Histochemical demonstration of the intestinal hetero-β-galactosidase (glucosidase)

Summary The histochemical demonstration of hetero--galactosidase (glucosidase) has been attempted in sections and zymograms of rabbit, monkey and human intestine and of rat kidney.The leakage of this enzyme from unfixed sections was prevented by the use of cold microtome sections adherent to semipermeable membranes. Methods with -D-glucosides and galactosides of 6-Br-2-naphthol (postincubation azocoupling with Fast Blue B as well as simultaneous azocoupling with hexazonium-p-rosaniline), of -naphthol (simultaneous azocoupling with hexazonium-p-rosaniline) and of 4-Cl-5-Br-3-indolyl (with ferricyanide, phenazonium methosulfate or nitro BT and without any oxidation agent) were used an evaluated concerning the specificity, localization ability and inhibition of enzyme activity. Pretreatment of sections with distilled water or saline and inhibition by p-Cl-mercuribenzoate, glucono- and galactono-lactones were used for the characterization of the demonstrated enzyme activity.6-Br-2-naphthyl--D-glucoside is the most specific substrate for hetero--galactosidase. It is not split by lactase and acid -galactosidase. Only lysosomal -glucosidase can interfere. Because the latter enzyme is membrane-bound the difference in color intensity between untreated and prewashed sections are due to hetero--galactosidase. Only localization on the cellular (not intracellular) level can be achieved, however.The simultaneous azocoupling method with -naphthyl--D-glucoside and hexazonium-p-rosaniline enables a very good localization of hetero--galactosidase in the rabbit intestine. Due to a great inhibition exerted by hexazonium-p-rosaniline on the enzyme activity the method is unsuitable for the detection of hetero--galactosidase in zymograms and in the human intestine. Interference of lactase (or lactase-phlorizine hydrolase complex) is to be considered. The lysosomal -glucosidase does not seem to interfere.Indigogenic methods are not sensitive either. With ferricyanide as an oxidation agent it was not possible to detect the activity of hetero--galactosidase in zymograms and in sections. This is possibly due to overoxidation of indigo. The same holds true for phenazonium methosulfate used for the processing of zymograms. However, it was possible to reveal the activity of hetero--galactosidase in sections of the rabbit and monkey intestine with phenazonium methosulfate as oxidation agent. Nitro BT enhanced the coloration both in zymograms and in sections. In the latter case diffusion artifacts cannot be prevented, however. The interference of lactase, lysosomal -galactosidase and possibly of lysosomal -glucosidase (depending on the glycoside used) is always to be considered.Hetero--galactosidase was localized in the cytoplasm (particularly in the supranuclear region) of differentiated enterocytes covering the villi of the rabbit (the highest activity), monkey and human (the lowest activity) intestine. In crypt enterocytes and in cells of Brunner's glands the activity was lower. The occurrence of a low activity of hetero--galactosidase in the brush border of enterocytes of the rabbit intestine was also demonstrated.A proximodistal gradient was observed in the rabbit and monkey intestine, the upper jejunum displaying the highest activity.In jejunal biopsies of patients with celiac sprue (in the acute stage of the disease) the activity of hetero--galactosidase was lowered. No changes of activity were observed in jejunal biopsies of patients with isolated deficiencies of lactase or sucrase.In the rat kidney the enzyme was demonstrated particularly in the cytoplasm of cells of proximal convoluted tubules.     

The immunofluorescent demonstration of cathepsin B1 in tissue sections

Summary A direct immunohistochemical method of high specificity is presented for the demonstration of sites of cathepsin B1. Antisera were raised in rabbits against pure enzyme preparations. The specific antibodies were subsequently isolated and labelled according to methods previously published. Sections from tissues specially rich in this enzyme like liver, kidney, intestinal epithelia and the active macrophages of talcum granulomas were studied. The results suggest a localization of this enzyme mainly to lysosomal organelles and phagocytic vacuoles, which are known from chemical data to contain this enzyme. The prerequisite for a positive tissue reaction is that the enzyme must be present in a form available for antigen-antibody interaction.     

