Chemical degradation and morphological instabilities during focused ion beam prototyping of polymers.

Focused ion beam processing of low melting materials, such as polymers or biological samples, often leads to chemical and morphological instabilities which prevent the straight-forward application of this versatile direct-write structuring method. In this study the behaviour of different polymer classes under ion beam exposure is investigated using different patterning parameters and strategies with the aim of (i) correlating local temperatures with the polymers' chemistry and its morphological consequences; and (ii) finding a way of processing sensitive polymers with lowest chemical degradation while maintaining structuring times. It is found that during processing of polymers three temperature regimes can be observed: (1) at low temperatures all polymers investigated show stable chemical and morphological behaviour; (2) very high temperatures lead to strong chemical degradation which entails unpredictable morphologies; and (3) in the intermediate temperature regime the behaviour is found to be strongly material dependent. A detailed look reveals that polymers which rather cross-link in the proximity of the beam show stable morphologies in this intermediate regime, while polymers that rather undergo chain scission show tendencies to develop a creeping phase, where material follows the ion beam movement leading to instable and unpredictable morphologies. Finally a simple, alternative patterning strategy is suggested, which allows stable processing conditions with lowest chemical damage even for challenging polymers undergoing chain scission.

[Recurrence of a rudimentary uterine horn rupture at 25 weeks of gestation: a case report]. / Récidive d'une rupture utérine sur corne rudimentaire d'un utérus pseudo-unicorne à 25 semaines d'aménorrhée : à propos d'un cas.

Pregnancy in a rudimentary uterine horn is a rare event which can be revealed by uterine rupture. Following the fetal extraction, some authors recommend the ablation of the rudimentary horn, in order to limit the risk of uterine rupture in case of subsequent pregnancy in the same horn. We report the obstetrical outcome of a patient with a history of rudimentary uterine horn rupture the treatment of which was conservative.

A nuclear localization signal targets proteins to the retrograde transport system, thereby evading uptake into organelles in aplysia axons.

The turnover of soluble proteins in axons and terminals is effected by replacing used proteins with newly synthesized constituents from the cell body. To investigate this complex process, which is especially important during nerve regeneration, we microinjected proteins into varicosities on axons of Aplysia neurons in vitro. When human serum albumin (HSA) coupled to rhodamine (r) was injected, it initially filled the varicosity; within seconds, however, it began to accumulate in packets and by 15 min was punctate. A similar pattern was observed after injecting soluble proteins from extruded axoplasm. In contrast, when we injected rHSA covalently attached to the SV-40 nuclear localization sequence (sp), the distribution was never punctate and the rHSA-sp was retrogradely transported from the varicosity to the cell body and into the nucleus. Electron microscopy of varicosities injected with HSA-gold showed that >90% of the particles were inside vacuoles and multivesicular bodies. These organelles probably function as storage rather than degradatory sites since they did not contain acid phosphatase. In contrast, when HSAsp-colloidal gold was injected, only 25% of the particles were in organelles. Thus, HSA and resident axonal proteins can be removed from axoplasm by uptake into organelles. The presence of a nuclear localization sequence (the sp) may avoid uptake by providing access to the retrograde transport/nuclear import pathway.

[Limit values for polycyclic aromatic hydrocarbons in soil of children's playgrounds--basic criteria and recommendations]. / Richtwerte für polyzyklische aromatische Kohlenwasserstoffe im Boden von Kinderspielplätzen--Ableitungskriterien und Empfehlungen.

Elevated concentrations of polycyclic aromatic hydrocarbons (PAK) are often found in the soil of former waste disposal sites, industrial areas, etc. It is desirable and useful to determine orientation values to facilitate and unify the evaluation of contaminations under the aspects of present or planned uses of an area, health protection and decision-making on remedial measures. In the present paper we wish to draw attention to, and discuss problems resulting from, particular characteristics of PAK, e.g. the toxicological property "complete carcinogens" or the necessity of taking into account oral, inhalative and dermal exposure of children on a playground. Based on the discussion, orientation values for benzo[a]pyrene and PAK ("normal" pattern) of 0.5 mg/kg soil and 5 mg/kg soil, respectively, are recommended for top soil of vegetation-free playgrounds. In comparison, deductions carried out by other working groups are presented.

