Observations on the micro-anatomy of crystallolithus hyalinus gaarder and markali

Summary The fine structure of Crystallolithus hyalinus (Gaarder and Markali), known to be the motile stage of Coccolithus pelagicus (Wallich) Schiller (Parke and Adams 1960), has been investigated for purposes of comparison with Chrysochromulina and Prymnesium, recently recognised as probably related to it. Anoptral contrast light microscopy of living cells and electron microscopy of thin sections and shadow-cast whole mounts show that there is a close resemblance to species of Chrysochromulina not only in cell structure but also in the details of haptonema structure and scale structure. The origin of the scales from internal vesicles has been traced as in other genera but the holococcoliths (crystalloliths) do not appear to arise in this way. The mode of origin of the crystalloliths is thus unknown, but it is suggested that they may be developed outside the cell from calcite secreted in solution into the appropriate cavity. Their constant position between a layer of scales and an outer enveloping skin is consistent with this and one unfamiliar cytoplasmic organelle, composed of peculiar thick-walled tubes within some large superficial vesicles, is described as perhaps involved in this process. Apart from these special features the resemblance to both Chrysochromulina and Prymnesium is sufficient to add considerable weight to Christensen's suggestion (Christensen 1962) that a new group should be defined to accommodate these genera.     

Phase contrast photomicrography of cellular behaviour in spongillid porocytes (Porifera: Spongillidae)

The combination of phase contrast photomicrography with a method for phase contrast observation of living sponges facilitated the initial photographic record of spongillid porocyte cellular behavior. Porocytes ofCorvomeyenia carolinensis Harrison (Spongillidae) exhibit true intracellular pores and are components of the incurrent system. The number of porocyte contractile vacuoles increases during pore dilation. This increase is possibly related to an increase in the amount of cell surface in contact with the hypotonic aqueous environment. The porocyte cell type appears closely related to, and is likely derived morphogenetically from, pinacocytes of the upper pinacoderm.     

Anastomoses and nuclear condition in Cephalosporium coccorum petch

Nuclear condition and formation of anastomoses inC. coccorum Petch have been reported. Preliminary study on the nuclear condition shows that the vegetative mycelium and conidia are uninucleate. Anastomoses in mycelium and conidia are discernible. Hyphal fusion through anastomosing bridges and nuclear migration through them show the possibility of heterokaryosis in the organism. Nutrient free medium is suitable for the development of anastomoses both in mycelium as well as in conidia.     

Fatal case of erythema nodosum and histoplasmosis

Summary In the post mortem examination of a patient, erythema nodosum of the upper extremities with fibrinoid arteritis was found in presence of primary pulmonary histoplasmosis with hematogenous dissemination. Sudden death is believed to have been the result of hypersensitivity to acute fungous spread.Prof. Titul. Direct. Inst. Anat. Patol., Universidad de los Andes, Merida Venezuela, Sen. Res. Assoc., Clinical Laboratories, Jewish Hospital, Cincinnati, Ohio (Dr.Jan Schwarz).     

Dynamics of l-valine in relation to the production of cyclosporin A by Tolypocladium inflatum

Summary We report the kinetics of endogenous l-valine in the fungus Tolypocladium inflatum, in an effort to understand the enhancing effect of externally supplemented l-valine on the production of the immunosuppressant cyclosporin A (CyA) in chemically defined medium. In a batch laboratory stirred reactor cultivation, the concentration of intracellular l-valine increased by up to four times between the end of the exponential phase and the beginning of the stationary phase when the medium was supplemented externally with 4 g/1 l-valine. The final CyA titre under these conditions was 710 mg/1 compared to only 130 mg/1 attained without l-valine supplementation. In contrast to substantial growth-associated production of CyA in unsupplemented culture, the formation of the immunosuppressant was prolonged during the stationary phase in l-valine-supplemented medium. As a result, the conversion yield of CyA on l-valine remained constant during the stationary phase at 0.27 g CyA/g l-valine.     

The Bacteroides glycocalyx as visualized by differential interference contrast microscopy

The glycocalyx of eight strains representing six species of Bacteroides was examined by differential interference contrast microscopy. Wet mounts in India ink were prepared from bacteria cultured in broth and on an agar medium; the wet mounts were observed by phase-contrast microscopy and differential interference contrast microscopy. With differential interference contrast microscopy, all bacteria demonstrated a glycocalyx, which included capsules surrounding single cells and microcolonies, strands of glycocalyx connecting cells and microcolonies, detached slime, and solid masses of glycocalyx in which innumerable bacteria were enmeshed. Bacteria showed comparable amounts of glycocalyx by visual observation with differential interference contrast microscopy whether grown on plates or in broth. Serial transfers of cultures did not diminish the amount of glycocalyx. Differential interference contrast microscopy proved to be a superior method to phase contrast for examining wet preparations of Bacteroides.     

