Receptor-mediated internalization of high density lipoprotein by rat sinusoidal liver cells: identification of a nonlysosomal endocytic pathway by fluorescence-labeled ligand

Rat sinusoidal liver cells possess the surface receptor for high density lipoprotein (HDL) (Murakami, M., S. Horiuchi, K. Takata, and Y. Morino. 1987. J. Biochem. (Tokyo) 101: 729-741). The present study was undertaken to determine whether cell surface-bound HDL underwent subsequent endocytic internalization by using 125I-labeled HDL and fluorescein isothiocyanate-labeled HDL (FITC-HDL). The cell-associated radioactivity obtained by a 40-min incubation with 125I-labeled HDL at 37 degrees C was released into the medium as acid-precipitable forms upon further incubation at 37 degrees C. When further incubated at 0 degree C instead of 37 degrees C, however, this release was significantly reduced. A similar phenomenon was observed after the cell-associated ligands had been treated with trypsin. The cell-associated ligands obtained after a 1-hr incubation with 125I-labeled HDL at 0 degree C were largely counted for by those bound to the outer surface of the cells, thus suggesting that HDL is internalized into cells at 37 degrees C but not at 0 degree C. Moreover, when cells were incubated with FITC-HDL at 0 degree C, the cell-associated ligands were found in a pH 7.2 +/- 0.1 compartment, whereas when incubated at 37 degrees C, its microenvironmental pH became much more acidic, exhibiting pH 6.2 +/- 0.1. Furthermore, this value returned to 7.1 +/- 0.1 upon treatment with carbonylcyanide m-chlorophenylhydrazone known to dissipate the total protonomotive force. These results suggest, therefore, that the internalization process does follow receptor-mediated binding of HDL in rat sinusoidal liver cells. This notion was also supported by fluorescence microscopic observations.     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.     

Breeding of Barbus bynni (Pisces,Cyprinidae) in Jebel Aulia Reservoir,Sudan

Barbus bynni begins to mature at Age IV. Ripening of gonads of mature fish starts in May when water temperature approaches the annual maximum. However, the spawning season coincides with the onset of the flood season in July. These facts, as well as the cyclic growth of the gonads, show that B. bynni spawns once a year. Fecundity varies with size of fish and gonads. However, this levels off in the middle size group. At this age the fecundity was estimated to be 1 424 693 eggs.     

Food and feeding habits of Labeo niloticus (Pisces,Cyprinidae) in Jebel Aulia Reservoir,Sudan

Basic knowledge on the feeding ecology of one of the common and commercially important fish species in Jebel Aulia Reservoir is provided.The structure of the feeding apparatus indicates that Labeo niloticus is a bottom feeder, depending on soft and decayed vegetation, organic debris and whatever small organisms found within. However, juveniles and fry are prone to explore all layers and depths of the river selectively for plankton. There is little evidence of seasonal selection of food. Changes in diet quality are governed by the availability of type of food. Variability of feeding activity is connected with climate and breeding season.     

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins

Intratumoral phenotypic diversity is well documented with regard to tumor associated carbohydrate antigens (TACA). The factors which control the expression of these cell-surface oligosaccharides on different cells of the same tumor are not understood. We investigated the expression of a panel of mucin associated oligosaccharides in cell lines growing at different surface densities (number of cells per cm² of growth flask). Results show that the apparent expression of extended Le^a-Le^x, Le^a and Le^x, sialyl Le^a, Tn and sialyl Tn varies with density of growth by an invasive human squamous cell lung carcinoma cell line (NU6-1), a benign variant (NE-18) and the human lung epithelial cell line BEAS-2B. The results indicate that one of the factors influencing the apparent expression of mucin-associated oligosaccharides is cell-cell interactions.Abbreviations Mab monoclonal antibody - FIT fluorescein isothiocyanate - TACA tumor associated carbohydrate antigen     

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.     

Polysaccharide production by Aureobasidium pullulans: factors affecting polysaccharide formation

Aureobasidium pullulans NRRL 6220 synthesized polysaccharide most actively in media containing sucrose, fructose or maltose with (NH₄)₂SO₄ (0.6 g/l) or ammonium acetate giving greatest yields of the polysaccharide. With (NH₄)₂SO₄ at 1.2 g/l, production of polysaccharide was decreased considerably. Polysaccharide production was highest with an initial pH of 6.5 while biomass formation was better below an initial pH of 5.5. Optimum phosphate concentration for polysaccharide production was 0.03 m.S.M. Badr-Eldin, H.G. El-Masry and O.A. Abd El-Rahman are with the Microbial Chemistry Department, National Research Center, Dokki, Cairo, Egypt; F.H.A. Mohamad is with the Chemical Engineering and Pilot Plant Department, National Research Center, Dokki, Cairo, Egypt. O.M. El-Tayeb is with the Microbiology Department, Faculty of Pharmacy, Cairo University, Egypt.     

