Death by apoptosis in salivary glands of females of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

The salivary glands of females of the tick Rhipicephalus sanguineus at three feeding stages: unfed, engorged, and at day three post-engorgement, were subjected to cytochemical methods of enzymatic analysis and cell viability. Comparing glands at these stages, was observed distinct staining patterns in cells of different types of acini, specially in degenerating types III, II, I, which were affected in this sequence by cell death. This study also revealed changes in: nuclei, staining intensity for acid phosphatase and ATPase activities, and permeability of the plasma membrane. Acid phosphatase activity was inversely proportional to that of ATPase, while ATPase activity was always proportional to membrane integrity. The glands of unfed females exhibited high metabolic activity and cells with intact nucleus and plasma membrane, suggesting that the presence of acid phosphatase detected in these individuals may participate in the normal physiology of some acini, as they were not undergoing degeneration. In acini I and II of engorged females, we observed cells with intact membranes, as well as changes characterized by nuclear changes, decrease in ATPase activity, and stronger acid phosphatase activity. At day three post-engorgement, degeneration progressed to more advanced stages, loss of membrane integrity was observed in most cells (of some type I acini, most type II acini, and all type III acini), as well as prominent nuclear changes, decrease in ATPase activity, and intense acid phosphatase activity, resulting in apoptotic bodies. During the death of cells nuclear changes preceded cytoplasmic ones in the following sequence: nuclear changes, loss of ATPase activity, loss of integrity of the plasma membrane, increase in acid phosphatase activity, and formation of apoptotic bodies. The presence of acid phosphatase with a secondary role (late) during cell death, degrading final cell remnants, characterized this process in the glands of R. sanguineus females as atypical or non-classic apoptosis.     

Morphofunctional study of the organization of the peripheral parenchyma of Acoela Oxyposthia praedator

The ultrastructure of undifferentiated cells in the peripheral parenchyma of Oxyposthia praedator was studied, along with the ways of their differentiation. The type I cells (3.5-4.0 microns in diameter) undergo mitotic division, while the type II cells (9 microns in diameter) produce specialized cells of the parenchyma. At the beginning of secretory cell differentiation one cistern of the rough endoplasmic reticulum (RER) is formed by the outer membrane of the nuclear envelope, the formation of other cisternae follows. The Golgi complex is formed simultaneously. The differentiated secretory cells are characterized by the abundance of RER cisternae and Golgi complexes. In the course of differentiation of other cell types RER cisternae are formed by several portions of the nuclear envelope. The Golgi complex appears in cells 12-14 microns long. The differentiation of digestive cells is characterized by autophagy. Autophagosomes are formed by RER cisternae. The consecutive stages of autophagosome formation are described. Using a cytochemical reaction revealing acid phosphatase the process of digestion of the autophagosome content was followed.     

Cytochemical approach to the development and behaviour of rat alveolar type II cells in culture

Summary The development and characteristics of rat alveolar type II cells were monitored by using various cytochemical techniques. Polarized light microscopy was found useful for observing live type II cells in culture. Cells progressively lose their birefringent granules starting from 48 h of cells in culture, indicating the disappearance of the phospholipids organized lamellae in the lamellar bodies. Similar results were obtained by using an immunocytochemical approach with antibodies raised against the apoprotein component of rat surfactant. A progressive decrease in immuno-staining corresponded to the disappearance of the lamellar bodies, and birefringence. Changes in lectins binding to the cultured type II cells were also observed. Freshly isolated and one day old cultured cells could bind Macula pomifera (MPA) but not Arachis hypogaea (PA) lectins. The reverse was found in 6–7 days old cultured cells which had the ability to bind PA but not MPA the advantage of using various cytochemical techniques for studying the development of type II cells in culture is being discussed.     

