Pre-lysosomal divergence of alpha 2-macroglobulin and transferrin: a kinetic study using a monoclonal antibody against a lysosomal membrane glycoprotein (LAMP-1)

We have used electron microscopic immunocytochemistry to compare the distribution of LAMP-1, a marker for lysosomal membranes, with the intracellular localization of alpha 2-macroglobulin (alpha 2-M) and transferrin at various time points after their endocytosis into cultured NIH 3T3 cells. The purposes of this study were (a) to determine how soon endocytic ligands reach lysosomal organelles, (b) to examine whether the intermediate endocytic vesicles gained lysosomal markers gradually or in a precipitous, discrete event, and (c) to examine the relationship, if any, between the pathway of recycling ligands and lysosomes. At early time points (0-5 min) after initiation of endocytosis, most structures containing alpha 2-M labeled with colloidal gold (receptosomes) were not labeled by anti-LAMP-1 detected using ferritin bridge or peroxidase immunocytochemistry. At late time points (greater than or equal to 15 min), the structures containing alpha 2-M (lysosomes) were strongly labeled by anti-LAMP-1. In contrast, transferrin that was directly labeled with ferritin was mostly located in LAMP-1-negative structures at all time points studied. The proportion of alpha 2-M-gold containing vesicles strongly labeled for LAMP-1 roughly paralleled the proportion of alpha 2-M-gold-containing structures positive for cytochemically detectable acid phosphatase. Our data indicate that ligands such as transferrin that are internalized through coated pits and receptosomes, but not delivered to lysosomes, do not traverse a lysosomal organelle compartment as marked by LAMP-1 content. Ligands such as alpha 2-M that are destined for lysosomal delivery reach a LAMP-1-positive organelle compartment only after they traverse LAMP-1-negative, non-lysosomal vesicles previously described as receptosomes.     

Mutations in Methanobacterium formicicum conferring resistance to anti-80S ribosome-targeted antibiotics

Summary Mutants of Methanobacterium formicicum resistant to the anti-80S ribosome-targeted inhibitor anisomycin were isolated and characterized. The resistance phenotype is correlated with a mutationally altered 50S ribosomal subunit. Anisomycin resistance in the mutants is accompanied by cross-resistance to other inhibitors of the 80S peptidyl-transferase centre like narciclasine, bruceantin, trichodermin and verrucarin A and by hypersensitivity to sparsomycin. This phenotype is identical to that reported for anisomycin-resistant mutants of yeast; it appears therefore, that the anisomycin interaction sites on the 70S ribosomes from M. formicicum bear the structural features typical of eukaryotic 80S organelles.     

Lateral diffusion of an 80,000-dalton glycoprotein in the plasma membrane of murine fibroblasts: relationships to cell structure and function

The lateral diffusion of an 80,000-dalton major cell surface glycoprotein of murine fibroblasts has been measured. This antigen, identified through the use of monoclonal antibodies, is an integral glycoprotein distributed through the plasma membrane as judged by immunofluorescence and immunoelectron microscopy (see preceding paper). Measurements of fluorescence recovery after photobleaching were performed on the antigen-antibody complex within the plasma membrane of C3H/10T1/2 and NIH/3T3 cells after labeling the monoclonal antibody with fluorescein. Measurements were performed as a function of temperature, for interphase, mitotic, and G0 C3H/10T1/2 cells. The mean lateral diffusion coefficients (D) for the antibody-protein complex in interphase cells were in the range of 0.7-3.5 X 10(-10) cm2/s between 9 degrees and 37 degrees C, while that for the lipid analog probe, dihexadecylindocarbocyanine was about two orders of magnitude greater. This comparison indicates that peripheral interactions other than bilayer fluidity limit the lateral mobility of the antigen. The mobile fraction of mitotic, G0, and interphase cells showed a monotonic increase with temperature with most of the antibody-antigen complexes being free to move about 25 degrees C. Semi-quantitative interpretations of both the slow glycoprotein diffusion and the immobile fraction are offered. Comparison of diffusion coefficients for cells in different phases of the cell cycle does not reveal striking differences. Mobile fractions for G0 cells at 25 degrees C or less are substantially lower than in interphase cells. In all cases, there was a remarkably broad range of the fluorescence recovery data between different cells, resulting in up to a 10-fold variation in diffusion coefficients, which is far greater than the precision limits of the experiment. Diffusion values and mobile fractions were generally well within a factor of two when measured at several arbitrary points on a single cell. The origins of this cellular heterogenity remain to be elucidated. Lateral mobility in cell fragments and specific regions of single cells was also examined. The glycoprotein was mobile in ventral surface cell fragments. Its mobility was not altered in regions of cell- cell underlapping. However, the diffusion coefficient was threefold higher near the leading edge of motile cells compared to the trailing region. This difference may reflect weaker coupling of the glycoprotein to the underlying cytoskeleton in the dynamic leading edge region.     

