Prolactin as a factor in the uterine response to progesterone in rabbits

The direct effect of prolactin on uteroglobin production and on uterine endometrial oestrogen and progesterone receptor concentrations was tested by using ovariectomized rabbits (at least 12 weeks) treated with prolactin; prolactin + progesterone; prolactin + oestradiol + progesterone; oestradiol + progesterone; or progesterone alone. Prolactin treatment produced a significant (P less than 0.05) increase in the concentration of cytosolic oestrogen and progesterone receptors, restoring the concentrations to values found at oestrus. However, the concentration of nuclear receptors remained low. In the remaining treatment categories there was no significant (P greater than 0.05) increase in the concentration of oestrogen and progesterone receptors compared with those in ovariectomized controls. However, the sequential treatment of ovariectomized animals with prolactin + progesterone stimulated uteroglobin production to a concentration equal to that found in intact rabbits on the 5th day of pregnancy. This was not achieved by prolactin or progesterone alone or with oestradiol. These results suggest that prolactin acts as an essential factor in the rabbit uterine response to progesterone, perhaps by the modulation of progesterone receptor activity.     

Intestinal disaccharidase and alkaline phosphatase activities in experimental rabbit mucoid enteropathy

Mucoid enteropathy was induced experimentally by ligation of the cecum, and the activities of mucosal disaccharidases and alkaline phosphatase were measured at different locations along the small intestine of the sick and control rabbits. In the duodenum of rabbits with mucoid enteropathy, the activity of acid beta-galactosidase II was elevated and hetero beta-galactosidase declined. In the jejunum, the activities of lactase, acid beta-galactosidase I and II, hetero beta-galactosidase, trehalase, sucrase and alkaline phosphatase were significantly lower in animals with mucoid enteropathy. In the ileum, acid beta-galactosidase II, hetero beta-galactosidase, maltase, trehalase, sucrase and alkaline phosphatase showed decreased activity in rabbits with mucoid enteropathy.     

Changes of Phospholipid-Metabolizing and Lysosomal Enzymes in Hypoglossal Nucleus and Ventral Horn Motoneurons During Regeneration of Craniospinal Nerves

In order to study the biochemical changes associated with the cell body response to axonal crush injury, two systems, hypoglossal nucleus and spinal cord ventral horn, were used. The time intervals chosen were 7, 14, and 28 days after unilateral crushing of the right hypoglossal nerve and cervicothoracic nerves of the rabbit. Non-crushed, contralateral nerves were used as controls. Three groups of enzyme activities were tested: (a) phospholipase A2, acyl CoA:2-acyl-sn-glycero-3-phosphocholine acyltransferase, and choline phosphotransferase, as indicators of phospholipid degradation and biosynthesis; (b) seven hydrolases, namely, beta-D-glucuronidase, beta-N-acetyl-D-hexosaminidase, arylsulfatase A, galactosylceramidase, GM1-ganglioside beta-galactosidase, and acid RNase, as indicators of lysosomal activity; and (c) free and inhibitor-bound alkaline RNase, as an index of RNA metabolism. Changes could be grouped into three distinct patterns. Compared to contralateral control, choline phosphotransferase showed a slight increase, whereas phospholipase A2 and most lysosomal hydrolases showed a significant increase of activity, especially evident in the ventral spinal cord neurons 14-28 days after crushing. These changes correlate with known increases of membrane and organelle numbers, including lysosomes, in motor and sensory neurons during peripheral regeneration. In contrast, free and acid alkaline RNase activity significantly decreased in the injured sides compared to the controls. This change can probably be correlated with a stabilization of RNAs needed for increased protein synthesis. No changes in total alkaline RNase and acyltransferase activities in either regeneration model were observed.     

