Use of substitute Nonidet P-40 nonionic detergents in intracellular tubulin polymerization assays for screening of microtubule targeting agents.

Shell Chemical Company Nonidet P-40 has been used for decades in many biochemical assays as a nonionic, nondenaturing detergent; however, Shell no longer manufactures this product. Four commercially available substitutes were investigated and their activities titrated in an intracellular tubulin polymerization assay. Although claimed by the supply companies to be identical to the Shell Nonidet P-40, all four substitutes were about 10-fold more potent and needed to be diluted accordingly. As microtubule targeting drugs are a major class of anticancer agent, and many researchers use the intracellular tubulin polymerization assay, this information is important to help troubleshoot assay development with the new substitutes. As the Shell Nonidet P-40 has been used in many biochemical buffers, these results will be of general interest to the biochemical, cell, and molecular research community.

Lack of recombinant factor VIII B-domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII.

Factor VIII (FVIII) is a multidomain blood plasma glycoprotein. Activated FVIII acts as a cofactor to the serine protease factor IXa within the membrane-bound tenase complex assembled on the activated platelet surface. Defect or deficiency in FVIII causes haemophilia A, a severe hereditary bleeding disorder. Intravenous administration of plasma-derived FVIII or recombinant FVIII concentrates restores normal coagulation in haemophilia A patients and is used as an effective therapy. In this work, we studied the biophysical properties of clinically potent recombinant FVIII forms: human FVIII full-length (FVIII-FL), human FVIII B-domain deleted (FVIII-BDD) and porcine FVIII-BDD bound to negatively charged phospholipid vesicles at near-physiological conditions. We used cryo-electron microscopy (Cryo-EM) as a direct method to evaluate the homogeneity and micro-organization of the protein-vesicle suspensions, which are important for FVIII therapeutic properties. Applying concurrent Cryo-EM, circular dichroism and dynamic light scattering studies to the three recombinant FVIII forms when bound to phospholipid vesicles revealed novel properties for their functional, membrane-bound state. The three FVIII constructs have similar activity, secondary structure distribution and bind specifically to negatively charged phospholipid membranes. Human and porcine FVIII-BDD induce strong aggregation of the vesicles, but the human FVIII-FL form does not. The proposed methodology is effective in characterizing and identifying differences in therapeutic recombinant FVIII membrane-bound forms near physiological conditions, because protein-containing aggregates are considered to be a factor in increasing the immunogenicity of protein therapeutics. This will provide better characterization and development of safer and more effective FVIII products with implications for haemophilia A treatment.

Allergic inflammation in the human lower respiratory tract affected by exposure to diesel exhaust.

