Mineral congregations, “spherites” in the midgut gland ofCoelotes terrestris (Araneae): Structure,composition and function

Summary Spherites in the digestive and secretory cells of the midgut gland of the agelenid spiderCoelotes terrestris were studied by electron microscopy and histochemical methods. Spherites measured 1–6 m in diameter and were characterized by alternating layers of electron dense and electron lucent material. The main-components of spherites were calcium phosphates and calcium carbonates. Guanine and barium, as well as aminopeptidase and alkaline phosphatase were also present. The matrix consisted of proteins and carbohydrates. Numerous spherites were found together with excretory products within the excretory vacuoles of the digestive cells.Spiders fed with food containing lead, showed deposition of the metall in the spherites. It is then proposed that spherites, aside from their role in storing calcium and other ions, may function in detoxification of heavy metals.     

Calcium reabsorption in the posterior caeca of the midgut in a terrestrial crustacean,Orchestia cavimana

Summary During molting, the epithelium of the posterior caeca (PC) of the midgut in the terrestrial crustacean, Orchestia cavimana, is active in calcium turnover. In the preexuvial period, epithelial cells that progressively differentiate into cell-type III secrete ionic calcium (originating from the old cuticle) from the base to the apex of the cell within a typical extracellular network of channels; the calcium is then stored in the PC lumen as calcareous concretions. Immediately after exuviation, the epithelial cells rapidly differentiate into cell-type IV, reabsorbing calcium from the concretions through successive generations of spherites which quickly appear, grow, and then disappear from the apex to the base of the same extracellular network. The PC epithelium is thus alternatively calcium-loaded and unloaded. When the calcium-reabsorbing process is complete (average 48 h after exuviation), the epithelial cells again differentiate into two different regional cellular types (cell-type I in the distal segment and cell-type II in the proximal segment) characteristic of the intermolt period.The dynamic changes in the PC epithelium during the postexuvial period are discussed, including the characteristic features of cell-type IV and of the reabsorption spherites.     

A study of the concentrically laminated concretions, 'spherites' in the regenerative cells of the midgut of Lepidopterous larvae

Concentrically laminated granules, spherites, are sometimes found in the regenerative cells of midgut of some species of lepidopterous larvae. The spherites are formed in cytoplasmic vesicles just before ecdysis and disappear during the differentiation of the regenerative cells to columnar and goblet cells. They function as intracellular stores of compounds used in the growth of the cell. Phosphates of magnesium and perhaps calcium are probable constituents. Spherites are sometimes also found in the degenerating columnar cells where they are excreted into the lumen with the exfoliating epithelium. The phenomenon of periodic precipitation which is the physical-chemical basis of the formation of spherites is discussed.     

Disturbance of the secretory pathway inMicrasterias denticulata by tunicamycin and cyclopiazonic acid

Summary Both tunicamycin, an inhibitor of N-linked glycosylation of proteins, and cyclopiazonic acid, which inhibits the Ca²⁺-dependent ATPase in the ER, influence the secretory pathway at the ER level and lead to a cessation of cell growth inMicrasterias. Electron microscopical investigations reveal that the mode of action of the two inhibitors differs. While tunicamycin treatment results in a disintegration of the Golgi bodies into small vesicles, cyclopiazonic acid prevents products being supplied from the ER, resulting in the dilatation of ER cisternae and a reduction in the number of Golgi cisternae, combined with a loss of dictyosomal activity. The disturbed cell wall formation under tunicamycin indicates that N-linked glycosylation of proteins is required for normal cell growth inMicrasterias. Moreover, our studies reveal that changes in cytoplasmic free calcium concentration, as a consequence of ATPase inhibition in the ER by cyclopiazonic acid, may inhibit wall material secretion by interrupting the normal ER-dictyosome association.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Fluorescence and histochemical studies of the calcification-initiating lipofuscin type pigment granules in the shell-repair membrane of the snail,Helix pomatia L.

Summary The pigment granules of lipofuscin type present in the amoebocytes and in the ground substance of the shell-repair membrane of the snail, Helix pomatia, have been investigated. These granules showed a bright, yellow primary fluorescence, which changed to orange-red when stained with fluorochrome acridine orange. Histochemical tests proved that the granules contained lipids, proteins and glycosaminoglycans. They showed acid-phosphatase activity. The lipofuscin granules showed that they had characteristics in common with lysosomes and appeared to be related to them. The function of these granules in the mineralizing matrix of the shell-repair membrane is discussed. They possess a complex structure, exhibit enzyme activity and appear to be engaged in the formation of the organic crystalline bodies. They incorporate calcium ions and are themselves transformed into the primary calcifying centra, i.e. the b-granules described previously. Common features in the mineralizing processes of the molluscs and the vertebrates were observed.This investigation was supported by grants from the Hierta-Retzius Stipendiefond and the Längmanska Kulturfond.     

