Cross-linking studies of the protein topography of rat liver microsomes

A cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP, was used to study the topography of the proteins in the endoplasmic reticulum membrane of rat liver. Reaction of untreated (control), phenobarbital- or 3-methylcholanthrene-induced microsomes with 0.5 mM DSP for 30 min at 0°C resulted in the cross-linking of a protein with a molecular weight of about 52 000 to form an apparent dimer. In phenobarbital microsomes, a smaller amount of a 52 000-dalton protein also appeared in a dimer in the absence of DSP if N-ethylmaleimide was not included during homogenization. In phenobarbital and 3-methylcholanthrene microsomes, a 48 000-dalton protein was cross-linked by DSP to a protein of about 57 000. In all three types of microsomes, a protein with an M_I of about 52 000 was also cross-linked to a protein of about 79 000. In phenobarbital and control microsomes, cross-linking resulted in an oligomeric protein of approximate molecular weight 180 000 which contained three proteins, two with M_r of about 52 000 and one about 79 000. Under the cross-linking conditions, little or no denaturation of cytochrome P-450 and NADPH-cytochrome c reductase was observed. The aryl hydrocarbon hydroxylase activity was significantly inhibited by the bifunctional cross-linking reagent, DSP, but not by the monofunctional reagent N-succinimidyl-3-(4-hydroxyphenyl) propionate. However, attempts to regenerate the aryl hydrocarbon hydroxylase activity by cleavage of the disulfide linkage with 2-mercapto-ethanol or dithiothreitol were not successful.     

Morphine sulphate induced histopathological and histochemical changes in the rat liver

In this study, the histopathological and histochemical changes due to chronic usage of morphine sulphate in liver were assessed in rats with both light and electron microscopes. Twenty male albino rats (Rattus norvegicus) (130-150 g) were included and divided into four groups. Normal saline (5 ml) was given orally as placebo in the control group (N = 5). Morphine groups (N = 5) received morphine orally at a single dose of 5 ml/kg/day for 10, 20 and 30 days (groups II, III and IV), respectively. Liver specimens from all groups were evaluated for histopathological and histochemical changes. Light microscopy revealed severe centrilobular congestion, portal fibrosis with bile ductal proliferation and an increased inflammatory infiltration and focal parenchymal necrosis. Histochemical study revealed a progressive depletion of general carbohydrates and an increase in total protein contents. These changes were confirmed at ultrastructural level, including the presence of accumulated lipid in the hepatocytes; deposits of a collagen-like fibrous material were seen in the space of Disse and a reduction in the number of endothelial cell fenestrations. Our findings pointed out the risk of increased lipid fibrosis and hepatic damage due to long-term use of morphine. Although opioids are reported to be effective in pain management, their toxic effects should be kept in mind during chronic usage.     

Increased gap junctional area in the rat liver after administration of dibutyryl cAMP

Summary Since cAMP has recently been reported to be a possible physiological modulator of cell-to-cell communication, we performed a quantitative freeze-fracture investigation on the hepatocyte gap junctions after administration of a membrane-permeant derivative of this cyclic nucleotide. For this purpose, male rats received two intraperitoneal injections of 100 mg dibutyryl cAMP/kg body weight with a time interval of 2.5 h. Litter mates were injected with saline only. Five hours after the start of the treatment, tissue blocks of the left lateral liver lobe were fixed by immersion and processed for freeze-fracture. By point counting on negatives projected on a square double-lattice test system the relative gap junctional area on contiguous hepatocyte membranes was determined. As compared to control animals, the proportion of the membrane area occupied by gap junctions in dibutyryl cAMP-treated liver parenchyma significantly increased from 4.9% to 6.1%. Within the gap junctions no changes in shape, particle density or packing pattern were observed. Possibly, the enlarged gap junctional area provides structural pathways for the integration of the response of hepatocytes to messages mediated by cAMP.This investigation was supported by grant nr. 3.0059.81 (to D.W.S.) from the Fund for Medical Scientific Research (Belgium).Research assistant of the National Fund for Scientific Research (Belgium).     

