Serum bactericidal activity of three different dosing regimens of colistin with implications for optimum clinical use.

Although colistin methanesulfonate (CMS) has been used extensively in critically ill patients infected with multidrug-resistant organisms, the optimum dosing regimen remains to be determined. Herein, we examined the pharmacokinetics of three different dosing regimens of CMS, 3 million units every 8 h (regimen A), 4.5 million units every 12 h (regimen B), 9 million units every 24 h (regimen C) and evaluated the bactericidal activity of serum containing various concentrations of colistin against Pseudomonas aeruginosa with a minimum inhibitory concentration (MIC) of 1 microg/ml. the means +/- SE serum C(max )of colistin for regimens A, B, and C were 3.34+/-0.35, 2.98+/-0.27, and 5.63+/-0.87 microg/ml, respectively. All serum samples containing colistin >4 microg/ml (serum concentration/MIC >4) eliminated P. aeruginosa whereas only 40% of samples containing colistin <4 microg/ml resulted in complete bacterial killing. these findings indicate that the currently used dosing regimens might not provide the most effective therapy with CMS and justify administering larger dosages in longer intervals.

Gene functional annotation by statistical analysis of biomedical articles.

BACKGROUND: Functional annotation of genes is an important task in biology since it facilitates the characterization of genes relationships and the understanding of biochemical pathways. The various gene functions can be described by standardized and structured vocabularies, called bio-ontologies. The assignment of bio-ontology terms to genes is carried out by means of applying certain methods to datasets extracted from biomedical articles. These methods originate from data mining and machine learning and include maximum entropy or support vector machines (SVM). PURPOSE: The aim of this paper is to propose an alternative to the existing methods for functionally annotating genes. The methodology involves building of classification models, validation and graphical representations of the results and reduction of the dimensions of the dataset. METHODS: Classification models are constructed by Linear discriminant analysis (LDA). The validation of the models is based on statistical analysis and interpretation of the results involving techniques like hold-out samples, test datasets and metrics like confusion matrix, accuracy, recall, precision and F-measure. Graphical representations, such as boxplots, Andrew's curves and scatterplots of the variables resulting from the classification models are also used for validating and interpreting the results. RESULTS: The proposed methodology was applied to a dataset extracted from biomedical articles for 12 Gene Ontology terms. The validation of the LDA models and the comparison with the SVM show that LDA (mean F-measure 75.4%) outperforms the SVM (mean F-measure 68.7%) for the specific data. CONCLUSION: The application of certain statistical methods can be beneficial for functional gene annotation from biomedical articles. Apart from the good performance the results can be interpreted and give insight of the bio-text data structure.

Impact of carbapenem administration on systemic endotoxemia in patients with severe sepsis and Gram-negative bacteremia.

In order to investigate the effect of carbapenems on systemic endotoxemia, 20 patients with severe sepsis due to ventilator-associated pneumonia and Gram-negative bacteremia were enrolled; 10 (group A) were administered 1 g t.i.d. of imipenem/cilastatin and 10 (group B) 2 g t.i.d. of meropenem. Blood was sampled at 0 time and after 1, 2, 4, 6, 12, 24, 36, 48, 60, 72, 84 and 96 hours for detection of endotoxins (LPS), interleukin-6 (IL-6), C-reactive protein (CRP) and drug levels. LPS were determined by the QCL-1000 LAL assay, IL-6 by an enzymeimmunoassay, CRP by nephelometry and carbapenem levels by a microbiological assay. We did not find that carbapenems had any effect on the kinetics of LPS and CRP; IL-6 of group A was lower than group B at 72 and 84 hours. No correlation was observed between drug levels of any carbapenem and LPS, IL-6 or CRP. It is concluded that in septic patients with Gram-negative bacteremia administration of either imipenem or meropenem did not affect systemic endotoxemia. The above data support the safe administration of both carbapenems in patients with severe sepsis.

