King Lau Chow

Synergistic toxicity of multiple heavy metals is revealed by a biological assay using a nematode and its transgenic derivative

Caenorhabditis elegans, a free-living nematode species, was adopted for a toxicity bioassay of 10 heavy metals. The lethal concentration (LC) of these metals was determined. Based on these data, we conducted pairwise and triple metal combination testing and demonstrated that these heavy metals displayed synergistic killing effects on C. elegans larvae. Drastic increases in mortality rate up to 100% could be observed at low metal concentrations. The results illustrate the complexity of toxicity tests in biological systems and show that physical-chemical monitoring of toxicants may underestimate biohazards in environmental samples. We also demonstrate that a transgenic derivative nematode strain, KC136, carrying a heat shock promoter driven gfp reporter gene could be used to reduce the duration of an assay so that the synergistic effects among toxicants could be revealed. This derivative strain allows rapid and frequent monitoring of environmental hazards, which usually requires the handling of a large number samples.

Study of hydroxyapatite osteoinductivity with an osteogenic differentiation of mesenchymal stem cells

Osteoinductivity of hydroxyapatite (HA) was investigated using uncommitted pluripotent mouse stem cells, C3H10T1/2 in an in vitro differentiation assay. For comparative analysis, the cells were cultured on substrates made of osteoinductive HA, with biocompatible titanium and plastics as the negative control. HA exhibited the ability to induce expression of osteo-specific genes in C3H10T1/2, including alkaline phosphatase (ALP), type I collagen, and osteocalcin; compared with its insignificant up-regulation of the same genes in osteoblast-like cells, Saos-2. HA osteoinductivity exhibited in C3H10T1/2 was comparable to that of a bone morphogenetic protein (BMP) with reference to the up-regulation of osteo-specific genes except the core binding factor 1 (Cbfa1, Runx). This result implies a difference in osteogenic induction pathway initiated by HA and BMP. Using this mesenchymal stem cells (MSC) culture assay, osteoinductivity was also demonstrated to be present in the conditioned medium derived from MSC cultured on HA substrates. This conditioned medium exhibited excellent ability to up-regulate ALP in the absence of HA and BMP. The results suggest that the HA can interact with the cells and generate potent inductive substance released into the medium. Such substance in turn is able to induce uncommitted cells to differentiate into the osteolineage.

The species, sex, and stage specificity of a Caenorhabditis sex pheromone

Four species in the ELEGANS group of subgenus the Caenorhabditis are distinguished by two very different mating systems: androdioecy in C. elegans and Caenorhabditis briggsae with males and self-fertilizing hermaphrodites and dioecy in Caenorhabditis remanei and Caenorhabditis sp. strain CB5161 with males and females. Using chemotaxis assays, we demonstrate that females secrete a potent sex pheromone that attracts males from a distance, whereas hermaphrodites do not. The female sex pheromone is not species-specific, with males of all four species attracted to both the C. remanei and Caenorhabditis sp. female sex pheromones. The pheromone is, however, sex-specific, with only females secreting the pheromone and attracting only males. Furthermore, the sex pheromone is stage-specific, with female secretion and male detection of the pheromone beginning near adulthood. Females lose their attractiveness immediately after mating but regain it several hours after mating ceases. Finally, the female somatic gonad is required for sex-pheromone production, and the male-specific cephalic neurons (CEM) are required for male response.

Cell culture medium as an alternative to conventional simulated body fluid.

Simulated body fluid (SBF) has been widely used for bioactivity assessment of biomaterials. Many different recipes have been developed, but there is still room for improvement. We have suggested the use of cell culture medium to overcome the drawbacks of conventional SBF. Compared with conventional SBF, cell culture medium is easy to prepare. The normal practices in cell culturing, such as filtering, can eliminate insoluble precipitates in the medium and incubation at 37 °C in an atmosphere with 5% CO(2) also better simulates the in vivo environment. After 4 days immersion in carbonate buffered Dulbeccos modified Eagles medium (DMEM), precipitates were found to have formed on the surfaces of hydroxyapatite (HA), α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP). In order to further verify the use of cell culture medium for SBF studies, we carried out computational thermodynamic and kinetic analyses of the precipitation reaction to reveal the effect of pH and ion concentrations on the driving force and nucleation rate of precipitation of different calcium phosphates (CaP). In general, a slight increase in pH of the cell culture medium from physiological pH (pH 7.4) would favor CaP precipitation thermodynamically and increase the rate, as in the case of r-SBF reported previously. [Ca] and [P] have more impact on precipitation compared with other ions, but the effect is consistent among different materials, indicating that other cell culture media with slightly different compositions may also be used. This study also shows that matching the buffer with the environment is required and fetal bovine serum (FBS) slows down surface CaP precipitation on HA.

SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS

Common dehydration methods of cells on biomaterials for scanning electron microscopy (SEM) include air drying, hexamethyldisilazane (HMDS) or tetramethysilane (TMS) treatment and critical point drying (CPD). On the other side, freeze-drying has been widely employed in dehydrating biological samples and also in preparing porous biomaterial scaffolds but not in preparing cells on three-dimensional (3D) biomaterials for SEM examination. In this study, we compare cells on porous hydroxyapatite (HA) prepared by air drying, HMDS and freeze-drying. The effects of fixation and using phosphate buffered saline (PBS) in the fixation were also assessed on three porous calcium phosphate (CaP) materials, namely, HA, α-tricalcium phosphate (α-TCP) and β-tricalcium phosphate (β-TCP) samples. There is no significant difference in samples prepared by HMDS treatment and freeze-drying viewed at low magnification. Besides, it is better not to use phosphate buffer in the fixation step for CaP materials to avoid undesirable spontaneous precipitation of CaPs. On the other hand, fewer exchanges of liquids are required for freeze-drying and hence chemical fixation may not be absolutely required for samples prepared by freeze-drying. Other technical details of the preparation were also investigated and discussed. This study suggests both HMDS and freeze-drying can be employed to dehydrate cells on 3D scaffolds for SEM examination.

Expression of ram‐5 in the structural cell is required for sensory ray morphogenesis in Caenorhabditis elegans male tail

Tissue morphogenesis requires complex cellular interaction and communication. The sensory ray in the Caenorhabditis elegans male tail has a simple cellular make‐up and a non‐essential function, thus providing an ideal model for studying the mechanisms guiding morphogenesis. We present here the analysis of a novel gene, ram‐5, mutations of which are characterized by abnormal lumpy rays in the male tail. Microscopic analysis and behavioral studies revealed that lumpy rays contain operational sensory neurons. However, abnormalities were observed in the hypodermis and structural cells as well as in appositions between these two cell types. Molecular cloning and expression studies revealed that the ram‐5 gene encodes a transmembrane protein localized in sensory ray support cells, the structural cells. Expression of ram‐5 in these cells is required for normal ray morphogenesis. ram‐5‐dependent cell–cell communication is implicated in organizing the structural cell and the hypodermis, potentially through adhesion at the structural cell–hypodermal cell border.

Expression of zebrafish mab21 genes marks the differentiating eye, midbrain and neural tube

Mab21 homolog in Caenorhabditis elegans is expressed in the sensory ray cells and determines the ray identity (Dev. Dyn. 221 (2001) 422). Similarly, vertebrate homologs of this gene have been shown to express in the craniofacial tissues, limb bud and neural tissues. We report here the cloning and expression analysis of two zebrafish mab21 genes. Both genes have a conserved open reading frame of 1080 nucleotides. Phylogenetic analysis suggested that the two family subgroups existed prior to the divergence of vertebrates. Their expression profiles revealed that mab21l1 was turned on in embryos as early as 8 h post-fertilization (hpf) while mab21l2 was first detected at around 11 hpf. In situ hybridization results showed that expression of these mab21 genes marked the early differentiating olfactory bulbs, eye primordia, midbrain and subsequently the branchial pouches and neural tube.

Postembryonic expression of Caenorhabditis elegans mab-21 and its requirement in sensory ray differentiation.

The male tail sensory rays in Caenorhabditis elegans are complex copulatory structures, the normal patterning of which requires a number of regulatory genes. Among them, mab‐21 specifies the identity of sensory ray 6. By using green fluorescent protein reporters, we identify multiple cis‐acting elements that control the developmental expression of mab‐21. Traced with a functional mab‐21:gfp gene driven by authentic regulatory sequences, mab‐21 expression could be detected in hypodermal, neuronal, muscle, and ray cells. We showed here that the expression of mab‐21 in the hypodermis and neuronal cells was dispensable for its function in ray 6. In contrast, its expression in the ray 6 structural cell and neurons as conferred by its 3′ enhancer was crucial for determining the correct ray 6 identity.

Developmental expression of Mab21l2 during mouse embryogenesis.

mab-21 has been identified as a critical component required for sensory organ identity establishment in Caenorhabditis elegans. [Chow, K.L., Emmons, S.W., 1994. Development 120, 2579-2592; Chow, E. L., Hall, D.H., Emmons, S.W., 1995. Development 121, 3615-3625]. Human and mouse homologs of this gene have been isolated and their transcripts are predominantly detected in the eye and cerebellum [Margolis, R.L., Stine, O.C., McInnis, M.G., et al., 1996. Hum. Mol. Genet 5, 607-616; Mariani, M., Corradi, A., Baldessari, D., et al., 1998. Mech. Dev. 79, 131-135. We report here the expression profile of a second murine mab-21 homolog, Mab21l2 [Wong, R.L.Y., Wong, H.T., Chow, K.L., 1999. Cyto. Cell Genet., [in press]. Whole mount in situ hybridization data from embryonic day 8.5 to day 15 revealed that Mab21l2 expression patterns partially overlapped with that of Mab21l1. In addition, its strong expression in the mid- and hindbrain, otic vesicle, optic vesicle, maxillary and mandibular process, paraxial mesoderm, dorsal midline, limb bud and developing digits suggest that Mab21l2 has more diverse functions in vertebrate development.

Mouse embryo cryopreservation utilizing a novel high-capacity vitrification spatula

Embryo cryopreservation is an indispensable technique in reproductive programs and in animal facilities where genetically modified mice are used extensively. Here we report the use of a vitrification spatula (VS) that can be readily homemade and has a large holding capacity to vitrify preimplantation mammalian embryos in a micro-drop employing ultra-rapid cooling in liquid nitrogen (LN2). Vitrified one-cell embryos and morulae have high survival rates after thawing, and the fertility of the derived progeny is comparable to that of the control unvitrified group. The large holding capacity (up to 50 embryos per VS) does not only allow rapid expansion of storage capacity for additional mouse strains but also opens up the possibility to streamline transgenic mice generation procedures in transgenic facilities.