Ionic derivatives of betulinic acid as novel HIV-1 protease inhibitors

Betulinic acid is a natural product possessing abundant and favourable biological activity, including anti-cancer, anti-malarial, anti-inflammatory and anti-HIV properties, while causing minimal toxicity to unaffected cells. The full biological potency of betulinic acid cannot be fully unlocked, however, for a number of reasons, a primary one being its limited solubility in aqueous and biologically pertinent organic media. Aiming to improve the water solubility of betulinic acid without disrupting its structurally related bioactivity, we have prepared different ionic derivatives of betulinic acid. Inhibition bioassays on HIV-1 protease-catalysed peptide hydrolysis indicate significantly improved performance resulting from converting the betulinic acid to organic salt form. Indeed, for one particular cholinium-based derivative, its water solubility is improved more than 100 times and the half maximal inhibitory concentration (IC(50)) value (22 μg mL(-1)) was one-third that of wide-type betulinic acid (60 μg mL(-1)). These encouraging results advise that additional studies of ionic betulinic acid derivatives as a therapeutic solution against HIV-1 infection are warranted.     

Optimization of condition(s) towards establishment of primary islet cell cultures from WNIN/Ob mutant rat

WNIN/Ob, a mutant rat strain, developed at the National Center for Laboratory Animal Sciences (NCLAS) facility of National Institute of Nutrition (NIN), is a new animal model to study the metabolic syndrome. These animals have 47% fat in their body and isolation of islets from these animals were compounded due to the formation of amorphous viscous and jelly like material which reduced the islet yield. However, islets isolated from WNIN adult (≥12 months) control rats gave a good islet recovery, under standard isolation procedures using collagenase digestion. In the present study we optimized culture conditions in WNIN/Ob rats to isolate islets with higher yield, and also established primary islet cell cultures from these mutant rats, retaining cellular integrity and functionality.     

Autotaxin-lysophosphatidic Acid axis is a novel molecular target for lowering intraocular pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.     

Regulation of adipocyte formation by GLP-1/GLP-1R signaling

Increased nutrient intake leads to excessive adipose tissue accumulation, obesity, and the development of associated metabolic disorders. How the intestine signals to adipose tissue to adapt to increased nutrient intake, however, is still not completely understood. We show here, that the gut peptide GLP-1 or its long-lasting analog liraglutide, function as intestinally derived signals to induce adipocyte formation, both in vitro and in vivo. GLP-1 and liraglutide activate the GLP-1R, thereby promoting pre-adipocyte proliferation and inhibition of apoptosis. This is achieved at least partly through activation of ERK, PKC, and AKT signaling pathways. In contrast, loss of GLP-1R expression causes reduction in adipogenesis, through induction of apoptosis in pre-adipocytes, by inhibition of the above mentioned pathways. Because GLP-1 and liraglutide are used for the treatment of type 2 diabetes, these findings implicate GLP-1 as a regulator of adipogenesis, which could be an alternate pathway leading to improved lipid homeostasis and controlled downstream insulin signaling.     

The metallomics approach: use of Fe(II) and Cu(II) footprinting to examine metal binding sites on serum albumins

Metal binding to serum albumins is examined by oxidative protein-cleavage chemistry, and relative affinities of multiple metal ions to particular sites on these proteins were identified using a fast and reliable chemical footprinting approach. Fe(ii) and Cu(ii), for example, mediate protein cleavage at their respective binding sites on serum albumins, in the presence of hydrogen peroxide and ascorbate. This metal-mediated protein-cleavge reaction is used to evaluate the binding of metal ions, Na(+), Mg(2+), Ca(2+), Al(3+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Zn(2+), Cd(2+), Hg(2+), Pb(2+), and Ce(3+) to albumins, and the relative affinities (selectivities) of the metal ions are rapidly evaluated by examining the extent of inhibition of protein cleavage. Four distinct systems Fe(II)/BSA, Cu(II)/BSA, Fe(II)/HSA and Cu(II)/HSA are examined using the above strategy. This metallomics approach is novel, even though the cleavage of serum albumins by Fe(II)/Cu(II) has been reported previously by this laboratory and many others. The protein cleavage products were analyzed by SDS PAGE, and the intensities of the product bands quantified to evaluate the extent of inhibition of the cleavage and thereby evaluate the relative binding affinities of specific metal ions to particular sites on albumins. The data show that Co(II) and Cr(III) showed the highest degree of inhibition, across the table, followed by Mn(II) and Ce(III). Alakali metal ions and alkaline earth metal ions showed very poor affinity for these metal sites on albumins. Thus, metal binding profiles for particular sites on proteins can be obtained quickly and accurately, using the metallomics approach.     

A duplication in chromosome 4q35 is associated with hereditary benign intraepithelial dyskeratosis

Hereditary benign intraepithelial dyskeratosis (HBID) is an autosomal dominant disorder characterized by elevated epithelial plaques on the ocular and oral mucous membranes. It has been reported primarily, but not exclusively, in individuals of American Indian heritage in North Carolina. We have examined and obtained DNA on two large families affected by HBID. Using genetic linkage analysis we have localized the HBID gene to chromosome 4 (4q35) with a peak LOD score of 8.97. Molecular analysis of these data reveals that all individuals affected with HBID in both families demonstrate the presence of three alleles for two tightly linked markers, D4S1652 and D4S2390, which map to the telomeric region of 4q35. This suggests the presence of a duplication segregating with the disease phenotype that is most likely involved in its causation.     