To improve understanding of human health risks from exposure to diesel exhaust particles (DEP*), we tested whether immunologic effects previously observed in the human nose also occur in the lower airways. Our overall hypothesis was that cell influx and production of cytokines, chemokines, immunoglobulin E (IgE), and other mediators, which would be measurable in sputum and blood, occur in people with asthma after realistic controlled exposures to diesel exhaust (DE). In Phase 1 we tested for direct effects of DE in subjects with clinically undifferentiated mild asthma. In Phase 2 we tested whether DE exposure would exacerbate response to inhaled cat allergen in subjects with both asthma and cat sensitivity. The exposure facility was a controlled-environment chamber supplied with DE from an idling medium-duty truck with ultra-low-sulfur fuel and no catalytic converter. We exposed volunteers for 2 hours with intermittent exercise to exhaust with DEP mass concentration near 100 microg/m3. Exposures to nitrogen dioxide (NO2) near 0.35 ppm (similar to its concentration in DE) and to filtered air (FA) served as controls. Blood was drawn before exposure on day 1 and again the next morning (day 2). Sputum was induced only on day 2. Bronchial reactivity was measured -1 hour after exposure ended. Supplementary endpoints included measures of blood coagulation status, cardiopulmonary physiology, and symptoms. Each phase employed 15 subjects with asthma; 3 subjects participated in both phases. In Phase 1, airway reactivity was measured with inhaled methacholine; in Phase 2, with inhaled cat allergen. We found little biologic response to DE exposure compared with exposure to control atmospheres. In Phase 1, interleukin 4 (IL-4) in sputum showed an estimated 1.7-fold increase attributable to DE exposure, which was close to statistical significance; airway resistance increased modestly but significantly on day 2 after DE exposure; and nonspecific symptom scores increased significantly during DE exposure. In Phase 2, indicators of airway inflammation in sputum showed a possibly meaningful response: polymorphonuclear leukocytes (PMNs) and eosinophils increased after DE exposure, whereas macrophages decreased. IgE in sputum and the bronchoconstrictive response to cat allergen varied significantly between atmospheres, but not in patterns consistent with our primary hypothesis. Symptom score changes relatable to DE exposure were smaller than those in Phase 1 and not statistically significant. Controlled exposures, lasting 2 hours with intermittent exercise, to diluted DE at a particle mass concentration of 100 microg/m3 did not evoke clear and consistent lower-airway or systemic immunologic or inflammatory responses in mildly asthmatic subjects, with or without accompanying challenge with cat allergen. Likewise, these DE exposures did not significantly increase nonspecific or allergen-specific bronchial reactivity. A few isolated statistically significant or near-significant changes were observed during and after DE exposure, including increases in nonspecific symptoms (e.g., headache, nausea) suggestive of subtle, rapid-onset systemic effects. It is possible the lower respiratory tract is more resistant than the nose to adjuvant effects of diesel particles on allergic inflammation, so that no meaningful effects occur under exposure conditions like these. Alternatively, the experimental conditions may have been near a threshold for finding effects. That is, important lower respiratory effects may occur but may be detectable experimentally with slightly higher DEP concentrations, longer exposures, more invasive testing (e.g., bronchoalveolar lavage), or more susceptible subjects. However, ethical and practical barriers to such experiments are considerable.

Effects of interdental cleaning devices in preventing dental caries and periodontal diseases: a scoping review.

To identify and map existing evidence on the effectiveness of interdental cleaning devices in preventing dental caries and periodontal diseases, a scoping review was carried out by electronically searching PubMed, Scopus and Embase. Studies on interdental cleaning devices, written in English, and published from January 2008 up to April 2019 were included in the review. Of 1860 studies identified, six systematic reviews (SR) were included in the review. One SR each was on flossing, interdental brushes, wood sticks and oral irrigation. Of two SR on multitude of interdental cleaning devices, one assessed comparative efficacy while the other both the individual and comparative efficacy. All reviews had assessed the heterogeneity and the methodological quality of studies included, and performed data extraction and meta-analysis where appropriate. Evidence ranged from weak to moderate with very low- to low-certainty for the adjunctive benefit of these devices to control plaque and gingivitis. It warrants long-term studies with sufficient power and those assessing the impact of interdental cleaning on interproximal caries to corroborate such evidence. Available evidence on the efficacy of interdental cleaning devices suggests that dental practitioners recommend patient-specific interdental cleaning devices that enable patients to achieve a safe and high standard of interdental cleaning.

Aortocavitary fistula secondary to vegetative endocarditis in a rabbit.

A 4-year-old intact male mini lop rabbit (Oryctolagus cuniculus) was presented with a 2-week history of severe progressive lethargy. A right parasternal continuous heart murmur and an irregular rhythm were detected on physical examination. Echocardiography identified vegetative aortic and tricuspid valve (TV) endocarditis. There was an aortocavitary fistula between the right sinus of Valsalva into the right ventricle, creating a left-to-right intracardiac shunt. Based on the echocardiographic findings, it was suspected that the infection originated in the aortic valve, eroded through the periannular tissue, and secondarily infected the TV. Pleural and peritoneal effusion secondary to right-sided congestive heart failure was also found during the echocardiogram. Atrial fibrillation, conducted with a left bundle branch block morphology, was identified using electrocardiography. Necropsy findings directly correlated with the echocardiographic diagnosis; marked periodontal disease was also identified. Aerobic culture of the aortic and tricuspid vegetations resulted in significant growth of Haemophilus parainfluenzae. Haemophilus spp. belong to a group of similar gram-negative coccobacillus bacteria (HACEK group), which can act as an uncommon cause of endocarditis in humans. HACEK endocarditis is most commonly associated with oral infection and/or dental procedures in people. This is the first case report of a rabbit with periannular complications of infective endocarditis. It remains unknown whether dental disease resulted in endocarditis in this patient.

