On the stainability of plant cell walls with alcian blue

This work is a continuation of a communication on the stainability of broad bean (Vicia faba L.) root tip cells with alcian blue, published some time ago. Following the standard method of staining with alcian blue, the cell walls are very strongly stained, the nuclei (except nucleolus) lightly, the nucleolus and cytoplasm are practically colourless. The weak dyeing of the nucleus is not equal throughout the whole section so that the comparison of stainability of cell walls and nuclei by itself cannot explain the staining with alcian blue. The results of this work on the staining of cell walls (if not including model experiments and experiments in vitro, which are not considered as decisive here) can be summarized as follows: the pH dependence of staining, the loss of stainability as a result of pectinase digestion, blocking of staining by methylation and regeneration of stainability by demethylation and, finally, the impossibility of staining in the presence of NaCl lead to the conclusion that the staining of the material studied in this work is primarily caused by the salt linkage of alcian blue with the free carboxyls of pectic substances. From the comparison of staining with alcian blue and with other basic dyes it follows that in the case of alcian blue some other factors may also take part and are the reason for the selectivity and firmness (fastness) of the staining of cell walls with this dye. Otherwise, the staining of plant cell walls with alcian blue corresponds quite well to the staining of carboxyls containing polysaccharides of animal tissues with this dye. By staining with alcian blue it was found impossible to distinguish between younger and older cell walls within the meristem. However, this staining is suitable for routine use when studying the meristematic tissue. It is often possible to use solutions of a higher pH than generally used.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

Alkali burns of the rabbit cornea. II. A histochemical study of glycosaminoglycans.

In alkali burned rabbit cornea the stainability of glycosaminoglycans in cold microtome setions was investigated. Staining by Alcian blue in 3% acetic acid, Alcian blue in various MgCl2 concentration and toluidine blue (pH 4.5) was employed. From the 1st to the 4th experimental day the intensity of reactions was decreased. This is most probably due to an increased hydration of the corneal stroma. On the 7th day hydration was markedly suppressed and reached nearly the normal level. In this time interval a decreased stainability of glycosaminoglycans was seen accompanied by a complete loss of staining in the marginal zone. On the 14th day the stainability in the traumatized area began to restore and in the marginal zone appeared. On the 32nd day the staining intensity of both areas was normalised, however when lower concentrations of MgCl2 were used; in the presence of higher concentrations of MgCl2 the decreased staining intensity persisted and points to a lower sulfatation of glycosaminoglycans. This was particularly remarkable in the area bordering the injured zone. This decrease runs parallel to the increased activities of acid glycosidases (especially of acid beta-galactosidase) which were reported previously.     

Histological staining as a measure of stress in collagen fibers

It has been reported previously that collagen fibers will stain either red or green by Masson's and other trichrome methods depending on whether they have been respectively stressed or relaxed prior to fixation. This was shown in skin [1, 2, 3] tendon [4, 5] bone [6] and films of collagen [7]. If this stain-stress dependence is of a unique quantitative nature, then staining could be used as a tension probe for collagen fibers. Relaxed and stressed collagen bundles of rat tail tendon and rat Achilles tendon have been stained using various staining periods, and results indicate that the change in staining may be associated with denser packing of the fibers in the bundle under stress rather than directly due to the stress itself. Denser packing may reduce the rate of penetration of the counterstain thus causing the staining differences. Since this rate of penetration is dependent on a number of other variables (unrelated to stress), it is concluded that collagen staining is not a reliable tension probe.     

