β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

Structure of the parathyroid glands,as revealed by different methods of fixation

Summary Stereology and semi-automatic image analysis were used with the aim of comparing the structure of parathyroid glands from untreated adult Mongolian gerbils fixed by immersion with those fixed by perfusion. Subclassification of the chief cells based upon the staining affinity or electron density of the cytoplasm was readily performed only in glands fixed by immersion, and so-called atrophic cells were observed only in these glands. The atrophic cells were often surrounded by light chief cells. In glands fixed by perfusion, light chief cells were only rarely encountered. A significant difference between glands fixed by immersion and those fixed by perfusion was found only with regard to the form of cells and nuclei, those fixed by perfusion being more spherical. When comparing individual cells within glands fixed by immersion, light chief cells were more spherical and had a significantly larger nuclear and cellular size, and a lower mitochondrial volume density than the intermediate/dark chief cells. Otherwise there were no significant differences in any of the parameters investigated. These data indicate that occurrence of socalled light chief cells and atrophic cells is a result of improper fixation. The results of this study do not favour the concept of a functional cycle with a simultaneous occurrence of active and inactive cells within parathyroid glands.     

Specific fluorescent bands on chromosomes produced by acridine orange after prestaining with base specific non-fluorescent DNA ligands

Metaphase chromosomes stained with acridine orange exhibit uniform yellow-green fluorescence. Chromosome preparations treated with the non-fluorescent A-T specific antibiotic distamycin A prior to acridine orange staining exhibit longitudinal fluorescent banding patterns similar to those produced by a number of fluorescent R-band techniques. Similarly, chromosome preparations treated with the non-fluorescent G-C specific antibiotic actinomycin D followed by acridine orange staining exhibit Hoechst-type banding patterns. Interactions of various ligand-DNA combinations in solution indicate that the base pair specific antibiotics induce banding patterns by selectively altering acridine orange binding sites in chromosomal regions rich in the particular base pair for which the antibiotic exhibits specificity.     

Cytoplasmic protein network in HeLa cells

Summary Monolayer HeLa culture nuclei isolated in situ with nonionic detergents remained attached to the glass as a nuclear monolayer. Searching for nuclear fixing structures a fine continous protein network was revealed around the isolated nuclei with fluorescence microscope after acridine orange staining.     

Partial mispairing and crossing-over between β0 and δ genes as the origin of the δ β0 thalassemia gene

Summary Two double heterozygous ⁰/⁰ thalassemic sibs of Mexican descent were studied. The father had a ⁰/⁰ genotype, while the mother, one sib and several maternal relatives were ⁰/⁰ heterozygotes. Parental consanguinity and an apparently low frequency of thalassemia among Mexicans suggested a possible common origin of both ⁰ and ⁰ genes. A hypothesis to explain such a possibility is proposed on the basis of a partial mispairing between ⁰ and genes followed by a crossing-over which would results in a ⁰ recombinant gene. This hypothesis could also be extended to explain the 22 gluala, 22 alaglu and 116 arghis Hb variants as recombinants from double crossing-over between and mispaired genes for which the name interstitial-Lepore is proposed.     

Mutant of Escherichia coli with unusual patterns of rpoB,C expression in response to rifampicin and acridine orange

Summary A mutation located near rpoB (89) in E. coli is responsible for unusual patterns of and (but not L7/L12) synthesis in response to the drugs rifampicin and acridine orange.     

Frequency and distribution of chiasmata in Syrian hamster spermatocytes studied by the BrdU antibody technique

The frequency and distribution of chiasmata and the nature of terminal associations was re-examined in Syrian hamster spermatocytes using the 5-bromo-2-deoxyuridine (BrdU) antibody technique (BAT) for differential chromatid labelling. Differential chromatid substitution was achieved by BrdU incorporation at the penultimate pre-meiotic S-phase followed by one of three different staining protocols: (i) fluorescence plus Giemsa (FPG), (ii) acridine orange staining or (iii) BAT. For analysis of chiasmata frequency and localization in the diplotene/diakinesis stages the resolution of FPG and acridine orange staining was comparable to that of BAT. In metaphase II chromosomes BAT was more informative than FPG and acridine orange staining and revealed small, terminal crossover exchanges. This finding proves that many terminal associations of meiotic chromosomes actually represent chiasmata at the end of the first meiotic division. Some crossover exchanges were localized in the constitutive heterochromatin of autosomes. Using BAT we also detected crossover exchanges in close vicinity to each other. This observation is reminiscent of the fact that crossing over interference means a reduction in frequency and does not imply total inhibition.     

