The ultrastructural basis for the identification of cell types in the pancreatic islets

Summary The pancreatic islets of rabbit, dog and opossum have been studied by light and electron microscopy. Silver-positive cells in the rabbit are predominantly sandwiched between the peripheral A and central B cells, and by electron microscopy are identified as D cells. Pancreatic islets in the tail of the dog pancreas have A, B, and D (silver-positive) cells, but the islets in the uncinate process of the dog pancreas lack phosphotungstic acid hematoxylin-positive A cells. By electron microscopy the characteristic D cells are found in both tail and uncinate process, but A cells are confined to the tail islets, confirming the identification of cell types. A unique cell type termed the F cell is found in the dog uncinate islets and it is characterized by secretory granules of angular profiles. In the opossum, the A cells contain considerable amounts of glycogen demonstrable by both light and electron microscopy. A unique cell type is also present in the opossum islets termed an E cell (Thomas, 1937), which has large secretory granules (400–500 m). The physiological implications of a multiplicity of cell types in pancreatic islets is discussed.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases. The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donahue.     

Salinity-dependent copy number change of endogenous plasmids in Synechococcus sp. strain PCC 7002

Marine cyanobacterium Synechococcus sp. strain. PCC 7002 has at least six endogenous plasmids. When it was cultured in 1 m NaCl medium, the copy numbers of the smallest (4.5 kb) and the second smallest (9.7 kb) plasmids decreased to one-third and one-tenth of those in control culture (0.3 m NaCl) respectively. In medium without NaCl, the copy numbers of those plasmids also decreased but the changes were much smaller. On the other hand, copy numbers of 15.4-kb, 30.0-kb, and 36.9-kb plasmids did not change among the three different NaCl concentrations (0 m, 0.3 m, 1 m). The copy number changes of the two plasmids were reversible. A similar copy number change was also observed in medium with 0.3 m NaCl+0.7 m KCl. These observations suggest that copy number controls are different among endogenous plasmids, and some of them are affected by salinity in the medium.     

Cytophotometric studies on the DNA content in diploid and polyploid enchytraeidae (Oligochaeta)

The amount of DNA in nerve nuclei was determined by Feulgen cytophotometry in 45 species and cytotypes. The DNA value of spermatids, spermatogonial nuclei and nerve nuclei in newly hatched worms was determined in some species. The total variation in DNA content ranges from a relative value of 0.40 to 4.85. The DNA content in diploid species ranges from 0.40 to 1.12. In two genera (Lumbricillus and Enchytraeus) wide diversification in chromosome numbers at the diploid level is accompanied by wide variation in DNA values, whereas other genera and species groups (Mesenchytraeus, Henlea and Marionina), which are conservative in this respect, are closer in DNA values. In the genus Fridericia constancy in chromosome number is associated with wide variation in DNA values, this may be due to an increase in DNA taking place in old individuals. In 9 out of 10 cases nearly identical ratios are found between chromosome numbers and DNA values in polyploids and related diploids or lower polyploids. Differential polyteny is recorded in two cases.Dedicated to Professor Dr. J. Seiler on the occasion of his eightieth birthday.     

Kinematographische Dokumentation einer amitotischen Zellteilung

Summary Identification of argyrophilic cells, in pancreatic islets of normal rabbits, is accomplished by light and electron microscopy in osmium-fixed plastic-embedded tissues.Fixative, pretreatment and pH of silver nitrate solution were essential for the light microscopic study to reveal argyrophilic cells in osmium-fixed plastic-embedded pancreatic islet tissue. The best result was obtained with Dalton's osmium fixation and buffered silver nitrate methanamine solution at pH 9.O. The cytoplasmic granules of argyrophilic cells generally are densely packed but some of the cells show only sparse silver impregnated granules in the cytoplasm. Occasionally there are some non-argyrophilic granular cells in which, after silver impregnation, the cytoplasm appears clear. There are three kinds of cells in the pancreatic islets, i.e., argyrophilic granular cells, non-argyrophilic granular (clear) cells, and beta cells (situated centrally in the islet and stained light yellow in silver impregnated sections).The cells known as argyrophilic cells in light microscopy can be identified as alpha cells in electron micrographs by comparison of consecutive sections of the same cell.The author would like to express his appreciation to professor Roy C. Swan for his generous guidance.     