Identification of G6PDH-active sinusoidal cells as Kupffer cells in the rat liver

Summary The aim of this study was to identify the G6PDH-active sinusoidal cells in the rat liver described by Rieder et al. (1978). Because of their number and distribution in the liver parenchyma, endothelial cells and pit cells could be excluded. Fat-storing cells were specifically marked by vital staining with vitamin A and identified by fluorescence microscopy. Kupffer cells could be detected after vital staining with carmine. Both staining methods allowed a subsequent incubation for the demonstration of G6PDH activity in the same unfixed cryostat section. Whereas more than 80% of the fluorescent particles were found outside the enzyme-positive cells, all G6PDH-active cells contained carmine particles. After counting the G6PDH-active cells, an estimation of 0.217 × 10⁸ cells/g liver tissue was obtained. The results indicate that high G6PDH activity is common to all Kupffer cells, and is therefore a highly specific marker enzyme for this class of sinusoidal liver cells.The essential parts of this study will be presented as an Inaugural-Dissertation to the Medical Faculty of the University of Freiburg by W. HosemannSupported by a grant from the SFB 46 (Molgrudent)     

Quantitative electron microscopic studies on the kinetics of secretory granules in G-cells

Summary Ultrastructural studies of secretory granules of rat antral G-cells and measurement of serum gastrin level were performed under the condition of fasting and administration of alkaline solution into the stomach. On electron micrographs, no qualitative difference was observed among those experimental groups. However, morphometrical analysis revealed significant quantitative differences. The population density of secretory granules of the rats treated once with alkali first increased and then decreased reaching that of the fasted group, while that of the repeatedly treated group remained nearly equal to the maximum value. The average sectioned surface area of secretory granules tended to decrease for 1.5h after the stimulation but the difference was not significant among those groups.From the results obtained at present, responding to chemical stimulation such as pH changes in the antrum, it seems probable that not only exocytosis but also migration of secretory granules from supra- and/or para-nuclear portion to the basal portion of the cell occurs rapidly in G-cells and that both these processes are inhibited immediately by antral acidification. Moreover, the present results apparently indicate that under the condition of no antral acidification G-cells have a capacity of secreting gastrin for a fairly long time, such as 4–8 h, responding to adequate stimulus. These findings are strongly suggestive of the existence of a capacious pool of granules in the supra- and/or para-nuclear cytoplasm or of fairly speedy production of secretory granules in the Golgi area.The author wishes to express thanks to Prof. R. Furihata, Department of Surgery, and Prof. T. Nagata, Department of Anatomy, Shinshu University School of Medicine, for their constant interest and guidance, and to Dr. F. Iida, Department of Surgery, who has followed the course of this work throughout     

Quantitative changes in aminoacylation of transfer RNA and in free amino acids during fructose-induced depletion of adenine nucleotides in rat liver

Fructose induces depletion of adenine nucleotides in liver and also strongly inhibits incorporation of radioactive amino acids into protein (Mäenpää, P.H., Raivio, K.O. and Kekomäki, M.P. (1968) Science 161, 1253–1254). In this study we have investigated the effects of fructose on aminoacylation of tRNA and on free amino acids in rat liver. 30 min after d-fructose (30 mmol/kg) was injected intraperitoneally into rats, liver ATP was reduced by 58%, ADP by 42%, AMP by 13%, the ATP/ADP ratio by 30%, and total adenine nucleotides by 48%. Using gas chromatography, the aminoacylation of tRNA was determined by quantifying the endogenous amino acids attached to tRNA in vivo. Aminoacylation was reduced by 31%. With different amino acids, reduction varied from 4% (asparagine plus aspartic acid) to 58% (arginine). On the other hand, the amount of free amino acids in the liver was increased by 24%. The most marked individual change was in alanine, which increased 5.7-times. This may have resulted from a combination of effects involving an increased production of alanine in muscle and liver and decreased hepatic gluconeogenesis from alanine caused by the ATP depletion.     

Quantitative analysis of the distribution of organelles in tobacco pollen tubes: implications for exocytosis and endocytosis

Summary The present study provides the first quantitative analysis on the distribution of organelles in pollen tubes ofNicotiana tabacum L. Organelles were studied on living pollen tubes by means of fluorescence confocal laser scanning microscopy and on cryo-fixed, freeze-substituted and serially sectioned material by electron microscopy. In the tip a 300 nm to 400 nm thick wall was secreted that proximately gradually separated into a wall with an opaque inner side and a more translucent, layered outer side. Tubular endoplasmic reticulum was particularly abundant in the tip of the tube, surrounding the region where secretory vesicles (SV) accumulated. Mitochondria were randomly distributed throughout the cytoplasm, no accumulations were present. Dictyosomes, however, showed an increased abundance at 25–30 m behind the tip. The accumulation of coated pits (CP) in a zone 6–15 m behind the tip identifies this zone as the major site of endocytosis: 50% of all CP occur in this zone. Quantification of exo- and endocytosis showed that only part of the membrane material of the SV can be retrieved after exocytosis. The typical zonation in endocytotic activity may serve to maintain a difference in membrane protein composition between the tip and the tube.     