ENZYMES OF THE LUNG : I. Detection of Esterase with a New Cytochemical Method

The esterases of rabbit lung have been investigated from two viewpoints, the cytochemical and the biochemical. To accomplish this objective, we designed and synthesized a series of ester substrates which provide both a cytochemical indicator of the location of the enzyme and a means of following the enzymatic activity in tissue homogenates and subfractions. The substrates are p-nitrophenylthiol esters which yield, upon hydrolysis, carboxylic acid and p-nitrothiophenol. The latter can react with aurous ions to give an electron-opaque deposit; in addition, the strong absorption of p-nitrothiophenol at 410 mµ permits continuous kinetic measurements. Thus, it is possible to correlate the intracellular site of action and the biochemical behavior of the esterases. The new substrates are the thiol analogues of the p-nitrophenyl esters frequently employed as esterase substrates. The rates of hydrolysis of the two series of esters are compared in vitro. During tissue fractionation, most of the esterase activity sediments with a particulate fraction. The effects of a number of common esterase inhibitors, such as diisopropyl phosphorofluoridate and eserine sulfate, are examined, and the effects of enzyme concentration and heat inactivation are shown with the use of the partially purified preparations. The cytochemical work shows that the esterase activity is most prominent in the lamellar bodies of the giant alveolar (type II, septal, or granular pneumatocyte) cells of the lung and to a lesser extent in squamous (type I, or membranous pneumatocyte) epithelial and endothelial cells. In both the cytochemical and biochemical studies, the enzymes are inhibited by diisopropyl phosphorofluoridate and phenyl methylsulfonyl fluoride but are insensitive to eserine sulfate.     

Correlative morphological and cytochemical observations on the nucleoli and nuclear bodies during avian oogenesis.

A correlative morphological and cytochemical study has been made of the nucleoli and nuclear bodies in the growing oocytes of the crow (Corvus splendens) and common myna (Acridotheres tristis). The nucleoli show morphological and cytochemical changes during oocyte growth, which are described in detail. In young oocytes at diplotene stage, two to six nucleoli, which are attached to condensed diplotene chromosomes, show RNA, lysine-rich histones and some non-histones, the proteins contain S-S and SH groups. In subsequent stages of oocyte growth, the cortex of nucleolus also develops arginine-rich histones whereas the medulla shows lysine-rich histones. The significance of both morphological and cytochemical changes of nucleoli has been discussed in relation to oocyte growth. Seven types of nuclear bodies are described. They are composed of proteins and carbohydrates. Their shape, size and structure vary during different stages of oocyte growth. Finally, their material is transported into the ooplasm.     

The alveolar epithelium of the newborn rat,and the significance of the osmiophilic lamellated bodies in cellular autophagy

Abstract

An attempt was made to determine the nature, origin, and fate of the membrane material of osmiophilic lamellated bodies, using lung tissue from neonate rats. The cytoplasm of the type II alveolar pneumonocyte contains centrioles, multivesicular bodies, and minute free vesicles similar to those in the multivesicular bodies. Autolysosomes, comprising membrane-bounded cytoplasmic regions and osmiophilic lamellated material, also occur in the type II pneumonocytes. The mitochondria often contain concentric membrane accumulations and membranous whorls. The type II alveolar cells are characterised by an intensive autophagy; this is apparently correlated with glycogenolysis, and with a radical cytodifferentiation by which the cells transform to the type I pneumonocyte. The osmiophilic lamellae of the autolysosomes are probably emptied isolation membranes. The mitochondria possibly serve as repositories for the massive membrane accumulations remaining after cytoplasmic lysis, which may invaginate into the organelles. The osmiophilic lamellated bodies typical of type II alveolar pneumonocytes may be mitochondrial membranes packed with the residual membranous material. Myeloid matter in the alveolar spaces (derived from the osmiophilic lamellated bodies) is best interpreted, not as an organised secretory product, but rather as a residue of cellular autophagy.     

