Emulsion polymerization of butyl methacrylate initiated by 2,2′-azoisobutyronitrile, 1. Kinetics and mechanism

The effect of 2,2′-azoisobutyronitrile (AIBN) on the kinetics and mechanism of emulsion polymerization of butyl methacrylate was studied in the presence of anionic emulsifier disodium dodecylphenoxybenzene disulfonate (Dowfax® 2A1) at 60°C. The ratio between the proportion of the polymerization in monomer droplets and that of the polymerization in the aqueous phase was determined for the overall initial rate of butyl methacrylate polymerization in the region of the increasing polymerization rate (interval I). Using the model of polymerization in discrete particles, the portion of the polymerization in monomer droplets with a diameter of 100 nm in the overall polymerization rate is 24,4%; the portion of the polymerization in the water phase is only 0,022% for a concentration of Dowfax® 2A1 of 5 · 10^?2 mol · dm^?3, and 60,4% and 0,054% for a Dowfax® 2A1 concentration of 1 · 10^?2 mol · dm^?3. The exponent of the emulsifier concentration in the equation for the polymerization rate is 0,56 for interval I and 0,36 for interval II; the exponent for the concentration of AIBN over the conversion range between 0 and 30% is 0,34. For the proposed reaction mechanism it is assumed that 2-cyanoisopropyl radicals, generated from AIBN in the water phase, are responsible for the initiation of polymerization in micelles swollen by monomer and in polymer/monomer particles. Polymer/monomer particles are formed also by co-precipitation of oligomer radicals, which in turn are formed by polymerization of monomer molecules present in the water phase. Polymerization within monomer droplets has no significant influence on the course of emulsion polymerization.     

Composite poly(methyl methacrylate-methacrylic acid-2-hydroxyethyl methacrylate) latex for immunoassay. The case of plasminogen

Poly(methyl methacrylate-methacrylic acid-2-hydroxyethyl methacrylate) latex (ACRYLAT) was synthesized by radical precipitation polymerization. The mass median diameter (MMD) and the geometical standard deviation (GSD) of the ACRYLAT particles were 138 nm and 1.2, respectively. The concentration of the titrable carboxylic groups in the surface layer of latex particles was equal to 8.41 × 10^-6 mol m^-2. Latex was able to bind up to 2.82 × 10^-7 mol of 1-aminopyrene per 1 m² of the surface of the latex particles due to the ionic interactions between carboxylate anions and ammonium cations of protonated 1-aminopyrene. ACRYLAT was able to immobilize covalently human serum albumin in amounts up to 0.23 mg m^-2. Aggregation of ACRYLAT with immobilized HSA, induced with specific antibodies (anti-HSA), was investigated turbidimetrically. The results indicated that in the model turbidimetric immunoassay, ACRYLAT coated with HSA can be used for the detection of anti-HSA in the goat anti-HSA serum diluted from 50 to 7000-fold. Immobilization of rabbit antibodies to plasminogen (anti-Plg) to ACRYLAT via the ε-aminocaproic acid linkers provided particles which were used for the development of the turbidimetric immunoassay for plasminogen. In this assay plasminogen could be detected in concentration ranging from 0.75 to 75μg ml^-1 in the blood plasma.     

Rapid simultaneous determination of regional blood flow and blood-brain glucose transfer in brain of rat1

A new method was developed and used in rat to measure regional and whole-brain blood flow and blood-brain glucose transfer simultaneously and in 20 s. This simple method consisted of i. v. bolus injection of labeled butanol and tracer glucose, determination of the average arterial tracer concentration and subsequent assay of cerebral tissue activity 20 s after bolus injection. The whole-brain blood flow rate averaged 129 ml (100 g)^-1 min^-1. The unidirectional blood-brain glucose transfer was twice as high as previously estimated in similar studies on rat, or 144 μmol (100 g)^-1 min^-1 at 10 mM glucose in plasma. The magnitude is sufficient to explain the high cerebral glucose consumption rates recently determined by means of the autoradiographic 2-deoxy-D-glucose method of Sokol off et al. (1977).     

