Staining and relative determination of elemental contents by the laser microprobe

Summary An improved laser microprobe procedure is developed and applied to the measurement of calcium content in microareas of right and left subepicardial muscles. The staining of canine cardiac muscles by Methylene Blue solution (1% w/v) was found to improve sampling efficiency. Elemental content is proportional to T ^–1/, where T is the transmittance of the characteristic emission line of the element of the photographic plate and is its contrast. In the present system, the calcium content is analyzed using T ^–3.2. We find that the staining of samples and the determination of T ^–1/ are useful procedures in the application of laser microprobe to the study of elemental content in biologic microareas.     

Redistribution of cell membrane probes following contraction-induced injury of mouse soleus muscle

Our aim was to study how mouse skeletal muscle membranes are altered by eccentric and isometric contractions. A fluorescent dialkyl carbocyanine dye (DiOC₁₈(3)) was used to label muscle membranes, and the membranes accessible to the dye were observed by confocal laser scanning microscopy. Experiments were done on normal mouse soleus muscles and soleus muscles injured by 20 eccentric or 20 isometric contractions. Longitudinal optical sections of control muscle fibers revealed DiOC₁₈(3) staining of the plasmalemma and regularly spaced transverse bands corresponding in location to the T-tubular system. Transverse optical sections showed an extensive reticular network with the DiOC₁₈(3) staining. Injured muscle fibers showed distinctively different staining patterns in both longitudinal and transverse optical sections. Longitudinal optical sections of the injured fibers revealed staining in a longitudinally-oriented pattern. No correlations were found between the abnormal DiOC₁₈(3) staining and the reductions in maximal isometric tetanic force or release of lactate dehydrogenase (P0.32). Additionally, no difference in the extent of abnormal staining was found between muscles performing eccentric contractions and those performing the less damaging isometric contractions. However, many fibers in muscles injured by eccentric contractions showed swollen regions with marked loss of membrane integrity and an elevated free cytosolic calcium concentration as observed in Fluo-3 images. In conclusion, a loss of cell membrane integrity results from contractile activity, enabling DiOC₁₈(3) staining of internal membranes. The resulting staining pattern is striking and fibers with damaged cell membranes are easily distinguished from uninjured ones.     

Distribution of calcium in a subset of chronic hibernating myocardium in man

Summary The structural correlates of chronic hibernating myocardium in man consist of myocardial cells which transformed from a functional state (rich in contractile material) to a surviving state (poor in contractile material, rich in glycogen). Since the calcium-handling organelles such as SR, sarcolemma and mitochondria underwent structural changes in cells so affected, the distribution of calcium was investigated in biopsies obtained from hibernating areas. The material was processed for microscopic localization of total calcium (laser microprobe mass analysis, LAMMA) and of exchangeable calcium (phosphate-pyroantimonate precipitation method, PPA). Subcellular distribution of total calcium as assessed by LAMMA revealed that in the structurally affected cells the areas in which sarcomeres were replaced by glycogen contained significantly more calcium than all other areas probed such as mitochondria, remaining sarcomeres at the cell periphery and subcellular areas of normally structured cells. Calcium precipitate, obtained after PPA assessment, was localized at the sarcolemma but was virtually absent in the mitochondria of affected cells. The high calcium content in the myolytic areas of affected cells most probably belongs to a pool of bound calcium. The observations that calcium is retained at the sarcolemma and that mitochondria are devoid of precipitate favour the hypothesis that cells structurally affected as such are not ischaemic and are still able to regulate their calcium homeostasis.     

The use of a laser microprobe to identify calcium oxalate in histological material

Summary Some birefringent renal deposits were found to give the usual histological reactions for calcium oxalate but were soluble in caustic alkalis. Comparison with known oxalate crystals using a laser microprobe mass analyser confirmed the presence of calcium oxalate. Similar crystals were found in liver tissue from a rat poisoned with ethylene glycol.     

Comparative study of inhibitory effects by murine interferon γ and a new bisphosphonate (alendronate) in hypercalcemic,nude mice bearing human tumor (LJC-1-JCK)

The inhibitory effect of murine interferon (muIFN) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), muIFN was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.     

Heterogeneity in the globin chain composition of a human minor fetal hemoglobin component

The first hemoglobin found to contain an acetyl blocking group was the minor human fetal hemoglobin, Hb FI, present as 10-15% of the total fetal hemoglobin in umbilical cord blood red cells. Acetylation occurs at the amino-terminal glycine of the gamma-globin chain. Assays for the acetyl group by two different methods gave values less than the 2 per tetramer expected for a fully acetylated hemoglobin. We have purified acetylated fetal hemoglobin FIc to homogeneity. The globin chain composition of Hb FIc has been examined by both globin chain separation on CM-cellulose and by tryptic peptide mapping by HPLC. The identities of the gamma globin chains and of the gamma T-1 peptides were confirmed by amino acid analysis. Globin chain separation profiles showed the presence of 22.3 +/- 7.0% of gamma 0 globin (of the total gamma globin) in Hb FIc. Accordingly, the tryptic peptide maps of Hb FIc tetramers also showed the presence of a similar amount of gamma 0T-1 peptide. The gamma 0T-1 peptide was not present in the maps of isolated gamma Ic globin. It is evident that column purified Hb FIc contains a certain percentage of non-acetylated gamma-globin chains, thus indicating a hybrid globin chain composition for this minor fetal hemoglobin component.     