Ultrastructural histochemical studies of secretory granule replenishment in rat submandibular granular tubules after cyclocytidine-induced secretion.

Rat submandibular glands have been examined electron microscopically at various times after degranulating the granular tubules by injecting cyclocytidine (75 mg/kg i.p.), to study events in the reformation of secretory granules in these cells. The changes were progressive but not synchronous in the cells. The first evidence of recovery was the re-appearance of glycogen particles 6 h after injection. Residual secretory granules were small and located periluminally at that time. More granules were present at 15 h after injection but they were still small and placed periluminally. There was more glycogen in the cells and some was present in aggregates. At 1 day after injection there were more secretory granules and they tended to be larger than previously. The secretory granules increased in size and number progressively thereafter and the cells appeared like normal controls by day 7. During the recovery, fusion profiles were seen between granules from 2 days onwards. Throughout, few Golgi complexes were detected and this may be related with the low glycosylation of the secretory proteins in these cells. The results confirm that the reformation of the secretory granules in granular tubule cells is a slow process that involves fusions of smaller granules.

Intranuclear virus-like particles of a Drosophila hybrid.

Intranuclear virus-like particles (VLPs) have been observed in different cell lines and adult tissues of Drosophila. In the present study, intranuclear VLPs have been found in larval tissues (salivary glands, midgut, fat body) as well as in adult tissues (midgut, genitals, fat body) of a rare interspecific hybrid (D. mauritiana x D. melanogaster) called 'mame'. The intranuclear VLPs were round or slightly elliptical with a diameter of 30 nm, and they were found mainly in highly organised clusters, forming large crystalline arrays, near the nucleolus and the polycene chromosomes. These particles were never observed in the cytoplasm of any mame's tissue. A few VLPs were also seen in the corresponding tissues of D. melanogaster, but they were never observed in any tissue of D. mauritiana. There is the intriguing possibility that these VLPs are related to transposable elements and probably contribute to the speciation process, in an unknown, so far, manner.

Ultrastructural changes in exocrine tissues of a DeltaF-508 CFTR mouse model.

Cystic fibrosis (CF) is characterized by abnormal secretion from epithelial cells. We wanted to detect changes in the ultrastructural characteristics of cells within a number of exocrine tissues, including the colon, submandibular and parotid salivary glands of DeltaF-508 CFTR animals. Therefore, in the present study a DeltaF-508 CFTR mouse model was compared to control, by applying conventional and complex carbohydrates staining techniques to tissue sections at the electron microscope level. The colon of DeltaF-508 CFTR mice contained thick mucous secretions that harbored many bacteria, along with cytoplasmic fragments and leukocytes. Leukocytes were also seen to infiltrate the cytoplasm of goblet cells. Tissues were taken before, 10 min after isoprenaline, and 30 min after a further injection of methacholine. In the submandibular gland, there is limited secretory activity after isoprenaline treatment, and this increases further with methacholine treatment. Depletion of the secretory granules of acinar cells is observed, following the combined isoprenaline and methacholine treatment, but no significant changes in granule numbers occurred in granular tubule cells. Glycogen, abundant before treatment, is reduced within 10 min of isoprenaline treatment and is completely exhausted by 30 min, especially in the convoluted granular tubule cells. A few secretory granules in acinar and in granular tubule cells of the DeltaF-508 CFTR submandibular glands displayed two electron densities. The secretory responses of the parotid gland cells were similar to those in submandibular gland cells, except that in these DeltaF-508 CFTR cells, secretory granules appeared more polymorphic in structure than those found in control animals.

Immunocytochemical localization of NOVH protein and ultrastructural characteristics of NCI-H295R cells.

