Combining steady state and temperature jump IR spectroscopy to investigate the allosteric effects of ligand binding to dsDNA.

Changes in the structural dynamics of double stranded (ds)DNA upon ligand binding have been linked to the mechanism of allostery without conformational change, but direct experimental evidence remains elusive. To address this, a combination of steady state infrared (IR) absorption spectroscopy and ultrafast temperature jump IR absorption measurements has been used to quantify the extent of fast (∼100 ns) fluctuations in (ds)DNA·Hoechst 33258 complexes at a range of temperatures. Exploiting the direct link between vibrational band intensities and base stacking shows that the absolute magnitude of the change in absorbance caused by fast structural fluctuations following the temperature jump is only weakly dependent on the starting temperature of the sample. The observed fast dynamics are some two orders of magnitude faster than strand separation and associated with all points along the 10-base pair duplex d(GCATATATCC). Binding the Hoechst 33258 ligand causes a small but consistent reduction in the extent of these fast fluctuations of base pairs located outside of the ligand binding region. These observations point to a ligand-induced reduction in the flexibility of the dsDNA near the binding site, consistent with an estimated allosteric propagation length of 15 Å, about 5 base pairs, which agrees well with both molecular simulation and coarse-grained statistical mechanics models of allostery leading to cooperative ligand binding.

Global REACH 2018: The influence of acute and chronic hypoxia on cerebral haemodynamics and related functional outcomes during cold and heat stress.

KEY POINTS: Thermal and hypoxic stress commonly coexist in environmental, occupational and clinical settings, yet how the brain tolerates these multi-stressor environments is unknown Core cooling by 1.0°C reduced cerebral blood flow (CBF) by 20-30% and cerebral oxygen delivery (CDO2 ) by 12-19% at sea level and high altitude, whereas core heating by 1.5°C did not reliably reduce CBF or CDO2 Oxygen content in arterial blood was fully restored with acclimatisation to 4330 m, but concurrent cold stress reduced CBF and CDO2 Gross indices of cognition were not impaired by any combination of thermal and hypoxic stress despite large reductions in CDO2 Chronic hypoxia renders the brain susceptible to large reductions in oxygen delivery with concurrent cold stress, which might make monitoring core temperature more important in this context ABSTRACT: Real-world settings are composed of multiple environmental stressors, yet the majority of research in environmental physiology investigates these stressors in isolation. The brain is central in both behavioural and physiological responses to threatening stimuli and, given its tight metabolic and haemodynamic requirements, is particularly susceptible to environmental stress. We measured cerebral blood flow (CBF, duplex ultrasound), cerebral oxygen delivery (CDO2 ), oesophageal temperature, and arterial blood gases during exposure to three commonly experienced environmental stressors - heat, cold and hypoxia - in isolation, and in combination. Twelve healthy male subjects (27 ± 11 years) underwent core cooling by 1.0°C and core heating by 1.5°C in randomised order at sea level; acute hypoxia ( PET,O2  = 50 mm Hg) was imposed at baseline and at each thermal extreme. Core cooling and heating protocols were repeated after 16 ± 4 days residing at 4330 m to investigate any interactions with high altitude acclimatisation. Cold stress decreased CBF by 20-30% and CDO2 by 12-19% (both P < 0.01) irrespective of altitude, whereas heating did not reliably change either CBF or CDO2 (both P > 0.08). The increases in CBF with acute hypoxia during thermal stress were appropriate to maintain CDO2 at normothermic, normoxic values. Reaction time was faster and slower by 6-9% with heating and cooling, respectively (both P < 0.01), but central (brain) processes were not impaired by any combination of environmental stressors. These findings highlight the powerful influence of core cooling in reducing CDO2 . Despite these large reductions in CDO2 with cold stress, gross indices of cognition remained stable.

Association of objectively measured physical activity and bone health in children and adolescents: a systematic review and narrative synthesis.

