Effects of short-term storage of gametes on fertilization of Pacific herring eggs

A method is described whereby arrays of samples ofClupea pallasi eggs may be stored during their preparation. The high fertilization potential retained by the eggs during short-term storage allows them to be fertilized synchronously when sample preparation is complete. A variation of the dry method of storage retained maximum fertilization potential (80–85%) of the eggs for about 1 hr, and of milt dilution (18 with 17 S sea water), about 7 hr. Following dry storage, eggs fertilized in salinities of 0–45 showed maximum rates of fertilization in salinities of 10–20, and fertilization rates 50% in salinities of 4.5–42. Salinities of fertilization influenced egg diameter, median hatching time, and larval length at hatching in egg samples transferred 21/2 hr after fertilization to an incubation salinity of 17 at 7°C. Fertilization rates were higher (90–95%) for eggs stored in 17 S at 7°C prior to fertilization. Under such wet storage conditions, maximum fertilization pontential was retained for about 2 hr. Highest fertilization rates (95–96%) were obtained for eggs stored and fertilized in salinities of 12–15. For the species and the area of origin considered (British Columbia), wet storage of eggs should result in maximum fertilization when the eggs are stored at 4°C for a period not greater than 2 hr prior to fertilization in the 12–15 S storage medium.Prepared under the auspices of the Canadian-German Scientific and Technical Cooperation Agreement.     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Sources of organic and inorganic carbon in a headwater stream: Evidence from carbon isotope studies

A combination of stable isotope studies and ¹⁴Cdating were used to identify the main sources andprocesses controlling streamwater DOC and TIC in atemperate non-forested watershed. ¹³Cvalues for terrestrial (–24.9 to –29.1) and aquatic(–30.5 to –33.5) plants were similar to valuesreported in the literature for similar ecosystems.¹³C values for DOC in soil solution andstreamwater were consistent with soil and terrestrialvegetation, indicating that the terrestrial ecosystemis the dominant source of aquatic DOC in thiswatershed. ¹³C values of soil atmosphereCO₂ (–17.2 to –25.2) were slightly lessnegative than would be expected for production viaaerobic soil microbial decomposition and rootrespiration. There was a close correspondence between¹³C values (–15.5 to –21.5) forstreamwater TIC and soil atmospheric CO₂ in thecentral part of the catchment where the stream drainsCO₂-rich peats. ¹⁴C dating showed thatalthough peat has been accumulating in the watershedfor at least 2700 years, DOC in soil pore water andstreamwater contains carbon of predominantly recentorigin (post-AD 1955).     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Prokaryotic endosymbionts in the chloroplast stroma of the dinoflagellateWoloszynskia pascheri

Summary Chloroplasts of the freshwater dinoflagellate,Woloszynskia pascheri, were found to contain small, double membrane-bound bodies that appear to be modified bacteria existing in this organelle as endosymbionts. These chloroplast endosymbionts (CESs) contain thin filaments, which observations on thin-sectioned and Feulgen-stained material indicate to be strands of naked DNA. They also possess putative prokaryotic-sized ribosomes. The outer of the two membranes that surround a CES may be expanded to form cisternae or tubules, which frequently connect with the outer membrane of adjacent CESs. Considering their appearance in relation to free-living bacteria, and their apparently benign presence in the dinoflagellate host, it is suggested that the CESs have been involved in a symbiotic relationship withW. pascheri for some time.     

On age morphological changes of males of Chydoridae (Cladocera)

Summary Young and adult males of 11 species of Chydoridae are studied, their figures being published here (fig. 1–15). The necessity is stressed to distinguish young forms of males and gynandromorphic individuals.Pleuroxus balatonicus is considered to be described from the population ofPleuroxus unicatus having under Balaton Lake conditions retarded transformation of young males into adult form, and accordingly having unusually numerous young males. \qO\qs\qn\qo\qv\qn\qy\ye \qr\ye\qz\qu\ql\Qj\qt\qa\qt\qy 11 (. 1–15). . , Pleuroxus uncinatus , Pleuroxus balatonicus.     