Die endokrinen drüsen im embryo von Oncopeltus fasciatus Dallas (Insecta,Heteroptera)

The differentiation of the endocrine glands in the embryo of Oncopeltus fasciatus is described. The function of these glands can be correlated with the embryonic moults. The nuclei of some tissues already become polyploid in the embryo. It is discussed whether the endomitotic growth is dependent upon the function of the endocrine glands.

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Abkürzungen in den Abbildungen A Ca Anlage des Corpus allatum - A Cc Anlage des Corpus cardiacum - A Maxd Anlage der Maxillendrüse - A Meth Anhang des Mesothorax - A Mth Anhang des Metathorax - A Pl Anlage des-Pleuropodiums - A Prd Anlage der Prothoraxdriise - A Pth Anhang des Prothorax - A Spd Anlage der Speicheldrüse - Am Amnion - Ant Antenne - Ao Aorta - Blz Blutzelle - Ca Corpus allatum - Cc Corpus cardiacum - Cul Cuticulinschicht - Cut Cuticula - Des Desmosom - dl denselayer - Do Dotter - Ent Entoderm - Ep Epidermis - Epk Epidermiskern - Fk Fettkörper - fl Do verflüssigter Dotter - Hyp Hypocerebralganglion - Hz Herzschlauch - Kl Kopflappen - La Labrum - Lab Labium - M Mitteldarm mit verschiedenen Abschnitten (I, II, III und IV) - Mal Malpighisches GefäB - Max Maxille - Md Mandibel - Mes Mesoderm - Mik Mikrovilli - Ms Muskel - Mst Mittelstrang - Nbl Neuroblast - NZ Neurosekretorische Zellen - Oen Oenocyt - Oes Oesophagus - Ogl Oberschlundganglion - p Cut Procuticula - Pcz Pericardialzelle - Pi Pleuropodium - Prd Prothoraxdrüse - Pyl Pylorus - Re Rectum - Rk Riesenkern - S Schlüpfakt - Sal Salivarium - Ser Serosa - Sg Segmentgrenze - Spd Speicholdrüse (I = vorderer Lappen, II = scitlicher Lappen, III = hinterer Lappen, IV = akzessorische Drüse und V = Hylus) - Spdg Speicheldrüsengang - St Stechborste - Std Stinkdrüse - Sti Stigma - Stom Stomodaeum - Stt Stechborstentasche - tb E tubuläre Einstülpung des Prothorax - Ugl Unterschlundganglion - V Vakuole Mit Unterstutzung der Deutschen Forschüngsgemcinschaft.     

The influence of pH on growth kinetics of yeasts in the presence of benzoate as a sole carbon source

The inhibitory effect and substrate properties of benzoic acid were estimated for 25 yeast strains belonging to generaCondida, Hansenula, Hypopichia, Rhodosporidium, Rhodotorula, Saitoella andTrichosporon. Benzoic acid can serve as a sole carbon source for growth of yeasts belong to generaRhodotorula, Rhodosporidium andSaitoella in synthetic mineral media. Specific growth rate is strongly dependent both on the concentration of benzoate and the pH value of the cultivation media. Maximum specific growth rate on benzoate is observed in alkaline cultivation media at pH 7.0–7.5 whereas those for growth on glucose in mildly acidic media at pH 5.0. Some of the strains showed weak growth on benzoate even at pH 8.5. Some carotenoid-containing yeasts of the generaRhodotorula andRhodosporidium lost their ability to synthesize carotenoid pigments during growth in alkaline benzoate media.     

Triplet state properties of the methylmercury(II)-tyrosine complex

CH₃Hg(II)OH forms complexes at pH 8 with tyrosine and with tyrosine ethyl ester (TEE) that are detected by ultraviolet difference absorption spectra. With K_f defined by CH₃HgOH + HB

CH₃HgB + H₂O, we find log K_f = 3.61 (tyrosine) and 3.36 (TEE). A heavy-atom effect is observed in frozen glasses of the complexes; this indicates a close interaction between Hg and the chromophore. No UV difference spectrum or heavy-atom effect is observed with N-acetyl tyrosine ethyl ester, indicating that complexing at the phenol O does not occur, and suggesting that binding occurs at the amine N. Zero field optically detected magnetic resonance (ODMR) measurements of the CH₃Hg(II)-tyrosine triplet state give (D, E) = (0.129, 0.047) or (0.134, 0.041) cm^?1 depending upon assignment of transitions. D of tyrosine is relatively unaffected, but E is reduced by CH₃Hg(II) complexing. Low-temperature kinetic measurements show that the shortest lived sublevel of the complex is T_z, where z lies along the phenol long axis in tyrosine. A dominant 11.6-msec component in the 77 K decay of the phosphorescence is consistent with the individual sublevel lifetimes obtained by ODMR.