Sumoylation of eIF4A2 affects stress granule formation.

Regulation of protein synthesis is crucial for cells to maintain viability and to prevent unscheduled proliferation that could lead to tumorigenesis. Exposure to stress results in stalling of translation, with many translation initiation factors, ribosomal subunits and mRNAs being sequestered into stress granules or P bodies. This allows the re-programming of the translation machinery. Many aspects of translation are regulated by post-translational modification. Several proteomic screens have identified translation initiation factors as targets for sumoylation, although in many cases the role of this modification has not been determined. We show here that eIF4A2 is modified by SUMO, with sumoylation occurring on a single residue (K226). We demonstrate that sumoylation of eIF4A2 is modestly increased in response to arsenite and ionising radiation, but decreases in response to heat shock or hippuristanol. In arsenite-treated cells, but not in hippuristanol-treated cells, eIF4A2 is recruited to stress granules, suggesting sumoylation of eIF4A2 correlates with its recruitment to stress granules. Furthermore, we demonstrate that the inability to sumoylate eIF4A2 results in impaired stress granule formation, indicating a new role for sumoylation in the stress response.

[PROPERTIES OF SACCHAROMYCES CEREVISIAE VOLUTIN GRANULES UNDER CONDITIONS OF THE CHANGE OF SPACE WEATHER].

In this work, the impact of space weather on the staining and the motility of volutin granules ("dancing bodies") in vacuoles of yeast Saccharoniyces cerevisiae with normal and changed phosphoric metabolism was studied. It was showed that the installed ear- lier relation between metachromatic reaction of volutin granules and galactic cosmic rays was confirmed by DAPI-staining of inorganic polyphosphates in cells S. cerevisiae UCM Y-517. The inverse correlation of efficiency of DAPI-staining of volutin granules with some solar activity indexes was observed. During the period of study, the rhythmicity of motile volutin granules in the studied cultures (S. cerevisiae UCM Y-517, S. cerevisiae CRY, S. cerevisiae CNX) was marked. The strains S. cerevisiae UCM Y-517 and CRY, both with unmodified phosphoric metabolism, showed the same rhythmicity of mobile volutin granules. However, this rhythmicity in the strain CNX, which cannot synthesize exopolyphosphatases PPX1 and PPN1 (CF 3.6.1.11), was changed. The volutin granule motion coincided with the rhythmicity of galactic cosmic rays within the period of 9 days. It can be suggested that "dancing bodies" are synchronized with rhythms of galactic cosmic rays. Observed differences between mutant and parental strains may indirectly indicate participation of polyphosphates in the reaction of cells on the changes of space weather pa- rameters. Thus, correlating variability of staining and motion of polyphosphate-containing volutin granules under action of cosmophysical factors may be an evidence of possible key role of phosphoric metabolism in sensitivity of yeast cells to space weather changes.

Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na(+) currents through intracellular Ca(2+) release.

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa ) densities by 62.5%. MT (5 µM) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca(2+) level, but it significantly elevated the intracellular Ca(2+) level evoked by the high K(+) stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca(2+) influx-induced Ca(2+) release.

Effects of adult-generated granule cells on coordinated network activity in the dentate gyrus.

Throughout the adult life of most mammals, new neurons are continuously generated in the dentate gyrus of the hippocampal formation. Recent work has documented specific cognitive deficits after elimination of adult hippocampal neurogenesis in rodents, suggesting that these neurons may contribute to information processing in hippocampal circuits. Young adult-born neurons exhibit enhanced excitability and have altered capacity for synaptic plasticity in hippocampal slice preparations in vitro. Still, little is known about the effect of adult-born granule cells on hippocampal activity in vivo. To assess the impact of these new neurons on neural circuits in the dentate, we recorded perforant-path evoked responses and spontaneous network activity from the dentate gyrus of urethane-anesthetized mice whose hippocampus had been focally X-irradiated to eliminate the population of young adult-born granule cells. After X-irradiation, perforant-path responses were reduced in magnitude. In contrast, there was a marked increase in the amplitude of spontaneous γ-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult-generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units.

Activity-dependent expression of RNA binding protein HuD and its association with mRNAs in neurons.