γδ T-cell receptor repertoire in human peripheral blood and thymus

T-cell clones expressing the T-cell receptor (Tcr) were generated from peripheral blood lymphocytes (PBLs) and from a thymus sample. In the panel of ten thymus-derived clones, four Tcr phenotypes [as defined by the reaction of monoclonal antibodies (mAbs) directed against known V and V regions] were identified. All the clones lacked expression of the V3 V region, while seven clones were V1+ . V1 was found in combination with V9 or with undefined VVregions. In addition, two other Tcr phenotypes were identified on these clones: V9⁺ V1^– V3^– and V9^– V1^– V3^– One of the clones expressed CD4 and another was CD8positive. The remaining clones were CD4^– CD8^–. In the panel of 76 PBL-derived, Tcr-bearing clones, five Tcr phenotypes could be identified. In contrast to the thymus-derived clones, 30% of the clones were V3⁺ whereas V1 was expressed by a minority of the clones only. One clone was CD4-positive and approximately 30% of the clones were CD8-positive. Four of the five mAb-defined Tcr phenotypes could be identified on both thymus and PBL-derived T-cell clones. However, biochemical analysis of the Tcrs demonstrates differences in the usage of Ct- and C2-encoded y chains by T cells derived from the thymus and PBLs. The results therefore indicate that, at the clonal level, similarities and differences exist between the Tcr repertoires expressed in the thymus and by PBLs. Furthermore, they indicate that combinatorial Tcr heterogeneity is larger than has so far been described. The receptor diversity, combined with the potential of Tcr+ cells to express CD4 or CD8, indicates that these cells are a heterogeneous population that might mediate a number of immune functions.     

Expression of integrin subunits αv and β3 in acute lung inflammation

Integrin subunits v and 3 form a dimer, v3, which is expressed on normal neutrophils and endothelium. We investigated the expression of integrin subunits v and 3 in acute lung inflammation in Sprague-Dawley rats (n=5 each) following intratracheal challenge with Escherichia coli or Streptococcus pneumoniae, which induce neutrophil recruitment through different mechanisms. Control rats (n=5) were given endotoxin-free saline. Both bacterial challenges induced similar levels of recruitment of neutrophils in lungs. Western blots showed lower expression of integrin subunits v and 3 in lungs challenged with E. coli compared to those given S. pneumoniae. Immunohistochemistry and immunogold electron microscopy localized both integrin subunits in neutrophils and endothelium in the control and treated rat lungs. Quantitative immunohistochemistry showed that E. coli-challenged rat lungs contained a lower percentage of neutrophils expressing integrin subunits v and 3 compared to those challenged with S. pneumoniae (P<0.05). We conclude that E. coli infection decreased the percentage of neutrophils expressing integrin subunits v and 3 compared to S. pneumoniae infection. These data lay the foundation for further characterization of these integrin subunits in neutrophil migration specifically in S. pneumoniae infection that utilizes molecules other than 2 integrins for neutrophil recruitment.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Bcl-2 blocks apoptotic signal of transforming growth factor-β in human hepatoma cells

Transforming growth factor- (TGF-) has been shown to induce apoptosis on normal hepatocytes and hepatoma cells both in vitro and in vivo. However, how the TGF- induces apoptosis is still not clear. We examined the expression of anti-apoptosis proteins and sensitivity to TGF- in three well differentiated human hepatoma cell lines. Two TGF- sensitive cell lines Hep3B and HuH7 totally lacked Bcl-2. In contrast, the TGF- resistant HepG2 cells expressed a substantial amount of Bcl-2. All three cell lines expressed equal amounts of Bcl-X_L, Bcl-X_S and Bax. Overexpression of Bcl-2 in Hep3B and HuH7 cells protected them from TGF--induced apoptosis. TGF- treatment increased intracellular peroxide production and suppressed the expression of glutathione-S-transferase in the Hep3B cells, and these effects were partially suppressed by the overexpression of Bcl-2. These results suggest that Bcl-2 may protect cell from TGF--F-induced apoptosis by interfering TGF- generated signals leading to induce reactive oxygen species production.     

Fine structure of early previtellogenic oocytes in Mugil (Liza) auratus Risso, 1810 (Teleostei,Mugilidae)

Summary Sequential cytological events at the onset of previtellogenesis were studied in oocytes from 12 m to 70 m in diameter of golden grey mullet. The main cytological changes observed (increase in size of cell, nucleus, nucleolus and increase in number of nucleoli, RNP particles, nuage and mitochondria) provide evidence for important synthetic processes in an early preparatory phase of oocyte development.Somatic cells (pre-follicle, follicle and thecal) are also described.     