Alterations in the fine structure of cytoplasmic organelles in the hepatopancreatic cells of shell-regenerating snail,Helix pomatia L.

Summary The investigation of hepatopancreatic cells following damage to the shell revealed hypertrophic alterations of several Cytoplasmic organelles. The network of endoplasmic reticulum was extensively developed within both digestive and calcium cells. The arrangement of tubules exhibited a peculiar hexagonal pattern. It is suggested that the reticulum is of agranular type and may be engaged in the transport of lipids and calcium ions. Remarkable alterations were observed in mitochondria, apparently as the result of their hyperfunction. Mitochondria with reduced cristae, with two-layered transverse septum, and with bleb-like protrusions, occurred frequently. In the calcium cells, helically entwined fibres appeared among the fragments of disintegrated calcium spherites. The results of the investigation and the possible presence of collagen in the fibres originating from disintegrated spherites are discussed.This investigation was supported by grants from the Swedish Natural Science Research Council, which are gratefully acknowledged. The author wishes to thank Mrs. I. Rehnberg for technical assistance.     

Nucleotide sequence of the cybB gene encoding cytochrome b561 in Escherichia coli K12

Summary The complete nucleotide sequence of the Escherichia coli cybB gene for diheme cytochrome b ₅₆₁ and its flanking region was determined. The cybB gene comprises 525 nucleotides and encodes a 175 amino acid polypeptide with a molecular weight of 20160. From its deduced amino acid sequence, cytochrome b ₅₆₁ is predicted to be very hydrophobic (polarity 33.7%) and to have three membrane spanning regions. Histidines, canonical ligand residues for protohemes, are localized in these regions, and the heme pockets are thought to be in the cytoplasmic membrane. No significant homology of the primary structure of cytochrome b ₅₆₁ with those of other bacterial b-type cytochromes was observed.     

Sequence, expression and localization of the immunity protein for colicin B

Summary Cells of Escherichia coli containing the cbi locus on plasmids are immune to colicin B which kills cells by dissipating the membrane potential through pore formation in the cytoplasmic membrane. The nucleotide sequence of the cbi region was determined. It contains an open reading frame for a polypeptide consisting of 175 amino acids. The amino acid sequence is homologous to the primary structure of the colicin A immunity protein. This, and the strong homology between the pore-forming domains of colicins A and B suggests a common evolutionary origin for both colicins. The immunity protein could be identified following strong overexpression of cbi. The electrophoretically determined molecular weight of 20 000 was close to the calculated molecular weight of 20 185. The protein contains four large hydrophobic regions. The immunity protein was localized in the membrane fraction and was mainly contained in the cytoplasmic membrane. It is proposed that the immunity protein inactivates the colicin in the cytoplasmic membrane.     

Changes in the activity of the Golgi apparatus during the cell cycle in antheridial filaments ofChara vulgaris L.

Summary The number of dictyosomes found in one central cell section in antheridial filaments ofChara vulgaris increases proportionally to the cell length during interphase. The activity of Golgi apparatus was expressed by a number of Golgi vesicles surrounding a single dictyosome. These vesicles are most numerous during mitosis and cytokinesis,i.e., prior to and during cell plate formation. In the middle and late S phase the number of Golgi vesicles decreases by about 25%; subsequently, during the early and middle G₂, it increases again. At the end of the G₂ phase, Golgi vesicles are the scarcest.The increase in the number of Golgi vesicles during the G₂ phase coincides with the period of intense cellular elongation, and, thus, it is probably related to the enhanced synthesis of cell wall components.Coated vesicles are most numerous in prophase, metaphase, and early telophase, and during interphase in both late S and G₂ phase. It was found that the number of coated vesicles is proportional to the degree of condensation of nuclear chromatin.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4.     

HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT CELLS IN THE DIGESTIVE GLAND OF APLYSIA PUNCTATA (CUVIER, 1803)

The crypt cells lining the Aplysia punctata digestive tubulescomprise of three types of cell; calcium, excretory, and thincells. The calcium cells play a role in osmoregulation, mineral storage,exocrine secretion, iron detoxification, and excretion processes.They possess well- developed microvilli and a basal labyrinth,suggesting a role in absorption. The Golgi apparatus is involvedin the production of two main components of calcium spherules;the fibrillar material and mineralized granules. Golgi complex,rough endoplasmic reticulum (RER), ribosomes, and altered mitochondriaare involved in the formation of calcium spherules. Secretoryactivity is indicated by the formation of dense granules containingiron and calcium salts. Lipofuscin pigment has been found inlarge concretions which may arise from cytoplasmic areas surrounded byendoplasmic reticulum, RER and Golgi tubules. There are threestages of excretory cells, called early, mature, and post-excretorycells. This study traces the development of granulofibrillarvacuoles up to the formation of the lipofuscin concretions andshows that excretory cells are in fact degenerating calciumcells. The fine structure of thin cells suggests that they areyoung calcium cells. (Received 29 December 1997; accepted 15 November 1998)     

X-ray microanalysis of the mineral components in the scales of an amoeba,Cochliopodium sp. (Testacea)

Summary Mineral components of the scales in an amoeba, Cochliopodium sp., were examined by energy-dispersive X-ray microanalysis. The precipitates in potassium antimonate-treated material detected calcium in the scales. Calcium was also clearly detected in freeze-substituted thin sections. Similar deposits of calcium antimonate were detected in scales in formation within vacuoles, and also in Golgi cisternae, Golgi vesicles and special granules near the nucleus. There were only minute amounts of magnesium and potassium. This suggests that calcium is the main mineral component of the scales and that it is added in the Golgi complex during scale formation.     

Agrobacterium tumefaciens 6bgenes are strain-specific and affect the activity of auxin as well as cytokinin genes

Summary The T-region located 6b gene of Agrobacterium tumefaciens has been found to interfere with cytokinin effects produced by the cotransferred ipt gene. We have compared the biological activity of three different 6b genes: A-6b from Ach5 (octopine, biotype 1), C-6b from C58 (nopaline, biotype 1) and T-6b from Tm4 (octopine, biotype III) by using different biological assays. Each 6b gene was inserted into a disarmed vector and tested on tobacco stems in coinfection experiments with the Ach5 cytokinin (ipt) gene (A-ipt). A-ipt/C-6b coinfections produced tumours with shoots, A-ipt/A-6b coinfections green tumours and A-ipt/T-6b coinfections tumours with a necrotic surface. The tumour phenotypes obtained were independent of the 6b/A-ipt coinfection ratios, indicating that the strain-specific 6b effects result from the activity of a non-diffusible 6b encoded product. Studies with ipt-less Tm4 mutants showed that 6b genes affect other tumour genes besides the ipt gene and pointed to an influence of T-6b on auxin effects resulting from the Tm4 iaa system. T-iaa/T-6b coinfection experiments showed that T-6b did indeed strongly increase tumour formation by the Tm4 iaa genes. The three 6b genes also have effects which do not require other T-region genes. The complex role of the 6b gene in crown gall induction and Agrobacterium host range will be discussed.     

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb ₅₆₁. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR ascorbate free radical - AO ascorbate oxidase - DTT dithiothreitol - FCCP carbonylcyanidep-trifluorome-thoxyphenylhydrazon - Hepes N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Oxonol VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol - PMSF phenylmethylsulfluoride     

The transmembrane domain of N –acetylglucosaminyltransferase I is the key determinant for its Golgi subcompartmentation

Golgi‐resident type–II membrane proteins are asymmetrically distributed across the Golgi stack. The intrinsic features of the protein that determine its subcompartment‐specific concentration are still largely unknown. Here, we used a series of chimeric proteins to investigate the contribution of the cytoplasmic, transmembrane and stem region of Nicotiana benthamiana N–acetylglucosaminyltransferase I (GnTI) for its cis/medial‐Golgi localization and for protein–protein interaction in the Golgi. The individual GnTI protein domains were replaced with those from the well‐known trans‐Golgi enzyme α2,6–sialyltransferase (ST) and transiently expressed in Nicotiana benthamiana. Using co‐localization analysis and N–glycan profiling, we show that the transmembrane domain of GnTI is the major determinant for its cis/medial‐Golgi localization. By contrast, the stem region of GnTI contributes predominately to homomeric and heteromeric protein complex formation. Importantly, in transgenic Arabidopsis thaliana, a chimeric GnTI variant with altered sub‐Golgi localization was not able to complement the GnTI‐dependent glycosylation defect. Our results suggest that sequence‐specific features in the transmembrane domain of GnTI account for its steady‐state distribution in the cis/medial‐Golgi in plants, which is a prerequisite for efficient N–glycan processing in vivo.     