The dendritic trafficking of RNA binding proteins (RBPs) is an important posttranscriptional process involved in the regulation of synaptic plasticity. For example, HuD RBP binds to AU-rich elements (AREs) in the 3' untranslated regions (3'UTR) of immediate-early gene (IEG) transcripts, whose protein products directly affect synaptic plasticity. However, the subcellular localization of HuD RBPs and associated mRNAs has not been investigated following neuronal stimulation. Immunofluorescence analysis revealed activity-dependent dendritic localization of HuD RBPs following KCl stimulation in hippocampal neurons, while immunoprecipitation demonstrated the association of HuD RBP with neuronal mRNAs encoding neuritin, Homer1a, GAP-43, Neuroligins, Verge and CAMKIIalpha. Activity-dependent expression of HuD involves activation of NMDAR as NMDA receptor 1 knockout mice (Nr1(neo-/-)) exhibited decreased expression of HuD. Moreover, translational regulation of HuD-associated transcripts was suggested by its co-localization with poly-A-binding protein (PABP) as well as the cap-binding protein (eIF4E). We propose that post-transcriptional regulation of neuronal mRNAs by HuD RBPs mediates protein synthesis-dependent changes in synaptic plasticity.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

UVA1 radiation (340-400 nm) interferes with the perforin-granule system of CD8hi+ cytotoxic T lymphocytes in vitro.

UVA1-irradiation was introduced as an innovative and effective phototherapy of atopic dermatitis (AD) and other skin diseases. In AD, a defect of a central apoptosis inducing effector system involved in immunoregulation and immune defense, i.e., the system of perforin-granules in cytotoxic T lymphocytes (CTL), was recently reported: perforin-reduction and perforin-hyperreleasability. We now investigated UVA1-effects on the perforin-granule system in vitro. Peripheral blood CTLs were exposed in vitro to 10-100 J/cm2 UVA1 (340-400 nm), and to 30-150 mJ/cm2 UVB (280-315 nm) as a control. A time-dependent perforin-granule release was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin. This release was inhibited dose-dependently by UVA1, but not by UVB. An UVA1-dose dependent pattern of sensitive (80%) and insensitive (20%) individuals was found. The kinetics of perforin release in AD-CTLs, i.e. hyperreleasability, was normalized by 50 J/cm2 UVA1 in vitro. Sodium azide as a quencher of reactive oxygen species prevented the UVA1-mediated inhibition of perforin-granule release. Our data demonstrate for the first time a dose- and wavelength-dependent UVA1-effect in vitro on a major effector system of cytotoxic lymphocytes, the system of perforin-granules. This might contribute to the further understanding of immunomodulatory UVA1-effects in vivo.

Photoprotective role of epidermal melanin granules against ultraviolet damage and DNA repair in guinea pig skin.

We previously developed a quantitative autoradiographic technique with special forceps for measuring unscheduled DNA synthesis (UDS) in mouse skin after treatment with ultraviolet light in vivo. By this method, we investigated the relationship between the protective role of melanin and UV-induced DNA repair in black-and-white guinea pigs. Flat areas containing a sharp border between pigmented and unpigmented skin were selected. The skin of the selected areas was shaved and irradiated with short-wave UV (254 nm) or UV-AB (270 to 440 nm, emission peak at 312 nm) at various doses. Immediately after irradiation, the skin was clamped off with forceps, and an isotonic aqueous solution of [methyl-3H]thymidine was injected s.c. into the clamped off portion. UDS was clearly demonstrated as silver grains in this portion of the skin after irradiation with 254 nm UV or UV-AB. Errors due to individual differences were avoided by comparing the intensities of UDS in basal cells from pigmented skin and unpigmented skin of the same animals. Unexpectedly, in groups of animals treated with 254 nm UV or UV-AB, no difference in UDS in pigmented and unpigmented skin was seen at any UV dose. These results suggested that epidermal melanin granules do not significantly protect DNA of basal cells against 254 nm UV or UV-AB irradiation. Results of a study on the effect of the wavelength of irradiation on the UDS response of albino guinea pigs are also reported.

Intramedullary megakaryocytes internalize released platelet factor 4 and store it in alpha granules.