Analysis of DNA single-strand breaks in human venous blood: A technique which does not require isolation of white blood cells

For DNA strand break analysis in human white blood cells, usually metrizoate-Ficoll centrifugation is used to isolate mononuclear cells. This procedure is time-consuming and requires at least 20 ml of blood per sample. Therefore, we developed a technique which does not require isolation of white blood cells prior to DNA strand break analysis by alkaline elution (direct method). The sensitivity of this new technique was compared to that of the standard method, which includes isolation of mononuclear blood cells. A statistically significant increase in sensitivity was observed using the direct method. After in vitro gamma-irradiation of venous blood, an increase in the elusion rate of 7.7 × 10⁻³ hr⁻¹/Gy was detected if mononuclear blood cells were isolated compared to 10.5 × 10⁻³ hr⁻¹/Gy with the new technique (P < 0.05). Incubation of venous blood with ethylene oxide for 1 hr caused an increase in the elution rate of 5.8 × 10⁻³ hr⁻¹/mM ethylene oxide for the standard and 12×10⁻³h⁻¹/mM for the direct method (P < 0.05). DNA single-strand breaks were detected in blood cells of 10 persons without any apparent genotoxic exposure. A mean normalized elution rate of 1.30 ± 0.38 (95% confidence interval) was detected in isolated mononuclear blood cells, and a similar mean normalized elution rate of 1.41 ± 0.50 was obtained using the direct method. The difference was not statistically significant. Five patients treated with a combination chemotherapy consisting of cyclophosphamide (750 mg/m² i.v.), doxorubicin (50 mg/m² i.v.), vincristine (1.4 mg/m² i.v.), and prednisolone (100 mg/m² p.o.) for non-Hodgkin's disease were analyzed for DNA single-strand breaks before and 16–18 hr after the application of chemotherapy. Increases in mean elution rate of 68% and 116% were detected using the standard and the direct methods, respectively. For the direct method, only 3 ml of venous blood were sufficient for analysis of one sample, compared to 25 ml needed if mononuclear cells were isolated, and about 4 hr of work per assay can be saved. Environ. Mol. Mutagen. 29:58–62, 1997 © 1997 Wiley-Liss, Inc.     

Phase transfer and characterization of poly(ε-caprolactone) and poly(L-lactide) microspheres

A method suitable for transfer of poly(ε-caprolactone) and poly(L-lactide) microspheres (synthesized by pseudoanionic dispersion polymerization of ε-caprolactone and L-lactide in heptane1,4-dioxane mixed solvent) from heptane to water was developed. This method consists of treating the microspheres with KOH-ethanol in the presence of surfactants (nonionic Triton X-405, anionic sodium dodecyl sulfate (SDS), and zwitterionic ammonium sulfobetaine-2 (ASB)). Partial hydrolysis of polyesters results in the formation of hydroxyl and carboxyl groups in the surface layer of microspheres and enhances their stability in water-based media. Minimal concentrations of surfactants, needed to obtain stable suspensions of particles, were equal to 3 × 10^-2, and 6 × 10^-2, and 3 × 10^-2 mol l^-1 for Triton X-405, SDS, and ASB, respectively. In the case of poly(ε-caprolactone) microspheres, suspensions in water were stable for all three surfactants for pH values ranging from 3 to 11. Suspensions of poly(L-lactide) were stable in the same range of pH values only for ASB. Surface charge density determined by electrophoretic mobility varied for poly(ε-caprolactone) microspheres from 2.6 × 10^-7 to 8.9 × 10^-7 mol m^-2, for particles stabilized with Triton X-405 and ASB, respectively. In the case of poly(L-lactide) microspheres, surface charge density varied from 3.9 × 10-7 (stabilizer: Triton X-405) to 7.4 × 10^-7 mol m^-2 (stabilizer: ASB). Carboxyl groups located in the surface layer of poly(L-lactide) microspheres were used for covalent immobilization of 6-aminoquinoline, a fluorophore with an amino group. Maximum surface concentration of immobilized 6-aminoquinoline was equal to 1.9 × 10^-6 mol m^-2. Poly(ε-caprolactone) microspheres transferred into water were loaded with ethyl salicylate. Loading up to 38% (w/w) was obtained.     

Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis

Summary Electron probe analysis of ultrathin cryosections with high spatial resolution was used to determinein situ the concentrations of Ca²⁺ and Mg²⁺ bound in the absence of ATP to myofilaments in the I and A-bands of skinned frog skeletal muscle. At 2.2×10^–11 m Ca²⁺ and 2.7×10^–9 m Mg²⁺, the inexchangeably bound Mg²⁺ in the I-band was equivalent to the amount of divalent cations known to be inexchangeably bound to F-actin, while the Ca²⁺ bound to the I-band was not significantly above zero. The bound Mg²⁺ in the I-band was not exchangeable with Ca²⁺ even when the skinned fibres were exposed to 10mm Ca²⁺ solution. These results clearly indicate that Mg²⁺, rather than Ca²⁺, is the divalent cation bound to F-actin in the thin filamentsin situ. In the presence of 1mm Mg²⁺, the exchangeable Ca²⁺ bound to the I-band was increased as a function of the free Ca²⁺, while that in the A-band was not significantly changed with [Ca²⁺] up to 2 × 10^–5 m, and increased to approximately 0.8 mol Ca²⁺ per mol myosin at 10^–4 m Ca²⁺. At a saturating free Ca²⁺ in Tris-Cl solution, the bound Ca²⁺ content (2–3 mol Ca²⁺ per mol troponin) of the nonoverlapping I-band was unexpectedly low; the replacement of Tris with Na⁺ enhanced Ca²⁺ binding to the level equivalent to 3–4 mol Ca²⁺ per mol troponin. The depressant effect of Tris on Ca²⁺ binding was greater in the absence of Mg²⁺. High concentrations of Tris also reduced the maximum tension induced by 10^–4 m Ca²⁺ buffered with 10mm EGTA. At 1.3×10^–7 m Ca²⁺, thought to be close to the cytoplasmic free Ca²⁺ in resting muscle, the I-band bound a significant amount of Ca²⁺: equivalent to about 1 mol Ca²⁺ per mol troponin. In rabbit myofibrils there was a significant amount (approximately 1.5 mol/mol myosin) of Ca²⁺ bound by the A-band at a free Ca²⁺ of 10^–4 m.     

Osmotic coefficients of aqueous polyelectrolyte solutions at low concentrations, 3. Dependence on macroion molecular weight

Eight sodium salts of polystyrenesulfonic acid (poly[1-(sulfophenyl)ethylene]) of different molecular weights were prepared similarly and purified in the same way. Their aqueous solutions were thus chemically equivalent, and differed only in their molecular weights, which were determined viscosimetrically. The osmotic coefficients (Φ) of the acid and its sodium, thallium, calcium and cadmium salt solutions were measured by a membrane osmometer for counterion molalities m_c = 10^?2 to 5·10^?4mol/kg, at 25°C. In the same concentration range, the activity coefficients of hydrogen counterions (γ_H) of acid solutions were calculated from electromotive force measurements and the osmotic coefficients of the acid solutions were determined by the cryoscopic method for two different molecular weights. The values obtained are presented graphically as functions of the measured average molecular weight for m_c = 10^?3 and 10^?2 mol/kg and are in good mutual agreement within experimental error. This agreement of results could be assumed to verify experimentally that the colligative properties of polystyrene-sulfonate polyelectrolytes at low concentrations are dependent on the molecular weight of the polyion. The obtained results together with some literature data, allow us to evaluate very approximately the activity coefficients of monovalent counterions (γ_c) for m_c = 10^?2 mol/kg as a function of the degree of polymerization (n) of linear oligoions or polyions, for values of n up to 10⁴.     

Double labelling of retinofugal projections in the cat: A study using anterograde transport of 3H-proline and horseradish peroxidase

Summary A new method is described for combining ³H-proline and horseradish peroxidase (HRP) as anterograde neuronal tracers. By this method the presence of both substances can be demonstrated in the same histological section. We developed this method to investigate the retinofugal projections from the two eyes in the cat. One eye was injected with ³H-proline the other with HRP. Cryostat sections of the brain were mounted on emulsion coated slides in the dark. Sections were first exposed to the emulsion for 2–3 weeks at –40° C and developed for autoradiography. Only then they were reacted for HRP-activity with tetramethylbenzidine (TMB). Keeping to this sequence autoradiographic procedures could not abolish the HRP-reaction product and silver grains and TMB can be visualized on the same slide.The wellknown projection pattern in the lateral geniculate nuclei was confirmed as a control for the new method. In the superior colliculus and in the pretectum a clear overlap of retinal terminals from the two eyes could be demonstrated for the first time.Supported by DFG-grant Ho 450/14     

Rapid steady-state analysis of blood-brain glucose transfer in rat

A new kinetic analysis of blood-brain glucose transport is described, based on a steady-state model that takes account of cerebral blood flow, mean capillary glucose concentration, and cerebral metabolic rate. The maximal rate (T_max) and half-saturation constant (K_m) of glucose transport from blood to brain were determined in rats by measuring the rate of blood-to-brain glucose transfer at different blood glucose concentrations. Each determination lasted 20 seconds. For whole-brain, T_max and K_m averaged 258±33 (S.E.) μmol (100 g)^-1 min^-1 and 5.9±1.6 (S.E.) mmol 1^-1, respectively. The regional variations were insignificant. The new approach permits kinetic parameters to be measured locally in brain in rapidly changing functional states.     