[Evaluation of wood preservative pollutants of indoor air]. / Beurteilung von Holzschutzmittelbelastungen in Innenräumen.

The evaluation of indoor pollution by wood preservatives and pesticides has been a matter of increasing interest for the past years. The present paper reviews actual knowledge. The so-called "wood preservative syndrome" is referred to. Basics and evaluation criteria concerning the investigation of external exposure (wood, dust, air) and human biomonitoring is summarized. Several substances, in particular pentachlorophenol, lindane and pyrethroids, and principle possibilities to decrease exposure are considered.

Axoplasm enriched in a protein mobilized by nerve injury induces memory-like alterations in Aplysia neurons.

Axon regeneration after injury and long-term alterations associated with learning both require protein synthesis in the neuronal cell body, but the signals that initiate these changes are largely unknown. Direct evidence that axonal injury activates molecular signals in the axon was obtained by injecting axoplasm from crushed or uncrushed nerves into somata of sensory neurons with uncrushed axons. Those injected with crush axoplasm behaved as if their axons had been crushed, exhibiting increases in both repetitive firing and spike duration, and a decrease in spike afterhyperpolarization 1 d after injection. Because similar changes occur in the same cells after learning, these data suggest that some of the long-lasting adaptive changes that occur after injury and learning may be induced by common axoplasmic signals. Since the signals in axoplasm must be conveyed to the cell soma, we have begun to test the hypothesis that at least some of these signals are proteins containing a nuclear localization signal (NLS). Axoplasmic proteins at the crush site and those that accumulated at a ligation proximal to the crush were probed with an antibody to an amino acid sequence (sp) containing a NLS that provides access to the retrograde transport/nuclear import pathway. One protein, sp97, displayed properties expected of an axonal injury signal: it responded to injury by undergoing an anterograde-to-retrograde change in movement and, when the ligation was omitted, it was transported to the cell bodies of the injured neurons.

Endogenous axoplasmic proteins and proteins containing nuclear localization signal sequences use the retrograde axonal transport/nuclear import pathway in Aplysia neurons.

When the nuclear localization signal peptide (sp) of the SV 40 large T antigen was coupled to human serum albumin (HSA), rhodaminated (r), and microinjected into axons of Aplysia neurons in vitro, the rHSA-sp was conveyed through the axon to the cell body and then into the nucleus (Ambron et al., 1992). But since rHSA-sp is an artificial construct, we needed to determine whether naturally occurring nuclear proteins use this pathway. We therefore injected calf thymus histone H-1 and Xenopus oocyte nucleoplasmin into axons. By 3 hr both were retrogradely transported and targeted into the nucleus, though histone H-1 less efficiently than rHSA-sp or nucleoplasmin. In contrast, neither rHSA, nor rHSA conjugated to a peptide with a random distribution of basic amino acids, was transported or imported. To see if proteins that use the pathway remain intact, we coupled sp to HRP. When injected into varicosities, the HRP-sp was transported/imported to the nucleus, where it was enzymatically active. A key issue was to determine whether endogenous proteins use this pathway. Consequently, axoplasm was extruded from Aplysia nerves and the proteins were fractionated by size. SDS-PAGE and Western blots showed that two fractions contained proteins that were recognized by an affinity-purified antibody to sp: fraction 3 included sp83, and fraction 4 contained sp75. In addition, these two proteins were found in nuclei isolated from neurons. To assess transport, the total proteins in the fractions were rhodaminated and injected into varicosities. Fraction 3, but not fraction 4, contained protein that was transported through the axon to the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

An 83 kDa O-GlcNAc-glycoprotein is found in the axoplasm and nucleus of Aplysia neurons.