The influence of day-to-day physical activity on bone in adolescence has not been well characterized. Forty articles were identified that assessed the relationship between accelerometry-derived physical activity and bone outcomes in adolescents. Physical activity was positively associated with bone strength in peri-pubertal males, with less consistent evidence in females. Physical activity (PA) is recommended to optimize bone development in childhood and adolescence; however, the influence of day-to-day PA on bone development is not well defined. The aim of this review was to describe the current evidence for objectively measured PA on bone outcomes in healthy children and adolescents. MEDLINE, Embase, Cochrane Library, Scopus, Web of Science, CINAHL, PsycInfo, and ClinicalTrials.gov were searched for relevant articles up to April 2020. Studies assessing the relationship between accelerometry-derived PA and bone outcomes in adolescents (6-18 years old) were included. Two reviewers independently screened studies for eligibility, extracted data, and rated study quality. Forty articles met inclusion criteria (25 cross-sectional, 15 longitudinal). There was significant heterogeneity in accelerometry methodology and bone outcomes measured. Studies in males indicated a significant, positive relationship between moderate to vigorous PA (MVPA) and bone outcomes at the hip and femur, particularly during the peri-pubertal years. The results for MVPA and bone outcomes in females were mixed. There was a paucity of longitudinal studies using pQCT and a lack of data regarding how light PA and/or impact activity influences bone outcomes. The current evidence suggests that objectively measured MVPA is positively associated with bone outcomes in children and adolescents, especially in males. However, inconsistencies in methodology make it difficult to determine the amount and type of PA that leads to favorable bone outcomes. Given that the majority of research has been conducted in Caucasian adolescents, further research is needed in minority populations.

Natural history of incomplete atypical femoral fractures in patients after a prolonged and variable course of bisphosphonate therapy-a long-term radiological follow-up.

Understanding the natural history of lateral femoral stress fractures helps to guide their management. Improvement in their radiographic characteristics is rare. Progression was generally sequential, most developing an incomplete fracture line before fracture displacement. Stopping bisphosphonates decreased the fracture rate, a feasible management option for lesions without incomplete fracture lines. INTRODUCTION: Retrospective study evaluating the natural history of lateral femoral stress fractures (FSF) by serial radiography over a variable period of time in a cohort of patients treated for some time with bisphosphonates for osteoporosis, whilst also identifying the fracture response in cases where bisphosphonates were discontinued. METHODS: The radiographs of 76 consecutive patients (92 femurs) with 161 FSF were reviewed to document their change over time. Femurs were classified into the following: A-normal, B-focal cortical thickening, C-dreaded black line and D-displaced fracture. Bisphosphonate history was recorded. RESULTS: 66.5% FSF showed group stability between the first and last radiographs: group B (79.1%), group C (45.7%). 28.6% progressed, mostly following an ordered sequence starting from group A, progressing to B, then C, before culminating in D. Progression rate was as follows: A-100% (11/11), B-18.3% (21/115), C-40% (14/35). Regression in FSF was uncommon-5.6% (8/161). 34.8% (32/92) sustained displaced fractures. Kaplan-Meier analysis showed statistically significant difference between the groups; median survival (95% CI): A-4189 (-), B-3383.0 (-), C-1807 (0.0-3788.6) and progression to displaced fracture when bisphosphonate had been stopped for at least 6 months. The group without recent bisphosphonates had a lower group progression rate (17.1%, 12/70). Nevertheless, 10.9% (5/46) progressed to displaced fracture. This group also had the highest proportion of stable (77.1%, 54/70) and regressive lesions (5.7%, 4/70). CONCLUSIONS: In FSF, there is natural progression from normal bone, to focal cortical thickening, to dreaded black line and eventually to displaced fracture. Most lesions persist, remaining static or progressing, especially if a dreaded black line is present and bisphosphonates are continued. Regression is uncommon and more frequent when bisphosphonates are discontinued. Despite stopping bisphosphonates, there remains a 10.9% risk of progression to displaced fracture.

Tuberculosis screening among HIV-infected patients: tuberculin skin test vs. interferon-gamma release assay.

National guidelines recommend screening for latent tuberculosis infection (LTBI) in all HIV-infected patients. Thus, the objective of this study was to measure protocol adherence to national guidelines regarding LTBI screening for HIV-infected patients entering care at an urban primary care clinic specializing in HIV care, identify clinical and other characteristics associated with adherence, and determine whether transitioning from the tuberculin skin test (TST) to the interferon-gamma release assay (IGRA) improved adherence. We conducted a retrospective study using protocol adherence to LTBI screening guidelines within twelve months of entering care at an HIV clinic as the primary outcome. Successful protocol adherence was defined as the placement and reading of a TST, performance of an IGRA, or a note in study clinic records documenting prior testing or treatment for tuberculosis in an outside setting. Multivariable modified Poisson regression models were used in analyses. Overall, 32% (n = 118/372) of patients received LTBI screening within twelve months of entering care. Protocol adherence to LTBI screening guidelines increased from 28% to 37% following the transition from TST to IGRA screening. IGRA screening [adjusted prevalence ratio: 1.45, 95% confidence limits: (1.07, 1.96)], male sex [1.47 (1.05, 2.07)], transfer patient status [1.51 (1.05, 2.18)], and greater than one year of clinic attendance [1.62 (1.06, 2.48)] were independently associated with protocol adherence. Among patients without prior LTBI screening or treatment, patients entering the clinic in 2013 under the IGRA screening protocol were more likely to be screened for LTBI compared to patients entering under the TST screening protocol (34.3% vs. 9.7%, p < 0.001). In conclusion, transitioning from TST to IGRA-based screening improved adherence to screening guidelines. However, further work on improving adherence to LTBI screening guidelines among HIV-infected patients is needed.

