Lanosterol Analogs: Dual-Action Inhibitors of Cholesterol Biosynthesis

Abstract

Deoxyribonucleic acid is one of the most interesting and also the most complex of all biological macromolecules. Paradoxically, this complexity arises from the simplicity of its basic subunit structure. A large eukaryotic chromosome probably contains a single chain of DNA with a molecular weight in excess of 10¹¹ daltons and is composed of a linear permutation of the four basic deoxyribonucleotides. Until recently, this fact posed considerable problems for the biochemist interested in isolating specific fragments of chromosomes as the methods available were nonspecific in nature. This is no longer so; the discovery of site specific endodeoxyribonucleases (restriction endo-nucleases) has opened new routes to the analysis of DNA structure and function and promises a revolution in molecular biology. A new field of genetic engineering is already being pioneered, and significant advances in many areas have been facilitated by the availability of these enzymes.     

Interplay between Clathrin and Rab5 Controls the Early Phagocytic Trafficking and Intracellular Survival of Brucella abortus within HeLa cells

Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.     

Influence of a skilled model on the behavior of conspecific observers in tufted capuchin monkeys (Cebus apella)

We analyze how the presence of a skilled juvenile capuchin monkey interacting with a mechanical puzzle requiring sequential actions affected the behavior of group-mates towards the puzzle. Using this study as an example, we suggest a methodological approach to the evaluation of social enhancement of activity and imitation. We suggest that this design could be useful in determining if social and demographic factors influence the occurrence of these phenomena. © 1995 Wiley-Liss, Inc.     

Lipid-lowering effect of berberine in human subjects and rats

Due to serious adverse effects and the limited effectiveness of currently available pharmacological therapies for obesity, many research efforts have focused on the development of drugs from natural products. Our previous studies demonstrated that berberine, an alkaloid originally isolated from traditional Chinese herbs, prevented fat accumulation in vitro and in vivo. In this pilot study, obese human subjects (Caucasian) were given 500 mg berberine orally three times a day for twelve weeks. The efficacy and safety of berberine treatment was determined by measurements of body weight, comprehensive metabolic panel, blood lipid and hormone levels, expression levels of inflammatory factors, complete blood count, and electrocardiograph. A Sprague-Dawley rat experiment was also performed to identify the anti-obesity effects of berberine treatment. The results demonstrate that berberine treatment produced a mild weight loss (average 5 lb/subject) in obese human subjects. But more interestingly, the treatment significantly reduced blood lipid levels (23% decrease of triglyceride and 12.2% decrease of cholesterol levels) in human subjects. The lipid-lowering effect of berberine treatment has also been replicated in the rat experiment (34.7% decrease of triglyceride and 9% decrease of cholesterol level). Cortisol, calcitriol, ACTH, TSH, FT4, and SHBG levels were not significantly changed following 12 weeks of berberine treatment. However, there was interestingly, an increase in calcitriol levels seen in all human subjects following berberine treatment (mean 59.5% increase, p=0.11). Blood inflammatory factors (CRP, IL-6, TNFα, COX-2) and erythrocyte sedimentation rate (ESR) were not significantly affected by treatment with berberine. Tests of hematological, cardiovascular, liver, and kidney function following berberine treatment showed no detrimental side effects to this natural compound. Collectively, this study demonstrates that berberine is a potent lipid-lowering compound with a moderate weight loss effect, and may have a possible potential role in osteoporosis treatment/prevention.     

Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA

Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.     

Population fragmentation and inter-ecosystem movements of grizzly bears in western Canada and the northern United States

