Biomedical subjects

V Grill

Publications and source records attributed to V Grill.

At least 91 records · Page 5Linked to original sources

PubMed1996

Expression and immunohistochemical localization of eight phospholipase C isoforms in adult male mouse cerebellar cortex.

By means of specific polyclonal or monoclonal antibodies we have investigated the expression and the localization of phospholipase C isoforms in the adult mice cerebellar cortex. Western-blot analysis revealed that mouse cerebellum expressed eight phospholipase C isozymes: -beta 1, -beta 2, -beta 3, -beta 4, -gamma 1, -gamma 2, -delta 1, -delta 2. Immunohistochemical analysis carried out on cryosections showed a distinct pattern of expression for each of the isoforms. Purkinje cells had high levels of -beta 1, -beta 3, -gamma 2 and -delta 2 isotypes. The -gamma 2 isozyme was the only one that was identified also in the dendrites of Purkinje cells. In the molecular layer we detected mostly -beta 1 and -gamma 1 isozymes whereas in the granular layer -gamma 1 and -gamma 2 isoforms prodominated. These results indicate a heterogeneity of the phospholipase C isoforms expressed in the layers of mouse cerebellar cortex conceivably due to the fact that these enzymes are coupled to different receptors and perform selective tasks in regulating cell signalling events taking place in the cerebellar cortex of mice.

Amino Acid Sequence↗

PubMed1996

Parathyroid hormone-related protein in hypercalcaemia associated with haematological malignancy.

The incidence of hypercalcaemia and its association with humoral mechanisms involving parathyroid hormone-related protein (PTHrP), parathyroid hormone (PTH), or 1.25(OH)2 vitamin D were assessed in a prospective study of patients admitted to a clinical haematology unit. Hypercalcaemia was detected in 18/165 patients, and was due to primary hyperparathyroidism in 3/17 patients in whom results of humoral mediator assessments were obtained. In the other patients, hypercalcaemia was associated in nine instances with myeloma, in five with B-cell non-Hodgkin's lymphoma (NHL), and in one with myeloid neoplasia. No evidence was obtained of a humoral mechanism involving 1.25(OH)2 vitamin D, but elevated circulating levels of PTHrP, comparable with those in humoral hypercalcaemia of malignancy, were present in 2/4 patients with NHL, and in 3/9 with myeloma. The relationship between presence or absence of elevated circulating PTHrP, and presence or absence of hypercalcaemia during the course of treatment, indicated PTHrP was involved in the production of hypercalcaemia. Such an association raises the possibility that PTHrP released by neoplastic cells in these disorders acts in a paracrine manner to produce local bone resorption, and when produced in greater amounts causes elevated circulating levels which make an additional humorally-mediated contribution to the development of hypercalcaemia.

Adult↗

PubMed1996

A fatty acid-induced decrease in pyruvate dehydrogenase activity is an important determinant of beta-cell dysfunction in the obese diabetic db/db mouse.

We studied the effects of fatty acid oxidation on insulin secretion of db/db mice and underlying molecular mechanisms of these effects. At 2-3 months of age, db/db mice were markedly obese, hyperglycemic, and hyperinsulinemic. Serum free fatty acid (FFA) levels were increased in 2-month-old (1.5 +/- 0.1 vs. 1.1 +/- 0.1 mmol/l, P < 0.05) and 3-month-old (1.9 +/- 0.1 vs. 1.2 +/- 0.1 mmol/l, P < 0.01) mice compared with the age and sex-matched db/+ mice serving as controls. Glucose-induced insulin release from db/db islets was markedly decreased compared with that from db/+ islets and was specifically ameliorated (by 54% in 2-month-old and 38% in 3-month-old mice) by exposure to a carnitine palmitoyltransferase I inhibitor, etomoxir (1 micromol/l). Etomoxir failed to affect the insulin response to alpha-ketoisocaproate. The effect of etomoxir on glucose-induced insulin release was lost after culturing db/db islets in RPMI medium containing 22 mmol/l glucose but no fatty acid. Culture of db/+ islets with 0.125 mmol/l palmitate led to a decrease in glucose-induced insulin secretion, which was partially reversible by etomoxir. Both islet glucose oxidation and the ratio of glucose oxidation to utilization were decreased in db/db islets. Etomoxir significantly enhanced glucose oxidation by 60% and also the ratio of oxidation to glucose utilization (from 27 +/- 2.5 to 37 +/-3.0%, P < 0.05). Pyruvate dehydrogenase (PDH) activity was decreased in islets of db/db mice (75 +/-4.2 vs. 91 +/- 2.9 nU/ng DNA, P < 0.01), whereas PDH kinase activity was increased (rate of PDH inactivation -0.25 +/- 0.02 vs. - 0.11 +/- 0.02/min, P < 0.0 1). These abnormalities were partly but not wholly reversed by a 2-h preexposure to etomoxir. In conclusion, elevated FFA levels in the db/db mouse diminish glucose-induced insulin secretion by a glucose-fatty acid cycle in which fatty acid oxidation inhibits glucose oxidation by decreasing PDH activity and increasing PDH kinase activities.