BACKGROUND: Megakaryocytes express and store platelet factor 4 (PF4) in alpha granules. In vivo, PF4 is a clinically relevant, negative regulator of megakaryopoiesis and hematopoietic stem cell replication. These findings would suggest a regulated source of free intramedullary PF4. OBJECTIVES: Define the source of free intramedullary PF4 and its intramedullary life cycle. METHODS: We interrogated both murine and human bone marrow-derived cells during megakaryopoiesis in vitro by using confocal microscopy and enzyme-linked immunosorbent assay. With immunohistochemistry, we examined in vivo free PF4 in murine bone marrow before and after radiation injury and in the setting of megakaryocytopenia and thrombocytopenia. RESULTS: Exogenously added human PF4 is internalized by murine megakaryocytes. Human megakaryocytes similarly take up murine PF4 but not the related chemokine, platelet basic protein. Confocal microscopy shows that internalized PF4 colocalizes with endogenous PF4 in alpha granules and is available for release on thrombin stimulation. Immunohistochemistry shows free PF4 in the marrow, but not another alphagranule protein, von Willebrand factor. Free PF4 increases with radiation injury and decreases with megakaryocytopenia. Consistent with the known role of low-density lipoprotein receptor-related protein 1 in the negative paracrine effect of PF4 on megakaryopoiesis, PF4 internalization is at least partially low-density lipoprotein receptor-related protein 1 dependent. CONCLUSIONS: PF4 has a complex intramedullary life cycle with important implications in megakaryopoiesis and hematopoietic stem cell replication not seen with other tested alpha granule proteins.

Effect of 8-methoxypsoralen plus long-wave ultraviolet (PUVA) radiation on mast cells. II. In vitro PUVA inhibits degranulation of rat peritoneal mast cells induced by compound 48/80.

Rat peritoneal mast cells incubated with a histamine liberator, compound 48/80, showed a significantly reduced capacity for releasing histamine following in vitro treatment with 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 1-5 J/cm2 of long-wave ultraviolet (UVA) irradiation (PUVA). No remarkable inhibition in histamine release was observed in the cells treated with 8-MOP only. Irradiation with 5 J/cm2 of UVA alone exerted an inhibitory effect on histamine release, to a lesser extent than PUVA. PUVA irradiation did not bring any decrease in cell viability or any spontaneous release of histamine from irradiated cells as shown by phase-contrast microscopy and by histamine assay, respectively. These results suggest that PUVA treatment may cause a noncytotoxic disturbance at mast cell membranes or on surface receptors, leading to a decreased capacity for secreting chemical mediators.

Protection against irradiation-induced damage to salivary glands by adrenergic agonist administration.

PURPOSE: Irradiation [IR]-induced damage to major salivary glands is an entity first described at the beginning of our century, yet its underlying mechanism is still enigmatic. Exposure of the salivary glands to IR is often inevitable when delivering radiotherapy for malignancies of the head and neck region. Frequently, this results in rapidly developing, life-long severe xerostomia for which no adequate prevention or treatment is available. The purpose of this study was to examine the role of secretion granules in serous cells of the parotid (P) and submandibular (SM) glands as mediators in the IR-induced salivary damage. Functional parameters (flow rate and gland weight), and total body weight were examined at both early term (4 days) and extended term (2 months) post-IR in male Wistar rats exposed to 15 Gy of head and neck irradiation following stimulation for granule secretion (degranulation). METHODS AND MATERIALS: At 4 days, it was demonstrated that IR reduced P flow rate, P gland weight, total body weight, and submandibular/sublingual gland weight by 89, 33, 30, and 32% (p < 0.01), respectively, while SM flow rate was not altered significantly. At 2 months, these parameters were reduced by 59, 37, 31, and 37%, respectively, and the SM flow rate was reduced by 39% (p < 0.01). RESULTS: Pilocarpine, a muscarinsic agonist which, albeit its efficacy as a salivary watery secretion stimulator, causes only limited degranulation, did not protect significantly any of the reduced parameters at either term. In contrast, cyclocytidine, an adrenergic agonist that is a very potent salivary degranulating agent, protected the P against the weight loss at 4 days and 2 months, and against the flow rate reduction at 2 months. The P weight and flow rate were protected to the extent that their values were not significantly different than those of the nonirradiated controls. Cyclocytidine also partially protected against the body weight reduction at 2 months. Our results emphasize the importance of secretion granules as mediatory agents in IR-induced P damage, and more so at the extended term. The demonstrated protective role of adrenergic agonists against IR damage to the P may be of importance in the clinical setting.

