The expression of ceruloplasmin in astrocytes is essential for postnatal myelination and myelin maintenance in the adult brain.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux. The absence of this protein in humans and rodents produces progressive neurodegeneration with brain iron accumulation. Astrocytes express high levels of Cp and iron efflux from these cells has been shown to be central for oligodendrocyte maturation and myelination. To explore the role of astrocytic Cp in brain development and aging we generated a specific conditional KO mouse for Cp in astrocytes (Cp cKO). Deletion of Cp in astrocytes during the first postnatal week induced hypomyelination and a significant delay in oligodendrocyte maturation. This abnormal myelin synthesis was exacerbated throughout the first two postnatal months and accompanied by a reduction in oligodendrocyte iron content, as well as an increase in brain oxidative stress. In contrast to young animals, deletion of astrocytic Cp at 8 months of age engendered iron accumulation in several brain areas and neurodegeneration in cortical regions. Aged Cp cKO mice also showed myelin loss and oxidative stress in oligodendrocytes and neurons, and at 18 months of age, developed abnormal behavioral profiles, including deficits in locomotion and short-term memory. In summary, our results demonstrate that iron efflux-mediated by astrocytic Cp-is essential for both early oligodendrocyte maturation and myelin integrity in the mature brain. Additionally, our data suggest that astrocytic Cp activity is central to prevent iron accumulation and iron-induced oxidative stress in the aging CNS.

Deletion of voltage-gated calcium channels in astrocytes decreases neuroinflammation and demyelination in a murine model of multiple sclerosis.

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

Simultaneous recovery of both bright and dim structures from noisy fluorescence microscopy images using a modified TV constraint.

The quality and information content of biological images can be significantly enhanced by postacquisition processing using deconvolution and denoising. However, when imaging complex biological samples, such as neurons, stained with fluorescence labels, the signal level of different structures can differ by several orders of magnitude. This poses a challenge as current image reconstruction algorithms are focused on recovering low signals and generally have sample-dependent performance, requiring tedious manual tuning. This is one of the main hurdles for their wide adoption by nonspecialists. In this work, we modify the general constrained reconstruction method (in our case utilizing a total variation constraint) so that both bright and dim structures can drive the deconvolution with equal force. In this way, we can simultaneously obtain high-quality reconstruction across a wide range of signals within a single image or image sequence. The algorithm is tested on both simulated and experimental data. When compared with current state-of-art algorithms, our algorithm outperforms others in terms of maintaining the resolution in the high-signal areas and reducing artefacts in the low-signal areas. The algorithm was also tested on image sequences where one set of parameters are used to reconstruct all images, with blind evaluation by a group of biologists demonstrating a marked preference for the images produced by our method. This means that our method is suitable for batch processing of image sequences obtained from either spatial or temporal scanning. LAY DESCRIPTION: Fluorescence microscopy images of complex biological samples contain a wide range of signal levels. This signal variation leads current reconstruction algorithms, which aim to enhance the quality of the raw images, to have sample-dependent performance. In this work, we design a new optimization that allows the reconstruction to "pay equal eqattention to" both bright and dim structures. In this way, we can simultaneously recover both bright and dim structures within a single image or image sequence, as validated when the algorithm was quantitatively tested on both simulated and experimental data. When our method was evaluated alongside current state of art algorithms by a group of biologists, our algorithm was considered qualitatively superior.

The medico-legal investigation of abandoned fetuses and newborns--a review of cases admitted to the Pretoria Medico-Legal Laboratory, South Africa.