Aging↗

PubMed1996

Impact of nicotinic acid treatment on insulin secretion and insulin sensitivity in low and high insulin responders.

The aim of the study was to evaluate the effect of nicotinic acid (NA) on glucose tolerance, insulin secretion and sensitivity in relation to perturbations of non-esterified fatty acids (NEFA) and previously characterized insulin responses. Healthy subjects (n = 12) were treated for 14 days with incremental doses of NA reaching 2 g day-1. Before NA and on day 14 a hyperglycaemic clamp (11 mmol l-1) was performed with arginine (5 g i.v.) stimulation before and during the clamp. Fasting serum levels of NEFA were evanescently decreased on day 3 (-38%; p < 0.01) and day 7 (-33%; p < 0.05), but not on day 14 (-14%; NS). NA treatment did not significantly affect levels of fasting blood glucose, insulin, C-peptide, proinsulin or glucagon. NA treatment lowered the amount of infused glucose necessary to achieve clamp levels 48 (8) vs. 61 (10) mumol kg-1 min-1 (p < 0.01). Incremental increases in fasting NEFA levels correlated (r = -0.72) with decreased insulin sensitivity as reflected by M/I ratios (the amount of glucose infused, minus glucosuria, divided by the mean insulin level) (p < 0.01). Insulin and glucagon responses to arginine and glucose were similar before and after NA in subgroups with initially low and high insulin responses to glucose. NA-induced insulin resistance in this study is (a) less than previously reported; (b) not associated with changes in insulin secretory responsiveness, but is (c) influenced by an individually variable NA effect on fasting NEFA levels. Our results do not indicate that NA treatment can be used to test the capacity of B cells to cope with insulin resistance.

Adult↗

PubMed1996

Role of the parasympathetic autonomic nervous system in digitalis intoxication: electrocardiographic and histological study.

The role of the parasympathetic autonomic nervous system (ANS) in digoxin intoxication was investigated in atropine pretreated conscious rabbits. Both the electrocardiographic registration and the histologic analysis indicated possible prevention of lethal arrhythmias by atropine pretreatment. The hearts of animals pretreated with atropine appeared less damaged, since they presented only a light cytoplasmic vacuolization. It was evident that the administration of digoxin without atropine pretreatment provoked dramatic signs of digitalis intoxication followed by animal death. Myocardia of these rabbits were highly damaged. On the other hand, our experimental data indicated that the abolishment of the vagal tone by atropine blockade caused only signs (i.e. inversion of the T wave) due to myocardial ischemia, to which the light cytoplasmic vacuolation could be correlated. Myocardial ischemia could be caused by constriction of coronary blood vessels, which could be consequential to both the prevalent activity of the orthosympathetic ANS, following parasympathetic blockade, and direct digoxin effects on vascular fibrecells, which both produce vasoconstriction. Therefore, it is reasonable to assume that the parasympathetic ANS plays a major role in digitalis intoxication in rabbits.

Animals↗

PubMed1996

Characteristics of insulin release in healthy subjects and in subjects with a glucokinase (Glu 256 Lys) Mutation.

Healthy subjects with previously observed low insulin responses to i.v. glucose have been further characterized. A low insulin response was relatively stable over decades. Adaptability to increased demands for insulin secretion was poor. Hence, dexamethasone treatment for 60 h induced insulin resistance but did not enhance insulin responses to glucose or to arginine. Birth weight was not associated with low insulin responses. Data are compatible with major influences of inherent factors on insulin secretion. Low insulin responses in healthy subjects were compared with responses in glucokinase-deficient subjects. Second phase of glucose-induced insulin secretion and glucose-dependent potentiation of arginine-induced secretion were decreased in both groups. However, the first phase response to glucose was higher in glucokinase-deficient subjects. Hence, decreased first phase insulin secretion is not specifically linked to deficiencies in beta-cell metabolism.

