Selective isolation of rat aortic wall layers and their cell types in culture—Application to converting enzyme activity measurement

The rat aorta, whose three wall layers can be separated by microdissection offers the rare possibility of comparing physiological characteristics of in vivo tissular cell components and corresponding cells after culture.We developed a technique allowing the dissociation of the three tunicae (intima, media and adventitia) of the rat aorta and the culture of their main cell types i.e: endothelial cells (EC) from intima, smooth muscle cells (SMC) from media and fibroblasts (Fib) from adventitia. Comparison between selected tunicae in vivo and their corresponding cells in vitro was performed via arterial angiotensin converting enzyme (ACE) activity measurements in Wistar rats.In vivo microsomial ACE activity for each tunica was as follows: 368.9 ± 34.3 (endothelium), 10.5 ± 1.9 (media) and 10.2 ± 4.9 (adventitia) pmol/mg protein/min. Corresponding cell primary culture values were 1.2 ± 0.1 (EC), 0.06 ± 0.02 (SMC) and 0.24 ± 0.01 (Fib) pmol/mg protein/min. Incubation of serum-deprived cells with Dexamethasone (10⁻⁷M) over 48 hr induced a statistically significant shift of total ACE activity from controls to stimulated cells of 2.9 ± 0.3 to 9.7 ± 1.0 in EC, 0.8 ± 0.1 to 32.1 ± 4.9 in SMC and 1.03 ± 0.65 to 57.2 ± 2.1 pmol/ mg prot/min in fibroblasts.In the rat aorta, ACE was present not only in the intimal endothelial cell lining, but also in the media and the adventitia. ACE activity levels in primary cultured vascular cells were about 100-fold less than those found in the ex vivo tissues. Nevertheless, ACE expression seems to be more constitutive in endothelial cells and more inducible in smooth muscle cells and fibroblasts. This methodological approach should be of interest in studying environmental or genetic regulation of protein expression in the three layers/three cell types of the vascular wall.     

Evaluation of prostaglandin-receptors in human and rat liver: Interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50: 2.5±1.7, (6.1±2.1) μM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9±0.9 (2.0±0.8) μM. The Scatchard analysis on [3H]PGE1- as well as on [3H]ilioprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3±5.2 (21.3±4.3) fmol/mg protein and a Kd of 2.1±1.8 (1.9±0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4±18.2 (86.1±13.2) fmol/mg protein and a Kd of 10.5±2.9 (15.3±3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4±13.9 (35.9±8.2) fmol/mg protein and a Kd of 4.1±1.2 (1.7±1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3±42.1 (142.9±17.8) fmol/mg protein and a Kd of 16.3±4.9 (9.2±7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4±51.9 (38.8±7.4) fmol/mg protein and a Kd of 16.2±3.2 (2.5±1.2) nM.It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are stable to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Survival of Brucella abortus Strain RB51 Lyophilized and as Liquid Vaccine under Different Storage Conditions

Brucella abortus strain RB51 (SRB51) is a new cattle vaccine that is approved for use in the U.S. for prevention of brucellosis. At the present time, other countries are implementing or considering the use of SRB51 vaccine in their brucellosis control programs. In the current study, the effect of three stabilizing media, two fill volumes (1 and 3 ml), and three storage temperatures (−25, 4 and 25°C) on the viability of lyophilized SRB51 over a 52 week period was determined. The effects of three concentrations of bacteria (5×10⁸, 1×10⁹, or 5×10⁹ cfu/ml) and two storage temperatures (4 or 25°C) on viability of liquid SRB51 vaccine were also determined. For lyophilized strain RB51 vaccine, fill volume did not influence viability (P> 0·05) during lyophilization. Although fill volume did not influence viability during storage in World Health Organization (WHO) media or media containing both WHO and Lactose Salt (LS) media, 1 ml fill volumes of SRB51 in LS media had greater (P< 0·05) viability when compared to 3 ml fill volumes. Lyophilized SRB51 vaccine stored at 25°C had a more rapid decline in viability (P< 0·05) when compared to vaccine stored at −25 or 4°C. With the exception of the 3-ml fill volumes of LS media, all three stabilizing media were similar in maintaining viability of SRB51 at −25°C storage temperatures. However, when compared to WHO or WHO/LS media, stabilization in LS media was associated with a more rapid decline in viability during storage at 4 or 25°C (P< 0·05). Initial SRB51 concentration in liquid vaccine did not influence (P> 0·05) viability during storage at 4 or 25°C. When compared to liquid SRB51 vaccine stored at 25°C, storage at 4°C was associated with a slower decline in viability (P< 0·05) during 12 weeks of storage. Biochemical and morphological characteristics of SRB51 were stable under the storage conditions utilized in the present study. This study suggests that viability of SRB51 can be readily maintained during storage as a lyophilized or liquid brucellosis vaccine.     