Protein kinase C (α, β, γ) in Pacinian corpuscle

Immunocytochemical demonstration of protein kinase C (PKC) subspecies (, , ) was carried out in Pacinian corpuscles of rat hind feet using monoclonal or polyclonal antibodies against each of these subspecies. The inner core cells and lamellae and the Schwann cell cytoplasm of the nerve fiber innervating the corpuscle were strongly positive for PKC -immunoreactivity (IR). In contrast, the axon terminal and the outer core did not display any positive -IR. Very weak PKC -IR was detected in the ultraterminal region of the axon terminal, while the trunk region showed no immunoreactivity. Very faint PKC -IR was found also in the lamellar cells located at the periphery of the inner core and the endoneurial fibroblasts in the intermediate layer. PKC -IR was not detected in any part of the corpuscle. The strong PKC -IR in the inner core and the presence of absence of PKC -, -, and -IR in the axon terminal are discussed from the point of view of the functional aspects of each part.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Die Feinstruktur der Zirbeldrüse normaler,trächtiger und experimentell beeinflußter Meerschweinchen

Zusammenfassung In der Meerschweinchenzirbeldrüse lassen sich elektronenmikroskopisch helle und dunkle Pinealzellen sowie einzelne Gliazellen nachweisen. In den bei weitem überwiegenden hellen Pinealzellen zeichnet sich ein Teil der vesicle-crowned rodlets (VCR) durch lokale Auftreibungen aus. Von VCR deutlich abzugrenzen sind die vesicle-crowned balls (VCB). Erstmalig beschrieben wird das Vorkommen von sog. Zylindern, die als Vorstufen von VCB aufgefaßt werden. In den relativ seltenen dunklen Pinealzellen, die sich durch chromatinreiche Kerne und elektronendichtes Zytoplasma auszeichnen, sind Vesikel, VCR, VCB und Zylinder seltener als in hellen Pinealzellen. Die reichlich vorhandenen marklosen Nervenfasern finden sich vor allem in perivasculären Räumen, seltener im Parenchym. Synapsen zwischen Nerven und Pinealzellen wurden nicht beobachtet. In den Zirbeldrüsen trächtiger Meerschweinchen zeichnen sich in der 2. Hälfte der Tragzeit die hellen Pinealzellen durch stärkere Lappung der Kerne, gehäuftes Auftreten von laktiven Zonen, Vermehrung von Mitochondrien, glattem ER, agranulären Vesikeln, VCR, VCB und Zylindern aus. Die dunklen Pinealzellen nehmen während der Tragzeit an Zahl zu. Post partum bilden sich diese Veränderungen innerhalb einer Woche zurück. Längerer Aufenthalt der Tiere in Dunkelheit führt zu einer Aktivierung der hellen Pinealzellen mit auffallender Vermehrung der VCR und zu einer Zunahme der dunklen Zellen. Unter Dauerbelichtung kommt es in den hellen Zellen zu einer Abnahme fast aller Zellorganellen und zu einer starken Vermehrung der VCR, die nach 70 Tagen auch Formveränderungen aufweisen. Nach Reserpinbehandlung beobachtet man eine Verminderung und degenerative Veränderungen der VCR. Es wird diskutiert, daß die VCR als prae- bzw. postsynaptische Strukturen der Erregungsübertragung von Nerven zu Pinealzellen bzw. von Pinealzellen untereinander dienen könnten.

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The fine structure of the pineal gland of normal, pregnant and experimentally affected guinea-pigs

Summary By means of electron microscopy light and dark pinealocytes can be distinguished in the guinea-pig pineal gland. Glial cells are rare. In the light pinealocyte. the most frequent cell type, some vesicle-crowned rodlets (VCR) show circumscribed thickenings. From these structures vesicle-crowned balls (VCB) have to be clearly distinguished. Furthermore cylinders occur, which, it is suggested, are precursors of VCB. Dark pinealocytes characterized by chromatin-rich nuclei and electron-dense cytoplasm are rare and contain fewer vesicles, VCR, VCB and cylinders than light pinealocytes. Numerous non-myelinated nerve fibres are situated within perivascular spaces, a few also in the parenchyma. Synapses between nerve fibres and pinealocytes were not observed. In the pineal gland of pregnant guinea-pigs the following changes can be observed in the second half of gestation. The light cells show many nuclear indentations and an increase of active zones, mitochondria, smooth ER, agranular vesicles, VCR, VCB, and cylinders respectively. The dark cells increase in number. After birth these changes reverse to normal within one week. Constant darkness leads to an activation of the light cells accompanied by an increase of the VCR and to an increase in number of the dark cells. Under constant illumination the light cells show a decrease of their organelles and a strong increase of the VCR. After 70 days the VCR also show a change in shape. Following reserpine treatment the VCR decrease in number and show signs of degeneration. It is discussed that the VCR function as pre- or postsynaptic structures and that they are involved either in transmitting impulses from nerve fibres to pinealocytes or from one pinealocyte to the other.