A photoreactive fluorescent marker for identifying eosinophils and their cytoplasmic granules in tissues.

Here we describe a simple histochemical technique that provides an improved approach to identifying eosinophil components in tissues through the formation of photoreactive complexes that produce stable fluorescent emissions. This method worked readily with histological tissue sections 6-60 microm thick, which were fixed in neutral buffered formalin (NBF), and with cell suspensions similarly fixed and unfixed. Deep red (>605 nm) fluorescent emissions were produced by eosinophil-specific granules when exposed to broadband excitation spectra from a 100-W mercury lamp source (510-590 nm), as well as single-wavelength excitations from both an argon laser (488 nm) and a UV-visible laser (514 nm). The fluorophore-granule complex emissions increased in intensity during the first minute of continuous photoexcitation, then remained stable (>10 min). All nonspecific autofluorescence phenomena associated with these tissues were photobleached in the first minute, including areas of background Biebrich scarlet binding where photoreactive complexes were not formed (i.e., collagen), indicating environmental influences on the fluorophore. This technique allows the visualization of eosinophil granules over a greater period of time than is usually permissible with standard fluorescent markers. Therefore, techniques such as confocal microscopy can be utilized to their fullest extent, providing much more detailed information on the location and distribution of the cytoplasmic contents of eosinophils.

The role of secretory granules in the radiosensitivity of rat salivary gland acini--a morphological study.

The aim of this study was to investigate the radiosensitivity of salivary gland tissue pretreated with isoproterenol to establish a status of depletion of secretory granules in acinar cells at the time of irradiation. Nuclear aberrations and cell lysis were taken as parameters for cell death. Local X irradiation with a single dose of 15 Gy induced comparable early morphological changes in the rat parotid and submandibular glands. During the first day after irradiation, the most obvious changes were degranulation of serous cells and induction of nuclear aberrations in both the secretory (serous as well as mucous) and intercalated duct compartment. Subsequently, progressive lysis occurred in secretory units but not in intercalated and striated ducts. Recovery of tissue integrity was observed from day 6. Early radiation-induced cell death was not reduced by isoproterenol-induced degranulation of acinar cells before irradiation. Subsequent recovery from radiation damage seemed to occur earlier in parotid glands but not in submandibular glands pretreated with isoproterenol. From the present study it is concluded that the radiosensitivity of serous salivary gland acini is not dependent on the presence of secretory granules at the time of irradiation. There was some evidence for a faster recovery from radiation damage observed after pretreatment with isoproterenol which may be the result of drug-induced stimulation of cell proliferation.

Irradiation-induced damage to the salivary glands: the role of redox-active iron and copper.

The mechanism of irradiation-induced hypofunction of the salivary glands is a process that is not fully understood. Here we examine the hypothesis that intracellular and redox-active ions of iron and copper, which are associated with the secretion granules, play a catalytic role in the irradiation-induced damage. Rats were subjected to head and neck irradiation (15 Gy X rays) and allowed to recover for 2 months. The function of the parotid and submandibular glands was then determined by pilocarpine-stimulated salivary secretion. A 45% decrease in the function of both glands was obtained when compared to nonirradiated controls. Treatment prior to irradiation (90 min) with cyclocytidine (200 mg/kg) led to a massive degranulation of the parotid gland and yielded nearly complete protection from radiation-induced damage. In contrast, pilocarpine stimulation prior to irradiation led to a marginal degranulation of the parotid gland and yielded only 13% protection. Neither agent caused degranulation of the submandibular gland mucous cells or yielded functional protection of this gland. Treatment with both agents yielded a marked increase in iron, copper and manganese levels in the parotid gland saliva. An analogous marked increase in the redox activity of iron and copper ions was recorded for the parotid saliva stimulated by pilocarpine and cyclocytidine. Pilocarpine-stimulated submandibular gland saliva contained metal levels similar to those of the parotid gland saliva. However, no redox activity and no increase in metal mobilization could be demonstrated in the submandibular gland saliva stimulated by both agents. The correlation between the patterns of gland degranulation, mobilization of redoxactive metals and the protection of gland function, for both parotid and submandibular glands, focuses attention on the catalytic roles played by transition metal ions in promoting free radical reactions, which likely participate in the process of injury to the tissue.