There is a dearth of literature on the extent of fetal or newborn abandonment or "dumping" and the medico-legal investigation procedures these cases require. This is despite the fact that these occurrences are a worldwide phenomenon and by definition involve criminal law concerns such as illegal abortion, concealment of birth, murder, or neonaticide, depending on the country concerned. This article contributes to current literature in both respects and provides a retrospective case audit for the period 2004-2008 pertaining to all abandoned newborns and fetuses admitted to the Pretoria Medico-Legal Laboratory (PMLL) in South Africa. Demographic details, scope, and nature of the medico-legal investigation as well as formulation of cause of death were recorded. A total of 289 cases were identified for inclusion in this study, 57% of which were considered to have been non-viable fetuses, while 45 of the viable fetuses were deemed to have been stillborn. These instances involve the crimes of concealment of birth and at times illegal abortion, yet prosecution of these cases are relatively unheard of. Signs of live birth were identified in 38 of the cases in the study. Of these infants, 9 were deemed to have died from injuries they have sustained, and in a further 9 cases, no anatomical cause of death could be identified. Homicidal cases should be brought in cases where death ensued as a result of abandonment; however, it is not known how many cases were prosecuted. A comparatively large number of cases were found to have been admitted to the Pretoria Medico-Legal Laboratory. This is alarming because South African abortion laws are liberal and services are free at point of access in the public health care sector. A substantial percentage of cases of abandoned infants were found to have shown signs of life after birth implying a homicidal manner of death or death by abandonment, but it seems these cases are merely shelved.

Epidemiology and factors associated with candidaemia following Clostridium difficile infection in adults within metropolitan Atlanta, 2009-2013.

We assessed prevalence of and risk factors for candidaemia following Clostridium difficile infection (CDI) using longitudinal population-based surveillance. Of 13 615 adults with CDI, 113 (0·8%) developed candidaemia in the 120 days following CDI. In a matched case-control analysis, severe CDI and CDI treatment with vancomycin + metronidazole were associated with development of candidaemia following CDI.

The regulated synthesis of a Bacillus anthracis spore coat protein that affects spore surface properties.

AIMS: Examine the regulation of a spore coat protein and the effects on spore properties. METHODS AND RESULTS: A c. 23 kDa band in coat/exosporial extracts of Bacillus anthracis Sterne spores varied in amount depending upon the conditions of sporulation. It was identified by MALDI as a likely orthologue of ExsB of Bacillus cereus. Little if any was present in an exosporial preparation with a location to the inner coat/cortex region established by spore fractionation and immunogold labelling of electron micrograph sections. Because of its predominant location in the inner coat, it has been renamed Cotγ. It was relatively deficient in spores produced at 37°C and when acidic fermentation products were produced a difference attributable to transcriptional regulation. The deficiency or absence of Cotγ resulted in a less robust exosporium positioned more closely to the coat. These spores were less hydrophobic and germinated somewhat more rapidly. Hydrophobicity and appearance were rescued in the deletion strain by introduction of the cotγ gene. CONCLUSIONS: The deficiency or lack of a protein largely found in the inner coat altered spore hydrophobicity and surface appearance. SIGNIFICANCE AND IMPACT OF THE STUDY: The regulated synthesis of Cotγ may be a paradigm for other spore coat proteins with unknown functions that modulate spore properties in response to environmental conditions.

Genetic diversity and population structure of a rare flowering tree endemic to Appalachia, Stewartia ovata.