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae,Saltatoria)

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.     

Cytochemical localization of cellulase activity in pollen mother cells of david lily during meiotic prophase I and its relation to secondary formation of plasmodesmata

Summary Cellulase activity was localized at the ultrastructural level in pollen mother cells (PMCs) of David lily [Lilium davidii var.willmottiae (Wilson) Roffill] at different stages of meiotic prophase I. The enzyme was observed to appear at the early leptotene stage and reached its highest level at the subsequent zygotene stage, and its subcellular distribution revealed by the presence of electron-dense deposits of reaction product was found to be restricted exclusively to the endoplasmic reticulum (ER), the vesicles derived from that, and the cell wall, especially at the sites of secondary plasmodesmata and cytoplasmic channels where the wall was being digested. Other cytoplasmic organelles, such as dictyosomes and Golgi vesicles, lacked such deposits of reaction product. After zygotene the enzyme activity decreased abruptly, and at the pachytene stage only very few deposits could be observed in the cell wall. Our results indicate that cellulase is synthesized on rough ER and secreted directly via the smooth ER and ER-derived vesicles into the cell wall by exocytosis, where it brings about local wall breakdown, leading to the secondary formation of plasmodesmata and cytoplasmic channels.     

Observations on the proventricular glands (‘organes racémiformes’) of the oribatid miteChamobates borealis (Acari,Oribatida): an organ of interest for studies on adaptation of animals to acid soils

The proventricular glands of the oribatid miteChamobates borealis (Trägårdh, 1902) were investigated by electron microscopy and histochemistry, and their function was tested in a laboratory experiment. Specimens of the same species collected during a field study also were investigated.The cells of the proventricular glands are characterized by great amounts of mineral spherites and seem to be sensitive to alterations of environmental pH and concentration of CaCO₃. It was demonstrated that a reduction of pH leads to a decrease of the spherites, whereas at a high pH the number of spherites increases.The proventricular glands with their spherites seem to play an important role in regulation of mineral budget, pH and detoxification of heavy metals.     

Perturbation of the secretory network inClosterium acerosum by Na+-selective ionophores

Summary Closterium acerosum possesses a well-defined, mucilage-secretory mechanism consisting of up to 100 Golgi bodies, two distinct vacuolar networks, and an active cytoplasmic-streaming network located in the cell periphery. Five different sodium-affecting agents were applied to actively secreting cells in order to determine the role, if any, of Na⁺ on this secretory mechanism. Significant effects to the endomembrane system and actin cytoskeleton were noted upon treatment with the Na⁺-specific ionophores monensin and SQl-Et. In particular, the following alterations were noted: incurling of Golgi cisternae and the formation of circular cisternal profiles at the trans face, swelling of the cis-medial cisternae, and dissociation of the Golgi body from the internal cytoplasm to the peripheral cytoplasmic zones. Immunogold labeling with a mucilage-specific polyclonal antibody reveals that mucilage production is diminished during longer ionophore treatments. Likewise, both the polar and peripheral vacuoles disintegrate into a series of smaller vacuoles. Cytoplasmic streaming ceases and the normal actin network of the peripheral cytoplasm transforms into irregularly spaced fibrillar bundles. Finally, multilaminate structures appear at the plasma membrane. No cytological effects could be observed with the Na⁺-channel blockers or Na⁺-current transducers QX-14, tetrodotoxin, or amiloride.Abbreviations DIC differential interference contrast - GA Golgi apparatus - LM light microscopy - TEM transmission electron microscopy - TGN trans Golgi network - WHM Woods Hole medium - DMSO dimethylsulfoxide     

Light and Electron Microscope Study of Dunaliella primolecta Butcher (Volvocida)*

SYNOPSIS. Light and electron microscope observations on Dunaliella primolecta Butcher from logarithmic and stationary phases of batch cultures are correlated. Except for the lack of a cell wall the fine structure has typical volvocid features. The transition from logarithmic to stationary phase is marked by changes in content and size of cytoplasmic vacuoles, accumulation of cytoplasmic lipid, accumulation of starch in the plastid matrix, and by the formation of autophagosome-like bodies. The organelles in stationary-phase flagellates are closely packed together because of the cytoplasmic lipid and starch-distended chloroplast. Organisms from logarithmic phase have an abundant ribosome-packed groundplasm supporting the organelles. In the cytochemical demonstration of acid phosphatase activity, Golgi cisternae and smooth and coated Golgi vesicles contain Gomori reaction product. The possible roles of the Golgi apparatus in this flagellate are discussed.