Ontogeny of tracheal system structure: a light and electron-microscopy study of the metathoracic femur of the American locust, Schistocerca americana

Does oxygen delivery become more challenging for insects as they increase in size? To partially test this hypothesis, we used quantitative light and electron microscopy to estimate the oxygen delivery capacity for two steps of tracheal oxygen delivery within the metathoracic femur (jumping leg) for 2nd instar (about 47 mg) and adult (about 1.7 g) locusts, Schistocerca americana. The fractional cross‐sectional areas of the major tracheae running longitudinally along the leg were similar in adults and 2nd instars; however, since the legs of adults are longer, the mass‐specific diffusive conductances of these tracheae were 4‐fold greater in 2nd instars. Diffusive gas exchange longitudinally along the leg is easily possible for 2nd instars but not adults, who have many air sacs within the femur. Mitochondrial content fell proximally to distally within the femur in 2nd instars but not adults, supporting the hypothesis that diffusion was more important for the former. Lateral diffusing capacities of the tracheal walls were 12‐fold greater in adults than 2nd instars. This was primarily due to differences in the smallest tracheal class (tracheoles), which had thinner epidermal and cuticular layers, greater surface to volume ratios, and greater mass‐specific surface areas in adults. Adults also had greater mitochondrial contents, larger cell sizes and more intracellular tracheae. Thus, larger insects do not necessarily face greater problems with oxygen delivery; adult grasshoppers have superior oxygen delivery systems and greater mass‐specific aerobic capacities in their legs than smaller/younger insects. J. Morphol. 262:800–812, 2004. © 2004 Wiley‐Liss, Inc.     

Bacterial cell disruption: a key unit operation in the recovery of intracellular products

The need for microbial cell disruption has hindered the large scale production of commercial biotechnological products of intracellular derivation. The intracellular nature of many recombinant products and the potential use of the bacterial storage product, PHB as a commodity thermoplastic have renewed interest in the improvement of this unit operation. This paper provides a review of processes of a mechanical, physical, chemical or biological nature used for cell disruption on both the laboratory and large scale. Applicability of the techniques to large scale operation is discussed. Modification of existing processes is suggested for the reduction of energy requirements and improved process economics. The requirements for the liberation of granular intracellular products such as inclusion bodies and virus-like yeast particles are distinguished from those for the liberation of soluble products, mainly proteinaceous in nature. The integrated nature of the process with both upstream and downstream processes is addressed. Finally, the recent approach of selective liberation of soluble products of interest is reviewed.     

Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli

A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step.     

Synthesis and degradation of fatty acid ethyl esters by cultured hepatoma cells exposed to ethanol

Fatty acid ethyl esters are a family of neutral lipids that are the products of esterification of fatty acids with ethanol. Unlike other pathways of ethanol metabolism, ethyl esters are present in numerous human organs which are the targets of ethanol-induced damage. In the present study, we have shown that fatty acid ethyl esters are synthesized by a hepatoma cell line in tissue culture when exposed to ethanol concentrations easily attained by man during social drinking. Unlike alcohol dehydrogenase, the enzyme(s) responsible for synthesis of ethyl esters are membrane-bound and concentrated in the microsomal fraction of rat hepatocytes. In addition, fatty acid ethyl esters are hydrolyzed to free fatty acids and ethanol by membrane-bound enzyme(s) that are enriched in the microsomal and mitochondrial-lysosomal fractions. Intracellular hydrolysis of fatty acid ethyl esters release free fatty acids which are preferentially incorporated into cellular cholesterol esters. Thus, we have shown that a hepatocellular line exposed to concentrations of ethanol easily achieved in man by social drinking utilize endogenous fatty acids to form long-lived ethanol metabolites, fatty acid ethyl esters. Importantly, this family of neutral lipids may act as biochemical mediators of ethanol-induced cell damage, including the changes in cholesterol metabolism noted in chronic alcoholics.     

The Enterococcus hirae copper chaperone CopZ delivers copper(I) to the CopY repressor

Expression of the cop operon which effects copper homeostasis in Enterococcus hirae is controlled by the copper responsive repressor CopY. Purified Zn(II)CopY binds to a synthetic cop promoter fragment in vitro. Here we show that the 8 kDa protein CopZ acts as a copper chaperone by specifically delivering copper(I) to Zn(II)CopY and releasing CopY from the DNA. As shown by gel filtration and luminescence spectroscopy, two copper(I) are thereby quantitatively transferred from Cu(I)CopZ to Zn(II)CopY, with displacement of the zinc(II) and transfer of copper from a non-luminescent, exposed, binding site in CopZ to a luminescent, solvent shielded, binding site in CopY.