Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells. A double immunocytochemical study

Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP anti-body producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin (mu or gamma chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin anti-body-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

C(alpha) chemical shift tensors in helical peptides by dipolar-modulated chemical shift recoupling NMR

The C chemical shift tensors of proteins contain information on the backbone conformation. We have determined the magnitude and orientation of the C chemical shift tensors of two peptides with -helical torsion angles: the Ala residue in G*AL (=–65.7°, =–40°), and the Val residue in GG*V (=–81.5°, =–50.7°). The magnitude of the tensors was determined from quasi-static powder patterns recoupled under magic-angle spinning, while the orientation of the tensors was extracted from C–H and C–N dipolar modulated powder patterns. The helical Ala C chemical shift tensor has a span of 36 ppm and an asymmetry parameter of 0.89. Its ₁₁ axis is 116° ± 5° from the C–H bond while the ₂₂ axis is 40° ± 5° from the C–N bond. The Val tensor has an anisotropic span of 25 ppm and an asymmetry parameter of 0.33, both much smaller than the values for -sheet Val found recently (Yao and Hong, 2002). The Val ₃₃ axis is tilted by 115° ± 5° from the C–H bond and 98° ± 5° from the C–N bond. These represent the first completely experimentally determined C chemical shift tensors of helical peptides. Using an icosahedral representation, we compared the experimental chemical shift tensors with quantum chemical calculations and found overall good agreement. These solid-state chemical shift tensors confirm the observation from cross-correlated relaxation experiments that the projection of the C chemical shift tensor onto the C–H bond is much smaller in -helices than in -sheets.     

Secretion of β-lactamase by Escherichia coli in vivo and in vitro: effect of cerulenin

The effect of cerulenin on the production of -lactamase and other periplasmic proteins was studied in Escherichia coli IA199 carrying plasmid pBR322. Cerulenin (10 to 25 g/ml) had almost no effect on the growth rate of E. coli but it decreased the amount of -lactamase and other periplamic proteins in shock fluid. Higher amounts of the antibiotic (40 to 100 g/ml)decreased turbidity and almost completely prevented synthesis of -lactamase and other periplasmic proteins. Cerulenin decreased incorporation of l-[³⁵S]methionine into membranes during growth as well. Spheroplasts secreted -lactamase into the external medium, but during a 3-h incubation in the presence of cerulenin (25 g/ml) this secretion was prevented by more than 90%. -Lactamase was secreted into the isolated membrane vesicles from E. coli IA199. However, only 5% of the total amount of pre--lactamase was secreted and processed by the membranes in vitro. Cerulenin did not prevent processing in vitro but the membranes prepared from the cells grown in the presence of cerulenin (25 g/ml) did not catalyze processing of pre--lactamase at all. Membrane preparations from Bacillus subtilis did not process pre--lactamase either in the absence or in the presence of cerulenin.     

Constitutive and elicitation induced metabolism of isoflavones and pterocarpans in chickpea (Cicer arietinum) cell suspension cultures

Constitutive phenolics of chickpea cell suspension cultures are the isoflavones formononetin and biochanin A, the isoflavanones homoferreirin and cicerin and the pterocarpans medicarpin and maackiain. They accumulate as vacuolar malonylglucosides. The biosynthetic pathways to isoflavones, pterocarpans and malonylglucoside conjugates together with their enzymes are explained. Elicitation of cell cultures leads to pronounced increases in the activities of biosynthetic enzymes with differential effects on the enzymes involved in conjugate metabolism. Low elicitor doses favour pterocarpan conjugate formation whereas high doses lead to pterocarpan aglycone accumulation accompanied by vacuolar efflux of formononetin and pterocarpan malonylglucosides. Elicitor-induced changes in enzyme activities and vacuolar efflux of conjugates are prevented by application of 10^-3M concentrations of cinnamic acid. Cinnamate is alternatively metabolized to a glucose ester, a S-glutathionyl conjugate and to cell wall bounds forms; these reactions are intensified by elicitation. Isoflavone and pterocarpan biosynthesis and conjugate metabolism as regulated by elicitation and cinnamate is depicted in a metabolic grid to explain the complex regulatory pattern of phenolic accumulation in chickpea cell cultures.Abbreviations AOPP L--aminooxy--phenylpropionic acid - BGM biochanin A 7-0-glucoside-6-0-malonate - FGM formononetin 7-0-glucoside-6-0-malonate - HPLC high performance liquid chromatography - MaGM maackianin 3-0-glucoside-6-0-malonate - MeGM medicarpin 3-0-glucoside-6-0-malonate     