Denervation does not change the ratio of collagen I and collagen III mRNA in the extracellular matrix of muscle

Denervation or inactivity is known to decrease the mass and alter the phenotype of muscle and the mechanics of tendon. It has been proposed that a shift in the collagen of the extracellular matrix (ECM) of the muscle, increasing type III and decreasing type I collagen, may be partially responsible for the observed changes. We directly investigated this hypothesis using quantitative real-time PCR on muscles and tendons that had been denervated for 5 wk. Five weeks of denervation resulted in a 2.91-fold increase in collagen concentration but no change in the content of collagen in the muscle, whereas in the tendon there was no change in either the concentration or content of collagen. The expression of collagen I, collagen III, and lysyl oxidase mRNA in the ECM of muscle decreased (76 +/- 1.6%, 73 +/- 2.3%, and 83 +/- 3.2%, respectively) after 5 wk of denervation. Staining with picrosirius red confirmed the earlier observation of a change in staining color from red to green. Taken with the observed equivalent decreases in collagen I and III mRNA, this suggests that there was a change in orientation of the ECM of muscle becoming more aligned with the axis of the muscle fibers and no change in collagen type. The change in collagen orientation may serve to protect the smaller muscle fibers from damage by increasing the stiffness of the ECM and may partly explain why the region of the tendon closest to the muscle becomes stiffer after inactivity.     

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phytochrome A regulates the intracellular distribution of phototropin 1-green fluorescent protein in Arabidopsis thaliana

It has been known for decades that red light pretreatment has complex effects on subsequent phototropic sensitivity of etiolated seedlings. Here, we demonstrate that brief pulses of red light given 2 h prior to phototropic induction by low fluence rates of blue light prevent the blue light-induced loss of green fluorescent protein-tagged phototropin 1 (PHOT1-GFP) from the plasma membrane of cortical cells of transgenic seedlings of Arabidopsis thaliana expressing PHOT1-GFP in a phot1-5 null mutant background. This red light effect is mediated by phytochrome A and requires approximately 2 h in the dark at room temperature to go to completion. It is fully far red reversible and shows escape from photoreversibility following 30 min of subsequent darkness. Red light-induced inhibition of blue light-inducible changes in the subcellular distribution of PHOT1-GFP is only observed in rapidly elongating regions of the hypocotyl. It is absent in hook tissues and in mature cells below the elongation zone. We hypothesize that red light-induced retention of the PHOT1-GFP on the plasma membrane may account for the red light-induced increase in phototropic sensitivity to low fluence rates of blue light.     

ANTIGEN-INDUCED CHANGES IN LYMPHOID CELL HISTONES : III. In Vitro and in Extract

Previous papers in this series have reported an acute, transitory effect of antigens on lymphoid cell nuclei. In the previous reports the effect was related to a change in ammoniacal silver (A-S) stainability of smears and cryostat sections. The variable substrate was identified as histone. This paper reports the results of an extended series of studies of histone and chromatin extracts from thymus glands exposed to antigen in vivo and in vitro. The antigen effect on A-S stainability is demonstrable not only in vitro but also in chromatin fibers representing a DNA-histone complex. However, it is not demonstrable in isolated histone fractions. The inference is drawn that the antigen-induced alteration in A-S stainability is brought about not by any quantitative change in histone, but by a biologically significant shift in histone binding, perhaps to DNA. It is suggested that alteration in DNA-histone binding during gene activation may alter A-S stainability of histones.     

Increased attachment and confluence of skin epidermal cells in culture induced by ascorbic acid: detection by permeation of trypan blue across cultured cell layers

In culture epidermal cells from the skin of newborn rats became attached to Millipore filters coated with type IV collagen much better than to filters coated with type I collagen. Ascorbic acid markedly increased the attachment and viability of epidermal cells seeded on type I collagen, but had no significant effect on cells seeded on type IV collagen. It was also found to enhance the synthesis of type IV collagen by the cells, which, we concluded, enabled the cells to become well attached to type I collagen. This conclusion was supported by studies on the penetration of trypan blue through the cell layers. There was a lag in penetration through cell layers cultured both with and without ascorbic acid on Millipore filters coated with either type I or IV collagen, indicating that the cells were confluent over the whole surface of the filters. The lag was much longer in the cultures with ascorbic acid, indicating greater confluence and tighter attachment of cells due to production of type IV collagen. The penetration was found to be due to destruction of the confluent cell layers by its cytotoxic effect. The time lag before penetration of trypan blue is a good index of the confluence and attachment of cultured cells to collagen layers.     

Changes in volume of the rhabdom in the compound eye of Aedes aegypti L.

