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Embryonal carcinoma (EC) cells provide a caricature of pluripotent embryonic stem (ES) cells and may be used as surrogates for investigating the mechanisms that regulate cell differentiation during embryonic development. NTERA-2 is a human EC cell line that differentiates in response to retinoic acid yielding cells that include terminally differentiated neurons. The expression of genes known to be involved in the formation of the vertebrate nervous system was examined during retinoic acid-induced NTERA-2 differentiation. Differentiation of these human EC cells into neurons could be divided into three sequential phases. During phase 1, in the first week of differentiation, hath1 mRNA showed a small transient increase that correlated with the rapid accumulation of nestin message, a marker of neuroprogenitors. Transcripts of nestin were quickly downregulated during phase 2 as expression of neuroD1, characteristic of neuroprogenitors exiting the cell cycle, was induced. A neural cell surface antigen, detected by the monoclonal antibody A2B5, was expressed by cells exiting the cell cycle, correlating with the expression of neuroD1 as the cells became post-mitotic. Markers of mature neural cells (e.g. synaptophysin and neuron-specific enolase) were subsequently increased during phase 3 and were maintained. This regulated pattern of gene expression and commitment to the neural lineage indicates that differentiation of NTERA-2 neurons in vitro follows a similar pathway to that observed by neural ectodermal precursors during vertebrate neurogenesis in vivo.

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability ofthe HepG2 cells they were cultured in 4-32 jiM copper(II) sulphate (CuS04)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 jiM Cu2 + but significantly declined at 64 MM compared to the controls. Lysosomal acid phosphatase showed a concen-tration dependent decline in activity at 72 h. Introduction Copper is a potent catalyst of processes generating reactive oxygen intermediates and its cellular up-take, transport and excretion are therefore tightly controlled. Elevated hepatic copper concentrations are associated with liver damage in Wilson's disease2, in Indian childhood cirrhosis3 (ICC), and in other copper-related cirrhoses of infancy. Nevertheless, the cellular consequences of copper toxicity remain to be understood in the context of the disease pathology. In Wilson's disease, biliary excretion of copper is impaired by mutations in a trans-Golgi P-type ATPase4 resulting in intracellular hepatic copper accumulation5 with progressive dysfunction, fibrosis, and liver failure. However, clinical variability of Wilson's disease and the lack of a clear genotype/ Cellls exposed to 64 MM Cu2 + had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 jiM Cu2 +, increases of necrosis up to 18% were seen whereas comparable levels ofapoptotic and necrotic cells (< 5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming effi-ciency at concentrations of 16 MM Cu2 + and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.

Response of virgin female Lutzomyia longipalpis Lutz & Nevia (Diptera: Psychodidae), contained in a 60-cm3 net cage, to sticky traps was significantly greater when the traps were baited with extract of male pheromone glands. Results confirm that the response of females to male pheromone does not necessarily depend upon the presence of a host.

Pheromone-disc contact experiments are described in which different populations of the sandfly Lutzomyia longipalpis from Brazil were exposed to each others male pheromone extracts. The Jacobina population reacted only to the conspecific pheromone. The Sobral 2S population, however, would respond to the pheromone of Jacobina males, but if given a choice showed a preference for conspecific extract. Mate choice experiments are also described using sandflies of two pheromone types from Sobral. One population preferentially selected conspecific sexual partners, but the other showed poor mating propensity.

A nylon net cage (260 x 40 x 40 cm) provided with unidirectional air flow was used to demonstrate the response of female sandflies, Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae) to a host and extract of male pheromone glands over a distance of 240 cm. Females responded more rapidly and in greater numbers to the host if male pheromone was present. Results suggest that one function of the pheromone may be to attract females from some distance to a common mating site, on or near the host.