Cytochemical approach to the development and behaviour of rat alveolar type II cells in culture

The development and characteristics of rat alveolar type II cells were monitored by using various cytochemical techniques. Polarized light microscopy was found useful for observing live type II cells in culture. Cells progressively lose their birefringent granules starting from 48 h of cells in culture, indicating the disappearance of the phospholipids organized lamellae in the lamellar bodies. Similar results were obtained by using an immunocytochemical approach with antibodies raised against the apoprotein component of rat surfactant. A progressive decrease in immuno-staining corresponded to the disappearance of the lamellar bodies, and birefringence. Changes in lectins binding to the cultured type II cells were also observed. Freshly isolated and one day old cultured cells could bind Macula pomifera (MPA) but not Arachis hypogaea (PA) lectins. The reverse was found in 6-7 days old cultured cells which had the ability to bind PA but not MPA the advantage of using various cytochemical techniques for studying the development of type II cells in culture is being discussed.     

Immunological localization of the major architectural protein associated with the nuclear envelope of the Xenopus laevis oocyte

The nuclear envelope (NE) of Xenopus laevis contains a major architectural protein which is resistant to extraction in high salt buffer and non-ionic detergent and is characterized by a polypeptide molecular weight (MW) of 68000. Two different antisera which showed specific binding of antibodies (IgG) to this polypeptide, as demonstrated by ‘immunoblotting’ techniques, were used for immunolocalization at the electron microscopic level. Whereas antibodies of serum I reacted only with the nuclear lamina structure, antibodies of serum II, which were raised against undenatured karyoskeletal protein from oocytes, showed additional strong reaction in nuclear pore complexes. This first positive localization of a polypeptide in the nuclear pore complex suggests that MW 68000 polypeptide contributes as a major karyoskeletal component to the structure of both the lamina and the pore complex.     

Cytochrome oxidase deficiency during development of amphibian nucleocytoplasmic hybrids.

The activity of mitochondrial cytochrome oxidase was investigated during the embryonic development of nucleocytoplasmic hybrids containing a nuclear genome derived from R. pipiens and a mitochondrial genome derived from R. palustris. Using a quantitative cytochemical approach, we found that the activity of cytochrome oxidase failed to increase during the development of these embryos. Control embryos containing a haploid chromosomal complement, derived from the same species as that from which the maternally inherited mitochondria is derived and hybrid crosses between R. palustris and R. pipiens, showed a significant increase in cytochrome oxidase activity during development. Oxygen uptake data from diploid and haploid R. pipiens embryos were in agreement with the data obtained by the cytochemical method. These results indicate that a normal pattern of cytochrome oxidase activity during embryonic development requires a nuclear genome which contains a haploid chromosomal complement derived from the same species as that from which the mitochondrial genome is derived.     

Propionibacterium from Liver and Lung of Pigs and Guinea Pigs. 1: Physiological Identification

A total of 231 Propionibacterium strains originated from animals were identified based on key physiological properties. They comprised 218 strains derived from livers of pigs with or without milk spots, eight from lungs of pigs used in parasitic infection experiments (including controls), and three from livers and two from lungs of guinea pigs in experimental parasitic infection. All identification tests were performed in cysteine-containing media (reduced state) and incubated in an aerobic atmosphere, except the seed cultures used for inoculation and cultures for catalase test which were incubated under pure carbon dioxide. Among these strains, 212 isolates were clearly identified as P. acnes, Eleven asP. granulosum , seven as P. avidum and one as Propionibacteriu m species. For P. acnes, the key identification properties were maltose- and sucrose-negative at species level; and sorbitol-positive for type I and erythritol-positive for type II at subspecies level. All P. acnes type II strains were sorbitol-negative, but some type I strains also showed the same reaction. The P. acnes type I strains showed growth on the surface of semi-solid sugar medium and type II strains showed deep growth in the same medium. These growth modes provided an important tool for type differentiation. By summarizing all the results of adonitol–mannitol–sorbitol (AMS) reaction pattern; erythritol–trehalose (ET) reaction pattern, gelatin liquefaction, indole production and the growth mode, 34 differential patterns were established. As a result, 139 strains were classified as type I (130 from pig liver, six from pig lung, one from guinea-pig liver, two from guinea-pig lung), 68 strains as type II (64 from pig liver, 2 from pig lung, 2 from guinea-pig liver), and five strains as untypable. The two species; P. granulosum and P. avidum were first characterized by being melezitose-, maltose- and sucrose-positive. Then they were differentiated by aesculin and gelatin reactions. P. granulosum was negative for both and P. avidum was positive. One strain of Propionibacterium sp. had a unique pattern different from the other species.     

