A hydrophilic resin-embedding method for light and electron microscopic detection of tissue anionic sites with cationic colloidal iron: as applied to mouse Paneth cells

A cationic colloidal iron method was introduced for electron microscopic detection of anionic sites in hydrophilic resin-embedded specimens, and the method was applied to Paneth cells of the mouse jejunum. Mouse jejunal blocks were embedded in hydrophilic acrylic resin (LR White), cut into ultrathin sections, stained with the diluted cationic colloidal iron, and exposed to osmium vapor. The jejunal tissues, including the Paneth cells, embedded in hydrophilic resin were reactive to the fine cationic colloidal iron. At pH value 1.5, fine electron dense colloidal iron deposited along the rims of the secretory granules and the Golgi apparatus of the Paneth cell. Colloidal particles distributed on the osmiophilic reticular structures in the rim and in dot-like fashion lined the border between the granular core and rim. At pH value 4.0, ribosomes reacted to cationic colloidal iron particles in addition to the granular rims and Golgi apparatus. At pH 7.0, even the cores of the secretory granules were stained. Semi-thin sections prepared from the LR White-embedded specimens and stained at pH 1.5 with the diluted (1:3 in volume) cationic colloidal iron showed sufficient Prussian blue reaction for light microscopy in the rims of Paneth granules and mucus of goblet cells. This method is therefore useful for correlative light and electron microscopic detection of tissue anionic sites, including sulfate, carboxyl and phosphate groups, at various pH values.     

The activity of HMG-CoA reductase and acetyl-CoA carboxylase in human apocrine sweat glands, sebaceous glands, and hair follicles is regulated by phosphorylation and by exogenous cholesterol

A unique property of human mast cell tryptase is its spontaneous inactivation, which may be relevant to the regulation of the activity of this enzyme in vivo. We have found, using size-exclusion chromatography, that the dissociation of the tetrameric active enzyme into the inactive monomer occurred immediately from the beginning of the inactivation process and at a rate significantly faster than that of the appearance of the inactive, tetrameric form. Eventually, a relatively long-lived state of apparent equilibrium between all three forms (active tetramer, inactive monomer, inactive tetramer) was reached. When tryptase was extensively cross-linked with several heterobifunctional photoactivatable reagents, this modified enzyme exhibited a long-term stability in low-ionic-strength buffer and at elevated temperature, unlike that of the native enzyme. Its is suggested that cross-linking prevents the spontaneous inactivation and dissociation of tryptase by 'freezing' the normal association state of the enzyme and supports the hypothesis that the dissociation of native tetrameric tryptase into inactive monomer is the primary event for the entire process of spontaneous inactivation of this enzyme.     

Tissue expression of lipocalins in human lacrimal and von Ebner's glands: colocalization with lysozyme

BACKGROUND: Tear-specific prealbumin is a group of proteins recently renamed as the tear lipocalins. These proteins were initially described as unique to lacrimal fluid. The tissue distribution and localization have never been thoroughly studied. METHODS: The distribution of purified tear lipocalins was studied in many human secretions and tissues by western blots, immunohistochemistry and immunoelectron microscopy. RESULTS: Tear lipocalin species of the same molecular weights were observed in western blot lanes loaded with tears, saliva, and protein extracts from the lacrimal and lingual von Ebner's glands. Lacrimal and von Ebner's glands contained tear lipocalins; other human tissues and secretions, including other salivary glands and taste buds, did not. Tear lipocalins colocalized with lysozyme in serous acinar cells of lacrimal and von Ebner's glands. Ultrastructurally, tear lipocalins were present on polyribosomes, endoplasmic reticulum, and Golgi areas. Lipocalins were concentrated in lacrimal secretory granules in amounts commensurate with a regulated pathway. CONCLUSION: Tear lipocalins are expressed and truncated similarly in lingual von Ebner's and lacrimal glands, but not at all in other human tissues. Lipocalins are expressed and secreted with lysozyme. Lipocalins are concentrated in secretory granules in an amount consistent with a regulated secretory pathway.     

