Regional and subcellular distribution of taurine-synthesizing enzymes in the rat central nervous system

The distribution of cysteine oxidase (CO) and cysteine sulfinate decarboxylase (CSD) was examined in 12 regions of the rat central nervous system (CNS). The distribution of CO activity, expressed as mol of cysteine sulfinate formed per h per g, was the following: hypothalamus, superior and inferior colliculi, 94–99 mol/h/g; olfactory bulbs, cerebral cortex, striatum, and hippocampus, 44–51 mol/h/g; cerebellum, 71 mol/h/g; pons-medula and spinal cord, 94 and 60 mol/h/g, respectively. The distribution of CSD activity expressed as mol of cysteine sulfinate decarboxylated per h per g was the following: hypothalamus and colliculi, 14–21 mol/h/g; olfactory bulbs, cerebral cortex, striatum, hippocampus, and cerebellum, 8–13 mol/h/g; pons-medulla, 7.3; and spinal cord, 3.6 mol/h/g. No CSD activity was detected in sciatic nerve. The subcellular distribution of CO and CSD activities was studied in hypothalamus, colliculi, and cerebral cortex. CO activity was localized in synaptosomes, mitochondria, and microsomes. CSD was primarily confined to the crude mitochondrial fraction and after subfraction, recovered mainly in the synaptosomal fraction.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

A chromosome study ofSchistocephalus solidus (Müller, 1776) (Cestoda: Pseudophyllidea)

Analysis of Giemsa-stained metaphase plates of plerocercoids ofSchistocephalus solidus revealed the mode diploid number of chromosomes for this species as 2n=18. Chromosome pairs 1, 5, 6, 8 and 9 are metacentric, pairs 2, 4 and 7 are subtelocentric, and pair 3 is subtelocentric to acrocentric. The karyotype is characterised by mean absolute chromosome length between 1.9 and 5.9 m and a total chromosome length of the haploid complement of 32 m. A comparison with other known karyotypes of related species of pseudophyllidean cestodes was made.     

Multiple shoot-bud formation and plantlet regeneration on Castanea sativa Mill. seeds in culture

Primordial initiation and development of shoot-buds has been accomplished by using shoots derived from chestnut (Castanea sativa Mill) seedlings cultured with added 6-benzylaminopurine (BAP). Germination of chestnut seeds in the presence of BAP (4 – 40 M) stimulated varying numbers of shoot-buds in those areas of the main axis that were favorably altered. When excised single shoots from these treated seeds were subcultured on a fresh medium containing BAP (4 – 40 M) continual shoot production was observed. Bud growth and shoot elongation were stimulated by transferring cultures to a reduced concentration of BAP (2 M) plus indole-3-butyric acid (IBA 0.4 M). Plant regeneration occurred in the presence of IBA (0.8 M) after a preconditioning treatment in which naphthaleneacetic acid (NAA 50 M) and kinetin (k 2 M) were applied to the tissue culture shoots for 7 days in light.     

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.     

Differents comportements de 22 souches d'Emmonsia Cifferi et Montemartini, 1959, champignon moniliace,dans les poumons de souris de laboratoire,Comparaison avec leur morphologie parasitaire es vitro

The 22 strains of Emmonsia Ciferri & Montemartini 1959, inoculated intranasally to laboratory mice are not equally virulent. One month after the inoculation, 15 of the strains had produced adiaspores 120 to 190 m in diameter in the lung. Another strain produced adiaspores measuring 44 m and 2 others measuring 20 m or 10 m. The remaining 4 strains did not develop in the lung tissue. Four thermophilic strains, which in vitro have adiaspores measuring 8 to 15 m, had adiaspores reaching 120–180 m in vivo. Neither budding nor endosporulation could be observed in any adiaspore.

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Differents comportements de 22 souches d'Emmonsia Cifferi et Montemartini, 1959, champignon moniliace, dans les poumons de souris de laboratoire, Comparaison avec leur morphologie parasitaire es vitro

Résumé Vingt deux souches d'Emmonsia Ciferri et Montemartini, 1959, inoculées par instillations nasales à des souris de laboratoire, ne présentent pas toutes la même virulence. Un mois après l'inoculation, quinze d'entre elles donnent dans le parenchyme pulmonaire des adiaspores de 120–190 m de diamètre, une souche, des adiaspores de 44 m, une autre, des adiaspores de 20 m et une autre, des adiaspores de 10 m. Les quatre dernières ne se développent pas dans les poumons. Quatre souches thermophiles, donnant in vitro de petites adiaspores de 8–15 m, différencient in vivo des adiaspores de 120–180 m. Aucun phénomène de bourgeonnement ou d'endosporulation n'est observé sur aucune adiaspore.

