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Cellular mechanophenotype is often a defining characteristic of conditions like cancer malignancy/metastasis, cardiovascular disease, lung and liver fibrosis, and stem cell differentiation. However, acquiring living cells based on mechanophenotype is challenging for conventional cell sorters due to a lack of biomarkers. In this study, we demonstrate a workflow for surface protein discovery associated with cellular mechanophenotype. We sorted heterogeneous adipose-derived stem/stromal cells (ASCs) into groups with low vs. high lamin A/C, an intracellular protein linked to whole-cell mechanophenotype. Proteomic data of enriched groups identified surface protein candidates as potential biochemical proxies for ASC mechanophenotype. Select surface biomarkers were used for live-cell enrichment, with subsequent single-cell mechanical testing and lineage-specific differentiation. Ultimately, we identified CD44 to have a strong inverse correlation with whole-cell elastic modulus, with CD44 lo cells exhibiting moduli three times greater than that of CD44 hi cells. Functionally, these stiff and soft ASCs showed enhanced osteogenic and adipogenic differentiation potential, respectively. The described workflow can be replicated for any phenotype with a known correlated intracellular protein, allowing for the acquisition of live cells for further characterization, diagnostics, or therapeutics., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Introduction: Antibodies are an essential research tool for labeling surface proteins but can potentially influence the behavior of proteins and cells to which they bind. Because of this, researchers and clinicians are interested in the persistence of these antibodies, particularly for live-cell applications. We developed an easily adoptable method for researchers to characterize antibody removal timelines for any cell-antibody combination, with the benefit of studying broad, hypothesized mechanisms of antibody removal., Methods: We developed a method using four experimental conditions to elucidate the contributions of possible factors influencing antibody removal: cell proliferation, internalization, permanent dissociation, and environmental perturbation. This method was tested on adipose-derived stem cells and a human lung fibroblast cell line with anti-CD44, CD90, and CD105 antibodies. The persistence of the primary antibody was probed using a fluorescent secondary antibody daily over 10 days. Relative contributions by the antibody removal mechanisms were quantified based on differences between the four culture conditions., Results: Greater than 90% of each antibody tested was no longer present on the surface of the two cell types after 5 days, with removal observed in as little as 1 day post-labeling. Anti-CD90 antibody was primarily removed by environmental perturbation, anti-CD105 antibody by internalization, and anti-CD44 antibody by a combination of all four factors., Conclusions: Antibody removal mechanism depended on the specific antibody tested, while removal timelines for the same antibody depended more on cell type. This method should be broadly relevant to researchers interested in quantifying an initial timeframe for uninhibited use of antibody-labeled cells., Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-021-00670-3., (© Biomedical Engineering Society 2021.)

Gene expression is used extensively to describe cellular characteristics and behaviors; however, most methods of assessing gene expression are unsuitable for living samples, requiring destructive processes such as fixation or lysis. Recently, molecular beacons have become a viable tool for live-cell imaging of mRNA molecules in situ. Historically, beacon-mediated imaging has been limited to fluorescence-based approaches. We propose the design and synthesis of a novel molecular beacon for magnetic resonance detection of any desired target nucleotide sequence. The biologically compatible synthesis incorporates commonly used bioconjugation reactions in aqueous conditions and is accessible for laboratories without extensive synthesis capabilities. The resulting beacon uses fluorine ( 19 F) as a reporter, which is broadened, or turned "off", via paramagnetic relaxation enhancement from a stabilized nitroxide radical spin label when the beacon is not bound to its nucleic acid target. Therefore, the 19 F NMR signal of the beacon is quenched in its hairpin conformation when the spin label and the 19 F substituent are held in proximity, but the signal is recovered upon beacon hybridization to its specific complementary nucleotide sequence by physical separation of the radical from the 19 F reporter. This study establishes a path for magnetic resonance-based assessment of specific mRNA expression, providing new possibilities for applying molecular beacon technology in living systems.