In this study we present biochemical and immunocytochemical results on NOVH protein secretion and localization in NCI-H295R cells, as well as results on the ultrastructural characteristics of NCI-H295R cells. NCI-H295R cells were characterized by small quantities of rough and smooth endoplasmic reticulum, many free ribosomes, large nuclei with prominent nucleoli, numerous elongated mitochondria, a few Golgi complexes, and a small number of lipid droplets. Large numbers of coated pits and coated vesicles were present, but no secretory granules or exocytotic profiles were seen. Best ultrastructural preservation of NCI-H295R cells was achieved when fixation was done directly on the culture dishes and the cells were detached by scraping. Our biochemical results showed that NCI-H295R cells secreted large amounts of NOVH protein. The immunocytochemical localization of NOVH protein showed that the protein was localized in the cytoplasm, the plasma membrane and the nuclear envelope. This localization pattern, along with the ultrastructural and biochemical findings raise interesting questions on the function(s) and the mode of secretion of NOVH protein.

Structural evidence for ion transport and tectorial membrane maintenance in the gerbil limbus.

Cells medial to the tunnel of Corti were examined to assess fine structural features relevant to their proposed role in cochlear K(+) homeostasis. A dense network of canaliculi referred to as canalicular reticulum (CR) resided in the foot body of inner pillar cells, where it bordered and could resorb ions released from inner radial and spiral nerves. Lateral interdental cells (IDCs) formed columns which connected the inner sulcus epithelium with the base of the tectorial membrane's (TM) middle zone. A spout-like neck in cells at the top of lateral IDC columns housed a dense concentration of CR which resembled that characteristic of ion transporting epithelia and appeared to be located here for transporting ions and fluid toward the TM. Clustered IDCs in the center of the limbus connected underlying limbal stroma with the TM's limbal zone and appeared capable of transporting ions from stroma to TM. Abundant CR in limbal stellate fibrocytes evidenced their capacity to transport ions and fluid, presumably from inner sulcus epithelium toward central IDCs. The most medial IDCs possibly function as the terminus of an ion cycling path from scala vestibuli to endolymph. Light fibrocytes situated between supralimbal fibrocytes and medial IDCs appeared to serve as a link in this pathway. The limbal zone of the TM overlying central IDCs consisted of three distinct regions which offered a structural basis for transformation of an amorphous matrix supplied by central IDCs into the protofibrils of the membrane's middle zone.

Ultrastructural histochemical studies of secretory processes in rat submandibular granular tubules during intermittent sympathetic nerve stimulation.

Secretory changes in the cells of granular tubules in rat submandibular glands have been studied sequentially during electrical stimulation of their sympathetic nerves. Results were assessed in a series of biopsied lobes from the same gland, taken at different times during the sympathetic stimulation. Changes were not synchronous between adjacent cells and it appeared that the time for the onset of secretory events differed between cells but, once set in action, a chain of similar events occurred. Nevertheless, some cells appeared to remain refractory throughout. Initially, some alignment of granules to the adjacent plasma membrane occurred and occasional evidence for classical exocytosis was seen. However, from early on microvesicles appeared in more luminally located granule membranes and were associated with granule fusions, that became common and led to the formation of large irregular aggregates. Most of the secretion of granule contents appeared to be through openings of aggregates into lumina. With granule fusions the intra-membrane microvesicles became internalised and tended to increase in size with time; they were commonly expelled with the contents of the aggregates. Fragments of cytoplasm also became incorporated in aggregate formation. Cytoplasm, often containing glycogen, also formed luminal blebs over some granular tubule cells and appeared to pass into the secretion by an apocrine process. At the end of stimulation multivesicular bodies were seen in association with redundant aggregates.

Ultrastructural studies on the effect of heat shock treatment on larval salivary gland cells of Drosophila auraria.

