Volume elasticity of the ocular avascular compartment in vivo and post mortem

By considering the frequency dependence of the ocular volume elasticity it is possible to locate the static volume elasticity function of the avascular compartment of the eye in vivo. The procedure used involved measuring the dynamic volume elasticity function E=f(P, v), where E=volume elasticity, P=intraocular pressure, and v=frequency, in vivo and post mortem at a frequency higher than the apparent upper mechanical response frequency of the intraocular vascular bed. In addition, post mortem measurements were made of the volume elasticity function at a frequency which was as low as experimentally possible. For practical purposes the latter volume elasticity function may serve as an estimate of the static elasticity function of the avascular compartment in vivo. This is possible in all cases because at the high frequency level the dynamic volume elasticity functions measured in vivo and post mortem are identical.Partly presented by the first author at the 4th Mackenzie Symposium, Stirling 1977Decaased 18.3. 1980     

Processing of snake venom l-amino acid oxidase during intracellular transport

The isoelectrofocusing patterns of l-amino acid oxidase (LAO) from venom gland homogenates and of the secreted venom of Vipera palaestinae have been compared. The LAO isozyme profile of actively synthesizing gland spans over a wider range of pIs (4.8–6.0) and includes more variants as compared with the profile of the secreted venom. A basic shift of the isoelectrofocusing pattern of LAO obtained by treatment of the gland homogenate or the venom with neuraminidase indicates that sialic acid residues are responsible for the changes in the electronegativity of the isozymes. Analyses of subcellular fractions show that the microsomal fraction of the venom gland homogenate exhibits the highest multiplicity of molecular forms of LAO, whereas the fraction including the secretory granules has an isozyme profile similar to the venom. Double labelling experiments show that the newly synthesized LAO include isozymes which span over a wide range of pIs, whereas later on labelling of the more acidic isozymes is prominent. The results obtained may suggest that the sialic acid residues which are attached to LAO during its transport serve as “markers” for secretion.     

Increased Noradrenergic Metabolism in the Cerebellum of the Mouse Mutant Dystonia Musculorum

Abstract: The neurological mouse mutant dystonia musculorum exhibits bizarre appendicular and truncal dystonia without known cerebellar histopathology. We evaluated striatal dopamine and cerebellar norepinephrine metabolism in this mutant and compared the results with those obtained in wild-type BALB/c and B6C3 controls. Tyrosine hydroxylase activity and dopamine metabolite levels (homovanillic acid and 3,4-dihydroxyphenylacetic acid) in the striatum of the mutant were similar to controls. Tyrosine hydroxylase activity and the steady-state level of 3-methoxy-4-hydroxyphenethyleneglycol, a metabolite of norepinephrine, in the cerebellum were 38% and 42-66%, respectively, greater in the mutant. However, the level of norepinephrine was similar (∼350 ng/g). Further, a Purkinje cell-specific marker, cGMP-dependent protein kinase, was unchanged in the mutant and no Purkinje cell pathology was observed with light microscopy. The lack of Purkinje cell derangement and similar levels of cerebellar norepinephrine and cGMP-dependent protein kinase activity suggest that increased norepinephrine metabolism in the cerebellum of this mutant is not a morphological response to gross target cell loss during morphogenesis. The observed changes may be a reaction to abnormal impulse traffic or altered input/output pathways to the mutant cerebellum during its development.     

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.     

Development of dopamine receptor denervation supersensitivity in the neostriatum of the senescent rat

Dopamine (DA) stimulated adenylate cyclase activity and [³H]-spiroperidol specific binding were assessed in the striata from mature and old rats lesioned in the left substantia nigra with 6-hydroxydopamine (6-OHDA). Rotational behavior following the DA releasing agent, amphetamine, and the DA receptor agonist, lergotril, was also examined at 7 and 30 days, respectively, after lesioning. Results indicated that while there were rotational behavioral deficits following amphetamine in the senescent animal, none were seen with respect to lergotril. Both old and young animals showed similar degrees of contralateral rotation (with respect to the lesion) following lergotril administration. This suggested that both old and young animals showed similar development of denervation supersensitivity in the DA receptors of the lesioned striatum. Subsequent biochemical confirmation of this hypothesis was provided by findings which showed comparable relative increases in DA stimulated adenylate cyclase activity and [³H]-spiroperidol specific binding in the striata from the lesioned hemispheres of young and old rats. Additionally, high positive correlations were found between rotation and [³H]-spiroperidol specific binding, while those between DA stimulated adenylate cyclase activity and rotation were lower and dependent upon the concentration of DA used to stimulate adenylate cyclase activity (1, 5 and 100 uM). Results are discussed in terms of the specificity of the age-related deficits seen in the striatum of the senescent animal.     

Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Mammalian hypotaurine aminotransferase: isethionate is not a product

We report that mammalian tissues posses hypotaurine (2-aminoethane- sulfinate) aminotransferase activity. One product of transamination, sulfinoacetaldehyde could theoretically undergo internal oxidation-reduction leading to isethionate (2-hydroxyethanesulfonate). This hypothesis was examined. No isethionate was formed $\text{in vitro}$.     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.     

Inhibition of insulin receptor binding by dimethyl sulfoxide.

Little is known of the effects of the solvent on hormone-receptor interactions. In the present study the effect of the polar solvent dimethyl sulfoxide on the binding of insulin to its surface receptors on cultured human lymphocytes of the IM-9 line was investigated. At concentrations exceeding 0.1% (v/v), dimethyl sulfoxide produced a dose-related inhibition of 125-I-labeled insulin binding. Insulin binding was totally abolished in 20% dimethyl sulfoxide. This inhibition was immediately present and was totally reversible. Analysis of the data of binding at steady state indicated that the decrease in binding of 125I-labeled insulin was due to a reduced affinity of the insulin receptor without noticeable change in the concentration of receptor sites. Kinetic studies showed that the decreased affinity could largely be accounted for by a decreased association rate constant; effects on dissociation and negative cooperativity of the insulin receptor was affected to a much lesser extent.