Vincristine saturation of cellular binding sites and its cytotoxic activity in human lymphoblastic leukemia cells: mechanism of inoculum effect

Vincristine (VCR) is an active agent in the treatment of acute lymphoblastic leukemia (ALL). We evaluated the relationship between the cytotoxic activity of VCR and the degree of VCR saturation of cellular drug binding sites, using the MOLT-3 ALL cell line. When MOLT-3 cells at a density of 1 x 10(6) or 1 x 10(8) cells/mL of pH-controlled medium were exposed to VCR for 1 hr, its cytotoxic activity on cells at high density was 10-fold less than on cells at low density (inoculum effect). The number of VCR binding sites measured by Scatchard analysis was 9.25 x 10(6)/cell. At high cell density, the saturation of VCR binding sites was one log order less than that at low density. Irrespective of cell density, curves of cell-kill versus the degree of VCR saturation of the cellular binding sites overlapped each other. Minimal cytotoxic activity was observed at 0.3% VCR saturation, and nearly maximal cytotoxic activity occurred at about 25% saturation, with the Ic50 at about 4% saturation. These data show that the VCR-induced cell-kill effect is dependent on the degree of saturation of VCR binding sites rather than on the extracellular VCR concentration. The lesser cell-kill on cells at high density can be explained by the lack of drug molecules to sufficiently saturate cellular binding sites. This phenomenon may be responsible, at least in part, for the poor chemotherapeutic outcome of ALL patients with high leukocyte counts at presentation.     

The interaction of leukocytes and their hydrolases with bacteria in vitro and in vivo: the modification of the bactericidal and bacteriolytic reactions by cationic and anionic macromolecular substances and by anti-inflammatory agents

Acid hydrolases from extracts of human blood leucocytes lyse Staph.aureus, Staph.albus and Strep.faecalis in vitro. The leucocyte enzymes can be substituted by a lytic mixture which contains crude trypsin, lysolecithin, phospholipase C and lysozyme, which lyse other bacterial species, e.g. E.coli and Listeria which are resistant to leucocyte enzymes. Bacteriolysis by the lytic agents is strongly inhibited by the anionic polyelectrolytes, heparin, chondroitin sulphate, DNA, dextran sulphate and other sulphated mucopolysaccharides, by the cationic materials, histone, protamine sulphate, leucocyte cationic proteins and polylysine. Other strong inhibitors are trypsan blue and congo red, the phospholipids phosphatidyl serine and ethanolamine, gold thiomalate, extracts of coffee and tea and the anti-inflammatory agents, ultracorten-H, and ultracortenol. Bacteriolysis is also strongly inhibited by normal human serum and by synovial fluids from patients with a variety of joint diseases. The inhibitors in these body fluids are associated with the globulin fractions. Since mixtures of anionic and cationic polyelectrolytes, at equimolar concentrations, failed to inhibit bacteriolysis by leucocyte enzymes, it is postulated that a delicate balance between positively and negatively charged inhibitors control the degradation of cell wall components of bacteria in inflamed areas. Such bacterial components, induce 'storage type' granulomas. The possible role played by polyelectrolytes in the control of the inflammatory process induced by leucocyte hydrolases will be discussed.     

Elongation and folding of nascent ricin chains as peptidyl-tRNA on ribosomes: the effect of amino acid deletions on these processes

This paper is an investigation of articulatory-acoustic correlations differing in degree of articulatory constraint. Data on F2 and on dorsopalatal contact (electropalatographic, EPG) were collected for the sequences /iCi/ and /aC./ with seven Catalan consonants differing in place and manner of articulation (velarised /l/, /n/, /n/, /s/, /f/, /l/, /p/). These consonants are characterised by different degrees of tongue dorsum constraint depending on their production requirements (dorsals > non-dorsals, fricatives > non-fricatives, etc.). The results showed an inverse relationship between vowel-dependent coarticulation and the degree of consonant-dependent articulatory constraint. F2 and dorsopalatal contact size were found to be positively correlated across consonants and speakers, and across consonants for each individual speaker. Correlation values were much lower for each consonant across speakers. These findings are discussed in the light of the acoustic theory of speech production and possible clinical applications are suggested.     

A study of effects of mouthwash on the human oral mucosae: with special references to sites, sex differences and smoking