In this study, the effect of heat shock treatment on Drosophila auraria late 3rd instar larval salivary glands was examined. Heat shock treatment was applied on whole animals and on isolated salivary glands. The fine structural changes were examined using transmission electron microscopy, after a temperature rise from normal (25 +/- 1degreesC) to 37 degreesC or 40 degreesC for various periods of time. The AcPace histochemical technique was used to demonstrate the acid phosphatase activity on lysosomal structures and x-ray microanalysis to determine the elemental composition of intramitochondrial granules. Our results indicate that the extent of heat shock damage on salivary gland cells depends on the heat shock intensity (temperature and duration). Three main changes were observed after heat shock treatment: a) appearance of lysosomal structures; b) alteration in the mitochondrial morphology and appearance of intramitochondrial granules and c) morphological alterations of secretory granules. Vesiculation of the Golgi complex and dilation of the rough endoplasmic reticulum were often seen. Irregular structures of unknown function were observed in the cytoplasm, which are referred to as x-structures. Rectangular secretory granules were observed in some cases, for the first time in a Drosophila species. These results are discussed in correlation with the heat shock effect on larval salivary glands of Drosophila.

Novel membranous structures in apical and basal compartments of inner hair cells.

Postfixation with a ferrocyanide-osmium tetroxide solution preserved a dense network of canaliculi extending from the apical to the upper lateral plasma membrane in cochlear inner hair cells (IHCs). Numerous Golgi bodies intermingled with this apical canalicular reticulum (CR). Osmium-ferrocyanide treatment also disclosed several previously unreported structures below the IHC nucleus. The first consisted of stacks of six or eight and sets of three parallel cisternae of rough endoplasmic reticulum spanning between clustered mitochondria. Some parallel cisternae ended with segmentation where they contacted mitochondria, and others terminated by transforming into blebs or continuing into canaliculi. A second feature was comprised of a complex of segmented cisternae and branching canaliculi with clustered mitochondria. Branching minicanaliculi with associated vesicles neighbored the complexes. A fourth entity consisted of synaptic-like vesicles that largely filled the subnuclear cytosol and congregated at synapses. An additional infranuclear structure was composed of slender canaliculi that collected near or streamed to plasmalemma, often next to a synapse. A paradoxical absence of rough endoplasmic reticulum above and Golgi zones below the nucleus provided evidence of atypical mechanisms for generating the membrane in CR and forming synaptic vesicles. The observations offer the view that IHCs are compartmentalized into an apical mechanoreceptor half and a basal half that affects neurotransmission. The apical CR provided a possible structural basis for sequestering the K+ known to influx apically and for directing its diffusion to the site of known efflux across the lateral plasmalemma. The codistribution of parallel cisternae, canalicular-mitochondrial complexes, and synaptic-like vesicles, all of which are unique to IHCs, implicated the cisternae and complexes in the genesis of the vesicles.

Structural and functional impairment of mitochondria in adriamycin-induced cardiomyopathy in mice: suppression of cytochrome c oxidase II gene expression.

The use of adriamycin (ADR) in cancer chemotherapy has been limited due to its cumulative cardiovascular toxicity. Earlier observations that ADR interacts with mitochondrial cytochrome c oxidase (COX) and suppresses its enzyme activity led us to investigate ADR's action on the cardiovascular functions and heart mitochondrial morphology in Balb-c mice i.p. treated with ADR for several weeks. At various times during treatment, the animals were assessed for cardiovascular functions by electrocardiography and for heart tissue damage by electron microscopy. In parallel, total RNA was extracted from samples of dissected heart and analyzed by Northern blot hybridization to determine the steady-state level of three RNA transcripts encoded by the COXII, COXIII, and COXIV genes. Similarly, samples obtained from the liver of the same animals were analyzed for comparative studies. Our results indicated that 1) treatment of mice with ADR caused cardiovascular arrhythmias characterized by bradycardia, extension of ventricular depolarization time (tQRS), and failure of QRS at high concentrations (10-14 mg/kg body weight cumulative dose); 2) the heart mitochondria underwent swelling, fusion, dissolution, and/or disruption of mitochondrial cristae after several weeks of treatment. Such abnormalities were not observed in the mitochondria of liver tissue; and 3) among the three genes of COX enzyme examined, only COXII gene expression was suppressed by ADR treatment, mainly after 8 weeks in both heart and liver. Knowing that heart mitochondria represent almost 40% of heart muscle by weight, we conclude that the deteriorating effects of ADR on cardiovascular function involve mitochondrial structural and functional impairment.

