A Precise Method for Replicating Suspension Cultures of Mammalian Cells

A simple, readily assembled shaker-culture system for the cultivation of mammalian cells is described. No specialized glassware and equipment were used in this system, which consists of an Erlenmeyer flask fitted with a breather-sampling assembly. This unit was employed to quantitate the effects of several variables, including medium ingredients, serial transfers, and freezing and storage on two variants of the L-cell line. This system is reproducible and precise and allows for growth of cells in suspension for extended periods of time. Large numbers of cells can be mass-produced. Many replicates can be run simultaneously to yield data for statistical analysis.     

Growth of Human Lymphoid Cells (Raji Strain) in a Five-Liter Fermentor

Procedures used to produce an established line of mammalian cells (L cell) in a New Brunswick fermentor were adapted to propagate human lymphoid cells in suspended culture. Control of pH within defined limits was more effective for regulation of cell metabolism than control of oxidation-reduction potential. An unusually high rate of agitation was required.     

Immunological characterization of embryonic cell surface antigens recognized by antiblastocyst serum

Stage-specific cell surface antigens expressed during mouse preimplantation development and detected by a rabbit antiserum prepared against mouse blastocysts (A-BL₂) have been characterized by serological and biochemical techniques. Immunofluorescence, immunoradiolabeling, and complement-mediated cytotoxicity assays reveal the expression of A-BL₂ surface antigens beginning at the 4-cell stage and reaching a maximum at the 8-cell to morula stages. At earlier times in development A-BL₂ antigens are not detectable, and there is a decline in expression at the blastocyst stage. No antibody reactivity is detected against adult mouse tissues or teratocarcinoma cell lines. The presence of A-BL₂ antibodies during in vitro embryo culture interferes with normal development. Treatment of embryos with β-N-acetylglucosaminidase, but not other glycosidases, proteases, or lipases, results in a quantitative decrease in the binding of A-BL₂ antibodies to surface antigens. Immunoprecipitation and electrophoretic analyses of A-BL₂ antigens demonstrate specific antibody activity against a pair of embryonic glycoproteins of 65,000 to 70,000 daltons which can be metabolically labeled with ³⁵S-methionine and ³H-glucosamine. Tunicamycin treatment alters the form of the A-BL₂ immunoprecipitate to a single 60,000-dalton protein.     

Behavioural approaches to demonstrate the ecological significance of exposure of juvenile Pacific salmon (genus Oncorhynchus) to the antisapstain fungicide TCMTB

Simulated stream conditions were used to expose underyearling Pacific salmon (genus Oncorhynchus) to sublethal doses of TCMTB (2-thiocyanomethylthio) benzothiazole; a fungicide used by the lumber industry to protect against discolouration by sapstain fungi. Observed changes in schooling, swimming, and respiration were studied in more detail utilizing progressively more complex and ecologically relevant experimental designs. Laboratory troughs (600 litre) were used to quantify innate shelter-seeking behaviour and susceptibility to predation during salinity stress. Swimming speed, schooling behaviour and responses to salinity and hypoxia were studied in a 4500-litre Water Column Simulator under vertically stratified conditions (fresh water overlying sea water). Finally a similarly stratified 15,500-litre outdoor tank was designed to compare susceptibility to predation during a volitional salinity challenge. Relative to controls, TCMTB-exposed underyearling salmon exhibited a reduction in cover response, were slower to seek shelter from bright light, were more erratic in their swimming behaviour and more likely to be eaten by marine predators. Videotape analyses confirmed that TCMTB reduced exploratory behaviour and swimming speed under both normoxic and hypoxic conditions in the WCS. The ecological consequences of these overt behavioural changes were experimentally linked to a clear increase in risk from predation by yellowtail rockfish (Sebastes flavidus) inhabiting the marine zone below the halocline of the 15,500-litre tank. These techniques illustrate our most recent efforts to correlate cumulative sublethal physiological stress with ecologically meaningful behavioural dysfunction.     

Effects of constant darkness and constant light on circadian organization and reproductive responses in the ram

The relationship between circadian rhythms in the blood plasma concentrations of melatonin and rhythms in locomotor activity was studied in adult male sheep (Soay rams) exposed to 16-week periods of short days (8 hr of light and 16 hr of darkness; LD 8:16) or long days (LD 16:8) followed by 16-week periods of constant darkness (dim red light; DD) or constant light (LL). Under both LD 8:16 and LD 16:8, there was a clearly defined 24-hr rhythm in plasma concentrations of melatonin, with high levels throughout the dark phase. Periodogram analysis revealed a 24-hr rhythm in locomotor activity under LD 8:16 and LD 16:8. The main bouts of activity occurred during the light phase. A change from LD 8:16 to LD 16:8 resulted in a decrease in the duration of elevated melatonin secretion (melatonin peak) and an increase in the duration of activity corresponding to the changes in the ratio of light to darkness. In all rams, a significant circadian rhythm of activity persisted over the first 2 weeks following transfer from an entraining photoperiod to DD, with a mean period of 23.77 hr. However, the activity rhythms subsequently became disorganized, as did the 24-hr melatonin rhythms. The introduction of a 1-hr light pulse every 24 hr (LD 1:23) for 2 weeks after 8 weeks under DD reinduced a rhythm in both melatonin secretion and activity: the end of the 1-hr light period acted as the dusk signal, producing a normal temporal association of the two rhythms. Under LL, the 24-hr melatonin rhythms were disrupted, though several rams still showed periods of elevated melatonin secretion. Significant activity rhythms were either absent or a weak component occurred with a period of 24 hr. The introduction of a 1-hr dark period every 24 hr for 2 weeks after 8 weeks under LL (LD 23:1) failed to induce or entrain rhythms in either of the parameters. The occurrence of 24-hr activity rhythm in some rams under LL may indicate nonphotoperiodic entrainment signals in our experimental facility. Reproductive responses to the changes in photoperiod were also monitored. After pretreatment with LD 8:16, the rams were sexually active; exposure to LD 16:8, DD, or LL resulted in a decline in all measures of reproductive function. The decline was slower under DD than LD 16:8 or LL.(ABSTRACT TRUNCATED AT 400 WORDS)     

Predation of incubator birds (Megapodius freycinet) by Komodo dragons (Varanus komodoensis)

A short study was made of the nesting habits of a species of megapode, Megapodius freycinet on the island of Komodo. The birds were found to build giant nesting mounds (0.95 × 7.15 m) in scrub and woodland close to the seashore or alongside river beds; 23 active and 19 abandoned mounds were located in an area of 250 ha.
The mounds were apparently added to from year to year, and consisted of dry sand and soil, plus leaves and sticks in most cases. The upper parts of the mounds were invariably exposed to the direct rays of the sun; the temperature within the active mounds was 33–37° C and slightly less in mounds not in use by the birds. According to the local people the nesting season of the megapodes extended from late August to November, and the chicks were hatched before the arrival of the rains in December. Up to four eggs had been recovered from a single mound.
The active mounds were all spaced apart by at least a 100 m, and the closest ones were usually separated by a river bed. This pattern suggested the species was territorial. One or two birds were often seen scraping at a mound or feeding close by, and their behaviour was recorded.
The Komodo dragon, Varanus komodoensis , was a frequent visitor to the nesting mounds of the megapodes, and was able to burrow into them and steal the eggs.