An elongated segment of DNA observed between two human α globin genes

Summary Detailed restriction enzyme analysis of the DNA from a Chinese female showed that one of her chromosomes had a >17.5 kb deletion of DNA, including the , 2, and 1 globin genes, which is present in many Southeast Asians with an -thalassemia-1 chromosome. Her normal chromosome had the expected cluster of -like globin genes (5----2-1-3), but the segment of DNA between the two globin genes was elongated by some 0.5–0.7 kb. Analyses of various restriction sites suggested that this normal variant of the human globin gene complex is due to a crossover between a normal chromosome with () and a chromosome with an -thalassemia-2 (–^3.7) and an -21-hybrid gene.     

Decrease of P-Glycoprotein Activity in K562/ADR Cells by MβCD and Filipin and Lack of Effect Induced by Cholesterol Oxidase Indicate That This Transporter Is Not Located in Rafts

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl--cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, light, heavy, and extra heavy fractions we show that no P-gp is found in the light fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.     

Interactions between interferon γ and retinoic acid with transforming growth factor β in the induction of immune recognition molecules

The cell-surface expression of major histocompatibility (MHC) antigens and the adhesion molecule intercellular adhesion molecule 1 (ICAM-1) is essential for target cell recognition by T lymphocytes. The expression of both classes of molecule is induced by various cytokines, notably interferon (IFN). Since transforming growth factor (TGF) has been recently reported to antagonise HLA-DR induction by IFN we have examined, using a number of murine and human cell lines, the effect of TGF on IFN-induced MHC class I and class II and ICAM-1 expression. All of the cell lines tested expressed elevated class I MHC following IFN treatment. Class II MHC induction was seen on most but not all of the cells, the exceptions being among a panel of human colorectal carcinoma cell lines. A striking difference between cells of different origin was noted in the response to TGF. TGF was found to antagonise IFN-induced class I and class II MHC expression on C3H 10T1/2 murine fibroblasts, early-passage BALB/c mouse embryo fibroblasts, a murine oligodendroglioma cell line, and on MRC5 human fibroblasts and two human glioblastoma cell lines. Class II MHC was much more strongly inhibited (sometimes completely) than class I MHC. TGF also inhibited induction of class I MHC expression by IFN. However, TGF did not inhibit class I or class II MHC induction by IFN in any of the nine colorectal carcinoma cell lines, although two of five of the lines tested were growth-inhibited by TGF. On the other hand, human ICAM-1 induction by IFN was not affected by simultaneous treatment with TGF in any of the cell lines. The down-regulation of IFN-induced MHC antigens by TGF is not, therefore, the result of a general antagonism of IFN. Retinoic acid has recently been reported to induce ICAM-1 expression on human tumour cells. We have confirmed this observation on MRC5, and the two human glioblastoma cell lines, however six colorectal carcinoma cell lines tested did not respond. In contrast to IFN-induced ICAM-1 expression, retinoic-acid-induced ICAM-1 expression was inhibited by TGF on two of the three responsive lines.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Über das Wachstum der Pankreaszellen von Säugetieren als Monolayer Cultures

Zusammenfassung Fetale menschliche Pankreaszellen bilden sowohl spontan auf zuerst gewachsenem Zellrasen (self-aggregation) als auch in Rollröhrchen (48 rph) (rotation-mediated-aggregation) histotypische Aggregate, die A- und B-Zellen enthalten. In der Nährlösung dieser 6–17 Tage lang gewachsenen Gewebestückchen konnte, Insulin (=IRI=immunoreactive-insulin) nachgewiesen werden.

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Monolayer cultures of pancreatic tissueII. Formation of histotypic aggregates from cell suspensions of fetal human pancreas

Summary Fetal Pancreas cells of man, whether in self-aggregation or rotation-mediated-aggregation, form spontaneously histotypical aggregates containing A- and B-cells. It is possible by means of an immunological method to determine insulin (=IRI=immuno-reactive-insulin) in the nutrient medium of these 6 to 17 days old tissue aggregates.