Cerebral homeostasis and orthostatic responses in residents of the highest city in the world.

Permanent residence at high-altitude and chronic mountain sickness (CMS) may alter the cerebrovascular homeostasis and orthostatic responses. Healthy male participants living at sea-level (LL; n = 15), 3800 m (HL3800m; n = 13) and 5100 m (HL5100m; n = 17), respectively, and CMS highlanders living at 5100 m (n = 31) were recruited. Middle cerebral artery mean blood flow velocity (MCAv), cerebral oxygen delivery (CDO2), mean blood pressure (MAP), heart rate variability and spontaneuous cardiac baroreflex sensitivity (cBRS) were assessed while sitting, initial 30 s and after 3 min of standing. Cerebral autoregulation index (ARI) was estimated (ΔMCAv%baseline)/ΔMAP%baseline) in response to the orthostatic challenge. Altitude and CMS were associated with hypoxemia and elevated hemoglobin concentration. While sitting, MCAv and LFpower negatively correlated with altitude but were not affected by CMS. CDO2 remained preserved. BRS was comparable across all altitudes, but lower with CMS. Within initial 30 s of standing, altitude and CMS correlated with a lesser ΔMAP while ARI remained unaffected. After 3 min standing, MCAv, CDO2 and cBRS remained preserved across altitudes. The LF/HF ratio increased in HL5100m compared to LL and HL3800m from sitting to standing. In contrary, CMS showed blunted autonomic nervous activation in responses to standing. Despite altitude- and CMS-associated hypoxemia, erythrocytosis and impaired blood pressure regulation (CMS only), cerebral homeostasis remained overall preserved.

Sequence-Dependent Melting and Refolding Dynamics of RNA UNCG Tetraloops Using Temperature-Jump/Drop Infrared Spectroscopy.

Time-resolved temperature-jump/drop infrared (IR) spectroscopy has been used to measure the impact of stem base sequence on the melting and refolding dynamics of ribonucleic acid (RNA) tetraloops. A series of three 12-nucleotide RNA hairpin sequences were studied, each featuring a UACG tetraloop motif and a double-stranded stem containing four base pairs. In each case, the stem comprised three GC pairs plus a single AU base pair inserted at the closing point of the loop (RNAloop), in the middle of the stem (RNAmid), or at the stem terminus (RNAend). Results from analogous DNA tetraloop (TACG) sequences were also obtained. Inclusion of AU or AT base pairs in the stem leads to faster melting of the stem-loop structure compared to a stem sequence featuring four GC base pairs while refolding times were found to be slower, consistent with a general reduction in stem-loop stability caused by the AU/AT pair. Independent measurement of the dynamic timescales for melting and refolding of ring vibrational modes of guanine (GR) and adenine (AR) provided position-specific insight into hairpin dynamics. The GR-derived data showed that DNA sequences melted more quickly (0.5 ± 0.1 to 0.7 ± 0.1 µs at 70 °C) than analogous RNA sequences (4.3 ± 0.4 to 4.4 ± 0.3 µs at 70 °C). Position-sensitive data from the AR modes suggests that DNA hairpins begin melting from the terminal end of the stem toward the loop while RNA sequences begin melting from the loop. Refolding timescales for both RNA and DNA hairpins were found to be similar (250 ± 50 µs at 70 °C) except for RNAend and DNAloop which refolded much more slowly (746 ± 36 and 430 ± 31 µs, respectively), showing that the refolding pathway is significantly impaired by the placement of AU/AT pairs at different points in the stem. We conclude that conformational changes of analogous pairs of RNA and DNA tetraloops proceed by different mechanisms.

SF-36 physical function and general health domains are independent predictors of acute hospital length of stay after hip fracture surgery.