Analysis of Variance↗

PubMed1996

Selective distribution of multiple protein kinase C isoforms in mouse cerebellar cortex.

An immunohistochemical study concerning the distribution of protein kinase C isoforms, a lipid-regulated serine/threonine kinase essential for signal transduction, was performed in mice cerebellar cortex, with particular emphasis on the localization of -iota and -lambda isozymes. By the means of immunoblotting analyses we detected the presence of 11 PKC subspecies in whole cerebellar extracts. Immunoreactivity on cryostat sections revealed, using polyclonal and monoclonal antibodies, that a few isoforms were widely but discretely distributed in all three cortical layers (molecular, granular and Purkinje cells) whereas other isozymes were present in a limited neuronal compartment. Overall, the distribution of several isoforms was in agreement with data obtained by other authors using rat cerebellum. As far as -iota and -lambda isozymes were concerned, we found them abundantly expressed in endothelial cells. Moreover, protein kinase C-lambda was also present in the body of Purkinje cell, conceivably associated with a 200-kDa neurofilament component. In all, these results hint at the possibility that in the cerebellar cortex at least some protein kinase C isoforms are involved in functions other than signal transduction at the synaptic level.

Animals↗

PubMed1995

Metabolic bone diseases.

Metabolic bone diseases often present in old age and some are more easily treatable than others. Osteoporosis is best managed by prevention, with maximisation of peak bone density and reduction of subsequent bone loss. Although hormone replacement therapy is most useful in prevention, it also has a role in established osteoporosis. Other treating agents include calcium, calcitriol, calcitonin and bisphosphonates. Osteomalacia in the elderly mainly results from vitamin D deficiency and supplementation should be considered in those at risk. The newer bisphosphonates show great promise in the treatment of Paget's disease, while surgery remains the only treatment option in primary hyperparathyroidism.

Aged↗

PubMed1995

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Antigens, Nuclear↗

PubMed1995

Distribution of the extended family of protein kinase C isoenzymes in fetal organs of mice: an immunohistochemical study.

Using isoenzyme-specific antisera, we have studied the distribution of protein kinase C isoforms in fetal mouse organs at the developmental age of 17 days. Two different sets of antibodies, produced by different manufacturers, were employed in this study. The specificity of the antisera was tested by immunoblotting experiments using whole fetal mouse extracts. Immunohistochemistry was carried out by means of an alkaline phosphatase-conjugated secondary antibody. Analysis of fetal mouse longitudinal cryostat sections stained with the antibodies demonstrated a distinct distribution of protein kinase C isoforms in the tissues. Protein kinase C-alpha and C-beta I were present in all tissues examined, whereas the C-beta II isoform was absent in the lung and the liver. Protein kinase C-gamma was identified in brain, spinal ganglia, and adrenal gland. The C-epsilon isoenzyme was abundantly expressed in spinal ganglia and in the smooth muscle cells of the bronchial wall. Antisera to C-zeta and C-eta isoforms heavily stained liver, kidney, and spinal ganglia, whereas the C-theta isozyme was mainly detected in brain, stomach and kidney. Thus, protein kinase C-alpha, C-beta I, C-beta II, C-zeta, C-eta and C-theta were the isoforms present in many of the organs investigated. The two sets of antibodies gave slightly different results that might be ascribed to the different epitopes recognized by the antisera. One set of antisera was employed to investigate the distribution of the isoforms in selected organs from an earlier developmental age (15 days) and from adult animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Relationship between insulin responses to D-glucose and to L-arginine in women with a history of gestational diabetes.