Population fragmentation compromises population viability, reduces a species ability to respond to climate change, and ultimately may reduce biodiversity. We studied the current state and potential causes of fragmentation in grizzly bears over approximately 1,000,000 km² of western Canada, the northern United States (US), and southeast Alaska. We compiled much of our data from projects undertaken with a variety of research objectives including population estimation and trend, landscape fragmentation, habitat selection, vital rates, and response to human development. Our primary analytical techniques stemmed from genetic analysis of 3,134 bears, supplemented with radiotelemetry data from 792 bears. We used 15 locus microsatellite data coupled with measures of genetic distance, isolation-by-distance (IBD) analysis, analysis of covariance (ANCOVA), linear multiple regression, multi-factorial correspondence analysis (to identify population divisions or fractures with no a priori assumption of group membership), and population-assignment methods to detect individual migrants between immediately adjacent areas. These data corroborated observations of inter-area movements from our telemetry database. In northern areas, we found a spatial genetic pattern of IBD, although there was evidence of natural fragmentation from the rugged heavily glaciated coast mountains of British Columbia (BC) and the Yukon. These results contrasted with the spatial pattern of fragmentation in more southern parts of their distribution. Near the Canada–US border area, we found extensive fragmentation that corresponded to settled mountain valleys and major highways. Genetic distances across developed valleys were elevated relative to those across undeveloped valleys in central and northern BC. In disturbed areas, most inter-area movements detected were made by male bears, with few female migrants identified. North–south movements within mountain ranges (Mts) and across BC Highway 3 were more common than east–west movements across settled mountain valleys separating Mts. Our results suggest that relatively distinct subpopulations exist in this region, including the Cabinet, Selkirk South, and the decades-isolated Yellowstone populations. Current movement rates do not appear sufficient to consider the subpopulations we identify along the Canada–US border as 1 inter-breeding unit. Although we detected enough male movement to mediate gene flow, the current low rate of female movement detected among areas is insufficient to provide a demographic rescue effect between areas in the immediate future (0–15 yr). In Alberta, we found fragmentation corresponded to major east–west highways (Highways 3, 11, 16, and 43) and most inter-area movements were made by males. Gene flow and movement rates between Alberta and BC were highest across the Continental Divide south of Highway 1 and north of Highway 16. In the central region between Highways 1 and 11, we found evidence of natural fragmentation associated with the extensive glaciers and icefields along the Continental Divide. The discontinuities that we identified would form appropriate boundaries for management units. We related sex-specific movement rates between adjacent areas to several metrics of human use (highway traffic, settlement, and human-caused mortality) to understand the causes of fragmentation. This analysis used data from 1,508 bears sampled over a 161,500-km² area in southeastern BC, western Alberta, northern Idaho, and northern Montana during 1979–2007. This area was bisected by numerous human transportation and settlement corridors of varying intensity and complexity. We used multiple linear regression and ANCOVA to document the responses of female and male bears to disturbance. Males and females both demonstrated reduced movement rates with increasing settlement and traffic. However, females reduced their movement rates dramatically when settlement increased to >20% of the fracture zone. At this same threshold, male movement declined more gradually, in response to increased traffic and further settlement. In highly settled areas (>50%), both sexes had a similar reduction in movements in response to traffic, settlement, and mortality. We documented several small bear populations with male-only immigration, highlighting the importance of investigating sex-specific movements. Without female connectivity, small populations are not viable over the long term. The persistence of this regional female fragmented metapopulation likely will require strategic connectivity management. We therefore recommend enhancing female connectivity among fractured areas by securing linkage-zone habitat appropriate for female dispersal, and ensuring current large source subpopulations remain intact. The fragmentation we documented may also affect other species with similar ecological characteristics: sparse densities, slow reproduction, short male-biased dispersal, and a susceptibility to human-caused mortality and habitat degradation. Therefore, regional inter-jurisdictional efforts to manage broad landscapes for inter-area movement will likely benefit a broad spectrum of species and natural processes, particularly in light of climate change. © 2011 The Wildlife Society.     

In situ chondrocyte deformation with physiological compression of the feline patellofemoral joint

The mechanical environment is an important factor affecting the maintenance and adaptation of articular cartilage, and thus the function of the joint and the progression of joint degeneration. Recent evidence suggests that cartilage deformation caused by mechanical loading is directly associated with deformation and volume changes of chondrocytes. Furthermore, in vitro experiments have shown that these changes in the mechanical states of chondrocytes correlate with a change in the biosynthetic activity of cartilage cells. The purpose of this study was to apply our knowledge of contact forces within the feline patellofemoral joint to quantify chondrocyte deformation in situ under loads of physiological magnitude. A uniform, static load of physiological magnitude was applied to healthy articular cartilage still fully intact and attached to its native bone. The compressed cartilage was then chemically fixed to enable the evaluation of cartilage strain, chondrocyte deformation and chondrocyte volumetric fraction. Patella and femoral groove articular cartilages differ in thickness, chondrocyte aspect ratio, and chondrocyte volumetric fraction in both magnitude and depth distribution. Furthermore, when subjected to the same compressive loads, changes to all of these parameters differ in magnitude and depth distribution between patellar and femoral groove articular cartilage. This evidence suggests that significant chondrocyte deformation likely occurs during in vivo joint loading, and may influence chondrocyte biosynthetic activity. Furthermore, we hypothesise that the contrasts between patella and femoral groove cartilages may explain, in part, the site-specific progression of osteoarthritis in the patellofemoral joint of the feline anterior cruciate ligament transected knee.     

Microarray Analysis of microRNA Expression during Axolotl Limb Regeneration

Among vertebrates, salamanders stand out for their remarkable capacity to quickly regrow a myriad of tissues and organs after injury or amputation. The limb regeneration process in axolotls (Ambystoma mexicanum) has been well studied for decades at the cell-tissue level. While several developmental genes are known to be reactivated during this epimorphic process, less is known about the role of microRNAs in urodele amphibian limb regeneration. Given the compelling evidence that many microRNAs tightly regulate cell fate and morphogenetic processes through development and adulthood by modulating the expression (or re-expression) of developmental genes, we investigated the possibility that microRNA levels change during limb regeneration. Using two different microarray platforms to compare the axolotl microRNA expression between mid-bud limb regenerating blastemas and non-regenerating stump tissues, we found that miR-21 was overexpressed in mid-bud blastemas compared to stump tissue. Mature A. mexicanum (“Amex”) miR-21 was detected in axolotl RNA by Northern blot and differential expression of Amex-miR-21 in blastema versus stump was confirmed by quantitative RT-PCR. We identified the Amex Jagged1 as a putative target gene for miR-21 during salamander limb regeneration. We cloned the full length 3′UTR of Amex-Jag1, and our in vitro assays demonstrated that its single miR-21 target recognition site is functional and essential for the response of the Jagged1 gene to miR-21 levels. Our findings pave the road for advanced in vivo functional assays aimed to clarify how microRNAs such as miR-21, often linked to pathogenic cell growth, might be modulating the redeployment of developmental genes such as Jagged1 during regenerative processes.