Calcium binding capacity and calcium sensitive protein content in denervated frog muscles

Total and ionic calcium content, calcium binding capacity of sarcoplasmic proteins and calcium insensitive proteins were examined in atrophying leg muscles of frog after 1-5 months period of denervation. Different muscles showed different levels of atrophy and the total calcium content varied with reference to the type of muscle. Ionic calcium levels doubled in the gastrocnemius muscle after three months denervation. Calcium binding capacity of proteins and calcium insensitive proteins decreased rapidly up to four months after denervation in the gastrocnemius muscle. However no significant changes in the levels of calcium binding capacity and calcium insensitive proteins were found with reference to the type of muscle. Since total calcium content remains constant and wet muscle mass (expressed as atrophy) decreased markedly, an apparent increase in calcium concentration occurs in each muscle on denervation.     

UNTERSUCHUNG DES CHEMISCHEN AUFBAUS VON MITTELKAMBRISCHEN OSTRACODENSCHALEN

A microprobe investigation demonstrated that the shell substance of archaeocope ostracodes consists mainly of calcium phosphate and contains subordinate quantities of iron and carbonaceous matter. The relative content of calcium phosphate increases towards the outer surface of the shell; the content of carbonaceous matter decreases in this direction.     

Laminin gamma3 chain binds to nidogen and is located in murine basement membranes

Recently a novel laminin gamma3 chain was identified in mouse and human and shown to have the same modular structure as the laminin gamma1 chain. We expressed two fragments of the gamma3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The gamma3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the beta2 VI/V fragment. The gamma3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the gamma1 III3-5 fragment. These data suggested that laminins containing the gamma3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against gamma3 VI/V and gamma3 III3-5 showed no cross-reaction with homologous fragments from the gamma1 and gamma2 chains of laminin and allowed the establishment of gamma chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the gamma3 chain compared with the gamma1 chain in various tissue extracts of adult mice. The expression of gamma3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the gamma3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the gamma3 chain to basement membranes of these tissues.     

Dopamine Release Evoked by Beta Scorpion Toxin,Tityus Gamma,in Prefrontal Cortical Slices is Mediated by Intracellular Calcium Stores

1. We have investigated the effect of tityus gamma (TiTX ) scorpion toxin on the release of [³H] dopamine in rat brain prefrontal cortical slices. The stimulatory effect of TiTX on the release of [³H]dopamine was dose/time-dependent with an EC₅₀ of 0.01M.2. Tetrodotoxin blocked the TiTX -induced release of [³H]dopamine, indicating the dependency for Na⁺ channels.3. EGTA had no effect on the TiTX -induced release of [³H]dopamine, indicating the process is independent of extracellular calcium. Release of [³H]dopamine evoked by TiTX was inhibited by 57% by BAPTA, a chelator of intracellular calcium.4. Xestospongin and 2-APB, putative blockers of IP₃-sensitive release of intracellular calcium stores, caused an equal and significant inhibition of 24% of the TiTX -induced release of [³H]dopamine, while the slight inhibition evoked by dantrolene, a putative blocker of ryanodine-sensitive calcium store was not significant.5. Nomifensine and ascorbic acid, blockers of dopamine transporter (DAT), caused an inhibition of 27 and 29%, respectively, on the toxin-induced release of [³H]dopamine suggesting that most of the TiTX -induced release of dopamine is not due to the reversal of Na⁺ gradient.6. In conclusion the majority of the TiTX -induced release of [³H]dopamine is exocytotic and mobilizes calcium from the intracellular IP₃-sensitive calcium stores.     

Orai and TRP channels in skeletal muscle cells

Modern data concerning expression, localization, biophysical properties, involvement in calcium regulation, and physiological functions of TRP and Orai channels in skeletal muscle cells are analyzed. In skeletal muscles TRPC1/2/3/4/5/6/7, TRPV2/4, TRPM2/7 and Orai1/3 channels are expressed. Activities of TRPC1/3 and TRPV4 facilitate maximal muscle contraction during tetanus. Orai1 channels provide recovery of intracellular calcium stores and are obligatory for proliferation of myoblasts and differentiation of skeletal muscles. TRPC1 knockout results in alterations of the development of skeletal muscles. Enhanced calcium influx via the channels is supposed to be a pathogenic factor of myodystrophy.     

Ultrastructural localization of acid protein polysaccharides and calcium in the vacuoles of the chicken thrombocyte.