Appearance of beta-hexosaminidase and other lysosomal-like enzymes in the uterine lumen of gilts, ewes and mares in response to progesterone and oestrogens

In one experiment, ovariectomized gilts were treated with corn oil (vehicle), progesterone, oestradiol-17 beta or both steroids. While oestradiol treatment did not stimulate enzyme activity in uterine flushings relative to vehicle-treated animals, gilts treated with progesterone had elevated amounts of all enzymes measured. Progesterone was less effective when co-administered with oestradiol-17 beta. Enzymes were not equally stimulated by progesterone. For example, there was a 909-fold increase in acid phosphatase activity in uterine flushings and a 304-fold increase in beta-N-acetylglucosaminidase, but only a 10-fold increase in beta-glucosidase. Endometrial explants from gilts synthesized and secreted radiolabelled beta-N-acetylglucosaminidase, suggesting that at least some lysosomal enzymes enter the uterus through secretory processes. In other experiments, changes in beta-N-acetyglucosaminidase in uterine fluids of mares and ewes treated with hormonal regimens similar to those given to the gilts were evaluated. Treatment with the combination of progesterone and oestrogen stimulated accumulation of the enzyme relative to that in vehicle-treated animals. The biochemical properties of porcine beta-N-acetylglucosaminidase were examined in detail. Properties of the uterine enzyme were similar to reported values for lysosomal hexosaminidase. These included molecular weight (82 000-89 000), pH optimum (pH 4.4), presence of two isomers (isoelectric points of 5.5 and 8.0) and ability to hydrolyse substrates for glucosaminidase and galactosaminidase. We conclude that steroids induce the accumulation of lysosomal enzymes in the uterine lumen. The degree of stimulation differed between enzymes, suggesting that those enzymes stimulated to the greatest extent may play an important role in pregnancy.     

Lysosomal hydrolases in reproductive organs during estrous cycle of the hamster

Changes in the total protein content and the activities of lysosomal hydrolases (arylsulfatase, acid phosphatases, β-glucuronidase, β-N-acetylhexosaminidase, α-L-fucosidase, and β-galactosidase) of the hamster genital tract during the 4 days of estrous cycle and in hormonally superovulated hamsters were measured. Levels of lysosomal hydrolases in uteri and uterine fluid changed significantly during the cycle. Similar changes were observed in uterine wet weight and uterine proteins. The pattern of enzyme activities in both the ovary and the oviduct were different from those in uteri. In the ovary, most enzyme activities and the total protein concentration remained elevated after ovulation. Protein concentration and enzyme activities were significantly higher in the ovary, oviduct, and uteri of superovulated hamsters as compared to controls.     

Histochemical and biochemical studies on the localization and activity of lysosomal enzymes in fracture callus

Summary Quantitative biochemical studies on the activities of four lysosomal hydrolases during different stages of fracture healing in the rat were performed, and the results obtained were integrated with those of histochemical observations relating to changes in the localization of acid phosphatase in the same tissue.The findings showed presence of all the four lysosomal enzymes assayed in the callus; during early callus formation the enzyme activities calculated on a DNA basis increased up to about 12 days after the fracture. The enzyme activities appeared to be roughly reflected histochemically by the acid phosphatase staining. The increasing activity during early callus formation seemed to depend on the presence of numerous macrophage-like cells in the tissue containing many large lysosomes. A decrease in enzyme activity was found after day 12. Comparison with the histochemical and ultrastructural findings suggested that this decrease was due to a reduction in the number of macrophage-like cells and a concomitant increase in osteogenic cells with a lower enzyme content.     

Comparative analysis of lysosomal hydrolases and natural glycoprotein substrates in the rat major salivary glands.

1. The activities of some lysosomal hydrolases and the concentrations of their natural substrates were studied in the submandibular and sublingual glands of male and female rats using biochemical procedures. 2. In sublingual gland enzyme activities and substrate concentrations show the highest values. 3. The enzyme activities appear, in general, lower and the natural substrate concentrations higher in the females with respect to males. 4. In both glands beta-galactosidase shows the highest activity and beta-glucosidase the lowest. 5. These findings suggest that metabolic turnover of glycoproteins is slower in females than in males, probably because the oestrogens control the activity of lysosomal hydrolases.     

A comparative biochemical research in the Schistocephalus solidus (Cestoda)--three-spined stickleback Gasterosteus aculeatus L. system

The activity of five lysosomal hydrolases (acid phosphatase, DNAase, RNAase, beta-glucosidase, beta-galactosidase), alkaline phosphatase and aldolase have been examined in tissues of the cestode Schistocephalus solidus (Müller, 1776) and the three-spined stickleback Gasterosteus aculeatus (L.) forming a stable parasite-host system. As a rule, the activity of enzymes was higher in a cestode body than in fish tissues. The acid and alkaline phosphatases were the exception. The activity and variation of lysosomal nucleases and aldolase in the parasite differed notably from those in both infested and healthy hosts. The paper discusses the role of lysosomal and cytoplasmic enzymes in a cestode adaptation to parasitism, as well as in the mechanisms of the host's chemical and immunological response to infection.     