OBJECTIVE: The demographics and co-morbidities of individuals may impact healthcare consumption, but it is less understood how premorbid physical and mental function may influence these effects. The aim of this study is to determine patient's pre-fracture quality of life and mobility affect acute hospital burden in the management of hip fracture, using length of stay (LOS) as a proxy for healthcare resource. MATERIALS AND METHODS: This is a retrospective study which investigated hip fracture patients who underwent surgery over the period of 2017-2020. Variables collected include LOS, age, gender, race, marital status, payer type, ASA score, time to surgery (TTS), type of surgery, fracture type, POD1 mobilization, discharge disposition, pre-fracture SF-36, EQ-5D and Parker mobility score (PMS) based on patient's recollection on admission. These variables were correlated with LOS using binary logistic regression on SAS. RESULTS: There were 1045 patients, and mean age was 79.5 + 8.57 (range 60-105) years with an average LOS 13.64 + 10.0 days (range 2-114). On univariate analysis, PMS, EQ-5D and all domains of SF-36 except bodily pain (BP), emotional role and mental health were associated significantly with LOS. Amongst the QOL and PMS scores, only the domains of SF-36 Physical Function (PF) (OR = 0.993, p = 0.0068) and General Health perception (GH) (OR 0.992, p = 0.0230) remained significant on the multivariate model. CONCLUSION: Our study showed that poor premorbid scores of SF36 PF and GH are independent factors associated with longer LOS in hip fracture patients after surgery, regardless of fracture type, age and ASA status. Hence, premorbid SF36 PF and GH can be used to identify patients that are at risk of prolonged hospital stay and employ targeted strategies to facilitate rehabilitation and discharge planning.

Early effects of acetazolamide on hemoglobin mass and plasma volume in chronic mountain sickness at 5100 m.

INTRODUCTION AND OBJECTIVES: Chronic Mountain Sickness (CMS) syndrome, combining excessive erythrocytosis and clinical symptoms in highlanders, remains a public health concern in high-altitude areas, especially in the Andes, with limited therapeutic approaches. The objectives of this study were to assess in CMS-highlanders permanently living in La Rinconada (5100-5300 m, Peru, the highest city in the world), the early efficacy of acetazolamide (ACZ) and atorvastatin to reduce hematocrit (Hct), as well as the underlying mechanisms focusing on intravascular volumes. MATERIALS AND METHODS: Forty-one males (46±8 years of age) permanently living in La Rinconada for 15 [10-20] years and suffering from CMS were randomized between ACZ (250 mg once-daily; N = 13), atorvastatin (20 mg once-daily; N = 14) or placebo (N = 14) uptake in a double-blinded parallel study. Hematocrit (primary endpoint) as well as arterial blood gasses, total hemoglobin mass (Hbmass) and intravascular volumes were assessed at baseline and after a mean (±SD) treatment duration of 19±2 days. RESULTS: ACZ increased PaO2 by +13.4% (95% CI: 4.3 to 22.5%) and decreased Hct by -5.2% (95% CI: -8.3 to -2.2%), whereas Hct remained unchanged with placebo or atorvastatin. ACZ tended to decrease Hbmass (-2.6%, 95% CI: -5.7 to 0.5%), decreased total red blood cell volume (RBCV, -5.3%, 95% CI: -10.3 to -0.3%) and increased plasma volume (PV, +17.6%, 95% CI: 4.9 to 30.3%). Atorvastatin had no effect on intravascular volumes, while Hbmass and RBCV increased in the placebo group (+6.1%, 95% CI: 4.2 to 7.9% and +7.0%, 95%CI: 2.7 to 11.4%, respectively). CONCLUSIONS: Short-term ACZ uptake was effective to reduce Hct in CMS-highlanders living at extreme altitude >5,000 m and was associated with both an increase in PV and a reduction in RBCV.

Measuring RNA UNCG Tetraloop Refolding Dynamics Using Temperature-Jump/Drop Infrared Spectroscopy.

