Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

The Reactions of Isolated Mucopolysaccharides to Several Histochemical Tests

The reaction to three histochemical tests of preparations of hyaluronic acid, chondroitin sulfate, heparin, the acidic mucopolysaccharides from cornea, gastric mucin, and dentine, and also of the neutral mucopolysaccharide from gastric mucin was studied. To 1% aqueous solutions of the acid mucopolysaccharides, equal volumes of 1% casein solution were added; drops of the resulting solutions were placed on slides and dried at 37 °C. The films were then fixed in acetic-alcohol (1:9). The technics employed were the periodic acid-Schiff (PAS) test, the metachromatic reaction and the Hale test. The relative acidity of the preparations was demonstrated by staining in dilute aqueous methylene blue at pH 3-6. With the exception of the preparation from dentine, the acid mucopolysaccharides stained only weakly with PAS; the neutral mucopolysaccharide stained strongly. It is concluded, therefore, that the use of the PAS technic for the histochemical demonstration of acid mucopolysaccharides is misleading, for many important members of this class of tissue component do not react appreciably. On the other hand, metachromasia was shown by all the acidic compounds studied, and the intensity of staining was approximately correlated with the acidity of the preparations. The Hale method was found to be nonspecific.     

A microfluorometric study of masked metachromasia in cells of the endocrine polypeptide (APUD) series

Synopsis Masked metachromasia can be demonstrated by staining with a metachromatic basis dye after a Feulgen-type hydrolysis of suitably fixed tissue, and is believed to be indicative of polypeptides with a high concentration of side-chain acidic groups and a random-coil conformation. In this investigation, the metachromatic fluorochrome Coriphosphine O was used. After staining, the degree of metachromasia under various conditions and in several tissues was assessed by microspectrofluorometric measurements of the ratio of metachromatic fluorescence (red) to orthochromatic fluorescence (green). This technique was employed, in the first instance, to determine the optimum staining conditions; details of the final staining method are presented. Measurements of metachromasia in different tissues under standardized conditions showed that the degree of metachromasia varied between different cell types in the APUD series.     

Role of protein dissociation in the transport of acidic amino acids by the Ehrlich ascites tumor cell.

The pH profile for the uptake of L-glutamic acid by the Ehrlich ascites tumor cell arises largely as a sum of the decline with falling pH of a slow, Na+-dependent uptake by System A, and an increasing uptake by Na+-independent System L. The latter maximizes at about pH 4.5, following approximately the titration curve of the distal carboxyl group. This shift in route of uptake was verified by (a) a declining Na+-dependent component, (b) an almost corresponding decline in the 2-(methylamino)-isobutyric acid-inhibitable component, (c) a rising component inhibited by 2-aminonorbornane-2-carboxylic acid. Other amino acids recognized as principally reactive with Systems A or L yielded corresponding inhibitory effects with some conspicious exceptions: 2-Aminoisobutyric acid and even glycine become better substrates of System L as the pH is lowered; hence their inhibitory action on glutamic acid uptake is not lost. The above results were characterized by generally consistent relations among the half-saturation concentrations of the interacting amino acids with respect to: their own uptake, their inhibition of the uptake, one by another, and their trans stimulation of exodus, one by another. A small Na+-dependent component of uptake retained by L-glutamic acid but not by D-glutamic acid at pH 4.5 is inhibitable by methionine but by neither 2-(methylamino)-isobutyric acid nor the norbornane amino acid. We provisionally identified this component with System ASC, which transports L-glutamine throughout the pH range studied. No transport activity specific to the anionic amino acids was detected, and the unequivocally anionic cysteic acid showed neither significant mediated uptake nor inhibition of the uptake of glutamic aic or of the norbornane amino acid. The dicarboxylic amino acids take the sequence, aspartic acid less than glutamic acid less than alpha-aminoadipic acid less than S-carboxymethylcysteine, in their rate of mediated, Na+-independent uptake at low pH. Diiodotyrosine and two dissimilas isomers of nitrotyrosine also show acceleration of uptake as the phenolate group on the sidechain is protonated, a result indicating that the acidic group need not be a carboxyl group and need not take a specific position in space to be accepted at the receptor site L. The presence of the carboxyl group does not upset the normal stereospecificity of System L until it falls on the beta-carbon in aspartic acid; even then it is the presence of the carbonyl group and not of the intact carboxyl group nor of its hydroxyl group that cancels out the stereospecificity, as was shown by the absence of normal stereospecificity for aspartic acid and asparagine and its presence in glutamic acid, homoserine and glutamine. In agreement, the uptak of aspartic acid is peculiarly sensitive to the presence of an alpha-methyl group or of other structures that modify the orientation of the sidechain.     