The youngest children, particularly in the first year of life, are the most vulnerable to physical abuse. Skeletal survey is the universal screening examination in children 24 months of age and younger. Fractures occur in over half of abused children. Rib fractures may be the only abnormality in about 30%. A repeat limited skeletal survey after 2 weeks can detect additional fractures and can provide fracture dating information. The type and extent of additional imaging for pediatric patients being evaluated for suspected physical abuse depends on the age of the child, the presence of neurologic signs and symptoms, evidence of thoracic or abdominopelvic injuries, and social considerations. Unenhanced CT of the head is the initial study for suspected intracranial injury. Clinically occult abusive head trauma can occur, especially in young infants. Therefore, head CT should be performed in selected neurologically asymptomatic physical abuse patients. Contrast-enhanced CT of the abdomen/pelvis is utilized for suspected intra-abdominal or pelvic injury. Particular attention should be paid to discrepancies between the patterns of injury and the reported clinical history. Making the diagnosis of child abuse also requires differentiation from anatomical and developmental variants and possible underlying metabolic and genetic conditions. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer-reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment., (Copyright © 2017 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Urinary tract infection (UTI) is common in young children and may cause pyelonephritis and renal scarring. Long-term complications from renal scarring are low. The role of imaging is to evaluate for underlying urologic abnormalities and guide treatment. In neonates there is increased risk for underlying urologic abnormalities. Evaluation for vesicoureteral reflux (VUR) may be appropriate especially in boys because of higher prevalence of VUR and to exclude posterior urethral valve. In children older than 2 months with first episode of uncomplicated UTI, there is no clear benefit of prophylactic antibiotic. Ultrasound is the only study that is usually appropriate. After the age of 6 years, UTIs are infrequent. There is no need for routine imaging as VUR is less common. In children with recurrent or complicated UTI, in addition to ultrasound, imaging of VUR is usually appropriate. Renal cortical scintigraphy may be appropriate in children with VUR, as renal scarring may support surgical intervention. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment., (Copyright © 2017 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

The cause of fever in a child can often be determined from history, physical examination, and laboratory tests; infections account for the majority of cases. Yet in 20%, no apparent cause can be found, designated as fever without source (FWS). The yield of chest radiography in children with FWS is low, and it is usually not appropriate. However, in children with respiratory signs, high fever (>39°C), or marked leukocytosis (≥20,000/mm(3)), chest radiography is usually appropriate, as it has a higher yield in detecting clinically occult pneumonia. In newborns with FWS, there is higher risk for serious bacterial infection, and the routine use of chest radiography is controversial. In children with neutropenia, fever is a major concern. In some clinical circumstances, such as after hematopoietic stem cell transplantation, chest CT scan may be appropriate even if the results of chest radiography are negative or nonspecific, as it has higher sensitivity and can demonstrate specific findings (such as lung nodule and "halo sign") that can guide management. In a child with prolonged fever of unknown origin despite extensive medical workup (fever of unknown origin), diagnosis is usually dependent on clinical and laboratory studies, and imaging tests have low yield. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment., (Copyright © 2016 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Osteonecrosis of the hip (Legg-Calvé-Perthes) is a common disease, with 10,000-20,000 symptomatic cases annually in the United States. The disorder affects both adults and children and is most frequently associated with trauma and corticosteroid usage. The initial imaging evaluation of suspected hip osteonecrosis is done using radiography. MRI is the most sensitive and specific imaging modality for diagnosis of osteonecrosis of the hip. The clinical significance of hip osteonecrosis is dependent on its potential for articular collapse. The likelihood of articular collapse is significantly increased with involvement of greater than 30%-50% of the femoral head area, which is optimally evaluated by MRI, often in the sagittal plane. Contrast-enhanced MRI may be needed to detect early osteonecrosis of the hip in pediatric patients, revealing hypoperfusion. In patients with a contraindication for MRI, use of either CT or bone scintigraphy with SPECT (single-photon emission CT) are alternative radiologic methods of assessment. Imaging helps guide treatment, which may include core decompression, osteotomy, and ultimately, need for joint replacement. The ACR Appropriateness Criteria(®) are evidence-based guidelines for specific clinical conditions that are reviewed every three years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment., (Copyright © 2016 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

More than 500,000 visits to the emergency room occur annually in the United States, for acute knee trauma. Many of these are twisting injuries in young patients who can walk and bear weight, and emergent radiographs are not required. Several clinical decision rules have been devised that can considerably reduce the number of radiographs ordered without missing a clinically significant fracture. Although a fracture is seen on only 5% of emergency department knee radiographs, 86% of knee fractures result from blunt trauma. In patients with a fall or twisting injury who have focal tenderness, effusion, or inability to bear weight, radiographs should be the first imaging study obtained. If the radiograph shows no fracture, MRI is best for evaluating for a suspected meniscus or ligament tear, or the injuries from a reduced patellar dislocation. Patients with a knee dislocation should undergo radiographs and an MRI, as well as an angiographic study such as a fluoroscopic, CT, or MR angiogram. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every three years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures, by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment., (Copyright © 2015 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Vomiting is a commonly reported symptom in infants less than three months of age. There are a multitude of pathologies to consider, both within and outside the gastrointestinal tract. In addition to conducting a thorough history and physical examination, a clinician formulates a reasonable differential diagnosis by consideration of two main factors: the infant's age and the characterization of the vomit as bilious or nonbilious. In this endeavor, the clinician is able to determine if an imaging study is needed and, if so, the urgency of the request. A review of the appropriate imaging evaluation of vomiting infants in the newborn to three-month-old age group is provided by organizing the discussion around the following three clinical scenarios: bilious vomiting, intermittent nonbilious vomiting since birth, and new-onset bilious vomiting. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every three years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment., (Copyright © 2015 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

