Poul Prentø

Metals and phosphate in the chloragosomes of Lumbricus terrestris and their possible physiological significance.

SummaryThe chloragog cells of the earthworm Lumbricus terrestris contain numerous granules, chloragosomes, which were analyzed for metals and phosphate by histochemistry, by use of an electron microscope X-ray microprobe (EMMA), and by chemical analysis of chloragosome preparations. Inorganic and organic phosphate each accounts for about 3% of the chloragosome dry mass, Ca for 2–3%, Zn for 1–3% and Mg for 0.2–0.4%. Carbonate is not present in chloragosomes. The average molar ratios Ca∶Mg∶Zn∶total PO4 are 1∶0.1∶0.3∶1. The Ca∶PO4 ratio is fairly constant (correlation coefficient 0.99), while the Zn∶PO4 ratio varies considerably. It is concluded that Ca is bound in the form of inorganic CaHPO4 and organic ROPO 3Ca (or possibly Ca-polyphosphate complexes). Mg may also be phosphate-bound, while Zn probably is not. Chemical analysis of the calciferous glands revealed a high concentration of Ca, small amounts of Mg and phosphate, but no Zn. It is concluded that Zn is not excreted through the calciferous gland.Storage of Ca in the chloragocytes and excretion of CaCO3 by the calciferous gland may be physiologically linked. Regulation of the concentrations of Ca and HCO3−ions in the blood and coelomic fluid may assist in equalization of osmotic pressures during dehydration and rehydration. This regulation may be a major function of the chloragosomes.The chloragosomes were discussed in relation to the “spherites” of various arthropods and molluscs and to the “cytosomes” of anoxia-tolerating molluscs.

Commercial formalin substitutes for histopathology.

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarkes ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200x, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarkes fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.

Distribution of 20 enzymes in the midgut region of the earthworm, Lumbricus terrestris L., with particular emphasis on the physiological role of the chloragog tissue

Abstract 1. 1. Lumbricus terrestris midgut was separated into peripheral chloragocytes, peripheral intestinal epithelium and typhlosole, and the distributions of 20 enzymes were assayed. 2. 2. The peripheral chloragog tissue contained ⩽4% of the midgut dehydrogenase activities, but about 44% or more of the total catalase, Superoxide dismutase, β- d -glucuronidase, alkaline phosphatase, esterase, (δ-aminolevulinate dehydratase and porphyrin synthetase activities. 3. 3. The midgut contained about 70% or more of the total midbody isocitrate dehydrogenase, glutaminate dehydrogenase and NADH- and NADPH-diaphorase activities. 4. 4. Most of the intermediary metabolism, including amino acid deamination, probably takes place in the midgut epithelium, which is also the site of the mixed function oxidase system. 5. 5. The chloragog tissue probably synthesizes the blood hemoglobin and is responsible for the destruction of Superoxide and hydrogen peroxide from the interaction between blood hemoglobin and molecular oxygen. 6. 6. The high alkaline phosphatase activity in ehloragog tissue may reflect transmembrane transport activity or may be involved in glycogen break-down. 7. 7. Neither xanthine oxidase nor uric acid, xanthine or hypoxanthine were present in detectable amounts.

Thermal balloon endometrial ablation : Safety aspects evaluated by serosal temperature, light microscopy and electron microscopy

OBJECTIVES Thermal balloon endometrial ablation is a new method for treating menorrhagia. The technique appears to be less difficult compared to standard hysteroscopic ablation techniques and to be significantly safer. The influence into the uterine wall of the thermal balloon ablation procedure was investigated with special reference to the ability of total destruction of the endometrium and the thermal action on the myometrium and the serosa. STUDY DESIGN Temperatures were measured at the uterine serosal surface during thermal balloon endometrial ablation for 8-16 min in eight patients. After subsequent hysterectomy the extent of thermal damage into the myometrium was assessed by light and electron microscopy. RESULTS The highest temperature measured on the uterine serosa was 39.1 degrees C. Coagulation of the myometrium adjacent to the endometrium could be demonstrated by light microscopy in all patients, with a maximum depth of 11.5 mm. By electron microscopy no influence of heat could be demonstrated beyond 15 mm from the endometrial surface. CONCLUSION Up to 16 min of thermal balloon endometrial ablation therapy can destroy the endometrium and the submucosal layers. The myometrium is only coagulated to a depth where full thickness necrosis or injury is unlikely.

Van Gieson's picrofuchsin. The staining mechanisms for collagen and cytoplasm, and an examination of the dye diffusion rate model of differential staining

The staining mechanism of van Giesons picrofuchsin was studied by use of simple protein model systems and tissue sections, and by spectrophotometry and dialysis experiments. At the endpoint of the staining reaction (equilibrium) cytoplasm is yellow. Dye dilution experiments demonstrated that the highest affinity in the tissue section — picrofuchsin system is between binding sites in cytoplasmic protein and acid fuchsin. Nevertheless sections that were first stained in acid fuchsin (AcF) and then in picrofuchsin ended up with cytoplasm stained yellow. It was concluded that differences in the dye diffusion rates and differences in the permeability of tissue components cannot be invoked to explain the differential staining result. Model experiments with dissolved proteins demonstrated a positive relationship between protein concentration and uptake of picric acid (PA) from picrofuchsin. From this and experiments with additives (sodium dodecylsulphate, urea etc.) and organic solvents, it is proposed that coagulant interchain cross-linking at the high protein concentration of the cytoplasm masks potential dye-binding sites. This affects high affinity dyes with multiple binding sites more than small dyes, and so puts AcF at a disadvantage compared to PA. Staining of non-collagen proteins is mainly by hydrophobic bonding, involving ionic attractions, apolar bonds, and release of water. This mode of binding is relatively strong, decreases swelling and leads to slow dye exchange. Dye binding to collagen is mostly by hydrogen bonds, but in aqueous dye solvent nonpolar residues and charged residues may also participate. This structure remains relatively open during and after dye-binding, and the bound dye ions are therefore easily exchanged for other dye ions.

Histochemistry of neurosecretion in the pars intercerebralis-corpus cardiacum system of the desert locust Schistocerca gregaria

The pars intercerebralis and corpus cardiacum of mature females of Schistocerca gregaria were investigated histochemically. It is concluded that the pars intercerebralis A and B cells are distinct cell types, each elaborating specific, predominantly proteinaceous products. The pars intercerebralis C cells did not show significant response to any of the histochemical procedures. The A cell material contains a high amount of cysteine, little or no tyrosine, tryptophan and dicarboxyl amino acids, and a moderate amount of basic amino acids. The B cell material contains a moderate amount of cystine, a very high amount of tyrosine, a low to moderate amount of tryptophan, a high amount of dicarboxyl amino acids, and a very high amount of basic amino acids. The secretory cells located in the corpus cardiacum elaborate a predominantly proteinaceous material with a moderate amount of cysteine, a very high tyrosine and tryptophan content, a low dicarboxyl amino acid content, and a moderate content of basic amino acids. In all three cases the dominant basic amino acid is probably histidine. Histochemically no change in the content of the A cell secretory material is observed during the axonic transport from the pars intercerebralis to the corpus cardiacum storage lobe. The neurosecretory cells in general contain very high amounts of ribonucleic acids. In the A cells variations in RNA content are especially distinct and seem to be correlated with phases in elaboration and storage of the secretory material.

Methyl green-pyronin Y staining of nucleic acids: studies on the effects of staining time, dye composition and diffusion rates

Since the introduction of the methyl green-pyronin Y procedure as a differential histological stain more than 100 years ago, the method has become a histochemical procedure for differential demonstration of DNA and RNA. Numerous variants of the procedure have been suggested, and a number of hypotheses have been put forward concerning kinetics and binding mechanisms. Using both filter paper models containing DNA, RNA or heparin and histological sections, we have attempted to evaluate the kinetics of staining and the role of staining time for methyl green and pyronin Y by applying the dyes individually, simultaneously and sequentially. The results are presented as color charts approximating the observed staining patterns using a computerized palette. Our results indicate unequivocally that the differential staining is not time-dependent, but that it is dictated by the relative concentrations of methyl green and pyronin Y and by the pH of the staining solution.

Uptake and long-time storage of natural and synthetic dyes by earthworm chloragocytes. In vivo and in vitro investigations

Abstract Cationic or anionic dyes adsorbed onto cellulose granulate were transported across the gut wall, bound to blood proteins, and accumulated by the chloragocytes. Solubility in water promoted accumulation. The dyes ended up mainly in the chloragosomes. Down to 20 μmol dye per litre soil water resulted in visible accumulation. Worms which after dye-exposure were kept dye-free for 5 months retained substantial amounts of dye in the chloragosomes. In vitro experiments indicate that the binding to chloragosomes of synthetic and natural phenolics is by ion exchange with calcium phosphate and with an uncharacterized matrix-bound calcium chelator, aided by hydrophobic interactions between the dye and constituents of the chloragosome matrix. The findings are relevant for the evaluation of the effects of constant or periodic soil contamination with industrial or agricultural organochemicals.

Cellular and intracellular localization of catalase and acid phosphatase in the midgut of Lumbricus terrestris L.: a cell fractionation study

Abstract 1. 1. Cellular and intracellular localization of catalase and acid phosphomonoesterase in the midgut of Lumbricus terrestris was studied by use of tissue fractionation. 2. 2. At least 60–70% of the catalase resides in the chloragocyte cytosol and the remaining 30–40% resides in gut epithelium peroxisomes. 3. 3. One of the main functions of the chloragocyte catalase is probably scavenging for H 2 O 2 arising from the interaction between blood heme-protein and oxygen. 4. 4. A simple method for the histochemical detection of cytosol catalase is proposed. 5. 5. About 10% of the gut acid phosphatase resides in chloragocyte lysosomes. The chloragosomes contain no acid phosphatase.

Blood sugar, sugar metabolism and related enzymes in the earthworm, Lumbricus terrestris L.

Abstract 1. 1. L. terrestris has no trehalose in the blood or coelomic fluid and is without trehalose activity. 2. 2. Glucose-6-phosphatase is also lacking and the concentration of glucose in the blood or coelomic fluid is very low (0.01–0.05 μg/μl normally). 3. 3. The body wall contains 80% of the lactate dehydrogenase activity of the worm and both the lactate formation and the reoxidation to pyruvate takes place in the body wall muscle itself. 4. 4. In accordance with this the glycogen-storing chloragocytes contain very little or no LDH. 5. 5. Thus the earthworm does not have the lactate-glucose exchange mechanism known from vertebrate liver and muscle and hormonal regulation of extracellular glucose is probably absent. 6. 6. Chloragocytes exhibit significant amylase- and maltase-activities, which may be responsible for glycogen break-down in the absence of the phosphorylase-glucose-6-phosphatase system. 7. 7. There are histological indications that chloragocytes, or their distal parts are released to the coelom to form trephocytes or eleocytes. 8. 8. They may then break up or be transported by the coelomic fluid to other parts of the body and their contents of glycogen and other materials released. 9. 9. Comparative and functional aspects of the above are discussed.