Pancreatic glucagon cells contain endorphin-like immunoreactivity

Summary On the basis of the histochemical activity of succinic dehydrogenase, only two fibre-types are distinguished in pigeon pectoralis major muscle. These are narrow Red and broad White. The histochemical activity of myofibrillar ATPase was studied in these two distinct fibre-types. Both fibre-types showed high activity for the ATPase. Red fibres of pigeon pectoralis were not alkali-labile, at incubation pH 9.4, as were the Type I fibres of both avian and mammalian muscles. Again unlike Type I fibres, the Red fibres of pigeon pectoralis lacked the characteristic activation of acid-preincubated ATPase reaction. Pigeon pectoralis Red fibres are known to posses some characteristics of fast-twitch fibres (e.g. high fat, considerable phosphorylase, fibrillenstruktur myofibrillar arrangement, focal en plaque pattern of nerve endings). It is emphasized, therefore, that the pigeon pectoralis Red fibres are not equivalent to Type I or slow-twitch, muscle fibres, but they are possibly fast-twitch fatigue resistent or Type II Red muscle fibres.     

Effect of triparanol (Mer 29) on rat liver lysosomes: cytochemical and biochemical study

Summary Rats treated with triparanol (MER-29) develop numerous membranous inclusions-myeloid bodies in the cytoplasm of liver cells. The myeloid bodies did not show cytochemically demonstrable acid phosphatase. Instead diffuse activity was observed throughout the cytoplasm. Biochemically, acid phosphatase was found in the liver lysosomal fraction obtained from triparanol treated rats. This fraction, however, did not show the structure-linked latency of acid phosphatase which is characteristic of normal lysosomes. It is suggested that myeloid bodies are lysosomes with altered membranes.     

Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate

Globo H (Fuc12Gal13GalNAc13Gal14Gal14Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc12Gal13GalNAc13Gal. An isomeric structure with an internal GalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.     

Competition between <Emphasis Type="Italic">Eucalyptus victrix</Emphasis> seedlings and grass species

Competition in a natural system may be interspecific or intraspecific. In semiarid ecosystems, competition for resources between established neighboring grass species and newly recruited seedlings is very high. To examine the effects of grass species density, growing space and time of establishment on Eucalyptus victrix seedlings (interspecific competition), and the effect of density and growing space within E.victrix (intraspecific competition) we conducted an experiment under controlled conditions. We tested four hypotheses (i) E.victrix seedling growth is not affected by grass density; (ii) there is no difference in E.victrix survival and growth between early and later grass establishment; (iii) interspecific competition is not more intense than intraspecific competition in E.victrix; and (iv) growth of E.victrix seedlings is not dependent on available growing space. In a monoculture of E.victrix, seedling mortality was higher (10%) in large pots. In mixed culture pots, where E.victrix seedlings and grass seedlings were planted on the same day, E.victrix seedlings survived for up to 4weeks, but started to die after week five in the smallest pots. However, mortalities occurred in pots of all sizes when grass was established before E.victrix seedlings. Results also indicated that the resources necessary for the growth of individual E.victrix seedlings were more limiting under conditions of increased density of neighboring grass species rather than intraspecific competition. In particular, photosynthetic area of E.victrix seedlings was drastically reduced in mixed cultures. Although density, pot size and time of planting had impacts on E.victrix seedlings, the patterns of these impacts were variable.     