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.     

Effects of forced migrations on spatial and temporal changes in genetic structure of two local populations in Bosnia

Ten qualitative traits were observed in two isolated human populations in Bosnia for prewar and postwar periods. Due to recent war in Bosnia and Herzegovina two million citizens was forced to migrate. Dramatic migration effects occurred in the investigated populations. Population characteristics found were geographic isolation, high levels of marital distance “zero”, patrilocality and propagation isolation. Relative recessive allele frequencies of ten qualitative traits were calculated. Average heterozigosity, genetic differentiation, total heterozigosity, within populations heterozigosity, genetic distance and relative measures based on results of genetic distance analysis were observed. The total number of observed individuals was 1875. We have observed 1209 individuals in prewar period (Bijela population 731 and Memici population 478) and 666 individuals in the postwar period (Bijela population 248 and Memici population 478). Results ofFst andGst analysis showed changed degree of genetic differentiation across observed loci. Significant changes in differentiation were recorded for crooked little finger and PTC tasting. Modest changes of gene differentiation were recorded for ear lobe type and thumb proximal extensibility. Genetic distance analisis between Bijela and Memici has lower value after then before the war, but genetic distance between Bijela-prewar and Bijela-postwar has higher value, as well as Memici-prewar and Memici-postwar genetic distance. Genetic distance between Bijela-prewar and Memiciprewar, as well as Bijela-postwar and Memici-postwar showed similarity. Constructed dendrogram based on results of genetic distance analyses indicated two clusters groups (1. Bijela-prewar, Memici-prewar; 2. Bijela-postwar, Memici-postwar). Changed genetic differentiation and results of genetic distance analyses indicated possibility of significant impact of forced migration in genetic structure of observed populations.     

Karyometric and cytophotometric study of hepatocyte nuclei of frogs exposed to cold and prolonged starvation

Summary Prolonged starvation of Rana pipiens at room temperature results in a decrease in the volume of the hepatocyte nuclei from that of recently fed individuals. A converse increase in the volume of the liver nuclei over the normal was noted in frogs exposed to low temperature for 10 days. The present work was conducted in an effort to determine which nuclear fraction is responsible for the observed changes in nuclear size.The amount of DNA, RNA and protein-bound sulfhydryl groups per nucleus was measured cytophotometrically and was found to be independent of changes in nuclear size. Total nuclear protein content, on the other hand, was found to vary in proportion to nuclear volume. It was concluded that the variation in nuclear size resulting from starvation and cold-treatment is due to differences in non-chromosomal protein content and cannot be attributed to changes in ploidy.Dedicated to Prof. Berta Scharrer in honor of her 60th Birthday. — Supported by U.S. Public Health Service Grant HD-1499-04 to Dr. Cowden.Work completed while a Postdoctoral Trainee under U.S. Public Health Training Grant to the Department of Pathology, University of Florida, NIH-GM-1142-03.Supported by a Career Development Award from the National Institute of Child Health and Human Development, K 3-HD-6176-04.     

Glutamate receptor changes in brain synaptic membranes from human alcoholics

Brains from human alcoholics and non-alcoholics were obtained shortly after death. The hippocampus was dissected, homogenized, and processed for the isolation of a synaptic membraneenriched fraction and the study ofl-[³H]glutamic acid and 3-((±)-2-carboxypiperazin-4-yl)-[1,2³H]propyl-l-phosphonic acid ([³H]CPP) binding sites. The pharmacological characteristics ofl-[³H]glutamic acid binding to synaptic membranes isolated from hippocampus corresponded to the labeling of a mixture of N-methyl-d-aspartate (NMDA), kainate and quisqualic acid receptor sites. Synaptic membranes prepared from the hippocampus of individuals classified as alcoholics had significantly higher density of glutamate binding sites than identically prepared membranes from non-alcoholic individuals. In addition, there was a clear definition of a population ofl-glutamate binding sites (approx. 10% of total) in the membranes from alcoholics that had a higher affinity for the ligand than the major set of sites labeled in membranes from both alcoholics and non-alcoholics. Neither the age of the individuals at the time of death nor the time that elapsed between death and processing of brain tissue were significant factors in determining either recovery of purified synaptic membranes from brain homogenates orl-[³H]glutamate binding to synaptic membranes. In order to determine whether some of the changes inl-[³H]glutamic acid binding were due to alterations in binding at the NMDA receptor subtype, we also measured binding of [³H]CPP to extensively washed crude synaptosomal membranes. Membranes from brains of alcoholics had higher affinity (3-fold) for [³H]CPP but lower binding capacity (3-fold) when compared with those of non-alcoholics. These observations suggest selective changes among different glutamate receptor subtypes in human brain under conditions of chronic alcohol intake.     

