Recombinant expression,purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV‐1 gp41

The transmembrane subunit (gp41) of the envelope glycoprotein of HIV‐1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high‐resolution X‐ray structures of some segments of the MPR were solved in the past, they represent the post‐fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR‐TM_TEV‐6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR‐TM_TEV‐6His was improved by fusion to the C‐terminus of Mistic protein, yielding ～1 mg of pure protein per liter. The isolated MPR‐TM_TEV‐6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR‐TM_TEV‐6His, which will be important for the structure‐based design of a mucosal vaccine against HIV‐1.     

Increased production of VLDL apoB-100 in subjects with familial hypercholesterolemia carrying the same null LDL receptor gene mutation

Early radiokinetic studies revealed that the classical metabolic defect in patients with familial hypercholesterolemia (FH) is hypocatabolism of LDL due to decreased LDL receptor activity. However, recent studies have suggested that hepatic oversecretion of apolipoprotein B-100 (apoB-100)-containing lipoproteins could also contribute to the markedly elevated plasma concentrations of LDL-cholesterol found in FH. The aim of this study was to examine the kinetics of apoB-100 labeled with a stable isotope (l-[5,5,5-D(3)] leucine) in five normolipidemic controls and in seven well-characterized FH subjects that included six FH heterozygotes and one FH homozygote carrying the same null LDL receptor gene mutation. As compared with controls, the VLDL apoB-100 production rate was increased by 50% in the FH heterozygotes and by 109% in the FH homozygote. Furthermore, FH subjects had significantly higher LDL apoB-100 pool size and lower LDL apoB-100 fractional catabolic rate than controls. These results indicate that the elevation of plasma LDL-cholesterol found in FH is attributable to both decreased clearance of LDL and increased hepatic production of apoB-100-containing lipoproteins.     

Structural evidence for cooperative microtubule stabilization by Taxol and the endogenous dynamics regulator MAP4

Microtubules (MTs) composed of αβ-tubulin heterodimers are highly dynamic polymers, whose stability can be regulated by numerous endogenous and exogenous factors. Both the antimitotic drug Taxol and microtubule-associated proteins (MAPs) stabilize this dynamicity by binding to and altering the conformation of MTs. In the current study, amide hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) was used to examine the structural and dynamic properties of the MT complex with the microtubule binding domain of MAP4 (MTB-MAP4) in the presence and absence of Taxol. The changes in the HDX levels indicate that MTB-MAP4 may bind to both the outside and the luminal surfaces of the MTs and that Taxol reduces both of these interactions. The MTB-MAP4 binding induces conformational rearrangements of α- and β-tubulin that promote an overall stabilization of MTs. Paradoxically, despite Taxol's negative effects on MAP4 interactions with the MTs, its binding to the MTB-MAP4-MT complex further reduces the overall deuterium incorporation, suggesting that a more stable complex is formed in the presence of the drug.     

IVICAT for the Masses: an Improved Technique for Permethylation of Peptides

To determine the levels of post-translational modifications, we needed a quantitative technique that would allow comparison of the amounts of acetylated versus mono-, di-, and tri-methylated lysines in histones. One method, IVICAT, generates trimethyl-amines and could be used, but is technically challenging. We have modified this technique to be used with standard laboratory equipment so that this chemistry is accessible to most proteomics laboratories.     

A multivariate search for pollination syndromes among penstemons

The seeming ubiquity of spatio-temporal variation in pollination regime suggests that flowers ought to be adapted to a wide range of pollinators, yet many comparative biologists perceive that in groups with complex flowers there is considerable specialization onto pollination syndromes. Statistical documentation of such syndromes has been presented for very few groups of flowers. Accordingly, we measured, for 49 species of Penstemon and close relatives, both the morphology of the flowers and visitation by pollinators. We describe the mechanics of pollination for representative species. Ordinations show a distinct difference between hummingbird-pollinated species and hymenopteran-pollinated species. Flower color is particularly good at separating hummingbird- from hymenopteran-flowers. Other characters are also correlated with this dichotomy. Within the hymenopteran-pollinated species, there are additional relationships between floral morphology and the size of the principal pollinators. Flowers frequented by large bees, such as Xylocopa , have large open vestibules and relatively short floral tubes. Flowers frequented by smaller bees, such as Osmia , have long narrow floral tubes. Unlike nectar-collecting bees, pollen-collecting bees tend to be attracted to flowers of the hummingbird syndrome. The overarching pattern was that syndrome characterizations were successful at predicting pollination by hummingbirds versus Hymenoptera, two types of animals that are profoundly different, but less successful at predicting visitation by one kind of bee versus another.     

Pathways by which interleukin 17 induces articular cartilage breakdown in vitro and in vivo.

Overexpression of interleukin (IL-)17 has recently been shown to be associated with a number of pathological conditions. Because IL-17 is found at high levels in the synovial fluid surrounding cartilage in patients with inflammatory arthritis, the present study determined the direct effect of IL-17 on articular cartilage. As shown herein, IL-17 was a direct and potent inducer of matrix breakdown and an inhibitor of matrix synthesis in articular cartilage explants. These effects were mediated in part by leukemia inhibitory factor (LIF), but did not depend on interleukin-1 activity. The mechanism whereby IL-17 induced matrix breakdown in cartilage tissue appeared to be due to stimulation of activity of aggrecanase(s), not matrix metalloproteinase(s). However, IL-17 upregulated expression of matrix metalloproteinase(s) in chondrocytes cultured in monolayer. In vivo, IL-17 induced a phenotype similar to inflammatory arthritis when injected into the intra-articular space of mouse knee joints. Furthermore, a related protein, IL-17E, was found to have catabolic activity on human articular cartilage. This study characterizes the mechanism whereby IL-17 acts directly on cartilage matrix turnover. Such findings have important implications for the treatment of degenerative joint diseases such as arthritis.     

Effect of chitin waste-based composts produced by two-phase composting on two oomycete plant pathogens

Biphasic composts were prepared by first mixing peat moss and sawdust with a nitrogen-rich biomass such as chitinous waste or cow manure and composting them until termination of the thermophilic phase. These partially stabilized composts were then amended with shrimp waste inducing a second thermophilic phase. Filter-sterilized water extracts obtained from two mature biphasic composts (SP₂W₂+S and MPW+S) reduced the growth of two oomycete plant pathogens, Phytophthora fragariae var. rubi and Pythium ultimum. Both SP₂W₂+S and MPW+S composts significantly reduce the incidence of cucumber damping-off caused by Pythium ultimum as compared to a commercial brand of compost made from shrimp waste and peat moss. Hydrolysis products of chitin were unlikely to be responsible for growth inhibition since no oligomeric forms of chitin were detected in SP₂W₂+S. The shrimp waste amendment carried out after the first thermophilic phase modified the microbial populations of biphasic composts. Following the amendment, the proportion of branched-chain microbial fatty acids typical of Gram-positive bacteria increased considerably suggesting that this group of bacteria became more prevalent within the total microbial population. These data suggests that the two-phase composting process promotes the proliferation of Gram-positive bacteria antagonistic to oomycete plant pathogens.     

Age-associated change in mitochondrial DNA damage

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline.

Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2'-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.