Regulation of hyaluronidase inhibition through its chemical modification

The modification of hyaluronidase by aldehydodextran regulates inhibition of the enzyme by heparin. A 70-90% modification of the surface amino groups of hyaluronidase results in sharp conformational changes and a substantial decrease of its inhibition by heparin, whereas hyaluronidase derivatives with a modification degree of 96-100% are practically uninhibited.     

Resistance of Dextran-Modified Hyaluronidase to Inhibition by Heparin

Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.     

Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity

Sulfated polysaccharides from the green algae Ulva conglobata were isolated and prepared by extraction in hot water, precipitation with ethanol and purification by ion-exchange and size-exclusion column chromatography. The characterizations of the sulfated polysaccharides were defined, and containing 23.04–35.20% sulfate ester groups, 10.82–14.91% uronic acid and 3.82–4.51% protein. Gas chromatography analysis shows that the sulfated polysaccharides from Ulva conglobata are mainly consisted of rhamnose with variable contents of glucose and fucose, trace amounts of xylose, glactose and mannose. The anticoagulant properties of the sulfated polysaccharides were compared with those of heparin by studying the activated partial thromboplastin time using normal human plasma. The sulfated polysaccharide from Ulva conglobata collected in Qingdao, China is the most potent among the sulfated polysaccharides tested. The mechanism of anticoagulant activity mediated by the sulfated polysaccharides is due to the direct inhibition of thrombin and the potentiation of heparin cofactor II.     

33-kDa C-terminal heparin binding fragment of fibronectin promotes attachment and spreading of hepatocytes

The possibility of interaction of hepatocytes with the heparin binding domain of Fibronectin was examined. Rat hepatocytes adhered to coverslips coated with the 33-kDa heparin binding fragment of the C-terminal region of plasma fibronectin. When different concentrations of the heparin binding fragment were used to coat coverslips and used as substratum, cell attachment showed saturation kinetics. Half the maximum attachment was observed at 30–40 min after seeding of cells. The cells became flat after 2–3 h indicating that they spread on the heparin binding domain as they do on intact fibronectin. Among the different glycosaminoglycans tested, maximum inhibition of attachment was observed for heparin. However it was not possible to completely inhibit attachment even at high concentrations. These results indicate that hepatocytes interact with fibronectin not only through the Arg-Gly-Asp-containing cell binding fragment, but also through the heparin binding domain of fibronectin and, further, that there exist heparin-dependent and heparin-independent mechanisms of interaction of cells with the 33-kDa heparin binding fragment of fibronectin     

Heparin Promotes Suspension Adaptation Process of CHO–TS28 Cells by Eliminating Cell Aggregation

While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO–TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P < 0.001). Heparin also exhibited a cell aggregation elimination role at all concentrations (P < 0.001). Furthermore, heparin promoted cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10⁴ cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P < 0.001) both occurring at 250 μg/ml heparin. When agitated, cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO–TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.     

Inhibition of hyaluronidase by fully O-sulfonated glycosaminoglycans.

We report a new flow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using fluorometric detection with the fluorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were confirmed by (1)H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC(50) values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects.     

Differential allelopathic expression of bark and seed of Tamarindus indica L.