Nitric oxide has been shown to decrease myocardial contractility and O2 consumption. This study was designed to evaluate the hypothesis that nitric oxide-mediated increases in cyclic GMP require elevated cyclic AMP to produce cardiac depression. Using isolated, Langendorff-perfused rat hearts, we determined the effects of intracoronary nitroprusside (NP, 1 and 10 mM) in the absence and presence of isoproterenol (ISO, 10(-8) M) on cardiac function, O2 consumption, cyclic GMP and cyclic AMP. ISO, with and without NP, increased cyclic AMP (from 287 +/- 21 to 477 +/- 33 pmol/g) without altering cyclic GMP. Left-ventricular pressure increased from 97 +/- 12 to 178 +/- 9 mm Hg and dP/dtmax from 1,786 +/- 275 to 4,049 +/- 354 mm Hg/s. NP increased cyclic GMP (from 4 to 30 pmol/g) in both the absence and presence of ISO, but NP did not alter cyclic AMP. Without ISO, NP insignificantly altered left-ventricular pressure; however, in the presence of ISO, NP significantly decreased left-ventricular pressure by -25 +/- 4 mm Hg and decreased dP/dtmax by -619 +/- 142 mm Hg/s. Isoproterenol increased O2 consumption, but the changes with NP were not significant. When this study was repeated in the presence of LY83583, a guanylate cyclase inhibitor, NP still produced cardiac depression in the presence of ISO. Therefore, cardiodepressant effects of NP were only observed against a background of inotropic stimulation with ISO. However, effects of NP on contractility were unrelated to increases in cyclic GMP or cyclic GMP-induced changes in cyclic AMP.     

Inhibition of paracrine angiotensin-converting enzyme in vivo: effects on interstitial glucose and lactate concentrations in human skeletal muscle

Microdialysis was used to selectively assess the effect of the paracrine renin-angiotensin system (RAS) on interstitial glucose and lactate concentration profiles in skeletal muscle of healthy volunteers (n = 8) during basal and insulin-stimulated conditions. Paracrine RAS was selectively inhibited by local retrodialysis with enalaprilate. Under basal conditions, local administration of enalaprilate (2 micrograms mL-1) increased interstitial dialysate glucose concentration from 0.71 +/- 0.14 mmol L-1 to 0.84 +/- 0.14 mmol L-1 and decreased the serum interstitial gradient (SIGglu) compared with baseline (P < 0.02). Under clamp conditions, enalaprilate, even at the lowest concentration (0.02 microgram mL-1), increased interstitial dialysate glucose concentration from 0.77 +/- 0.11 mmol L-1 to 1.02 +/- 0.09 mmol L-1 and decreased SIGglu compared with baseline (P < 0.01). Interstitial lactate concentrations slightly increased during basal as well as during clamp conditions (P < 0.05 vs. baseline). Selective inhibition of paracrine muscle angiotensin-converting enzyme (ACE) increases interstitial glucose and lactate concentrations and decreases SIGglu in muscle by facilitating transcapillary glucose transport. This effect is more pronounced during hyperinsulinaemia and may be of clinical relevance in diabetic patients treated with therapeutic doses of enalapril.     

Interaction of human gallbladder mucin with calcium hydroxyapatite: binding studies and the effect on hydroxyapatite formation

Calcium hydroxyapatite (HAP) crystals formed in vitro in the presence of polymeric human gallbladder mucin (1.0 mg/mL) were smaller (0.75 +/- 0.39 microns) than control crystals (7.86 +/- 2.76 microns), but the mucin did not affect the kinetics of crystal formation or alter the amount of mineral phase present at equilibrium. In contrast, glycopeptide subunits produced by proteolysis of the native mucin had no effect on HAP crystal size. Both native mucin and glycopeptides bound to mature HAP crystals, but the glycopeptides were much more readily displaced by phosphate ions. Therefore, in experiments where HAP was being formed, the phosphate ions inhibited the interaction of glycopeptides with the nascent HAP. These results indicate that gallbladder mucin may modulate HAP formation in vivo, and that this ability may be altered during pathological states, such as neutrophil infiltration or bacterial colonization, that may cause the release of proteinases capable of digesting mucin.     

In vitro study of the effect of miglitol on carbohydrate digestion and intestinal metabolism in normal and non-insulin-dependent diabetic rats

The effect of miglitol was studied (20 mg/kg body weight), administered intraduodenally alone or together with maltose, on the absorption and intestinal metabolism of glucose during its translocation from the lumen of the intestine to the blood, using in vitro perfused preparations of complete small intestine-pancreas, proximal small intestine alone, or distal small intestine alone, isolated from normal and non-insulin-dependent diabetic rats. In the absence of a luminal administration of maltose in normal rats, the glucose uptake from the vascular perfusate was greater in the presence (0.52 +/- 0.04 mmol/h) than in the absence (0.39 +/- 0.02 mmol/h) of miglitol (p < 0.05). In diabetic rats, no significant variations were observed in glucose uptake from the vascular perfusate as an effect of miglitol, but the glucose uptake in the presence of this drug was significantly less (p < 0.05) than that observed in normal rats. Portal lactate was significantly greater (p < 0.05) in diabetic than in normal rats and, after administration of miglitol, rose in both normal and diabetic rats, the rise being significantly greater in normal than in diabetic rats (p < 0.01). When maltose was administered luminally (2 g/kg body weight), the values of portal glucose in both normal and diabetic rats were significantly less in the presence of miglitol in the complete as well as in the distal and proximal small intestine preparations (p < 0.05); the glucose uptake from luminal administered maltose was greater in the presence of miglitol in diabetic (p < 0.05) and in normal (p < 0.05) rats except in the complete small intestine of normal rats; and no significant differences were observed in portal lactate levels between normal and diabetic rats in the presence of miglitol. In conclusion, our results show that miglitol administered luminally at the doses employed here, as well as reducing the transport of glucose from the lumen of the intestine into the blood supply, significantly stimulate intestinal glucose metabolism.     

Bradykinin-induced vasodilation of human coronary arteries in vivo: role of nitric oxide and angiotensin-converting enzyme

Voltage-clamped GABA(A fast) and GABA(A slow) inhibitory postsynaptic currents (IPSCs) were selectively elicited in hippocampal area CA1 pyramidal neurons. Clinically relevant concentrations of halothane (1.2 vol.%) prolonged both GABA(A fast) and GABA(A slow) IPSC decay times approximately 2.5 fold, while having little to no effect on current amplitudes or rise times. Current-voltage analysis revealed that IPSC reversal potentials (-70 to -75 mV) remained constant in the presence of halothane. Under control conditions, GABA(A slow) IPSC decay times increased linearly with membrane depolarization, and this IPSC decay time voltage dependence was not significantly altered by halothane. These results confirm the existence of separable GABA(A fast) and GABA(A slow) IPSCs in hippocampus, and further elucidate the effects of halothane on these currents.     

Demonstration of peripheral fucose units in N-linked glycans of human immunodeficiency virus type 1 gp 120: effects on glycoprotein conformation

Fucosylated N-linked glycans are important constituents of membrane glycoproteins, owing to their significance as biologically active ligands for several selectins and their role in modulating protein conformation of viral glycoproteins. The human immunodeficiency virus type 1 (HIV-1) glycoprotein contains more than 30 different glycan structures but so far fucose was found associated solely with the innermost GlcNAc of N-linked glycans. In the present report we determined whether fucose units also were linked to the distal GlcNAc via alpha(1-3) or alpha(1-4) linkages in N-linked glycans of gp 120. [3H]-fucose labelled gp 120 was subjected to endoglycosidase F digestion, releasing diantennary complex type N-linked glycans, but leaving the inner polypeptide-bound carbohydrates, GlcNAc and possibly associated fucose units, intact. Gel filtration of the digested material revealed that [3H]-fucose label was released from gp 120 by this treatment, indicating presence of peripheral fucose units. Furthermore, [3H]-focuse label was also released by treatment of the labelled gp 120 with an alpha-L-fucosidase specifically removing fucose in alpha(1-3) and alpha(1-4) linkages. Altogether the results indicated presence of fucose units linked to peripheral GlcNAc of gp 120 N-linked glycans. We have earlier shown that other peripheral carbohydrate determinants, i.e. beta(1-4)-galactose on N-linked glycans, maintain a correct antigenic conformation of gp 120. Using a coupled ELISA system, where changes in antigenic behaviour of a viral glycoprotein were correlated to stepwise elimination of peripheral monosaccharides from N-linked glycans, we found that treatment of gp 120 with a pan-specific alpha-fucosidase as well as an enzyme specific for alpha(1-3)- or alpha(1-4)-linked fucose disclosed a hidden linear epitope situated in the gp 120 C2 region. The effects of the general fucosidase on epitope exposure was more prominent than those obtained with the enzyme with narrow specificity, suggesting that peripheral and inner fucose units co-operate in the maintenance of gp 120 conformation.     

Detection of gluten in human sera by an enzyme immunoassay: comparison of dermatitis herpetiformis and celiac disease patients with normal controls

We have developed a triple sandwich enzyme immunoassay to detect circulating gluten in human sera. With human sera containing known amounts of added gluten as controls, the assay was sensitive in the range of 0.75 to 75 micrograms of gluten per ml of serum. Forty-one control subjects were compared to 21 patients with dermatitis herpetiformis and 11 patients with celiac disease. The dermatitis herpetiformis and celiac disease patients had significant elevation of serum gluten values over the control subjects. Circulating gluten antigenemia is a previously unrecognized feature which may be important in understanding the pathogenesis of dermatitis herpetiformis and celiac disease.     