Sialogogue-related radioprotection of salivary gland function: the degranulation concept revisited.

To investigate whether secretory granules play a role in the radiosensitivity of the salivary glands of rats, parotid acinar cells, submandibular acinar cells and/or submandibular granular convoluted tubule (GCT) cells were degranulated prior to irradiation. Degranulation of GCT cells was obtained by pretreatment with phenylephrine (5 mg/kg, t = -60 min) and methacholine (3.75 mg/kg, t = -120 min). Degranulation of acinar cells was attained by pretreatment with isoproterenol (5 mg/kg, t = -90 min). Combinations of pretreatments were also tested. Irradiation was performed with a single dose of 15 Gy of X rays. Samples of parotid and submandibular/sublingual saliva were collected 4 days prior to and 1, 3, 6, 10 and 30 days after irradiation. Pretreatment with phenylephrine, isoproterenol and methacholine plus phenylephrine resulted in less radiation damage to parotid gland function as indicated by the lag phase and flow rate. Since the pretreatment with phenylephrine and phenylephrine plus methacholine did not degranulate parotid gland acinar cells, the observed protective effect on this gland cannot be explained by the "degranulation concept." Furthermore, salivary gland function was significantly greater 3 days after irradiation as a result of pretreatment with phenylephrine and phenylephrine plus methacholine compared to rats given only radiation. This may indicate recovery from damage rather than a reduced amount of initial damage. The sparing was most obvious for the later effects (6-30 days). Submandibular/sublingual gland function was improved significantly after pretreatment with methacholine plus phenylephrine, although no increase in degranulation of GCT cells was observed compared to pretreatment with phenylephrine alone, again not favoring the degranulation concept. The results indicate that the secretory granules do not play the often-assumed important role in the radiosensitivity of the salivary gland. The mechanism underlying the observed improvement of salivary gland function may involve second messenger-induced increases in proliferation of salivary gland cells resulting in recovery of tissue after the irradiation.

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: evoked release of glutamate, GABA, aspartate and glutamate decarboxylase activity in control and degranulated rat hippocampus.

Using discontinuous density gradient centrifugation in isotonic Percoll sucrose, we have characterized two subcellular fractions (PII and PIII) enriched in mossy fiber synaptosomes and two others (SII and SIII) enriched in small synaptosomes. These synaptosomal fractions were compared with those obtained from adult hippocampus irradiated at neonatal stage to destroy granule cells and their mossy fibers. Synaptosomes were viable as judged by their ability to release aspartate, glutamate and GABA upon K+ depolarization. After irradiation, compared to the control values, the release of glutamate and GABA was decreased by 57 and 74% in the PIII fraction, but not in the other fractions and the content of glutamate, aspartate and GABA was also decreased in PIII fraction by 62, 44 and 52% respectively. These results suggest that mossy fiber (MF) synaptosomes contain and release glutamate and GABA. Measurement of the GABA synthesizing enzyme, glutamate decarboxylase, exhibited no significant difference after irradiation, suggesting that GABA is not synthesized by this enzyme in mossy fibers.

Acute and protracted radiation effects on small intestinal morphological parameters.

PURPOSE: To compare the responses of small intestinal morphological parameters after acute and protracted doses of radiation. MATERIALS AND METHODS: Male C57BL6 mice were examined 6, 24 and 72 h after whole body gamma-irradiation, given either as an acute 5 Gy dose, or as a protracted (continuous) dose of 20 cGy per day for 25 days to a total dose of 5 Gy. Many different structural parameters at both the light microscopical and ultrastructural levels were assessed quantitatively. RESULTS: At different time points following both schedules there were changes in the number of villous enterocytes, goblet cells, lamina propria cells and mitotic figures. Ultrastructural changes occurred in the epithelium. Many of the parameters that showed changes following the protracted schedule appeared to be returning to normal within 3 days of the cessation of radiation, a finding which was in contrast with the acute dose. The protracted schedule produced increases in the number of Paneth cells and in the length of enterocyte microvilli. CONCLUSIONS: Many of the responses that occurred after the protracted schedule suggest that adaptive mechanisms may be being triggered following persistent exposure to radiation.

Radiation induced cell loss in rat submandibular gland and its relation to gland function.

PURPOSE: To understand early and late radiation-induced loss of function of the submandibular gland, changes in cell number were documented and correlated with data on gland function. Modulation of the radiation effect by sialogogues was used to investigate possible mechanisms of action. MATERIALS AND METHODS: Rats were irradiated with a single dose of 15 Gy of X-rays after pre-treatment with either saline, the muscarinic receptor agonists methacholine or pilocarpine, the adrenergic receptor agonist phenylephrine or methacholine plus phenylephrine. Before and 1-240 days after irradiation, submandibular saliva flow rate was measured. At the same time points and from comparable animals submandibular glands were carefully extirpated, weighed and prepared for light microscopic examination. RESULTS: Soon after irradiation (<30 days) no significant loss of cells was observed, whereas the gland function was severely compromised. Sialogogue pre-treatment attenuated the radiation-induced loss of gland function. At later intervals a considerable loss of acinar cells and to a lesser extent loss of granular convoluted tubule cells were observed. Gland function subsequently declined slowly. Pre-treatment with sialogogues gave transient protection against cell loss and loss of gland function. CONCLUSIONS: The lack of cell loss observed soon after irradiation indicates that the observed reduction in gland function was caused by a compromised functioning of the acini. The later loss of cells is probably due to death of cells that normally proliferate, leading to a further reduced secretory capacity. Protection of gland morphology and function by sialogogues at later times must therefore involve resistance of progenitor cells to radiation-induced cell death.

Effect of 24 hours light on circadian rhythms of secretory enzymes and morphology of rat von Ebner's glands.

Von Ebner's glands of the rat are minor salivary serous glands in the posterior portion of the tongue. They secrete two digestive enzymes, lingual lipase and amylase. In this investigation, circadian rhythm in feeding was established under a normal 12 h light/12 h dark cycle, with the rats eating primarily during the dark period. At lights on, the size of the acinar cells and the area of the inclusive secretory granules, and the amount of digestive enzyme activity (lingual lipase and amylase) remaining in the gland was significantly less than in the mid-afternoon, after very little daylight food consumption. However, after 7 days of continuous light the circadian rhythm was altered: the food consumption during the normal night-time hours (5 p.m. to 8 a.m.) went from 88% of total 24 h food consumption to 45%, and during normal daylight hours (8 a.m. to 5 p.m.) from 12% to 55%. These changes were correlated with histometric findings of a near reversal of the areas of acinar cells and secretory granules of a.m. and p.m. samples under continuous light. Lingual lipase activity in the glands went from 35% under 12 h light to 61% under continuous light in the a.m. and from 65% to 39% in the p.m. Amylase activity also showed nearly a reversal in activity remaining in the gland, from 36% at 12 h light to 58% at 24 h light in the a.m. and 64% to 41% for the p.m. samples. These results indicate that the von Ebner's glands of the rat have a circadian rhythm of secretion and storage of secretory proteins that is subject to light entrainment similar to that seen in other exocrine glands such as the parotid and pancreas.

Irradiation of rabbit submandibular glands. Histology and morphometry after 15 Gy.

Irradiation of rabbit submandibular glands with a single absorbed dose of 15 Gy caused tissue damage which persisted 4 and 10 months later. The injuries were studied by histology, measurement of gland weights and histomorphometry. Reduction of gland weight due to hypoplasia and missed weight gain were seen and the proportions of the three major intralobular compartments of the glands were altered being dependent on the interval between radiation exposure and observation time. The size of the lobules was reduced and extralobular fibrosis in the hilar region increased. The seromucous acini were partly atrophied, with successively changed architecture and reduced size of the granules. The serous tubules showed pronounced reduction of the granules at 4 months and a remarkable adenomatous regeneration at 10 months post irradiation. The striated ducts were almost unaffected during the observation time. Arteriolar changes were slight to moderate, and there was scarcely any capillary damage. The numbers of intra- and extralobular plasma cells were increased.