This ACR Appropriateness Criteria article offers imaging triage guidance for several variants of patients presenting with acute foot trauma. Patients meeting inclusion criteria for the Ottawa Rules should undergo a 3-view radiographic series. Diabetic patients with peripheral neuropathy should undergo radiography, even though they do not meet the Ottawa Rules inclusion criteria. Patients with suspected midfoot and/or Lisfranc injury should undergo 3-view radiographs with weight bearing on at least the anterior-posterior view. Patients with suspected Lisfranc injury and normal radiographs should be considered for MRI and CT on a case-by-case basis. MRI or ultrasound could confirm cases of suspected acute tendon rupture. Radiography is the initial imaging modality for suspected plantar plate injury after metatarsal-phalangeal joint injury. Weight-bearing anterior-posterior, lateral, and sesamoid axial views may detect proximal migration of the hallux sesamoids. Ultrasound or MRI can directly evaluate the capsuloligamentous complex, specifically the plantar plate. Radiography can detect radiopaque penetrating foreign bodies, and ultrasound can be helpful in detecting those that are nonradiopaque. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 3 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures, by the panel. In instances in which evidence is lacking or is not definitive, expert opinion may be used to recommend imaging and treatment., (Copyright © 2015 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Acute ankle injuries are frequently diagnosed and treated in emergency departments and outpatient clinics. Recent evidence-based clinical treatment guidelines and systematic review of economic analyses support the use of 3-view (anteroposterior, lateral, and mortise) radiographic evaluation of patients meeting the criteria of the Ottawa ankle rules. Cross-sectional imaging has a limited secondary role primarily as a tool for preoperative planning and as a problem-solving technique in patients with persistent symptoms and suspected of having occult fractures. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 3 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment., (Copyright © 2015 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Background: Anthocyanin pigments aid in reproduction and provide ultraviolet protection to land plants. We have examined the phylogenetic relationships among the five primary enzymes responsible for producing anthocyanin pigment in its three major forms. Dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), Flavonoid 3'glucosyltransferase (F3GT), flavonoid 3'hydroxylase (F3'H), and flavonoid 3'5' hydroxylase (F3'5'H) are responsible for the final steps in anthocyanin pigment production., Results: We were interested in how conserved the anthocyanin pathway genes may be among land plants, and evolutionarily how far back into the plant lineage anthocyanin production may be traced. The DFR, ANS, F3GT, and F3'H genes date back 450 million years to the first land plants. Mosses, spike mosses, and ferns express these four products, although there is no evidence of sequence orthologues for these genes in algae. Additionally, F3'5'H is not evident in organisms that predated gymnosperms., Conclusion: Our findings support the hypothesis that "blue" anthocyanin pigments did not evolve until 300-350 mya along with the gymnosperms, although the "red" anthocyanin pigments may be as ancient as the mosses (~450 mya).

Head trauma is a frequent indication for cranial imaging in children. CT is considered the first line of study for suspected intracranial injury because of its wide availability and rapid detection of acute hemorrhage. However, the majority of childhood head injuries occur without neurologic complications, and particular consideration should be given to the greater risks of ionizing radiation in young patients in the decision to use CT for those with mild head trauma. MRI can detect traumatic complications without radiation, but often requires sedation in children, owing to the examination length and motion sensitivity, which limits rapid assessment and exposes the patient to potential anesthesia risks. MRI may be helpful in patients with suspected nonaccidental trauma, with which axonal shear injury and ischemia are more common and documentation is critical, as well as in those whose clinical status is discordant with CT findings. Advanced techniques, such as diffusion tensor imaging, may identify changes occult by standard imaging, but data are currently insufficient to support routine clinical use. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 3 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment., (Copyright © 2014 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