Calcium-dependence of catecholamine release from bovine adrenal medullary cells after exposure to intense electric fields

Summary By subjecting isolated adrenal medullary cells to intense electric fields of brief duration it is possible to gain access to the cell interior without impairing the ability of the cell to undergo exocytosis. After a single exposure to a field of 2 kV/cm, =200 sec, adrenal medullary cells behave as if their plasma membrane contains two pores of effective radius 2 nm. At 37°C these equivalent pores remain patent for up to 1 hr. The formation and stability of these pores is not affected by the Ca content of the bathing solution. The pores permit externally applied catecholamine and Ca-EGTA to equilibrate rapidly with the cell water.Cells rendered leaky in K glutamate medium containing 5mm Mg-ATP and EGTA to give an ionized Ca close to 10^–8 m release less than 1% of their total catecholamine. These same cells can release up to 30% of their catecholamine when exposed to 10^–5 m Ca. This Ca-dependent release is unaffected by Ca-channel blockers such as D600. Catecholamine release in response to a calcium challenge only seems to occur during the first few minutes whilst the Ca concentration is changing, and the extent of release depends on the final Ca concentration achieved. Half-maximal release occurs at about 1 m Ca, and this value is independent of the EGTA concentration used to buffer the ionized Ca. The relation between ionized Ca and catecholamine release is best fitted by a requirement for 2 Ca ions.Calcium-evoked release of catecholamine is associated with the release of dopamine--hydroxylase (DH) but not lactate dehydrogenase. The ratio DH/catecholamine released is the same as that in stimulated intact cells and perfused glands. The time course of appearance in the external medium of DH and catecholamine is identical. Transmission electron microscopy of leaky cells exposed to 10^–8 m Ca reveals no marked differences from unstimulated intact cells. The cytoplasm of leaky cells exposed to 10^–5 m Ca contains large membrane-bounded vacuoles. When secretion is caused to take place in the presence of horseradish peroxidase, this marker is found within the vacuoles.Ca-dependent release of both catecholamine and DH requires Mg-ATP. Cells equilibrated with Ca in the absence of Mg-ATP can be triggered to undergo exocytosis by the addition of Mg-ATP. In the absence of Mg, ATP alone is ineffective. Of a variety of other nucleotides tested, none is as effective as ATP. Mg-ATP affects the extent of exocytosis and not its apparent affinity for Ca.Replacement of glutamate as the major anion by chloride results in a marked reduction in Ca-dependent release of both catecholamine and DH. Chloride causes a small increase in Ca-independent release of catecholamine, a large reduction in the extent of exocytosis, and a decrease in the apparent affinity of exocytosis for Ca. Of a variety of anions examined, their order of effectiveness at supporting Ca-dependent exocytosis is glutamate^–>acetate^–>Cl^–>Br^–>SCN^–.Exocytosis is not obviously affected by replacing K by Na or sucrose or by altering the pH over the range pH 6.6 to 7.8. Raising the free Mg concentration reduces the extent of Ca-dependent exocytosis and also its apparent affinity for calcium. Calcium-dependent exocytosis in leaky cells is largely unaffected by (i) a variety of agonists and antagonists of the nicotinic receptor; (ii) agents that disrupt microtubules and microfilaments; (iii) phalloidin; (iv) vanadate; (v) inhibitors of anion permeability; (vi) protease inhibitors; and (vii) agents that dissipate the vesicle pH gradient and potential. It is partially inhibited by (i) certain antipsychotic drugs; (ii) a rise in osmotic pressure, (iii) lowering the temperature below 20°C, and (iv) N-ethyl maleimide.     

Possible linkage between uncoiler chromosome Un 1 and mylase polymorphism Amy 2 loci

Summary A family has been investigated in which 7 from 12 members bear the uncoiler chromosome number 1 pair. In all these members with uncoiler chromosome 1 heterozygous amylase polymorphism variant Amy 2^A2^B has been estimated (with exception of one 3-month-old boy in whom the phenotype of amylase variant is not yet fully developed). This pedigree represents a typical double back-cross family which enables to suppose very probably the close linkage between both gene loci Un 1 and Amy 2 and also to assign Amy 2 locus to chromosome 1.