Staining and osmotic properties of young gametophytes ofPolypodium vulgare L. and their bearing on rhizoid function

Summary The rhizoids of gametophytes ofPolypodium vulgare L. rapidly absorb vital stains whereas the protonemal cells are impermeable to these stains, which can only enter the cells from the rhizoids. The protonemal cells which bear rhizoids were found to have a slightly higher osmotic equivalent than did the rhizoids or the protonemal cells on either side. From the results of several staining procedures it was demonstrated that the rhizoid walls contain free carboxyl groups and thus possess cation exchange properties. Most of the carboxyl groups are probably present in a yellow-brown wall matrix substance, which shows high resistance to acid and alkali extraction. The precise nature of this substance has not been determined but it could be an acid mucopolysaccharide. Carboxyl groups are detectable in the protonemal cell walls only after saponification and are probably esterified in the untreated wall. Several other chemical and physiological differences were found between the rhizoids and the protonemal cells and it was concluded that the specific properties of the rhizoids are related to their function as organs of uptake.     

Morphochemical analysis of mucous cells in the skin and slime glands of hagfishes

Summary The mucous cells of the epidermis and slime glands in three species of Pacific hagfishes were studied histochemically to determine the presence or absence of acidic and neutral mucosubstances. Most mucous cells contained diastase-resistant PAS reactivity that varied in intensity. A few mucous cells contained diastase-labile substances exclusively.Acidic groups were detectable due to their stainability by several basic dyes which were utilized singly or in combination. Considerable species diversity of hagfish mucins was noted in their affinity toward azure A at pH 1.0, 2.0, and 3.0 depending on whether sections were viewed wet from the staining jar or viewed after dehydration and mounting. At pH 1.0, a few mucous cells were metachromatic under both conditions while the majority were unstained in both types of section. As the pH was raised, the majority of cells were metachromatic when viewed wet or mounted. Most mucous cells were reactive toward alcian blue at pH 1.0, aldehyde fuchsin and the high iron diamine reagents. Each of these reactions was absent following exposure of sections to acidic (0.1N HCl) methanol for 4 hours at 60°C while control sections exposed to unacidified methanol or acidified isopropanol for the same time and temperature resulted in no loss of staining.Sialidase-labile acidic groups were detected in the epidermal mucous cells of one species of hagfish. Following prior treatment of sections with Vibrio cholerae sialidase for 16–24 hours at 39° C, there was a reduction of metachromasia of the mucous cells produced by azure A at pH 3.0. Although confirmatory autoradiographic and biochemical data on hagfish mucosubstances are lacking, the histochemical staining results and their subsequent modifications by enzyme and chemical treatment indicate that the majority of these mucins contain sulfomucin while a few are composed of sialomucin. Similar results of previous histochemical and autoradiographic studies of epithelial secretions from higher animals correlated, in some instances, with the biochemical data for those mucins.Supported by Research Grant AM — 11064 of the United States Public Health Service.Recipent of a Lederle Medical Faculty Award, 1968–1971.     

The electrophoretic mobility of normal and leukaemic cells of mice

1. The pH-mobility relationships for saline-washed cells from a mouse strain of acute lymphoblastic leukaemia were examined before and after treatment with lower aldehydes, diazomethane and neuraminidase (EC 3.2.1.18). 2. The content of sialic acid released into the supernatant fluid of neuraminidase-treated cells was measured. 3. The stability of the charge-determining structures to temporary changes in environment (pH and ionic strength) was established. 4. Similar measurements were made on lymph-node cells obtained from non-leukaemic mice (a resistant and a leukaemia-susceptible strain were examined). 5. It is deduced that both the malignant and the non-malignant cell possess two dissociable acid functions at the cell surface, a carboxyl group of sialic acid and another acidic group(s), probably carboxyl, of pK 3.0-4.5. The malignant cells, however, have a basic dissociable function not present in the non-malignant types. 6. Suggestions are made as to how the difference in surface chemistry may be related to the problem of malignancy.     

A study on the structure-function relationship of lipopeptide biosurfactants

Arthrofactin (AF) and surfactin (SF) are the most effective cyclic lipopeptide biosurfactants ever reported. Linear AF and linear SF were prepared by saponification of lactone ring. The oil displacement activities decreased to one third of their respective original values. When residues of both an aspartic acid and a glutamic acid of SF were methylated or amidated, the activity increased by 20%, although their water solubility was lost. When these amino acid residues were modified by aminomethane sulfonic acid, the activity was drastically decreased probably owing to charge repulsion and structural distortion inhibiting micelle formation. Both AF and SF expressed higher activity under alkaline conditions than acidic conditions. AF was more resistant to acidic conditions than SF and it kept high activity even under pH 0.5. Although SF drastically reduced its activity under acidic conditions, surfactin-Asp/Glu-amido ester and surfactin-Asp/Glu-methyl ester retained similar activities irrespective of the pH change. A couple of conformers of SF prepared by reverse-phase HPLC showed the same oil displacement activity but different surface tension-reducing activity. AF was produced as a series of different fatty acid chain lengths (from C8 to C12). Among them, AF with fatty acid chain length of C10, which was the main product of the strain, showed the highest activity.     

