The isolation of barley-aleurone protoplasts

Summary A procedure is described for the isolation of large numbers of viable aleurone protoplasts. After treatment with Onozuka cellulase protoplasts were obtained which were surrounded by a thin, Onozuka-resistant wall. These cells were termed spheroplasts. Treatment of spheroplasts with Glusulase digested away the residual wall, yielding naked protoplasts. Measurements of respiration using a Clarke-type oxygen electrode indicated that aleurone cells isolated by this procedure were viable.Work supported by National Science Foundation grant GB-27468.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

T cells in murine lupus: propagation and regulation of disease

MRL/Mp-lpr/lpr mice develop a spontaneous lupus syndrome, including hypergammaglobulinemia, autoantibodies, glomerulonephritis, and lymphadenopathy. To investigate the role of lymphocyte subsets in the pathogenesis of disease, lupus-prone MRL mice deficient in T cells, T cells, or both were generated. Mice deficient in T cells developed a partially penetrant lupus syndrome, characterized by lymphadenopathy, elevated levels of class-switched immunoglobulins, an increased incidence of antinuclear antibodies, and immune deposits in kidneys which progressed to renal insufficiency over time. In comparison to wild type animals, T cell-deficient animals developed an accelerated and exacerbated disease phenotype, characterized by accelerated hypergammaglobulinemia and enhanced autoantibody production and mortality. Repertoire analysis of these latter animals identified polyclonal expansion (V) of CD4+B220-cells. Mice lacking both and T cells failed to generate class-switched autoantibodies and immune complex renal disease. First, these findings demonstrate that murine lupus in the setting of Fas-deficiency does not absolutely require the presence of T cells, and they also suggest that a significant basis for MRL/lpr disease, including renal disease, involves T cell-independent, T cell dependent, polyreactive B cell autoimmunity, upon which T cell-dependent mechanisms aggravate specific autoimmune responses. Second, these data indicate that T cells partake in the regulation of systemic autoimmunity, presumably via their effects on CD4+B220-T cells that provide B cell help. Finally, these results demonstrate that MRL/lpr B cells, despite their intrinsic abnormalities, cannot per se cause tissue injury without T cell help.Abbreviations snRNPs small nuclear ribonucleoprotein particles     

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae

Summary A 2 m circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in high-copy and low-copy number cells was determined. High-copy number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.     

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.     

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.     

Induction,polarity and spatial control of cytokinesis in some abnormal subsidiary cell mother cells ofZea mays

Summary Cytokinesis in the subsidiary cell mother cells (SMCs) ofZea mays leaves grown in the presence of 5 mM of caffeine solution is usually partially inhibited. A continuous wall strip, resembling a portion of the subsidiary cell (SC) wall, is laid down in the preprophase microtubule band (PMB) cortical zone. Sometimes, the incomplete SC (SC) wall grows centripetally in the absence of a phragmoplast and the gap becomes smaller or closes. The SC nucleus escapes through the SC wall gap into the larger SMC compartment and may fuse with the other nucleus.The aberrant SMCs (a-SMCs) pass through another division cycle, reattempting to produce a SC. A typical PMB is found in the SC space, in the site of the previous PMB. Moreover, in some preprophase SMCs, the cytoplasm adjacent to the SC wall is traversed by a small number of microtubules. The preprophase nuclei are partly or totally separated from the PMB by the perforated SC wall and may lie far from the latter.Usually, one mitotic spindle is assembled. The cycling paired polarized nuclei appear to synchronize and their chromosomes line up together on a single metaphase plate. Although the mitotic spindle axis is diversely oriented, one of its poles tends to be stabilized in the proximity of the SC wall gap. These divisions separate abnormal cells. Most or all the cell plate edges fuse with wall regions far from the PMB cortical zone. However, when some of them approach the SC wall strips, they are attracted and intersect their rims. In rare occasions the cell plate, invading the SC space is guided by the PMB cortical zone to create a SC-like curved wall portion, in absence of a daughter nucleus.Observations show that the cell plate arrangement in redividing aberrant SMCs is not subjected to a strict spatial control. The disorder of polarization sequence generated by the SC wall ring and especially the perturbation of the spatial (and functional?) relationship between PMB-PMB cortical zone and the nucleus—mitotic spindle is a causal factor of the variable cell plate arrangements.     

A cyto-biochemical study of the ‘canal’ formation in the yeast cell wall

Summary A distinction between the chemical composition of ultrastructurally modified regions and the rest of the cell wall (canals) of the yeast Schwanniomyces occidentalis was shown by cytochemical staining of cell wall polysaccharides. The formation of canals was induced by cultivation of yeasts on hydrocarbons and was parallelled by the enhancement of -glucosidase, -glucanase and -mannosidase activities which were all capable of degrading cell wall polysaccharides. The presence of cycloheximide prevented canal formation. We assume that these hydrolases modified definite cell wall regions transforming them into canals.     

