The coelomic elements of sea urchins (Strongylocentrotus)

Summary The four coelomocyte classes of the red sea urchin,Strongylocentrotus franciscanus, described by light-microscope studies, are confirmed and the fine structure described. Material examined included fresh, non-aggregated cells; partially aggregated ones that had been heldin vitro up to four days; and aggregated cells heldin vitro for 40 days. Leukocytes from youngin-vitro preparations differed from most fresh leukocytes by having enlarged dense nucleoli and enlarged rough endoplasmic reticulum, which was often filled with secretion, and sometimes connected to the perinuclear cisterna. Leukocytes held 40 daysin vitro were mainly plasmodial. Unlike cells held a limited timein vitro, the 40-day leukocytes had nuclei much like those in fresh preparations.The three classes of spherule-bearing cells (vibratile cells, red spherule cells, and colorless spherule cells) differed greatly in ultrastructure, and varied in appearance according to the fixative and pH present during fixation. Vibratile-cell spherules were of biphasic construction, suggesting the condition of certain vertebrate mast cells. Red spherule cells occurred in two forms. The most common form in fresh preparations had despherulated,i.e., lacked material in the spherules; and the spherules of the second type were filled with either granular or homogeneous material. Colorless spherule cells had evenly and finely granular material in the spherules. Colorless spherule cells were uncommon or missing in material that had been heldin vitro. Certain unidentifiable spherule cells occurred in some preparations.Although samples are small, it is notable that in May and June, recognizable glycogen was present only in leukocytes that had been heldin vitro, not in any fresh cells. Glycogen occurred in fresh cells of all classes from samples taken in December and February (during or shortly before the normal spawning season ofS. franciscanus). Unlike the cells in fresh preparations made in May, June, and December, fresh leukocytes and vibratile cells taken in February often had extremely lobed nuclei and considerably developed rough endoplasmic reticulum.This investigation was supported by Public Health Service Research Grant No. 9296 (to P. T.Johnson) from the National Institute of Allergy and Infectious Diseases.     

Variant forms ofα-2-l-fucosyltransferase in human submaxillary glands from blood group ABH “secretor” and “non-secretor” individuals

The properties of the -2-l-fucosyltransferases in submaxillary gland preparations from blood group ABH secrefors and non-secretors were compared. The level of activity in the non-secretor gland homogenates amounted to about 5% only of that found in the secretor gland preparations. The enzymes from the two sources differed in solubility properties, charge and affinities for donor and acceptor substrates. The enzyme from secretor glands showed a preference for acceptors with Type 1 [d-galactosyl(1–3)-N-acetyl-d-glucosamine] structures whereas the enzyme from non-secretor glands had a preference for Type 2 [d-galactosyl(1–4)-N-acetyl-d-glucosamine] structures.These results demonstrate that expression of the secretor gene (Se) is associated with a molecular form of the -2-l-fucosyltransferase that is different from the species present in the same tissue when theSe gene is not expressed.     

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic β-cells and HIT cells

Summary ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic -cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 m and as high as 500 m, with maximal activation between 10 and 100 m ADP in mouse -cell membrane patches. At concentrations greater than 500 m, ADP inhibited K(ATP) channels while 10 mm virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mm ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of -cell membrane potential, increasing ADP in the 10 and 100 m range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize -cells.The results suggest that (i) K(ATP)-channel activity is not well predicted by the ATP/ADP ratio, and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10–100 m range as suggested by biochemical studies.We would like to thank Mr. Louis Stamps for expert technical assistance and Dr. Wil Fujimoto and Ms. Jeanette Teague for generously providing HIT cells obtained from Dr. Robert Santerre at Eli Lilly. We would also like to thank Dr. Michel Vivaudou for providing the program ALEX. Support was provided by the NIH and the Department of Veterans Affairs.     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Purine glucosylating activity in cell suspension cultures ofSolanum tuberosum L.

Cell suspension cultures ofSolanum tuberosum L. cv. Adretta were established from leaf-derived calluses. In the search for purine glucosylating activity, the metabolism of 6-benzylaminopurine was studied. The main metabolite of BA was isolated and identified as 6-benzylaminopurine 7--d-glucopyranoside indicating the occurrence of purine glucosylating activity.Abbreviations BA 6-Benzylaminopurine - [3G]BA BA 3--d-glucopyranoside - [7G]BA BA 7--d-glucopyranoside - [9G]BA BA 9--d-glucopyranoside - RA Radioactivity - R _T Retention Time     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

On anixiopsis stercoraria (hansen) hansen, 1897 and its imperfect stage: Keratinomyces ajelloi vanbreuseghem, 1952

Summary It is herein reported on the discovery of the imperfect stage of an ascomycete,Anixiopsis stercoraria (Hansen)Hansen, 1897, which is also the imperfect stage of another ascomycete,Arthroderma uncinatum,Dawson &Gentles, 1961:Keratinomyces ajelloi Vanbreuseghem, 1952. Thus, the same imperfect form belongs to two different perfect forms.The discovery was made onMicrosporon sp. infected hairs in a case of typical tinea microsporina. The present investigation encompasses 16 strains, all cultured from tinea microsporina. There were both single and double infections.Nine of the cases yieldedAnixiopsis stercoraria along with its imperfect form:Keratinomyces ajelloi Vanbreuseghem, 1952.Four cases revealed a double infection,Keratinomyces ajelloi on one hand and,Arthroderma uncinatum as well asAnixiopsis stercoraria, on the other.The three final cases yieldedM. audouinii andKeratinomyces ajelloi with its perfect form,Anixiopsis stercoraria. K. ajelloi produces only macroconidia, never microconidia. What was mistakenly construed as the microconidia ofK. ajelloi, belongs as microconidia toAnixiopsis stercoraria. Out of these microconidia the development of true macroconidia was observed.Both the relationship of the perfect and imperfect forms ofAnixiopsis stercoraria and cultural characteristics of the perfect form and its pathogenicity are discussed.

