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The study aimed at improving the protocol for cryopreservation of Atlantic cod sperm. Five diluents, four cryoprotectants, two time intervals between thawing and activation of spermatozoa, and two sperm collection times prior to freezing were tested for their effect on post-thaw motility and fertilization ability. Minimum sufficient sperm:egg ratio was determined. The study was expanded by quantitative assessment of cod sperm using computer-assisted sperm analysis ( CASA). Fertilization success obtained with fresh sperm was generally higher than with the cryopreserved sperm. Hanks' solution + 10% hen's egg yolk + 10% glycerol or 10% dimethyl actetamide ( HBSS+ EY+ DMA and HBSS+ EY+Gly, respectively) were the best among the tested variants and resulted in average hatching success of embryos produced with use of cryopreserved sperm (44 and 36%, respectively) not differing from control (36%). A 30 min delay between thawing and activation did not have a significant effect on post-thaw spermatozoa motility in 15 out of 20 tested variants. Hatching success of embryos produced with the use of cryopreserved sperm stored for 3 days prior to freezing, in two out of three individual sperm samples, did not differ significantly from control. . Percentage and straight-line velocity of motile spermatozoa were found to be good predictors for the fertilization potential of cryopreserved sperm, correlating highly and positively ( r > 0.80) with fertilization and hatching success of embryos obtained with cryopreserved sperm, particularly when low sperm:egg ratios were applied. When using cryopreserved sperm, the minimum sperm:egg ratio required to maximize fertilization succes was 300 000 : 1. [ABSTRACT FROM AUTHOR]

Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19.Following an oral dose administration in Long-Evans rats, 14 C-emvododstat-derived radioactivity was widely distributed throughout the body, with the highest distribution in the endocrine, fatty, and secretory tissues and the lowest in central nervous system.Following a single oral dose of 14 C-emvododstat in rats, 54.7% of the dose was recovered in faeces while less than 0.4% of dose was recovered in urine 7 days post-dose. Emvododstat was the dominant radioactive component in plasma and faeces.Following a single oral dose of 14 C-emvododstat in dogs, 75.2% of the dose was recovered in faeces while 0.5% of dose was recovered in urine 8 days post-dose. Emvododstat was the dominant radioactive component in faeces, while emvododstat and its two metabolites ( O -desmethyl emvododstat and emvododstat amide bond hydrolysis product) were the major circulating radioactivity in dog plasma.

The Multifactor Leadership Questionnaire (MLQ 5X Short) is the most frequently used leadership measure in scholarship and organizational practice. However, so far it has not been validated in the Polish context. Therefore, the present study aimed to validate and shorten the MLQ (5X Short) in the Polish organizational setting. A total sample of 1,065 employees (572 women and 493 men) from different organizations took part in two sessions of an online study. Respondents were between 18 and 70 years old ( M = 40.1; SD = 12.9) with an average job tenure of 17.00 years ( SD = 12.1). In subsample 1 ( n = 539), using exploratory factor analysis, a three-factor structure of the MLQ full form (MLQ-FF) was established, comprising transformational-supportive, inspirational goal-oriented, and passive-avoidant leadership. Based on qualitative (i.e., content analysis) and quantitative criteria (psychometric parameters), we constructed an 18-item MLQ short form (MLQ-SF). Both forms were supported by the confirmatory factor analysis in subsample 2 ( n = 526). The MLQ-FF and MLQ-SF factors displayed acceptable to high levels of item-related parameters (e.g., intra-class, inter-item, and item-total correlations), as well as scale-related reliability (e.g., internal consistency, temporal stability). Both forms indicated high convergent and predictive validity examined by correlations with authentic leadership and employee's work outcomes (i.e., work satisfaction, work effectiveness, work engagement, and organizational commitment) (subsample 3; n = 691). Our study provided the full and the short form of the MLQ as reliable and valid instruments, potentially suitable to measure leadership styles in academic research and organizational practice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bajcar and Babiak.)

