The role of vocal individuality in conservation

Identifying the individuals within a population can generate information on life history parameters, generate input data for conservation models, and highlight behavioural traits that may affect management decisions and error or bias within census methods. Individual animals can be discriminated by features of their vocalisations. This vocal individuality can be utilised as an alternative marking technique in situations where the marks are difficult to detect or animals are sensitive to disturbance. Vocal individuality can also be used in cases were the capture and handling of an animal is either logistically or ethically problematic. Many studies have suggested that vocal individuality can be used to count and monitor populations over time; however, few have explicitly tested the method in this role. In this review we discuss methods for extracting individuality information from vocalisations and techniques for using this to count and monitor populations over time. We present case studies in birds where vocal individuality has been applied to conservation and we discuss its role in mammals.     

The TrkB-Shc site signals neuronal survival and local axon growth via MEK and P13-kinase

To determine how signals emanating from Trk transmit neurotrophin actions in primary neurons, we tested the ability of TrkB mutated at defined effector binding sites to promote sympathetic neuron survival or local axon growth. TrkB stimulated signaling proteins and induced survival and growth in a manner similar to TrkA. TrkB mutated at the Shc binding site supported survival and growth poorly relative to wild-type TrkB, whereas TrkB mutated at the PLC-gamma1 binding site supported growth and survival well. TrkB-mediated neuronal survival was dependent on P13-kinase and to a lesser extent MEK activity, while growth depended upon both MEK and P13-kinase activities. These results indicate that the TrkB-Shc site mediates both neuronal survival and axonal outgrowth by activating the P13-kinase and MEK signaling pathways.     

The relevance of sweat testing for the diagnosis of cystic fibrosis in the genomic era

Cystic fibrosis (CF) is the most common inherited disorder of childhood. The diagnosis of CF has traditionally been based on clinical features with confirmatory evidence by sweat electrolyte analysis. Since 1989 it has been possible to also use gene mutation analysis to aid the diagnosis. Cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has advanced our understanding of CF, in particular the molecular basis of an expanded CF phenotype. However, because there are over 1000 mutations and 200 polymorphisms, many without recognised effects on CFTR, the molecular diagnosis can be troublesome. This has necessitated measurement of CFTR function with renewed interest in the sweat test. This review provides an overview of the clinical features of CF, the diagnosis and complex genetics. We provide a detailed discussion of the structure and function of CFTR and the classification of CFTR mutations. Sweat electrolyte analysis is discussed, from the physiology of sweating to the rigours of a properly performed sweat test and its interpretation. With this information it is possible to understand the relevance of the sweat test in the genomic era.     

Reduced thermotolerance in aged cells results from a loss of an hsp72- mediated control of JNK signaling pathway

Aged organisms exhibit a greatly decreased ability to induce the major heat shock protein, Hsp72, in response to stresses, a phenomenon that can also be observed in cell cultures (Heydari AR, Takahashi R, Gutsmann A, You S and Richardson A (1994) Hsp70 and aging. Experientia 50: 1092–1098). Hsp72 was shown to protect cells from a variety of stresses. The protective function of Hsp72 has been commonly ascribed to its chaperoning ability. However, recently we showed that Hsp72 protects cells from heat shock by suppression of a stress-kinase JNK, an essential component of the heat-induced apoptotic pathway (Gabai VL, Meriin AB, Mosser DD, Caron AW, Rits S, Shifrin VI and Sherman MY (1997) Hsp70 prevents activation of stress kinases. A novel pathway of cellular thermotolerance. J Biol Chem 272: 18033–18037). Here we demonstrate that because of the diminished inducibility of Hsp72 in aged cells, Hsp72-mediated control of JNK signaling pathway is compromised. This results in increased rate of apoptotic cell death following heat shock. We show that forced expression of Hsp72 in aged cells from an adenovirus-based vector completely suppresses activation of JNK by heat shock and consequently protects from heat-induced apoptosis. We also demonstrate for the first time that it is possible to restore endogenous expression of Hsp72 in aged cells. This can be achieved by treatment with the proteasome inhibitor MG132. Induction of Hsp72 in aged cells under these conditions leads to suppression of JNK activation by a heat shock and restoration of thermotolerance manifested in a lower rate of apoptosis.     

