Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-l-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.     

Structure–activity relationships amongst 4-position quinoline methanol antimalarials that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum

Utilizing mefloquine as a scaffold, a next generation quinoline methanol (NGQM) library was constructed to identify early lead compounds that possess biological properties consistent with the target product profile for malaria chemoprophylaxis while reducing permeability across the blood–brain barrier. The library of 200 analogs resulted in compounds that inhibit the growth of drug sensitive and resistant strains of Plasmodium falciparum. Herein we report selected chemotypes and the emerging structure–activity relationship for this library of quinoline methanols.     

DNA Is Taken Up by Root Hairs and Pollen,and Stimulates Root and Pollen Tube Growth

Phosphorus (P) enters roots as inorganic phosphate (P_i) derived from organic and inorganic P compounds in the soil. Nucleic acids can support plant growth as the sole source of P in axenic culture but are thought to be converted into P_i by plant-derived nucleases and phosphatases prior to uptake. Here, we show that a nuclease-resistant analog of DNA is taken up by plant cells. Fluorescently labeled S-DNA of 25 bp, which is protected against enzymatic breakdown by its phosphorothioate backbone, was taken up and detected in root cells including root hairs and pollen tubes. These results indicate that current views of plant P acquisition may have to be revised to include uptake of DNA into cells. We further show that addition of DNA to P_i-containing growth medium enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with P_i only. Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs. Our results indicate that DNA is not only taken up and used as a P source by plants, but ironically and independent of P_i supply, DNA also induces morphological changes in roots similar to those observed with P limitation. This study provides, to our knowledge, first evidence that exogenous DNA could act nonspecifically as signaling molecules for root development.Phosphorus (P) is an essential macronutrient that limits plant growth in many situations due to a low availability in soils (for review, see Schachtman et al., 1998; Raghothama, 1999; Vance et al., 2003; Lambers et al., 2008). P enters plant roots as orthophosphates (P_i) via active transport across the plasma membrane (Smith et al., 2003; Park et al., 2007; Xu et al., 2007). Concentrations of P_i in soil solution are generally very low (<10 μm; Bieleski, 1973) and plants have evolved root specializations to access P from inorganic and organic sources (Raghothama, 1999; Hinsinger, 2001; López-Bucio et al., 2003; Vance et al., 2003; Lambers et al., 2008). Roots exude enzymes and chemicals to mobilize P directly from soil compounds or indirectly via enhanced activity of soil microbes, and form symbioses with P-mobilizing mycorrhizal fungi (Schachtman et al., 1998; Raghothama, 1999; Bucher, 2007).However, similar to other nutrients, notably nitrogen, research on P nutrition of plants has focused on inorganic sources although organic P (P_org) in soil can account for 40% to 80% of the total P pool of mineral and organic soils, respectively (Bower, 1945; Raghothama, 1999; Vance et al., 2003). P_org compounds in soils are derived from plant residues, soil biota, and from synthesis by soil microbes (Jencks et al., 1964). Soil P_org is composed primarily of phospholipids, nucleic acids, and phytin (Dyer and Wrenshall, 1941). Phytic acid (inositol hexaphosphate) and its salts phytate, account for a large proportion of the P_org pool of soils (Anderson, 1980). Nucleic acids (RNA, DNA) represent approximately 1% to 2% of the soil P_org pool (Dalal, 1977). It can be released from prokaryotic and eukaryotic cells after death and protected against nuclease degradation by its adsorption on soil colloids and sand particles (Pietramellara et al., 2009).Although P_org can be a substantial constituent of the soil P pool, its contribution to the P nutrition of plants is poorly understood. P_org can be converted to P_i via root-exuded enzymes (Tarafdar and Claassen, 1988; Marschner, 1995; Vance et al., 2003). Secretion of nucleolytic enzymes and breakdown of nucleic acid were considered the reason for the observed growth of axenic Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum) on nucleic acid substrates as the sole P source (Chen et al., 2000; Richardson et al., 2000).Whether plants take up intact DNA has not been reported. We recently showed that roots take up protein, possibly via endocytosis (Paungfoo-Lonhienne et al., 2008). We hypothesized that roots may take up DNA by a similar process and grew Arabidopsis in the presence of phosphorothioate oligonucleotides (S-DNA) labeled with Cy3-fluorescent dye. S-DNA has a sulfur backbone and cannot be digested by plant nucleases, allowing tracking DNA of known size into cells (Spitzer and Eckstein, 1988). We examined if S-DNA of 25 nucleotides in length enters root hairs and pollen tubes as both types of cells are strongly elongated and have similar polarized growth (Schiefelbein et al., 1993; Hepler et al., 2001). We also assessed if addition of DNA to the growth medium affects the morphology of roots and pollen tubes. Here, we present evidence that plants take up DNA and demonstrate that the presence of DNA in the growth medium enhances lateral branching of roots, and the length of root hairs and pollen tubes, irrespective of P_i supply.     