Emvododstat was identified as a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of acute myeloid leukaemia and COVID-19. The objective of this paper is to evaluate the metabolism, pharmacokinetics, and drug interaction potentials of emvododstat.Emvododstat showed high binding to plasma protein with minimal distribution into blood cells in mouse, rat, dog, monkey, and human whole blood. O -Demethylation followed by glucuronidation appeared to be the major metabolic pathway in rat, dog, monkey, and human hepatocytes. CYP2C8, 2C19, 2D6, and 3A4 were involved in O -desmethyl emvododstat metabolite formation. Both emvododstat and O -desmethyl emvododstat inhibited CYP2D6 activity and induced CYP expression to different extents in vitro .Emvododstat and O -desmethyl emvododstat inhibited BCRP transporter activity but did not inhibit bile salt transporters and other efflux or uptake transporters. Neither emvododstat nor O -desmethyl emvododstat was a substrate for common efflux or uptake transporters investigated.Emvododstat is bioavailable in mice, rats, dogs, and monkeys following a single oral dose. The absorption was generally slow with the mean plasma T max ranging from 2 to 5 h; plasma exposure of O -desmethyl emvododstat was lower in rodents, but relatively higher in dogs and monkeys.

Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [ 14 C]-ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was also observed in the incubation in human intestinal and kidney microsomes, but not in human pulmonary microsomes. UGT1A9 was found to be the major uridine diphosphate glucuronosyltransferase (UGT) responsible for ataluren glucuronidation in the liver and kidney microsomes. Enzyme kinetic analysis of the formation of ataluren acyl glucuronide, performed in human liver, kidney, and intestinal microsomes and recombinant human UGT1A9, found that increasing bovine serum albumin (BSA) levels enhanced the glucuronidation Michaelis-Menten constant (K m ) and ataluren protein binding but had a minimal effect on maximum velocity (V max ) of glucuronidation. Due to the decreased unbound Michaelis-Menten constant (K m,u ), the ataluren unbound intrinsic clearance (CL int,u ) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7-fold more active than human liver microsomes, in terms of CL int,u /mg protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Ataluren is a unique small molecule developed for the treatment of diseases caused by nonsense mutations, which result in premature termination of ribosomal translation and lack of full-length protein production. This study investigated the in vivo metabolism and disposition of ataluren in mice, rats, dogs, and humans. After single oral administration of [ 14 C]ataluren, the overall recovery of radioactivity was ≥93.7%, with approximately 39%, 17%-21%, 12%, and 55% in the urine and 54%, 70%-72%, 80%, and 47% in the feces from intact mice, rats, dogs, and humans, respectively. In bile duct-cannulated (BDC) rats, approximately 10%, 7%, and 82% of the dose was recovered in the urine, feces, and bile, respectively, suggesting that biliary secretion was a major route for the elimination of ataluren in the rats. Ataluren was extensively metabolized after oral administration, and the metabolic profiles of ataluren were quantitatively similar across all species. Unchanged ataluren was the dominant radioactive component in plasma. Ataluren acyl glucuronide was the most prominent metabolite in plasma of all species and the dominant metabolite in BDC rat bile and human urine, whereas the oxadiazole cleavage products were the major or prominent metabolites in the feces of all species. Overall, the results indicate that phase I metabolism is negligible and that the pathway largely involves glucuronidation. No other circulatory conjugation metabolite was detected across investigated species. SIGNIFICANCE STATEMENT: Ataluren is a novel carboxylic acid-containing small molecule drug for treating nonsense mutation Duchenne muscular dystrophy. In vivo metabolism and disposition after a single dose of the drug were investigated in mice, rats, dogs, and humans. Phase I metabolism of ataluren was negligible, and the pathway largely involves glucuronidation. No other circulatory conjugation metabolite was detected across investigated species., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Objectives: The aim of this paper is a presentation of the adaptation process of the Cyberchondria Severity Scale (SCC) to measure cyberchondria and verification of reliability and construct validities of the test in the Polish population., Methods: The study included 380 participants (203 women and 177 men) aged 19-68 (M = 26.5; SD = 11.1). The CSS-PL was used to measure cyberchondria, the Short Health Anxiety Inventory (SHAI) for measuring hypochondria, and the Dimensional Obsessive-Compulsive Scale (DOCS) to verify obsessive-compulsive tendencies., Results: Four-factor structure of cyberchondria measured with the CSS-PL was supported. Internal consistency indices of the CSS-PL were between 0.87 and 0.95, test-retest reliability results were 0.58-0.76. This study demonstrated construct validity of the CSS-PL via its correlations with health anxiety (r = 0.31-0.56) and obsessive-compulsive symptoms (r = 0.17-0.48)., Conclusions: The CSS-PL is an initial standardized version of an instrument for measuring cyberchondria and meets the psychometric criteria of reliability and validity for psychological testing tools. The CSS-PL may be used both in diagnostic and scientific research.