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Zusammenfassung Wir berichten über eine Familie, in welcher 7 unter insgesamt 12 untersuchten Familienmitgliedern ein asymmetrisches Chromosom des ersten Chromosompaares mit den sehr verlängerten langen Armen (uncoiler chromosome) gefunden wurde. Bei allen diesen Familienmitgliedern mit, uncoiler chromosome 1 wurde auch gleichzeitig die heterozygote Variante des Amylase-Polymorphismus Amy 2^A2^B gefunden (mit Ausnahme eines 3 Monate alten Knaben, bei welchem das Phenotyp der Amylase-Variante noch nicht völlig entwickelt wurde). Dieser Stammbaum stellt eine typische double back-cross-Familie vor, aus welcher mit großer Wahrscheinlichkeit die linkage zwischen beiden Genorten Un 1 und Amy 2 und damit auch die Lokalisation des Genortes Amy 2 auf dem Chromosom 1 hervorgeht.

     

Morphological and functional changes in the tight junctions of the bile canaliculi induced by bile duct ligation

Summary Thin sections after bile duct ligation showed that the depth of tight junctions appeared to increase and that the distance between individual punctate contacts appeared to become irregular and wider than in controls. The freeze fracture replicas clearly demonstrated these changes in the tight junction morphology. Changes were noted most conspicuously in the tight junction three weeks after ligation. Measurements of the junctional morphology in control and ligated specimens showed that the junctional depth had increased two fold in the latter, whereas the number of strands had scarcely changed. Lanthanum tracer experiments showed that the tight junctions did not permit the passage of the tracer in normal nor ligated rats. It was concluded that the mechanism of obstructive jaundice could not be related to changes in junctional morphology causing increased junctional permeability.Tight junction depth in this paper is synonymously used with Tight junction width or Tight junction thickness     

Notes on the Mechanism of ATP Synthesis

The most commonly quoted mechanism of the coupling between the electrochemical proton gradient and the formation of ATP from ADP and P_i assumes that all states of the F₁ portion of the ATP synthase have subunits in tight, loose, and open conformations. Models based on this assumption are inconsistent with some of the available experimental evidence. A mechanism that includes an additional subunit conformation, closed, observed in the rat liver structure overcomes these difficulties.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Over expression of integrin α 5 β 1 in human hepatocellular carcinoma cell line suppresses cell proliferation in vitro and tumorigenicity in nude mice

Integrin 5 1 and 2 1 are the major integrin receptors in human hepatocytes. However, in human hepatocellular carcinoma cells it was found that the expression of integrin 5 1 was decreased and another integrin 6 1 increased. In this study, the SMMC7721 human hepatocellular carcinoma cells cotransfected or singlely transfected with integrin 5 and/or 1 cDNAs were established, and designated 5 1.6-7721, 5.3-7721, and 1.6-7721 cell lines, respectively. Transfection with cDNAs of integrin 5 and 1 subunits resulted in the overexpression of each integrin and modified biological properties, including a slowed growth rate, changes in the cell cycle from 15.5% of control cells in the G2/M phase to 12.1%, 9.6% and 9.4% in 5.3-7721, 1.6-7721, 5 1.6-7721, respectively, and a decrease in the Cell Mitosis Index from 1.6 in controls to 0.96, 0.95, and 0.72, and 34%, 28% and 52% derived from colony forming ability, respectively. Tumorigenicity was also tested in nude mice with inoculation of cells subcutaneously. Tumor masses growing in nude mice following inoculation with 1.6-7721,and 5 1.6-7721 cells weighed only 52% or 31% those of control cells. These results indicated that deletion or low expression of integrin 5 1 may play an important role in the development of hepatocellular carcinoma. Therefore, induction of expression of the integrin 5 1 in malignant cells could be a potential means of treating hepatocellular carcinoma.     