Untersuchung unter Leitung von Univ.-Doz. Dr. L. Vollrath.     

An electron microscopical investigation of the follicle gland (cerebral gland) and of some neurosecretory cells in the lateral lobe of the cerebral ganglion of the pulmonate gastropod Lymnaea stagnalis L.

Summary The ultrastructure of the follicle gland and of some Gomori-positive neurosecretory cells in the lateral lobes of the cerebral ganglia of Lymnaea stagnalis is described.The follicle wall consists of epithelial cells containing secretion granules, and of processes of lateral lobe nerve cells bearing cilia, including the processes of bipolar neurosecretory B-cells. The ultrastructure of the follicle of Ancylus fluviatilis and Planorbarius corneus appeared to be very similar to that of L. stagnalis. From the ultrastructure of the follicle wall and lumen, no conclusions can be drawn as to its formerly proposed possible functions (sense organ, endocrine organ, neuroendocrine organ). Of three neurosecretory cell types (B-cells, canopy cells, droplet cells), the positivity to chrome-haematoxylin and paraldehyde-fuchsin appeared to be due to elementary granules of different size and appearance. This finding supports in a particular sense the view that results obtained with the neurosecretory stains, should be judged carefully. Also in ordinary neurons elementary granules can be found, at times in great numbers. Several types of elementary granules (at least 6) could be distinguished. Their character and function (neurohormones, neurotransmitters) are not clear.     

Bacterial regeneration of ammonium and phosphate as affected by the carbon:nitrogen:phosphorus ratio of organic substrates

The effect of carbonnitrogenphosphorus (CNP) ratio of organic substrates on the regeneration of ammonium and phosphate was investigated by growing natural assemblages of freshwater bacteria in mineral media supplemented with the simple organic C, N, and P sources (glucose, asparagine, and sodium glycerophosphate, respectively) to give 25 different substrate CNP ratios. Both ammonium and phosphate were regenerated when CN and NP atomic ratios of organic substrates were 101 and 161, respectively. Only ammonium was regenerated when CN and NP ratios were 101 and 10–201, respectively. On the other hand, neither ammonium nor phosphate was regenerated when CN and NP ratios were 151 and 51, respectively. In no case was phosphate alone regenerated. As bacteria were able to alter widely the CNP ratio of their biomass, the growth yield of bacteria appeared primarily dependent on the substrate carbon concentration, irrespective of a wide variation in the substrate CNP ratio.     

Histochemical investigations on the human fetal subcommissural organ

Summary A histochemical and ultrastructural study was carried out on subcommissura organs from 42 human embryos and fetuses in order to characterize some large granulesTypical granules make their appearance in the rostral hypendymal region of the subcommissural organ (SCO) in fetuses of about 50 mm CRL. Although they appear in other SCO-regions later, the highest number of granules is always located towards the pineal gland.Typical granules are of spherical shape with a diameter of about 2 microns. The various histochemical reactions reveal a reactivity which differentiates the shell of the granules from the granule interior. Nucleoproteins are present in the shell together with phospholipids and/or lipoproteins. The interior of the granules can contain different materials such as glycogen or lipid or neurosecretory substance. Ultrastructural observations show that a granule consists of whorls of endoplasmic reticulum sparsely studded with ribosomes surrounding an interior containing either lipid or lipoprotein inclusions, large amounts of glycogen or simply cytoplasm.It is suggested that the concentric lamellar organelle (CLO) is a morphological entity that might be involved in secretory processes rather than being the secretory granules themselves.This work was supported by a grant from Statens almindelige Videnskabsfond, Copenhagen.