PTC299 was identified as an inhibitor of VEGFA mRNA translation in a phenotypic screen and evaluated in the clinic for treatment of solid tumors. To guide precision cancer treatment, we performed extensive biological characterization of the activity of PTC299 and demonstrated that inhibition of VEGF production and cell proliferation by PTC299 is linked to a decrease in uridine nucleotides by targeting dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme for de novo pyrimidine nucleotide synthesis. Unlike previously reported DHODH inhibitors that were identified using in vitro enzyme assays, PTC299 is a more potent inhibitor of DHODH in isolated mitochondria suggesting that mitochondrial membrane lipid engagement in the DHODH conformation in situ is required for its optimal activity. PTC299 has broad and potent activity against hematologic cancer cells in preclinical models, reflecting a reduced pyrimidine nucleotide salvage pathway in leukemia cells. Archived serum samples from patients treated with PTC299 demonstrated increased levels of dihydroorotate, the substrate of DHODH, indicating target engagement in patients. PTC299 has advantages over previously reported DHODH inhibitors, including greater potency, good oral bioavailability, and lack of off-target kinase inhibition and myelosuppression, and thus may be useful for the targeted treatment of hematologic malignancies., (©2018 American Association for Cancer Research.)

Objective: To evaluate a recently implemented procedure of discharge medication reconciliation and patient counseling completed by pharmacists at a nursing facility., Setting: This is a 138-bed nursing facility that houses long-term care residents as well as patients for subacute rehabilitation., Practice Description: Discharge process involves the medical team (geriatrician, medical resident, medical students), social workers, and nurse coordinators., Practice Innovation: Pharmacists are incorporated in the discharge process by completing medication reconciliation, patient counseling, and telephone follow-up, to improve patient understanding and satisfaction., Main Outcome Measurements: Medication discrepancies identified by pharmacists via medication reconciliation, number of patients who were counseled by pharmacist, and number of patients encountered for telephone follow-up., Results: Fifty-four patients were discharged during the study period. A total of 200 discrepancies were identified after discharge medication reconciliation by the pharmacist. On average, we found that there were 4 discrepancies per patient (range 0 to 16). Most of the discrepancies that were found were medication additions and omissions. Forty-five patients (83.3%) agreed to counseling and were then counseled by a pharmacist. Patients were often not counseled because of last-minute discharge, and no encounter was made., Conclusion: Involving pharmacists in patient transitions of care may be beneficial as previous studies have demonstrated; however, additional studies in a nursing facility setting are needed to validate these benefits.