The aquatic insect fauna of salt marshes is poorly characterized, with the possible exception of biting Diptera. Aquatic insects play a vital role in salt marsh ecology, and have great potential importance as biological indicators for assessing marsh health. In addition, they may be impacted by measures to control mosquitoes such as changes to the marsh habitat, altered hydrology, or the application of pesticides. Given these concerns, the goals of this study were to conduct the first taxonomic survey of salt marsh aquatic insects on Long Island, New York, USA and to evaluate their utility for non-target pesticide impacts and environmental biomonitoring. A total of 18 species from 11 families and five orders were collected repeatedly during the five month study period. Diptera was the most diverse order with nine species from four families, followed by Coleoptera with four species from two families, Heteroptera with three species from three families, then Odonata and the hexapod Collembola with one species each. Water boatmen, Trichocorixa verticalis Fieber (Heteroptera: Corixidae) and a shore fly, Ephydra subopaca Loew (Diptera: Ephydridae), were the two most commonly encountered species. An additional six species; Anurida maritima Guérin-Méneville (Collembola: Neanuridae), Mesovelia mulsanti White (Heteroptera: Mesovelidae), Enochrus hamiltoni Horn (Coleoptera: Hydrophilidae), Tropisternus quadristriatus Horn (Coleoptera: Hydrophilidae), Dasyhelea pseudocincta Waugh and Wirth (Diptera: Ceratopogonidae), and Brachydeutera argentata Walker (Diptera: Ephydridae), were found regularly. Together with the less common Erythrodiplax berenice Drury (Odonata: Libellulidae), these nine species were identified as the most suitable candidates for pesticide and environmental impact monitoring due to abundance, position in the food chain, and extended seasonal occurrence. This study represents a first step towards developing an insect-based index of biological integrity for salt marsh health assessment.

Background: In many parts of the world, salt marshes play a key ecological role as the interface between the marine and the terrestrial environments. Salt marshes are also exceedingly important for public health as larval habitat for mosquitoes that are vectors of disease and significant biting pests. Although grid ditching and pesticides have been effective in salt marsh mosquito control, marsh degradation and other environmental considerations compel a different approach. Targeted habitat modification and biological control methods known as Open Marsh Water Management (OMWM) had been proposed as a viable alternative to marsh-wide physical alterations and chemical control. However, traditional larval sampling techniques may not adequately assess the impacts of marsh management on mosquito larvae. To assess the effectiveness of integrated OMWM and marsh restoration techniques for mosquito control, we analyzed the results of a 5-year OMWM/marsh restoration project to determine changes in mosquito larval production using GIS and geostatistical methods., Methods: The following parameters were evaluated using "Before-After-Control-Impact" (BACI) design: frequency and geographic extent of larval production, intensity of larval production, changes in larval habitat, and number of larvicide applications. The analyses were performed using Moran's I, Getis-Ord, and Spatial Scan statistics on aggregated before and after data as well as data collected over time. This allowed comparison of control and treatment areas to identify changes attributable to the OMWM/marsh restoration modifications., Results: The frequency of finding mosquito larvae in the treatment areas was reduced by 70% resulting in a loss of spatial larval clusters compared to those found in the control areas. This effect was observed directly following OMWM treatment and remained significant throughout the study period. The greatly reduced frequency of finding larvae in the treatment areas led to a significant decrease (approximately 44%) in the number of times when the larviciding threshold was reached. This reduction, in turn, resulted in a significant decrease (approximately 74%) in the number of larvicide applications in the treatment areas post-project. The remaining larval habitat in the treatment areas had a different geographic distribution and was largely confined to the restored marsh surface (i.e. filled-in mosquito ditches); however only approximately 21% of the restored marsh surface supported mosquito production., Conclusion: The geostatistical analysis showed that OMWM demonstrated considerable potential for effective mosquito control and compatibility with other natural resource management goals such as restoration, wildlife habitat enhancement, and invasive species abatement. GPS and GIS tools are invaluable for large scale project design, data collection, and data analysis, with geostatistical methods serving as an alternative or a supplement to the conventional inference statistics in evaluating the project outcome.

Culex salinarius is considered one of the most likely bridge vectors involved in the human transmission cycle of West Nile virus (WNV) and eastern equine encephalomyelitis virus (EEEV) in the northeastern USA. The larval habitats of this species in the coastal region of New York State are currently poorly known. Between 2005 and 2007, a larval survey was carried out to identify and characterize possible larval habitats in Suffolk County, encompassing natural and man-made freshwater wetlands, artificial containers, and salt marshes. Only relatively undisturbed salt marsh yielded Cx. salinarius larvae in considerable numbers from several sites over a period of 2 years. The immature stages of this species were found associated with Spartina patens and S. alterniflora of the upper marsh at salinities ranging from 4.3 to 18.8 parts per thousand. Both heavily impacted and relatively undisturbed salt marshes produced several hundreds of adult Cx. salinarius per Centers for Disease Control and Prevention (CDC) light trap per night, an order of magnitude higher than CDC light traps deployed at upland sites. The ability of Cx. salinarius to use both heavily impacted and relatively undisturbed salt marshes for reproduction has significant repercussions for marsh restoration and vector control practices.

Preoperative imaging studies and operative inspection may provide insufficient information to appropriately manage certain complex pancreatic pseudocysts. Intraoperative ultrasound accurately identifies and localizes peripancreatic fluid collections, cyst wall thickness, parenchymal and ductal anatomy, and relationships to adjacent visceral and vascular structures. Adjunctive use of intraoperative ultrasonography altered the surgical management in the clinical case described herein and is advocated for assessment of problematic pancreatic pseudocysts.