Whole genome sequence and phenotypic characterization of a Cbm+ serotype e strain of Streptococcus mutans.

We report the whole genome sequence of the serotype e Cbm+ strain LAR01 of Streptococcus mutans, a dental pathogen frequently associated with extra-oral infections. The LAR01 genome is a single circular chromosome of 2.1 Mb with a GC content of 36.96%. The genome contains 15 phosphotransferase system gene clusters, seven cell wall-anchored (LPxTG) proteins, all genes required for the development of natural competence and genes coding for mutacins VI and K8. Interestingly, the cbm gene is genetically linked to a putative type VII secretion system that has been found in Mycobacteria and few other Gram-positive bacteria. When compared with the UA159 type strain, phenotypic characterization of LAR01 revealed increased biofilm formation in the presence of either glucose or sucrose but similar abilities to withstand acid and oxidative stresses. LAR01 was unable to inhibit the growth of Strpetococcus gordonii, which is consistent with the genomic data that indicate absence of mutacins that can kill mitis streptococci. On the other hand, LAR01 effectively inhibited growth of other S. mutans strains, suggesting that it may be specialized to outcompete strains from its own species. In vitro and in vivo studies using mutational and heterologous expression approaches revealed that Cbm is a virulence factor of S. mutans by mediating binding to extracellular matrix proteins and intracellular invasion. Collectively, the whole genome sequence analysis and phenotypic characterization of LAR01 provides new insights on the virulence properties of S. mutans and grants further opportunities to understand the genomic fluidity of this important human pathogen.

Submarine watch schedules: underway evaluation of rotating (contemporary) and compressed (alternative) schedules.

INTRODUCTION: With a desire to increase health, cognitive performance effectiveness, and quality of life for submarine watch-standers underway, we performed an evaluation comparing an alternative, compressed-work (ALT) schedule, designed to enhance circadian rhythm entrainment and sleep hygiene, to the contemporary submarine (SUB) forward rotating schedule, aboard the ballistic-missile submarine, USS Henry M. Jackson (SSBN-730 Gold). METHODS: We assessed a compressed close-6 watch-schedule ("ALT") relative to the existing backward rotating 6-hr on, 12-hr off 18-hr watch schedule ("SUB") employed underway aboard submarines. We monitored 40 subjects' sleep, and temperature and salivary cortisol from 10 of the 40 for approximately two weeks on each respective schedule underway. RESULTS: The cortisol cosinor mesors (midline estimating statistic of rhythm), and amplitudes did not differ significantly between conditions. The temperature cosinor mesors, and the cosinor amplitude were not significantly different, while the cosine curve fit accounted for significantly more variance in the ALT condition than in the SUB condition. The SUB schedule garnered significantly more sleep (7.1 +/- 0.2 hours) than that of the ALTMID schedule (6.3 +/- 0.3 hours). Surveys revealed that 52% of respondents preferred the SUB schedule, 15% preferred the ALT, and 33% were either indifferent or submitted uninterpretable surveys. CONCLUSIONS: The ALT schedule was not superior to the existing SUB schedule by physiological or subjective measures and was incompatible to accommodating operational constraints.

Collagen-binding proteins of Streptococcus mutans and related streptococci.