Glycoproteins containing O-linked N-acetylglucosamine (O-GlcNAc) are present in axons of Aplysia neurons (Gabel et al., 1989) and among transcription factors and other proteins in the nucleus of eukaryotic cells (Jackson and Tjian, 1988). A recently discovered pathway in neurons transports proteins through the axon and then into the nucleus (Ambron et al., 1992). If any of the axonal O-GlcNAc glycoproteins use this pathway, then the axon and the nucleus will have these glycoproteins in common. We addressed this issue by using galactosyltransferase and UDP-3H-galactose to label and identify the glycoproteins in three regions of Aplysia neurons: axoplasm, extruded from nerves; nuclei, isolated by manual dissection of single neurons; and cytoplasm, obtained after removal of nuclei. At least 21 glycoproteins were labeled by this procedure; several, at 200, 180, 83, 76, and 66 kDa, from the nucleus and axoplasm comigrated after SDS-PAGE. Radiolabeled galactosyl-N-acetylglucosaminitol was released from the glycoproteins by base/borohydride, thereby verifying the presence of O-GlcNAc. Comparison of the 83 kDa glycoprotein from the nucleus and axoplasm revealed that both were soluble, had multiple O-GlcNAcs, and were bound to WGA. Thus, the 83 kDa constituent is a good candidate to use the axonal transport/nuclear import pathway.

Intracellular sodium activity and its regulation in guinea-pig atrial myocardium.

1. Intracellular Na+ activity (aNai) and membrane resting potential were studied in quiescent guinea-pig atrial and papillary muscles by means of Na(+)-sensitive and conventional microelectrodes. The effects of the cardioactive steroid dihydroouabain (DHO) on aiNa, force of contraction and sarcolemmal Na+, K(+)-ATPase activity were also investigated. 2. In thirty atria and twenty-two papillary muscles, aNai amounted to 8.0 +/- 0.2 and 4.7 +/- 0.3 mM, respectively (mean +/- S.E.M.). When both tissues were from the same animal, with the same ion-sensitive microelectrode mean aNai values of 7.9 +/- 0.2 and 5.1 +/- 0.5 mM (P < 0.01) were obtained from eight atrial and eight papillary muscles, respectively. 3. Membrane resting potentials (Em) were significantly (P < 0.001) more negative in the papillary muscles (-83.5 +/- 0.7 mV; n = 8) than in the atrium (-78.1 +/- 0.5 mV; n = 8). Deviation of Em from EK (determined by K(+)-sensitive microelectrodes) was 3.0 +/- 0.2 mV in ventricular (P < 0.05) and 6.1 +/- 0.3 mV in atrial preparations (P < 0.05). 4. Inhibition of the Na+ pump by DHO increased aNai of the atrium within 10 min by 0.6 +/- 0.1 (n = 7), 1.3 +/- 0.1 (n = 5) and 3.2 +/- 0.2 mM (n = 5) at 5, 10 and 30 microM, respectively. In the papillary muscle, 10 microM DHO was without effect while aNai rose by 1.0 +/- 0.1 (n = 5) and 2.9 +/- 0.2 mM (n = 6) at 30 and 120 microM DHO. 5. Consistent with the aNai measurements, the potency of DHO to increase force of the isometric contraction was three times higher in atrium than in papillary muscle (stimulation frequency 0.2 Hz). 6. Hydrolytic activity of sarcolemmal Na+,K(+)-ATPase isolated from atria amounted to only one third of that detected in ventricles (0.07 +/- 0.01, n = 6, versus 0.2 +/- 0.01 mumol phosphate released min-1 (g tissue)-1, n = 5). The inhibitory potencies of DHO on sarcolemmal Na+,K(+)-ATPase preparations were found to be identical in the enzymes from either tissue. 7. It is concluded that a lower Na+ pump density is responsible for the higher aNai and for the lower resting membrane potential in atrial as compared to ventricular cells. The regulation of cellular Na+ homeostasis in atrial muscle appears to be closer to the limits of its capacity than in ventricle, explaining the higher sensitivity of the atrium to interventions which impede Na+ pump activity.

A signal sequence mediates the retrograde transport of proteins from the axon periphery to the cell body and then into the nucleus.