Type I and II β-turns prediction using NMR chemical shifts

A method for predicting type I and II β-turns using nuclear magnetic resonance (NMR) chemical shifts is proposed. Isolated β-turn chemical-shift data were collected from 1,798 protein chains. One-dimensional statistical analyses on chemical-shift data of three classes β-turn (type I, II, and VIII) showed different distributions at four positions, (i) to (i + 3). Considering the central two residues of type I β-turns, the mean values of C_ο, C_α, H_N, and N_H chemical shifts were generally (i + 1) > (i + 2). The mean values of C_β and H_α chemical shifts were (i + 1) < (i + 2). The distributions of the central two residues in type II and VIII β-turns were also distinguishable by trends of chemical shift values. Two-dimensional cluster analyses on chemical-shift data show positional distributions more clearly. Based on these propensities of chemical shift classified as a function of position, rules were derived using scoring matrices for four consecutive residues to predict type I and II β-turns. The proposed method achieves an overall prediction accuracy of 83.2 and 84.2 % with the Matthews correlation coefficient values of 0.317 and 0.632 for type I and II β-turns, indicating that its higher accuracy for type II turn prediction. The results show that it is feasible to use NMR chemical shifts to predict the β-turn types in proteins. The proposed method can be incorporated into other chemical-shift based protein secondary structure prediction methods.     

Lectin binding patterns to terminal sugars of rat lung alveolar epithelial cells.

We used post-embedding cytochemical techniques to investigate the lectin binding profiles of rat lung alveolar epithelial cells. Sections from rat lung embedded in the hydrophilic resin Lowicryl K4M were incubated either directly with a lectin-gold complex or with an unlabeled lectin followed by a specific glycoprotein-gold complex. The binding patterns of the five lectins used could be divided into three categories according to their reactivity with alveolar epithelial cells: (a) the Limax flavus lectin and Ricinus communis I lectin bound to both type I and type II cell plasma membranes; (b) the Helix pomatia lectin and Sambucus nigra L. lectin bound to type II but not type I cells; and (c) the Erythrina cristagalli lectin reacted with type I cells but was unreactive with type II cells. The specificity of staining was assessed by control experiments, including pre-absorption of the lectins with various oligosaccharides and enzymatic pre-treatment of sections with highly purified glycosidases to remove specific sugar residues. The results demonstrate that these lectins can be used to distinguish between type I and type II cells and would therefore be useful probes for investigating cell dynamics during lung development and remodeling.     

CYTOCHEMICAL STAINING OF MULTIVESICULAR BODY AND GOLGI VESICLES

To investigate the origin and nature of vesicles found within multivesicular bodies (mvb), the cytochemical staining properties of mvb vesicles were compared with those of other cytoplasmic vesicles, i.e. those associated with the Golgi complex and endocytic vesicles found near the apical cell surface. Rat epididymal tissue was stained in unbuffered OsO₄ for 40–48 hr, and the distribution of stain was compared to that of reaction products for acid phosphatase (AcPase) to mark lysosomal vesicles, or thiamine pyrophosphatase (TPPase) to mark certain Golgi vesicles, or infused with peroxidase (HRPase) to demonstrate endocytic vesicles. Mvb vesicles were stained only by OsO₄; AcPase, TPPase, and HRPase reaction products stained the mvb matrix. OsO₄ also stained certain vesicles along the convex surface of the Golgi complex. The findings suggest that mvb vesicles in epididymal epithelium are not lysosomes and are not involved in protein uptake. The majority of these vesicles have cytochemical reactions in common with vesicles located along the convex surface of the Golgi complex and may be derived therefrom. A minority are derived from the mvb-limiting membrane.     