Stewartia ovata (cav.) Weatherby, commonly known as mountain stewartia, is an understory tree native to the southeastern United States (U.S.). This relatively rare species occurs in isolated populations in Virginia, Kentucky, Tennessee, North Carolina, South Carolina, Georgia, Alabama, and Mississippi. As a species, S. ovata has largely been overlooked, and limited information is available regarding its ecology, which presents obstacles to conservation efforts. Stewartia ovata has vibrant, large white flowers that bloom in summer with a variety of filament colors, suggesting potential horticultural traits prized by ornamental industry. However, S. ovata is relatively slow growing and, due to long seed dormancy, propagation is challenging with limited success rates. This has created a need to assess the present genetic diversity in S. ovata populations to inform potential conservation and restoration of the species. Here, we employ a genotyping-by-sequencing (GBS) approach to characterize the spatial distribution and genetic diversity of S. ovata in the southern Appalachia region of the eastern United States. A total of 4475 single nucleotide polymorphisms (SNPs) were identified across 147 individuals from 11 collection sites. Our results indicate low genetic diversity (He = 0.216), the presence of population structure (K = 2), limited differentiation (F ST = 0.039), and high gene flow (Nm = 6.16) between our subpopulations. Principal component analysis corroborated the findings of STRUCTURE, confirming the presence of two distinct S. ovata subpopulations. One subpopulation mainly contains genotypes from the Cumberland Plateau, Tennessee, while the other consists of genotypes present in the Great Smoky Mountain ranges in Tennessee, North Carolina, and portions of Nantahala, Chattahoochee-Oconee national forests in Georgia, highlighting that elevation likely plays a major role in its distribution. Our results further suggested low inbreeding coefficient (F IS = 0.070), which is expected with an outcrossing tree species. This research further provides necessary insight into extant subpopulations and has generated valuable resources needed for conservation efforts of S. ovata.

The surgical technique of minimally invasive transforaminal lumbar interbody fusion.

We aim to describe the minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) technique. The MI-TLIF procedure was developed to achieve the same goal of neural decompression and interbody arthrodesis as the traditional, open TLIF techniques. MI-TLIF has been utilized in the treatment of an array of lumbar pathologies, while offering the advantages of reducing soft tissue trauma, decreasing postoperative pain, and reducing the rate of complication when compared to the open techniques. The surgical technique of MI-TLIF is described in a step-by-step fassion. A technical review of this novel minimally invasive procedure was performed. Additionally, data collected through our experience with this procedure is reported. Data was collected retrospectively from patients between January 2008 and December 2009 who underwent MI-TLIF. The mean preoperative VAS score was 6.12±2.02 compared to 2.11±2.69 postoperatively. The mean ODI score dropped from 38.29±13.19 preoperatively to 16.00 ±16.598 postoperatively. Eighty-four patients who underwent MI-TLIF between October 2007 and December 2010 were divided based on age (over or under 65 years) and intraoperative estimated blood loss (EBL) was compared. The mean EBL for the young age group was 93.37±102.16 mL compared to 100 ±61.24 mL for the older group. Operation times for the MI-TLIF procedure has decreased from 3-4 hours to approximately 2 hours throughout our experience with this technique. The MI-TLIF technique is a safe and effective procedure offering the advantages of less tissue damage, less blood loss, and reduced postoperative hospitalization over the open techniques.

Enhancing the Measure of Participation Burden in Protocol Design to Incorporate Logistics, Lifestyle, and Demographic Characteristics.

BACKGROUND: Growing interest in improving patient participation convenience and the feasible execution of clinical trials has increased demand for new approaches to leverage patient input in the protocol design process. METHODS: This study builds on prior work conducted by the Tufts Center for the Study of Drug Development in collaboration with ZS. A comprehensive participant burden algorithm based on protocol procedures, participation requirements and lifestyle preferences was developed and tested. Clinical trial preferences and perceptions from 3002 global patients were analyzed to inform and derive the algorithm. It was next tested against a convenience sample of 266 completed protocols. Descriptive statistics, significance tests, and regression analyses were performed. RESULTS: Mean participant burden scores were highly associated with, and predictive (p < 0.01) of, screen failure rates, overall clinical trial duration and the number of substantial protocol amendments; and predictive (p < 0.05) of protocol treatment duration. Of 11 subgroups assessed, those that most influenced the algorithm and drove higher overall burden scores included disease condition, caregiver reliance, race, prior experience as a clinical trial participant and participant age. Geographic area and participant sex showed only minimal influence. CONCLUSION: This study presents advancement and refinement in measuring participation burden that will assist drug development teams and protocol authors in retrospectively understanding clinical trial performance outcomes and in prospectively informing protocol design decisions.

Cross-sectional prevalence of SARS-CoV-2 antibodies in healthcare workers in paediatric facilities in eight countries.