A study was conducted to determine the prevalence of perinatal substance use in Missouri. The population sample studied was selected according to a multi-stage probability-proportional-to-size sampling. The weighted prevalence for perinatal exposure to alcohol, tobacco or illicit substances was 31.9%, 10.8% for illicit substances, 21.9% for tobacco use, and 7.9% for self-reported alcohol use. In 1993, an estimated 23,925 perinatal exposures to licit and illicit substances occurred.

The possible involvement of acyl-coenzyme A:1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles in regulating the mitochondrial permeability transition have been examined by direct methods. 1-Acyllysophospholipid acyltransferase activity found in mitochondrial preparations obtained by differential centrifugation is inhibited by several transition-inducing agents and by glutathione disulfide. However, marker enzyme analysis employing mitochondria prepared by Percoll density gradient centrifugation or fractionated by a shear force-dependent method indicate that this activity is associated with contaminating microsomes and not with mitochondria. The absence of phospholipid acylation-deacylation cycles in isolated mitochondria is demonstrated by the absence of 18O incorporation from H2(18)O into phospholipid acylester carbonyl groups, confirming conclusions arrived at from marker enzyme data by a definitive independent approach. Mitochondria prepared by differential centrifugation and Percoll density gradient centrifugation are shown to be equivalent in requirements for induction of the permeability transition and the apparent rate of this process. It is concluded that 1-acyllysophospholipid acyltransferase activity and phospholipid acylation-deacylation cycles are not factors regulating the transition in isolated mitochondria. However, mitochondrial phospholipase A2 activity remains as a potential regulating factor, whereas the action of transition-inducing agents on microsomal 1-acyllysophospholipid acyltransferase may be important in mechanisms of cell injury.

The possibility that carrier proteins are involved in bile acid synthesis was investigated using rat liver homogenates. The 105 000 X g supernatant fraction was found to contain heat stable proteins that bound the bile acid precursor, 7alpha-hydroxy-4-cholesten-3-one, and increased the amount of 7alpha, 12alpha-dihydroxy-4-cholesten-3-one formed by the microsomal enzyme, 12alpha-hydroxylase. Subsequent studies were carried out to determine if squalene and sterol carrier protein or albumin, two lipid binding proteins present in the 105 00 X g supernatant fraction of rat liver homogenates, may be responsible for the effects seen with this fraction. Squalene and sterol carrier protein bound several water insoluble bile acid precursors, including 7alpha-hydroxy-4-cholesten-3-one, and increased the apparent activity of 12alpha-hydroxylase. Squalene and sterol carrier protein, however, did not bind either cholic acid or chenodeoxycholic acid. Rat serum albumin also bound 7alpha-hydroxy-4-cholesten-3-one and increased the apparent activity of 12alpha-hydroxylase. Kinetic analysis indicated that the apparent stimulation of 12alpha-hydroxylase by squalene and sterol carrier protein and albumin was due to increased solubilization of the substrate, 7alpha-hydroxy-4-cholesten-3-one. Thus, these studies indicate that bile acid precursor carrier proteins are present in the 105 000 Xg supernatant fraction of rat liver homogenates and suggest that squalene and sterol carrier protein or albumin may participate as carrier proteins in bile acid synthesis.

Studies on sterol synthesis in cultured human renal cell carcinoma were conducted in an attempt to elucidate the mechanism by which clear cell tumors accumulate cholesterol in the cytoplasm. Cultured renal cell carcinoma requires serum lipoproteins for optimal growth. In some renal cell carcinomas cellular cholesterol content was significantly higher than that present in cultured kidney cells under similar conditions. Renal cell carcinoma synthesizes sterols from labeled acetate. The rate of sterol synthesis is inversely proportional to the levels of exogenous cholesterol in the growth medium, suggesting the presence of a negative feedback mechanism. In addition, the conversion of 27-carbon-atom sterol precursors to cholesterol appears to be slow and inefficient under the presnt conditions. We conclude that an aberrant regulation of cholesterol synthesis cannot be invoked necessarily to explain cholesterol accumulation in renal cell carcinoma.