Hypothermic coagulopathy in trauma: effect of varying levels of hypothermia on enzyme speed, platelet function, and fibrinolytic activity

Comparative study of protein metabolism in neurons of layers III and V of the sensorimotor cortex was carried out in two groups of Wistar rats, which differed in learning results: "bad" (60% of population) and "good" learners (40%). It was found out that the associative neurons (layer III) were most sensitive to cognitive load. In "bad" learners, an increase in nuclear and cytoplasmic dimensions and rise in protein concentration and content took place in these neurons, while in the efferent neurons (layer V) the protein content increased only in the cytoplasm. In "good" learners, the cognitive load led to a decrease in all the cytochemical parameters in neurons of the layer III while in the neurons of the layer V the content and concentration of proteins increased both in nuclei and cytoplasm. It is suggested that the character of protein metabolism changes produced by information load can be considered as a reflection of individual peculiarities of cognitive activity, and the extent of cytochemical changes as a reflection of complicity of a cognitive task.     

Effects of saturation and esterification of fat sources on site and extent of digestion in steers: ruminal fermentation and digestion of organic matter, fiber, and nitrogen

Extracellular oxygen radicals produced by H9c2 rat heart cells in monolayer cultures during ischemia and subsequent reoxygenation were monitored using the luminol-horseradish peroxidase-enhanced chemiluminescence technique. As expected, the photon count diminishes during ischemia but again rapidly attains normal values following reoxygenation. In the presence of superoxide dismutase, this photon emission is repressed, as is also the case in the presence of diphenylene iodonium, a specific inhibitor of NADPH-oxidase activity. Thus, the conclusion seems justified that H9c2 rat heart cells in monolayer cultures produce superoxide radicals extracellularly due to an NADPH oxidase-like action. In order to characterize this extracellular superoxide-generating system, we determined its sensitivity to increased temperatures, inhibition of protein synthesis and perturbations of cytoskeletal structures. Heat shocks result in a delayed inactivation of the NADPH oxidase activity followed by recovery, the kinetics of which depend on the imposed heat shock temperature. This inactivation is independent of protein synthesis and actin cytoskeletal structures, but the recovery of the enzyme's activity is dependent on these entities.     

Expression of mannose-ligand receptors on human spermatozoa: effect of lecithin and association with sperm binding to the zona pellucida

OBJECTIVE: To evaluate the change in the expression of mannose-ligand receptors and sperm binding capacity after the incubation of sperm cells with lecithin liposomes. DESIGN: A randomized, blinded-controlled experiment. SETTING: Andrology laboratory at the Lis Maternity Hospital. PATIENT(S): Fifteen fertile sperm donors and 10 subfertile men. INTERVENTION(S): Incubation of sperm samples with either control medium or 1 mg/mL of liposomal lecithin for 2 hours. MAIN OUTCOME MEASURE(S): Expression of mannose-ligand receptors as evaluated by mannosylated bovine serum albumin-fluorescein isothiocyanate and sperm binding to the zona pellucida as evaluated by the hemizona assay. RESULT(S): The mean +/- SE percentages of spermatozoa with patterns I, II, and III were 86% +/- 4.8%, 11% +/- 3.4%, and 3% +/- 1.6%, respectively, after treatment with control medium and 71% +/- 5.7%, 22% +/- 3.5%, and 7% +/- 2.5%, respectively, after treatment with lecithin. The same effect of lecithin was observed in the 10 sperm samples from subfertile men. The mean +/- SE numbers of sperm that bound to hemizonae after treatment with control medium or lecithin were 116 +/- 32.4 and 176 +/- 29.6, respectively. Statistically significant correlations were observed between the shift in patterns II and III and the enhancement of sperm binding after lecithin treatment (r = 0.44 and 0.6, respectively). CONCLUSION(S): Lecithin shifts the expression of mannose-ligand receptors to the capacitated and acrosoine-reacted patterns and enhances the binding capacity of the sperm cells.     

Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration

Five human diseases are due to an excessive number of CAG repeats in the coding regions of five different genes. We have analyzed the repeat regions in four of these genes from nonhuman primates, which are not known to suffer from the diseases. These primates have CAG repeats at the same sites as in human alleles, and there is similar polymorphism of repeat number, but this number is smaller than in the human genes. In some of the genes, the segment of poly(CAG) has expanded in nonhuman primates, but the process has advanced further in the human lineage than in other primate lineages, thereby predisposing to diseases of CAG reiteration. Adjacent to stretches of homogeneous present-day codon repeats, previously existing codons of the same kind have undergone nucleotide substitutions with high frequency. Where these lead to amino acid substitutions, the effect will be to reduce the length of the original homopolymeric stretch in the protein.