Structural and physiological features of sterols necessary to satisfy bulk membrane and sparking requirements in yeast sterol auxotrophs

A variety of sterols and stanols have been analyzed for their ability to satisfy bulk membrane and high-specificity (sparking) functions in three yeast sterol auxotrophs. While many sterols and stanols satisfied bulk membrane requirements, only those possessing a C-5,6 unsaturation or capable of being desaturated at C-5 fulfilled the high-specificity sparking requirement. Unsaturation of the A-ring or beta-saturation of a C-5,6 double bond rendered both sterol and stanol unsuitable for either function. The C-28 methyl group of ergosterol, while not required for growth, allowed for greater ease of desaturation at C-5 in vivo. As a result some sterols and stanols lacking the C-28 methyl were incapable of satisfying the sparking requirement while identical compounds possessing the C-28 methyl were able to fulfill the sparking function(s). These data are extended to hypothesize a role for the C-28 methyl group of ergosterol in yeast.     

Characterization of bimodal cell death of insect cells in a rotating‐wall vessel and shaker flask

In previous publications, we reported the benefits of a high‐aspect rotating‐wall vessel (HARV) over conventional bioreactors for insect‐cell cultivation in terms of reduced medium requirements and enhanced longevity. To more fully understand the effects that HARV cultivation has on longevity, the present study characterizes the mode and kinetics of Spodoptera frugiperda cell death in this quiescent environment relative to a shaker‐flask control. Data from flow cytometry and fluorescence microscopy show a greater accumulation of apoptotic cells in the HARV culture, by a factor of at least 2 at the end of the cultivation period. We present a kinetic model of growth and bimodal cell death. The model is unique for including both apoptosis and necrosis, and further, transition steps within the two pathways. Kinetic constants reveal that total cell death is reduced in the HARV and the accumulation of apoptotic cells in this vessel results from reduced depletion by lysis and secondary necrosis. The ratio of early apoptotic to necrotic cell formation is found independent of cultivation conditions. In the model, apoptosis is only well represented by an integral term, which may indicate its dependence on accumulation of some factor over time; in contrast, necrosis is adequately represented with a first‐order term. Cell‐cycle analysis shows the percent of tetraploid cells gradually decreases during cultivation in both vessels. For example, between 90% and 70% viability, tetraploid cells in the HARV drop from 43 ± 1% to 24 ± 4%. The data suggests the tetraploid phase as the likely origin for apoptosis in our cultures. Possible mechanisms for these changes in bimodal cell death are discussed, including hydrodynamic forces, cell–cell interactions, waste accumulation, and mass transport. These studies may benefit insect‐cell cultivation by increasing our understanding of cell death in culture and providing a means for further enhancing culture longevity. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 14–26, 1999.     

Familiar soil conditions help Pinus ponderosa seedlings cope with warming and drying climate

Changes in temperature and moisture as a result of climate forcing can impact performance of planted trees. Tree performance may also be sensitive to new soil conditions, for example, brought about by seeds germinating in soils different from those colonized by ancestral populations. Such “edaphic constraint” may occur with natural migration or human‐assisted movement. Pinus ponderosa seedlings, sourced from one location (“home” site), were grown across a field environmental gradient in either their original home soil or in soils from two different “away” sites. Seedlings were inoculated with site‐specific soil organisms by germinating seeds in living soil. After 6 months, the inoculated seedlings were transplanted into sterilized soils from the home or away sites. This experimental design allowed us to uncouple the importance of abiotic and biotic soil properties and test (1) how biotic and abiotic soil properties interact with climate to influence plant growth and stress tolerance, and (2) the role of soil biota in facilitating growth in novel environments. Seedlings grew least in hotter and drier away sites with away soil biota. Home soil biota ameliorated negative impacts on growth of hotter and drier away sites. Measurements of photosynthetic rate, stomatal conductance, and chlorophyll florescence (Fv/Fm) suggest that edaphic constraint reduced growth by increasing tree water stress. Results suggest that success of Ponderosa pine plantings into warming environments will be enhanced by pre‐inoculation with native soil biota of the seed source.     

A deliberate approach to screening for initial crystallization conditions of biological macromolecules

A method to rationally predict crystallization conditions for a previously uncrystallized macromolecule has not yet been developed. One way around this problem is to determine initial crystallization conditions by casting a wide net, surveying a large number of chemical and physical conditions to locate crystallization leads. A facility that executes the rapid survey of crystallization lead conditions is described in detail. Results and guidelines for the initial screening of crystallization conditions, applicable to both manual and robotic setups, are discussed.     

Tissue microenvironment and the genetic control of hair pigment patterns in mice

This study was conducted to assess microenvironmental variability within integumental tissue of genetically identical mice with respect to a specific cellular response: cyclic synthesis of yellow and black pigment by hair bulb melanocytes. Crosses were performed within and between inbred strains of mice that were isogenic with the exception of a single gene substitution at the agouti locus. Agouti locus genes included the A^vy, A^w, A, a^td, a^t, a^x, a^m, and a alleles. The pigment patterns of dorsal, flank, and ventral hairs of the first and third hair generations and of hairs growing in special integumentary areas such as the pinna, tail, and hind foot were studied. It was found that the amount of yellow pigment synthesized by hair bulb melanocytes within genetically identical mice is both agedependent and conditioned by the integumentary environment. Furthermore, the special integumentary regions produce hairs with a variety of pigment patterns in which the distribution and relative amounts of black and yellow pigments do not necessarily conform to dominance relationships expected among agouti locus alleles as judged by their effects on the pigmentation of the dorsal pelage. We conclude that within genetically uniform integumental tissues, microenvironmental differences occur and are reflected as alterations in the metabolic pattern of differentiated cells.     

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ss-tubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils.     

Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

Background

Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment.

Methods

Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS.

Results

607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16).

Conclusions

Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of treatment.