The relationship between insulin responses to glucose and to arginine was studied in non-obese women with previous gestational diabetes (PGD). One group, n = 10, had normal glucose tolerance (NGT) by WHO criteria and another, n = 8, had impaired glucose tolerance (IGT). A third group of women without PGD, n = 12, was also studied. A hyperglycaemic clamp (blood glucose level 11 mM) and an arginine stimulation test (150 mg/kg L-arginine followed by 10 mg/kg.min) were performed on separate days. The ratios of arginine to glucose responses 0-10 min differed: they were 1.00 for non-PGD, 1.29 for NGT and 1.46 for IGT (P < 0.02 vs non-PGD). A further difference between groups was the ratio between first- and second-phase glucose-induced insulin secretion, which was significantly decreased in IGT, 0.72, compared with NGT, 0.98 (P < 0.01), and non-PGD, 1.05 (P < 0.005). However, within each group insulin responses 0-10 min to glucose and arginine were strongly correlated: for NGT (r = 0.75, P < 0.05), for IGT (r = 0.85, P < 0.01) and for women without PGD (r = 0.69, P < 0.05). Insulin sensitivity, as assessed by the M/I ratio, was non-significantly decreased in IGT (0.18 +/- 0.03 mg/kg.min per mU/l vs 0.26 +/- 0.03 in NGT and 0.28 +/- 0.03 in non-PGD, P < 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗

PubMed1995

Protein kinase C isoenzymes in mouse harderian gland. Differential expression of the alpha- and epsilon-isoforms during pregnancy. Protein kinase C-OC.

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-alpha, -epsilon, -zeta and -eta. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the alpha- and epsilon-isoforms. The 80-kDa native from of PKC-alpha almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C-epsilon appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant in the Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Mandibular growth rates in human fetal development.

A morphometric analysis of changing proportions in the developing mandible was undertaken in 18 human embryos and fetuses of both sexes (developmental age from 8 to 14 weeks, crown-rump length, CRL, from 34 to 110 mm), previously cleared and stained with a specific method for bone (alizarin red S). Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), superior symphyseal point (SSP), for measuring linear dimensions, i.e. Pcl-GN, Pcl-Pco, Pco-GN, GO-GN, SSP-GN. The gonial (Pcl-GO-GN) and the (Pcl-GN-Pcl) angles were also measured. All linear dimensions were correlated with the CRL by bivariate allometry (1n y = 1n a+b 1n x): they all grew with positive allometry, except GO-GN with isometry. The mandibular ramus grew relatively faster than the body, both in length and height, and the greatest growth rate was found for ramus height. The relation between mandibular shape and the craniofacial structures was investigated using scale drawings obtained from photographs of fetal skulls in lateral view. In the youngest fetuses the mandible was prognathic, then became retrognathic. During the period investigated the zygomatic process and squama of the temporal bone were in a lower and more inclined position in relation to the transverse plane passing through the zygomatic arch than in the newborn and adult. This study identifies parameters fitting changing trends in height, length and shape of the human mandible during the prenatal period (8-14 weeks); moreover, it emphasizes that the mandibular growth patterns differ significantly from those of successive development periods.

Adult↗

PubMed1995

Hypercalcaemia.

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Diagnosis, Differential↗

PubMed1995

Use of R-beta-[1-11C]hydroxybutyrate in PET studies of regional cerebral uptake of ketone bodies in humans.

A method for determining regional cerebral utilization of ketone bodies in humans is described. After a bolus injection of R-beta-[1-11C]hydroxybutyrate, the time course of the tracer in the brain was measured with positron emission tomography in five healthy volunteers. The regional cerebral blood flow was measured separately. The tracer uptake in the brain could be well described by a single rate constant, indicating that the concentration of unmetabolized ketone bodies in the brain is very low and that transport across the blood-brain barrier is the rate-limiting step. At an average plasma concentration of beta-hydroxybutyrate of 0.043 mumol/ml, the utilization rate was estimated to be 0.48 nmol.ml-1.min-1. In accordance with previous animal studies, the utilization rate was found to increase almost linearly with increasing plasma concentration of beta-hydroxybutyrate. Furthermore, the utilization was higher in gray than in white matter. Finally, the ratio between the utilization in the basal ganglia and the brain as a whole was lower for ketone bodies than for glucose.

3-Hydroxybutyric Acid↗

PubMed1995

Deficiency of pyruvate dehydrogenase activity in pancreatic islets of diabetic GK rats.