     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Induction of apoptosis by gemcitabine in BG-1 human ovarian cancer cells compared with induction by staurosporine, paclitaxel and cisplatin

A variety of chemotherapeutic agents induce cell death via apoptosis. We had shown previously that gemcitabine (2,2-difluorodeoxycytidine) induced an atypical apoptosis in BG-1 human ovarian cancer cells; therefore, further studies were conducted to characterize more precisely gemcitabine-induced apoptosis in BG-1 cells compared to a general inducer of apoptosis, staurosporine. BG-1 cells exposed to 0.5, 1.0 and 10 M gemcitabine for 8 h, or staurosporine (1.0 M) for 6 h, exhibited high molecular weight DNA fragmentation (50 kbp); however, only staurosporine treatment produced internucleosomal DNA fragments (200 bp) in a laddered pattern on the agarose gel. Staurosporine (1.0 M) rapidly induced phosphatidylserine plasma membrane translocation that increased linearly with time as measured by annexin V-FITC binding, and similar kinetics were observed for caspase activation by staurosporine in BG-1 cells. In contrast, 10 M gemcitabine increased phosphatidylserine expression in a small fraction of cells (5–10%) vs. untreated controls over the course of 48 h and significant caspase activity was detected within 12 h of drug exposure. Time-lapse video microscopy of BG-1 cells exposed to 1.0 M staurosporine or 10 M gemcitabine for up to 72 h showed that the morphologic changes and kinetics of cell death induced by these agents differed significantly. We also evaluated the apoptosis induced by paclitaxel (a mitotic poison) and cisplatin (an agent not dependent on cell cycle functions) in BG-1 cells by these methods because these drugs are used clinically to treat ovarian cancer. Our findings demonstrate that the kinetics of apoptotic cell death induced by gemcitabine and other chemotherapeutic agents should be taken into account when designing treatment strategies for ovarian cancer.     

Zur Feinstruktur der Elektrozeptorepidermis der Mormyridae (Teleostei,Pisces)

Zusammenfassung Die schwachelektrischen Mormyridae haben eine dreischichtige Epidermis, deren innere Schicht aus nur etwa 0,22 m dicken sechseckigen Zellen von ca. 60 m Durchmesser besteht. Die etwa 2 m dicken, linsenförmigen Kerne von 7,6 m Durchmesser liegen am Zellrand. Die Zellen sind zu Säulen aufgeschichtet. Ihr Rand ist ausgezackt und dort, wo er die Säulengrenze erreicht, auf etwa 0,34 m verdickt. In der Nähe der Säulengrenzen sind die Zellen über Desmosomen mit den Nachbarn in der eigenen und in der angrenzenden Säule verbunden. Diese Epidermisschicht ist auf die Körperpartien beschränkt, in denen auch Elektrorezeptoren ausgebildet sind.Die beiden anderen Epidermisschichten haben den üblichen Aufbau einer Fischepidermis, abgesehen vom Fehlen der Becherzellen.

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Ultrastructure of the electroceptor epidermis of the Mormyridae (Teleostei, Pisces)

Summary The weakly electric fish of the family Mormyridae have a three layered epidermis, with a medium layer consisting of hexagonal cells of only 0.22 m in thickness and about 60 m in diameter. The lens-shaped nuclei are about 2 m thick and 7.6 m in diameter and are situated near the border of the cells. The cells are piled up to hexagonal columns. Their margin is serrate and where it reaches the boundary of the column, it has a thickness of about 0.34 m. Close to the boundaries of the columns, the cells are linked to their neighbours within the column and of the adjoining column by desmosomes. This layer of the epidermis is confined to those regions of the body surface which also contain electroreceptors.The other layers of the epidermis have a structure as usual in fish, except for the lack of goblet cells.

     

Triazole treatment of explant source provides stress tolerance in progeny of Geranium (Pelargonium hortorum Bailey) plants regenerated by somatic embryogenesis

Thehypothesis that chemically induced stress tolerance in plants can betransferredto a larger clonal population regenerated by somatic embryogenesis wasevaluatedusing the triazole compound paclobutrazol as a chemical inducer of stresstolerance in Geranium (Pelargonium horturum Bailey). Seedswere imbibed in 3.4, 10.2 or 17.0 M (1, 3, 5 mgL^–1) paclobutrazol for 24 h and germinatedfor 7 days. Hypocotyl explants were cultured in vitro toinduce somatic embryogenesis. Plants regenerated from somatic embryos wereexposed to heat stress at 56°C. Explants treated with3.4 M paclobutrazol yielded a substantially higher number ofsomatic embryos compared with untreated explants. In contrast, 17.0M paclobutrazol treatment inhibited embryogenesis producing asignificantly lower number of somatic embryos. There was no difference in theembryo number between control and 10.2 M treatment. Somaticembryos derived from 3.4 and 10.2 M paclobutrazol treatedexplants developed into plants at a faster rate than the control and 17.0M treatments. Plants derived from paclobutrazol-treatedexplants displayed a greater tolerance to heat stress compared with thecontrols. Observations in this study provide a technique for regeneratingplantsin tissue/cell culture with additional desirable traits such as stresstolerancewith minimal chemical contamination of the environment.     