BACKGROUND: Healthcare workers (HCWs) have been disproportionately affected by coronavirus disease 2019 (COVID-19), which may be driven, in part, by nosocomial exposure. If HCW exposure is predominantly nosocomial, HCWs in paediatric facilities, where few patients are admitted with COVID-19, may lack antibodies to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and be at increased risk during the current resurgence. AIM: To compare the seroprevalence of SARS-CoV-2 amongst HCWs in paediatric facilities in seven European countries and South Africa (N=8). METHODS: All categories of paediatric HCWs were invited to participate in the study, irrespective of previous symptoms. A single blood sample was taken and data about previous symptoms were documented. Serum was shipped to a central laboratory in London where SARS-CoV-2 immunoglobulin G was measured. FINDINGS: In total, 4114 HCWs were recruited between 1st May and mid-July 2020. The range of seroprevalence was 0-16.93%. The highest seroprevalence was found in London (16.93%), followed by Cape Town, South Africa (10.36%). There were no positive HCWs in the Austrian, Estonian and Latvian cohorts; 2/300 [0.66%, 95% confidence interval (CI) 0.18-2.4] HCWs tested positive in Lithuania; 1/124 (0.81%, 95% CI 0.14-4.3) HCWs tested positive in Romania; and 1/76 (1.3%, 95% CI 0.23-7.0) HCWs tested positive in Greece. CONCLUSION: Overall seroprevalence amongst paediatric HCWs is similar to their national populations and linked to the national COVID-19 burden. Staff working in paediatric facilities in low-burden countries have very low seroprevalence rates and thus are likely to be susceptible to COVID-19. Their susceptibility to infection may affect their ability to provide care in the face of increasing cases of COVID-19, and this highlights the need for appropriate preventative strategies in paediatric healthcare settings.

Endogenous and monoclonal antibodies to the rat pancreatic acinar cell Golgi complex.

Normal, unimmunized mouse serum from several strains (BALB/c, C57/b, DBA/2, NZB, SJL, CD/1) contains an endogenous IgG antibody that localizes to the Golgi complex of rat pancreatic acinar cells. Treatment of pancreatic acini with 5 microM monensin resulted in the swelling and vacuolization of the Golgi cisternae, and in a corresponding annular staining by the mouse serum as observed by immunofluorescence, suggesting that the antigen recognized is on the Golgi complex cisternal membrane. The antiserum did not react with pancreatic secretory proteins, and its binding to smooth microsomal membranes was retained following sodium carbonate washing, supporting a Golgi membrane localization. Advantage was taken of the existence of the endogenous murine antibody for the isolation of monoclonal antibodies directed to the Golgi complex of the rat pancreas. Two antibodies, antiGolgi 1 and antiGolgi 2, are described. Both antibodies are IgMs that recognize integral membrane proteins of the trans-Golgi cisternae, with lighter and patchy staining of the pancreatic lumen membrane, as observed both by light and electron microscopy. AntiGolgi 1 recognizes predominately a protein of molecular weight 103,000-108,000, whereas antiGolgi 2 shows a strong reaction to a 180-kd band as well as the 103-108-kd protein.

mip causes hyperinnervation of a retinotopic map in Drosophila by excessive recruitment of R7 photoreceptor cells.

The two central photoreceptor neurons of the Drosophila eye, R7 and R8, form a retinotopic map in the optic lobe of the fly brain. We have developed a technique that allows us to visualize the projections of these neurons with high resolution. Using this technique, we have identified a new mutant, mip (more inner photoreceptors), in which this map shows a striking hyperinnervation. The extra terminals in the brain derive from an excessive recruitment of sevenless-independent R7 photoreceptor cells during eye development. The original R7, however, remains sevenless responsive. The behavior of this gene suggests that recruitment to the R7 pathway, and possibly to multiple programs in ommatidial assembly, is partially regulated by inhibition.

Anabolic payout of terminal implant alters adipogenic gene expression of the longissimus muscle in beef steers.