Quantitative studies of postnatal changes in synapses in rat superficial motor cerebral cortex

Summary Quantitation of synapses at different postnatal ages has been undertaken in the cerebral cortex of the rat. In this study axial ratios of presynaptic bags, proportion of cortex occupied by presynaptic bags and numbers of synapses per unit volume of cortex have been estimated. Observations on synaptic vesicle packing densities have also been made.Synaptic bags become increasingly spherical up to 7 days of age and become more elongated thereafter. The proportion of cortex occupied by presynaptic bags increases rapidly up to 7 days of age and then at a decelerated rate up to maturity. The number of synapses per unit volume increases slowly over the first four days after which there is a rapid increase to 14 days, followed by a decelerated rate.The average presynaptic bag shows marked changes in volume with increasing age which indicate the probability of two stages of synaptic development. This two stage development is further reflected in the estimates on vesicle packing densities. The implications of the results are discussed in relationship to changes in functional activity of the cortex during postnatal development.The authors wish to express their thanks to Mr. R. Birchenough and Mr. J. Manston for much technical assistance.     

Effect of phlebotomy on the ferritin iron content in the rat liver as determined morphometrically with the use of electron energy loss spectroscopy

Summary Phlebotomy of untreated and iron-loaded rats results in a significant decrease in total liver iron. In ironloaded rats a marked decrease in iron-containing particles is observed ultrastructurally in lysosomes and cytoplasm of hepatic sinusoidal cells but not in parenchymal cells. This remarkable phenomenon was further investigated in a morphometric study, based on element-specific (iron) distribution images made in situ in the parenchymal cell by means of electron energy loss spectroscopy. With the use of this technique it could be shown that in spite of phlebotomy the ferritin iron content of the iron-loaded liver parenchymal cell is not decreased.     

Quantitative morphology of stimulation-induced damage in rabbit fast-twitch skeletal muscles

Summary The purpose of this study was to examine the contention that stimulation-induced damage, resulting in degeneration with subsequent regeneration, plays a major role in the transformation of fibre type brought about by chronic electrical stimulation. Data from histological and histochemical sections of 9-day-stimulated rabbit fast-twitch muscles were analysed with multivariate statistical techniques. Fibre degeneration and regeneration varied non-systematically between sample areas at any given cross-sectional level. In the extensor digitorum longus muscle, but not in the tibialis anterior, there was more degeneration in proximal than in distal portions of the muscle. The extensor digitorum longus muscle consistently showed more degeneration than the tibialis anterior muscle. Degeneration was less extensive for an intermittent pattern of stimulation that delivered half the aggregate number of impulses of continuous stimulation. Degeneration and regeneration varied markedly between individual rabbits in each of the groups. Sections that revealed the most degeneration and regeneration also had more fibres that reacted positively with an anti-neonatal antibody. Rigorous analysis of different sources of variation has helped to explain apparent conflicts in the literature. The incidence of muscle fibre damage in the stimulated tibialis anterior muscle is low, showing that the contribution of degenerative-regenerative phenomena to fibre type conversion in this muscle is insignificant.     

Learned helplessness in the rat: effect of response topography in a within-subject design

Three experiments investigated learned helplessness in rats manipulating response topography within-subject and different intervals between treatment and tests among groups. In Experiment 1, rats previously exposed to inescapable shocks were tested under an escape contingency where either jumping or nose poking was required to terminate shocks; tests were run either 1, 14 or 28 days after treatment. Most rats failed to jump, as expected, but learned to nose poke, regardless of the interval between treatment and tests and order of testing. The same results were observed in male and female rats from a different laboratory (Experiment 2) and despite increased exposure to the escape contingencies using a within-subject design (Experiment 3). Furthermore, no evidence of helplessness reversal was observed, since animals failed to jump even after having learned to nose-poke in a previous test session. These results are not consistent with a learned helplessness hypothesis, which claims that shock (un)controllability is the key variable responsible for the effect. They are nonetheless consistent with the view that inescapable shocks enhance control by irrelevant features of the relationship between the environment and behavior.     

Histochemical studies on the morphology of the golgi apparatus and its relationship to catecholamine biosynthesis in the locus coeruleus of the rat

Summary Detailed histochemical studies have been performed on the morphology of the Golgi apparatus (GA) by application of the thiamine pyrophosphatase (TPPase) method (Novikoff and Goldfischer, 1961) to the neurons of the locus coeruleus (LC) of normal and catecholamine biosynthesis inhibitors (fusaric acid and D, L--methyl-p-tyrosine methylester HCl) given adult healthy male Wistar strain rats. The neurons were classified into five categories on the basis of the morphology of the Golgi apparatus. The number of cells in individual categories was counted to evaluate the percentage of each category in the whole nucleus.The majority of cells belongs to Types II, III, and IV whose GA goes through cyclic activity, but the remaining neurons belong to Types I and V which may have a strong tendency to be different from the former in character. The latter neurons correspond formally with Types I and V of the rabbit LC, but they do not respond to the drugs administered. The rat LC is very similar to the dorsal vagal nucleus of the rabbit in regard to the dominant category. The present results indicate that the majority of the rat LC neurons may work vigorously and they may be motor neurons.Administration of the drugs caused reduction of TPPase activity, augmentation of disintegration and the budding-off process of the GA of Type IV, a decrease in the percentage of Type IV and an increase in that of Type II. Administration of 100 mg/kg fusaric acid caused maximal morphological change of the GA at the 90th minute; however, administration of 200 mg/kg fusaric acid showed more marked change of the GA, having two peaks and two valleys. The GA revealed much more intense reaction to D,L--methyl-p-tyrosine methylester HCl than to fusaric acid. The present results indicate that tyrosine hydroxylase may be the rate-limiting enzyme in the catecholamine biosynthesis.These noticeable changes of GA caused by administration of the drugs were completely restricted to the neurons of LC and the neurons of the mesencephalic nucleus of the trigeminal nerve did not show any morphological changes of the GA. These results strongly suggest that the GA of the rat LC neurons may have ability to synthesize catecholamine whereas the GA of the rat mesencephalic nucleus of the trigeminal nerve may be completely devoid of this ability and that the role of the GA may be different depending on the anatomical regions.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Long-term cell culture of two differentiated cell types from the liver of larval and adult Xenopus laevis

Summary Two distinct types of cells were derived from organ cultures of liver from adult and larval Xenopus laevis. Each type was isolated in clonal cell culture. Several media were compared with respect to support of epithelioid outgrowths from explants and support of growth of epithelioid colonies in cell culture. Ultracentrifuged embryo extract promotes the growth of all cell types, but the particulate fraction is also required for the maintenance of the epithelioid morphology of larval cells. In these media it was possible to maintain some epithelioid cell cultures for over 6 months. The identity and retention of some specialized functions of both cell types were demonstrated on larval cells. One cell type contained PAS-stainable, amylase-sensitive granules that increased in amount after treatment with glucocorticoids. This same type was shown by histochemical methods to contain phosphorylase, glucose-6-phosphatase, and dexamethasone-inducible tyrosine aminotransferase, and is considered to be a hepatocyte. The second type appears to be a sinusoidal cell, since it phagocytosed trypan blue and stained positively for acid phosphatase.     

Silicon in rat liver organelles: Electron probe microanalysis

Summary Electron probe microanalysis of unfixed freeze-substituted rat liver tissue embedded in Spurr's low viscosity epoxy resin demonstrated the occurrence of Si as well as P, S, and Cl in the nucleus, nucleolus, mitochondria, and rough endoplasmic reticulum. Chemical analysis confirmed that the Si in the organelles did not originate from instrumental contaminants. This suggests that Si may be involved in the biochemistry of these subcellular organelles.Supported by Grant GM-08229-12-13 from the National Institutes of Health, USPHS.We are grateful to the Kevex Corporation and Mr. Glenn W. Meyer, Sales Engineer, for the use of the Kevex X-ray spectrometer; we wish to thank as well the Perkin-Elmer Corporation and their Western Branch Manager, Mr. Michael E. Mullen and Senior Microscopist, Mr. Minoru Shinorhara, for use of and assistance with the Hitachi HU 12 A transmission electron microscope. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and the X-ray photographs, and Darlene Lum for technical assistance in the laboratory.     