The presynaptic terminal and axon of neurons can undergo structural changes in response to environmental signals. Since these changes require protein synthesis in the cell body, the needs of the periphery must somehow be communicated to the cell soma. To look for such a mechanism, we used artificial protein constructs with properties expected of a signal that is transported from the axon to the nucleus. One construct consisted of the nuclear import signal peptide (sp) of the SV40 large T antigen, coupled to human serum albumin (HSA) and rhodamine (r). When injected into the axoplasm of Aplysia californica neurons in vitro, the rHSA-sp was transported in the retrograde direction through the axon to the cell body and then into the nucleus. Little, if any, moved in the anterograde direction toward growth cones. The retrograde movement of injected rHSA-sp was rapid (greater than 25 mm/d) and depended upon intact microtubules. The sp portion of rHSA-sp provided access to both the retrograde transport system and the nuclear import apparatus. Thus, rHSA was not transported at all, but accumulated in organelles near the injection site. Also, rHSA-sp containing an sp with a Lys to Thr substitution, which is known to reduce nuclear import markedly, was transported only poorly. To look for endogenous molecules that use this system, we affinity-purified a rabbit polyclonal antibody to the signal sequence. The antibody recognized an 83 kDa polypeptide on Western blots of Aplysia nervous tissue. These data indicate that Aplysia neurons contain the machinery to convey macromolecules from the axon periphery to the nucleus.

Intracellular Na+ activity and positive inotropic effect of sulmazole in guinea pig ventricular myocardium. Comparison with a cardioactive steroid.

Recent studies suggest that inhibition of Na+,K(+)-ATPase may contribute to the positive inotropic action of the imidazopyridine sulmazole. Therefore, we investigated the effect of sulmazole and its stereoisomers and for comparison the effect of the cardioactive steroid dihydroouabain (DHO) on intracellular Na+ activity by means of Na(+)-sensitive microelectrodes. In the resting papillary muscle of the guinea pig, (+/-)-sulmazole increased intracellular Na+ activity (aiNa) within 15-20 minutes by 0.5 +/- 0.1 (n = 3), 1.3 +/- 0.1 (n = 7), 2.7 +/- 0.2 (n = 6), and 4.9 +/- 0.5 (n = 6) mM at 60, 100, 300, and 1,000 microM, respectively. (+)-Sulmazole was more effective than the racemate; aiNa was increased by 1.2 +/- 0.3, 2.1 +/- 0.3, and 4.0 +/- 0.2 mM at 60, 100, and 300 microM, respectively (n = 2 for each concentration). In the contracting papillary muscle (0.2 Hz), (+)- and (+/-)-sulmazole (600 and 1,000 microM) produced a maximum positive inotropic effect that exceeded that of DHO by 11% and 8%, respectively. As an inotropic agent, (+)-sulmazole was almost twice as potent as the racemate. The maximum direct inotropic effect of (-)-sulmazole (1,000 microM) amounted to only 14% of the DHO maximum and was, in contrast to the racemate and (+)-sulmazole, antagonized by 3 microM carbachol. (-)-Sulmazole did not affect aiNa.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic receptor stimulation and cyclic AMP-dependent effects in guinea-pig ventricular myocardium.

1. The effect of carbachol on force of contraction, contraction duration, intracellular Na+ activity and cyclic AMP content was studied in papillary muscles of the guinea-pig exposed to isoprenaline or the phosphodiesterase inhibitor 3-isobutyl, 1-methyl xanthine (IBMX). The preparations were obtained from reserpine-pretreated animals and were electrically driven at a frequency of 0.2 Hz. 2. Isoprenaline (10 nM) and IBMX (100 microM) produced comparable positive inotropic effects of 9.8 and 9.7 mN, respectively. Carbachol (3 microM) attenuated the inotropic effects by 82% (isoprenaline) and by 79% (IBMX). The shortening of contraction duration which accompanied the positive inotropic effect of isoprenaline (by 14.9%) and of IBMX (by 22.4%) was not significantly affected by 3 microM carbachol. 3. The positive inotropic effect of 10 nM isoprenaline and of 100 microM IBMX was accompanied by an increase in cellular cyclic AMP content of 58 and 114%, respectively. Carbachol (3 microM) failed to reduce significantly the elevated cyclic AMP content of muscles exposed to either isoprenaline or IBMX. 4. In the quiescent papillary muscle, isoprenaline (10 nM) and IBMX (100 microM) reduced the intracellular Na+ activity by 28 and 17%, respectively. This decline was not influenced by the additional application of 3 microM carbachol. 5. The results demonstrate that muscarinic antagonism in guinea-pig ventricular myocardium exposed to cyclic AMP-elevating drugs is restricted to force of contraction. The underlying mechanism does not apparently involve the cytosolic signal molecule cyclic AMP.