Selective Isolation of Bacillus sphaericus from Soil by Use of Acetate as the Only Major Source of Carbon

A simple chemically defined medium containing sodium acetate (37 or 74 mM) as the only major source of carbon was inoculated with soil from various locations. The bacteria which grew most rapidly at 30°C were almost entirely strains of Bacillus sphaericus and Arthrobacter species. Pasteurization of the soils made the medium selective for B. sphaericus. Several new strains were isolated, and the peptidoglycans of their cell walls were examined.     

Improvement of recombinant protein production with the human adenovirus/293S expression system using fed-batch strategies

The human adenovirus/293S cell expression system is used for the production of either recombinant protein or adenovirus vectors for use in gene therapy. In this work, the production of protein tyrosine phosphatase (PTP1C) was used as a model for the scale-up of both applications. Maximum specific production of 30 to 45 mug of active protein/10(6) cells was maintained upon infection with adenovirus vectors at cell densities between 2 x 10(6) to 3 x 10(6) cells/mL in a 3.5-L bioreactor. This was achieved by resuspending the culture in fresh medium at infection time. The pH was kept at 7.0 throughout the experiment and, at 24 h postinfection, glucose and essential amino acids were added. Attempts to replace the complete change of medium at the time of infection with nutrient supplementation of the used medium led to lower production levels, suggesting that protein expression was limited not by the absence of a key nutrient but by inhibitory factors. Two potentially inhibitory factors were investigated: lactic acid accumulation and increased osmolarity. Medium acidification such as that which would be brought about by lactic acid accumulation was shown to depress PTP1C production. The lactate molecule itself decreased the cell viability when added in concentrations of 20 mM or more. But the specific productivity was affected at higher lactate concentrations of 40 mM or more. Additions of glucose, amino acids, and NaHCO(3) used to control pH, led to increases in osmolarity. Osmolarities above 400 mOsm lowered cell density. However, specific production was not significantly affected below 500 mOsm. But, at 500 mOsm, PTP1C production peak was shifted from 48 to 72 hpi. Because of the cell loss, this per cell yield increase did not translate into higher volumetric production. When glucose concentrations was kept at 5 mM by fed-batch addition, lactate production and increases in osmolarity were reduced. In shake flasks, this method permitted maximum production with cells resuspended either in fresh or spent medium at infection. This fed-batch process was implemented successfully at the 3.5-L scale. Fed-batch with glucose may provide a means to increase infected-cell density beyond 3 x 10(6) cells/mL.     

Culture of insect cells in helical ribbon impeller bioreactor

An 11-L helical ribbon impeller (HRI) bioreactor was tested for the culture of Spodoptera frugiperda (Sf-9) cells. This impeller and surface baffling ensured homogeneous mixing and high oxygen transfer through surface aeration and surface-induced babble generation. Serum-supplemented and serum-free cultures, using TNMFH and IPL/41 media, respectively, grew a similar specific growth rates(0.031 and 0.028 h(-1)) to maximum cell densities of 5.5 x 10(6)-6.0 x 10(6) cells. mL(-1) with viability exceeding 98% during exponential growth phase. Growth limitation coincided with glucose and glutamine depletion and production of significant amounts of alanine. The bioreactor was further tested under more stringent conditions by infecting a serum-free medium culture with a recombinant baculovirus. Heterologous protein production of approximately 35 mug per 10(6) cells was comparable to yields obtained in serum-free cultures grown in spinner flasks and petri dishes. Average specific oxygen up-take and carbon dioxide production rates of the serum-free culture prior to infection as measured by on-line mass spectroscopy were 0.20 mumol O(2)mu.(10(6) cells)(-1) h(-1) and 0.22 mumol CO(2) . (10(6) cells)(-1)h(-1) and increased by 30-40% during infection. Therefore, the mixing and oxygenation conditions of this bioreactor were suitable for insect cell culture and recombinant protein production, with limitation being mainly attributed to nutrient depletion and toxic by-product generation.