Visualization of the uptake of high-density lipoprotein by rat aortic endothelial cells and smooth muscle cells in vitro

High-density lipoproteins (HDL) were conjugated to Fluorescein 1,1-dioctadecyl 3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) or colloidal gold for the investigation of ultrastructural aspects of binding and uptake of HDL by cholesterol-loaded cultured endothelial and smooth muscle cells from rat aorta. When cells were incubated for 2h at 4°C, HDL–DiI and HDL–gold conjugates were seen only on the cell surface. When cells were returned to incubation at 37°C for 5min, HDL–DiI appeared in the cytoplasm and colocalized with the fluorescent cholesteryl ester tag BODIPY-FL-C₁₂. HDL–gold conjugates appeared in the plasmalemmal invaginations and plasmalemmal vesicles. After incubation for 15min, most of the HDL–gold conjugates reappeared on the cell surface. After incubation for 30min, only a few conjugates were observed and they localized in lysosomal-like bodies. Quantitative data indicated that when the cholesterol-loaded cells were incubated at 4°C for 2h, the numbers of HDL–gold associated in clusters on the endothelial cell surface was 1.18 clusters/m. When cells were returned to incubation at 37°C for 5min, this value decreased to 0.7, increased again to 1.13 at 15min, and decreased to 0.29 at 30min. The numbers of clusters in the plasmalemmal invaginations were 0.06 clusters/m at 4°C for 2h, increased to 0.34 at 37°C for 5min and decreased gradually to 0.19 and 0.04 at 15 and 30min, respectively. The incidence of clusters in the plasmalemmal vesicles per non-nuclear cytoplasm was 0.01 clusters/m² at 4°C for 2h, increased significantly to 1.08 at 37°C for 5min, and decreased to 0.43 and 0.14 at 15 and 30min, respectively. This work supports that the plasmalemmal invaginations and plasmalemmal vesicles are linked to the HDL uptake in cholesterol-loaded aortic endothelial cells and smooth muscle cells.     

Electron microscopic evidence for the existence of an intercellular substance in rat cerebral cortex

Summary The intercellular spaces of rat cerebral cortex are filled with a dense material, demonstrable by electron microscopy. This intercellular substance is in part preserved by chemical fixation with formaldehyde and osmium tetroxide but is solubilized and largely lost during subsequent dehydration with ethyl alcohol. Dehydration with acetone or Durcupan favors the preservation of the intercellular substance, which is preserved also by freezing and drying. Whether the intercellular substance demonstrated here is part of the outer leaflets of apposing plasma membranes (glycocalyx) or truly an intercellular substance similar to connective tissue ground substance is not known. The probability of the latter is discussed with regard to proposed physiological mechanisms.This work was supported by USPHS Research Grants NB 05175 and AM 06998.     

Congruent evolution between whiteflies (Homoptera: Aleyrodidae) and their bacterial endosymbionts based on respective 18S and 16S rDNAs

Whiteflies (family Aleyrodidae) possess heritable eubacterial endosymbionts sustained in specialized organ-like structures called mycetomes. Comparisons of distances between the ash whitefly,Siphoninus phillyreae, and two biotypes (A and B) of the sweetpotato whitefly,Bemisia tabaci, based on sequence analysis of genes for 18S rRNAs (rDNAs), were equivalent to the distances represented by the 16S rDNAs of their respective endosymbionts. This finding indicates that evolutionary divergence in whitefly hosts and their endosymbionts is congruent. The nucleotide sequences of the 18S rDNAs and endosymbiont 16S rDNAs indicate the two biotypes ofB. tabaci are the same species.     

A model for light adaptation: Producing Weber's law with bleaching-type kinetics

An adaptation model having two stages is introduced and its mathematical properties are examined. The two stages are the adaptive process (parameter K _b), which has bleaching-type kinetics, and the response function (parameters K _r and n), which incorporates response saturation. In order to study the increment threshold functions generated by the adaptation model the concept of a detector is required. It is demonstrated that without an adaptive process the compression hypothesis, in the form of the difference equation, produces increment threshold functions which saturate and do not obey Weber's law. It is then shown that an adaptive process with bleaching-type kinetics can prevent saturation and produce Weber's law behavior provided that the adaptive strength of the system exceeds the detector sensitivity.     

Genetics of Ia-like alloantigens in chickens and linkage withB major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens (Ia) and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the la antigens are determined by a single codominant locus and that theEa-B and Ia loci are on the same chromosome. Failure to detect a single recombinant between theEa-B and Ia loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The Ia genes are thus included in theB major histocompatibility complex of the chicken.