Improvement of Induction Remission Rate by Modifying the Dose of Idarubicin for Relapsed Childhood Acute Lymphoblastic Leukemia

Relapse is the major cause of treatment failure in acute lymphoblastic leukemia (ALL), yet there is no established treatment for relapsed ALL. To improve the induction remission rate, we modified the dose of idarubicin in the original Children''s Cancer Group (CCG)-1884 protocol, and retrospectively compared the results. Twenty-eight patients diagnosed with relapsed ALL received induction chemotherapy according to the CCG-1884 protocol. Complete remission (CR) rate in all patients after induction chemotherapy was 57%. The idarubicin 10 mg/m²/week group showed CR rate of 74%, compared with the 22% CR rate of the idarubicin 12.5 mg/m²/week group (p=0.010). Remission failure due to treatment-related mortality (TRM) was 44% and 5.2% in the idarubicin 12.5 mg/m²/week and 10 mg/m²/week groups, respectively (p=0.011). Overall survival (OS) and 4-yr event-free survival (EFS) were 12.8% and 10.3%, respectively. OS and 4-yr EFS were higher in the idarubicin 10 mg/m²/week group (19.3% and 15.6%) than in the 12.5 mg/m²/week group (0% and 0%). In conclusion, a modified dose of idarubicin from 12.5 mg/m²/week to 10 mg/m²/week resulted in an improved CR rate in the treatment of relapsed ALL, which was due to lower TRM. However, despite improved CR rate with modified dose of idarubicin, survival rates were unsatisfactory.     

Effect of bradykinin and histamine on the membrane voltage,ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (V _m), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37°C. The mean value of V _m was –41±0.5 mV (n=189). BK (10^–6 mol/l, n=29) and Hist (10^–5 mol/l, n= 55) induced a rapid transient hyperpolarization by 15±1 mV and 18±1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6±1 mV (BK 10^–6 mol/l) and 7±1 mV (Hist 10^–5 mol/l). The ED₅₀ was about 5×10^–8 mol/l for BK and 5×10^–7 mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K⁺ concentration from 3.6 to 30 mmol/l depolarized V _m by 8±1 mV and completely inhibited the hyperpolarizing effect of both agents (n=11). Reduction of extracellular Cl^– concentration from 145 to 30 mmol/l led to a depolarization by 2 ±1 mV (n=25). In 30 mmol/l Cl^– the depolarizations induced by BK (10^–7 mol/l) and Hist (10^–6 mol/l) were augmented to 9±2 mV (n=14) and to 10±2 mV (n=11), respectively. Ba²⁺ (5 mmol/l) depolarized V _m by 19±5 mV (n=6) and completely inhibited the hyperpolarization induced by BK (10^–6 mol/l, n=3) and reduced that of Hist (10^–5 mol/l) markedly (n=3). Preincubation with the K⁺ channel blocker charybdotoxin (1–10 nmol/l) for 3 min had no significant effect on V _m, but reduced markedly the BK(10^–6 mol/l, n=11) and Hist-(10^–5 mol/l, n=6) induced hyperpolarizations. In 10 out of 31 experiments in the cell attached nystatin patch configuration big K⁺ channels with a conductance of 247±17 pS were found. The open probability of these K⁺ channels was increased 3- to 5-fold during the hyperpolarization induced by BK (10^–7 mol/l) or Hist (10^–5 mol/l, both n= 4). In excised inside/out patches this K⁺ channel had a mean conductance of 136±8.5 pS (n=10, clamp voltage 0 mV). The channel was outwardly rectifying and its open probability was increased when Ca²⁺ on the cytosolic side was greater than 0.1 mol/l. The data indicate that BK and Hist activate a and a in hGEC. The hyperpolarization is induced by the activation of a Ca²⁺-dependent maxi K⁺ channel.     

Development of fluorescent nanoparticle-labeled lateral flow assay for the detection of nucleic acids

The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)₃ ²⁺-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)₃ ²⁺-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimethoxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when “dipstick” format was used.     