Allelopathic performance of the bark and seed of Tamarindus indica L. tree was evaluated through bioassay-guided studies using seven common agronomic crops (asparagus, cucumber, lettuce, radish, sesame, tomato and welsh onion) and seven weed species (barnyard grass, Chinese milk vetch, perennial ryegrass, phacelia, timothy grass, white clover and wild ginger) under laboratory conditions. As demonstrated by a sandwich method, the bark of the tamarind tree caused strong growth inhibition (compared to the corresponding controls) in both radicles and hypocotyls of the species tested, and the inhibitory effect was highest in barnyard grass (52–65%) and lowest in welsh onion (19–13%). The crude-water soluble extracts of bark at different concentrations (1, 5 and 10%) (w/v) exhibited a strong growth inhibition in all the plant species tested, and a proportional increase in the percentage of growth inhibition was observed with an increase in the concentrations of the extracts. The magnitude of inhibition in weed species was higher (5–60%) than those of agronomic crop species (3–40%). The growth of all the weed species tested was strongly inhibited (17–56%), while the agronomic crop species showed both inhibited (5–21%) and stimulated (5–27%) growth due to the effect of crude-water soluble exudates of tamarind seed. Among the agronomic crop species tested, lettuce (22–27%) followed by radish (20–25%) and sesame (5–8%) showed stimulatory growth with the crude-water soluble exudates of seed. In the pot culture experiments using four agronomic crops (lettuce, radish, tomato and cucumber) and two weed species (barnyard grass and white clover), spraying of crude-water soluble extracts of tamarind seed-coat at three different concentrations (1, 5 and 10%) (w/v) showed that the growth of lettuce (35–62%) and radish (32–56%) was stimulated, while all other species tested showed growth inhibition (29–61%). When the spraying of crude extracts of seed-coat was turned off, the growth of both lettuce and radish continued to be stimulated (4–7%) and all other previously inhibited species recovered well, the recovery percentage ranging between 78 and 82%. However, when spraying of crude extracts of seed-coat was continued, growth increased (10–14%) in lettuce and radish, and reduced (37–76%) in four other species tested. The inhibitory or stimulatory effects of the crude extracts on agronomic crop and weed species were higher in the radicle than the hypocotyl and reached a peak with 10% (w/v) concentrations. These results clearly demonstrated the differential allelopathic effects (inhibitory and excitatory) of bark and seed of tamarind tree in the species tested. Thus, it is evident that these two organs contain certain biologically active true growth regulator(s) and are either additively or synergistically involved in the plant-specific expression, particularly by the seed-coat.     

Transforming growth factor-β1 blocks the enhancement of tumor necrosis factor cytotoxicity by hyaluronidase Hyal-2 in L929 fibroblasts

Background  

Functional antagonism between transforming growth factor beta (TGF-β) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-20 counteracts TGF-β1-mediated growth inhibition of epithelial cells. PH-20 sensitizes various cancer cells to tumor necrosis factor (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domain-containing oxidoreductase (WOX1). TGF-β1 blocks PH-20-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-β1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined.     

PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells

PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor. In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt) expression, an increase in p27^Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27, and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the proliferation of endometrial carcinoma cells through the induction of contact inhibition.     

Role of the glycosaminoglycan microenvironment of hyaluronidase in regulation of its endoglycosidase activity

The glycosaminoglycan microenvironment of testicular hyaluronidase was simulated by multipoint covalent attachment of the enzyme to glycans as a result of benzoquinone activation. The efficiency of their binding was assessed using gel chromatography, ultrafiltration, titration of surface amino groups of the enzyme, electrophoresis, as well as judging by the value of residual endoglycosidase activity and its inhibition with heparin. Copolymer glycosaminoglycans, such as dermatan sulfate and heparin, inactivated the endoglycosidase activity as a result the C-5 epimerization of hexuronic acid. It was shown that glucuronic acid and, to a lesser extent, N-acetylglucosamine determine the specificity of hyaluronidase. The chondroitin-sulfate microenvironment made the enzyme resistant to heparin inhibition because the equatorial orientation of the OH groups is similar to that in hyaluronic acid. Model experiments with dextran and dextran sulfate showed that sulfation of the glycan chain increased its rigidity, thus hampering the stabilizing effect on hyaluronidase. The effect of chondroitin sulfate on the endoglycosidase activity of hyaluronidase had additive character and did not directly affect the small fragment of the active site of the enzyme located at the bottom of a groove. The glycosaminoglycan microenvironment of hyaluronidase, containing an iduronic acid residue, the 1-3 and 1-4 glycosidic bond, inactivated the hyaluronidase activity of the enzyme, whereas simple polymers (such as gluco- and galactoaminoglycans) potentiated it due to a similar way of linking—(1e-4e) and (1e-3e). To understand the nature of these interactions in detail, the effect of oligomeric glycosaminoglycan fragments and their derivatives on hyaluronidase should be studied.     