The ability of Streptococcus mutans to interact with collagen through the expression of collagen-binding proteins (CBPs) bestows this oral pathogen with an alternative to the sucrose-dependent mechanism of colonization classically attributed to caries development. Based on the abundance and distribution of collagen throughout the human body, stringent adherence to this molecule grants S. mutans with the opportunity to establish infection at different host sites. Surface proteins, such as SpaP, WapA, Cnm and Cbm, have been shown to bind collagen in vitro, and it has been suggested that these molecules play a role in colonization of oral and extra-oral tissues. However, robust collagen binding is not achieved by all strains of S. mutans, particularly those that lack Cnm or Cbm. These observations merit careful dissection of the contribution from these different CBPs towards tissue colonization and virulence. In this review, we will discuss the current understanding of mechanisms used by S. mutans and related streptococci to colonize collagenous tissues, and the possible contribution of CBPs to infections in different sites of the host.

Pegylation of a chlorin(e6) polymer conjugate increases tumor targeting of photosensitizer.

Photodynamic therapy is emerging as a viable modality for the treatment of many cancers. A limiting factor in its use against intracavity tumors such as disseminated ovarian cancer is insufficient selectivity of the photosensitizer for tumor compared with normal tissue. We report on an approach to improve tumor targeting by exploiting differences between cell types and by chemical modification of a photosensitizer conjugate. Attachment of polyethylene glycol (pegylation) to a polyacetylated conjugate between poly-l-lysine and chlorin(e6) increased the relative phototoxicity in vitro toward an ovarian cancer cell line (OVCAR-5) while reducing it toward a macrophage cell line (J774), compared with the nonpegylated conjugate. Surprisingly, the increased phototoxicity of the pegylated conjugate correlated with reduced oxygen consumption. Pegylation also reduced the tendency of the conjugate to aggregate and reduced the consumption of oxygen when the conjugates were illuminated in solution in serum containing medium, suggesting a switch in photochemical mechanism from type II (singlet oxygen) to type I (radicals or electron transfer). Pegylation led to more mitochondrial localization as shown by confocal fluorescence microscopy in OVCAR-5 cells, and, on illumination, produced a switch in cell death mechanism toward apoptosis not seen with J774 cells. Conjugates were injected i.p. into nude mice bearing i.p. OVCAR-5 tumors, and the pegylated conjugate gave higher amounts of photosensitizer in tumor and higher tumor:normal tissue ratios and increased the depth to which the chlorin(e6) penetrated into the peritoneal wall. Taken together, these results suggest that pegylation of a polymer-photosensitizer conjugate improves tumor-targeting and may increase the efficacy of photodynamic therapy for ovarian cancer.

Glycosylphosphatidylinositol-anchored proteins are not required for crosslinking-mediated endocytosis or transfection of avidin bioconjugates into biotinylated cells.

Even though glycosylphosphatidylinositol (GPI)-anchored proteins lack direct structural contact with the intracellular space, these ubiquitously expressed surface receptors activate signaling cascades and endocytosis when crosslinked by extracellular ligands. Such properties may be due to their association with membrane microdomains composed of glycosphingolipids, cholesterol and some signaling proteins. In this study, we hypothesize that GPI proteins may be required for crosslinking-mediated endocytosis of extracellular bioconjugates. To test this hypothesis, we first biotinylated the surface membranes of native K562 erythroleukemia cells versus K562 cells incapable of surface GPI protein expression. We then compared the entry of fluorescently labeled avidin or DNA condensed on polyethylenimine-avidin bioconjugates into the two biotinylated cell populations. Using fluorescence microscopy, nearly 100% efficiency of fluorescent avidin endocytosis was demonstrated in both cell types over a 24 h period. Surprisingly, plasmid DNA transfer was slightly more efficient among the biotinylated GPI-negative cells as measured by the expression of green fluorescence protein. Our findings that GPI proteins are not required for the endocytosis of avidin bioconjugates into biotinylated cells suggest that endocytosis associated with general membrane crosslinking may be due to overall reorganization of the membrane domains rather than GPI protein-specific interactions.

Fragmentation of 1 GeV/nucleon iron ions in thick targets relevant for space exploration.

We have measured charged nuclear fragments produced by 1 GeV/nucleon 56Fe ions interacting with aluminium, polyethylene and lead. These materials are relevant for assessment of radiation risk for manned space flight. The data will be presented in a form suitable for comparison with models of nuclear fragmentation and transport, including linear energy transfer (LET) spectrum, fluence for iron and fragments, event-tack- and event-dose-averaged LET, total dose and iron contribution to dose.

Validation of the HZETRN code for laboratory exposures with 1A GeV iron ions in several targets.

A new version of the HZETRN code capable of validation with HZE ions in either the laboratory or the space environment is under development. The computational model consists of the lowest order asymptotic approximation followed by a Neumann series expansion with non-perturbative corrections. The physical description includes energy loss with straggling, nuclear attenuation, nuclear fragmentation with energy dispersion and downshift. Measurements to test the model were performed at the Alternating Gradient Synchrotron and the NASA Space Radiation Laboratory at Brookhaven National Laboratory with iron ions. Surviving beam particles and produced fragments were measured with solid-state detectors. Beam analysis software has been written to relate the computational results to the measured energy loss spectra of the incident ions for rapid validation of modeled target transmission functions.

The quality of radiographs accompanying endodontic referrals to a health authority clinic.

AIM: To assess the quality of radiographs accompanying endodontic referrals, from general dental practitioners, to a health authority clinic. METHODS: A total of 200 conventional film and digital radiographs accompanying referrals were assessed and rated as 'excellent', 'diagnostically acceptable' or 'unacceptable' according to the National Radiographic Protection Board (NRPB) guidelines. Statistical analyses of the results included inter- and intra-observer agreement to achieve a kappa score and the chi-squared test. RESULTS: Out of the 200 radiographs assessed, 38 (19%) were conventional film and 162 (81%) were digital. Of the conventional film radiographs, 55% were rated 'excellent' and 37% were 'diagnostically acceptable', whilst 27% of digital radiographs were rated 'excellent' and 40% were 'diagnostically acceptable'. In the 'unacceptable' category, 33% were digital and 8% were conventional film radiographs (p <0.001). CONCLUSIONS: The quality of digital radiographs was significantly lower compared with conventional film radiographs. The percentage of 'unacceptable' digital radiographs was above the target according to the NRPB guidelines. Hence, there is a need for improvement in quality to avoid repeat radiographs and unnecessary ionising radiation exposure. Instead of hard, printed copies, digital radiographs accompanying referrals should, within the parameters of information governance, be supplied electronically so that they may be optimised, if necessary, for better diagnostic value.

Photosensitizer delivery for photodynamic therapy of choroidal neovascularization.

The present review examines the importance of improving photosensitizer delivery for choroidal neovascularization (CNV) in light of the clinical impact of photodynamic therapy (PDT) for CNV. An overview of the classes of available photosensitizers is provided and the properties governing photosensitizer uptake and circulation in serum are discussed. Current delivery systems, for example liposomal formulations as well as the use of the promising strategy of antibody targeted delivery as a strategy to improve PDT selectivity and efficiency for CNV treatment are described. A summary of the work using Verteporfin, tin ethyl purpurin and Lu-Tex--photosensitizers currently in clinical trials for CNV--is given.

Progesterone synthesis in developing rabbit corpora lutea in the absence of follicular estrogens.