Electrolytes and plasma expanders

Summary The effect of Gey's balanced salt solution deficient in NaCl on cells in tissue culture led to a study of various solutions employed as vehicles for plasma expanders.The behavior of epithelium from the adult human mucosa, tonsils from children, and mouse kidney papilla was recorded in a perfusion chamber with phase contrast, time-lapse cinematography. After perfusion for one to two hours with fluid nutrient medium, test solutions were brought in contact with the cells for one hour. The fluid system was then generally replaced with Gey's balanced salt solution and, or, the proteinaceous nutrient medium.So-called normal saline produced marked injury both when employed alone or in combination with 6% Dextran. Damage resulting from 5% dextrose either alone or in combination with 6% Dextran was characterized by vacuole formation. Dextran at 6% in Gey's balanced salt solution or in Ringer's solution appeared to produce little injury to cells under the conditions of these experiments.Work in progress is designed to examine the lifesaving value of the various solutions in the treatment of partially exsanguinated dogs.This investigation was supported by the Medical Research and Development Board, Office of the Surgeon General, Department of the Army, under Contract No. DA-49-007-MD-32.     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

Hepatotoxicity studies with primary cultures of rat liver cells

Summary A method for preparing primary monolayer cultures of postnatal rat hepatocytes has been developed in our laboratory. Growing cultures in arginine-deficient medium inhibits fibroblast overgrowth, and relatively pure cultures of parenchymal hepatocytes are obtained. This cell culture system has been used to study the cytotoxicity of two hepatotoxic agents, tetracycline and norethindrone. Caffeine was evaluated as an agent thought to be relatively nontoxic to liver. Cytotoxicity was evaluated by phase-contrast microscopy of cellular morphology and by measurement of leakage of intracellular enzymes [arginosuccinate lyase (ASAL), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and acid phosphatase (AP)] into the culture medium. Hepatic cultures were treated with each of the agents in concentrations ranging from 5×10⁻⁶ to 1×10⁻³ m and for durations from 1 to 24 hr. ASAL was found to be the most sensitive in predicting early cell injury and AP the least sensitive; the other three enzymes tested were intermittent in value and equally sensitive in evaluating cytotoxicity. Treatment of the cultures with tetracycline (5×10⁻⁴ m) for 6 hr resulted in ASAL leakage that was 400% of control values; and norethindrone (5×10⁻⁴ m) for 6 hr caused a 250% increase relative to controls. The hepatotoxic agents demonstrated a dose- and timedependence of cytotoxicity in the cultures. In contrast, caffeine was relatively nontoxic to the cultures. Part of this investigation was presented orally at the 17th Annual Meeting of the Society of Toxicology, San Francisco, March 13, 1978.     

Combined fluorescence histochemistry and 3h-noradrenaline measurements of adrenergic nerves

Summary A method is described which combines the histochemical fluorescence technique of Falck and Hillarp with isotope measurements in the same pieces of tissue. Tissue pieces incubated in isotope solutions were treated for fluorescence microscopy and examined. They were then removed from the microscopical slides, and the radioactivity determined. It was shown that NA¹ content and estimated fluorescence intensity were well correlated. The procedure devised is of special value when isotope measurements are needed of structures which can be safely identified only in the fluorescence microscope, and it has been used for quantitative estimations of adrenergic innervation.Abbreviations used Na noradrenaline - cpm counts per minute - dpm desintegrations per minute     

Behaviour of chromosomal spindle fibres in living cells

Summary Mitosis in endosperm of Haemanthus katherinae was studied in vitro by the use of 16 mm time-lapse microcinematography. In several cells, chromosomal fibres were seen in phase contrast microscope. The fibres are more convergent in late metaphase and anaphase than in prometaphase. There exist two distinct fibre attachments at each daughter kinetochore, i.e. there are four in metaphase chromosome.Details of the division of a nearly cytoplasm-free mitotic apparatus are also reported as well as some data concerning the elongation of the spindle and activity of the phragmoplast.Dedicated to Professor H. Bauer on the occasion of his sixtieth birthday.     