Endometrial protein secretion during early pregnancy in entire and ovariectomized ewes

The secretion and synthesis of protein in vitro by explants of endometrium were examined in entire ewes during the first 10 days of the oestrous cycle and during an equivalent interval in ovariectomized ewes which received injections of oestradiol and progesterone. The schedule of steroid injections given was designed to simulate endogenous ovarian secretion of progesterone during the luteal phase before oestrus, of oestradiol around oestrus and of progesterone during the luteal phase after oestrus. The rate of protein synthesis and tissue RNA:DNA and protein:DNA ratios in intercaruncular and caruncular endometrium were generally higher in entire than in ovariectomized ewes. In ovariectomized ewes oestradiol increased these activities at 2-4 days after oestrus, whereas progesterone preceding oestradiol caused increases at oestrus, but not thereafter. In entire ewes and in ovariectomized ewes receiving the full steroid treatment regimen, protein secretion was high at oestrus and declined markedly during the next 4-6 days. In ovariectomized ewes not receiving progesterone before oestradiol, secretion increased between 4 and 6 days after oestrus, or during the equivalent stage of treatment in ewes which did not show oestrus. The omission of this progesterone did not modify secretion by caruncular endometrium. Oestradiol increased protein secretion by both tissues. The data suggest that progesterone given before oestradiol (or its equivalent in entire ewes) inhibits the secretion, at about 4-7 days after oestrus, of uterine proteins which may impair embryo development in ovariectomized ewes which do not receive this progesterone.     

Induction of liver lysosomal enzymes during the autophagic phase following phenobarbital treatment of rat

Phenobarbital was given to male rats as a single injection and as repetitive injections for 7 days. The effects of treatment on the lysosomal hydrolases acid phosphatase, cathepsin D, and aryl sulfatase were analyzed at different intervals ranging from 1 to 15 days after seven injections, and from 1 to 48 h after a single injection. In both cases, microsomal protein and NADPH-cytochrome c reductase were measured to ensure proper induction. After a single injection, a slight decrease in hydrolytic activities was observed. Repetitive administration of phenobarbital gave rise to a marked decrease of lysosomal enzyme activities 1 day after cessation of treatment. This decrease was followed by a continuous increase in activity up to day 3 and 4. One or 2 weeks after treatment, enzyme activities declined to control values. The increase in activity of lysosomal hydrolytic enzymes was correlated with the onset of induced autophagy of endoplasmic reticulum membranes described as occurring in liver upon cessation of phenobarbital exposure. It is concluded that phenobarbital treatment per se decreases lysosomal enzyme activities, whereas the induced autophagy following cessation of exposure is associated with enhanced levels of lysosomal hydrolases in rat liver.     

Studies on the nephrotoxicity of p-nitrophenylarsonic acid: changes in rat kidney and urinary enzyme activities following the administration of p-nitrophenylarsonic acid.

Histological studies showed that the administration of p-nitrophenylarsonic acid to rats resulted in renal tubular necrosis. The nephrotoxin was administered intraperitoneally and doses greater than 30 mg/kg were found to be fatal. The severity of the renal lesion depended on the amount of the nephrotoxin used. Elevated serum urea levels, urinary protein and volume were recorded over an 8-day period following the injection of the nephrotoxin. These changes were paralleled by an increase in the activity of lactate dehydrogenase, acid and alkaline phosphatase, N-acetyl-beta-glucosaminidase and beta-glucosidase in the urine. beta-Glycosidase activities increased in kidney homogenates, immediately after the injection of the nephrotoxin, but this eventually fell to well below the normal range. Subcellular fractions were prepared from sucrose homogenates by differential centrifugation and beta-glycosidases and cytochrome oxidase were used as enzyme markers. Only minor changes in the activity of cytochrome oxidase activity resulted from the administration of p-nitrophenylarsonic acid. One of the earliest indications of renal damage was a decrease in lysosomal latency. The activities of the lysosomal and soluble enzymes were elevated above normal during the first two days after the injection of p-nitrophenylarsonic acid, but they fell to values, significantly lower than normal, on the third day. The isoenzymic forms of beta-galactosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase in normal and damaged kidneys were studied, using starch gel electrophoresis. The activities of both the lysosomal and the soluble forms of these enzymes decreased following the injection of the nephrotoxin, confirming the results obtained with whole homogenates. The relationship between the changes in renal enzyme activity and urinary enzyme excretion during the nephrotoxic process is discussed.     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r     