We have investigated the role of 17beta-estradiol in the early development of the rabbit corpus luteum. Ectopic corpora lutea were established in all animals by autotransplanting preovulatory follicles beneath the kidney capsule 6.5 to 8 h after mating (day 0). At this time, the rabbits were bilaterally ovariectomized and in some of these rabbits a Silastic capsule containing crystalline 17beta-estradiol was implanted SC. Mean serum concentration of estradiol in rabbits with an estradiol impant was 16.4 pg/ml. In rabbits without an estradiol implant, the estradiol concentration averaged 1 pg/ml despite the presence of transplanted luteinized follicles. Daily blood samples were analyzed for progesterone by radioimmunoassay. Ectopic corpora lutea developed in rabbits with or without estradiol treatment. Serum progesterone concentrations in the two groups increased above castrate values and were not significantly different from one another through day 5. After day 5, progesterone concentrations steadily increased in rabbits treated with estradiol and reached a value of 4.8 ng/ml by day 10. In contrast serum progesterone steadily decreased after day 5 in rabbits without estradiol treatment to a level of 450 pg/ml by day 10. Hysterectomy on day 0 did not prevent this decline in progesterone, indicating that a uterine luteolytic agent was not involved. Total luteal weight on day 10 was positively correlated with serum progesterone concentration (r equals .92; P greater than 0.01). These results indicate that for a period of approximately 5 days afte r ovulation, the development of the rabbit ectopic corpus luteum and the secretion of progesterone are autonomous from estradiol secreted by ovarian follicles. After this time, there is an absolute requirement for estrogen which permits further development of the corpus luteum and the continuation of progesterone synthesis.

Estradiol suppression of luteinizing hormone (LH)/human chorionic gonadotropin receptors and LH-sensitive adenylyl cyclase without decreased adenosine 3',5'-monophosphate content in rabbit corpora lutea.

It has been observed that elevated concentrations of estradiol, the principle luteotropin in the rabbit, reduce LH-stimulated adenylyl cyclase activity in corpora lutea during midpseudopregnancy without suppressing serum and tissue progesterone concentrations. If LH modulates intraluteal cAMP levels in this species, this suggests that in the presence of exogenous estradiol, progesterone synthesis may be independent of cAMP. To test this possibility and to investigate the physiological significance of LH in regulating the rabbit corpus luteum, the concentrations of cAMP and progesterone and the activity of adenylyl cyclase were measured in luteal homogenates, the numbers of LH/hCG receptors were estimated in crude membrane preparations, and the concentrations of progesterone and estradiol were measured in serum on days 2-12 of pseudopregnancy in rabbits treated with or without estradiol. Throughout days 2-12 of pseudopregnancy, estradiol treatment increased LH-stimulated adenylyl cyclase activity on day 2, but reduced its activity after day 4 by up to 50%, reduced the number of LH/hCG receptors after day 2 by up to 50%, and had no effect on the activities of basal, epinephrine-stimulated, or NaF-stimulated adenylyl cyclase. Tissue cAMP levels were not altered by estradiol treatment, nor were serum progesterone concentrations (except for an increase on day 2). Since LH receptors and LH-stimulated adenylyl cyclase were both reduced by day 5 of pseudopregnancy without a concomitant decrease in luteal cAMP or serum progesterone concentrations, our data suggest that LH is not a physiological regulator of luteal cAMP or serum progesterone during days 4-12 of pseudopregnancy. We propose that basal adenylyl cyclase activity, which is not reduced by estradiol treatment, may play a more significant role than LH-stimulated adenylyl cyclase activity in regulating tissue cAMP levels and the progesterone synthetic capacity of the rabbit corpus luteum.

Premature regression of corpora lutea in pseudopregnant rabbits following the removal of polydimethylsiloxane capsules containing 17 beta-estradiol.