The volume of the rhabdom in compound eyes of mosquitoes decreases upon illumination. This decrease is probably mediated by a bleaching of the visual pigment, since blue light is most effective in producing the change and red light is least effective. The reduction in rhabdom volume appears to be a result of rhabdomal membrane loss to coated vesicles and multivesicular bodies. These organelles were seen most frequently in blue adapted eyes, markedly less frequently in red adapted eyes, and only rarely in dark adapted eyes.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin-stretching device

A controlled, quantitative histochemical study was performed in five piglets to establish changes in undermined and not undermined stretched skin. The skin was stretched with a stretching device for 30 minutes to close a large skin defect. On each flank of the piglet, at a standard position, 9 x 9-cm wounds were created under general anesthesia. On one flank, the surrounding skin was undermined cranially and caudally over a 10-centimeter area. Sections of skin biopsies obtained during stretching were stained with picrosirius red and studied with routine light microscopy and polarized light microscopy in combination with image analysis. The length of collagen fibers was analyzed as a parameter of changes in the dermis resulting from skin stretching. This newly developed quantitative method appeared to be valid, specific, and reproducible, allowing for objective determination of changes in the length of the fibers in the plain of the sections. Changes in the orientation of collagen fibers in the dermis as a result of skin stretching were thereby determined. Epidermal thickness did not change significantly under the influence of stretching forces in both undermined and not undermined skin. However, the orientation of the collagen fibers changed significantly as a result of skin stretching. In undermined wounds, parallel alignment and elongation of the fibers in the plane of the sections was already observed after 15 minutes of stretching. The fibers became aligned in the direction of the stretching force, perpendicular to the wound margin. After 30 minutes of stretching, the mean major axes of the collagen fibers were longest in the plane of the sections (p < 0.001). This meant that elongation and parallel alignment of the collagen fibers had occurred. Stretching of not undermined skin for 15 minutes resulted in significantly stronger parallel alignment in the plane of the sections as compared with undermined skin. This was less well defined after 30 minutes of stretching in not undermined skin. It is concluded that skin stretching with a skin-stretching device for 30 minutes results in significant histomorphological changes of collagen fibers in the dermis of both undermined and not undermined skin. The fibers realign rapidly as a result of stretching forces and become aligned in the direction of the stretching force, perpendicular to the wound margin. These dynamic changes in collagen fibers explain the significantly decreased wound closing tension resulting from skin stretching and explain how skin stretches beyond its inherent extensibility.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

An improved staining method for intervertebral disc tissue

The objective of this study was to design a new staining procedure for human disc tissue for visualizing both collagen and proteoglycan-matrix components on the same histology section. Weigert's hematoxylin, alcian blue and picrosirius red were combined to produce distinctive staining of collagen (red), proteoglycans (blue) and cellular elements of the intervertebral disc. This novel stain reveals sharp details of collagen composition in the perilacunar, territorial and intraterritorial extracellular matrix, and concomitantly demonstrates the presence of proteoglycan accumulations around cells in the lacunar spaces and in the extracellular matrix. These details reveal variations within the tissue that would not be apparent with routine stains.     

Skin Collagen and Thickness in Simple Obesity

The effects of stretching of the skin on its collagen content and thickness have been studied in a group of subjects with chronic obesity. Despite the increase in skin surface a normal skin thickness, collagen content, and density were maintained. It is concluded that the skin stretching induced by prolonged obesity led to hypertrophy of collagen and that this had maintained both skin thickness and collagen content. It is not known whether this is due to enhanced synthesis or decreased degradation.     

