Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65–70% of the rate with NADH. Added cytochrome b₅ stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b₅ and cytochrome b₅ reductase determine the rate of methemoglobin reduction in hemolysates.     

Cytochemical localization and biochemical evaluation of a lysosomal serine protease in lung: dipeptidyl peptidase II in the normal rat

Dipeptidyl peptidase II (DPP II) in normal rat lung was evaluated by the enzymes' ability to hydrolyze Lys-Ala or Lys-Pro derivatives of 4-methoxy-2-naphthylamine (MNA). For visualization of this activity, the liberated MNA was coupled with fast blue B for light microscopy (LM) or hexazotized pararosaniline with osmication for electron microscopy (EM). Granular to diffuse reaction product was noted in many lung cells in frozen sections for LM, including alveolar and tissue macrophages, fibroblasts, chondrocytes, bronchial and bronchiolar epithelial cells and mast cells. Reaction product at the EM level was seen in the lysosomal structures of the above cells, although lysosomal heterogeneity with regard to reactivity was noted. Cellular content of reaction product by EM correlated with LM staining intensity. Additional structures, not obviously reactive by LM, such as the lamellar bodies of type II cells and lysosomes in other cell types, were seen to contain reaction product ultrastructurally. A modified biochemical assay for the quantitation of DPP II in tissue homogenates was used to determine the activity of the enzyme in rat lung. Enzyme activity in polyacrylamide isoelectric focusing gels indicate that Lys-Ala-MNA was the more specific substrate but, by virtue of its rapid hydrolysis, Lys-Pro-MNA was more sensitive.     

The use of 2-thionaphthyl acetate as a substrate for the localization and characterization of nonspecific esterase activity in rat alveolar and peritoneal macrophages

Summary A 2-thionaphthyl acetate substrate was utilized to assess the subcellular distribution of nonspecific esterases in rat pulmonary alveolar and peritoneal macrophages. The enzymatically liberated 2-thionaphthol was visualized at pH 7.1 by utilizing gold as a capture agent. Glutaraldehyde-fixed macrophages derived from healthy animals using standard lavage techniques exhibited a high affinity for the substrate and reaction times were thus relatively short (30–60 min). Alveolar macrophages had heavy reaction product on the external surface of the plasma membrane and membranes limiting cisternae of rough endoplasmic reticulum, Golgi complex and mitochondria. Only a thin layer of reaction density was observed associated with the limiting membranes of lysosomes and phagosomes. Peritoneal macrophages were similarly but much less intensely reactive, although they generally lacked or had very little plasma membrane-associated staining. The 2-thionaphthyl acetate esterase activities in both alveolar and peritoneal macrophages were sensitive to diisopropylfluorophosphate (DFP), while only the latter was inhibited by sodium fluoride. Polyacrylamide gel isoelectric focusing of whole cell homogenates indicated that the 2-thionaphthyl acetate esterase activity was the same as that for -naphthyl acetate in these cells. The data indicate that a significantly different distribution of nonspecific esterase activity results with use of a 2-thionaphthyl acetate substrate in the presence of gold ions than that previously reported with other methods. The rapid penetrability and sensitivity of this substrate make it a potentially useful tool for evaluating subcellular localization of esterase activity and probing characteristics of cellular organelles.     

Differences in basement membrane-associated microdomains of type I and type II pneumocytes in the rat and rabbit lung