The coexistence of acid protein polysaccharides and calcium in the vacuoles of chicken thrombocytes were studied by means of ultrahistochemical methods and electron probe X-ray microanalysis. The thrombocytes possessed large vacuoles of a surface connected membrane system. After both ruthenium red staining and tannic acid fixation the innersurface coat of vacuoles was always strongly and continously visualized. Electron microscopic X-ray microprobe analysis of antimonate precipitates in thrombocytes fixed in K-antimonate-OsO4 revealed calcium localization on the innersurface of vacuoles. From these facts it seems likely that the vacuoles of the surface connected membrane system may take part in the pool or the transport of calcium.     

Enhanced inactivation of salmonella and pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log(10) units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.     

Calcium signalling in the regulation of PGC-1alpha, PDK4 and HKII mRNA expression

The role of calcium signalling and specific intracellular calcium signalling pathways in regulating skeletal muscle tissue peroxisome proliferator-activated receptor gamma co-activator (PGC)-1alpha, hexokinase (HK)II and pyruvate dehydrogenase kinase (PDK)4 mRNA was examined. Cultured primary rat skeletal muscle cells were incubated for 6 h in caffeine or ionomycin. Because PGC-1alpha mRNA clearly showed greater induction with ionomycin, the latter was chosen for the main experiments, whereby cells were incubated for 6 h with either ionomycin alone or in combination with either cyclosporin A or KN-62. The PGC-1alpha mRNA level was increased (p<0.05) approximately six-fold and HKII mRNA content approximately two-fold by ionomycin relative to the corresponding controls, whereas the PDK4 mRNA content remained unaffected. Cyclosporin A abolished (p<0.05) and KN-62 reduced (p<0.1) the ionomycin-induced increase in PGC-1alpha mRNA. Electrical stimulation of in vitro incubated rat EDL muscle increased (p<0.05) PGC-1alpha mRNA by 2.2-fold after 4 h of recovery relative to a resting control, and this increase was absent when muscles were incubated with KN-62 or cyclosporin A. The present data strongly suggest that calcium signalling is involved in regulating the PGC-1alpha and HKII genes, but not PDK4. Both calcineurin and CaMK signalling seem to be involved in the calcium- and contraction-mediated PGC-1alpha up-regulation in skeletal muscle.     

γ-Tubulin in tobacco pollen tubes: association with generative cell and vegetative microtubules

-Tubulin was localized in tobacco pollen tubes using an antibody raised against a peptide conserved in all known -tubulins. Antibody staining occurs in a primarily punctate pattern along the length of the microtubule bundles in generative cells and along cortical microtubules in the vegetative cytoplasm. During generative cell division, -tubulin is localized in the forming mitotic apparatus. By metaphase, it is present along kinetochore fibers except at their plus ends located at the kinetochores. By telophase, staining is observed in the phragmoplast, where it again avoids the plus ends of microtubules at the cell plate. -Tubulin is also present at the periphery of the sperm nuclei. A patch of intense staining on the distal side of each nucleus marks the site of assembly of a new population of sperm microtubules. No specific fluorescence is present in control pollen tubes treated with preimmune IgG. These localization patterns bear similarities to those seen in somatic cells and in addition may help explain changes in microtubule arrays between generative cells and sperm.     

Ultrastructural localization of potassium and calcium in an insect ommatidium as demonstrated by X-ray microanalysis

Summary Ultrastructural localization of potassium and calcium in the ommatidium of the house-cricketGryllus domeslicus L. was studied by X-ray microprobe analysis using samples prepared as thin sections (2 or 5 m) of freeze-dried and embedded tissue. Real resolution was limited by the size of ice crystals (Fig. 2) and estimated as about 1 m.Average values for potassium, calcium, sodium and phosphorus in different cells of the compound eye are given in Table 1.Striking non-uniformity in distribution of these elements over the cells and their compartments was found by probe scanning (Figs. 3, 4, 5). The highest potassium and calcium concentrations were measured in the pigmented zones of photoreceptors and pigment cells. The pigment granules are thought to be the ionic depots of the eye.Potassium and sodium are fully accessible to water in sections of embedded tissue, whereas all the calcium and half of the phosphorus are not.The functional significance of the non-uniformity discovered is briefly discussed.     

Acetylcholine receptor in a C2 muscle cell variant is retained in the endoplasmic reticulum

We have examined the properties and intracellular localization of acetylcholine receptors in the C2 muscle cell line and in a variant (T-) that accumulates AChR intracellularly. On immunoblots, the subunit structures of the AChR from wild-type and T- cells were similar except that the gamma and delta subunits of the variant AChR had altered mobilities. Digestion with endoglycosidases H and F demonstrated that this difference results from a failure of high-mannose N-linked oligosaccharides on AChR subunits to be processed to complex forms in the variant. N-linked glycosylation of other proteins in the variant was normal. When examined by immunocytochemistry, the distribution of internal AChR in wild-type cells was consistent with a location both in the endoplasmic reticulum and in the Golgi. Variant cells, however, showed no evidence of Golgi staining. Subcellular fractionation experiments also demonstrated AChR in the Golgi fractions of wild-type cells, but not in those derived from T- cells. We conclude that in T- myotubes most of the AChR fails to be transported out of the endoplasmic reticulum.