Photocontrol of Chlorophyll Loss in Papaya Leaf Discs

Both red and blue light pulses are separately shown to retarddark-stimulated chlorophyll loss of papaya leaf discs suggestingparticipation of phytochrome and blue light photoreceptors inregulating the pigment loss. The red light effect is fully reversibleby far-red light. The partial failure of far-red pulses to reversethe action of blue light suggests that blue light effect maynot be entirely through the phytochrome action. The apparentineffectiveness of continuous white light to check the chlorophyllloss is attributed to a balance of photooxidation and photoprotectionof the pigment. The interaction of blue light and kinetin at its different concentrationssuggests that the effect of interactions is additive. The bluelight effect in retarding chlorophyll loss is partly independentof the hormone level. (Received December 10, 1985; Accepted August 25, 1986)     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

Kinetics of Initiation of Germination of Bacillus pumilus Spores by Hydrostatic Pressure

The kinetics of initiation of germination and inactivation by hydrostatic pressure of phosphate-buffered Bacillus pumilus spores is shown to be a consecutive first-order process at 25 C. The effect of increasing pressure at constant temperature was studied, and rate constants were derived by using the criteria of heat resistance, refractility, and stainability. The calculated volume change of activation (DeltaVdouble dagger) was -139 +/- 6 cm(3)/mole for loss of heat resistance, -158 +/- 8 cm(3)/mole for the loss of refractility, and -153 +/- 4 cm(3)/mole for the change in permeability to dilute stains for the pressure range 800 to 1,010 atm at 25 C. It is suggested that the spore exists as a Donnan phase and that pressure triggers germination by influencing the equilibrium.     

A study of the effect of low-intensity laser radiation of the blue, green, and red spectral regions on the healing of experimental skin wounds in rats

The effect of low-intensity laser radiation of the blue (441.2 nm), green (532 nm), and red (632.8 nm) spectral regions on the healing of experimental skin wounds in rats has been studied. The effect of the traditionally applied laser radiation in the red region has been compared with the effect of laser radiation in the other spectral regions, assuming that, upon irradiation of wounds by lasers emitting in the blue and green regions, a similar effect can be achieved at lower doses. The following parameters characterizing the healing of experimental wounds were used: the functional activity of phagocytes of wound exudates, which was determined by luminol-dependent chemiluminescence, and their number; the antioxidant activity of wound exudates; and the rate of healing, which was determined as a change in the wound area. It was shown that irradiation with laser accelerated the healing of wounds in all cases. The exposure to laser radiations in the red (1.5 J/cm), blue, and green (0.75 J/cm2) spectral regions shortened the time of wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes after the exposure increased on day 5 after the infliction of the wound, whereas in the control it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after the operation. A maximum increase in the superoxide dismutase activity occurred after the exposure to laser radiation in the red region at a dose of 1.5 J/cm and in the blue and green spectral regions at a dose of 0.75 J/cm2.     

AT2-AT3-profiling: a new look at synonymous codon usage

The teleology of synonymous codon usage (SCU) still awaits a unifying concept. Here the 2nd codon letter of human mRNA-codons was graphically, aided by a computer program, put in relation to the 3rd codon letter, the carrier of SCU: AT2, the density of A+T in 2nd codon position, behaves to AT3, the analogous density of the 3rd codon position, mostly in an inverse fashion that can be expressed as typical figures: mRNAs with an overall AT-density below 50% have a tendency to produce bulky figures called "red dragons" (when redness is attributed to graph-areas, where AT3< AT2), while mRNAs with an AT-density above 50% produce a pattern called "harlequin" consisting of alternating red and blue (blueness, in analogy, when AT3>AT2) diamonds. With more diversion of AT3 from AT2, the harlequin patterns can assume the pattern of a "blue dragon". By analysing the mRNA of known proteins, these patterns can be correlated with certain functional regions: proteins with multiple transmembrane passages show bulky "red dragons", structural proteins with a high glycine- and proline content such as collagen result in "blue dragons". Non-coding mRNAs tend to show a balance between AT2 and AT3 and hence "harlequin patterns". Signal peptides usually code red due to a low AT3 with an AT2-density at the expectance level. With this technique DNA-sequences of as yet unknown functional meaning were scanned. When stretches of harlequin patterns appear interrupted by red or blue dragons, closer scrutiny of these stretches can reveal ORFs which deserve to be looked at more closely for their protein-informational content. At least in humans, SCU appears to follow protein-dependent AT2-density in a reciprocal fashion and does not seem to serve the purpose of influencing mRNA secondary structure which is discussed in depth.