The basement membrane-associated microdomains of type I pneumocytes in rat and rabbit pulmonary alveoli were found to be uniquely different from those of type II pneumocytes in the specific distribution of cytochemically detectable sulfate esters as demonstrated with the high iron diamine (HID) technique at the electron microscopic level. Aldehyde-fixed frozen or Vibratome sections of neonatal and adult lungs were treated with a mixture of the meta and para isomers of N,N-dimethyl-phenylenediamine-HCl in the presence of ferric chloride, which at low pH (1.0) has been previously shown to be highly specific for sulfate esters of glycosaminoglycans and glycoproteins. Reaction product was subsequently enhanced with a thiocarbohydrazide-silver proteinate, postembedding sequence for electron microscopy. Samples of lung parenchyma treated in this fashion were observed to have discrete, electron-dense silver grains associated with the various microanatomical components of pulmonary basement membranes. In the region of the alveolar basement membrane, the lamina rara externa associated with type I cells was observed to contain an abundance of regularly disposed, cytochemically detectable sulfate esters, while the lamina densa and lamina rara interna were diffusely and sparsely reactive by comparison. Quantitatively, 62% of all reactive sites found in the basement membrane region of type I cells were localized in the lamina rara externa. By contrast, the lamina rara externa of type II cells had less than half as many reactive foci indicative of sulfate esters as the same region of type I cell basement membranes. HID-reactive sulfate esters were found evenly distributed within the laminae associated with the basement membrane of type II cells. This cytochemically detectable difference in the sulfate ester composition of basement membrane-associated sulfate ester composition of basement membrane-associated microdomains of type I compared with that of type II pneumocytes may be highly significant when considering known patterns of epithelial renewal in pulmonary alveoli. Since type II cells are known to divide and either remain type II cells or differentiate into type I cells, regional differences in the molecular composition of the alveolar basement membranes and their associated structures may be key determinants of cell-specific processes of cytodifferentiation in the pulmonary alveolus.     

Subcellular localization of complex carbohydrates in rat macrophages and monocytes.

Methods for visualization of complex carbohydrates ultrastructurally were employed to study specific organelles of the rat monocyte and macrophage. Vicinal glycols of glycoconjugates were demonstrated with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) postembedding sequence and acid groups were delineated by the dialyzed iron (DI) and high iron diamine (HID) preembedding techniques. Lysosomal bodies were generally found reactive with all three methods, although those of monocytes from the bone marrow and peripheral blood were notably lacking in acidic groups. The Golgi complex was consistently PA-TCH-SP-reactive, as were associated vesicles and occasional cisternal expansions, possibly related to GERL. Numerous cytoplasmic vesicles and small granulated structures and cisternae of the rough endoplasmic reticulum were also PA-TCH-SP-reactive.     

Tannic acid-metal salt sequences for light and electron microscopic localization of complex carbohydrates.

Use of tannic acid (TA), in sequence with ferric chloride, uranyl acetate or gold chloride resulted in staining of selective but sometimes different sites in paraffin sections. TA-uranyl acetate of TA-ferric chloride stained sites rich in complex carbohydrates, wherease TA-gold chloride stained the collagen of various connective tissues different shades of red-purple to gray-black. Applied to epoxy-embedded thin sections of tissues fixed with glutaraldehyde and not post-osmicated, TA-uranyl acetate and TA-ferric chloride imparted density to subcellular sites known to contain a high concentration of mucosubstances, such as secretory granules and cisternae of the Golgi complex of certain cells. TA-gold chloride proved unsatisfactory for ultracytochemistry because of its tendency to form globular precipitates on thin sections. The effect of blockage procedures at the light microscopic level indicated that vicinal glycols are not required for binding of TA to tissue sites. Electrostatic forces were shown to be of minimal significance, whereas hydrogen bonding appeared to play a part in both TA-tissue and TA-metal binding mechanisms.     

Alterations in cytoskeletal and immune function-related proteome profiles in whole rat lung following intratracheal instillation of heparin

Background

Heparin has been shown to modify fundamental biologic processes ranging from blood coagulation and cell proliferation to fibrogenesis and asthma. The goal of this study was to identify specific or broad biologic responses of the rat lung to intratracheal instillation of heparin by targeted proteomic analysis.

Methods

Rats were given either aerosolized 500 μg heparin in 250 μl saline or saline alone. Lungs were harvested at 0, 24, or 96 hours post-treatment and isolated proteins analyzed by two-dimensional gel electrophoresis. Proteins which increased and decreased significantly in treated groups above controls were then selected for identification by mass spectrometry.

Results

Although heparin treatments resulted in a general reduction in cytosolic protein expression, there were significant increases within members of discrete groups of proteins. At 24 hours, proteins which function in cytoskeletal organization and in calcium signaling were up-regulated between 2- and 27-fold above baseline and untreated controls. Increased proteins include annexins V and VI, septin 2, capping G protein, actin-related protein 3, moesin, RhoGDP dissociation inhibitor, and calcyclin. A group of proteins relating to immune response and tumor suppressor function were either up-regulated (tumor suppressor p30/hyaluronic acid binding protein-1, Parkinson disease protein 7, proteosome 28 subunit/interferon-γ inducible protein, and proteosome subunit macropain α-1) or strongly down-regulated (transgelin). At 96 hours, most proteins that had increased at 24 hours remained elevated but to a much lesser degree.