System y+L-like Activities Account for High and Low Amino-Acid Transport Phenotypes in Chicken Erythrocytes

The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal population studied (male Leghorn chickens, 6–14 weeks old) was found to consist of two groups presenting either low (LT, 19 individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration of l-lysine of 1 μm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general features of system y⁺L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y⁺L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects, the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective breeding experiments of chicken with high and low amino-acid transport activity. Received: 12 February 2001/Revised: 11 June 2001     

Occurrence of B-chromosomes in diploid Allium stracheyi Baker and their elimination in polyploids

Summary In a population of Allium stracheyi Baker (2n=14) growing in Darjeeling both diploids and polyploids occur. The diploids contain B-chromosomes varying from 2–10 in number. Polyploids are conspicuous by absence of B-chromosomes. These in diploids are found in the pollen mother cells and also in the pollen, and some are provided with subterminal constriction.Diploid individuals when brought from Darjeeling to Calcutta (i. e. from temperate to tropical regions) became polyploid within a month and the B-chromosomes were simultaneously lost. In order to confirm this unexpected result, the transfer experiment has been repeated thrice with fresh collections in each case and selection of diploid bulbs after cytological observation. In all cases the result has been the same. In rare cases one or two B-chromosomes were found in the polyploid cells which might represent intermediate steps of the disappearance.B-chromosomes in diploids possibly help the individual to compete with polyploids by enlarging the adaptive capacity.The sudden polyploidisation by transfer from the mountains to the plains might have been the result of a shock due to the temperature difference. The high temperature may be deleterious for the reproduction of B-chromosomes, and their degeneration products possibly contribute to cytoplasmic changes and the spindle disturbances which effect polyploidisation.     

Chromosome numbers and reproduction inRubus subgen.Malachobatus

A review of current knowledge of chromosome numbers and modes of reproduction in the genusRubus L. is presented. Chromosome numbers from some species of subg.Malachobatus Focke together with results of crossing experiments are reported for the first time.     

Relations of enzymes inAspergillus repens grown under sodium chloride stress

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2m sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2m NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na⁺ (as NaCl) when added up to 100mm in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.     

Empirical assessment of software efficiency and accuracy to detect introgression under variable stocking scenarios in brook charr (<Emphasis Type="Italic">Salvelinus fontinalis</Emphasis>)

Stocking wild populations with domesticated fish is a common practice that promotes variable levels of introgression depending on the stocking intensity. The detection of hybridization and introgression has recently benefited from the application of Bayesian techniques implemented in various software. However, few studies have assessed their efficiency under various scenarios of stocking in the wild. The objective of this study is to assess quantitatively the effects of using two of the most widely distributed software, Structure and NewHybrids, on the level of introgression detected in wild brook charr (Salvelinus fontinalis) subjected to variable stocking intensities. We first found differences in the efficiency of software assignments based on simulated individuals, with Structure performing better than NewHybrids. However, NewHybrids showed higher assignments accuracy than Structure for the same sets of individuals. Thus, our results suggest that these software should be used in combination to assess the effects of stocking. Indeed, Structure is particularly relevant to evaluate the presence of hybrids in wild populations, whereas NewHybrids might be preferred to accurately assess the number of hybrids present in a sample. When applied to wild populations, Structure assigned more individuals than NewHybrids to the wild category. Moreover, the proportions of assigned domestic and hybrid individuals were higher in more intensively stocked lakes, whereas the opposite trend was observed for wild individuals.     

