Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

A hypothesis on p34cdc2 sequestration based on the existence of Ca(2+)-coordinated changes in H+ and MPF activities during Xenopus egg activation [corrected]

The entry into, and exit from, mitosis are controlled by a universal M-phase promoting factor (MPF) composed of at least p34cdc2 and a cyclin. Embryonic systems are convenient for studying the association and dissociation of the active MPF complex because oocytes and eggs are naturally arrested at a specific point of the cell cycle until progression to the next point is triggered by a hormonal signal or sperm. In amphibians, eggs prior to fertilization are arrested at metaphase 2 of meiosis due to the presence of a stabilized MPF complex. Fertilization (egg activation) produces a transient increase in intracellular free Ca2+, a propagating Ca2+ wave, that specifically triggers the destruction of cyclin, leading to MPF inactivation and entry into the first embryonic inter-phase. We have recently shown that intracellular pH (pHi) variations in amphibian eggs, a large increase at fertilization and small oscillations during the embryonic cell cycle, were temporally and functionally related to the corresponding changes in MPF activity. In addition, the recent finding that the pHi increase at fertilization in Xenopus eggs is a propagating, Ca(2+)-dependent pH wave which closely follows the Ca2+ wave, together with the absence in the egg plasma membrane of pHi-regulating systems responsible for that pHi increase, suggest the existence of cortical or subcortical vesicles acidifying in the wake of the Ca2+ wave, thus producing the pH wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.     

Construction of a new universal vector for insertional mutagenesis by homologous recombination

We describe here the construction of a vector (pSSC-9) which can be used for the insertional mutagenesis of any gene for which genomic sequences have been cloned. This vector contains a neomycin-resistance-encoding gene (neo^R) which is driven by a modified thymidine kinase (tk) promoter for positive selection. Flanking neo^R are two tk genes driven by their own promoters for negative selection of nonhomologous insertions. The neo^R and tk cassettes are separated by four unique cloning sites on the right-hand side of the neo^R cassette and three unique sites on the left-hand side. The vector also includes two SfiI sites, one on each side of the tk cassettes, for the excision of the cloned genomic DNA fragments along with the selectable markers. Electroporation of pSSC-9 into mouse embryonic stem (ES) cells and cultured diploid mouse adrenal Y-1 cells conferred resistance to G418 and sensitivity to ganciclovir in both cell lines. These results illustrate the expression of the positive and negative selectable markers in two different cell lines and thus suggest that the vector could be used in ES cells, as well as in cultured somatic cells.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

A free-radical hypothesis for the instability and evolution of genotype and phenotypein vitro

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., anin vitro evolution, which includes crisis, spontaneous transformation (immortalization), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by thein vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause ofin vitro evolution. We propose an oxyradical hypothesis to explain each of the stages ofin vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.ORR, CDER, FDA Mod-1, Room 2023, 8301 Muirkirk Road, Laurel MD 20708, USA     

In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

Isolation of third, fourth, and fifth instar larval midgut epithelia of the moth, Trichoplusia ni

A new tissue isolation technique was used to create intact midgut epithelial wholemounts from three Trichoplusia ni (Lepidoptera: Noctuidae) larval instars. The protease, dispase, removed the basal lamina and associated connective tissue and allowed for high resolution light microscopy of entire epithelia. Columnar, goblet, differentiating, and stem cells were characterized by double fluorescent labelling of f-actin and nuclei. A comparison of cell populations by digital image analysis revealed significant regional and temporal changes in the density and number of differentiating and stem cells. Growth of the midgut epithelium from third to fourth instar, and from fourth to fifth instar, was accomplished by both cell differentiation and cell division. Cell division however, was greatly reduced from fourth to fifth instar with a concomitant sharp decrease in the stem cell population.