Contribution of adenosine 5′-diphosphoglucose pyrophosphorylase to the control of starch synthesis is decreased by water stress in growing potato tubers

Water stress stimulates sucrose synthesis and inhibits starch and cell-wall synthesis in tissue slices of growing potato (Solanum tuberosum L. cv. Desirée) tubers. Based on the analysis of fluxes and metabolites, Geigenberger et al. (1997, Planta 201: 502–518) proposed that water deficits up to −0.72 MPa stimulate sucrose synthesis, leading to decreased starch synthesis as a result of the resulting decline of phosphorylated metabolite levels, whereas more-severe water deficits directly inhibit the use of ADP-glucose. Potato plants with decreased expression of adenosine 5′-diphosphoglucose pyrophosphorylase (AGPase) have been used to test the prediction that the contribution of AGPase to the control of starch synthesis should decrease in severely water-stressed tuber material. Freshly cut slices from wild-type and antisense tubers were incubated at a range of mannitol concentrations (20, 300 and 500 mM) and the metabolism of [¹⁴C]glucose was analysed. A 86–97% reduction of AGPase activity led to a major but non-stoichiometric inhibition of starch accumulation in intact growing tubers attached to the plant (40–85%), and an inhibition of starch synthesis in non-stressed tuber slices incubated in 20 mM mannitol (60–80%). The inhibition of starch synthesis was accompanied by a 2- to 8-fold increase in the levels of sugars in intact tubers and a 2- to 3-fold stimulation of sucrose synthesis in tuber slices, whereas respiration and cell-wall synthesis were not significantly affected. The strong impact of AGPase on carbon partitioning in non-stressed tubers and tuber slices was retained in slices subjected to moderate water deficit (300 mM mannitol, corresponding to −0.72 MPa). In discs incubated in 500 mM mannitol (corresponding to −1.2 MPa) this response was modified. A 80–97% reduction of AGPase resulted in only a 0–40% inhibition of starch synthesis. Further, the water stress-induced stimulation of sucrose synthesis was abolished in the transformants. The results provide direct evidence that the contribution of AGPase to the control of starch synthesis can be modified by environmental factors, leading to a lower degree of control during severe water deficits. There was also a dramatic decrease in the labelling of cell-wall components in wild-type tuber slices incubated with 300 or 500 mM mannitol. The water stress-induced inhibition of cell-wall synthesis occurred independently of AGPase expression and the accompanying changes in starch and sucrose metabolism, indicating a direct inhibition of cell-wall synthesis in response to water stress. Received: 24 February 1999 / Accepted: 28 May 1999     

Heparin and the phenotype of adult human vascular smooth muscle cells

Summary To study mechanisms controlling growth and phenotype in human vascular smooth muscle cells, we established culture conditions under which these cells proliferate rapidly and achieve life-spans of 50–60 population doublings. In medium containing heparin and heparin-binding growth factors, growth rate and life-span of human vascular smooth muscle cells increased more than 50% relative to cultures with neither supplement, and more than 20% compared to cultures supplemented only with heparin-binding growth factors. In contrast to observations made in rat vascular smooth muscle cells, smooth muscle-specific α-actin in the human cells was expressed only in the presence of heparin and colocalized with β/γ nonmuscle actins in stress fibers, not in adhesion plaques. Heparin, in the presence of heparin-binding growth factors, also caused more than 170% stimulation of tracer glucosamine incorporation into hyaluronic acid and a 7.5-fold increase in hyaluronic acid accumulation. In comparison, total sulfate incorporation into sulfated glycosaminoglycans increased by less than 40%. In light of our previous findings that heparin suppresses collagen gene expression, we conclude that heparin induces human vascular smooth muscle cells exposed to heparin-binding growth factors to remodel their extracellular matrix by altering the relative rates of hyaluronic acid (HA) and collagen synthesis. The resulting hyaluronic-acid-rich, collagen-poor matrix may enhance infiltration of CD44/hyaluronate-receptor-bearing T-lymphocytes and monocytes into the vascular wall, an early event in atherogenesis.     

Optimization and scale-up of a new photobleaching agar extraction process from Gracilaria lemaneiformis

An eco-friendly photobleaching extraction process for agar extraction from the red alga Gracilaria lemaneiformis was developed for the benefit of workers’ health and environmental safety. Here we report the optimization of key process parameters (alkali modification concentration, photobleaching duration, algal length and screen filter opening size) in order to scale up this new technique. The optimal conditions were found to be modification by 3–5% NaOH, photobleaching for 5 h, using algal fragments 2 –4 cm in length, and a filter screen with a 6 μm opening. A 20-L agar extraction reactor was thus constructed, and the scale-up of the agar extraction process was tested in six batch experiments. The resulting agar quality was similar to that of the laboratory-scale extraction. In addition, batch-to-batch reproducibility was excellent. The results demonstrate the excellent scale-up ability and potential application of this new photobleaching agar extraction process on a commercial scale. The agar yield and gel strength for 5% NaOH modified agar were 26.8% and 1,897 g cm⁻², while those for 3% NaOH modified agar were 28.2% and 1,287 g cm⁻², respectively. It is clear that the agar yield and quality can be manipulated via alkali modification in this new eco-friendly extraction to meet market demands.     