Scanning electron microscopic studies of cilia

Summary A simple method for the preparation of ciliated epithelia for study with the scanning electron microscope is described. Ciliary groups are well preserved and it is possible to discern individual cilia and work out their numbers and orientation. Following scanning electron microscopical study some of the material was prepared for transmission electron microscopy and the ultrastructure of the tissue was found to be surprisingly well preserved. The tracheal epithelium of the rabbit, the olfactory epithelia of the goldfish and the rabbit, and the sensory epithelia in the statocyst of a cephalopod mollusc were examined with the scanning electron microscope to demonstrate the possibilities of the method. Acknowledgements. We would like to thank Professor J. Z. Young for his continued interest and support. The scanning electron microscope was purchased with a grant provided by the Science Research Council to Dr. Boyde, Mr. R. Willis helped in the initial stages of the study, Mr. G. Savage provided help with the goldfish material, Mr. S. Waterman provided much photographic assistance, and Mrs. N. Finney the secretarial assistance.     

Estimation of the biomass of plankton

Summary A mathematical function is demonstrated in the numbers of individuals of the various species in a planktonic biocoenosis. The logarithms of the numbers form a Gauss or normal probability curve. A similar probability relation is found in the volumes of the individuals of the various species as well as in the biomass of the various populations.This relationship in the numbers is caused by the effect of the numerous ecological factors influencing the rate of proliferation of the various plankton species. The cause of this relationship concerning the volumes of the various species is not understood. The relationship between the various biomasses is the mathematical product of number and mean volume.An approximate hyperbolic function can be derived from the population volumes and with the aid of a simple equation the plankton biomass is calculated. A modus operandi is given to abbreviate the work necessary to determine the plankton biomass with Lohmann's method. Only ten or twenty of the most dominant populations out of all species present in a plankton sample, have to be counted and measured.The biomasses of the populations in various plankton samples may easily be compared using the hyperbolic or the probability relationship.The biomasses of plankton in various habitats may easily be compared in a graphic way. The logarithms of the biomasses found during the year follow a probability curve and may be plotted and compared on a cumulative logarithmic probability graph.The number of organisms of each species to be counted depends on the degree of accuracy and has to be about a hundred. A chance determined spread is always found in plankton counts.The spatial distribution of most plankters shows a very broad spread. Therefore, sampling at ten places and working with the mean of the ten samples is compulsory.Some gregariously living zooplankters form bunches in the water. A reliable mean may be calculated using the hyperbolic function which seems to describe their densities at the various places.From the existing methods of collection of plankton the rotary, electric pump is chosen. A translucent hose with a special and moving mouth is let down into the water. First the water passes through the plankton-net and after that through the pump and the water-meter. A series of 7 samples of increasing decimal volumes is drawn in this way. From these samples the plankton is concentrated and fractionated by means of two sedimentation chambers, four small plankton sieves and three plankton nets. The sieves and nets have various standardized meshes.Square counting chambers of 10 cm² area are used. These chambers have a thin glass bottom and a broad rim. The sedimentation chambers and the small plankton sieves fit on and into the chambers thus minimizing the loss of organisms.The plankton organisms are enumerated by means of an inverted microscope projecting the image on a ground glass which makes counting easier. Only those organisms seen within a measured square on the ground glass are counted.By standardization of the sample volumes, the magnifications of the microscope and the dimensions of the squares the conversion factors are so simple that only zeros or a decimal point have to be placed in the number counted to obtain the result.International standardization of the method of estimation of the biomass of plankton and the expression of the results is proposed.     

Changes in the flora of some Aegean islets 1968–2000

The flora of 17 islets was studied during the period 1968–2000, with thorough inventories from 1971. At each visit the species present and their population sizes were noted. The flora of the smallest islets of hard rock consisted mainly of perennials, and remained unchanged in terms of species present throughout the period of study. In the larger and more complex islets the major part of the flora consisted of islet-specialised taxa staying for long periods of time. To them are added some other mostly casual species from the close large islands, introduced by human influence, birds or winds and standing for most of the turnover in the islets flora. Few changes were observed in the plant communities of rock crevices, the spray zone of the shores and other constant habitats. The dominant perennial species forming layer of shrubs or subshrubs also mostly remained the same. Most of the changes concerned soil spaces opened by animals or erosion. Most annual species were found to survive by an extreme seed bank strategy. Many taxa common on the neighbouring islands were lacking or casual on the islets. Some others reached dominance or high frequencies that have not been observed on islands or in mainland areas. The conditions for the flora on the Aegean islets are considered to differ fundamentally from that on large isolated islands because they are situated comparatively close among each other and to islands and mainland areas. They are also vulnerable towards catastrophic events and therefore frequently presenting opened targets for invasions. The islet-specialised taxa are found to retain good ability for dispersal over moderately long distances. The grazing of the islets is stated to be an important factor for the evolution of islet adapted and specialised taxa.     