Quality of the Entomopathogenic NematodeSteinernema carpocapsaeProduced on Different Media

The characteristics of morphometric (body length and width), biochemical (fatty acid content), motility, and penetration rate of infective juveniles ofSteinernema carpocapsaeBeijing strain reared on four different culture media [e.g., plant protein medium (I), animal protein medium (II), plant and animal protein medium (III), andin vivoculture (IV) were systematically compared in this research. The results showed that the average lengths of infective juveniles were 497.4 ± 0.09, 514.3 ± 0.08, 525.7 ± 0.09, and 556.6 ± 0.09 μm, the average widths were 24.9 ± 0.006, 25.6 ± 0.005, 26.1 ± 0.006, and 27.9 ± 0.004 μm, and the average dry weights per million infective juveniles were 49.2 ± 2.2, 58.6 ± 2.4, 59.6 ± 1.8, and 80.7 ± 1.7 mg cultured by media I, II, III, and IV, respectively. The highest relative content of fatty acid of infective juveniles was obtained from medium IV at 15.4 ± 1.2 × 10⁵μV/s, and the lowest one was 6.76 ± 0.3 × 10⁵μV/s from medium I and 11.8 ± 0.2 × 10⁵and 13.7 ± 0.3 × 10⁵μV/s from media II and III, respectively. The numbers of nematodes that moved a vertical distance of 5 cm in sand column within 48 h were 24 ± 3.6, 75 ± 11.6, 69 ± 9.7, and 92 ± 13.2 and the penetration rates into theGallerialarva within 24 h were 2.8 ± 0.45, 6.0 ± 1.14, 6.4 ± 0.74, and 6.0 ± 0.7% from media I to IV, respectively. The results indicated that the quality of entomopathogenic nematode was influenced by the cultural medium component. The animal protein, especially from insects which were presented in media II, III, and IV, has a strong positive effect on nematode quality.     

A morphological comparison of aortic elastin from five species as seen with the scanning electron microscope

The elastic laminae were extracted from thoracic aortas of adult animals including sheep, dogs, rabbits, cats and rats by treating them in hot alkaline solution (0.1 N NaOH at 75 degrees C) and observed with a scanning electron microscope. The elastic laminae are comprised of sheet-like internal elastic lamina, fibrous and membraneous elastin in tunica media, interlamellar fibers and hollow spaces which we presume were formerly filled with smooth muscle cells in the tunica media. These structures are the same in all five species except that the number of layers and the total thickness of the wall differs.     

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h⁻¹) is almost twice that of digestion without stretch (0.069±0.028 h⁻¹). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Solid-state (13)C NMR reveals effects of temperature and hydration on elastin.