Inhibition of <Emphasis Type="Italic">Naja naja</Emphasis> Venom Hyaluronidase by Plant-Derived Bioactive Components and Polysaccharides

The inhibitory effect of several bioactive compounds on the activity of hyaluronidase enzyme purified from Naja naja venom was investigated in vitro. Compounds were found to inhibit the hyaluronidase activity dose dependently. Among glycosaminoglycans, heparin, heparan sulfate, and dermatan sulfate showed maximum inhibition compared to chondroitin sulfates. Different molecular forms of chitosan inhibit the enzyme, and inhibition appears to depend on the chain length. In addition, plant-derived bioactive compounds also inhibited the activity of hyaluronidase dose dependently. Among those tested, aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone exhibited inhibition, with aristolochic acid and quercetin completely inhibiting the enzyme activity. It is concluded that the inhibitors of hyaluronidase could be used as potent first aid agents in snakebite therapy. Furthermore, these inhibitors not only reduce the local tissue damage but also retard the easy diffusion of systemic toxins and hence increase survival time.     

Conformation of heparin studied with macromolecular hydrodynamic methods and X-ray scattering

The hydrodynamic characteristics of heparin fractions in a 0.2 M NaCl solution have been determined. Experimental values varied over the following ranges: the sedimentation coefficient (at 20.0 °C), 1.3<s₀×10¹³<3.2 s; the Gralen coefficient (sedimentation concentration-dependence parameter), 10<k_s<70 cm³ g^–1; the translational diffusion coefficient, 3.9<D₀×10⁷<15.4 cm² s^–1; the intrinsic viscosity, 7.9<[]<40 cm³ g^–1. Combination of s₀ with D₀ using the Svedberg equation yielded molecular weights in the range 3.9<M×10^–3<37 g mol^–1. The value of the mass per unit length of the heparin molecule, M_L, was determined using the theory of hydrodynamic properties of a weakly bending rod, giving M_L=570±50 g nm^–1 mol^–1. The equilibrium rigidity, Kuhn segment length (A=9±2 nm) and hydrodynamic diameter (d=0.9±0.1 nm) of heparin were evaluated on the basis of the worm-like coil theory without the excluded volume effect, using the combination of hydrodynamic data obtained from fractions of different sizes. Small-angle X-ray scattering for three heparin fractions allowed an estimate for the cross-sectional radius of gyration as 0.43 nm; from the evolution with the macromolecule contour length of the radius of gyration, a value for the Kuhn segment length of 9±1 nm was obtained. A good correlation is thus observed for the conformational parameters of heparin from hydrodynamic and X-ray scattering data. These values describe heparin as a semi-rigid polymer, with an equilibrium rigidity that is essentially determined by a structural component, the electrostatic contribution being negligible in 0.2 M NaCl.Presented at the conference for Advances in Analytical Ultracentrifugation and Hydrodynamics, 8–11 June 2002, Grenoble, France     

Bioassay as a tool for assessing susceptible and resistant plant species for field contaminated with industrial effluent

A bioassay technique was used to select plant species that were able to germinate and grow in a site contaminated with flash torch and battery manufacturing industrial effluents. Three varieties each of rice, namely Surya-52, Jaya-14 and Pant-10; three varieties of pulses [Gram (Bahar), Mung (K-851) and Lens (T-9)] and three varieties of oilseeds (Mustard-RS-30, Mustard-B-9 and Mustard T-69) were used for the determination of phytotoxicity by bioassay technique. The average % phytotoxicity for rice was 18.03% (14.34–22.7%), for pulses was 15.76% (8.75–26.64%) and for oil seed was 11.09% (6.42–15.24%). Accumulation of metals (Cu, Fe, Mn, Cd, Cr and Pb) was estimated in the root, shoot and edible parts of different crop varieties grown in pot culture up to maturity and treated with industrial effluent. The concentration of different metal ions in root, shoot and edible parts were in the range of Cu: 9.25–45.25, 7.25–42.35 and 5.65–35.26; Fe: 60.66–212.25, 45.24–155.62; Mn: 7.12–38.30, 6.25–27.27.23 and 4.25–24.45; Cd: 0.80–2.45, 0.75–2.12 and 0.45–1.95, Cr: 6.54–28.25, 5.36–24.45 and 4.35–16.32; and Pb: 0.95–3.75, 0.78–2.25 μg/g d.w. respectively. A higher concentration of Cd was found in Surya-52 rice variety and in Gram (Bahar) pulse variety and of Pb was detected in Surya-52 rice variety. Cd and Pb are non-essential metal ions and highly toxic to plants. Accumulation of toxic metal ions like Pb and Cd in the edible parts of oil a seed variety may not exceed the recommended daily intake limits. Percentage phytotoxicity and inhibition of root and shoot length was also less in the oil seed variety. Thus these plant varieties can be considered for cultivation in fields contaminated by waste from the flash-torch and battery-manufacturing industry.     