The ultrastructure of the nuclei and the behaviour of the sex chromosomes of human spermatogonia

Summary The nuclear structure of human spermatogonia has been studied with electron microscopical and histochemical methods. Type B spermatogonia have chromatin clumps without any special ultrastructure and several nucleoli. Five different types of nuclear bodies, and besides, a nuclear vacuole, have been observed in type A spermatogonia. Type I bodies are typical nucleoli consisting of three regions: amorphous, fibrillar and granular. Type II, III and V are considered to be atypical nucleoli. Type IV bodies are small chromatin condensations. Type I bodies are the only ones in which RNA was demonstrated by light histochemical techniques and no PAS positive material was found inside the nuclei. The absence of any special ultrastructure in the chromatin from spermatogonia, and the small mass of the chromatin condensations, show that the human X chromosome and perhaps the Y chromosome are not heteropycnotic in the interphasic nuclei of human spermatogonia.Abbreviations Used RNA ribonucleic acid - gonia spermatogonia This work has been supported by a grant (No. 2623) of the Consejo Nacional de Investigaciones Cientificas y Tecnicas, and partially by a grant (C.M. 6522) from the Population Council.We wish to thank Professor R. E. Mancini for his suggestions during this investigation and his support for its achievement, and to Dr. J. C. Lavieri for providing the biopsies.     

Type I photosynthetic reaction centres: structure and function

We review recent advances in the study of the photosystem I reaction centre, following the determination of a spectacular 2.5 A resolution crystal structure for this complex of Synechococcus elongatus. Photosystem I is proving different to type II reaction centres in structure and organization, and the mechanism of transmembrane electron transfer, and is providing insights into the control of function in reaction centres that operate at very low redox potentials. The photosystem I complex of oxygenic organisms has a counterpart in non-oxygenic bacteria, the strictly anaerobic phototrophic green sulphur bacteria and heliobacteria. The most distinctive feature of these type I reaction centres is that they contain two copies of a large core polypeptide (i.e. a homodimer), rather than a heterodimeric arrangement of two related, but different, polypeptides as in the photosystem I complex. To compare the structural organization of the two forms of type I reaction centre, we have modelled the structure of the central region of the reaction centre from green sulphur bacteria, using sequence alignments and the structural coordinates of the S. elongatus Photosystem I complex. The outcome of these modelling studies is described, concentrating on regions of the type I reaction centre where important structure-function relationships have been demonstrated or inferred.     

Chiral tetradentate amine and tridentate aminocarbene ligands: Synthesis, reactivity and X-ray structural characterizations

(1R,2R)-diaminocyclohexane (1) and (1R,2R)-diaminodiphenylethylenediamine (2) were used as building blocks for the synthesis of chiral tetradentate diquinolyl-diamine and related diquinolyl-dihydroimidazolium salts. A neutral chiral palladium(II) complex was synthesized by reaction of palladium acetate with the tetradentate diquinolyl diamine derived from 2 and used as a homogeneous catalyst for the Heck reaction between styrene and haloarenes. A chiral tridentate aminocarbene was generated in situ by deprotonation of the dihydroimidazolium salt derived from 1 and allowed to react with CuI to give a new chiral quinolyl-carbene copper(I) complex.     

Characterization of nuclear matrix proteins of Physarum polycephalum and mammalian cells

Analysis of the nuclear matrix of Physarum polycephalum and renal epithelial cells in culture (LLC-PK1) reveals a complex protein pattern. Applying various experimental protocols we observe only negligible differences in the final nuclear matrix protein pattern, in Physarum as well as in LLC-PK1 cells. Immunoblotting with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies demonstrates the presence of intermediate filament proteins as components of the nuclear matrix. Preparation of type I and type II matrix structures does not yield different protein compositions, neither in Physarum nor in differentiated LLC-PK1 cells; therefore in both systems a distinction between these two types of matrices is questionable.     

Temperature dependent conformational changes at the active site of pyruvate kinase. A li nuclear magnetic resonance study.

