The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.     

The effect of N-terminal acetylation on the structure of an N-terminal tropomyosin peptide and alpha alpha-tropomyosin.

We have used a synthetic peptide consisting of the first 30 residues of striated muscle alpha-tropomyosin, with GlyCys added to the C-terminus, to investigate the effect of N-terminal acetylation on the conformation and stability of the N-terminal domain of the coiled-coil protein. In aqueous buffers at low ionic strength, the reduced, unacetylated 32mer had a very low alpha-helical content (approximately 20%) that was only slightly increased by disulfide crosslinking or N-terminal acetylation. Addition of salt (> 1 M) greatly increased the helical content of the peptide. The CD spectrum, the cooperativity of folding of the peptide, and sedimentation equilibrium ultracentrifugation studies showed that it formed a 2-chained coiled coil at high ionic strength. Disulfide crosslinking and N-terminal acetylation both greatly stabilized the coiled-coil alpha-helical conformation in high salt. Addition of ethanol or trifluoroethanol to solutions of the peptide also increased its alpha-helical content. However, the CD spectra and unfolding behavior of the peptide showed no evidence of coiled-coil formation. In the presence of the organic solvents, N-terminal acetylation had very little effect on the conformation or stability of the peptide. Our results indicate that N-terminal acetylation stabilizes coiled-coil formation in the peptide. The effect cannot be explained by interactions with the "helix-dipole" because the stabilization is observed at very high salt concentrations and is independent of pH. In contrast to the results with the peptide, N-terminal acetylation has only small effects on the overall stability of tropomyosin.     

Gastric colonization withCandida albicans

This is the first report on the isolation ofCryptococcus neoformans from pigeon droppings in China and their serotypes.C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified asC. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that onlyC. neoformans var.neoformans strains could be isolated from pigeon droppings, although the varietygattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells ofC. neoformans strains in same samples of pigeon droppings were observed.     

A tooth at the border of two morphogenetic fields

A tooth at the border between two morphogenetic fields (mandibular canine and honing premolar) may become morphologically similar to and/or functionally incorporated with the teeth of either field. In light of this, observations of the morphology and occlusion of female anthropoid C1s from 58 extant species are presented to assess whether and to what extent they exhibit incisor-like form and function. Female C1s in 74% of the taxa observed exhibit well developed incisor-like traits which may reflect field border phenomena. In another 9%, incisor traits are present but they are not examples of field border phenomena. Interspecies variation in female C1 morphology is related to behavior, function, natural selection and phyletic inertia. A selection model, derived from the data, is used to explain C1 sexual dimorphism and the evolution of male and female human canines. The data's relevance to the field vs clone theory debate is also discussed.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Effect of thyroidectomy and adrenalectomy on changes in liver glutathione and malonaldehyde levels after acute ethanol injection

At low concentrations ethanol is metabolized largely by alcohol dehydrogenase to acetaldehyde, while at higher concentrations a microsomal ethanol oxidising system (MEOS) is involved, namely cytochrome P450 IIE1, which also probably generates free radical species. In hyperthyroidism hepatic glutathione stores are depleted and net superoxide anion production occurs. In contrast, in hypothyroidism hepatic glutathione may be increased and thus renders the liver less sensitive to alcohol generated free radical production. Steroid hormones inhibit lipid peroxidation. Sixty male Wistar rats either underwent thyroidectomy, adrenalectomy, or sham procedures. Twenty control animals were pair fed with thyroidectomized animals, whilst another twenty fed ad libitum. An intraperitoneal injection of alcohol (75 mmol/kg) was given 2.5 h prior to sacrifice to half the animals in each group, the remainder receiving saline. The total hepatic glutathione contents of the pair fed and the ad libitum groups were not different, but were significantly increased by thyroidectomy (p = <0.001). This effect was significantly reduced by alcohol (p < 0.01). The sham procedures and dietary restrictions had no effect. The ethanol alone reduced total hepatic glutathione, but this only reached statistical significance in the thyroidectomized and sham-adrenalectomized groups. Hepatic malonaldehyde (MDA) levels were significantly reduced in the thyroidectomy group but alcohol had no effect on them. We conclude that hypothyroidism increased hepatic glutathione status, presumably by reducing radical production by enzyme systems, which would otherwise consume this important scavenger. Long term exposure to ethanol with induction of MEOS is probably required for it to generate toxic levels of free radical species.     

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.