Current anti-VEGF (Vascular Endothelial Growth Factor A) therapies to treat various cancers indiscriminately block VEGF function in the patient resulting in the global loss of VEGF signaling which has been linked to dose-limiting toxicities as well as treatment failures due to acquired resistance. Accumulating evidence suggests that this resistance is at least partially due to increased production of compensatory tumor angiogenic factors/cytokines. VEGF protein production is differentially controlled depending on whether cells are in the normal "homeostatic" state or in a stressed state, such as hypoxia, by post-transcriptional regulation imparted by elements in the 5' and 3' untranslated regions (UTR) of the VEGF mRNA. Using the Gene Expression Modulation by Small molecules (GEMS™) phenotypic assay system, we performed a high throughput screen to identify low molecular weight compounds that target the VEGF mRNA UTR-mediated regulation of stress-induced VEGF production in tumor cells. We identified a number of compounds that potently and selectively reduce endogenous VEGF production under hypoxia in HeLa cells. Medicinal chemistry efforts improved the potency and pharmaceutical properties of one series of compounds resulting in the discovery of PTC-510 which inhibits hypoxia-induced VEGF expression in HeLa cells at low nanomolar concentration. In mouse xenograft studies, oral administration of PTC-510 results in marked reduction of intratumor VEGF production and single agent control of tumor growth without any evident toxicity. Here, we show that selective suppression of stress-induced VEGF production within tumor cells effectively controls tumor growth. Therefore, this approach may minimize the liabilities of current global anti-VEGF therapies., Competing Interests: We have the following interests: The authors are employed by and received monetary compensation from PTC Therapeutics, Inc. Liangxian Cao, Marla Weetall, Jenelle Bombard (former employee), Hongyan Qi (former employee), Tamil Arasu (former employee), William Lennox, Jean Hedrick, Josephine Sheedy, Nicole Risher, Panayiota Trifillis, Christopher Trotta, Young-Choon Moon, John Babiak, Neil G Almstead, Joseph M Colacino, Thomas W Davis (former employee) and Stuart W Peltz are or were employed by and hold or held financial interests in PTC Therapeutics, Inc. PTC Therapeutics hold the patents pertaining to the results presented in this paper as below: Liangxian Cao and Panayiota Trifillis, Methods for Identifying Compounds that Modulate Untranslated Region-Based Regulation, CA2514184 and US8460864. Christopher R Trotta and Liangxian Cao, Methods and Agents for Screening for Compounds Capable of Modulating VEGF Expression, CA2567111, EP2400038 and US8426194. Liangxian Cao; William Lennox; Hongyan Qi; Young-Choon Moon and Nadarajan Tamilarasu, Tetra-Cyclic Carboline Derivatives Useful in the Inhibition of Angiogenesis, MX286760 and US8940896. Young-Choon Moon; William Lennox and Hongyan Qi, Tetrahydrocarbazoles as Active Agents for Inhibiting VEGF Production by Translational Control, US8946444 and US9271960. There are no additional patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

PTC299 is a novel small molecule that specifically blocks the production of protein from selected mRNAs that under certain conditions use noncanonical ribosomal translational pathways. Hypoxia, oncogenic transformation, and viral infections limit normal translation and turn on these noncanonical translation pathways that are sensitive to PTC299. Vascular endothelial cell growth factor (VEGF) is an example of a transcript that is posttranscriptionally regulated. Single doses of PTC299 (0.03 to 3 mg/kg) were administered orally to healthy volunteers in a phase 1 single ascending-dose study. In a subsequent multiple ascending-dose study in healthy volunteers, multiple-dose regimens (0.3 to 1.2 mg/kg twice a day or 1.6 mg/kg 3 times a day for 7 days) were evaluated. PTC299 was well tolerated in these studies. As expected in healthy volunteers, mean plasma VEGF levels did not change. Increases in Cmax and AUC of PTC299 were dose-proportional. The target trough plasma concentration associated with preclinical efficacy was achieved within 7 days at doses of 0.6 mg/kg twice daily and above. These data demonstrate that PTC299 is orally bioavailable and well tolerated and support clinical evaluation of PTC299 in cancer, certain viral infections, or other diseases in which deregulation of translational control is a causal factor., (© 2016, The Authors. Clinical Pharmacology in Drug Development Published by Wiley Periodicals, Inc. on behalf of The American College of Clinical Pharmacology.)