Involvement of the Golgi apparatus in sorting of materials to opposite ends of frog rod retinal photoreceptors.

We have studied the rod cells of retinas of Rana pipiens by phosphatase cytochemistry and immunocytochemistry. We find that the Golgi apparatus of these cells, although different in its intracellular distribution from that of other neurons, has a cis-trans organization like that of other neurons as regards morphological features and the distribution of phosphatase activities. Antibodies against opsin bind to several sacs of the rod Golgi apparatus, especially those at the trans side of the Golgi stack. This suggests that Golgi involvement in the packaging of opsin for eventual delivery to the photoreceptive outer segments of the cell involves passage through trans Golgi systems. Proteins destined for the opposite end of the cell--the presynaptic terminal--also seem to pass through trans Golgi systems, as is indicated both by immunocytochemical localization of the synaptic vesicle protein p38 (synaptophysin) and by the presence of thiamine pyrophosphatase activity in some of the synaptic vesicles. Our findings suggest that sorting of membrane proteins destined for opposite ends of the photoreceptor takes place in systems at or near the trans Golgi face.

Differential effects of (-)-norepinephrine and of (+/-)-isoproterenol on cardiac contraction, an uptake phenomenon.

Concentration-dependent inotropic effects of (-)-norepinephrine and of (+/-)-isoproterenol were compared in isometrically contracting guinea pig papillary muscles stimulated at a frequency of 0.2 Hz. (-)-Norepinephrine (0.1-100 mumol/l) elicited a positive inotropic effect that was not antagonized by carbachol, failed to produce a positive inotropic staircase after resumption of stimulation and shortened relaxation time in a concentration-dependent fashion.(+/-)-Isoproterenol had a dual action: at low and moderately effective concentrations (1-30 nmol/l), the positive inotropic effect was antagonized by carbachol, a positive inotropic staircase was elicited and time to peak force was shortened. At (+/-)-isoproterenol concentrations exceeding an EC80 for the positive inotropic effect (greater than 30 nmol/l), relaxation time became shorter, staircase and carbachol-induced antagonism became less pronounced until at 300 nmol/l inotropic effects of (+/-)-isoproterenol resembled closely those of (-)-norepinephrine. Cyclic AMP derivatives and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine mimicked (+/-)-isoproterenol in their inotropic actions. Impairment of neuronal uptake caused (-)-norepinephrine 1) to produce (+/-)-isoproterenol-like effects on the isometric contraction, 2) to induce a positive inotropic staircase and 3) to become sensitive to carbachol-induced antagonism. The results are compatible with the concept that neuronal uptake produces a distribution of (-)-norepinephrine within the papillary muscle which allows predominantly high concentrations of (-)-norepinephrine to become effective in the receptor compartment.(ABSTRACT TRUNCATED AT 250 WORDS)

A phosphatase activity and a synaptic vesicle antigen in multivesicular bodies of frog retinal photoreceptor terminals.

Previous work has suggested that multivesicular bodies participate in endocytosis and membrane cycling at nerve terminals, including the presynaptic terminals of retinal photoreceptors. We now have found that multivesicular bodies located in the presynaptic terminals of photoreceptors in retinae of Rana pipiens show reaction product in preparations incubated to demonstrate phosphatase activity at pH 5, using cytidine monophosphate as the substrate. Evidently, multivesicular bodies in photoreceptors can possess at least some hydrolytic enzymes during their sojourn in the terminals. We have also found that the multivesicular bodies in frog retinal photoreceptor terminal stain, immunocytochemically, for the presence of SV2, an antigen of synaptic vesicles. This observation supports the suggestion that, along with the extensive, repeated reuse of membrane components for synaptic vesicle recycling, there is some incorporation of the components into structures that are potentially degradative.