A glass bead treatment facilitating the fixation and infiltration of yeast and other refractory cells for electron microscopy

Summary Physical removal of the cell wall of yeast and other fungal cells, by rapid mixing of the cells with glass beads after preliminary fixation in glutaraldehyde or formalin, removes the cell wall barrier to fixation and/or infiltration of the cells for electron microscopy. The technique has been used on a variety of fungal cells which have been difficult to fix for electron microscopy, and appears to have wide applicability.     

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the δ isoform during neural differentiation

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC , PIC , PIC . These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxyUridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC was detected in the nucleus whereas PIC was only cytoplasmic; PIC was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the isoform was concentrated near the nucleus, while the immunoreactivity of the form remained constant and the form was increased. After 10 days of treatment with NGF, PIC was hardly detectable and PIC immunostaining was considerably decreased. On the contrary, PIC progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC and PIC were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC and and the increase of PIC isozyme associated with the decrease of cell proliferation.     

A novel oxylipin-associated ‘ghosting’ phenomenon in yeast flocculation

Research on the distribution of oxylipins (3-hydroxy fatty acids) in flocculant strains of the yeast Saccharomyces cerevisiae led to the uncovering of a novel ghosting phenomenon observed during assumed lectin-mediated aggregation. We found that intracellular oxylipin-containing osmiophilic layers migrate through yeast cell walls in a ghostlike fashion without visually affecting the cell wall structure or the layers. This migration resulted in the binding of these layers to cell walls of adjacent cells. Consequently, ghosting seems a prerequisite for flocculation to occur. However, ghosting alone may not be sufficient to ensure flocculation.     

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.     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

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.     

Isolation of a β-glucosidase binding and activating polysaccharide from cell walls of Trichoderma reesei

The extracellular -glucosidase from the filamentous fungus Trichoderma reesei QM 9414 is mainly bound to the cell wall of the fungus and only partially released into the medium. Isolation of the cell walls and its hydrolysis by enzymatic treatment with Aspergillus niger cellulase released -glucosidase, which appeared tightly associated with a cell wall polysaccharide. This polysaccharide was purified by gel filtration and ion exchange chromatography and was shown to consist of mannose, galactose, glucose, galacturonic acid and glucuronic acid. It was devoid of protein and phosphate. It reassociated both with extracellular -glucosidase as well as -glucosidase released from the fungus' cell wall. Addition of the polysaccharide to the -glucosidase in vitro increased the enzyme's activity against 4-nitrophenyl--glucoside twofold. These findings suggest, that the isolated polysaccharide functions as an anchor glycan for the -glucosidase in Trichoderma reesei.     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.     

Involvement of β 1,4 galactosyltransferase 1 and Galβ 1→4GlcNAc groups in human hepatocarcinoma cell apoptosis

1,4 galactosyltransferase 1 ( 1,4GT1) synthesizes Gal 14GlcNAc groups in N-linked sugar chains of animal glycoproteins, which have been demonstrated to play an important role in many biological events, including sperm-egg interaction, cell migration and mammalian embryonic development. In this study, the mRNA level of 1,4GT1 was found to increase greatly during the 7721 hepatocarcinoma cells apoptosis induced by cycloheximide. Ricinus Communis Agglutinin-I staining indicated generous increase of Gal 14GlcNAc groups during apoptosis. Further study showed that the 7721 hepatocarcinoma cells transiently transfected with 1,4GT1 were more susceptible to the apoptosis induced by cycloheximide. The increased susceptibility was in accordance to the transfection concentration of 1,4GT1, which also led to the increased Gal 14GlcNAc groups on the transfected cell surface. All the observations suggested that 1,4GT1 and Gal 14GlcNAc groups might be associated with the apoptosis of human hepatocarcinoma cells.     

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.     

Biosynthesis regulation of the β-glucosidase produced by a yeast strain transformed by genetic engineering

The biosynthesis of the -glucosidase enzyme was studied in a transformed yeast obtained by cloning in Saccharomyces cerevisiae the structural gene coding for -glucosidase in Kluyveromyces fragilis. The enzyme biosynthesis was found to be non-adaptative, and repressed by glucose. These features are similar to those observed in K. fragilis. -Glucosidase activity in the transformed yeast was much higher than in K. fragilis. We attempted to ferment cellobiose with the transformed yeast: practically no cellobiose was consumed, growth and ethanol production were negligible. Warburg experiments showed that cellobiose fermentation did not occur when the respiratory chain was not functioning.