Rat liver sterol carrier protein (SCP) is a major intracellular protein regulating lipid metabolism and transport. During a dark-light cycle, SCP undergoes a dramatic diurnal variation in synthesis and level, reflecting translational events. Several hormones participate in the control of SCP synthesis. Insulin was implicated when the circadian rhythm of SCP was lost in both diabetes and fasting, states where insulin is low. After a 12-h fast the amplitude of the diurnal rhythm is diminished; after a 48-h fast it disappears, although SCP synthesis and level remain high. When endogenous insulin secretion is increased in fasted rats by glucose administration, SCP increases 2-fold in less than 30 min. When food intake is manipulated, but the dark-light cycle is unchanged, the circadian rhythm of SCP corresponds to feeding patterns and not light cycling. During feeding, increases in SCP are triggered following the expected increase in serum insulin. However, SCP is rapidly and significantly elevated in response to insulin only when glucocorticoids are normally high or increased by injection of the synthetic glucocorticoid, dexamethasone. Hepatocyte SCP levels are also induced by a combination of insulin and dexamethasone (2.3-fold) or insulin alone (1.3-fold). Dexamethasone alone causes a striking depression of SCP (2.4-fold). Thus, insulin is a major regulator of the diurnal variation of SCP synthesis. Glucocorticoids and other hormones (e.g. triiodothyronine) are also essential for maximum induction of SCP but play permissive roles.

Squalene and sterol carrier protein (SCP) levels and sterol/phospholipid molar ratios of whole cells and plasma membranes were measured in cultured primary tumor and metastatic cell lines. SCP is abundant in all cell lines. However, metastatic lines have significantly lower SCP levels and plasma membrane sterol/phospholipid ratios than do primary lines. The results indicate that extremely malignant, metastatic cells are unable to produce or maintain adequate levels of both SCP and plasma membrane sterols when grown in lipoprotein deficient media. This defect, in vivo, probably causes excess uptake of SCP and lipid.

Rat liver sterol carrier protein (SCP), a major regulator of lipid metabolism and transport, undergoes a rapid turnover and dramatic circadian variation in amount. The level of SCP was quantitated by a specific immunochemical assay using an antibody to homogeneous liver SCP. During a 12-h dark, 12-h light cycle, liver exhibits a biphasic pattern in SCP level. A 7-fold increase in SCP (i.e. from 1 to 7 mg/g of liver) occurs in the dark period, peaking at the midpoint and returning to basal levels by the beginning of the light period. A similar but smaller pattern of variation in SCP amount occurs in the light cycle. To elucidate the basic mechanism responsible for these changes in SCP level, the relative synthetic rate of SCP and mRNA functional activity for SCP were measured during the dark-light cycle. Alterations in the rate of SCP synthesis can account for the variations in SCP concentration. Although large changes occur in relative synthetic rate, no significant changes were found in the level of mRNA for SCP. Therefore, the circadian rhythm in SCP synthesis and amount does not reflect variations in the concentration of mRNA for SCP, but instead is caused by some mechanism controlling the efficiency of translation of SCP mRNA.

To date there have been no in vivo studies of the known propensity of human renal cell cancer (RCC) to accumulate large amounts of cholesterol. Caki-1, a human RCC line, was grown in the cheek pouches of immunosuppressed golden Syrian hamsters. Analysis of cholesterol levels revealed 2 to 4 times as much cholesterol in the heterotransplants as in any of the host tissues. After intraperitoneal injection of tumor-bearing hamsters with 4-14C-cholesterol, the tumors accumulated less radioactivity than did hamster liver and no more radioactivity than did hamster kidney or cheek pouch. It appears that accumulation of cholesterol by human RCC is not dependent on extensive uptake of extracellular cholesterol. Instead, an intrinsic defect in the cellular regulation of cholesterol synthesis and efflux is more likely to be responsible.

Sterol carrier protein (SCP) is a highly abundant, ubiquitous, low molecular weight protein that has a rapid turnover and multifunctional roles in lipid metabolism and transport. It is also known as the fatty acid-binding protein. These and other characteristics led to studies on the possible role of SCP in mediation of steroidogenic responses of the adrenal gland to corticotropin. To quantitate the level of SCP in adrenal tissue and subcellular fractions, a specific immunochemical assay was developed using an antibody to homogeneous liver SCP. SCP is a major protein in adrenal cells (greater than 100 micrograms/mg of total protein). The bulk of it is present in the soluble (60%) and mitochondrial (35%) fractions. Nearly all mitochondrial SCP is present in the inner membrane. Adrenal SCP undergoes a dramatic diurnal variation, varying from 2% to 12% of total protein. When corticotropin was administered in vivo in the presence of aminoglutethimide, an inhibitor of steroidogenesis, there was a parallel movement of SCP with cholesterol to the inner mitochondrial membrane. Other work showed mitochondrial SCP levels increase only in situations where there is an increase in cholesterol levels. These findings strongly suggest that one of the functions of adrenal SCP is to participate in teh corticotropin-stimulated movement of cholesterol to the inner mitochondrial membrane for steroidogenesis.