Determining the structural dynamics of RNA and DNA is essential to understanding their cellular function, but direct measurement of strand association or folding remains experimentally challenging. Here we illustrate a temperature-jump/drop method able to reveal refolding dynamics. Time-resolved temperature-jump/drop infrared spectroscopy is used to measure the melting and refolding dynamics of a 12-nucleotide RNA sequence comprising a UACG tetraloop and a four-base-pair double-stranded GC stem, comparing them to an equivalent DNA (TACG) sequence. Stem-loop melting occurred an order of magnitude more slowly in RNA than DNA (6.0 ± 0.1 µs versus 0.8 ± 0.1 µs at 70 °C). In contrast, the refolding dynamics of both sequences occurred on similar time scales (200 µs). While the melting and refolding dynamics of RNA and DNA hairpins both followed Arrhenius temperature dependences, refolding was characterized by an apparent negative activation energy, consistent with a mechanism involving multiple misfolded intermediates prior to zipping of the stem base pairs.

The cosmopolitan maternal heritage of the Thoroughbred racehorse breed shows a significant contribution from British and Irish native mares.

The paternal origins of Thoroughbred racehorses trace back to a handful of Middle Eastern stallions, imported to the British Isles during the seventeenth century. Yet, few details of the foundation mares were recorded, in many cases not even their names (several different maternal lineages trace back to 'A Royal Mare'). This has fuelled intense speculation over their origins. We examined mitochondrial DNA from 1929 horses to determine the origin of Thoroughbred foundation mares. There is no evidence to support exclusive Arab maternal origins as some historical records have suggested, or a significant importation of Oriental mares (the term used in historic records to refer to Middle East and western Asian breeds including Arab, Akhal-Teke, Barb and Caspian). Instead, we show that Thoroughbred foundation mares had a cosmopolitan European heritage with a far greater contribution from British and Irish Native mares than previously recognized.

Shorter acute hospital length of stay in hip fracture patients after surgery predicted by early surgery and mobilization.

Time to surgery, early mobilization, fracture type, and ASA grades independently affect acute hospital length of stay after hip fracture surgery. Modifiable factors can be audited to reduce length of stay, and non-modifiable factors can be used for consideration of a tiered bundled payment reimbursement model. INTRODUCTION: As hip fracture incidence rises with our ageing global population, there will be an increase in consumption of healthcare resources. We hypothesized that hospital management and patient factors can affect healthcare burden load. Using length of stay (LOS) as a surrogate for consumption, the aim of this study is to elucidate the effect of hospital management and patient-related factors on length of stay (LOS) for patients after hip fracture surgery. We studied modifiable and non-modifiable factors influencing LOS, and identification of these modifiable factors accords opportunities for mitigating these factors. METHODS: This retrospective study examines hip fracture data from a large tertiary hospital in Singapore over the period of 2017 to 2020. Data collected on the electronic medical record included age, gender, race, marital status, payer type, ASA score, TTS, type of surgery, fracture type, POD1 mobilization, discharge position, and presence of pressure sores, and they were correlated with LOS using binary logistic regression on SAS. RESULTS: A total of 1045 patients were included in this study with 704 females and 341 males. The mean age was 79.5 ± 8.57 years (range 60-105) with an average LOS 13.64 ± 10.0 days (range 2-114). On binary logistic regression, ASA and trochanteric fracture remains a significant non-modifiable factor for LOS with OR = 1.486 (95% CI 1.106, 1.996, p = 0.0086) and OR 1.522 (95% CI 1.149, 2.015, p = 0.0034) respectively. Significant modifiable factors were TTS > 48 h (OR = 1.819, 95% CI 1.205, 2.746, p = 0.0044) and POD1 mobilization (OR = 0.441, 95% CI 0.257, 0.756, p = 0.0029). CONCLUSIONS: Our analysis showed TTS and POD1 are significant modifiable factors for LOS, and resources can be diverted towards them for the management of hip fracture patients and pre-empting the increasing load on our healthcare system.

Emission of methane from plants.

It has been proposed that plants are capable of producing methane by a novel and unidentified biochemical pathway. Emission of methane with an apparently biological origin was recorded from both whole plants and detached leaves. This was the first report of methanogenesis in an aerobic setting, and was estimated to account for 10-45 per cent of the global methane source. Here, we show that plants do not contain a known biochemical pathway to synthesize methane. However, under high UV stress conditions, there may be spontaneous breakdown of plant material, which releases methane. In addition, plants take up and transpire water containing dissolved methane, leading to the observation that methane is released. Together with a new analysis of global methane levels from satellite retrievals, we conclude that plants are not a major source of the global methane production.

Immunological aspects of axon injury in multiple sclerosis.

