A comparative analysis of various methods used to evaluate α-adrenergic blocking activity of neuroleptic drugs

Eight neuroleptic drugs were studied for α-adrenergic blocking activity using three different pharmacologic methods: (1) protection against norepinephrine lethality in rats (NEL), (2) inhibition of WB-4101 binding in rat brain (WB), and (3) antagonism of pressor effects to norepinephrine in perfused rat hindquarters (DR₁₀). Results from each of the methods were subjected to Spearman Rank correlation analysis in order to determine if NEL and WB would accurately predict α-adrenergic blocking activity in the cardiovascular system. Both the NEL and WB assays were found to correlate significantly with results from the rat perfusion experiments. These results support the concept that WB-4101 and NEL screening methods are valid predictors of cardiovascular α-adrenergic antagonist activity.     

Evidence for increased amyloid enhancing factor activity in Alzheimer brain extract

Summary Soluble brain extracts containing 0.1 to 16 mg of protein from 3 normal human brain and 11 patients with Alzheimer's disease, Down's syndrome and other neurological disorders were assayed for amyloid enhancing factor (AEF) activity in the mouse bioassay. At the 0.1 mg dosage, five of seven brain extracts from amyloid-positive samples and only one of four amyloid-negative samples demonstrated AEF activity. Marginal AEF activity was detected in the normal brain extracts at 8 or 16 mg protein dosage. Alzheimer-AEF was aggregated by exhaustive dialysis against 0.01 M phosphate buffer, pH 6 or distilled water and the solubilized aggregate was fractionated on a BioGel P-60 column. Of the two protein peaks, AEF activity was present only in the low mol · wt second fraction, which on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining showed two discrete and three minor peptide bands between 60 and 66 kDa and one of these was periodic acid-Schiff positive, and three fuzzy bands near 14 kDa. Pretreatment of the crude and second fraction with 10 mM phenylmethylsulfonyl fluoride (PMSF) nearly completely abolished the in vivo AEF bioactivity. It is suggested that (a) a higher AEF concentration is present in amyloid-positive brain samples than those negative for amyloid or normal brain tissues, (b) AEF-positive fraction contains at least five dominant peptides ranging between 14 to 66 kDa, and (c) abolition of PMSF-treated Alzheimer-AEF activity, similar to that of murine AEF, might be due to its serine/thiol proteinase nature. To our knowledge, this is the first time that AEF activity has been demonstrated in Alzheimer brain samples.Supported by a grant (MA-10172) from the Medical Research Council of Canada     

Determination and interpretation of hydrocarbon toxicity to ciliate protozoa

Acute toxicity threshold concentrations of 17 hydrocarbons to two species of ciliate protozoa were measured in a conventional ‘open’ system and in a novel ‘closed’ bioassay system consisting of a gas-tight syringe. Colpidium colpoda was employed as the test organism in the former system while Tetrahymena elliotti was used in the latter case. Partitioning analysis of the results highlighted the importance of controlling or eliminating the air space during the bioassay procedure, and of considering the quantity of hydrocarbon which may partition into test organisms. Although the toxicity thresholds, expressed as aqueous concentrations, varied by a factor of 1300, the corresponding biotic concentrations calculated from the partitioning analysis were relatively constant, thus providing a capability to predict toxicity effects from the solubility, octanol-water partition coefficient, or HPLC retention time of a compound. Some solid hydrocarbons, because of their fugacity ratios, were unable to establish a sufficient aqueous concentration to exert an acute toxic effect. The mechanism of toxicity and the applicability of the test methods and partitioning analysis to other organisms and compounds are discussed.     

Human Sperm Penetration into Zona-Free Hamster Oocytes as a Test to Evaluate the Sperm Fertilizing Ability

Penetration menschlicher Spermatozoen in Zona pellucida-freie Hamster-Oozyten. Test zur überprüfung der Fertilisation von Spermatozoen
Der Eintritt menschlicher Spermatozoen in pellucidafreie Oozyten von Hamstern wurde verwandt, um die Fertilität menschlicher Samenproben einer andrologischen Klinik zu untersuchen. Die Chromatin-Dispersion des Spermas, im Phasenkontrastmikroskop festgestellt, wurde als Kriterium für den Spermatozoeneintritt genommen. Die Ultrastrukturstudie zeigte, daß das Verhalten der Gametenmembran während der Verschmelzung sich nicht wesentlich von normalen Fertilitätsvorgängen unterscheidet.
Vierundachtzig Proben wurden als normal (bezogen auf das Spermiogramm) eingeschätzt, aber nur 62 (74%) erzielten ein positives Ergebnis im Vergleich zu 30 (32%) positiven Ergebnissen von 95 Proben pathologischer Spermiogramme. Achthundertzehn Oozyten, die mit Spermatozoen von normalen Proben befruchtet wurden, erreichten eine Penetration von 25%, während 1046 Oozyten, die mit pathologischen Samenproben befruchtet wurden, eine Penetration von 7% erlangten. Der Normalzustand der Hamsteroozyten wurde durch eine gleichzeitige Befruchtung mit Spermatozoen von Menschen und Hamstern nachgewiesen. Während der Prozentsatz menschlicher Spermatozoenpenetration niedrig blieb, war die der Hamster über 70%.
Der vorliegende Bioassay wird als zusätzlicher Parameter zu den Spermiogrammen zur Untersuchung menschlicher Zeugungsfähgikeit empfohlen.     

The effect of cytokines on the in-vitro erythropoietin bioassay

Summary. The effects of various concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (γIFN) and interleukins 1 α (IL-1α), 1β (IL-1β) and 3 (IL-3) on the anaemic mouse spleen cell bioassay for erythropoietin (EPO) were investigated. Addition of IL-3 and GM-CSF at various concentrations had no effect on EPO stimulated ³H thymidine incorporation. However the addition of IL1-α, IL1-β and γIFN (3.3 × 10^-8 gl^-1) caused a significant (P < 0.01) inhibition of EPO stimulated thymidine incorporation. This suggests that the EPO bioassay may be influenced by variable levels of some inflammatory cytokines in serum. Previous studies have shown that the bioassay is influenced by serum transferrin levels and thus serum immunoassays remain the technique of choice for specific estimates of EPO. Since EPO bioassays are not specific, they should be reserved for situations in which an estimate of the total erythropoietic activity of serum is required.     

A dose-dependent effect of recombinant erythropoietin on the reticulocyte population of rats

Summary The effect of human recombinant erythropoietin (rEPO) on the reticulocyte count and the reticulocyte maturation distribution in rats was measured with an automatic reticulocyte analysis system (AURAS). The strongest reaction was found on the third day after a single application of rEPO with respect to reticulocyte count, maturation distribution, and the count of immature reticulocytes. At this time these parameters show a distinct dose dependency. The results indicate a possible basis for a simple rEPO bioassay using normocytemic rats.     

Bioassay detection of mouse nerve growth factor (mNGF) in the brain of adult mice

Two pools of seven brains each from adult Swiss-Webster mice were homogenized, and supernatants were collected for bioassay. PC-12 cells were placed in a bioassay plate at time zero, at a concentration of 10⁴ cells per well, and primed for 48 hours in a medium containing 50 ng/ml of mNGF. The PC-12 cell bioassay for neurite outgrowth was conducted after primed cells were exposed to an NGF-free medium for 24 hours. Suitable controls for serum toxicity and cell viability were established. The sensitivity of the bioassay approximates 100 pg NGF/ml. The results showed 80–100% neurite outgrowth in wells exposed to brain pool supernatant (BPS) alone, and control level outgrowth (3–8%) in wells containing BPS and specific anti-β-NGF antibody. Therefore, the brains of Swiss-Webster adult mice contain an NGF-like substance which promotes neurite outgrowth in PC-12 cells. The substance probably is NGF itself, since the effect is blocked by specific NGF antiserum.     

The quantitative bioassay of nerve growth factor: use of frozen ‘primed’ PC12 pheochromocytoma cells

A quantitative bioassay for nerve growth factor (NGF) has been previously described which employs PC13 rat pheochromocytoma cells. The basis of this assay is that rapid neurite regeneration by PC12 cells pretreated ('primed') with NGF for at least a week is quantitatively related to the amount of NGF present in the culture medium. The present experiments demonstrate that NGF-pretreated PC12 cells retain their priming for over a year when appropriately frozen and stored and that such frozen cells can be employed for the quantitative bioassay of NGF. The major advantage of this assay is that large numbers of cells can be primed at a time (in suspension culture) and stored in aliquots until future use. The newly described bioassay employs the frozen primed cells essentially as reagents and avoids the need to have freshly primed cultures on hand.     

Comparison between rat prolactin radioimmunoassay and bioassay values under different experimental and physiological conditions

Serum rat PRL concentrations were compared using values determined by RIA and the Nb2 lymphoma cell bioassay (Nb2BA). Rat serum samples were obtained under different physiological conditions and after the administration of pharmacological agents known to affect PRL secretion. Of the treatments examined, estrogen, morphine bromocriptine and haloperidol significantly altered the relationship between Nb2BA and RIA estimates of PRL. The estrogen-induced increase in PRL levels of ovariectomized females and the proestrus surge of PRL in intact females led to higher bioassay than RIA estimates of PRL. Treatment with haloperidol, bromocriptine and morphine altered the relationship, favoring immunoreactive more than bioactive hormone, and reversing the pretreatment Nb2BA/RIA ratio. No discrepancies between estimates of PRL by the two assays were noted in untreated males, diestrous, estrous and ovariectomized females, or following ether or TRH administration. These results confirm previous observations of discrepancies between rat serum bioassay and RIA estimates, and the data suggest that different forms of prolactin are present in the circulation at different times.     

Sublethal effects of treated liquid effluent from a petroleum refinery. IV. Respiratory movements and coughing of rainbow trout

Rainbow trout increased the frequency of gill irrigation in linear relation to the concentration of treated refinery effluent. Full-strength effluent resulted in a significantly higher frequency than in clean water, but 50% and lower did not, because of variation in response. Coughing rate increased sharply and was a less variable measure of response. A concentration of 50% effluent caused significantly more coughs than in clean water, while 25% did not. The eight samples of effluent caused little lethality at full strength, and average physico-chemical characteristics were near or below Canadian regulatory limits, except for elevated oil and grease. Coughing rate shows promise as a rapid initial method for sublethal screening or monitoring of refinery effluents.