Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle

Summary Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.     

Decreased lysosomal protease content of skeletal muscles from streptozotocin-induced diabetic rats: a biochemical and histochemical study

Summary The activity of four lysosomal proteases in soleus and extensor digitorum longus muscles was studied in streptozotocin-induced diabetic rats using newly developed fluorescence histochemical and biochemical techniques. The results indicate that the content of lysosomal protease in skeletal muscle cells was decreased three weeks after the induction of diabetes. The reduction was most pronounced in the extensor digitorum longus for all the proteases tested, but in the soleus only cathepsin B and dipeptidyl peptidase II showed a decrease. Biochemical assays on total muscle homogenates and muscle extracts confirmed the histochemical observations that protease activity was significantly lower in diabetic muscles. This decrease in activity varied with the duration of diabetes beginning as early as 48 h for the soleus. In conclusion, myofibre-specific decreases in lysosomal proteases occur following diabetes.     

Transmission of Megatrypanum Trypanosomes to Cervus dama by Tabanidae1

Four fallow deer, Cervus dama, became infected with Trypanosoma (Megatrypanum) sp. by oral application of triturated guts from tabanids collected in an area with deer but without any cattle; four control calves remained negative. Upon challenge with triturated guts from tabanids from an area with pastured cattle, the four calves became infected with Trypanosoma (M.) theileri. The prepatent period in deer was five days or less. Haematopota spp. and Tabanus spp. were identified as vectors of the deer trypanosomes. It is concluded that the trypanosomes of C. dama belong to a Megatrypanum species that is not identical with T. theileri.     

On the ultrastructure and the supposed function of the mineralizing matrix coat of sea urchins (Echinodermata,Echinoida)

Summary Echinoderm ossicles are part of the mesenchyme. Their formation and growth, with respect to the underlying tissues, is studied using echinoid spines and teeth and applying different methods of fixation. The calcification process in echinoderms is strictly intracellular and needs (1) syncytial sclerocytes which completely enclose (2) a vacuolar cavity which in turn contains (3) an organic matrix coat. Strictly speaking, each ossicle is nothing but the calcified vacuolar space of a single syncytium of sclerocytes. In fully grown parts, however, the continuous sheath may split open and the matrix-coated mineral may come into contact with the extracellular space. According to biochemical analyses the matrix consists of insoluble components, but most (95%) of its constituents are soluble in EDTA or weak acids. If routine transmission electron microscope methods are used the soluble components are lost and the matrix at best looks electron light. If tannic acid is added to the fixative the soluble matrix components are preserved and reveal further ultrastructural details of the biomineralization process in echinoderms. The matrix coat looks extremely electron dense. Further soluble material is to be found within the vacuolar space or attached to the vacuolar surface of the cytoplasmic sheath. The results lead to the opinion that the matrix coat consists of a hydrophobic framework of insoluble components that contains soluble components which guide the Ca through pores in the hydrophobic layers into the interior of the matrix-coated space. It is only within this space that the mineral is deposited.     

Physical mapping of three fruit ripening genes：Endopolygalacturonase,ACC oxidase and ACC synthase from apple（Malus x domestica） in an apple rootstock A106（Malus sieboldii

The apple rootstock,A106(Malus sieboldii),had 17 bivalents in pollen mother cells at meiotic metaphase 1,and 17 chromosomes in a haploid pollen cell.Karyotypes were prepared from root-tip cells with 2n=34 chromosomes,Seven out of 82 karyotypes(8.5%) showed one pari of satellites at the end of the short arm of chromosome 3.C-bands were shown on 6 pairs of chromosomes 2,4,6,8,14,and 16 near the telomeric regions of short arms.Probes for three ripening-related genes from Malus x domestica:endopolygalacturonase(EPG,0.6kb),ACC oxidase(1.2kb),and ACC synthase(2kb)were hybridized in situ to metaphase chromosomes of A106.Hybridization sites for the EPG gene were observed on the long arm of chromosome 14 in 15 out of 16 replicate spreads and proximal to the centromere of chromosomes 6 and 11.For the ACC oxidase gene,hylridization sites were observed in the telomeric region of the short arm of chromosomes 5 and 11 in 87% and 81% of 16 spreads respectively,proxiaml to the centromere of chromosome 1 in 81% of the spreads,and on the long arm of chromosome 13 in 50% of the spreads. Physical mapping of three fruit ripening genes in an apple rootstock A106.Twenty five spreads were studied for the ACC synthase gene and hybridization sites were observed in the telomeric region of the short arm of chromosome 12 in 96% of the spreads.chromosomes 9 and 10 in 76% of the spreads,and chromosome 17 in 56% of the spreads.     