Enzyme cytochemistry combined with electron microscopy, pharmacokinetics, and clinical chemistry for the evaluation of the effects of steady-state valproic acid concentrations on the mouse

A number of organs from adult female mice were investigated after continuous application of the anticonvulsant drug valproic acid (VPA) by enzyme cytochemistry, light and electron microscopy, pharmacokinetics and clinical chemistry. VPA plasma levels were maintained between 55 micrograms/ml and 67 micrograms/ml for three days following subcutaneous implantation of drug reservoirs. Effects detectable by enzyme cytochemical or electron microscopical means were mainly observed in liver, kidney, thymus and spleen. A strict concentration-dependency of drug effects could not be found. In the liver, the activities of some surface-membrane hydrolases were increased at the biliary pole; the activities of other hydrolases were decreased or unchanged. Electron microscopically, number and length of microvilli of hepatocytes were increased and many of them showed fat inclusions, mitochondrial swellings and autophagic vacuoles. In some of the proximal convoluted tubules of the kidney, the reaction product originating from microvillous and lysosomal hydrolases was diffusely distributed and its amount lowered. This was paralleled by tubular cells with an increased number of fat droplets and swollen mitochondria or destroyed tubular cells, as demonstrated by electron microscopy. Additionally, peritubular endothelial cells were arranged in a garland-like pattern. Alkaline phosphatase was activated in the straight portion of the proximal tubules. Increased glucose, creatinine and total protein concentrations and increased gamma-glutamyl transpeptidase and alkaline phosphatase activities in the urine reflected well the damage of the proximal renal tubules. Cortical and medullary morphology varied considerably in the thymus. In extreme cases, the cortical zone was either reduced in size or the medulla showed a cortex-like structure or vice versa (inverted type of thymus). The thymic cortical reticular cells showed increased aminopeptidase A activity accompanied by a generalized aminopeptidase M and alkaline phosphatase reaction. Our data indicate that--in addition to the liver--also the kidney, thymus and spleen are target organs of VPA-induced toxicity in the mouse.     

Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.     

Ultrastructural localization of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium of rat

The effect of ovarian hormones on the activities of glucose-6-phosphatase and alkaline phosphatase in the vaginal epithelium was studied in immature and ovariectomized rats, using ultracytochemical techniques. Comparative studies were done on normal rats at the luteal phase and on day 14 of pregnancy. Various vaginal cells show different degrees of response to progesterone and diethylstilbestrol (DES) with regard to glucose-6-phosphatase activity. Intense glucose-6-phosphatase activity was observed in the cisternae of granular endoplasmic reticulum (rER), Golgi saccules and vesicles, and nuclear envelope of both basal cells and stromal cells of progesterone treated rats, whereas in the basal cells and stromal cells of DES-treated and control animals the enzyme was totally lacking. Detectable glucose-6-phosphatase activity was also observed, however, in the rER cisternae and Golgi complex of keratohyalin-secreting squamous intermediate cells of the vaginal epithelium of DES-treated rats. Alkaline phosphatase was also found on the limiting membranes of secretory granules of mucocytes in animals at the luteal phase and during pregnancy. DES and progesterone in the doses used did not affect alkaline phosphatase activity in the rat vagina. Overall, progesterone enhances glucose-6-phosphatase activity in basal cells of the rat vagina prior to completion of mucification. Alkaline phosphatase was found in all cells involved in mucin secretion.     

Lysosomal hydrolase secretion by Tetrahymena: a comparison of several intralysosomal enzymes with the isoenzymes released into the medium.