Glycosyltransferase alterations are cell type related when human promyelocytic leukemia (HL-60) cells are treated with various inducers of differentiation.

We have assayed glycosyltransferase activities during the granulocytic and macrophage-like differentiation of human promyelocytic leukemia (HL-60) cells. Functional granulocytic differentiation was assayed by the decarboxylation of 2-deoxyglucose in addition to nitroblue tetrazolium reduction. Dimethylsulfoxide (DMSO) treated HL-60 cells, induced to granulocytic differentiation, had higher 2-deoxy-glucose decarboxylation activity, and contained less sialyltransferase (ST), more fucosyltransferase (FT), and more N-acetylglucosaminyltransferase (NGT) activities than untreated cells. HL-60 cells treated with another granulocytic differentiator, retinoic acid, also had higher 2-deoxyglucose decarboxylation activity, and contained less ST, more FT, and more NGT activities than untreated cells. In contrast, cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reported to differentiate HL-60 to macrophage-like cells, but did not show an increased level of 2-deoxyglucose decarboxylation activity, but contained more galactosyltransferase (GT) and FT activities as compared to untreated cells. These findings suggest that the alterations of glycosyltransferase levels during the differentiation of precursor cells may not depend upon different inducers, but are characteristic of the phenotypic expression of the mature cell type.

Serum protein binding of drugs is not altered in patients with severe chronic cardiac failure.

The aim of this study was to investigate whether serum protein binding of drugs is altered in patients with severe chronic cardiac failure. A total of 27 patients of the cardiac unit participated in the study. One group comprised 15 subjects with chronic cardiac failure (grade III-IV according to the New York Heart Association); 12 patients served as controls (grade I-II). The extent of binding was determined in the therapeutic concentration range by means of equilibrium dialysis at pH 7.4 and 37 degrees C. The binding of six marker drugs shows no difference between controls and patients with chronic cardiac failure. Furthermore, measured free fractions were in the range reported in the literature for healthy, untreated individuals. Our selection of drugs comprised substances that are representative of the three major drug-binding sites on human albumin (diazepam-digitoxin-warfarin/phenytoin). Furthermore, propranolol and imipramine represent examples of drugs binding mainly to lipo- and glycoproteins. The results suggest that the binding of most drugs encountered in clinical practice will be unchanged in patients with chronic cardiac failure.

Characterization of galactosyltransferase released from human hepatoma cells.

A human hepatoma cell line (SK-H-MA) released a large amount of sialyltransferase (ST) and galactosyltransferase (GT) into the culture medium, whereas cells derived from normal human liver (Chang) released a large amount of GT but very little ST. The characteristics of hepatoma GT were studied since an abnormal GT isoenzyme has been associated with human gastrointestinal neoplasms. Both hepatoma and Chang medium GT activities had an absolute requirement for MnCl2 (25 mmol/l) and a broad optimal pH between 6.5 and 7.0, and were not affected by 0.1% Triton X-100. These two enzyme preparations were inhibited to the same extent by N-acetylglucosamine and N-acetylgalactosamine, while N-acetylglucosamine was 100 times more potent than N-acetylgalactosamine. Various nucleotides inhibited both enzyme activities equally well. Uracil-containing nucleotides were better inhibitors than thymine-containing nucleotides, and other nucleotides were only slightly inhibitory. The most effective inhibitor was UDP. More of the GT activity in hepatoma medium (65%) as compared to Chang medium (35%) bound to concanavalin A-Sepharose, and was eluted with 2.5% alpha-methylmannoside. These results suggest that the GTs from hepatoma and Chang media are not different in their enzymatic activity but may differ in their carbohydrate contents, which may be another manifestation of the neoplastic nature of the hepatoma cell line.