     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

A model for neural representation of binocular disparity in striate cortex: distributed representation and veto mechanism

A model in striate cortex is proposed for a distributed neural representation of binocular disparity with a simple cell. In the model, disparity is represented by far, near and tuned inhibitory simple cells. However, the representation will be vetoed by model cells where disparity is excessively large. The veto mechanism consists of a neural network of the model cell which received output from simple cells and which interacts with neighbors. The mechanism is necessary, the model cell responds like a simple cell, and the network is physiologically plausible in the brain. Computer simulation on the neural network model with random dot stereography indicates reasonable performance.     

Role of the Na,K-ATPaseβ-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors

Summary No functional role could yet be established for the glycosylated -subunit of the Na,K-ATPase. In this study, we describe the intracellular processing of the -subunit as a glycoprotein in toad bladder cells and the consequences of its structural perturbation with glycosylation inhibitors on the cellular expression of the - and -subunits and on the structural and functional maturation of the enzyme. Controlled trypsinolysis of homogenates from pulse-labeled cells reveals that the -subunit is subjected to glycosylation-dependent structural rearrangements during its intracellular routing. Inhibition of correct terminal glycosylation of the -subunit with deoxynojirimycin or swainsonine has no effect on the trypsin sensitivity of the -subunit, its ability to perform cation-dependent conformation changes or the cellular Na,K-ATPase activity. Acquisition of core-sugars is sufficient for the enzyme to assume its catalytic functions. On the other hand, complete inhibition of glycosylation with tunicamycin leads to a destabilization of both the - and the -subunits as judged by their higher trypsin sensitivity. In addition, tunicamycin treatment results in a decrease of the amount of newly synthesized - and -subunit indicating that a glycoprotein, possibly the -subunit itself, plays a role in the efficient accumulation of the -subunit in the endoplasmic reticulum.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Effects of D‐mannoheptulose upon D‐glucose metabolism in tumoral pancreatic islet cells

D[³H]mannoheptulose was recently reported to be poorly taken up by tumoral pancreatic islet cells of the RINm5F and INS1 lines. We have now investigated the effects of Dmannoheptulose upon Dglucose metabolism in these two cell lines. Dmannoheptulose (1.0–10.0 mM) only caused a minor decrease of Dglucose metabolism in RINm5F cells, whether at low (1.1 mM) or higher (8.3 mM) Dglucose concentration. A comparable situation was found in INS1 cells examined after more than 20 passages. In both cases, however, the hexaacetate ester of Dmannoheptulose (5.0 mM) efficiently inhibited Dglucose metabolism. In the INS1 cells, the relative extent of the inhibitory action of Dmannoheptulose upon Dglucose metabolism increased from 12.4 ± 2.6 to 38.3 ± 3.8% as the number of passages was decreased from more than 20 to 13–15 passages, the latter percentage remaining lower, however, than that recorded in INS1 cells also examined after 13–15 passages but exposed to Dmannoheptulose hexaacetate (66.9 ± 2.2%). These findings when compared to our recent measurements of D[³H]mannoheptulose uptake, reinforce the view that the entry of the heptose into cells and, hence, its inhibitory action on Dglucose metabolism are dictated by expression of the GLUT2 gene.     

The structure of the incompatibility factors of Schizophyllum commune: Constitution of the three classes of B factors

Summary The B factors of Schizophyllum commune are of 3 classes: The high recombining class I has 7 alleles and 7 alleles; the low recombining classes are class II with 7 allels and probably 2 alleles and class III with probably 2 (or also 2) alleles and 7 allels. A fourth hypothetical class (-) was not found and either does not exist or is indistinguishable from class III by the tests employed. The and alleles differ from and by either (a) mutations affecting both mating specificity and recombination frequency, or (b) deletions involving most of the B region.The research was supported by a grant from the Atomic Energy Commission of the U.S. No. (30-1)-3875 and was performed at the Biological Laboratories, Harvard University, Cambridge, Mass., U.S.A.