Facilitation of actin polymerization by interfilamentous factors

Summary The polymerization of actin in low ionic strength buffer at 0° C in the presence of 0.25mm Mg²⁺ was studied by viscometry, turbidity and absorbance at 232 nm. Under these conditions, significant polymerization occurred only in the viscometer and not in isotropic mixtures. The polymerization rate with 0.25mm MgCl₂, as judged from shear viscosity, was equal to or greater than that observed with 0.1mm KCl and 0.25 mm MgCl₂ at 0° C, and was characterized by a longer nucleation period. Measurements of the turbidity at 350 nm (detecting filament formation and aggregation) and the absorbance at 232 nm (detecting conformational changes of the G-F transition) showed no evidence for polymerization or nucleation in a bulk solution at 0° C when Mg²⁺ was added to 0.25mm and, furthermore, F-actin nuclei were ineffective as seeds under these conditions. However, nucleation and polymerization by these criteria could be induced by raising the temperature to 20° C. These results demonstrate the existence of narrow conditions when elongation of F-actin is dissociated from nucleation of oligomeric acceptor nuclei, even if monitored on the sub-molecular level (absorbance at 232 nm). Under these conditions, elongation appears to require anisotropic F-Actin, I.e. that filaments are ordered by laminar flow.     

Measurement of airborne rat urinary allergen in an epidemiological study

The suitability of radioallergosorbent test (RAST) inhibition to quantify occupational exposure to rat urinary aeroallergen (RUA) has been assessed. When using a constant pool of rat allergic sera, the reproducibility of the assay over 1 year was comparable to that reported for other immunoassays; at 50% RAST inhibition the inter-assay coefficient of variation (CV) was 7.0% and the intra-assay CV was 3.0%. The assay was highly specific for rat urine; mouse urine was 1100-fold less potent at inhibiting the rat urine RAST system. Significant inter-assay variation in the ‘high’ control was not due to batch variation and was relatively small when compared with the variation in RUA concentrations in the occupational environment. Measurement of workplace RUA exposure demonstrated that those directly involved in the care of rats experienced the highest RUA exposure of the nine occupational groups studied (animal technicians GM = 23.10/μg/m³) followed in decreasing order by those working with soiled litter (e.g. cage cleaners GM = 4.20 μg/m³), dead animals (e.g. post mortem GM = 1.60 μg/m³, scientists GM = 0.67 μg/m³) and rat tissue (e.g. slide production GM = 0.04μg/m³). In view of the complexity of rat allergens, RAST inhibition is an appropriate method for the quantification of occupational exposure to rats.     

NIR photothermal therapy using polyaniline nanoparticles

Developing a biocompatible and efficient photothermal coupling agent with appropriate size is a prerequisite for the development of near-infrared (NIR) light-induced photothermal therapy (PTT). In the present study, polyaniline nanoparticles (PANPs) with a size of 48.5 ± 1.5 nm were fabricated and exhibited excellent dispersibility in water by a hydrothermal method and further surface functionalization by capping with F127. The developed F127-modified PANPs (F-PANPs) had a high molar extinction coefficient of 8.95 × 10⁸ m⁻¹ cm⁻¹, and high NIR photothermal conversion efficiency of 48.5%. Furthermore, combined with NIR irradiation at 808 nm and injection of F-PANP samples, in vivo photothermal ablation of tumor with excellent treatment efficacy was achieved. In vitro transmission electron microscopy (TEM) images of cells, methyl thiazolyl tetrazolium (MTT) assay, histology, and hematology studies revealed that the F-PANPs exhibit low toxicity to living systems. Therefore, F-PANPs could be used as PTT agents for ablating cancer, and the concept of developing polyaniline-based nanoparticles can serve as a platform technology for the next generation of in vivo PTT agents.     

Does stimulation of NaCl secretion in in vitro perfused rectal gland tubules of Squalus acanthias increase membrane capacitance?