This experiment evaluated the dose and payout pattern of trenbolone acetate (TBA) and estradiol-17ß (E) on LM mRNA expression of adenosine monophosphate-activated protein kinase-ɑ (-ɑ), ß, G protein-coupled receptor 41(), G protein-coupled receptor 43 (), γ, and stearoyl CoA desaturase () in finishing feedlot steers as indicators of adipogenesis and marbling development. British × Continental steers (n = 168; 14 pens/treatment; initial BW = 362 kg) were used in a randomized complete block design. Treatments included: no implant (NI), Revalor-S (REV-S; 120 mg TBA + 24 mg E), or Revalor-XS (REV-X; delayed release implant: 80 mg TBA + 16 mg E [uncoated], 120 mg TBA + 24 mg E [coated], 200 mg TBA + 40 mg E [total]). Steers were fed 1 time daily for an average of 164 d. The LM biopsies were collected (1 steer/pen) on d -1, 27, 55, and 111 relative to timing of implant. Total RNA was isolated from each sample and real-time quantitative PCR was used to measure quantity of -ɑ, ß, , ,it, γ, and mRNA. No implant × day interactions were detected ( ≥ 0.19) in this experiment. Day impacted the mRNA expression of all adipogenic genes ( ≤ 0.02). The main effect of implant tended ( = 0.09) to influence expression of -ɑ, REV-X had an 8.8% increase over NI and an 18.7% increase over REV-S. Implant influenced ( = 0.03) mRNA expression of , expression of for the REV-X treatment was not different ( > 0.10) from NI, and both were greater ( ≤ 0.05) than REV-S (1.13, 1.00, and 0.67 ± 0.224 arbitrary units) for REV-X, NI, and REV-S, respectively. Implant also influenced ( = 0.02) expression of , expression of for REV-X was not different ( > 0.10) from NI, and both were greater ( ≤ 0.05) than REV-S (1.27, 1.07, and 0.72 ± 0.234 arbitrary units) for REV-X, NI, and REV-S, respectively. Implant influenced ( = 0.02) mRNA expression of γ in LM tissue, expression of γ for REV-X was not different ( > 0.10) from NI, and both were greater ( ≤ 0.05) than REV-S (1.09, 1.02, and 0.69 ± 0.195 arbitrary units) for REV-X, NI, and REV-S, respectively. The REV-X steers received the greatest anabolic dose of TBA + E without detriment to marbling scores. The increased mRNA expression of adipogenic genes for REV-X steers suggest that the delayed and gradual release of anabolic stimulants associated with REV-X might have mitigated decreases in marbling generally attributed to multiple combined TBA + E implants.

Secretion of matrix metalloproteinases and their inhibitors (tissue inhibitor of metalloproteinases) by human prostate in explant cultures: reduced tissue inhibitor of metalloproteinase secretion by malignant tissues.

Unregulated secretion of matrix metalloproteinases (MMPs) or their endogenous protein inhibitors (tissue inhibitor of metalloproteinases, TIMPs) has been implicated in tumor invasion and metastasis. Species of MMPs and TIMPs secreted by epithelial cultures of normal, benign, and malignant prostate were identified and their levels were compared. Fragments of fresh tissue were cultured in a serum-free medium that supported the outgrowth of prostatic epithelial cells. Biochemical analysis of the conditioned media by gelatin zymography and enzyme assays showed that both normal and neoplastic tissues secreted latent and active forms of both M(r) 72,000 type IV collagenase (MMP-2) and M(r) 92,000 gelatinase (MMP-9). However, conditioned media from malignant prostate explants contained a higher proportion of the active form of MMP-2. Significant amounts of free TIMPs were secreted by normal juvenile and adult prostates, but they were either markedly reduced or not detectable in conditioned media from neoplastic tissues. These findings suggest that there is an imbalance of secretion between MMPs and TIMPs in prostatic carcinoma.

Ionophore strategy affects growth performance and carcass characteristics in feedlot steers.

One hundred ninety-two steers (BW = 354 ± 23.5 kg) were used in a randomized block design to evaluate the effects of ionophore and ractopamine hydrochloride (RH) supplementation strategies on performance and carcass characteristics. Twelve pens of 4 steers were assigned to each of the following treatments: unsupplemented control (CON), laidlomycin propionate (12.1 mg/kg DM) with or without RH (LPRH and LP, respectively), and monensin sodium (36.4 mg/kg DM) with RH (MSRH). Steers were fed for 151 d, of which respective treatments received RH (Actogain; Zoetis, Florham Park, NJ) at a rate of 300 mg/(animal · d) for the final 32 d. Laidlomycin was removed from the LPRH treatment during this period, as no combination feeding has been approved. Upon harvest, carcass data were collected by trained personnel, and subsequent analysis of the LM was conducted to estimate tenderness using Warner-Bratzler shear force (WBSF). Prior to RH supplementation, both LP and LPRH had greater ADG ( ≤ 0.02) and G:F ( < 0.01) than CON, whereas MSRH was intermediate. During the final 32 d, MSRH improved G:F ( ≤ 0.02) compared to all other treatments and tended to increase ADG over unsupplemented controls ( = 0.05). Cattle receiving LP without RH had significantly greater BW at d 151 than CON ( = 0.02), whereas both RH treatments tended to improve final BW ( ≤ 0.09). Ionophores improved ADG ( ≤ 0.03) and G:F ( < 0.01) for the entire feeding period, and although LP-supplemented cattle had greater DMI for the final 32 d than both RH treatments ( ≤ 0.01), intakes for the 151-d trial were similar among treatments. Carcass weights were greater ( = 0.04) in cattle fed LP with no RH than CON, where cattle yielded an average of 12 kg more HCW. Ractopamine increased LM area in MSRH-supplemented cattle ( = 0.03) and tended to increase LM area for steers receiving LPRH ( = 0.07). Longissimus steaks of MSRH-supplemented cattle had greater WBSF values than CON ( = 0.04) after 7 d of postmortem aging and greater WBSF values than LPRH steaks after 28 d ( = 0.03). All other carcass and WBSF measurements were similar among treatments. The results of this study indicate that LP supplementation without RH may yield a performance similar to and carcass responses associated with the administration of a ß-agonist. These results also suggest that performance and carcass characteristics for cattle fed LP are similar to those of cattle fed monensin throughout the feeding period.

Contributing factors in the trafficking of [3H]arachidonate between phospholipids.

Cultured human promyelocytic leukemia cells (HL-60), depleted of arachidonic acid by continued growth in serum-free media, were used as a model system to examine various factors that control the incorporation and distribution of [3H]arachidonic acid into classes and subclasses of cellular lipids. Increasing the culture media concentration of [3H]arachidonic acid from 1 x 10(-8) M to 1 x 10(-5) M caused a greater percentage of the cellular tritium to be distributed into triacylglycerols (from less than 1% at 1 x 10(-8) M to 38% at 1 x 10(-5) M) with a corresponding decrease in cellular [3H]diradylglycerophosphoethanolamine (from 53% at 1 x 10(-8) M to 12% at 1 x 10(-5) M) during 2 h incubations. A greater proportion of the tritium present in diradylglycerophosphoethanolamine and diradylglycerophosphocholine, at the higher media concentration of [3H]arachidonic acid (1 x 10(-5) M), was found in the diacyl subclasses of these two lipids than was observed at the lower concentrations (less than 1 x 10(-6) M) of [3H]arachidonic acid. Significant amounts of diarachidonoyl molecular species were found in the phosphatidylethanolamine (10%) and phosphatidylcholine (15%) of HL-60 cells that were labeled for 2 h with 1 x 10(-5) M [3H]arachidonic acid. This was the only molecular species of phosphatidylcholine to completely disappear when prelabeled cells were placed in arachidonate-free media for 22 h. Prelabeling-chase experiments with 1 x 10(-5) M [3H]arachidonic acid were consistent with movement of [3H]arachidonate from triacylglycerols into diradylglycerophosphatides and from diacylphospholipids into ether-linked phospholipids. Increasing the concentration of HL-60 cells in the incubations influenced the distribution of [3H]arachidonic acid in cellular lipid classes in a manner analogous to decreasing the concentration of [3H]arachidonic acid in the media. Increasing the endogenous level of cellular arachidonate in phospholipid classes with supplements of unlabeled arachidonic acid changed the subsequent lipid class distribution of a low concentration (1 x 10(-8) M) of [3H]arachidonic acid to resemble results obtained with a much higher mass level of [3H]arachidonate in arachidonate depleted cells. HL-60 cells differentiated into granulocytes by treatment with dimethyl sulfoxide incorporated less [3H]arachidonic acid but had a greater proportion associated with alkylacylglycerophosphocholine and alk-1-enylacylglycerophosphoethanolamine than undifferentiated HL-60 cells.