The role of immune-mediated axonal injury in the induction of nonremitting functional deficits associated with multiple sclerosis is an area of active research that promises to substantially alter our understanding of the pathogenesis of this disease and modify or change our therapeutic focus. This review summarizes the current state of research regarding changes in axonal function during demyelination, provides evidence of axonal dysmorphia and degeneration associated with demyelination, and identifies the cellular and molecular effectors of immune-mediated axonal injury. Finally, a unifying hypothesis that links neuronal stress associated with demyelination-induced axonal dysfunction to immune recognition and immunopathology is provided in an effort to shape future experimentation.

Enhancement of Sendai virus-mediated cell fusion by cupric ions.

The effect of divalent cations on cell fusion by concentrated Sendai virus, inactivated by beta-propiolactone, was investigated using Vero and mouse L-929 cells in monolayers. With both cell lines, which are normally resistant to exogenous viral fusion, Cu(2+) in sublethal concentrations was found to promote polykaryon formation to a marked degree. The simultaneous presence of Cu(2+) and virus was required for this effect, which was thought to be related to the cytotoxic action of Cu(2+) on the cell membrane. Accordingly, under standard conditions and in the absence of virus, leakage of isotopically labeled intracellular protein was shown to bear a quantitative relationship to Cu(2+) concentration. Concomitant changes in the membrane were seen electron microscopically to consist of loss of microvilli and the appearance of numerous vesicles on, or adjacent to, the membrane. The relationship of enhanced fusibility to these toxic changes was not further elucidated. The fusion-promoting effect of Cu(2+) far exceeded that of Ca(2+); and other cations tested had no effect.

Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells.

One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 kdaltons which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. We have treated isolated, membrane-intact brush borders with ATP and obtained solubilization of the 110-kdalton protein, calmodulin (CM), myosin, and lesser amounts of several other cytoskeletal proteins. Electron micrographs of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with "ballooning" of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with calcium and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110-kdalton protein. This result suggests that associated CM is necessary for the ATP-induced solubilization of the 110-kdalton protein. A 110-kdalton protein-CM complex, with 110-kdalton protein: CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110-kdalton protein-CM complex is an irregular, elongated molecule that ranged in size from 5 X 8 nm to 8 X 14 nm, with a Stokes' radius of 6.1 nm. This 110-kdalton protein-CM complex exhibited no Mg++-ATPase activity and no detectable myosin light chain kinase activity. In co-sedimentation assays, the 110-kdalton protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110-kdalton protein were calcium-independent. Negative stains of F-actin and 110-kdalton protein-CM in the absence of ATP showed loosely organized aggregates of actin with the 110-kdalton protein-CM complex coating the surface of the filaments. On the basis of our data, and in agreement with previous calculations (Matsudaira, P.T., and D.R. Burgess, 1979, J. Cell Biol. 83:667-673), we suggest that the lateral bridge of the microvillus is composed of a dimer of the 110-kdalton protein with four associated calmodulins.

Brush-border calmodulin. A major component of the isolated microvillus core.

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle. Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure. The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons. The supernate from the brush-border preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons. The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca++, and (c) 4--5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca++. With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin. In the presence of Ca++ (greater than 5 x 10(-7) M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable from. The dissociation of calmodulin was reversed by removal of Ca++. If microvillus cores were pretreated with phalloidin, the Ca++-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1:10 in the demembranated brush border and 1:2-3 in the microvillus core. No calmodulin was detected in the detergent-solubilized brush-border membrane fraction.

Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.

Mechanisms of cytoskeletal regulation: modulation of membrane affinity in avian brush border and erythrocyte spectrins.

The spectrins isolated from chicken erythrocytes and chicken intestinal brush border, TW260/240, share a common alpha subunit and a tissue-specific beta subunit. The ability of these related proteins to bind human erythrocyte inside out vesicles (IOVs) and human erythrocyte ankyrin in vitro have been quantitatively compared with human erythrocyte spectrin. Chicken erythrocyte spectrin binds human IOVs and human ankyrin with affinities nearly identical to that for human erythrocyte spectrin. TW260/240 does not significantly bind to either IOVs or ankyrin. These results demonstrate a remarkable tissue preservation of ankyrin-binding capacity, even between diverse species, and confirm the role of the avian beta-spectrins in modulating this functionality. Avian brush border spectrin may represent a unique spectrin which serves primarily as a filament cross-linker and which does not interact strongly with membrane-associated proteins.

Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen.

Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B in injected into athymic mice, metastatic hepatocellular carcinomas appear. These cell lines provide experimental models for investigation of plasma protein biosynthesis and the relation of the hepatitis B viru genome to tumorigenicity.