Rapid formation of myometrial gap junctions during parturition in the unilaterally implanted rat uterus

Summary In uterine smooth muscles, gap junction plaques rapidly form during the final stages of gestation. To investigate the related mechanisms, regional differences in myometrial gap junction development in rat uterus were examined quantitatively during delivery, using thin-section and freeze-fracture techniques in combination with light- and electron microscopy.Examination of implanted and nonimplanted horns in the unilaterally ligated rat bicornuate uteri, revealed no differences in the occurrence of gap junction plaques, but after 2 to 4 pups had been delivered, the contracted segments contained more gap junction plaques than did noncontracted segments examined immediately before delivery. In all segments, gap junctions were found more frequently in the circular muscle layers than in the longitudinal muscle layers. Gap junctions ranged in size from 0.002 m² to 0.52 m², but two-thirds were less than 0.1 m². The frequency of small gap junction plaques (less than 0.1 m²) was higher in the noncontracted segment.These results suggest that gap junctions are dynamic structures, and that their formation is controlled not only by general hormonal factors, possibly involved in gap junction increases in the myometrium before delivery, but also by local factors, possibly related to the contraction, that may accelerate an increase in gap junction formation during delivery.     

Ultrastructure and membrane topography of special ciliary organelles in the ciliate Eufolliculina uhligi (Protozoa)

Summary In Eufolliculina uhligi and other folliculinid ciliates, a territory has been identified that differs ultrastructurally from other areas of the cell, and that is especially sensitive to mechanical stimuli. This territory is located around the anterior oral apparatus of the loricate trophont and posterior to the membranellar spiral of the swarmer. Each cilium in this territory is closely apposed to a small membrane-covered pin that is supported by transverse microtubules of the cilium. In front of the pin, the base of the cilium bulges out; the ciliary membrane is interconnected with the axoneme by filamentous material. Freeze-fractured cilia show a large rectangular particle array at the site of the basal swelling. Only scattered particles have been observed in the pin membrane. It is suggested that the cilium and the pin act as a unit, which has therefore been named the ciliumpin-complex. Comparison with ciliary organelles of unicellular and multicellular organisms indicates that, because of their polar organization, the complexes are involved in the transduction of oriented, presumably mechanical, stimuli.     

Quantitative analysis of rod-cored vesicles and dense granules of large granular lymphocytes in the liver,spleen, and peripheral blood of rats

Large granular lymphocytes (LGL) comprise a natural defense system in the liver and exert an inhibitory effect on tumor cell metastasis. In order to demonstrate the maturation of LGL in the liver from the morphological aspect, we evaluated electron-microscopically the frequency of 0.2 m vesicles (rod-cored and empty vesicles) and dense granules in LGL from the liver, spleen, and peripheral blood of the rat. Both of these cell organelles are characteristic to LGL and may relate to natural killer-mediated cytolysis. On the average, there were 12.7 of the 0.2 m vesicles and 4.3 rod-cored vesicles (RCV) per cell section in the liver, 6.6 0.2 m vesicles and 1.6 RCV in the spleen, and 8.6 0.2 m vesicles and 0.9 RCV in the peripheral blood. The number of 0.2 m vesicles per cell section ranged from 0 to 19 with the exception of a few higher instances. Therefore, LGL were divided into vesicle-rich(>9 0.2 m vesicles per cell section) and vesicle-poor (<8 per cell section) populations. Hepatic LGL consisted mainly of a vesicle-rich population while splenic LGL consisted mainly of a vesicle-poor population, and peripheral blood contained equal proportions of both populations. In addition to diversity with regard to the number of 0.2 m vesicles, LGL obtained from various organs also displayed heterogeneity in the number and size of dense granules. Since the number of dense granules per cell section usually ranged from 1 to 13, LGL were diveded into 2 populations, i.e., LGL with many (>7 per cell section) granules and those with a few(<6 per cell section) granules. Specifically, splenic LGL had a few small (average diameter, less than 400 nm) dense granules, while sections of LGL from the liver and peripheral blood displayed many small dense granules and a few large (>400 nm) ones, respectively, in addition to the populations seen in the spleen. Thus, the present study has demonstrateda difference in the distribution of 0.2 m vesicles in LGL based on the tissue of origin. The present study has revealed the difference in the distribution of 0.2 m vesicles of LGL by tissue and indicated that immature LGL are predominant in the spleen, while hepatic LGL are generally more mature as defined by the number of vesicles. These data suggest that the microenvironment of the liver may contribute to the increased expression of these vesicles in LGL.     

Biphasic effect of fructose 2,6-bisphosphate on the liver fructose-1,6-bisphosphatase: mechanistic and physiological implications

Fructose 2,6-bisphosphate has been claimed to be both a substrate analogue and an allosteric inhibitor of fructose-1,6-bisphosphatase. The results reported here show that fructose 2,6-bisphosphate can be both an inhibitor and an activator of the enzyme, depending on the substrate concentration. This biphasic behaviour at saturating concentrations of substrate can only be due to an allosteric effect. In addition to the mechanistic implication it is possible that this finding may have physiological meaning.