Corpuscular and trilaminar appearance of cultivated chick embryo fibroblast plasma membranes

Summary Electron microscope examination of chick embryo fibroblasts grown in tissue culture and fixed in a variety of osmium fixatives as well as in fixatives not containing osmium revealed a limiting cell membrane with a corpuscular substructure. A trilaminar appearance was more difficult to demonstrate in plasma membranes of cells fixed in osmium or glutaraldehyde solutions followed by osmication, while many extensive regions of membranes with this appearance were observed after fixation in buffered potassium permanganate.A study of the conditions favorable to the demonstration of a trilaminar appearance indicated that the level of focus of the electron microscope image and alteration of the intrinsic structure of the summits within the membrane played significant roles in producing this appearance.This work was aided by Research Grant GB 2129 from the National Science Foundation. Some of the equipment used was purchased with funds from the National Institutes of Health Grant 2 TI GM 326. I wish to thank Dr. Robert M. Dougherty from the Department of Microbiology who grew and supplied me with the chick embryo fibroblast cultures used in these studies, and Mrs. Ursula Feller and Miss Leslie Hammack for their technical assistance.     

Activity manifestations of Hassall's corpuscles in vitro as revealed by cinematography

Summary Thymic explants from newborn rats were cultivated in Rose chambers under dialysis membranes. With the use of phase contrast, time-lapse cinematography, the following activity manifestations were observed: 1. Several corpuscles showed rotatory movements; 2. nuclear rotation was observed in some cells of the corpuscles; 3. some individual cells in the 15-day culture showed pulsatile activity which might be involved in the mechanism of the rotatory movements of the cellular aggregates; 4. the corpuscles increased in size by the addition of cells and/or by mitosis; 5. the elements at the periphery of the more mature corpuscles (7- and 9-day cultures) were viable, while the central area appeared to be composed of necrotic tissue or hyalinized material.Grateful acknowledgement is made to Dr. C. M. Pomerat for encouragement in the course of this study. Mr. Charles Raiborn aided in the preparation of cultures and Messrs. C. G. Lefeber and Robert Olson rendered indispensable assistance with the photographic work.Aided by an American Cancer Society Student Fellowship (USC-IDC) and in part by Grant No. G-14091 from the National Science Foundation administered by C. M. Pomerat.     

l-Lysine production in continuous culture of an l-lysine hyperproducing mutant of Corynebacterium glutamicum

Summary l-Lysine production was studied in and volumetric productivity were 105 g/l and 5.6 g/l per hour, respectively. The latter corresponded to a 2.5-fold higher value than that of a fed-batch culture. Higher agitation or use of oxygen-enriched air was critical for high productivity. This indicates that oxygen supply greatly affects l-lysine productivity in continuous culture.     

A new principle of continuous flow cell cultivation

Summary A completely liquid-filled culture chamber with gas exchange across a synthetic membrane (Larsen andNilsson 1985) was incorporated into an automatic continuous flow system. The absence of an airliquid interface in the system permits removal of cell samples, and addition of fresh medium, under strictly sterile conditions. In this system,Tetrahymena pyriformis can be kept under optimal growth conditions in a rich nutrient medium and any defined cell density may be maintained for extended periods of time by varying the dilution rate of the culture. Furthermore, it has been possible to demonstrate, in the slope of the growth curve, even small changes which are difficult to detect in batch cultures since the duration of these changes is short. In the continuous flow system, the relative cell volume distribution and the food vacuole forming capacity of the cells were unaltered; however, all cells contained small refractive granules. The system permits the culture volume to be varied, but a standard volume of 20 ml was maintained in most experiments. Since the culture volume is small, the system requires less than one liter of fresh medium per week to maintain the cells in the exponentially multiplying growth phase.     

Koinzidenzfilter mit kurzen Impulsen

Summary Coincidence filters consist of one or more threshold elements (e.g. neurons or monostable multivibrators, extended by multiple input gates). They permit the propagation of an impulse train applied to the input only if its repetition rate does not exceed an upper and a lower boundary value. The difference between the upper and the lower boundary value may be defined as the functional bandwidth of the coincidence filter. The functional bandwidth is one of the most interesting characteristic figures of a coincidence filter. By means of this definition, the coincidence filter may be described as a device selecting quickly those impulse trains the repetition rates of which lie within the functional bandwidthThe functional bandwidth depends on the parameters of the impulse trains and of the coincidence filter. This gives rise to the question, which minimal bandwidth could be realized by coincidence filters.On the initiation by Tischner the properties of coincidence filters operated by rectangular impulses have been investigated by Schie f and by Kosel. Rectangular impulses have the advantage, that moderate variations of the amplitudes do not disturb the coincidence. In this case however very small impulse durations are required for the realization of small bandwidth.In the present paper the operation of coincidence filters by non rectangular impulses has been considered. Having the shape of an excitatory post-synaptic potential of motoneurons, the impulses are completely determined by the rising phase and the falling phase. These impulses have been termed short impulses in contrast to the rectangular impulses, which are long, compared to the duration of their rising and falling phases. The width of the short impulses decreases with increasing measuring level. Close to the amplitude the width becomes very small, which theoretically provides a very small functional bandwidth. The practical realization of very small functional bandwidth is heavily limited by the big variations which will be caused by minute alterations of the amplitudes as introduced by noise. The variation of the functional bandwidth caused by 1% alteration of the amplitude has been termed the error factor. In the present paper some relationships between the following four quantities have been worked out: realizable functional bandwidth, tolerable variation of functional bandwidth, error factor, and given variation of the amplitudes and thresholds (noise).The short impulses have been piecewise approximated by analytical functions (parabolic and hyperbolic) which in general permits an analytical treatment of the problems.     