Elastin is the principal protein component of the elastic fiber in vertebrate tissue. The waters of hydration in the elastic fiber are believed to play a critical role in the structure and function of this largely hydrophobic, amorphous protein. (13)C CPMAS NMR spectra are acquired for elastin samples with different hydration levels. The spectral intensities in the aliphatic region undergo significant changes as 70% of the water in hydrated elastin is removed. In addition, dramatic differences in the CPMAS spectra of hydrated, lyophilized, and partially dehydrated elastin samples over a relatively small temperature range (-20 degrees C to 37 degrees C) are observed. Results from other experiments, including (13)C T(1) and (1)H T(1 rho) measurements, direct polarization with magic-angle spinning, and static CP of the hydrated and lyophilized elastin preparations, also support the model that there is significant mobility in fully hydrated elastin. Our results support models in which water plays an integral role in the structure and proper function of elastin in vertebrate tissue.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

In vitro development of bovine embryos cultured with stem cell factor or insulin-like growth factor-I following IVF with semen of two bulls having different field fertility

The usefulness of IVF as a potential tool to evaluate the field fertility of bulls is equivocal and growth factor addition to culture media research is needed to delineate components needed for providing defined environments for embryos. The overall aim was to evaluate the in vitro development of embryos derived using a serum supplemented and serum-free production systems and semen from two bulls of different field fertility. The study was conducted to determine the combinatorial effect of stem cell factor (SCF) and/or insulin-like growth factor-I (IGF-I) in culture on subsequent embryo development in cattle. Oocytes were aspirated separately from ≥3 to <3 mm follicles to test different follicle size populations and were matured in TCM-199 supplemented with LH, FSH, estradiol and BSA (Fraction V). Matured oocytes were fertilized in BSA supplemented synthetic oviductal fluid (SOF)-IVF medium. Presumptive zygotes were cultured for 8 d (in humidified 5% CO₂ at 38.5 °C) in BSA supplemented SOF-in vitro culture (IVC) medium. SOF-IVC medium was supplemented with fetal bovine serum (4%), IGF-I (100 ng/mL), SCF (50 ng/mL) or IGF-I (100 ng/mL) + SCF (50 ng/mL). The development competence of embryos did not differ between the bulls and among the culture environments. Nevertheless, there was an effect of follicle size on cleavage rate (P < 0.05) and a greater cleavage rate resulted from oocytes aspirated from ≥3 mm follicles (71.0 ± 1.5%) compared to those collected from <3 mm follicles (64.8 ± 1.6%). The overall cleavage rate (%); blastocyst formation (%); and expanded/hatched blastocyst formation (%) were 68.2 ± 1.5 and 67.7 ± 1.7; 29.4 ± 1.4 and 28.6 ± 1.5; and 18.6 ± 1.2 and 18.5 ± 1.1, respectively, for the bull of above and below average field fertility. The results indicate that follicle size for oocyte aspiration is effective for determining IVC success and that IVF may not discriminate among bulls of different field fertility.     

Prostaglandin F2α, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: Effects of oxytocin, luteinizing hormone, prostaglandin F2α and estradiol-17β

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 × 10⁵ cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F₂α (PGF), oxytocin (OT), estradiol-17β (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 ± 66.2, 111.1 ± 37.8, 57.7 ± 15.4 and 124.3 ± 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P<0.01) than on Day 8, 14 and 18 (rmOT: 17.5 ± 2.6 versus 5.6 ± 0.7, 6.0 ± 1.4 and 3.1 ± 0.4 pg/ml; P: 138.9 ± 19.5 versus 23.2 ± 7.5, 35.4 ± 6.5 and 43.6 ± 8.1 ng/ml, respectively). Oxytocin increased (P<0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17β stimulated (P<0.05) PGF secretion on Days 8, 14 and LH increased (P<0.01) PGF production only on Day 14. Prostaglandin F₂α, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P<0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P<0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Ontogenetic changes in biochemical composition during larval and early postlarval development of Lepidophthalmus louisianensis, a ghost shrimp with abbreviated development