Tetrahymena pyriformis were grown in proteose-peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three isoenzymes of acid phosphatase, three of acid protease, and two of beta-N-acetylhexoseaminidase were found, as well as single peaks of alpha-mannosidase, beta-galactosidase, and beta-fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM-cellulose. Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, contered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of beta-fucosidase, beta-galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only alpha-mannosidase activity, was detected just inside the gradient. Measurements were made of the effect of heat (10 minutes at 66 degrees) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two beta-N-acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1-hour starvation period or after secretion.     

Effects of oestradiol, the oestrous cycle and pregnancy on weight, metabolism and cytosol receptors in the oviduct and vaginal complex of the brushtail possum (Trichosurus vulpecula)

Weight, RNA, DNA and protein content of the oviduct, vaginal cul-de-sac, lateral vagina and urogenital sinus and oestradiol and progesterone cytosol receptor concentrations in vaginal cul-de-sac, lateral vagina and urogenital sinus were examined after administration of oestradiol to ovariectomized animals and on days 0, 5, 9 and 13 of the non-pregnant cycle and on day 13 of the pregnant cycle. In ovariectomized animals, oestradiol induced an increase in weight, RNA:DNA and protein:DNA ratios and a decrease in DNA:tissue weight ratio for each organ and in addition an increase in total DNA in vaginal cul-de-sac and urogenital sinus. There was no effect of oestradiol on oestradiol cytosol receptor concentration but there was a significant increase in progesterone cytosol receptor concentration in all organs that were examined. During the oestrous cycle, changes in the wet weight of each organ showed a common pattern with maximum weight at day 0 followed by variable rates of decline until day 13. In oviduct and vaginal cul-de-sac, the decrease in weight was paralleled by a decrease in RNA:DNA and protein:DNA ratios whereas the DNA: tissue weight ratio showed the opposite pattern and total DNA remained unchanged. The changes in the lateral vagina and urogenital sinus were similar except that a significant decline in total DNA was also seen after day 0 and the DNA:tissue weight and protein:DNA ratios in the urogenital sinus and the lateral vagina respectively showed no significant changes. Progesterone cytosol receptor concentration in the lateral vagina and urogenital sinus were high on day 0 and then declined until day 13. In contrast, there were no consistent effects on oestradiol receptor concentration.     

Azoindoxyl methods for the investigation of hydrolases. IV. Suitability of various diazonium salts (author's transl)]

Using fresh frozen, freeze-dried or cryostate sections from aldehyde fixed rat tissues 13 diazonium salts were tested as simultaneous coupling reagents for the localization of acid, neutral and alkaline hydrolases with azo indoxyl methods. Hexazotized new fuchsine and/or Fast blue B are the diazonium salts of choice for the demonstration of acid beta-galactosidase, neuraminidase, beta-N-acetylglucosaminidase, acid phosphatase, and non-specific esterase followed by hexazotized p-rosaniline. Fast blue VB, BB and RR and Fast violet B are recommended for the investigation of alkaline phosphatase and lactase, Fast garnet GBC for acid beta-galactosidase, glucosaminidase and lactase. Fast red B, RC, RL and TR and Fast black K can only be employed for lactase studies. The exact concentration of the coupling reagent depends on the activity of the enzyme and the organ imvestigated. On the average 0.01-0.02 ml unstable diazonium salt/ml and 0.3--1 microgram stable diazonium salt/ml are sufficient for the correct localization of these hydrolases. Freeze-dried cryostat sections yield the best results in the demonstration of lactase and alkaline phosphatase independent on the coupling reagent used. Sections from formaldehyde or glutaraldehyde fixed organs are superior for the localization of the other hydrolases; an exception is the investigation of acid beta-galactosidase and glucosaminidase with Fast garnet GBC. Then, excellent results are obtained also with freeze-dried material. Fresh frozen sections are suitable for the localization of lactase with hexazotized new fuchsine or p-rosaniline and of alkaline phosphatase with Fast blue VB and BB or violet B. The total activity of acid, neutral and alkaline hydrolases can be investigated using semipermeable membranes in combination with all unstable and stable diazonium salts of choice. Reliable osmification of the azoindoxyl dye is only possible if hexazotized p-rosaniline is employed for coupling; without further posttreatment all azoindoxyl dyes are extracted by ethanol, isopropanol or xylol. 7 incubation media are given for the demonstration of hydrolases with azoindoxyl methods at the level of light microscopy for routine studies and typical examples for the application of these methods are presented. A modified procedure is described for the freeze-drying of cryostat sections with the Edwards-Pearse tissue dryer EPD3.     

Changes in the activity of some lysosomal enzymes and in the fine structure of submandibular gland due to experimental diabetes

The aim of this study was to establish and quantify changes in the activities of the some lysosomal enzymes and to determine the type of changes in the ultrastructure of the submandibular gland in rabbits caused during progression of diabetes. The experiment was conducted on 89 New Zealand rabbit males. Diabetes was induced by the intravenous administration of 10% alloxan solution at a dose of 10-mg/kg-body weight. On the seventh day after alloxan administration, the level of glucose in blood was determined. Rabbits were divided into five groups: intact (n=18), 21-day diabetes (n=18), 42-day diabetes (n=17), 90-day diabetes (n=19) and 180-day diabetes (n=17). From killed animals in each group, the submandibular glands were removed and fixed or stored. Enzyme activities were assayed by spectrophotometric methods using substrates (Sigma) which release 4-methyloumbeliferol when they react with the proteases. Fixation procedure was done according to standard methods. Semi-thin and ultra-thin specimens were prepared by use of clearly visible after 42 days of diabetes. Mitochondria were damaged, accumulation of large amounts of lipids in the intracellular spaces was observed. After 90 days the presence of vacuoli and swollen lysosomes were observed, some cells also contained myelin figures. After 180 days the greatest changes were observed in the blood vessels, which had thickened walls and were often occluded. We concluded that the total activity of acid phosphatase and beta-N-acetyl-glucosaminidase in the submandibular gland was correlated with the level of glucose but there was no correlation between total beta-galactosidase activity and the serum concentration level of glucose has been detected during course of diabetes. The activities of the free fractions of acid phosphatase, beta-galactosidase and beta-N-acetyl-glucosaminidase in the submandibular gland were higher than the bound fractions in all groups of rabbits. The changes in the ultrastructure of the submandibular gland were correlated with changes in serum glucose level and with lysosomal enzymes activities during progression of experimental diabetes in rabbits.     

Histochemical response of mice to mistletoe lectin I (ML I)

Summary The acute toxicity of lectin ML I from the toxic drug, mistletoe, was demonstrated in previous experiments. Because the reason for this extremely high toxicity is not yet clear, mice were studied histochemically at different times after treatment with various doses of ML I, ML I A or ML I B chain separately, or recombinations of ML I A and ML I B. Various plasma membrane-associated hydrolases as well as Golgi apparatus-and endoplasmic reticulum-linked hydrolases, peroxisomal and extraperoxisomal oxidases, lysosomal hydrolases, mitochondrial dehydrogenases, the cytoskeletal proteins keratin and vimentin as well as iron, glycogen and lipids were analysed in all organs and tissues of female mice. Irrespective of the dose, a clear-cut response was only observed in the liver. After ML I treatment, glycogen disappeared completely from all hepatocytes, and this effect did not depend on the ML I-concentration and exposure time. The increase in activity of Golgiassociated thiamine pyrophosphatase in hepatocytes and of non-specific alkaline phosphatase in the sinusoidal endothelial cells depended on the applied ML I concentration and the time of treatment. Doses of 600 or 900 ng ML I/kg drastically increased the phosphatase activities. These clear-cut changes of glycogen and enzyme activities were not observed after administration of the ML I B chain alone, and less so when the mice were treated only with the ML I A chain, or were treated with a recombination of ML I A and ML I B even at concentrations higher than that of ML I. The glycogen as well as thiamine pyrophosphatase and non-specific alkaline phosphatase responses after ML I administration were highly reproducible. None of the other investigated cell constituents responded to treatment with ML I, irrespective of the organs, tissues and cells analysed. This was also true for mice treated with ML I concentrations higher than the LD₅₀, after which the animals died with 2–3 days. In conclusion, application of ML I induces clear-cut liver cell changes, which are not observed after treatment with individual constituents of the ML I molecule either singly or in recombination. However, these changes do not explain the deaths of the animals.Supported by WTZ (Vereinbarung über wissenschaftlich-technische Zusammenarbeit: Projekt 13)