A method to mitigate or possibly eliminate reproduction in farmed fish is highly demanded. The existing approaches have certain applicative limitations. So far, no immunization strategies affecting gonadal development in juvenile animals have been developed. We hypothesized that autoimmune mechanisms, occurring spontaneously in a number of diseases, could be induced by targeted immunization. We have asked whether the immunization against specific targets in a juvenile zebrafish gonad will produce an autoimmune response, and, consequently, disturbance in gonadal development. Gonadal soma-derived factor (Gsdf), growth differentiation factor (Gdf9), and lymphocyte antigen 75 (Cd205/Ly75), all essential for early gonad development, were targeted with 5 immunization tests. Zebrafish (n = 329) were injected at 6 weeks post fertilization, a booster injection was applied 15 days later, and fish were sampled at 30 days. We localized transcripts encoding targeted proteins by in situ hybridization, quantified expression of immune-, apoptosis-, and gonad-related genes with quantitative real-time PCR, and performed gonadal histology and whole-mount immunohistochemistry for Bcl2-interacting-killer (Bik) pro-apoptotic protein. The treatments resulted in an autoimmune reaction, gonad developmental retardation, intensive apoptosis, cell atresia, and disturbed transcript production. Testes were remarkably underdeveloped after anti-Gsdf treatments. Anti-Gdf9 treatments promoted apoptosis in testes and abnormal development of ovaries. Anti-Cd205 treatment stimulated a strong immune response in both sexes, resulting in oocyte atresia and strong apoptosis in supporting somatic cells. The effect of immunization was FSH-independent. Furthermore, immunization against germ cell proteins disturbed somatic supporting cell development. This is the first report to demonstrate that targeted autoimmunity can disturb gonadal development in a juvenile fish. It shows a straightforward potential to develop auto-immunization-based technologies to mitigate fish reproduction before they reach maturation. However, the highly variable results between treatments and individuals suggest significant optimization should be performed to achieve the full potential of this technology.

Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA., (Copyright © 2014, American Association for the Advancement of Science.)

Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.

A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis. The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid. Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations. These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors. One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml). This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.

High throughput screening (HTS) involves using automated equipment to test a large number of samples against a defined molecular target to identify a reasonable number of active molecules in a timely fashion. Major factors which can influence priorities for the limited resources of the HTS group are projects, productivity, progress and people. The challenge to the HTS group is to provide excellent and timely screening services, but still devote efforts to new technologies and personnel development. This article explains why these factors are so important.

It is well recognized within the pharmaceutical industry that high throughput screening is a valuable and rapid tool to identify novel chemical compounds that may lead to tomorrow's drugs. High throughput screening involves testing as many chemical compounds as quickly as possible against a defined molecular or cellular 'target' (for example an enzyme) in the hope that interacting compounds may provide significant therapeutic benefits.At Wyeth-Ayerst Research, a Robotics and Automation Research Core Group has been established which serves as the in-house resource for high throughput screening. The robotics group has three missions: (1) develop and perform high throughput screens for customers in all therapeutic departments in the company; (2) educate customers in issues related to screen design; and (3) help customers to bring automated workstations into their laboratories. The mission, therefore, requires the effective use of automation, as well as building a strong collaboration with customers.THE CHALLENGES THAT HAVE BEEN FACED FALL INTO TWO CATEGORIES: technology limiting and customer relations. Technological challenges arise because it is necessary to develop and implement assays with very different formats and biochemical endpoints within extremely shortened time frames. The primary means to meet these challenges is with flexible robotics and flexible people. Challenges in the area of customer relations include setting realistic expectations, maintaining a sense of collaboration (and not merely service), educating investigators as to how to deal with the huge amount of data generated and seeking feedback. Effective and frequent communication, and an awareness of each individual's perspective, are essential to provide the most appropriate service.

Hydatidiform moles can be subclassified based on their ploidy. In general, complete moles are diploid, and partial moles are triploid. The standard method for the determination of DNA content is flow cytometric analysis. In this study, the authors investigated whether static cytometric analysis with the CAS 200 Image Analyzer (Cell Analysis Systems, Inc., Elmhurst, IL) with a software program designed for quantitation of nuclear DNA content in tissue sections can be used to classify moles. Tissue sections from 17 moles were analyzed with this system, and the results were compared with those obtained with flow cytometric analysis. It was found that cell selection was an important factor. A high proportion of the hyperplastic trophoblast was in G2M. Exclusion of these areas and measurement of the trophoblast lining the villi only led to reliable results, and complete agreement between the results of the two methods was obtained. The findings indicate that cytometric analysis on tissue sections is a reliable alternative to flow cytometric analysis for the designation of moles as diploid or triploid.