Arachidonate-containing triacylglycerols: biosynthesis and a lipolytic mechanism for the release and transfer of arachidonate to phospholipids in HL-60 cells.

When HL-60 cells are incubated in media containing 10 microM [3H]arachidonic acid the label is immediately incorporated into both triacylglycerols and phospholipids. About one-half of the cellular tritium was associated with triacylglycerols after 2 h of incubation and this [3H]arachidonate was then transferred to phospholipids as soon as the labeled cells were placed in arachidonate-free media. A technique was devised to analyze the stereospecific distribution of [3H]arachidonate at the three sn-positions of glycerol in order to identify the mechanism(s) responsible for the biosynthesis of the labeled triacylglycerols. [3H]Arachidonate was found to be distributed in nearly equal amounts among all three glycerol positions of the triacylglycerols. In addition, analysis of intact triacylglycerols containing [3H]arachidonate revealed that 24% of the tritium eluted from reverse-phase HPLC with triarachidonoylglycerol. Both of these findings would be expected if a significant portion of the arachidonate-containing triacylglycerols were synthesized de novo. Homogenates prepared from [3H]arachidonate prelabeled HL-60 cells were capable of hydrolyzing the endogenous [3H]arachidonate-containing triacylglycerols to produce mainly free fatty acids and smaller amounts of monoacylglycerols. The relatively small amount of monoacyl- and diacylglycerols produced by the lipolytic activity of the homogenates indicated that [3H]arachidonate was hydrolyzed from all three sn-positions of the [3H]triacylglycerols. This lipase activity had a pH optimum of 4.5 and was associated to a greater extent with the soluble fraction than in the total membrane fraction. Although it is not known whether this lipolytic activity is the same as that expressed in the intact cells, the activity of the cell-free triacylglycerol lipase was of sufficient magnitude to have easily accounted for the decrease in [3H]triacylglycerols that was observed after transfer of the intact HL-60 cells (prelabeled with [3H]arachidonate) to fresh media. The data suggest that transfer of arachidonate from triacylglycerols to phospholipids probably occurs through an acyltransferase utilizing a lysophospholipid and arachidonoyl-CoA.

The CoA-independent transacylase in PAF biosynthesis: tissue distribution and molecular species selectivity.