Effects of donor age on proteasome activity and senescence in trabecular meshwork cells

The mechanisms involved in the progressive malfunction of the trabecular meshwork (TM) in glaucoma are not yet understood. To study age-related changes in human TM cells, we isolated primary TM cell cultures from young (ages 9, 14, and 25) and old (ages 66, 70, and 73) donors, and compared levels of oxidized proteins, autofluorescence, proteasome function, and markers for cellular senescence. TM cells from old donors showed a 3-fold increase in oxidized proteins and a 7.5-fold decrease of proteasome activity. Loss of proteasome function was not associated with decreased proteasome content but with partial replacement of the proteolytic subunit PSMB5 with the inducible subunit LMP7. Cells from old donors also demonstrated features characteristic of cellular senescence associated with phosphorylation of p38MAPK but only a modest increase in p53. These data suggest that age-related proteasome inhibition and cellular senescence could contribute to the pathophysiological alterations of the TM in glaucoma.     

Dry mass production and water use of non- and drip irrigated <Emphasis Type="Italic">shape Thuja occidentalis</Emphasis> ‘Brabant’: Field experiments and modeling

Generally, irrigation increases dry mass production (DM) on sandy soils of horticultural crops and at the same time increases the risk of percolation losses of water and chemicals to below the root zone. However, the effects of irrigation are highly site-specific and not easily determined, which hampers the development of proper management tools and guidelines. A two-dimensional soil-water balance model combined with a crop growth model was parameterized and validated, and used to investigate DM and water use of Thuja occidentalis Brabant in a field trial under non- and drip irrigated conditions. Measured leaf DM and leaf area index (LAI) were not affected by irrigation but irrigation increased stem DM and the specific leaf area. Simulated DM and LAI were in good agreement with the measurements. Simulated pressure head followed the measured pressure head, although models performance was better under dry than under wet conditions. Simulation experiments indicated that increasing irrigation threshold levels increased DM production and leaching relatively to no irrigation, when the irrigation threshold level was measured at 0.25m depth.     

Pathogenesis and Pathology of Invasive Aspergillosis

Purpose of Review

Invasive aspergillosis occurs in immunosuppressed individuals and is associated with high morbidity and mortality. Understanding the host defenses and pathogen characters is important in the causation of disease.

Recent Findings

Neutropenia and/or corticosteroid administration increase the risk of invasive infections and majority are due to Aspergillus fumigatus. The size of the conidia, thermotolerance, hydrophobins and melanin on conidial surface, adaptability to host environment, and angioinvasive nature contribute to pathogenecity. The large conidial size, hot and humid environment, and constant exposure to high spore content are implicated in the pathogenesis of chronic invasive infections in a normal host due to Aspergillus flavus. Pathology depends on the immune status and varies from granuloma with fibrosis or suppuration to abscess or infarction.

Summary

The risk factors and the interplay of host pathogen in the pathogenesis of invasive aspergillosis are reviewed. Genetic predisposition and identification of such genetic factors are necessary for prevention and treatment.

     

Diurnal export and carbon economy in an expanding source leaf of cucumber at contrasting source and sink temperature

Effects of contrasting temperatures of an expanding leaf (source) and of remaining plant parts (sink) on diurnal export and distribution of carbon were studied in seedlings of Cucumis sativus L., cv. Farbio. The time course of the rate of export was calculated by measuring simultaneously the exchange of ¹⁴CO₂ and the amount of ¹⁴C in the source leaf by means of a Geiger-Müller detector using a steady-state labelling technique. In all treatments average export rate during the night (16 h) was maximally 50% of the average rate during the 8-h day. Temperature affected the diurnal course of export via the source leaf and the sink in different ways. At a source leaf temperature of 25 or 30°C export stopped 12 h after start of the night, whereas at 20°C export continued throughout the night. However, the total amount of carbon exported during a 24 h cycle, expressed as a proportion of the amount of carbon assimilated, was the same at source leaf temperatures of 20 or 30°C. Thus source leaf temperature did not affect the distribution of assimilates between source and sink, in contrast to sink temperature. After 24 h at a sink temperature of 30°C, 20% more ¹⁴C was exported to plant parts below the source leaf than with a sink temperature of 20°C, at the expense of carbon remaining in the source. During the day less starch and more structural dry matter was formed at a source leaf temperature of 30°C than at 20°C. After a complete day/night cycle, however, there was no difference between the treatments. Starch was the primary carbon source during the night, and the decline in the rate of export coincided with the depletion of starch. Thus the decline in the rate of export at a source leaf temperature of 25 or 30°C at 12 h after the start of the night was due to the depletion of starch at that time. Similarly, at 20°C export could continue until the end of the night as the starch degradation supplied assimilates during the whole night.