Metabolism of dynorphin A1–13 in human CSF

In-vitro incubation of human cerebrospinal fluid (CSF) obtained from patients ranging from 22–78 years with 10 μM of dynorphin A1–13 (Dyn A1–13) resulted in several cleavage products. Dyn A1–12 and A2–13 were identified as the major CSF metabolites by matrix-assisted laser desorption mass spectrometry (LD-MS). Further metabolites were Dyn A1–6, A2–12 and A4–12. LD-MS further suggested the formation of Dyn A1–8, A1–7, A1–10, A7–10, A3–12, A7–12, A3–13, A7–13 and A8–13. The metabolic half-life of Dyn A1–13 at 37°C was approximately 2.5 h (range 1.75–8.5 h), compared to less than one minute in plasma. The half-life of Dyn A1–13 decreased markedly with age or age-associated processes (n=20, r²=0.498). Noncompartmental kinetic analysis in the absence or presence of enzyme inhibitors (leucinethiol 10 μM, captopril 100 μM and GEMSA 20 μM) suggested that Dyn A1–13 is mainly metabolized by carboxypeptidase to A1–12 (51%) and by aminopeptidases to A2–13 (35%). The generation of A1–6 (13%) was only detected under enzyme inhibition. The extent of conversion into the main metabolites did not follow an age-associated trend, thus over-all enzyme levels but no specific enzymatic systems are elevated with age.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

Histochemical demonstration of acidic glycosaminoglycans in the cell nuclei of the iris and other tissues.

Using histochemical methods, the presence of acidic glycosaminoglycans in the cell nuclei of 51 human irides and a series of monkey organs was demonstrated. In general, these substances are sensitive to testicular hyaluronidase and chondroitinase ABC and also to Streptomyces hyaluronidase, when using special staining methods. The specificity of testicular hyaluronidase was tested by inhibition with heparin. By simultaneously staining with alcian blue and Feulgen, acidic glycosaminoglycans can be distinguished from the nucleic acids. Sporadically, hyaluronidase-resistant substances with a specific acidic glycosaminoglycan stainability occur. We assume the existence of various acidic glycosaminoglycans in the cell nuclei. Aging changes were not traceable with constancy.     

GABAergic disinhibition changes the recovery cycle of bat inferior collicular neurons

This study examines the contribution of GABAergic inhibition to the discharge pattern and recovery properties of 110 bat inferior collicular neurons by means of bicuculline application to their recording sites. When stimulated with single pulses, 74 (67%) neurons discharged one or two impulses (phasic responders), 19 (17%) discharged three to ten impulses (phasic bursters) and 17 (16%) discharged impulses throughout the entire stimulus duration (tonic responders). Bicuculline application changed phasic responders into phasic bursters or tonic responders, increased the number of impulses by 10–2000% and shortened the response latency of most neurons. When stimulated with pairs of sound pulses, the recovery cycles of these neurons can be described as: (1) long inhibition (n = 49, 45%); (2) short inhibition (n = 41, 37%); and (3) fast recovery (n = 20, 18%) based upon the 50% recovery time that was either longer than 20 ms, between 10 and 20 ms or shorter than 10 ms. Bicuculline application shortened the 50% recovery time of most neurons by 11–2350% allowing them to respond to pairs of sound pulses at very short interpulse intervals. These data demonstrate that GABAergic inhibition contributes significantly to auditory temporal processing. Accepted: 18 April 1997