Maternal age as a source of variation in the ability of an aphid to produce dispersing forms

Summary Apterous parthenogentic females of the pea aphid,Acyrthosiphon pisum (Harris), begin to produce alate offspring soon after they have been subjected to crowding. Females which were born early in their own parent’s reproductive period respond most strongly to crowding, producing much larger numbers of alatae than their late-born sisters. In contrast, the early-born daughters of most alate females do not produce winged offspring after being crowded. Some of their later-born sisters may produce a few winged individuals, resembling in this respect the late-born daughters of the apterous females. Control of the production of alatae thus begins in the grandparental generation. Risk-spreading by means of differential dispersal becomes a less uncertain venture when local populations can modify their responses to environmental changes by utilizing past as well as present signals from their surroundings.     

The rhabdomeric microvilli of several arthropod compound eyes kept in darkness

Summary Electron microscopical studies were made on the fine structure of the rhabdomeric microvilli of the compound eyes of seven species of arthropods (Procambarus, Neocaridina, Caridina, Potamon, Artemia, Diestrammena, Drosophila) raised in complete darkness for 1–8 months or for successive generations, using various fixation techniques.The rhabdomeric microvilli of the individuals kept in darkness for a long period were regularly arranged as in normal eyes in the material prepared by double fixation with glutaraldehyde and OsO₄, whereas in those fixed solely by OsO₄ various forms of vesiculations were seen. The structural changes of the rhabdomere in darkness, which have been reported by several workers, were conceived to be an artefact caused by OsO₄ fixation.This work is supported by a grant from the U.S. Army Research and Development Group (Far East), Department of the Army (DA-CRD-AG-S92-544-67-G61).The authors wish to express their gratitude to Dr. S. Mori and Mr. T. Kuramoto for their help in supplying some of the materials. We are also indebted to Dr. C. J. C. Rees for correcting English of this paper.     

Regulation of calcium fluxes in rat pancreatic islets: Calcium extrusion by sodium-calcium countertransport

Summary The mechanisms by which glucose regulates calcium fluxes in pancreatic endocrine cells were investigated by monitoring the efflux of⁴⁵Ca from prelabeled and perifused rat pancreatic islets. In the absence of both extracellular calcium and glucose, partial or total removal of extracellular sodium decreases the efflux of⁴⁵Ca from prelabeled islets. Glucose also reduces the efflux of⁴⁵Ca from islets perifused in the absence of extracellular calcium. This inhibitory effect of glucose on⁴⁵Ca efflux is decreased by half when the extracellular concentration of sodium is lowered to 24mm. In the absence of extracellular calcium but presence of glucose, partial or even total removal of extracellular sodium fails to decrease the efflux of⁴⁵Ca. At normal extracellular calcium concentration (1mm) partial removal of extracellular sodium dramatically increases⁴⁵Ca efflux from pancreatic islets. This increase in⁴⁵Ca efflux is partially but not totally suppressed by either 16.7mm glucose or cobalt. It is totally suppressed by 4.4mm glucose or by the combination of 16.7mm glucose and cobalt. At normal extracellular calcium concentration, glucose initially reduces and subsequently increases⁴⁵Ca efflux. The initial fall is unaffected by tetrodotoxin but decreased by 50% at low extracellular sodium concentration (24mm). The present results suggest the existence in pancreatic endocrine cells of a glucose-sensitive process of sodium-calcium counter-transport. By inhibiting such a process, glucose may decrease the efflux of calcium from islet cells. The effect of glucose is not mediated by an increase in intracellular sodium concentration. It could contribute to the intracellular accumulation of calcium which is thought to trigger insulin release.This paper is the IVth in a series.