We investigated the effect of temperature, variant hemoglobins, and hyperlipidemia on determination of glycosylated hemoglobin by an electrophoretic method (Clin. Chem. 26: 1598-1602, 1980). We found that: (a) temperature variations ranging from 4 to 30 degrees C were without effect on results obtained by electrophoresis; (b) concurrent determination of glycosylated hemoglobin by electrophoresis and column-chromatography in blood specimens from 150 diabetic patients yielded almost identical mean values for both procedures when operations were carried out at 22 degrees C; (c) electrophoretic determination of glycosylated hemoglobin in whole-blood hemolysate was not affected by concentration of triglycerides; and (d) unlike column-chromatographic procedures, which underestimate the percentage of glycosylated hemoglobin in patients with hemoglobin S and C, the electrophoretic method accurately determined the proportion of glycosylated hemoglobin in these hemoglobinopathies. Evidently, electrophoresis on agar gel is an excellent alternative to cation-exchange column-chromatographic methods for glycosylated hemoglobin.

The fluorescent sterol analogue delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was synthesized and purified by reverse-phase high-performance liquid chromatography. Dehydroergosterol in aqueous solution had a critical micelle concentration of 25 nM and a maximum solubility of 1.3 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of dehydroergosterol with purified rat liver squalene and sterol carrier protein (SCP). SCP increased the maximal solubility of dehydroergosterol in aqueous buffer. The fluorescence emission spectrum of dehydroergosterol was blue shifted upon addition of SCP. The fluorescence lifetime of dehydroergosterol in aqueous buffer was 2.3 ns; addition of SCP resulted in the appearance of a second lifetime component near 12.4 ns. The SCP increased the fluorescence polarization of monomeric dehydroergosterol in aqueous buffer from 0.033 to 0.086. Scatchard analysis of the binding data indicated that dehydroergosterol interacted with purified rat liver SCP with an apparent KD = 0.88 microM and Bmax = 4.8 microM. At maximal binding, 1.0 mol of dehydroergosterol was specifically bound per mole of SCP. The close molecular interaction of dehydroergosterol with SCP was also demonstrated by energy-transfer experiments. The intermolecular distance between SCP and bound dehydroergosterol was evaluated by fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene series of double bonds in dehydroergosterol. The transfer efficiency was 36%, and R, the apparent distance between the tyrosine energy donor and the dehydroergosterol energy acceptor, was 19 A. The significance of these data obtained in vitro for dehydroergosterol interaction with SCP was also tested in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.

An enzymatic method for cholesterol in serum [Clin. Chem. 20, 470 (1974)] was initially found to be unsatisfactory for measuring cholesterol in high-density-lipoprotein fractions prepared by precipitation with Mn2+. A fine precipitate formed in the cuvette and cholesterol values were falsely increased. We describe a simple, convenient method for circumventing these problems. An ethylenediaminetetraacetate solution is used to reconstitute the enzymatic reagent. Cholesterol values by this procedure correlated with those obtained by the Lipid Research Clinic's procedure for the same lipoprotein fraction preparations (regression slope, .998; Y-intercept, 8.9 mg/liter; correlation coefficient, .984; standard error of the estimate, 16.8 mg/liter). Precision of the assay, including the precipitation step, was calculated. The SDwithin day was 9.7 mg/liter and SDoverall was 23.7 mg/liter. Results for total cholesterol with the modified reagent were linearly related to concentrations exceeding 4 g/liter, thereby permitting determination of high-density-lipoproteins and total cholesterol in a single run.

This report summarizes our recent studies on the protein known as sterol carrier protein (SCP) or fatty acid binding protein (FABP). SCP is a highly abundant, ubiquitous protein with multifunctional roles in the regulation of lipid metabolism and transport. SCP in vitro activates membrane-bound enzymes catalyzing cholesterol synthesis and metabolism, as well as those catalyzing long chain fatty acid metabolism. SCP also binds cholesterol and fatty acids with high affinity and rapidly penetrates cholesterol containing model membranes. Studies in vivo showed SCP undergoes a remarkable diurnal cycle in level and synthesis, induced by hormones and regulated in liver by translational events. SCP rapidly responds in vivo to physiological events and manipulations affecting lipid metabolism by changes in level. Thus SCP appears to be an important regulator of lipid metabolism. Preliminary evidence is presented that SCP is secreted by liver and intestine into blood and then taken up by tissues requiring SCP but incapable of adequate SCP synthesis.

Sterol carrier protein (SCP), also known as liver fatty acid binding protein, is a major adrenal protein localized in the cytosol and inner mitochondrial membrane. SCP is synthesized in liver and intestine and then rapidly secreted into blood, where it associates primarily with the high density lipoprotein fraction. Studies using steroidogenesis inhibitors showed that corticotropin has a specific effect on the uptake of SCP and its movement with cholesterol to the inner mitochondrial membrane. Thus, SCP appears to be intimately involved in the mechanism(s) of adrenal steroidogenesis from cholesterol.