Cytologic evidence for mechanisms of K+ transport and genesis of Hensen bodies and subsurface cisternae in outer hair cells.

UNLABELLED: A system commonly termed the tubulocisternal endoplasmic reticulum (TCER), but designated here the canalicular reticulum (CR), occurs selectively in ion-transporting epithelia, in which it is interpreted as facilitating the transcellular diffusion of ions. Mechanoelectrical transduction in the cochlear outer hair cells (OHCs) depends on the apical influx and the subsequent basolateral efflux of K+. Cytologic structures that possibly mediate K+ transport in gerbil OHCs were investigated here. METHODS: Cochleas were fixed primarily with glutaraldehyde and secondarily with reagents for demonstrating TCER or were fixed with a ferrocyanide-osmium tetroxide solution to preserve intracellular membranes. The distribution of membranous structures retained with these techniques was examined by using electron microscopy. RESULTS: Secondary fixation with osmium tetroxide-ferrocyanide permitted ultrastructural demonstration in OHCs of increased numbers of Hensen bodies and newly detected membranous systems, including CR, linear cisternae, small clusters of cytosolic vesicles and complexes of canaliculi, segmented cisternae, and mitochondria. CR filling an apical stratum beside and below the cuticular plate and contacting laterally the uppermost subsurface cisternae (SSC) was situated to sequester and transport the apical K+ influx that attends the acoustically generated receptor potential and the silent current. The close association of CR with numerous, highly developed Golgi bodies exclusively in the apex of the cell suggested genesis of CR from Golgi cisternae. Nonbranching, linear cisternae occupied a lower cell stratum and spread from CR laterally to a more inferior region of the SSC. Small clusters of vesicles in the central cytosol resembled Hensen bodies in their envelopment by branching canaliculi and segmented cisternae in close association with mitochondria. Viewing the vesicles in Hensen bodies and the small clusters as functioning like most other cytoplasmic vesicles in transport of cell membrane permitted the interpretation that these vesicles move nascent membrane from the canalicular-mitochondrial complex to the SSC. Other small clusters of vesicles contacted the innermost layer of the SSC, often at cisterna-depleted foci in which the vesicles appeared to either replenish the SSC or arise in the course of its turnover. Proximity of multivesicular bodies and lysosomes to small vesicle clusters in foci of depleted SSC implicated the lysosomes in digesting vesicles released from the SCC. Populations of unique, large, lysosome-like bodies and of small, dense bodies in the upper cytosol of OHCs appeared to be involved in different catabolic pathways mediating the turnover ofSSC, CR, and other structures. CONCLUSIONS: Cochlear OHCs contain previously unrecognized membranous organelles that facilitate ion transport and presumably contribute thereby to mechanoelectrical transduction. Vesicles in small clusters and Hensen bodies arise from complexes of canaliculi, cisternae, and mitochondria and contribute membrane to the genesis of the SSC.

Age-related thickening of basement membrane in stria vascularis capillaries.

Ultrastructural examination was undertaken to investigate the pathogenesis of age-related atrophy of the stria vascularis (StV). Basement membrane (BM) thickness was increased in 65-85% of strial capillaries in gerbils aged 33 months or older and often exceeded by several-fold that observed in young controls. In an early stage of thickening the BM expanded slightly around the full capillary profile, after which nodular expansions of BM encircling slender cell processes were often observed at or near one or both poles of the elliptical vessel profile. As widening progressed, the BM consisted of 2-3 layers separated by cell processes in the nodules but fewer strata elsewhere. Association of slender processes of both endothelial cells and pericytes with focal thickening outside the process suggested their participation in genesis of the capillary lesion. In later stages of atrophy, pericytes degenerated and disappeared, while endothelial cells remained intact. Eventually, thick multilayered BM devoid of endothelial cells surrounded a narrow lumen occluded by debris. The age-related change in BM in the inner ear was confined to StV capillaries. Degenerative changes in StV epithelial cells occurred apparently as a secondary consequence of the capillary lesion. The pathologic alterations in marginal cells included extrusion of blebs from the luminal surface, separation and loss of basolateral interfoldings, alteration and depletion of mitochondria and nuclear pyknosis. At the end-stage of degeneration, the StV consisted of a simple or multiple layer of squamous cells lining the scala media.

Immunoglobulin deposition in thickened basement membranes of aging strial capillaries.

The presence of immunoglobulins in the thickened basement membrane (BM) of aging strial capillaries was investigated as a possible indicator of autoimmunity in the genesis of atypical BM. Cochleas from young and old Mongolian gerbils raised in quiet were examined by immunostaining at the light microscopic level for IgG and IgM and for the BM components laminin (La) and type IV collagen (IV-C). Another age-graded series of cochleas was stained for IgG at the ultrastructural level. No immunoreactive IgG was detected in specimens from animals less than 6 months old. In contrast, 2 of 12 cochleas from 20- to 28-month-old gerbils and 11 of 20 cochleas from gerbils 30 months or older showed positive staining for IgG in strial capillary BM. IgM was not detected at any age. At the electron microscope level, no immunoreactive IgG was detected in the stria of cochleas younger than 30 months. However, labeling demonstrative of IgG was observed in the thickened BM of some strial capillaries in all six cochleas from gerbils older than 33 months. Lysosome-like granules in endothelial cells and the superiormost marginal cells also stained for content of IgG as did fibrillar material in edematous regions in the intrastrial space. In addition to showing accumulation of IgG, the findings confirm our prior demonstration of increased La deposition in the thickened strial capillary BM of all cochleas from old gerbils. The BM alterations appear confined to strial capillaries in old gerbils, since morphological observations and immunostaining for La and IgG failed to detect changes in BMs at any other site in a wide survey of aged gerbil organs including vessels in other regions of the affected cochleas. The results point more towards the development of an age-dependent permeability to IgG selectively in strial capillaries than to autoimmunity as an explanation of the IgG in BM.

Increased laminin deposition in capillaries of the stria vascularis of quiet-aged gerbils.

The distribution of laminin (LA) and type IV collagen (IV-C) in the gerbil inner ear was investigated by light and electron microscopic immunohistochemistry. Changes in protein expression were assessed from birth to old age to determine the relation of these constituents to maturation of the cochlea and development of presbyacusis. The distribution of LA paralleled that of IV-C during postnatal development, and both were visualized in the basement membrane (BM) of endothelial, epithelial and spiral ganglion cells in neonatal and young adult gerbils. Immunopositive BM underlying the stria vascularis disappeared at 8-12 days after birth coincident with the development and maturation of the strial capillaries. Immunoreactivity for LA afforded an index to the thickness of the BM and was found to increase with age only in the BM of strial capillaries. At 6 months of age, occasional strial capillaries in the apex of the cochlea showed thickening of the LA-positive BM. Abnormal deposition of LA in strial capillary BM spread to lower turns and increased in prevalence with advancing age, affecting apical and basal more than middle cochlear turns. Thickening of the capillary BM appeared to precede capillary obstruction which eventuated in complete strial atrophy. Staining for IV-C in the walls of the strial capillaries did not increase with age. The data show that LA and IV-C play important roles in postnatal development of the cochlea and that LA deposition increases with age only in the BM of strial capillaries.

Localization of organ of Corti protein II in the adult and developing gerbil cochlea.