The inherent radiosensitivity of tumor biopsies obtained from a series of patients with carcinoma of the uterine cervix or endometrium has been characterized. Early passage cell lines were irradiated and assayed for cell survival using a clonogenic assay system. Survival curves were generated using the alpha/beta model and the surviving fraction at 2 Gy (SF2) was estimated. A wide range of SF2 values was observed among histologically similar tumors. The mean (+/- SD) SF2 value was 0.29 +/- 0.12 (range = 0.11-0.59) for the cervical biopsies and 0.30 +/- 0.13 (range = 0.11-0.67) for the endometrial biopsies. No correlation between inherent radiosensitivity and tumor DNA index or histopathology was observed. Patient accrual continues with the expectation that these results may help to determine whether SF2 values are of clinical value in predicting the response of individual patients to treatment with radiotherapy.

Flow cytometric DNA analysis of paraffin-embedded tumors is an important diagnostic and prognostic tool in clinical pathology. The technique is limited, however, by the time-consuming multistep procedure for dewaxing and rehydrating tissue. The authors developed an automated procedure to complete the dewaxing and rehydration of tissue using a routine histologic tissue processor with a 24-hour timer. This technique provided excellent tissue recovery and reproducible DNA histograms comparable to those obtained by manual methods. Subsequently, the authors analyzed the DNA content of 93 paraffin-embedded breast cancer tissues. The automation of a significant portion of the routine processing required for paraffin-embedded tissue makes cytometric DNA analysis a more practical procedure in the laboratory.

The plasma lipoproteins are the primary means of transport of cholesterol among tissues. In particular, the apo B-containing lipoproteins (VLDL, IDL and LDL) are important for the delivery of cholesterol from the liver to peripheral tissues, while HDL appear to mediate the reverse process of movement of cholesterol from tissues back to the liver. Both of these transport processes are necessary for efficient whole body cholesterol homeostasis, because the liver is the major site of both the production and excretion of cholesterol. However, deviations from a proper balance of transport of cholesterol, either increases in LDL levels or decreases in HDL cholesterol flux, may result in accumulation of cholesterol in extrahepatic tissues. Increased risk of atherosclerosis and CHD may be associated with elevation in the number of LDL particles, increase or decrease in LDL particle size, or changes in the composition of plasma LDL. These modifications of plasma LDL may be brought about following perturbation of one of several aspects of LDL metabolism. These include decreased LDL receptor activity, increased VLDL production and cholesterol enrichment of the liver-derived VLDL. The events in the arterial wall that make some LDL particles apparently atherogenic are not well understood. In the case of nonhuman primates, large-size LDL are associated with an increased risk of CHD. One characteristic of these LDL is that their core lipids are rich in saturated cholesteryl esters and their transition temperatures are frequently above body temperature. The liquid crystalline cholesteryl ester cores of such LDL may modulate the conformation of apo B on the surface and thereby affect the interaction of these LDL with cellular receptors or connective tissue matrix proteoglycans. It is likely, though, that changes in LDL particle number, LDL particle size and LDL particle composition may each contribute to progression of atherosclerosis. The presumed metabolic events that make HDL protective against atherosclerosis have been termed reverse cholesterol transport, and suggest that small HDL that are deficient in free cholesterol acquire this lipid from cell membranes. The HDL cholesterol is esterified by LCAT in the circulation, forming large HDL that can then deliver the cholesteryl ester to the liver by both direct and indirect means. In most circumstances, it is assumed that an increase in plasma HDL cholesterol concentration reflects an increase in the rate at which HDL is removing cholesterol from tissues and, consequently, a decrease in atherosclerosis.(ABSTRACT TRUNCATED AT 400 WORDS)