We investigated the role of islet pyruvate dehydrogenase (PDH) enzyme activity and fatty acid oxidation in the impaired insulin secretion in spontaneously diabetic GK rats. Blood glucose levels were elevated in 2- to 3-month-old GK rats (8.7 +/- 0.5 vs. 6.5 +/- 0.3 mM in control Wistar rats; P < 0.01), whereas serum insulin levels were comparable to those in control rats. Insulin and DNA contents were similar in freshly isolated islets from GK and control rats, whereas insulin responses to 27 mM glucose from GK islets were reduced by 52%. The effect of acetate or pyruvate on insulin responses evoked by succinate monomethylester (SAM) were compared to indirectly assess deficient generation of acetyl-coenzyme A from pyruvate. Acetate potentiated SAM-induced insulin secretion similarly in GK and control islets, whereas 10 mM pyruvate (which supplies acetyl-coenzyme A through PDH enzyme activity) failed to normally potentiate insulin secretion in GK islets (92% of SAM-induced response in GK vs. 154% in control islets). The PDH activity (active form) was decreased in GK islets by 35% (P < 0.001). The proportion of active form PDH to total PDH activity was reduced in GK islets (56% vs. 71% in control islets; P < 0.01). The activity of PDH kinase (which inactivates PDH by phosphorylation) was increased in GK islets, the rate of ATP-dependent inactivation of PDH was -0.29 +/- 0.02 vs. -0.19 +/- 0.02/min in control islets (P < 0.05). Culturing GK islets for 48 h at 5.5 mM glucose failed to correct the impaired insulin response to glucose and the decreased PDH activity. Serum FFA levels and islet triglyceride contents did not differ between GK and control rats. Etomoxir (1.0 and 10 microM), a carnitine palmitoyl transferase I inhibitor, failed to enhance glucose-induced insulin release in GK islets. The following conclusions were reached: 1) a kinase-mediated decrease in PDH activity in islets of GK rats may in part account for the decreased ratio of oxidized to utilized glucose and impaired insulin release in these islets; and 2) impaired insulin release in the GK rats is not linked to an inhibitory influence of islet fatty acid oxidation.

Acetates↗

PubMed1995

Long term exposure to fatty acids and ketones inhibits B-cell functions in human pancreatic islets of Langerhans.

We previously demonstrated in the rat that long term exposure to fatty acids inhibits B-cell function in vivo and in vitro. To further assess the clinical significance of these findings, we tested in human islets the effects of fatty acids on glucose-induced insulin release and biosynthesis and on pyruvate dehydrogenase (PDH) activity. Human islets were obtained from the beta-Cell Transplant Unit (Brussels, Belgium). Exposure to 0.125 mmol/L palmitate or oleate for 48 h during tissue culture (RPMI-1640 and 5.5 mmol/L glucose) inhibited the postculture insulin response to 27 mmol/L glucose by 40% and 42% (P < 0.01 for difference). Inhibition was partly prevented by coculture with 1 mumol/L etomoxir, a carnitine-palmitoyl-transferase-I inhibitor (P < 0.05 for effect of etomoxir). Inhibitory effects on glucose-induced insulin secretion by previous palmitate were additive to the inhibitory effects exerted by previous high glucose (11 and 27 mmol/L). Palmitate-induced inhibition of insulin secretion was evident after exposure to 25 mumol/L added fatty acid. The insulin content of islets exposed to fatty acids was significantly reduced, and glucose-induced proinsulin biosynthesis was inhibited by 59% after palmitate addition and by 51% after oleate exposure (P < 0.01). These effects were partly prevented by etomoxir (P < 0.05). The activity of PDH in mitochondrial extracts of islets preexposed for 48 h to palmitate was decreased by 35% (P < 0.05) vs. that in control islets, whereas the activity of PDH kinase (which inactivates PDH) was significantly increased in the same preparations (P < 0.05). The effects of ketones were tested by 48-h exposure to beta-hydroxybutyrate (beta-D-OHB). Ten millimoles of D-beta-OHB per L inhibited the subsequently tested insulin response to 27 mmol/L glucose by 56% (P < 0.001). Half-maximal inhibitory effects of D-beta-OHB on insulin secretion and insulin content were seen at concentrations between 0.5-2.5 mmol/L. Inhibition by D-beta-OHB was partially reversed by etomoxir, whereas exposure to D-beta-OHB failed to affect PDH and PDH kinase activities. We conclude that fatty acids as well as ketone bodies diminish B-cell responsiveness to glucose in human islets by way of a glucose-fatty acid cycle. Increased plasma concentrations of fatty acids and ketones are likely to be important factors behind the negative influences on B-cell function exerted by a diabetic state in both type 1 and type 2 diabetes.

3-Hydroxybutyric Acid↗

PubMed1995

A simple and rapid staining technique for plastic embedded cartilage and bone.

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semithin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na2 CO3, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.

Animals↗