Changes in growth and biochemical composition during the transition from egg through zoea to decapodid in the ghost shrimp, Lepidophthalmus louisianensis (Schmitt, 1935), were documented in terms of dry weight, lipid classes, fatty acid composition, and carbon to nitrogen (C:N) ratios. Larvae of the ghost shrimp were mass-reared in the laboratory (28°C; 20‰ S) from hatching to the decapodid stage. Iatroscan lipid class analysis revealed that major lipid classes in recently produced eggs were phospholipids (80.8±1.3%) and triglycerides (16.0±1.1%), which decreased during the incubation period. Polar lipids (zoea I: 77.4±1.7%; zoea II: 77.5±2.1%; decapodid: 80.0±1.7%) and neutral lipids, of which free fatty acids (zoea I: 10.5±2.7%; zoea II: 13.1±5.2%; decapodid: 7.8±2.1%) were dominant, represented the major lipid classes in the zoeal and decapodid stages. Triglycerides were present in small amounts. The predominant fatty acids of L. louisianensis eggs, zoeae and decapodids were palmitic (16:0), stearic (18:0), eicosapentaenoic (20:5ω3), oleic (18:1ω9), and arachidonic (20:4ω6). Elemental composition of eggs, larvae, and the decapodid stage revealed conspicuous changes in the C:N ratio, with N being relatively stable during larval development but C decreasing during the decapodid stage. These data suggest independence of newly hatched L. louisianensis on external energy resources. This combined with the ability to incorporate saturated fatty acids into polar lipids provides a selective advantage for fast development of new tissue and growth, characteristic of decapod crustacean larvae with lecithotrophic development.     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.     

The Histological Components of the Phoniatrical Body-Cover Model in Minipigs of Different Ages

Pigs are models in human phoniatry. However, features of maturation and ageing have not been considered with regard to the so-called body-cover model in this species. Therefore, the glottis of “young” (2–3 months; n = 6) and “old” (4–7 years; n = 6) minipigs was investigated. Their cranial (CraF) and caudal (CauF) vocal folds were histomorphometrically and stratigraphically analysed with emphasis on their amounts of collagen structures and elastic fibres. A dense subepithelial layer (SEL) was a distinct feature of CraF and CauF of both age groups; it was spread upon the underlying loose, flexible “cover” like a fibro-elastic membrane. The “cover” was characterised by the so-called superficial layer (SL), which was distinctly loose in the “young” minipigs, but had a much denser texture in the “old” minipigs. Here, the SL was dominated by elastic fibres in the CraF, but was of mixed qualities (collagenous and elastic) in the CauF. The structural requirements for the SL’s function as a loose “cover” were thus met only in the “young” animals. A clearly demarcated intermediate layer (IL)—characterised by high amounts of elastic fibres (as in humans)—was only found in the CraF of the “young” animals. In the “old” animals, it had lost its demarcation. In the depth of the CraF of the “old” animals, many thick collagen fibre bundles were detected in a location equivalent to that of the vocal muscle in the CauF. The development of their large diameters was interpreted as part of the maturation process, thereby supporting the hypothesis of their functional importance as a component of the “body.” In the CauF, the amounts of collagen structures increased throughout the entire lamina propria, resulting in a loss of demarcated stratigraphical subdivisions in the “old” minipigs. This situation resembled that described in the vocal fold of geriatric humans.     

Lactate Oxidation for the Detection of Mitochondrial Dysfunction in Human Skin Fibroblasts

To screen fibroblasts for defects in lactate/pyruvate oxidation, cells were grown to confluence in 25-cm² flasks, rinsed, and incubated in glucose-free media containing 25 μM L-lactate and 0.1 μCi [D,L-1-¹⁴C]lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of ¹⁴CO₂ generated /mg protein/min. Fibroblasts from patients with mitochondrial or peroxisomal disorders had decreased lactate oxidation compared to the control (CON): CON, 1.9 ± 0.13 nmol/mg/min; neonatal adrenoleukodystrophy (NALD), 0.45 ± 0.01 (P < 0.001); rhizomelic chondrodysplasia punctata (RCDP), 0.13 ± 0.002 (P < 0.001); mitochondrial defect of unknown etiology (MIT), 0.77 ± 0.003 (P <0.001); pyruvate dehydrogenase (PDH) deficiency, 0.98 ± 0.02 (P < 0.001). This method is useful for screening fibroblasts for defects in lactate oxidation in patients with mitochondrial or peroxisomal disorders. Confirmation of the site of the defect may then be investigated with specific assays, e.g., PDH, in cellular homogenates: CON, 0.93 ± 0.02 nmol/mg/min; NALD, 0.55 ± 0.02; RCDP, 0.44 ± 0.02; MIT, 0.53 ± 0.03; PDH deficiency, 0.19 ± 0.02.     