Application of Modeling to Scale-up Dissolution in Pharmaceutical Manufacturing

Liquid mixing scale-up in pharmaceutical industry has often been based on empirical approach in spite of tremendous understanding of liquid mixing scale-up in engineering fields. In this work, we attempt to provide a model-based approach to scale-up dissolution process from a 2 l lab-scale vessel to a 4,000 l scale vessel used in manufacturing. Propylparaben was used as a model compound to verify the model predictions for operating conditions at commercial scale that would result in similar dissolution profile as observed in lab scale. Geometric similarity was maintained between both of the scales to ensure similar mixing characteristics. We utilized computational fluid dynamics (CFD) to ensure that the operating conditions at laboratory and commercial scale will result in similar power per unit volume (P/V). Utilizing this simple scale-up criterion of similar P/V across different scales, results obtained indicate fairly good reproducibility of the dissolution profiles between the two scales. Utilization of concepts of design of experiments enabled summarizing scale-up results in statistically meaningful parameters, for example −90% dissolution in lab scale at a given time under certain operating conditions will result in 75–88% at commercial scale with 95% confidence interval when P/V is maintained constant across the two scales. In this work, we have successfully demonstrated that scale-up of solid dissolution can be done using a systematic process of lab-scale experiments followed by simple CFD modeling to predict commercial-scale experimental conditions.     

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.     

Persistent activation of STAT3 by latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma, primary effusion lymphoma, and plasmablastic multicentric Castleman's disease. STAT3 has been shown to be important for the maintenance of primary effusion lymphoma cells in culture and is chronically activated in many tumor cell lines. However, little is known about the role of KSHV in the activation of STAT3 or the role of STAT3 in KS tumors. We demonstrate that STAT3 is activated by KSHV infection of endothelial cells, the KS tumor cell type, in a biphasic fashion. Viral binding and entry activate STAT3 in the first 2 h after infection, but this activation dissipates by 4 h postinfection. By 12 h after KSHV infection, concomitant with the expression of latent genes, STAT3 is once again activated, and this activation persists for as long as latent infection is maintained. Activated STAT3 translocates to the nucleus, where it can bind to STAT3-specific DNA elements and can activate STAT3-dependent promoter activity. Conditioned medium from KSHV-infected endothelial cells is able to transiently activate STAT3, indicating the involvement of a secreted factor and that a latency-associated factor in KSHV-infected cells is necessary for sustained activation. KSHV upregulates gp130 receptor expression, and both gp130 and JAK2 are required for the activation of STAT3. However, neither human nor viral interleukin-6 is required for STAT3 activation. Persistent activation of the oncogenic signal transducer, STAT3, by KSHV may play a critical role in the viral pathogenesis of Kaposi's sarcoma, as well as in primary effusion lymphomas.     

Visual documentation of ovine pituitary gland development with magnetic resonance imaging following zeranol treatment

The objective of the current study was to determine whether magnetic resonance imaging (MRI) could be successfully utilized to document the effect of an oestrogenic anabolic agent on pituitary gland growth. The experimental animals consisted of two 1/2 sibling Suffolk wethers (castrated rams), which received either no implant (control, n = 1) or a 24 mg zeranol implant at day 0 and day 42 (zeranol; n = 1). Animals were anaesthetized with propofol and supported with oxygen during the MRI procedure. A mobile MRI unit with a 0.5 tesla (T), superconducting magnet was used to obtain 3 mm thick, non-contrast enhanced, T1-weighted (TR 500-600, TE25) sagittal, transverse and dorsal images of the pituitary gland. Sagittal images were recorded only when the mesencephalic aqueduct and infundibulum were distinctly visible in the same image. Pituitary glands were imaged at 14-day intervals for 70 days to determine if and when the anabolic effects of zeranol on pituitary gland growth could be visualized using MRI techniques. Three separate measurements of the pituitary gland dimensions made with the on-screen cursor were averaged to calculate pituitary gland dimensions and volume. A computer-assisted image analysis system and laser film images were used to determine pituitary gland area. Increases in pituitary gland volume for control and zeranol-treated animals were evident within 14 days, and by the end of the 70-day study, the increase in pituitary volume for the zeranol-treated animal was three times greater than that of the control animal. Overall, our results indicate that MRI technology can be successfully used to document the development of the pituitary gland in vivo. Application of knowledge gained from this novel approach to study the growth, development and function of endocrine glands over time, and within the same animal, will enhance human and animal endocrine diagnostic procedures.     

Differential activation of insulin receptor substrates 1 and 2 by insulin-like growth factor-activated insulin receptors

The insulin-like growth factors (insulin-like growth factor I [IGF-I] and IGF-II) exert important effects on growth, development, and differentiation through the IGF-I receptor (IGF-IR) transmembrane tyrosine kinase. The insulin receptor (IR) is structurally related to the IGF-IR, and at high concentrations, the IGFs can also activate the IR, in spite of their generally low affinity for the latter. Two mechanisms that facilitate cross talk between the IGF ligands and the IR at physiological concentrations have been described. The first of these is the existence of an alternatively spliced IR variant that exhibits high affinity for IGF-II as well as for insulin. A second phenomenon is the ability of hybrid receptors comprised of IGF-IR and IR hemireceptors to bind IGFs, but not insulin. To date, however, direct activation of an IR holoreceptor by IGF-I at physiological levels has not been demonstrated. We have now found that IGF-I can function through both splice variants of the IR, in spite of low affinity, to specifically activate IRS-2 to levels similar to those seen with equivalent concentrations of insulin or IGF-II. The specific activation of IRS-2 by IGF-I through the IR does not result in activation of the extracellular signal-regulated kinase pathway but does induce delayed low-level activation of the phosphatidylinositol 3-kinase pathway and biological effects such as enhanced cell viability and protection from apoptosis. These findings suggest that IGF-I can function directly through the IR and that the observed effects of IGF-I on insulin sensitivity may be the result of direct facilitation of insulin action by IGF-I costimulation of the IR in insulin target tissues.