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.     

Structural characterization of proteoglycans produced by testicular peritubular cells and Sertoli cells

The structural characteristics of proteoglycans produced by seminiferous peritubular cells and by Sertoli cells are defined. Peritubular cells secrete two proteoglycans designated PC I and PC II. PC I is a high molecular mass protein containing chondroitin glycosaminoglycan (GAG) chains (maximum 70 kDa). PC II has a protein core of 45 kDa and also contains chondroitin GAG chains (maximum 70 kDa). Preliminary results imply that PC II may be a degraded or processed form of PC I. A cellular proteoglycan associated with the peritubular cells is described which has properties similar to those of PC I. Sertoli cells secrete two different proteoglycans, designated SC I and SC II. SC I is a large protein containing both chondroitin (maximum 62 kDa) and heparin (maximum 15 kDa) GAG chains. Results obtained suggest that this novel proteoglycan contains both chondroitin and heparin GAG chains bound to the same core protein. SC II has a 50-kDa protein core and contains chondroitin (maximum 25 kDa) GAG chains. A proteoglycan obtained from extracts of Sertoli cells is described which contains heparin (maximum 48 kDa) GAG chains. In addition, Sertoli cells secrete a sulfoprotein, SC III, which is not a proteoglycan. SC III has properties similar to those of a major Sertoli cell-secreted protein previously defined as a dimeric acidic glycoprotein. The stimulation by follicle-stimulating hormone of the incorporation of [35S]SO2(-4) into moieties secreted by Sertoli cells is shown to represent an increased production or sulfation of SC III (i.e. dimeric acidic glycoprotein), and not an increased production or sulfation of proteoglycans. Results are discussed in relation to the possible functions of proteoglycans in the seminiferous tubule.     

Effects of osmotic conditions on ultrastructure of rat mammary epithelium

Summary Effects of osmotic conditions on secretion of milk serum were examined using standard transmission electron microscopy. Rat mammary glands were infused with hyper-, iso-, and hypo-osmotic solutions. The intramammary infusion of these agents elicited distinct and repeatable morphological responses from lactating epithelial cells. The response to hyperosmolarity was an increase in compound exocytotic figures and an increase in secretory vesicle size (¯x=1.65 m in diameter). Glands infused with hypo-osmolar solutions exhibited the opposite response; reduction in compound exocytotic figures and reduced vesicle size (¯x=0.34 n in diameter). The response to iso-osmotic solutions was indistinguishable from untreated control tissue. The ratio of vesicular projections to depressions (vesicle membrane/plasma membrane interactions) could be experimentally altered through the intramammary infusion of solutions with different osmotic potentials. These observations support the suggestion that osmotic conditions may influence exocytosis of milk serum.     

Detection of an antigenic cell wall layer inHistoplasma capsulatum

Histoplasma capsulatum yeast cells have been studied by immunoelectron microscopy using rabbit polyclonal antisera and a biotin-avidin-peroxidase detection system. An antigenic surface layer has been visualized in the cell wall of immunostained organisms. This layer was not seen in samples prepared by standard electron microscopic methods or in negative controls used with the immunocytochemical technique. Without immunostaining the cell wall ofHistoplasma appeared almost transparent. In contrast, after immunoperoxidase staining the cell wall was conspicuous, bounded by the darkly stained outer layer. This electron dense layer, appeared to be a reservoir of surface antigens that were recognized by anti-Histoplasma antibodies.Abbreviations CHHA Cystine-heart-hemoglobin agar - PBS phosphate buffered saline - Ig immunoglobulin - TBS Tris buffered saline - DAB 3,3-diaminobenzidine tetrachloride - FITC fluorescein isothiocyanate - M199 tissue culture medium 199, according to Morgan et al. (1950)     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

Summary The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4° C some LDL-or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8° C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37° C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37° C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice

Summary In vivo localization of a mouse monoclonal antibody (F₂-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab)₂ fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radiolocalization studies were performed by using ¹²⁵I- or ¹³¹I-labeled IgM monoclonal antibody or its F(ab')₂ fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab)₂ fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of ¹²⁵I-radiolabeled F(ab)₂ fragments and ¹²⁵I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab)₂ fragments was lower than with IgM, and so -camera imaging was workable with F(ab)₂ fragments without background substraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab)₂ fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab)₂ fragments make them hardly suitable as carriers of toxic drugs. Abbreviations used are: MEM Minimum essential medium; SDS sodium dodecylsulfate; PAGE polyacrylamide gel electrophoresis     

Chronobiological aspects of the host-parasite relationships between Biomphalaria glabrata and Schistosoma mansoni: cercarial production and infectivity, and growth kinetics of the host

In the Biomphalaria glabrata-Schistosoma mansoni association, variations in cercarial production, in cercarial infectivity, and in the growth of infected snails are rhythmic. These chronobiological aspects are correlated with the dynamics of the intramolluscan larval stages of the parasite during the course of the infection. Rhythmic variations in the growth kinetics of infected snails are interpreted in terms of host-parasite metabolic exchanges.     

Involvement of the globular domain of histone H1 in the higher order structures of chromatin

We have attacked H1-containing soluble chromatin by α-chymotrypsin under conditions where chromatin adopts different structures.Soluble rat liver chromatin fragments depleted of non-histone components were digested with α-chymotrypsin in NaCl concentrations between 0 mm and 500 mm. at pH 7, or at pH 10, or at pH 7 in the presence of 4 m-urea. α-Chymotrypsin cleaves purified rat liver histone H1 at a specific initial site (CT) located in the globular domain and produces an N-terminal half (CT-N) which contains most of the globular domain and the N-terminal tail, and a C-terminal half (CT-C) which contains the C-terminal tail and a small part of the globular domain. Since in sodium dodecyl sulfate/polyacrylamide-gel electrophoresis CT-C migrates between the core histones and H1, cleavage of chromatin-bound H1 by α-chymotrypsin can be easily monitored.The CT-C fragment was detected under conditions where chromatin fibers were unfolded or distorted: (1) under conditions of H1 dissociation at 400 mm and 500 mm-NaCl (pH 7 and 10); (2) at very low ionic strength where chromatin is unfolded into a filament with well-separated nucleosomes; (3) at pH 10 independent of the ionic strength where chromatin never assumes higher order structures; (4) in the presence of 4 m-urea (pH 7), again independent of the ionic strength. However, hardly any CT-C fragment was detected under conditions where fibers are observed in the electron microscope at pH 7 between 20 mm and 300 mm-NaCl. Under these conditions H1 is degraded by α-chymotrypsin into unstable fragments with a molecular weight higher than that of CT-C. Thus, the data show that there are at least two different modes of interaction of H1 in chromatin which correlate with the physical state of the chromatin.Since the condensation of chromatin into structurally organized fibers upon raising the ionic strength starts by internucleosomal contacts in the fiber axis (zig-zag-shaped fiber), where H1 appears to be localized, it is likely that in chromatin fibers the preferential cleavage site for α-chymotrypsin is protected because of H1-H1 contacts. The data suggest that the globular part of H1 is involved in these contacts close to the fiber axis. They appear to be hydrophobic and to be essential for the structural organization of the chromatin fibers. Based on the present and earlier observations we propose a model for H1 in which the globular domains eventually together with the N-terminal tails form a backbone in the fiber axis, and the nucleosomes are mainly attached to this polymer by the C-terminal tails.