Glucose transport in Entamoeba histolytica

That the uptake of glucose by the parasitic amoeba Entamoeba histolytica occurs by an equilibrative transport system is supported by the following observations. 1. The rate of glucose uptake is several orders of magnitude greater than the uptake by pinocytosis. 2. The uptake of glucose exhibits saturation kinetics, with K(m)=1.6mm and V(max.) ranging from 2 to 5mumol/min per ml of cells at 37 degrees C. 3. The glucose analogues 2-deoxyglucose, 3-O-methylglucose and d-xylose are transported by the glucose system although with much less affinity. Competitive inhibition was observed between pairs of substrates, with K(i) values for any sugar closely coincident with the corresponding K(m). 4. d-Xylose, a sugar not metabolized by the cells, equilibrated with 80% of the amoebal cell water. 5. Cells equilibrated with xylose exhibited countertransport of this sugar against its concentration gradient when another substrate was added to the medium. 6. Blocking of glycolysis by iodoacetate or F(-) has no immediate effect on transport. The presence of a glucose-transport system in E. histolytica contrasts with the situation found in the non-parasitic amoeba, where pinocytosis seems to be the only mechanism of solute uptake.     

Pulsed NMR studies on Na + binding to simple carbohydrates

Pulsed NMR spectroscopy has been used to study Na⁺ binding to several simple carbohydrates in aqueous solution. Changes in the ²³Na spin-lattice relaxation time (T₁) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na⁺ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, $\text{c i s}$-inositol forms the strongest complexes with the stability constant about 1.2 M^?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na⁺ and Ca²⁺ was done, indicating that both cations have the same binding sites.     

Time-dependence of auxin and ethrel effects on flowering in chenopodium rubrum L.

IAA, NAA and ethrel (1 × 10^-4M and 3 × 10^-4M) was applied to the plumula of Chenopodium plants at different time after the start of photoperiodic treatment and the flowering response was investigated. The inhibitory effect was found with all the applications during the first two days, whereas a stimulatory one on the third and fourth day. We assume this dual effect reflects the differences attained in developmental phase and in the degree of shoot apex differentiation.     

Intracytoplasmatic inclusions in cells of lettuce leaves with mosaic-like symptoms

Ultrathin sections were prepared from the tissues of lettuce leaves with mosaic-like symptoms and thickened nervature which were studied by means of electron microscopy. Intracellular inclusions surrounded by a membrane were found in the cytoplasm of parenchym cells of the investigated lettuce leaves(Lactuca saliva L. provar.capitata L. nid.jaggeri Helm., cv. Pra?an). Crystals with a distinctly apparent hexagonal lattice could be observed in the inclusions. No crystal containing inclusions were found in the tissues from the leaves without mosaic-like symptoms and in those from thickened nervature.     

The extent and differences in mitotic activity of the root tip ofVicia faba L.

Mitotic activity does not stop for different meristematic cells of the root apex at the same distance from the initials. The differences are connected with the functional heterogeneity of the apical meristem of the root. The arrangement of vascular bundles,i.e. the alternation of independent xylem and phloem groups, is of major importance. In broad bean roots, the protophloem sieve elements stop dividing first. The centre of the stelei. e. late metaxylem elements stop dividing next. Division in the stele gradually ceases centrifugally, while it ceases centripetally in the peripheral part of the root. The cylindrical region with prolonged cell division includes internal layers of the cortex including endodermis, pericycle and adjoining cells of the stele. Proximally apical meristem is reduced to isolated strands of cells adjacent to the protoxylem poles. Pericycle cells stop dividing last at a distance of approx. 9–10 mm from the initials. The number of the division cycles is limited and is specific for individual cell types. Epidermal and cortical cells divide in broad bean roots transversely approximately seven times, cells of late metaxylem approximately five times. Root apical meristem is an asynchronous cell population with a different duration of the mitotic cycle. We determined local variations in the duration of the mitotic cycle in the apical meristem of broad bean root by means of colchicine-induced polyploidy. The cells of the quiescent centre had the longest mitotic cycle after colchicine treatment. The region of the proper root adjacent to the quiescent centre was mixoploid (2n and 4n). Isolated cells with a long cycle occurred also in the cortex and in the central cylinder. Cells with a division cycle of 18h were found in the root cap, in the epidermis, in the cortex and in the central cylinder. Relatively numerous cells with the shortest division cycle, approx. 12 h, occurred farther of the quiescent centre in the epidermis, in the cortex, in the pericycle, and in adjacent layers of the stele through-out the entire meristematic region. The results derived from the analysis of the apical meristem are discussed in connection with the ontogenesis of different types of cells taking part in the primary structure of the root.