 NaCl secretion in rectal gland tubules (RGT) of Squalus acanthias requires the activation of Cl^– channels in the luminal membrane. The RGT and its mechanism of activation are an early evolutionary paradigm of exocrine secretion. The respective Cl^– channels probably resemble the shark equivalent of the cystic fibrosis transmembrane conductance regulator (CFTR). Activation of these Cl^– channels occurs via cAMP. It has been hypothesized that the activation of CFTR occurs via exocytosis or inhibited endocytosis. To examine this question directly by electrical measurements we have performed whole-cell patch-clamp analyses of in vitro perfused RGT. NaCl secretion was stimulated by a solution (Stim) containing forskolin (10 μmol/l), dibutyryl-cAMP (0.5 mmol/l) and adenosine (0.5 mmol/l). This led to the expected strong depolarization and an increase in membrane conductance (G _m). The membrane capacitance (C _m) was measured by a newly devised two-frequency synchronous detector method. It was increased by Stim significantly from 5.00±0.22 to 5.17±0.21 pF (n=50). The increase in C _m correlated with the increase in G _m with a slope of 51 fF/nS. Next the effect of furosemide (500 μmol/l) was examined in previously stimulated RGT. Furosemide was supposed to inhibit coupled Na⁺2Cl^–K⁺ uptake and to reduce cell volume but not membrane trafficking of Cl^– channels. Furosemide reduced G _m slightly (due to the fall in cytosolic Cl^– concentration) and C _m to the same extent by which Stim had increased it. Both changes were statistically significant, and the slope of ΔC _m/ΔG _m was similar to that caused by Stim. Inhibitors of microtubules or actin (colchicine, phalloidin and cytochalasin D added at 10 μmol/l to the pipette solution and dialysed for >10 min) did not alter cell voltage, G _m or C _m, nor did these inhibitors abolish the stimulatory effect of cAMP. These data suggest that the small C _m changes observed with Stim reflect a minor cell volume increase and an ”unfolding” of the plasma membrane. The present data do not support the exocytosis/endocytosis hypothesis of cAMP-mediated activation of Cl^– channels in these cells. Received: 11 March 1998 / Received after revision 15 April 1998 / Accepted: 20 April 1998     

DNA-protein crosslinking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5–7 DPC per 10¹⁰ dalton were induced in these cells by either 150 kJ/m² of sunlamp UV > 315 nm plus photoreactivating light (PRL) or 10 kJ/m² of sunlamp UV > 295 nm. The irradiated cells were then incubated for 0–24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV > 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV > 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.     

Nuclear size as a cell-kinetic marker for osteoblast differentiation

A nuclear morphometic assay for preosteoblasts is introduced as a cell-kinetic technique, applicable to routine histological preparations of mineralized tissue. Because this method is a morphological marker for osteoblast precursor cell differentiation, it provides a new dimension for determining the mechanism of osteoblast histogenesis. Osteoblast precursors of the periodontal ligament are a mixed population of progenitors, kinetically separable into two distinct groups according to nuclear size. Preosteoblasts, the immediate proliferating precursors of osteoblasts, have large nuclei (>170 μm³) and are derived from relatively undifferentiated fibroblastlike cells, which have smaller nuclei (<80 μm³). Increase in nuclear volume, during G ₁ phase of the cell cycle, is apparently a morphological manifestation of change in genomic expression. This key event in preosteoblast differentiation is related to mechanical stress/strain and may be an important rate-limiting step in osteoblast histogenesis.     

Formation of new quasi-crystalline ordered aggregates by gizzard myosin

Summary Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 ihm KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl₂ and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-Si and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2m KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg²⁺ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 him MgCl₂, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.     

Interaction between polynucleotides in aqueous NaCl solution, 4. Poly(riboadenylic acid) — poly(2-riboinosinic acid) system

Solution properties of poly(A) and poly(I) solution at a mole ratio of poly(A) to poly(I) of 1/2 were studied by means of calorimetry and a modified differential scanning calorimetry (DSC) with the help of circular dichroism (CD) and ultraviolet (UV) spectral methods. It was found that the heat of mixing, ΔH^M, depends sharply on the ionic strength. At [NaCl] < 10^?2 mol · dm^?3 ΔH^M is nearly zero. At higher ionic strength ΔH^M decreases and reaches a minimum value at [NaCl] ≈? 2 · 10^?2 mol ? dm^?3. Above this ionic strength ΔH^M increases again. This behavior is explained on the basis of the formation of a triplex poly(A) · poly(2I) and the transition of poly(I) from a disordered to an ordered structure with increasing ionic strength. The heat of formation, ΔH, of the poly(A) · poly(21) triplex was estimated to be ca ?41 kJ per mole of base triplet. Above an NaCl concentration of 2 · 10^?1 mol · dm^?3 the triplex formation may be represented by the following scheme: