Molecular cloning of cDNA for lysenin,a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle

Lysenin, which causes contraction of rat vascular smooth muscle, is a protein that was isolated from the earthworm Eisenia foetida. A cDNA encoding lysenin was isolated by use of a partial cDNA probe that had been generated by the PCR with a primer designed by reference to an internal peptide sequence of lysenin. This clone had an ORF encoding 297 amino acid residues. The amino acid sequence deduced from the cDNA revealed the absence of any significant homology to those of previously characterized vasoactive substances. The recombinant lysenin was produced in Escherichia coli. This protein and native lysenin isolated from the earthworm had similar contractive activities when tested on rat aorta. Northern blot analysis of the RNA from various tissues of the earthworm indicated that lysenin is produced by the coelomocytes.     

Immunochemical evidence for met-enkephalin-like and leu-enkephalin-like peptides in tissues of the earthworm, Lumbricus terrestris

The presence of met- and leu-enkephalin-like immunoreactive materials in nerve, gut, seminal vesicle and body wall tissues of the earthworm Lumbricus terrestris has been demonstrated by means of radioimmunoassay technique. The greatest activity of met- and leu-enkephalin-like immunoreactivity in earthworm gut appears in regions of high digestive enzyme activity and gastrin-like immunoreactivity where it presumably plays a role in regulation of gut function. In all tissues studied the levels of met-enkephalin-like immunoreactivity were higher than that of leu-enkephalin-like immunoreactivity. Dual localization of met- and leu-enkephalin-like immunoreactivity in earthworm gut and nerve tissues follows the pattern observed of peptide hormones in vertebrates which are common to both endocrine and non-endocrine tissues.     

Rapid fixation and embedding for electron microscopy

A method has been developed for rapid processing of animal tissues for electron microscopy. The whole process of fixation staining dehydration, infiltration and embedding including polymerization is completed in less than 4 hr. A variety of human and animal tissues such as liver, spleen, muscle, kidney and embryonic chick heart were processed by this method and the results were excellent. The rapid fixation and embedding method is strongly recommended when relatively soft tissues are to be studied. This method is especially useful for examining pathological tissues for rapid diagnostic purposes.     

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.     

Immunochemical evidence for a gastrin-like peptide in the intestinal tissues of the earthworm Lumbricus terrestris

• 1.1. The presence of gastrin-like immunoreactive materials in intestinal tissues of the earthworm Lumbricus terrestris has been demonstrated by means of radioimmunoassay technique.
• 2.2. The greatest activity coincides with the areas of the intestines with high digestive enzyme activities. No gastrin-like activities were detected in nerve tissues.
• 3.3. The results also indicate that earthworm gut tissues may contain different forms of gastrin or gastrin-like peptides.
• 4.4. The absence of gastrin-like activity in earthworm nerve tissues may have significant phylogenetic implication for the proposed gastrin-cholecystokinin origin in neuronal elements of invertebrates.

     

Immunochemical evidence for substance P-like peptide in tissues of the earthworm Lumbricus terrestris: action on intestinal contraction

The presence of a substance P-like peptide in intestinal and body wall tissues, ventral nerve fiber and seminal vesicles of the earthworm Lumbricus terrestris has been demonstrated by means of a radioimmunoassay technique. The greatest substance P-like immunoreactivity was measured in intestinal tissues where it stimulates the rate of spontaneous contraction. This effect is inhibited by the substance P antagonist (D-pro2, D-trp7,9)-SP suggesting a possible involvement of receptor mechanisms. Dual localization of substance P-like immunoreactivity in earthworm intestinal and nerve tissues follows the pattern observed of peptidal hormones in vertebrates which are common to both endocrine and non-endocrine tissues.     

4种蚯蚓肠道微生物对砷毒性的响应差异研究

蚯蚓肠道是微生物多样性的一个潜在存储库。砷对蚯蚓肠道微生物群落的影响已被证实,但砷在不同蚯蚓肠道菌群中生物转化的差异仍不清楚。为了进一步阐述土壤中广泛存在的低浓度砷(浓度为5,15,25 mg/kg)对不同种类蚯蚓肠道微生物影响的差异,将4种典型蚯蚓暴露于砷污染土壤后,测定其肠道微生物组成变化,并分析砷对不同蚯蚓肠道内砷富集、形态和砷生物转化基因的影响。结果显示,所有蚯蚓组织内均存在明显的砷富集,其富集系数由高到低依次为:安德爱胜蚓(1.93)>加州腔蚓(0.80)>通俗腔蚓(0.78)>湖北远盲蚓(0.52),蚯蚓组织和肠道内砷形态主要以无机砷为主,其中As(III)含量比例> 80%,部分蚯蚓组织内还发现少量有机砷。4种蚯蚓肠道微生物群落在门水平上主要以变形菌、厚壁菌和放线菌为主,并与周围土壤细菌群落组成存在显著差异。同时,在土壤和肠道内共检测到17个砷转化基因,其中蚯蚓肠道内As(V)还原和砷转运相关基因相对丰度较高,而砷(去)甲基化基因丰度较低。此外,低浓度砷污染对蚯蚓生长无显著影响,却能引起蚯蚓肠道微生物群落的紊乱。蚯蚓种类和砷污染是引起蚯蚓肠道微生物...     

Dark hair cells in the inner ear of the rainbow trout. A study of the influence of different fixation methods

The occurrence of dark staining cells in different tissues has been suggested to be artefactual and caused during the fixation process. In inner ear sensory epithelia, dark hair cells (DHC) have been suggested to be apoptotic cells. We have examined whether dark cells represent dying cells or whether they are the results of fixation artefacts. The effects of buffer osmolarity and different fixation methods on the incidence of dark hair cells in the inner ear macula sacculi of the rainbow trout (Oncorhynchus mykiss) were investigated by light and electron microscopy. Glutaraldehyde in phosphate buffer with osmolarities of 0, 135, 225, 425, and 560 mosmol were used for fixation by immersion. For comparison, fixation by vascular perfusion as well as the effects of mechanical injury and delayed fixation were studied. DHC were found in all examined saccular maculae except for the delayed fixation protocol where almost all the sensory cells were lost. The number of DHC accounted for 2.5–12.9‰ of the sensory cells. Neither the buffer osmolarity nor the fixation method had significant effects on the frequencies of DHC. Mitotic cell division events were seen exclusively in the apical cell strata of the sensory epithelium. The DHC are suggested to be associated with apoptosis rather than fixation artefacts.     

Fixation and decalcification of adult zebrafish for histological, immunocytochemical, and genotypic analysis

To facilitate the molecular analysis of tissues in adult zebrafish, we tested eight different fixation and decalcification conditions for the ability to yield DNA suitable for PCR and tissue immunoreactivity, following paraffin embedding and sectioning. Although all conditions resulted in good tissue histology and immunocytochemistry, only two conditions left the DNA intact as seen by PCR. The results indicate that zebrafish fixed in either 10% neutral buffered formalin or 4% paraformaldehyde, followed by decalcification in 0.5 M EDTA, is an easy and reliable method that allows molecular experiments and histology to be performed on the same specimen. The fixation and decalcification by Dietrich's solution permitted the PCR amplification of DNA fragments of 250 but not 1000 bp. Therefore, a protocol of formalin or paraformaldehyde fixation followed by decalcification with EDTA is broadly applicable to a variety of vertebrate tissues when excellent histological, immunocytochemical, and genotypic analyses may be simultaneously required.     

Appraisal of the electrical octet method for estimating earthworm populations in arable land

Quantitative methods are needed for the assessment of the size and composition of earthworm communities. A poorly documented electrical sampling method, Thielemann's octet method, was compared with two long‐established methods, formalin extraction and soil hand sorting, in conventional and direct‐drilled wheat cropping systems at two sites with medium to heavy textured soils in Ireland. Under all agronomic conditions tested, the electrical method extracted significantly higher earthworm numbers than formalin, but earthworm biomasses were not significantly different. When used routinely over two years during periods of high earthworm activity, the electrical method yielded community estimates that were comparable in both size and species composition to those obtained by soil hand sorting (25 cm depth), except in recently ploughed land. However, Murchieona minuscule, a minute endogeic species, was underestimated by electrical extraction. It is concluded that the electrical octet method can be a reliable and useful alternative to other dynamic methods for estimating earthworm populations, especially in situations where minimum soil disturbance is desirable.     

Occurrence of ceramide-glycanase in the earthworm, Lumbricus terrestris

We have detected the presence of ceramide-glycanase in the earthworm, Lumbricus terrestris. We have also devised a simple method for the preparation of this enzyme from the earthworm. This enzyme cleaved the linkage between the ceramide and the glycan chain in LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, GM3, GM2, GM1 and GD1a. By using tritium-labeled GM2 as substrate, the optimum pH of this enzyme was found to be between pH 4 and 4.5. In the earthworm, the ceramide-glycanase was mainly found in the muscle. The intestine was found to contain a very low level of this enzyme. Because of their easy availability, earthworms should become a convenient source for the preparation of ceramide-glycanase.     

A new antigen retrieval technique for human brain tissue

Immunohistochemical staining of tissues is a powerful tool used to delineate the presence or absence of an antigen. During the last 30 years, antigen visualization in human brain tissue has been significantly limited by the masking effect of fixatives. In the present study, we have used a new method for antigen retrieval in formalin-fixed human brain tissue and examined the effectiveness of this protocol to reveal masked antigens in tissues with both short and long formalin fixation times. This new method, which is based on the use of citraconic acid, has not been previously utilized in brain tissue although it has been employed in various other tissues such as tonsil, ovary, skin, lymph node, stomach, breast, colon, lung and thymus. Thus, we reported here a novel method to carry out immunohistochemical studies in free-floating human brain sections. Since fixation of brain tissue specimens in formaldehyde is a commonly method used in brain banks, this new antigen retrieval method could facilitate immunohistochemical studies of brains with prolonged formalin fixation times.     