An IkappaB-beta COOH terminal region protein is essential for the proliferation of CHO cells under acidic stress

CHO-K1 cells were able to proliferate and maintain pHi homeostasis at pH 6.3. A novel acidic sensitive mutant, AS-5B, which proliferated at pH 7.4 but failed to either proliferate or maintain pHi homeostasis at pH 6.3, was derived from CHO-K1 using a replica method. The acidic-sensitivity of AS-5B was not due to deficiencies in sodium proton exchangers, HCO3- (co)transporters or H+-ATPases. A cDNA clone encoding a COOH terminal region of IkappaB-beta conferred partial acidic-resistance on AS-5B, and the encoded protein was present in CHO-K1, but was nearly absent from AS-5B. Our data demonstrated that the expression of this small protein was essential for the proliferation of CHO cells under acidic stress.     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells.

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate. Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I, suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid could be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8, and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydrofolate, lower affinity for N5-methyl-tetrahydrofolate, and no apparent affinity for N5-formyltetrahydrofolate or methotrexate.     

Conformational properties of gastrin fragments of increasing chain length

The conformational properties of a series of biologically active gastrin peptides of increasing chain length have been investigated in TEE solution by spectroscopic techniques. It was found that elongation of the glutamic acid sequence from 1 to 5 residues at the N-terminal portion of the molecules causes a cooperative change of the conformation of the peptide backbone. The environment of the biologically important C-terminal sequence-Trp-Nle-Asp-Phe-NH₂ monitored by the near-uv chiroptical propertical properties is alos affected by chain elongation. However, the change of the structure of the C-terminal portion does not parallel the conformational change of the peptide backbone. In fact, the final folded structure at the C-terminus is almost reached in the fragment with a sequence of 4 glutamic acid residues, while an additiona, relevent conformational change of the backbne is observed on further elongation of the chain to minigastrin and little gastrin. The ability of the fragments to fold into an ordered conformation on chain elongation parallels the increase of biolocical potency tested in vivo, reported in the literature, and suggests a correlation between these two facts. Ionization of the carboxyl side chains is without effect on the structure of the fragments with 2, 3, and 4 glutamic acid residues, while an effect is observed in minigastrin and little gastrin. From analysis of the CD properties and from their dependence upon side-chain ionization a structural model is proposed for the hormones minigastrin and little gastrin. This tentative model includes a β-bend located in the sequence Ala-Tyr-Gly-Trp- and a short helical section at the N-terminal portion of the hormones.     

Sialomucin in Paget cells of extramammary Paget's disease

The cytoplasmic sialomucin in Paget cells of extramammary Paget's disease was examined by means of a battery of histochemical techniques. The staining methods used involved an electrolyte--Alcian Blue (pH 5.8), periodic acid--Schiff and azure A at selected pH levels. Methylation, saponification, borohydride reduction, acid hydrolysis, and digestion with diastase, neuraminidase (Vibrio cholerae) or chondroitinase ABC, were also employed. The cytoplasmic mucin was found to exhibit positive reaction for the above staining which were variously altered by the chemical modification procedures and diminished in intensity or abolished by digestion with neuraminidase. These results suggest that the cytoplasmic mucin is sialomucin without side-chain substituent in extramammary Paget's disease located in axillary or genital area, and with a substituent at C7 in the disease located in perianal area.     

Indole reactions of mast cells and enterochromaffin cells related to fixations with and without aldehydes

Summary Survey of a considerable number of rat, mouse and hog tissues which presented large numbers of mast cells in preparations stained with toluidine blue and other metachromatic or basic dyes at low pH levels, revealed numbers of oval bodies of about the same size as mast cells which reacted weakly or even moderately to the postcoupled benzylidene indole reaction. The numbers of these were always less than that of mast cells in toluidine blue sections of the same blocks. They never occurred in clusters of perhaps 15–20 in a single high power field, as mast cells often do. Smooth and especially striated muscle which often formed the background tissue where most mast cells are found with metachromatic stains, regularly present indole reactions due to protein tryptophan. This is usually equal to or stronger than that in the supposed mast cells.Indole reactive bodies whose morphology suggests mast cells are also present in similar numbers in formaldehyde and glutaraldehyde fixed tissue as well as with aldehyde free fixations. Glutaraldehyde and formaldehyde are known to inhibit the benzylidene reaction of 5-HT in vitro (30 min for glutaraldehyde, 3 h for formaldehyde) (Lillie, 1977). This action was avoided in mercury and lead heavy metal fixations and in acetone, Carnoy, chloroform methanol and similar fixations.The mast cell-like bodies are best explained as tangential or oblique sections of individual muscle fibers. We have described the same phenomenon with the ferric ferricyanide (Golodetz-Unna, 1909) reaction (Lillie et al., 1978a), and the PCB reaction is that of tryptophan in these muscle cell sections.In contrast to the DMAB type reaction failure acid diazosafranin successfully demonstrated mast cells with both aldehyde and aldehyde free fixations. This reaction has been shown to occur with 5-HT and 5-HTP (Lillie et al., 1973).     