Intestinal microflora in the deep-sea isopodBathynomus giganteus

Observations with the scanning electron microscope showed that the deep-sea isopodBathynomus giganteus harbors a dense microflora within the digestive tract. The gut microflora is composed of a diversity of microorganisms, including an unusually large bacterial morphotype that predominates almost exclusively in the anterior end of the hindgut. The majority of these large bacteria measured 1.9 m×8.5 m and many reached a size of 2.0 m×10.0 m.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Ultrastructural analysis of the sperm cells of mature pollen of maize,Zea mays

Summary Ultrastructural analysis of the mature viable unhydrated pollen of maize,Zea mays from dehiscent anthers shows that the sperm cells are physically distant, each bounded by an envelope comprising their own plasma membrane and the inner plasma membrane of the vegetative cell. The chondriome is unusual in containing one or more filamentous complexes, up to 12m in length appressed to the side of the sperm nucleus. The extensions at each end of the elongate sperm cells contain longitudinally-oriented arrays of endoplasmic lamellae. In a three-dimensional reconstruction of serial thin sections, there is a long J-shaped sperm, c. 35 × 5m and up to 1m in thickness, sited within pointed evaginations of the vegetative nucleus and a second shorter sperm c. 20 × 5m and up to 3.5m in thickness.Abbreviations PA-TCH-SP periodic acid-thiocarbohydrazide-silver proteinate - DAPI 4,6-diamino-2-phenylindole - SC sperm cell - Sn sperm nucleus - Ua-Pb Uranyl acetate-lead citrate staining - ER endoplasmic reticulum     

Freeze-fracture studies on the cockroach hemocyte membrane complex: Symmetric and asymmetric membrane particle distributions

Summary Freeze-fracture studies were conducted on the membranes of normal cockroach hemocytes. The plasmalemma is asymmetric with the A fracture face containing 80–100 Å membrane intercalated particles at a concentration of 2500/². The B fracture face contains 120–150 Å particles with a relatively low density (800/²). The nuclear envelope displays an asymmetry with the A fracture face containing 1500 particles/² and the B face containing 300/ ². No significant particle size differences were observed in nuclear envelope fracture faces. Two types of symmetric membranes were also found in these cells. Both A and B fracture faces of the membrane surrounding the numerous cytoplasmic inclusion bodies contain particle sizes and concentrations similar to the B face of the plasmalemma. A second type of symmetry was observed in cells apparently engaged in exocytosis. Vesicles (0.1 D) from this process were completely particle free on both fracture faces. Such particle free vesicles could be found in the cytoplasm, attached to the plasmalemma, or completely separated from the cell.Supported by a Pharmaceutical Manufacturers Association Foundation Fellowship.The author wishes to thank Ms. Annalena K. Charla for assistance in plate preparation, Dr. Julius Schultz and the Papanicolaou Cancer Research Institute for use of the freeze-etch device, and Dr. David Smith for the electron microscope facilities.     

Myosatellite cells associated with different muscle fibre types in the Atlantic hagfish (Myxine glutinosa,L.)

Summary The incidence of myosatellite cells associated with white and red muscle fibres of the parietal muscle and red fibres of the craniovelar muscle was estimated by quantitative electron microscopy in the Atlantic hagfish (Myxine glutinosa, L.). Myosatellite cell nuclei constitute 3, 11 and 23 % of the total number of nuclei inside the basal lamina of the three types of muscle fibres, respectively. However, the total number of nuclei is highest in white fibres, most of the nuclei belonging to striated muscle cells. Myosatellite cell profiles in transverse sections constitute 23, 41 and 61 % of the number of muscle fibre profiles of the three types, respectively. The intervals between adjacent myosatellite cells are 135 m in white fibres, 55 m in red parietal fibres, and only 25 m in craniovelar fibres. Since craniovelar fibres are also comparatively thin, myosatellite cells constitute a significant fraction of the volume inside the basal lamina in these fibres. The myosatellite cells are 30–50 m long and up to 5 m thick. Some myosatellite cells possess few organelles, whereas others appear to contain many free ribosomes, granular endoplasmic reticulum, prominent Golgi apparatus and lysosome-like bodies.This investigation was supported by the Norwegian Research Council for Science and the Humanities (NAVF grant No. C20.30–37). The authors are indebted to Jorunn Line Vaaland and Berit Branil for technical assistance, and to Dr. Finn Walvig, Biological Station, University of Oslo, Drøbak, for supplying the hagfish     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Determination of copper, manganese and zinc in human liver