17-beta-Estradiol, which is luteotropic in rabbits, was administered during pseudopregnancy via polydimethylsiloxane (Silastic) implants to determine the effects on serum progesterone concentrations. Implants which released estradiol at a rate of approximately 2 mug/day were place beneath the skin the day after sterile mating and ovulation (day 0). Blood (3 ml) was obtained from the marginal ear vein on days 3, 6, 9, 10, 11 and 12. Serum estradiol levels, determined by radioimmunoassay, were 2- to 3-fold higher in estradiol-treated rabbits (11.7 plus or minus 1.2 pg/ml) than in untreated pseudopregnant controls (5.9 plus or minus 1.4 pg/ml). Weights of corpora lutea in treated and control rabbits were not different at the conclusion of the experiment on day 12. Serum progesterone concentrations, also determined by radioimmunoassay, were not significantly different between treated and control animals. However, when estradiol implants were removed from other rabbits on day 10, a rapid decline in serum progesterone occurred, from 14.0 plus or minus 2.4 to 2.6 plus or minus 0.8 ng/ml 24 h later. By comparison, serum progesterone concentrations in rabbits with estradiol implants left in place and in untreated rabbits on day 12 were similar (similar to 12 ng/ml). The premature decline in serum progesterone was accompanied by a decrease in the wet weight of corpora lutea. Other experiments revealed: 1) a precipitous fall in serum estradiol to basal values within 2 h after estradiol implants were removed, preceding the decline in serum progesterone by approximately 6 to 10 h; 2) reduced levels of estradiol in ovarian venous blood, but elevated levels of estradiol in peripheral arterial blood of rabbits with estradiol impants. The inability to elevated estradiol to increase serum progesterone or weights of corpora litea suggests that the luteotropic effect is maximal when estradiol is present at physiological concentrations. Following the continuous administration of estradiol, ovarian secretion of estradiol appears diminished and the corpora lutea become dependent upon the exogenous estradiol for luteotropic support. Although the ovaries continue to release measureable quantities of estradiol, this is inmeasurable quantities of estradiol, this is insufficient to prevent regression of corpora lutea when exogenous estradiol is rapidly withdrawn from the circulation.

Seizures induced by playing music.

A 67-year-old organist and minister with diabetes mellitus had stereotyped focal seizures of the left lower face, jaw, and neck. Attacks occurred spontaneously or were induced when he played a specific hymn on the organ. The seizures were not induced by reading, singing, hearing, or playing the hymn silently. The patient had interictal weakness of the left lower face and left side of the tongue. Focal seizures were recorded on an electroencephalogram (EEG) at the right temporofrontal area. This patient illustrates partial seizures induced by playing music.

3-Alkyl-3-hydroxyglutaric acids: a new class of hypocholesterolemic HMG CoA reductase inhibitors. 1.

Derivatives of 3-hydroxy-3-methylglutaric acid (HMG), a portion of the substrate for HMG CoA reductase, were prepared and tested for their inhibitory action against rat liver HMG CoA reductase and for their hypocholesterolemic activity. Structure-dependent competitive inhibition was observed. Optimal structures had a free dicarboxylic acid with an alkyl group of 13-16 carbons at position 3. 3-n-Pentadecyl-3-hydroxyglutaric acid (3j) (IC50 = 50 microM) reduced serum cholesterol in the Triton-treated rat and HMG CoA reductase activity in the 20,25-diazacholesterol-treated rat.

Stable expression of green fluorescent protein after liposomal transfection of K562 cells without selective growth conditions.

Little is known about the durability of plasmid DNA transgene expression in mammalian cells in the absence of growth selection. For this purpose, we have begun the study of liposomal transfer and expression of plasmid DNA encoding green fluorescent protein (GFP) in human erythroleukemia K562 cells. Detection and selection of GFP expression were accomplished visually and by flow cytometry. GFP expression was noticeable in cells within 4 h of transfection. In nine separate transfections, approximately 20% of the transfected cells expressed GFP with a mean fluorescence 40-50x that of control cells (15 fluorescent units [FU] vs. 0.3 FU) during the first five days after transfection. The percentage of GFP positive cells dropped rapidly to 0.1% by day 14 post-transfection, but fluorescence activated cell sorting on this day resulted in the identification of stable transfectants expressing GFP for an additional 6-12 months in culture. GFP expression is adequate for the identification, isolation and monitoring of stable transfection events after lipid-mediated transfection of eukaryotic cells.