPH₂ → methylviologen) in a low-salt thylakoid suspension is inhibited by Mg²⁺ both under light-limited and the light-saturated conditions, the magnitude of inhibition being the same. The 2,6-dichlorophenol indophenol (DCIP) concentration dependence of the light-saturated rate in the presence and in the absence of Mg²⁺ shows that the overall rate constant of the photoreaction is not altered by Mg²⁺. With N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethylphenylenediamine as electron donor only the light-limited rate, not the light-saturated rate, is inhibited by Mg²⁺ and the magnitude of inhibition is the same as with DCIP as donor. The results are interpreted in terms of heterogeneous Photosystem I, consisting of two types, PS I-A and PS I-B, where PS I-A is involved in cation-regulation of excitation energy distribution and becomes unavailable for DCIPH₂ → methyl viologen photoelectron transfer in the presence of Mg²⁺.     

Synthesis and secretion of cobalamin binding proteins by opossum kidney cells

Opossum kidney epithelial cells synthesize and secrete two Cobalamin (Cbl) binding proteins of Mr 66,000 and 43,000. When grown on culture inserts, the apical medium contained both these proteins while the basolateral medium contained only the 43 kDa Cbl binder. Colchicine, a microtubule disruptive drug, increased two fold the apical but not the basolateral secretion of the Cbl binding proteins. Although the opossum Cbl binders did not cross react with anti-serum raised to Cbl binders from other species, the identity based on Cbl binding and size suggest that the 66 kDa and 43 kDa proteins are haptocorrin and transcobalamin II.     

Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

Mycobacterium tuberculosis and Escherichia coli nucleoside diphosphate kinases lack multifunctional activities to process uracil containing DNA

E. coli nucleoside diphosphate kinase (EcoNDK) is an important cellular enzyme required to maintain balanced nucleotide pools in the cells. Recently, it was reported that EcoNDK is also a multifunctional base excision repair enzyme, possessing uracil-DNA glycosylase (UDG) and AP-DNA processing activities. We investigated for the presence of such activities in M. tuberculosis NDK (MtuNDK), which shares 45.2% identity, and 52.6% similarity with EcoNDK. In contrast to the robust uracil excision activity reported for EcoNDK, MtuNDK preparation exhibited very poor excision of uracil from DNA. However, this activity was undetectable when MtuNDK was purified from an ung(-) strain of E. coli, or when the assays were performed in the presence of extremely low amounts of a highly specific proteinaceous inhibitor, Ugi which forms an extremely tight complex with the host Ung (UDG), showing that MtuNDK preparation was contaminated with UDG. Reinvestigation of uracil processing activity of EcoNDK, showed that even this protein lacked UDG activity. All preparations of NDK were shown to be active by their autophosphorylation activity. Ugi neither displayed a physical interaction with EcoNDK nor did it affect autophosphorylation of NDKs. Further, neither of the NDK preparations processed the AP-DNA generated by UDG treatment of the uracil containing DNA duplexes. However, partially purified preparations of NDK did process such DNA substrates.     

Method for lipase-catalyzed carbonate synthesis via one- and two-step alkoxycarbonylation reactions

Lipase-catalyzed alkoxycarbonylation methods offer potential advantages over the currently practiced industrial scale chemical synthesis of carbonates. We report a method for synthesis of organic carbonates via lipase-catalyzed alkoxycarbonylation between diphenyl carbonate and various alcohols in hexane. This method utilizes precursors that are readily available and does not involve extensive purification of the intermediate. In a two-step process, the two phenyl groups of diphenyl carbonate were substituted by two alcohol nucleophiles. The approach was demonstrated for two-step synthesis of 14 different disubstituted carbonate products. The rates of reaction for the two steps were much slower if the order of nucleophile addition was reversed. Under optimal conditions, complete conversion of diphenyl carbonate occurred within 8-15 h at 50 degrees C, which is a significant improvement from 50-90 h at 24 degrees C. A kinetic model for the alkoxycarbonylation reaction was derived based on the Michaelis-Menten equation, which simplified to first-order kinetics at low and equimolar concentration of substrates.     

Association between plasma zinc concentration and zinc kinetic parameters in premenopausal women

The objective of this study was to measure relationships between plasma zinc (Zn) concentrations and Zn kinetic parameters and to measure relationships of Zn status with taste acuity, food frequency, and hair Zn in humans. The subjects were 33 premenopausal women not taking oral contraceptives and dietary supplements containing iron and Zn. Main outcomes were plasma Zn concentrations, Zn kinetic parameters based on the three-compartment mammillary model using 67Zn as a tracer, electrical taste detection thresholds, and food frequencies. Lower plasma Zn was significantly (P < 0.01) associated with smaller sizes of the central and the lesser peripheral Zn pools, faster disappearance of tracer from plasma, and higher transfer rate constants from the lesser peripheral pool to the central pool and from the central pool to the greater peripheral pool. The break points in the plasma Zn-Zn kinetics relationship were found between 9.94 and 11.5 micromol/l plasma Zn. Smaller size of the lesser peripheral pool was associated with lower frequency of beef consumption and higher frequency of bran breakfast cereal consumption. Hypozincemic women with plasma Zn <10.7 micromol/l or 700 ng/ml had decreased thresholds of electrical stimulation for gustatory nerves. Our results based on Zn kinetics support the conventional cutoff value of plasma Zn (10.7 micromol/l or 700 ng/ml) between normal and low Zn status.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

Global Spectrum of Copy Number Variations Reveals Genome Organizational Plasticity and Proposes New Migration Routes

Global spectrum of CNVs is required to catalog variations to provide a high-resolution on the dynamics of genome-organization and human migration. In this study, we performed genome-wide genotyping using high-resolution arrays and identified 44,109 CNVs from 1,715 genomes across 12 populations. The study unraveled the force of independent evolutionary dynamics on genome-organizational plasticity across populations. We demonstrated the use of CNV tool to study human migration and identified a second major settlement establishing new migration routes in addition to existing ones.     

Design of a Novel Low Cost Point of Care Tampon (POCkeT) Colposcope for Use in Resource Limited Settings

Introduction

Current guidelines by WHO for cervical cancer screening in low- and middle-income countries involves visual inspection with acetic acid (VIA) of the cervix, followed by treatment during the same visit or a subsequent visit with cryotherapy if a suspicious lesion is found. Implementation of these guidelines is hampered by a lack of: trained health workers, reliable technology, and access to screening facilities. A low cost ultra-portable Point of Care Tampon based digital colposcope (POCkeT Colposcope) for use at the community level setting, which has the unique form factor of a tampon, can be inserted into the vagina to capture images of the cervix, which are on par with that of a state of the art colposcope, at a fraction of the cost. A repository of images to be compiled that can be used to empower front line workers to become more effective through virtual dynamic training. By task shifting to the community setting, this technology could potentially provide significantly greater cervical screening access to where the most vulnerable women live. The POCkeT Colposcope’s concentric LED ring provides comparable white and green field illumination at a fraction of the electrical power required in commercial colposcopes. Evaluation with standard optical imaging targets to assess the POCkeT Colposcope against the state of the art digital colposcope and other VIAM technologies.

Results

Our POCkeT Colposcope has comparable resolving power, color reproduction accuracy, minimal lens distortion, and illumination when compared to commercially available colposcopes. In vitro and pilot in vivo imaging results are promising with our POCkeT Colposcope capturing comparable quality images to commercial systems.

Conclusion

The POCkeT Colposcope is capable of capturing images suitable for cervical lesion analysis. Our portable low cost system could potentially increase access to cervical cancer screening in limited resource settings through task shifting to community health workers.