A conserved glutamic acid bridge in serine carboxypeptidases, belonging to the alpha/beta hydrolase fold, acts as a pH-dependent protein-stabilizing element.

Serine endopeptidases of the chymotrypsin family contain a salt bridge situated centrally within the active site, the acidic component of the salt bridge being adjacent to the catalytically essential serine. Serine carboxypeptidases also contain an acidic residue in this position but it interacts through a short hydrogen bond, probably of low-barrier type, with another acidic residue, hence forming a "glutamic acid bridge." In this study, the residues constituting this structural element in carboxypeptidase Y have been replaced by site-specific mutagenesis. It is demonstrated that the glutamic acid bridge contributes significantly to the stability of the enzyme below pH 6.5 and has an adverse effect at pH 9.5. Carboxypeptidase WII from wheat contains 2 such bridges, and it is more stable than carboxypeptidase Y at acidic pH.     

Tunicate Endostyles: Histochemistry of Acidic Glycoproteins Re-examined in Ciona intestinalis and Styela plicata

Previous reports of tunicate endostyles have suggested that they contain little or no acidic glycoproteins in the glandular zones. The endostyles of Ciona intestinalis and Styela plicata were examined after anhydrous fixation with cyanuric chloride. Polyanions were stained with alcian blue (AB) at pH 2.5 or azure A, while sulfomucins were stained with high-iron diamine (HID) or AB at pH 1.0. Endostyles were also tested for sensitivity to acid hydrolysis (AH) and saponification. In Ciona zones 2 and 4 sometimes demonstrated positive HID and AB 1.0 responses. Almost invariably zone 6 was AB+ at pH 2.5; zones 2 and 4 were frequently responsive to AB, but less intense. Each of these 3 zones, when AB+, was sensitive to AH. Responses by zones 3 and 5 to AB (pH 2.5), azure A and saponification suggest that these zones contain mostly nuclear material. In secretory zones 2, 4 and 6 histological responses are consistent with the histochemistry of sialomucins. Zones 1 and 8 had sulfated material in the apical edges in both animal groups. Among the fixatives used for Ciona, only anhydrous fixation demonstrated most of the positive responses to polyanion-sensitive stains.     

Proton equilibria in the minor groove of DNA.

Poisson-Boltzmann calculations by Pack and co-workers suggest the presence of regions of increased hydrogen ion density in the grooves of DNA. As an experimental test of this prediction, we have attached proton-sensitive probes, with variable linker lengths, to random-sequence DNA at G sites in the minor groove. The amino groups of beta-alanine, gamma-aminobutyric acid (GABA), and epsilon-aminocaproic acid have been coupled at pH 5, via a formaldehyde link, to the exocyclic amino group of guanine, utilizing a reaction that has been extensively investigated by Hanlon and co-workers. The resulting adducts at pH 5 retained duplex B form but exhibited typical circular dichroism (CD) changes previously shown to be correlated with the presence of a net positive charge in the minor groove. Increases in the solvent pH reversed the CD spectral changes in a manner suggesting deprotonation of the carboxylic acid group of the adduct. These data were used to calculate an apparent pK(a) for the COOH. The pK(a) was increased by 2.4 units for beta-alanine, by 1.7 units for GABA, and by 1.5 units for epsilon-amino caproic acid, relative to their values in the free amino acid. This agrees well with Poisson-Boltzmann calculations and the energy minimization of the structures of the adducts that place the carboxyl groups in acidic domains whose hydrogen ion density is approximately 2 orders of magnitude greater than that of bulk solvent.     

Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Combination of Certain Fluorane Derivative dyes with Bacterial cells at Different Hydrogen on Concentrations

While acid dyes have not been widely used for staining bacteria, several suggestions for their use have been advanced (Conn and Holmes, 1926; Maneval, 1941). In general these procedures call for adding an acid to the stain to intensify the combination of the dye with the bacterial cell. McCalla and Clark (1941) have demonstrated greater adsorption of acid dyes at lower pH levels. Conn and Holmes (1926) list a number of dyes derived from fluorane and compare their relative acidic tendencies, color intensities, and other properties. In a study of the influence of the acidic properties of dyes on their combination with bacterial cells at various hydrogen-ion concentrations, the data herein reported were obtained.