Evaluation of scanning resolution,detector choice and detector orientation to be used for accurate and time-efficient commissioning of a 6MV clinical linear accelerator

The present study is aimed at exploring different scanning parameters, detectors and their orientations for time-efficient and accurate commissioning of a 6 MV clinical linear accelerator (LINAC). Beam profiles and percentage depth dose (PDD) curves were measured with a PTW dosimetry diode, a PTW Semiflex and a PinPoint ion chamber in different orientations. To acquire beam data, equidistant (step size of 0.5 mm, 1 mm, 2 mm and 3 mm) and fanline (step size of 2–0.5 mm, 2–1 mm, 3–0.5 mm and 3-1 mm) scanning modes were employed and data measurement time was recorded. Scan time per measurement point was also varied (0.2 s, 0.5 s and 1.0 s) to investigate its effect on the accuracy and acquisition time of beam data. Accuracy of the measured data was analyzed on the basis of the variation between measured data and data modeled by a treatment planning system. Beam profiles (particularly in penumbra region) were found to be sensitive to variation in scanning resolution and showed an improved accuracy with decrease in step size, while PDD curves were affected negligibly. The accuracy of beam data obtained with the PTW dosimetry diode and the PinPoint ion chamber was higher than those obtained with the PTW Semiflex ion chamber for small fields (2?×?2 cm² and 3?×?3 cm²). However, the response of the PTW diode and the PinPoint ion chamber was significantly indifferent in these fields. Furthermore, axial orientation of the PTW Semiflex ion chamber improved accuracy of profiles and PDDs as compared to radial orientation, while such a difference was not significant for the PinPoint ion chamber. It is concluded that a scan time of 0.2 s/point with a fanline scanning resolution of 2–1 mm for beam profiles and 3 mm for PDDs are most favorable in terms of accuracy and time efficiency. For small fields (2?×?2 cm² and 3?×?3 cm²), a PinPoint ion chamber in radial orientation or a dosimetry diode in axial orientation are recommended for both beam profiles and PDDs. If a PinPoint ion chamber and a PTW dosimetry diode are not available, a Semiflex ion chamber in axial orientation may be used for small fields.

     

Direct molecular analysis of the fragile X syndrome in a sample of Egyptian and German patients using non-radioactive PCR and Southern blot followed by chemiluminescent detection

Molecular genetic analysis of individuals from 6 Egyptian and 33 German families with fragile X syndrome and 240 further patients with mental retardation was performed applying a completely non-radioactive system. The aim of our study was the development of a non-radioactive detection method and its implementation in molecular diagnosis of the fragile X syndrome. Furthermore, we wanted to assess differences in the mutation sizes between Egyptian and German patients and between Egyptian and German carriers of a premutation. Using non-radioactive polymerase chain reaction (PCR), agarose gel electrophoresis and blotting of the PCR products, followed by hybridisation with a digoxigenin-labelled oligonucleotide probe (CGG)₅ and chemiluminescent detection, we identified the fragile X full mutation (amplification of a CGG repeat in the FMR-1 gene ranging from several hundred to several thousand repeat units) in all patients. We observed no differences in the length of the CGG repeat between the Egyptian and German patients and carriers, respectively. However, in one prenatal diagnosis, we detected only one normal sized allele in a female fetus using the PCR-agarose assay, whereas Southern blot analysis with the digoxigenin labelled probe StB 12.3 revealed presence of a full mutation. Our newly established nonradioactive genomic blotting method is based on the conventional radioactive Southern blot analysis. Labelling of the probe StB 12.3 with digoxigenin via PCR allowed the detection of normal, premutated and fully mutated alleles. For exact sizing of small premutated or large normal alleles, we separated digoxigenin labelled PCR products through denaturing poly-acrylamide gelelectrophoresis (PAGE) and transfered them to a nylon membrane using a gel dryer. The blotted PCR-fragments can easily be detected with alkaline phosphate-labelled anti-digoxigenin antibody. The number of trinucleotide repeat units can be determined by scoring the detected bands against a digoxigenated M13 sequencing ladder. Our newly developed digoxigenin/chemiluminescence approach using PCR and Southern blot analysis provides reliable results for routine detection of full fragile X mutations and premutations.