The Feulgen-Picric-Acid block Stain Additional Data

Additional experiments on staining of tissues in the block by means of picric acid and the Feulgen reaction (Lhotka and Davenport, 1947) show that a variety of tissues from both plant and animal sources will respond favorably. An attempt to combine fixation and staining into a single step was not successful for mammalian tissues, but gave fair results on frog. Fixation in sublimate-acetic was unsatisfactory for block staining, and conversely, fixation in sulfosalicylic-picric gave poor Feulgen stains on the slide. Soxhlet extraction of tissue with absolute alcohol removed Feulgen positive, non-nuclear material with the exception of that in vascular endothe-lium, cartilaginous matrix and elastic tissue. After such extraction, similar results were obtained with both block and slide methods of staining.     

Specific features of nitrogen transformation in the gut and coprolites of earthworms

It has been found that nitrogenase activity in the guts and coprolites of three earthworm species (Lumbricus terrestris, Aporrectodea rosea, and Aporrectodea caliginosa) is one to three orders of magnitude higher than that in the control soil. In A. caliginosa earthworms, the actual nitrogenase activity remains high upon keeping them in soils of different types. However, it reaches a peak in the gut lumen and decreases to a minimum on its wall, which is evidence for a major role of transitional microflora in intestinal nitrogen fixation. Nitrogen fixation activity in freshly excreted coprolites decreases to one-half or one-third of that in the gut and then increases again, reaching a peak on days 3–5 of exposure in the soil. According to the results of multisubstrate testing, the functional diversity of nitrogen-fixing soil microorganisms increases in the course of passage through the earthworm gut. Thus, the microbial community in coprolites retains its functional potential and, within a few days, shows the second peak of activity in the soil. Due to a short-term increase in the rate of nitrogen fixation, coprolites contain a pool of bound amino acids, which become involved in the formation of new humus substances.     

Kupffer cells and PIMs in acute experimental African swine fever.

An ultrastructural study of Kupffer cells and pulmonary intravascular macrophages (PIMs) of healthy and African Swine Fever (ASF)-infected pigs was carried out. A vascular perfusion method was performed in order to obtain an optimal intravascular morphology and tissue fixation. The infection developed acute ASF lesions in both organs. Both Kupffer cells and PIMs were studied at different stages of infection. The differences observed in both macrophagic cells from uninfected and infected tissues are shown and discussed.     

Elemental analysis of histochemically defined cells in the earthworm Lumbricus terrestris.

A method for preparation of biological specimens for electron probe X-ray microanalysis is described, that aims at retaining the original elemental distribution within the tissue at the cellular level. The tissue is without any chemical fixation, quench-frozen, and 16-micron sections are prepared with a conventional cryomicrotome, transferred to a carbon specimen holder and freeze-dried. Adjacent serial sections, collected on glass slides and stained with various histological procedures, are used to correlate the data obtained by X-ray microanalysis with other histochemical information on the same cell or tissue. To demonstrate the possibilities of the method, sections of the earthworm Lumbricus terrestris were analyzed. In the chloragogenous cells, high concentrations of Ca, Zn and P were found. The inner and outer muscle layer show slightly different properties, both with regard to elemental composition and to myofibrillar ATPase activity.     

New freeze-dry and vapor fixation method for immunohistochemistry of soluble proteins: subcellular location of the progesterone receptor

We describe a new application of freeze-drying and vapor fixation for immunohistochemical location of soluble proteins. The method avoids the liquid phase, which eliminates the possible diffusion of soluble proteins. Two vapor fixatives, paraformaldehyde and p-benzoquinone, were tested and p-benzoquinone was found to preserve antigenicity of progesterone receptor (PR) and ovalbumin better than paraformaldehyde. The method proved to be highly sensitive, since higher concentrations of antigen were found in some tissues and some tissues found to be antigen negative by earlier liquid fixation methods proved to contain antigen. The location of PR as a highly soluble protein was studied. With the present method, both unoccupied and occupied PR were located in the nuclei, a similar finding as with the earlier liquid fixation method. The results further support the concept that PR is an intranuclear protein independent of its ligand occupation. PR was detected in a few cells inside the follicles of the bursa of Fabricius and in the smooth muscle cell nuclei of the small intestine, observations not previously made owing to the insensitivity of the earlier methods.     

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.     

Fixation of neural tissue for electron microscopy with an electronically controlled perfusion pump

Many attempts to improve the perfusion of mammalian tissues aim at changes of the osmotic pressure. We describe a method for fixation of nervous tissues controlling both the hydrostatic pressure and the flow rate of a perfusion solution. The constancy of these parameters is guaranteed by an electronically controlled perfusion pump. Thus, a more uniform and complete preservation can be achieved. Further advantages of this method include provision for a rapid succession of rinsing and fixation solution and a continuous control of the hydrostatic pressure during perfusion.