Autopsied liver tissue samples collected from 42 males and 31 females were analyzed for copper, manganese and zinc using atomic absorption spectrometry (AAS). With the exception of two liver samples for which the copper levels were determined to be 74.8 and 104.0 g/g (dry weight), hepatic copper concentrations were found to range from 1.7 to 32.4 g/g with a mean concentration of 14.2 g/g and standard deviation of 7.0 g/g. Manganese concentrations (with the exception of one sample having 12.9 g/g) ranged from 0.22 to 4.6 g/g with a mean of 2.26 ± 1.00 g/g. Hepatic zinc levels averaged 118.3 ± 44.4 g/g and ranged from 38.5 to 231.3 g/g. There were no apparent trends for the levels of any metals versus age nor were there any differences in average hepatic metal concentrations for males and females. © Rapid Science 1998.     

Lead‐related effects on rat fibroblasts

Lead (Pb) is an environmental toxicant that can induce structural and functional abnormalities of multiple organ systems, including the central nervous and the immune systems. The aim of this study was to evaluate the effects of extracellular Pb supplementation on the cellular content of the metal and on the proliferation and the survival of normal rat fibroblasts.We found that the concentration of Pb in the culture medium was 0.060 M and the normal Pb concentration in rat fibroblasts was 3.1 ± 0.1 ng/10⁷ cells. Then we exposed the cells to increasing concentration of Pb (as Pb acetate) from 0.078–320 M. We observed a dosedependent inhibition of cell proliferation after 48 h, which was already apparent at a concentration of 0.312 M (p = 0.122) and became statistically significant for concentration higher than 0.625 M (p = 0.0003 at 5 M). Cell proliferation was completely compromised at 320 M Pb total inhibition of cell proliferation.To investigate the mechanisms of Pbmediated inhibition of cell proliferation, we evaluated the occurrence of apoptosis in the same cells and found that cytosolic DNA fragments, hallmark of apoptotic cell death, increased significantly at Pb concentrations from 2.5–10.0 M. The occurrence of apoptosis was also confirmed by FACS analysis which showed the appearance of a subdiploid peak at Pb concentrations from 5–20 M. The distribution of cells in the cell cycle showed a dosedependent accumulation of cells in the G₀/G₁ phase mainly compensated by a decrease in the percentage of cells in the S phase. In conclusion, our results demonstrate that induction of apoptosis contributes to the Pbinduced inhibition of cell proliferation in rat fibroblasts.     

Modelling of sulfur oxidation from elemental sulfur

An elemental S oxidation model has been developed which combines a maximum S release rate with modifiers for temperature and soil moisture conditions. This model has been combined with a pasture growth and CNSP nutrient cycling model to match S oxidation rate to pasture S demand. In two Southern Australian enviroments, 100m elemental S was superior to 200m particles whilst in Northern Australia the 200m particles were superior. These models can be used to match S release to plant demand.     

Ultrastructural changes in the mitochondria of brown adipose cells during the hibernation cycle of Citellus lateralis

Summary The mitochondrial structure in the brown adipose cells of the golden mantled squirrel, Citellus lateralis, was examined throughout the year in biopsy samples. The mitochondria showed remarkable and apparently reversible changes in size and internal structure related to the physiologic activity of the animal. In the active animal the size of the largest mitochondria was 2.4 m × 1.5 m; during hibernation it increased to 7 m × 2.5 m; and during arousal it reached 11.2m × 5.3 m. The cristae of the mitochondria in the brown adipose cells of the animals in hibernation phase formed loops, whorls and mesh-like interconnections. During the arousal phase they underwent further configurational changes. The most remarkable structure was associated with mitochondria of most unusual proportions which by dissolution gave rise to a new generation. This was a common finding during arousal but did not occur in any other phase of the hibernation cycle. The new mitochondria were virtually indistinguishable from those of brown adipose cells of any active animal.Supported by a grant from the Medical Research Council of CanadaThe author is grateful to colleagues, Dr. G. Dempster and Dr. W.A. Spencer, for many valuable suggestions in the course of the work