To determine whether altered hepatic secretion of HDL is part of the mechanism by which polyunsaturated fat lowers plasma HDL concentration, we have studied HDL secretion in the isolated perfused livers of African green monkeys fed an atherogenic diet containing either safflower oil as the polyunsaturated fat or butter as the saturated fat. During recirculating perfusion with a lipoprotein-free medium, livers from safflower oil-fed animals produced 21% less HDL mass on the average than those from butter-fed animals. Newly secreted hepatic HDL were characterized after their isolation and subfractionation by a combination of agarose column chromatography and density gradient ultracentrifugation. In both diet groups the HDL were heterogeneous in size, morphology, and composition and consisted of discoidal particles ranging in diameter from greater than 200 A to as little as 50 A. Large, discoidal particles that were rich in apoE and apoA-I were separated from small particles that were poor in apoE but rich in apoA-I. All hepatic HDL subfractions contained only small amounts of cholesteryl ester and triglyceride. The hepatic particles resembled in composition and structure the large variety of HDL particles found in the plasma of patients with the familial deficiency of lecithin:cholesterol acyltransferase. Accordingly, perfusate LCAT activity was measured and found to be 2% or less than that in monkey plasma. We conclude that the perfused monkey liver produces a variety of nascent HDL that are relatively unmodified by the post-secretory metabolic events which normally occur in blood plasma in vivo, and that livers of polyunsaturated fat-fed monkeys secrete fewer plasma HDL precursor particles than do those of saturated fat-fed monkeys.

The effect of the type of dietary fat on the concentrations and compositions of high density lipoprotein (HDL) subpopulations was studied in groups of African green monkeys consuming 40% of calories as fat supplied as saturated fat (P/S = 0.3) or polyunsaturated fat (P/S = 2.2) in the presence of either 0.8 mg or 0.03 mg cholesterol/kcal. Plasma HDL cholesterol concentrations were lower in polyunsaturated fat-fed animals. The distribution of mass among HDL subfractions was assessed by analytic ultracentrifugation (AnUC), density gradient ultracentrifugation (DGUC), and polyacrylamide gradient gel electrophoresis (GGE). This made it possible to characterize and quantitate the HDL subpopulations HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c (arranged in order of decreasing particle size and decreasing cholesterol content). Polyunsaturated fat-fed animals had lower concentrations of the large, cholesterol-rich HDL2b subpopulation, as well as higher concentrations of intermediate size HDL (HDL2a and HDL3a on the high cholesterol diet; HDL3a and HDL3b on the low cholesterol diet). Consistent with the observed fat-related redistribution of HDL mass, the saturated fat-fed monkeys had higher apo A-I/apo A-II ratios. The larger HDL often contained detectable apo E; however, the concentration of apo E in HDL was low in both saturated and polyunsaturated fat-fed animals. Thus, compared to saturated fat, dietary polyunsaturated fat induced the formation of smaller size HDL subpopulations and, therefore, an overall lower cholesterol content per particle for plasma HDL.

Seventy-three adult female cynomolgus monkeys fed an atherogenic diet were studied to determine the effect of two different combination contraceptive steroid preparations containing equivalent amounts of estrogen but different progestin components on plasma lipids and lipoproteins. Our hypothesis was that any high density lipoprotein (HDL) lowering effect of the contraceptive steroid preparations was proportional to the rise in total serum cholesterol caused by the progestins. For 2 years, one group (Ovral [Wyeth Laboratories], n = 23) received 75 micrograms norgestrel and 7.5 micrograms ethinyl estradiol daily, while another (Demulen [Searle & Co.], n = 25) received 150 micrograms ethynodiol diacetate and 7.5 micrograms ethinyl estradiol daily. The control group (n = 24) received no treatment. On average, the two oral contraceptive groups had higher total serum cholesterol and triglyceride concentrations but lower HDL cholesterol concentrations and smaller low density lipoproteins (LDL) compared with the control group. There was an inverse relationship between total serum cholesterol and HDL cholesterol for all three groups, but at any given total serum cholesterol concentration between 350 and 500 mg/dl, the Ovral group had HDL cholesterol concentrations that averaged 37% and 14% lower than the control and Demulen groups, respectively. The decrease in HDL concentrations with oral contraceptive treatment was associated with a sharp decrease in (HDL2b)gge protein (82% for Ovral and 59% for Demulen) and a corresponding increase in (HDL3b,c)gge protein as determined by gradient gel electrophoresis. Of 23 animals in the Ovral group, six had HDL subfractions greater than 10 nm diameter (HDL2b)gge compared with 22 of 24 animals in the control group. Although LDL size, on average, was smaller and plasma triglycerides were greater with oral contraceptive treatment compared with controls, there was no apparent relationship between LDL size and plasma triglyceride concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)