Conclusion

These cumulative observations demonstrate that whole lung heparin treatment results in significant up-regulation of selected groups of proteins, primarily those related to cytoskeletal reorganization and immune function, which may prove to be relevant biomarkers useful in analysis of lung exposures/treatments as well as in system biology studies.     

Histochemical localization of cathepsin B, dipeptidyl peptidase I, and dipeptidyl peptidase II in rat bone

The histochemical distribution of the thiol proteases cathepsin B and dipeptidyl peptidase I and the serine protease dipeptidyl peptidase II was examined in rat bone and joint using amino acid derivatives of 4-methoxy-2-naphthylamine (MNA). The liberated MNA was then visualized by simultaneous coupling with fast blue B. Cathepsin B was examined with CBZ-Arg-Arg-MNA, dipeptidyl peptidase I (DPP I) with Gly-Arg- or Pro-Arg-MNA, and dipeptidyl peptidase II (DPP II) with Lys-ALA- or Lys-Pro-MNA. Bright red reaction product indicative of proteolytic activity was observed in most cell types associated with bone and its surrounding connective tissues, including osteocytes, osteoblasts, chondrocytes, chondroblasts, fibroblasts, and macrophages. Surprisingly, protease activity in osteoclasts could not be established with certainty, and it was concluded that these enzymes are either absent, present in very low amounts, or secreted as soon as they are synthesized rather than stored within the cell. The cells of the resting zone of the growth plate were intensely reactive for DPP II but were only moderately reactive for cathepsin B and DPP I. The reverse was true of the proliferating and hypertrophic layers. The protease activity observed in bone, cartilage, tendon, ligament, and synovium would be expected to contribute significantly to normal protein metabolism as well as to pathological destruction in these tissues.     

MARCKS‐dependent mucin clearance and lipid metabolism in ependymal cells are required for maintenance of forebrain homeostasis during aging

Ependymal cells (ECs) form a barrier responsible for selective movement of fluids and molecules between the cerebrospinal fluid and the central nervous system. Here, we demonstrate that metabolic and barrier functions in ECs decline significantly during aging in mice. The longevity of these functions in part requires the expression of the myristoylated alanine‐rich protein kinase C substrate (MARCKS). Both the expression levels and subcellular localization of MARCKS in ECs are markedly transformed during aging. Conditional deletion of MARCKS in ECs induces intracellular accumulation of mucins, elevated oxidative stress, and lipid droplet buildup. These alterations are concomitant with precocious disruption of ependymal barrier function, which results in the elevation of reactive astrocytes, microglia, and macrophages in the interstitial brain tissue of young mutant mice. Interestingly, similar alterations are observed during normal aging in ECs and the forebrain interstitium. Our findings constitute a conceptually new paradigm in the potential role of ECs in the initiation of various conditions and diseases in the aging brain.     

Switching on the light: using metagenomic shotgun sequencing to characterize the intestinal microbiome of Atlantic cod

Atlantic cod (Gadus morhua) is an ecologically important species with a wide-spread distribution in the North Atlantic Ocean, yet little is known about the diversity of its intestinal microbiome in its natural habitat. No geographical differentiation in this microbiome was observed based on 16S rRNA amplicon analyses, yet such finding may result from an inherent lack of power of this method to resolve fine-scaled biological complexity. Here, we use metagenomic shotgun sequencing to investigate the intestinal microbiome of 19 adult Atlantic cod individuals from two coastal populations in Norway–located 470 km apart. Resolving the species community to unprecedented resolution, we identify two abundant species, Photobacterium iliopiscarium and Photobacterium kishitanii, which comprise over 50% of the classified reads. Interestingly, the intestinal P. kishitanii strains have functionally intact lux genes, and its high abundance suggests that fish intestines form an important part of its ecological niche. These observations support a hypothesis that bioluminescence plays an ecological role in the marine food web. Despite our improved taxonomical resolution, we identify no geographical differences in bacterial community structure, indicating that the intestinal microbiome of these coastal cod is colonized by a limited number of closely related bacterial species with a broad geographical distribution.