Microsomal membranes from six different rat tissues (spleen, lung, kidney, brain, testis, and liver) were found to possess CoA-independent transacylase activity that could both acylate lyso-[3H]PAF (1-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine) and then deacylate the 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine product via the transacylation of added exogenous 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Platelet-activating factor (1-[3H]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) was produced when acetyl-CoA was added to the spleen microsomes during generation of lyso-[3H]PAF by the transacylases. More extensive studies with subcellular fractions from spleen revealed that, in addition to microsomes, the transacylase activities were also present in the 15,000 x g membrane fraction but not in the cytosol. Analysis of molecular species of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine before and after addition of 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine as the acyl acceptor demonstrated a high selectivity for polyunsaturated fatty acids (> 3 double bonds/acyl group) in both the acylation and deacylation processes that occurred in testicular microsomal membranes. The transfer of acyl groups by the transacylase appeared to be equally effective for either arachidonic or docosapentaenoic(n - 6) fatty acids, whereas linoleic and oleic fatty acids were not transferred from 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine following the addition of 1-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine. Similar experiments with the membrane fraction of undifferentiated HL-60 cells showed that arachidonic acid supplementation of intact cells enhanced both the CoA-independent transacylation of lyso-[3H]PAF and the subsequent deacylation of 1-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine caused by addition of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. Differentiation of the HL-60 cells into a neutrophil-like form had no effect on the transacylase activity. Our results indicate the PAF-related transacylase is widely distributed among tissues and, although highly selective for polyunsaturated acyl groups, does not discriminate selectively among the polyunsaturates.

O-alkyl-O-methylglycerophosphocholine, an antineoplastic lipid, undergoes spontaneous redistribution between biological membranes prepared from HL-60 cells.

Cytotoxic actions of the unnatural phospholipid 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholine (alkylmethyl-GPC) appear to be targeted to the plasma membrane of sensitive cells. We analyzed the distribution of [3H]alkylmethoxy-GPC in subcellular membranes isolated from human promyelocytic leukemia (HL-60) cells. [3H]Alkylmethyl-GPC added to intact cells selectively labels plasma membrane-enriched fractions of the postnuclear supernatant, but the labeling profile is independent of the temperature and duration of the incubation, and concentration of the molecule. Also, an identical distribution pattern is obtained when [3H]alkylmethyl-GPC is directly added to postnuclear supernatants. Moreover, [3H]alkylmethyl-GPC translocates between subcellular membranes in a manner that does not depend on membrane-adsorbed or cytosolic transfer proteins. These results indicate that the subcellular localization studies reported for alkylmethyl-GPC and structurally-related molecules must be interpreted with caution.

Molecular species of ethanolamine plasmalogens and transacylase activity in rat tissues are altered by fish oil diets.

Effects of dietary fish oil ethyl esters and alkyldiacetylglycerols (an ether-linked lipid) on the distribution of subclasses of choline- and ethanolamine-glycerophospholipids as well as effects on highly unsaturated molecular species of ethanolamine plasmalogens from brain, spleen, kidney, lung, and testis of rats were examined. Supplementation of ethyl ester concentrates of n-3 fatty acids had no effect on the distribution of subclasses in any of the tissues. However, the supplements of 1-O-octadec-9'-enyl-2,3-diacetyl-sn-glycerol (diacetates of selachyl alcohol) caused significant increases in the alkylacylglycerophosphocholine and alkylacylglycerophosphoethanolamine subclasses from spleen and lung and in the alkylacylglycerophosphoethanolamine subclass from kidney. Dietary supplements of fish oil ethyl esters reduced the arachidonate-containing species of ethanolamine plasmalogens whereas molecular species having 20:5(n-3), 22:6(n-3), and/or 22:5(n-3) acyl groups were increased in the spleen, lung, and kidneys, but not brain. In testicular tissue from rats fed the fish oil diets, the molecular species of ethanolamine plasmalogens containing 22:5(n-6) acyl groups were reduced. An increase of ethanolamine plasmalogens with 18:1 alk-1-enyl moieties paired with highly unsaturated sn-2 acyl groups were found in the tissues of rats fed the fish oil plus selachyl alcohol diacetate supplements. Rats on the diet containing fish oil ethyl esters had significantly lower [3H]alkyllysoglycerophosphocholine CoA-independent transacylase activity in spleen microsomes than controls. This suggests that supplements of n-3 fatty acids interferes with the transacylation of arachidonate, an event that could seriously impair the release of arachidonate and lysophospholipids (e.g., lyso-PAF) that are precursors of potent bioactive lipid derivatives.