The interaction of Li⁺, a weak activator of pyruvate kinase, with substrate and inhibitor complexes of the enzyme has been investigated by magnetic resonance techniques. Proton relaxation rate (PRR) titrations indicate that the dissociation constant of Li⁺ from the ternary enzyme-Mn(II)-phosphoenolpyruvate (P-enolpyruvate) complex is 15 mm at 5 °C and 17 mm at 30 °C. The electron paramagnetic resonance spectrum of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex is the superposition of spectra for two distinct species (Reed, G. H., and Cohn, M. (1973) J. Biol. Chem.248, 6436–6442). Low temperatures favor the form giving rise to the more nearly isotropic spectrum, whereas high temperatures favor the species giving rise to the anisotropic “K⁺-like” spectrum. ⁷Li nuclear magnetic resonance data are consistent with a model in which the two forms observed by epr correspond to differing Mn(II) to Li(I) distances. The form giving rise to the anisotropic spectrum is characterized by a Mn(II) to Li(I) distance of 4.7 Å, and in the more isotropic form this distance is approximately 9 Å. The 4.7 Å separation of the Mn(II) and Li(I) in the anisotropic form of the complex compares favorably with the 4.9 Å separation of Mn(II) and T1(I) (Reuben, J., and Kayne, F. J. (1971) J. Biol. Chem.246, 6227–6234) in the P-enolpyruvate complex, although T1⁺ is a much better activator of the pyruvate kinase reaction. Thus, a change in the distance between the monovalent and divalent cations does not account quantitatively for the lower activation by Li⁺, inasmuch as more than 50% of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex has the “active” conformation with respect to the separation of the cations and the epr spectrum of the complex. As reported previously (Reed, G. H., and Morgan, S. D. (1974) Biochemistry13, 3537–3541), the dissociation constant of oxalate and the epr spectrum for the ternary complex of pyruvate kinase with Mn(II) and oxalate are not influenced by the species of monovalent cation present. The nuclear relaxation rates of Li⁺ are increased in the presence of the ternary oxalate complex, although the separation of the Mn(II) and Li(I) appears to be much greater than for the “anisotropic” form of the P-enolpyruvate complex.     

Structural components of the nuclear body in nuclei of Allium cepa cells

INTRODUCTIONSmall spherical nucleax bodies have long beenobserved in both hamal and plain interphasenuclei. In the case of animal cells, these nuclear bodies are generally called coiled bodies[1].As for plant cells, they have been vaxiously described as coiled bodies, ~somes, micronucleolior nucloolus-associated bodies because they sometimes appeared in the vicinity of nucleolusl2-4].Eaxly cytologists noted that nuclear bodies in platcells appeared as a tangle of coiled threads forming a …     

Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts

The lateral distribution of the main chlorophyll-protein complexes between appressed and non-appressed thylakoid membranes has been studied. The reaction centre complexes of Photosystems I and II and the light-harvesting complex have been resolved by an SDS-polyacrylamide gel electrophoretic method which permits most of the chlorophyll to remain protein-bound.

The analyses were applied to subchloroplast fractions shown to be derived from different thylakoid regions. Stroma thylakoids were separated from grana stacks by centrifugation following chloroplast disruption by press treatment or digitonin. Vesicles derived from the grana partitions were isolated by aqueous polymer two-phase partition. A substantial depletion in the amount of Photosystem I chlorophyll-protein complex and an enrichment in the Photosystem II reaction centre complex and the light-harvesting complex occurred in the appressed grana partition region. The high enrichment in this fraction compared to grana stack fractions derived from press or digitonin treatments, suggests that the grana Photosystem I is restricted mainly to the non-appressed grana end membranes and margins, and that the grana partitions possess mainly Photosystem II reaction centre complex and the light-harvesting complex.

In contrast, stroma thylakoids are highly enriched in the Photosystem I reaction centre complex. They possess also some 10–20% of the total Photosystem II reaction centre complex and the light-harvesting complex.

The ratio of light-harvesting complex to Photosystem II reaction centre complex is rather constant in all subchloroplast fractions suggesting a close association between these complexes. This was not so for the ratio of light-harvesting complex and the Photosystem I reaction centre complex.

The lateral heterogeneity in the distribution of the photosystems between appressed and non-appressed membranes must have a profound impact on current understanding of both the distribution of excitation energy and photosynthetic electron transport between the photosystems.