The distribution of organ of Corti protein II (OCP-II) was assessed in the developing and mature gerbil cochlea by light and electron microscopic immunohistochemistry. In the adult cochlea, OCP-II was expressed only in certain epithelial cells which included all supporting cells of the organ of Corti, inner and outer sulcus cells and interdental cells. Inner and outer hair cells lacked immunoreactivity. The highest gold particle labeling density was seen overlying intracellular regions devoid of organelles. In the developing inner ear, OCP-II was first detected at 2 days after birth (DAB) with the strongest staining in immature Deiters, inner phalangeal and pillar cells. Immunostaining intensity increased gradually in cells lying laterally and medially to the more centrally located supporting cells and reached adult levels in all reactive cell types around 18 DAB. The results demonstrated conclusively that OCP-II is a cytosolic protein and fail to support its role as a transcription factor postulated on the basis of its homology with p15 or a role in the control of the cycle as suggested by its near-identity with p19Skp1, a cyclin A/CDK2-associated protein. The continued high level of expression in the mature cochlea argues against OCP-II's involvement in regulating the development and differentiation of epithelial cells. The protein's unique distribution and its gradual increase in expression prior to and during the onset and maturation of hearing, however, support its potential function in the recycling of K+ effluxed from hair cells and neurons back to endolymph.

Exocytosis from rat submandibular granular tubules during cyclocytidine stimulation shows unusual features, including changes in the granule membrane.

Sequential secretory changes in granular tubule cells caused by the secretagogue cyclocytidine (75 mg/kg i.p.) were studied at the ultrastructural level, in perfusion (n = 5 animals) and immersion (n = 8 animals) fixed rat submandibular glands, using the periodic acid-thiocarbohydrazide-silver proteinate technique (PA-TCH-SP). The onset of secretion varied from 45 to 75 minutes after administering the cyclocytidine. During the initial stages of overt secretion, structural changes occurred irregularly in a progressive fashion with: (1) an increase in granule membrane staining with PA-TCH-SP and a parallel alignment of the secretory granules with the adjacent apical plasma membrane, which developed a honeycomb-like appearance; (2) docking of these secretory granules to the apical plasma membrane; (3) early secretion of some secretory granules in a semiclassical exocytotic fashion (but this was rarely witnessed). During stages (1) and (2), the cytochemical characteristics of the membrane of the secretory granules, as well as of the plasma membrane, suggest a priming process is occurring. After these initial preparatory phases, further structural changes occurred in the granule membranes with a gradually progressive formation of microvesicles and granule fusions; secretion continued in an explosive manner with proteinaceous material being transferred to lumina in at least three different ways: (1) by typical exocytosis (but it was infrequent); (2) from granules fused intracellularly into aggregates (compound exocytosis); and (3) some apocrine-type of secretion through bleb formation. The formation of these intracellular aggregations was associated with the microvesicles in the granule membranes and some aggregates became very large. Secretion of their contents into lumina occurred through elongated membrane channels. The material secreted included microvesicular forms that had become interiorised in the granular aggregates, and any cytoplasm that may also have been entrapped.

The fate of glycogen in granular tubule cells of rat submandibular glands during secretory events.

Glycogen, studied electron microscopically in granular tubule cells by means of the periodic acid-thiocarbohydrazide-silver proteinase technique, was found to be scattered abundantly throughout the cytoplasm. Parasympathetic nerve stimulation caused no detectable change in the glycogen. Degranulation of the granular tubule cells after either sympathetic nerve stimulation or cyclocytidine injection caused loss of the glycogen from the cells. Study of tubule cells undergoing secretion in the early stages after cyclocytidine injection indicated that glycogenolysis was occurring. Glycogen had reaccumulated in the cells within 24 h, before extensive reformation of the secretory granules had occurred, and remained abundant throughout the subsequent granule formation. It is concluded that glycogen provides an important source of energy during secretory degranulation of the granular tubule cells.