The striking changes in amount of rat liver SCP (sterol carrier protein) during a 24-h dark-light cycle are due to alterations in the relative synthetic rate of SCP. However, functional SCP mRNA, measured by a cell-free translational assay, does not fluctuate in the dark-light cycle. Since cell-free translational assays do not always reflect the actual abundance of an mRNA molecule, a specific cDNA hybridization assay was used to directly quantitate SCP mRNA sequences. The cDNA probe was selected from a rat liver library by hybridization to a mixture of synthetic oligonucleotides containing a portion of the sequence of SCP mRNA. The relative amount and size distribution of the SCP mRNA species (approximately 700-800 nucleotides) does not change during the diurnal cycle. To explore possible mechanisms of this translational control, the polysomal distribution of SCP mRNA was compared at the maximum and minimum points of SCP synthesis. No significant amounts of SCP mRNA were present in nonpolysomal ribonucleoprotein particles. Furthermore, no alteration in the relative level of SCP mRNA associated with polysomes or in polysome size occurs at the maximum and minimum points of SCP synthesis. Thus, changes in total SCP mRNA levels or its polysomal distribution cannot account for the diurnal variation in SCP synthesis.

The rat liver enzyme system, which catalyzes reduction of delta 5,7,24-cholestatrien-3 beta-ol to cholesterol (delta 5-cholesten-3 beta-ol), converted radiolabeled delta 5,7,22-cholestatrien-3 beta-ol to delta 5,22-cholestadien-3 beta-ol, but not to cholesterol. This enzyme system thus contains membrane-bound delta 7- and delta 24-reductase and no delta 22-reductase. Kinetic and competition studies showed that the enzyme system contains a single delta 5,7-sterol delta 7-reductase, which is not influenced by unsaturation at the delta 22-position of the sterol side chain. The identity of delta 5,22-cholestadienol was established by chromatographic, spectral and chemical analyses. Use of the enzyme system and readily available delta 5,7,22-cholestatrienol provides a facile procedure for specific production of delta 5,22-cholestadien-3 beta-ol in quantity.

An enzymatic triglyceride method has been shown to be a suitable alternative to the Lipid Research Clinics' extraction/fluorometry method in long-term population studies. Correlation of results obtained with this method by this laboratory (y-axis) and by the Minneapolis Lipid Research Clinic Laboratory (x-axis) during a nine-week standardization period produced a curve with an intercept of -72 mg/liter, a slope of 1.019, and a correlation coefficient of r=0.996 (n=47). During this standardization period certain methodological problems were observed and corrected. An increase in background in certain clinical specimens, caused by spontaneous degradation of NADH, was observed, accurately measured, and taken into account when appropriate.

The immunosuppressive peptide cyclosporin A is a powerful inhibitor of the Ca2+-dependent permeability transition in rat liver mitochondria. When swelling is used to monitor the transition, the inhibitor is effective regardless of whether N-ethylmaleimide, Hg2+, WY-14643, t-butyl hydroperoxide, oxalacetate, rhein, phosphate, phosphoenolpyruvate, or ruthenium red plus uncoupler is used as the inducing agent. Twenty-five to fifty pmol/mg protein of cyclosporin A reduces the swelling response by 50% with complete inhibition obtained at about 150 pmol/mg protein. The compound, which does not inhibit Ca2+ uptake or mitochondrial phospholipase A2, is effective when added before or after the transition promoting agent. These findings, together with the shape of the inhibition dose-response curve, suggest that cyclosporin A essentially titrates a mitochondrial component which is present at 80-90 pmol/mg protein. It is proposed that this component is a solute unselective, regulated pore or a factor involved in controlling such a structure.

The overall conclusion to be made from the information presented here is that for many reasons SCP is a highly unusual protein. Some of these reasons are, first, SCP serves as cofactor for a number of different membrane-bound enzymes catalyzing specific steps in lipid metabolism. Second, SCP is involved in intracellular transport or movement of both cholesterol and fatty acids. Third, SCP is remarkably abundant and ubiquitous; its structure is conserved throughout nature. Fourth, SCP is exported to the blood stream from its site of synthesis by some, perhaps unique, mechanism and then rapidly taken up by specific tissues, e.g., the adrenal. Fifth, SCP is free in the cytosol and can also move to the inner mitochondrial membrane, where it is tightly bound. Sixth, SCP undergoes a dramatic diurnal variation in amount, reflecting changes in synthetic rate. Its half-life is less than an hour. Seventh, the diurnal variation in amount is triggered by feeding and influenced by several hormones. The diurnal variation is lost but a high level of SCP is maintained in the face of debilitating conditions, i.e., starvation, diabetes. Eighth, malignant cells exhibit defects in the uptake, synthesis, or turnover of SCP. Ninth, the synthesis of SCP is regulated by the efficiency of translation of its ever abundant mRNA. Tenth, there is much more to be learned about the functions and regulation of SCP.