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Projections of the guinea-pig paracervical ganglion to pelvic viscera

Summary The uterine cervix, urinary bladder and rectum of guinea pigs were injected with Fast Blue dye for retrograde transport studies. Dye-laden neuronal perikarya were detected for each viscus in the paracervical ganglion. These same perikarya also exhibited immunoreactivities for tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine -hydroxylase, neuropeptide Y, or vasoactive intestinal peptide, though the perikarya projecting to the urinary bladder did not exhibit immunoreactivity for aromatic amino acid decarboxylase. The results of this study indicate that the guinea-pig paracervical ganglion projects to viscera in addition to the uterus, and that the ganglion contains a range of immunoreactivities related to adrenergic and non-adrenergic neurotransmitters.     

The lantern of Aristotle: organization of its coelom and origin of its muscles (Echinodermata,Echinoida)

Summary Comparative ultrastructural analyses of the muscles that work the lantern of Aristotle support the opinion that the muscles in question are myoepithelially organized or derivatives of myoepithelia. They are part of the epithelium of the peripharyngeal cavity (=lantern coelom). The coelom epithelium may become multiplelayered in certain regions and is composed of (1) a layer of muscle cells that vary in number and size, (2) nerve cells and their processes that are interspersed between the muscle layer and (3) monociliated adluminal cells that build a continuous cell lining and completely cover the muscle layer. According to their complexity, the lantern muscles exhibit consecutive stages of myoepithelial variations and may finally simulate subepithelial musculature. The results of this study support the hypothesis of a histological development of subepithelial musculature from simple myoepithelia, although both epithelial and mesenchymal musculature may occur in the Echinodermata. Detailed knowledge of the organization of the lantern's coelom space was a prerequisite for the present study. In contrast to previous examinations the lantern coelom is not a continuous space, but is subdivided into several cavities that are partially completely separated from each other. On the one hand, this subdivision is probably caused by the sophisticated arrangement of the lantern's ossicles and on the other by the septa that give rise to muscles that fulfill different functions. lanter's ossicles and on the other by the septa that give     

The cytokineplast: purified, stable, and functional motile machinery from human blood polymorphonuclear leukocytes

We examined the formation of motile, chemotactically active, anucleate fragments from human blood polymorphonuclear leukocytes (PMN, granulocytes), induced by the brief application of heat. These granule-poor fragments are former protopods (leading fronts, lamellipodia) that become uncoupled from the main body of the cell and leave it, at first with a connecting filament that breaks and seals itself. The usual random orientation of such filaments can be controlled by preorientation of cells in a gradient of the chemotactic peptide, N-formylmethionylleucylphenylalanine (F-Met-Leu-Phe) (2x10(-9) M- 1x10(-8)). Cytochalsin B, 2.5-5 μg/ml, prevents fragment formation; colchicine, 10(-5) M, does not. In scanning electron micrographs, fragments are ruffled and the cell body rounded up and rather smooth. In transmission electron micrographs, fragments contain microfilaments but lack centrioles and microtubules. Like intact cells, both bound and free fragments can respond chemotactically to an erythrocyte destroyed by laser microirradiation (necrotaxis); the free, anucleate fragments may do so repeatedly, even after having been held overnight at ambient temperatures. We propse the name cytokineplast for the result of this self-purification of motile apparatus. The exodus of the motile machinery from the granulocyte requires anchoring of the bulk of the cell to glass and uncoupling, which may involve heat-induced dysfunction of the centrosome. In ultrastructural studies of the centrosomal region after heat, centriolar structure remains intact, but pericentriolar osmiophilic material appears condensed, and microtubules are sparse. These changes are found in all three blood cell types examined: PMN, eosinophil, and monocyte. Of these, the first two make fragments under our conditions; the more sluggish monocyte does not. Uncoupling is further linked to centrosomal dysfunction by the observation that colchicines-treated granulocytes (10(-5)M, to destroy the centrosome’s efferent arm) make fragments after less heat than controls. If motive force and orientation are specified mainly from the organelle-excluding leading front, then endoplasmic streaming in PMN is a catch-up phenomenon, and microtubules do not provide the vector of locomotion but rather stabilize and orient the “baggage” (nucleus, granuloplasm)—i.e., they prevent fishtailing. Moreover, constraints emanating from the centrosome may now be extended to include, maintenance of the motile machinery as an integral part of the cell.     

Further studies on a unique T-tubular acid phosphatase in avian skeletal muscle

Summary Whith the unique observation, using conventional cytochemistry, of acid phosphatase reaction production in the T-tubules of the posterior latissimus dorsi muscle of the chicken, the possibility of andocytosis of lysosomal enzymes by muscle cells came into question. After testing the substrate specificity of this T-tubular phosphatase, it was clear that the enzyme was not 5-nucleotidase for a typical lysosomal acid phosphatase. The T-tubular enzyme hydrolysed glucose 6-phosphate and -glycerophosphate at pH 5.0 but not cytidine-5-monophosphate which was hydrolysed by dense bodies and autophagic vacuoles. The cytochemical evidence points to a mique phosphatase present on mucle cell membranes which apparently does not belong to the vacuolar apparatus of skeletal muscle and is not 5-nucleotidase.