An electron microscopical study of the ventral nerve cord of the leech

Summary Three types of fibrillar structure can be seen with the electron microscope in nerve cells of the vental nerve cord of the leech: the neurofibrillar bundles, the tubules and the tonofibrils. In neuroglial cells only the tonofibrils are present. The three types are structurally distinct, and, contrary to past suggestions, there is no evidence that neurofibrillar bundles may consist of tightly packed or badly fixed tubules.In vertebrates the electron microscope reveals bundles of discrete neurofilaments that form the basis for the argyrophilic neurofibrillae seen by light microscopy. Each neurofilamentous unit appears as a dot in cross section. In contrast, in the leech, the electron microscope shows compact fibrillar bundles that clearly correspond to the neurofibrils described by light microscopists. These bundles are made up of closely packed units rather than discrete filaments and where the units occur singly they are seen to have an angular or stellate outline in cross section. To make this distinction clear these have been termed neurofibrillar bundles rather than neurofilaments.Attachment plaques occur in both neurons and neuroglia. These plaques have tonofibrils attached, and the glial tonofibrils are far more numerous than the neuronal tonofibrils. The glial fibrils are identical with the tonofibrils in the glial cells.The attachment plaques are invariably related to an extracellular space that contains material identical with the basement membrane. This material is continuous, by a complex system of channels and diverticulae, with the outer basement membrane in the neuron packets, but forms isolated patches in the other parts of the nervous system.We are grateful to Prof. J. Z. Young, F. R. S., for his encouragement to Mrs. Astafiev for the drawings, to Miss B. Shirra and Mr. K. Watkins for technical assistance and to Mr. S. Waterman for photography.     

Karyotype evolution in a tumour derived plant tissue culture analysed by Giemsa C-banding

Summary A hyperdiploid aneuploid karyotype, consisting of 7 chromosomes, has been found in a tumorous suspension cell culture ofCrepis capillaris (2 n=6). Giemsa C-banding has revealed that these 7 chromosomes show consistent patterns of differential staining in all dividing cells. This stable karyotypic situation has persisted during 18 months of cytological monitoring of the culture. Comparison with the diploid C-banded complement of the root tip indicates that numerous structural rearrangements must have occurred during the formation of the aneuploid complement. A likely pathway for evolution of this karyotype involves initial tetraploidy followed by chromosome loss. Such a mechanism has previously been proposed for a plant tissue culture system (Bayliss andGould 1974) and commonly occurs in animal systems, particularly in animal tumours (Terzi andHawkins 1975). An alternative mechanism, which does not necessarily involve tetraploidy, is also proposed.     

Bacterial flagella: Do they rotate or do they propagate waves of bending?

Conclusion Whether the flagellum is rigid or nearly so or whether there are moving helical lines of contraction (Astbury et al. 1955,Lowy andSpencer 1968), incompatible sets of bonds at different radii (Klug 1967), subunits which alter their configurational states (Asakura 1966, 1970) or slip between subunits carried by edge dislocations (Harris andScriven 1971, 1973), the prevention of apparent rotation of the flagellum will result in actual rotation of the body. The differences between the two classes of mechanisms have a scale beyond the power of resolution of the light microscope. Because of this it is most unlikely that direct observation of a moving bacterium will provide evidence favouring any one of the mechanisms. The mechanism of movement of bacterial flagella remains unknown.     

Effect of 2-deoxy-d-glucose on maintenance in culture of neonatal B cell of rat

Summary The effect of 2-deoxy-d-glucose on maintenance in culture of B cells of the neonatal rat was examined by supplementation of Medium 199 containing 5.5 mM glucose with 1 mM 2-deoxy-d-glucose. Islets maintained in medium with 5.5 mM glucose (basal medium) for 7 d underwent remarkable decreases in glucose sensitivity, and the levels of insulin in the medium dropped. By contrast, addition of 2-deoxy-d-glucose promoted a higher insulin content in medium and an increase in the glucose-induced insulin release and biosynthesis. Moreover, the addition of the deoxysugar caused a selective deletion of fibroblasts and prevented the deterioration of islet cells in basal medium, yielding clusters mostly consisting of islet cells at the end of culture.