Uptake of biotin by human hepatoma cell line,Hep G2: A carrier-mediated process similar to that of normal liver

Little is known about the cellular and molecular regulation of the uptake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G₂. Uptake to biotin by Hep G₂ cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na⁺ gradient-dependent as indicated by studies of Na⁺ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of ³H-biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G₂ cells with sulfhydryl group inhibitors (e.g., p-chloromer-curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G₂ cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na⁺ uptake in Hep G₂ cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na⁺-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G₂ cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work, and as such, is in the public domain in the United State of America

.     

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

Evidence for the role of glycoprotein G of respiratory syncytial virus in binding of Neisseria meningitidis to HEp-2 cells

Abstract Viral glycoproteins G and F are expressed on the surface of cells infected with respiratory syncytial virus (RSV). We investigated the role of these proteins in the previously reported enhanced binding of Neisseria meningitidis to RSV-infected HEp-2 cells. Virus particles attached to bacteria were detected by immunofluorescence with flow cytometry. Binding of FITC-labelled bacteria to RSV-infected cells was significantly inhibited by monoclonal antibody against glycoprotein G. Unlabelled bacteria interfered with binding of the anti-G monoclonal antibody to these cells. These interactions were not found with a monoclonal antibody against glycoprotein F. We propose that glycoprotein G of RSV expressed on the surface of infected cells might act as an additional receptor for meningococci.     

Three research priorities for just and sustainable urban systems: Now is the time to refocus

Now is the time to refocus efforts in urban research and design. A changing climate and extreme weather events are presenting unique challenges to urban systems around the world. These challenges illuminate the social barriers that accompany disruptive events such as resource inequities and injustices. In this perspective, we provide three research priorities for just and sustainable urban systems that help to address these matters. The three research priorities are: (1) social equity and justice, (2) circularity, and (3) digital twins. Conceptual context and future research directions are provided for each. For social equity and justice, the future directions are mandatory equity analysis and inclusionary practices, understanding and reconciling historical injustices, and intentional integration with diverse community stakeholders. For circularity applications, they are better metrics for integration, more robust evaluation frameworks, and dynamic modeling at multiple spatial and temporal scales. Future directions for digital twins include developing principles to reduce complexity, integrating model and system components, and reducing barriers to data access. These research priorities are core to meeting several of the United Nations Sustainable Development Goals (i.e., 1—No Poverty, 8—Decent Work and Economic Growth, 10—Reduced Inequalities, and 11—Sustainable Cities and Communities). Useful social and technical matters are discussed throughout, where we highlight the importance of prioritizing localized research efforts, provide guidance for community-engaged research and co-development practices, and explain how these priorities interact to align with the evolving field of industrial ecology.     

High-temperature stress in crops: male sterility,yield loss and potential remedy approaches

Global food security is one of the utmost essential challenges in the 21st century in providing enough food for the growing population while coping with the already stressed environment. High temperature (HT) is one of the main factors affecting plant growth, development and reproduction and causes male sterility in plants. In male reproductive tissues, metabolic changes induced by HT involve carbohydrates, lipids, hormones, epigenetics and reactive oxygen species, leading to male sterility and ultimately reducing yield. Understanding the mechanism and genes involved in these pathways during the HT stress response will provide a new path to improve crops by using molecular breeding and biotechnological approaches. Moreover, this review provides insight into male sterility and integrates this with suggested strategies to enhance crop tolerance under HT stress conditions at the reproductive stage.     

Flow injection assays for NADH and ethanol using photosensitized rose bengal and luminol–copper (II) chemiluminescence system

The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10⁻⁸ and 2.17 × 10⁻⁵ M, and linear range 1.3 × 10⁻⁷ to 2.5 × 10⁻⁵ M (R² = 0.9998, n = 6) and 0.11–2.17 × 10⁻³ M (R² = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h⁻¹ with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly.     

Environmentally friendly protocol for the determination of sitagliptin phosphate in pharmaceutical preparations and biological fluids using l-tyrosine as a fluorescence probe

A responsive spectrofluorometric method was developed for the determination of sitagliptin phosphate using l -tyrosine as a fluorescence probe. The fluorescence intensity of l -tyrosine was quenched with sitagliptin phosphate. The fluorescence intensity was recorded at 307 nm using a 272 nm excitation wavelength. The calibration plot between fluorescence intensity and the concentration of drug was linear in the range of 0.1 to 2.0 mM with a good correlation value of 0.997. The limit of detection and quantification were established to be 3.7 × 10⁻⁴ and 1.23 × 10⁻³ mM, respectively. Commonly used excipients did not interfere with sitagliptin phosphate measurement. The proposed method was used to measure the sitagliptin phosphate in its standard type, dosage form, and biological samples. The percent recovery ranged from 97.41–103.36%. The static quenching was shown to be responsible for quenching as indicated by the Stern–Volmer plot. The method was validated using ICH guidelines and profitably applied for the content uniformity test, resulting in a high percent recovery and small relative standard deviation. The proposed approach is effortless, susceptible, selective, economic, and provides a high precision and accuracy, and can be used to determine sitagliptin phosphate in the pharmaceutical industry.     

New branched Porolithon species (Corallinales,Rhodophyta) from the Great Barrier Reef,Coral Sea,and Lord Howe Island

Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity.