Specific inhibition of caspase-8 and -9 in CHO cells enhances cell viability in batch and fed-batch cultures

In an attempt to investigate the molecular mechanism that leads to apoptotic death in Chinese hamster ovary (CHO) cells in batch and fed-batch cultures, we cloned caspase-2, -8 and -9 from a CHO cDNA library. Recombinant Chinese hamster caspase-2 and -9 expressed in Escherichia coli show highest activities towards commercial peptide substrates Ac-VDVAD-pNA and Ac-LEHD-pNA, the designated commercial substrates for human caspase-2 and -9, respectively. However, Chinese hamster caspase-8 shows a broad specificity profile and it cleaves the caspase-9 substrate more efficiently than it cleaves the caspase-8 substrate. The commercially available fluoromethyl ketone type of caspase inhibitors, such as Z-LEHD-fmk, Z-IETD-fmk, Z-VDVAD-fmk and Z-DEVD-fmk, were shown to completely lack specificity in inhibiting these caspases. The reversible aldehyde form of inhibitors for human caspase-8 and -9, Ac-LEHD-CHO and Ac-IETD-CHO, are equally efficient in inhibiting Chinese hamster caspase-8. Therefore, the wildly used method of utilizing the "caspase-specific" inhibitors to track the role of individual caspases in dying cells can be inaccurate and thus misleading. As an alternative, we stably expressed dominant negative (DN) mutants of Chinese hamster caspase-2, -8 and -9 to specifically inhibit these enzymes in CHO cells. Our results showed that inhibition of either endogenous caspase-8 or caspase-9 enhanced the viability of the CHO cells in both batch and fed-batch suspension cultures, but the inhibition of caspase-2 had minimal effects. These results suggest that caspase-8 and -9 are possibly involved in the apoptotic cell death in batch and fed-batch cultures of CHO cells, whereas caspase-2 is not. These findings can be valuable in the development of strategies for genetically engineering CHO cells to counter apoptotic death in batch and fed-batch cultures.     

Susceptibility of green leaves and green flower petals of CAM orchid <Emphasis Type="Italic">Dendrobium</Emphasis> cv. Burana Jade to high irradiance under natural tropical conditions

Photosynthetic rates of green leaves (GL) and green flower petals (GFP) of the CAM plant Dendrobium cv. Burana Jade and their sensitivities to different growth irradiances were studied in shade-grown plants over a period of 4 weeks. Maximal photosynthetic O₂ evolution rates and CAM acidities [dawn/dusk fluctuations in titratable acidity] were higher in leaves exposed to intermediate sunlight [a maximal photosynthetic photon flux density (PPFD) of 500–600 μmol m⁻² s⁻¹] than in leaves grown under full sunlight (a maximal PPFD of 1 000–1 200 μmol m⁻² s⁻¹) and shade (a maximal PPFD of 200–250 μmol m⁻² s⁻¹). However, these two parameters of GFP were highest in plants grown under the shade and lowest in full sun-grown plants. Both GL and GFP of plants exposed to full sunlight had lower predawn F_v/F_m [dark adapted ratio of variable to maximal fluorescence (the maximal photosystem 2 yield without actinic irradiation)] than those of shade-grown plants. When exposed to intermediate sunlight, however, there were no significant changes in predawn F_v/F_m in GL whereas a significant decrease in predawn F_v/F_m was found in GFP of the same plant. GFP exposed to full sunlight exhibited a greater decrease in predawn F_v/F_m compared to those exposed to intermediate sunlight. The patterns of changes in total chlorophyll (Chl) content of GL and GFP were similar to those of F_v/F_m. Although midday F_v/F_m fluctuated with prevailing irradiance, changes of midday F_v/F_m after exposure to different growth irradiances were similar to those of predawn F_v/F_m in both GL and GFP. The decreases in predawn and midday F_v/F_m were much more pronounced in GFP than in GL under full sunlight, indicating greater sensitivity in GFP to high irradiance (HI). In the laboratory, electron transport rate and photochemical and non-photochemical quenching of Chl fluorescence were also determined under different irradiances. All results indicated that GFP are more susceptible to HI than GL. Although the GFP of Dendrobium cv. Burana Jade require a lower amount of radiant energy for photosynthesis and this plant is usually grown in the shade, is not necessarily a shade plant.     

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.     

The Involvement of the Myelin-Associated Inhibitors and Their Receptors in CNS Plasticity and Injury

The limited capacity for the central nervous system (CNS) to repair itself was first described over 100 years ago by Spanish neuroscientist Ramon Y. Cajal. However, the exact mechanisms underlying this failure in neuronal regeneration remain unclear and, as such, no effective therapeutics yet exist. Numerous studies have attempted to elucidate the biochemical and molecular mechanisms that inhibit neuronal repair with increasing evidence suggesting that several inhibitory factors and repulsive guidance cues active during development actually persist into adulthood and may be contributing to the inhibition of repair. For example, in the injured adult CNS, there are various inhibitory factors that impede the outgrowth of neurites from damaged neurons. One of the most potent of these neurite outgrowth inhibitors is the group of proteins known as the myelin-associated inhibitors (MAIs), present mainly on the membranes of oligodendroglia. Several studies have shown that interfering with these proteins can have positive outcomes in CNS injury models by promoting neurite outgrowth and improving functional recovery. As such, the MAIs, their receptors, and downstream effectors are valid drug targets for the treatment of CNS injury. This review will discuss the current literature on MAIs in the context of CNS development, plasticity, and injury. Molecules that interfere with the MAIs and their receptors as potential candidates for the treatment of CNS injury will additionally be introduced in the context of preclinical and clinical trials.     

Can artificial substrates enriched with crustose coralline algae enhance larval settlement and recruitment in the fluted giant clam (<Emphasis Type="Italic">Tridacna squamosa</Emphasis>)?

Habitat recognition and selection can greatly increase the early-life survival of sessile reef organisms. This study describes the settlement and recruitment responses of the fluted giant clam, Tridacna squamosa, to concrete tablets and tiles containing different concentrations of crustose coralline algae covered coral rubble (CCACR). Crustose coralline algae is known to induce settlement in a variety benthic animals, but it has not been used previously as an aggregate in concrete—potentially a way of encouraging colonization of man-made structures erected on or near coral reefs. After being given the choice of small tablets made with 0%, 30% or 60% CCACR for 4 days, 11 days old larvae preferred the substrate containing the most CCACR. Recruitment responses of juvenile clams to larger tiles made with the same three CCACR concentrations were also tested. These tiles were further divided into rough and smooth surface textures. After 6 weeks, more juvenile clams had recruited to the rough surfaced tiles than the smooth ones, but no significant differences among the CCACR treatments were found. Thus, even though concrete made with CCACR is initially attractive to larvae, it has no effect on recruitment of juvenile T. squamosa. Handling editor: I. Nagalkerken     

Experimental investigation of failure load and fracture patterns of C2 (axis).

The injury mechanism and magnitude of failure load of C2 fractures are important in the clinical treatment of its fixation. The magnitudes of the failure load of C2 and the mechanism of injury in vivo are uncertain. Accordingly, nine C2 vertebrae obtained from cadaver spines, ranging in age from 51 to 80 years, were used for the study. Special restraint conditions were applied to yield specific fracture of C2. With the posterior element potted postero-anteriorly up to one-quarter of the inferior facet, posterior shear force ranging from 840 to 1220N was required to cause fracture across the pars interarticularis. For odontoid fracture study, a special rig was fabricated to encapsulate the body of C2 in a cell using ISOPON, and a thin layer of ISOPON sandwiched between the inferior facets and two lateral plates. The assembled rig permits slight sagittal movement of C2 about the cup lateral pivot supports. Failure load of between 900 and 1500N was recorded for odontoid fracture. These values are in agreement with published data. The experiment carried out under these two different restraint conditions had specifically resulted in different fractures of C2. In reality, depending on factors such as the inclination of this force vector applied to the head, the precise posture at the time of trauma, the spinal geometry, and the physical properties, different types of fracture patterns of C2 may be produced. This additional data will be useful in the biomechanical study of C2 vertebra using analytical approaches, and in surgical anterior/posterior fixation using screws.     

The abundance and geographic distributions of two species of ticks in South Australia: Bundey Bore revisited

Perhaps one of the central questions in ecology has been what limits the geographic distribution of species. The geographic distributions of the reptile-ticks Amblyomma limbatum, Bothriocroton hydrosauri and Amblyomma albolimbatum are a classic example of this problem: where these ticks meet, narrow regions-of-overlap often occur. Despite studies of interactions among these ticks, and of these ticks and their environment, we still do not understand what limits their geographic distributions. Many hypotheses have been proposed and tested, but all have seemingly been rejected. Our aim was to account for the abundance and the geographic distributions of A. limbatum and B. hydrosauri at Bundey Bore Station, South Australia, where these species come in contact and have been studied for over 35 years, leading to over 55 000 records of reptiles and their ticks using a capture-mark-recapture approach. We constructed models which had combinations of temperature and moisture indices from different months of the year to determine the relationship between climate and tick abundance, and between climate and the shifting local geographic distribution of A. limbatum and B. hydrosauri at Bundey Bore Station. We found that climate together with the abundance of ticks in the previous year and the abundance of the lizard host, accounted for 54%–77% of the variation in the abundance of A. limbatum and B. hydrosauri among years. We also found that the climate in late autumn to winter, together with the abundance of ticks and their host, accounted for 18%–84% of the variation in the geographic distributions of these ticks among years. Climate was central to the abundance and the geographic distributions of A. limbatum and B. hydrosauri at Bundey Bore Station. We speculate that the same mechanisms account for the geographic distributions of A. limbatum, B. hydrosauri and A. albolimbatum elsewhere in Australia; so, climate may explain the three-tick problem in Austral ecology.