Culture medium improvement for Isaria fumosorosea submerged conidia production

Submerged conidia and blastospores of the entomopathogenic fungus Isaria fumosorosea are produced in several liquid culture media. However, yields and the ecological fitness of these propagules vary according to culture media composition. In most culture media, hyphae, blastospores and submerged conidia are white but we found that in some media they develop a brown pigmentation. A dark pigment was extracted from brown-pigmented propagules and analyzed by IR spectroscopy. Adsorption bands coincided to those characteristics of melanins.Hadamard's matrices were employed in order to increase submerged conidia yields and brown pigmentation of fungal propagules. Media containing 20–30 mg/l of FeSO₄·7H₂O and 6–12 mg/l of CuSO₄·5H₂O allowed reaching the highest pigmentation (9 in a hedonic scale). A maximal concentration of submerged conidia of 1.0 (±1.2) × 10¹² cell/l was achieved after 120 h of liquid culture in a improved culture medium, containing 25 ml/l of Polyethylene glycol (MW 200), substance which enhanced submerged conidia production, reducing free mycelia or mycelial pellets formation. In the improved medium, it was estimated that more than 60% of produced biomass corresponded to submerged conidia and blastospores, while in other media, mycelia were the main product (80–97%).     

Mechanical properties of the human red blood cell membrane at −15 °C

The most common method for measuring the mechanical behavior of the human red blood cell (RBC) membrane is micropipette aspiration, because it can be used to apply both a low uniaxial stress at a small part of the membrane or high two-axial stresses to the whole membrane [E.A. Evans, R.E. Waugh, Mechano-chemical study of red cell membrane structure in situ, in: Kroc Foundation Series, vol. 13, Erythrocyte Mechanics and Blood Flow, Alan R. Liss. Inc., New York, 1980, pp. 31–56 (Chapter 3); H.J. Meiselman, Measures of blood rheology and erythrocyte mechanics, in: Kroc Foundation Series, vol. 13, Erythrocyte Mechanics and Blood Flow, Alan R. Liss. Inc., New York, 1980, pp. 75–117 (Chapter 5)]. The elastic shear moduli and area changes of the human RBC published to date were calculated by means of this technique. However, a main drawback of the method is its impracticability at subzero temperatures. Experiments at below 0 °C are of interest because it is at these temperatures that RBC lysis occurs during freezing and thawing after cryopreservation, via a mechanism that may be mechanical.A method for circumventing this limitation is deforming the cell membranes by applying an electric ac field to a supercooled suspension. In a previous study, we applied this technique to human RBCs down to −15 °C [M. Krueger, F. Thom, Deformability and stability of erythrocytes in high-frequency electric fields down to subzero temperatures, Biophys. J. 73 (1997) 2653–2666]. In this technique, the electrical dimensions must be translated into those of mechanics. We provided a formula for these calculations, which demonstrated excellent concordance with known mechanical measurements at room temperature [F. Thom, H. Gollek, Calculation of mechanical properties of human red cells based on electrically induced deformation experiments, J. Electrostat. 64 (2006) 53–61]. Using this formula, we have now calculated the shear moduli and stress–strain diagram for our deformation experiments at −15 °C and present the results below.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy

Metabolism of [³H] arachidonic acid ([³H] AA) and synthesis of prostaglandins were examined with ovine conceptuses and endometrial slices collected on various days after mating. Tissues were incubated for 24 hr with or without 5 μCi of [³H] AA and with 200 μg radioinert AA. In experiment 1, results of chromatography indicated that conceptuses collected on days 14 and 16 after mating metabolized [³H] AA to PGE₂, PGF_2α, PGFM, 6-keto-PGF_1α, and to unidentified compounds in three chromatographic regions. One of these regions (region 1) contained triglycerides. Endometrial slices metabolized only small amounts of the [³H] AA to prostaglandins. In experiment 2, results of radioimmunoassays indicated that day 14 conceptuses released somewhat similar amounts (ng/mg tissue) of PGF_2α (32.1 ± 17.9), PGFM (8.4 ± 6.2), PGE₂ (12.3 ± 7.5) and 6-keto-PGF_1α (41.4± 4.8), whereas day 16 conceptuses released more (P<.05) PGF_2α (9.0 ± 4.1) and 6-keto-PGF_1α (15.9 ± 2.7) than PGE₂ (0.9 ± 0.2) or PGFM (0.5 ± 0.08). Day 14 and 16 endometrial slices released (ng/mg tissue) more (P<.05) PGFM (3.0 ± 0.2) and 6-keto-PGF_1α (4.0 ± 0.4) than PGF_2α (0.5 ± 0.08) or PGE₂ (0.05 ± 0.02). In experiment 3, conceptuses were recovered on days 16, 20 and 24 of pregnancy and incubated with [³H] AA to determine the effects of indomethacin on [³H] AA metabolism. In general, indomethacin (Id; 4 × 10⁻⁴ M) reduced (P<.05) the percentage of total dpm recovered as prostaglandins, but Id increased the release of chromatographic region I. Experiment 4 was conducted with day 16, 20 and 24 conceptuses to evaluate the time course of metabolism of [³H] AA, and the appearance of region I and of prostaglandins. In general, the percentage of total dpm in region I increased as the percentage of dpm as [³H] AA decreased. The percentage of dpm as prostaglandins increased as the percentage of dpm in region I decreased. Prostaglandins, probably essential for embroynal survival and development, were synthesized in vitro by ovine conceptuses.     

NAD(P)H oxidase/nitric oxide interactions in peroxisome proliferator activated receptor (PPAR)α-mediated cardiovascular effects

Activation of peroxisome proliferator activated receptor (PPAR)α and its protective role in cardiovascular function has been reported but the exact mechanism(s) involved is not clear. As we have shown that PPARα ligands increased nitric oxide (NO) production and cardiovascular function is controlled by a balance between NO and free radicals, we hypothesize that PPARα activation tilts the balance between NO and free radicals and that this mechanism defines the protective effects of PPARα ligands on cardiovascular system. Systolic blood pressure (SBP) was greater in PPARα knockout (KO) mice compared with its wild type (WT) litter mates (130 ± 10 mmHg versus 107 ± 4 mmHg). l-NAME (100 mg/L p.o.), the inhibitor of NO production abolished the difference between PPARα KO and WT mice. In kidney homogenates, tissue lipid hydroperoxide generation was greater in KO mice (11.8 ± 1.4 pM/mg versus 8.3 ± 0.6 pM/mg protein). This was accompanied by a higher total NOS activity (46 ± 6%, p < 0.05) and a 3 fold greater Ca²⁺-dependent NOS activity in kidney homogenates of untreated PPARα WT compared with the KO mice. Clofibrate, a PPARα ligand, increased NOS activity in WT but not KO mice. Bezafibrate (30 mg/kg) reduced SBP in conscious rats (19 ± 4%, p < 0.05), increased urinary NO excretion (4.06 ± 0.53–7.07 ± 1.59 μM/24 h; p < 0.05) and reduced plasma 8-isoprostane level (45.8 ± 15 μM versus 31.4 ± 8 μM), and NADP(H) oxidase activity (16 ± 5%). Implantation of DOCA pellet (20 mg s.c.) in uninephrectomized mice placed on 1% NaCl drinking water increased SBP by a margin that was markedly greater in KO mice (193 ± 13 mmHg versus 130 ± 12 mmHg). In the rat, DOCA increased SBP and NAD(P)H oxidase activity and both effects were diminished by clofibrate. In addition, clofibrate reduced ET-1 production in DOCA/salt hypertensive rats. Thus, apart from inhibition of ET-1 production, PPARα activation exerts protective actions in hypertension via a mechanism that involves NO production and/or inhibition of NAD(P)H oxidase activity.