---

Zusammenfassung Es wird über die Entdeckung der Konidialform eines Ascomyceten berichtet,Anixiopsis stercoraria (Hansen)Hansen, 1897, die zugleich die Konidialform eines anderen Ascomyceten ist,Arthroderma uncinatum,Dawson undGentles, 1961:Keratinomyces ajelloi Vanbreuseghem, 1952. Demzufogle gehört dieselbe Konidialform zwei verschiedenen Ascomyceten an.Die Entdeckung erfolgte an durchMikrosporon infizierten Haaren eines Falles einer klassischen Tinea microsporina. Die gegenwärtige Untersuchung umfasst 16 Fälle, alle von Tinea microsporina der behaarten Kopfhaut herstammend. Die Tinea microsporina war durch einfache und doppelte Infektion verursacht.Neunmal lieferte die KulturAnixiopsis stercoraria zusammen mit der Konidialform:Keratinomyces ajelloi.Viermal lag eine Doppelinfektion vor:Keratinomyces ajelloi, einerseits, undArthroderma uncinatum sowieAnixiopsis stercoraria, andererseits.Endlich lieferten die Kulturen in drei FällenM. audouinii undK. ajelloi zusammen mitAnixiopsis stercoraria. K. ajelloi entwickelt nur Makrokonidia und niemals Mikrokonidia. Was irrtümlicherweise als Mikrokonidia vonK. ajelloi aufgefasst wurde, gehört als MikrokonidiaAnixiopsis stercoraria an. Die Entwicklung von Makrokonidien aus diesen Mikrokonidien wurde beobachtet.Die Wechselbeziehung der Konidial- und Perithecialformen vonAnixiopsis stercoraria, ihr kultureller Charakter und ihre Pathogenität sind einer Diskussion unterzogen.

---

Résumé On s'y rapporte à la découverte de la forme conidiale d'un ascomycète,Anixiopsis stercoraria (Hansen)Hansen, 1897, laquelle est, en même temps, la forme conidiale d'un autre ascomycète,Arthroderma uncinatum,Dawson etGentles, 1961:Keratinomyces ajelloi Vanbreuseghem, 1952. Conséquemment, la même forme conidiale appartient aux deux ascomycètes différents.La découverte a été effectuée sur des poils infectés parMicrosporon sp. dans un cas d'une teigne microsporique typique. La présente investigation embrasse 16 cas, provenant tous des cas des teignes microsporiques du cuir chevelu. Des teignes microsporiques ont été causées ou par une simple ou par une double infection.Neuf fois on a obtenu des cultures deAnixiopsis stercoraria avec sa forme conidiale:Keratinomyces ajelloi.Quatre fois il y avait d'infection double:Keratinomyces ajelloi, d'une part, etArthroderma uncinatum etAnixiopsis stercoraria, d'autre part.En fin, les cultures ont démontré en trois cas,M. audouinii etK. ajelloi avecAnixiopsis stercoraria. K. ajelloi ne developpe que des macroconidies et jam ais des micronidies. Ce qu' on avait conçu par erreur comme microconidies deK. ajelloi, appartient comme microconidies à l'Anixiopsis stercoraria. Le développement des macroconidies à partir de ces microconidies a été observé.La relation des formes conidiale et périthéciale del'Anixiopsis stercoraria, son charactère culturel et sa pathogénie ont été discutés.

     

Electron microscopy of taste buds of the rat

Summary The taste buds of the circumvallate papillae have been examined by electron microscopy in OsO₄-fixed, PTA stained material or after KMnO₄ fixation. The microvilli of the receptor cells have terminal dilatations which presumably give an increased surface area for transduction. The extracellular spaces at the necks of the receptor cells near the bases of the microvilli are interrupted by closed contacts.The synapses have a well defined synaptic cleft suggesting a chemical rather than an electrical mode of transmission. Synaptic membrane specialisations differ from the membrane thickenings of other types of synapse. Presynaptic dense projections are present but there is no well define postsynaptic thickening. Vesicles occur in both pre- and postsynaptic components, but it is debatable whether or not they should be termed synaptic vesicles. Acknowledgements. We are indebted to Professor J. Z. Young, F. R. S., for his stimulating support, and to Mr. S. Waterman for skilled photography.     

Comparative studies on the localization of esteroproteases and kallikrein-like activity in primate organs

Summary Various organs of three species of monkey were screened histochemically for esteroproteases usingN-acethyl-l-methionine--naphthylester ( N-O-met) as the substrate and also for enzymes with kallikrein-like activity usingd-Val-Leu-Arg-4-methoxy-2-naphthylamide as the substrate. Characteristic differences were found in the localization of the reaction products obtained with both substrates. In the main salivary glands, esteroproteases ( N-O-met reactivity) were found in mucous cells (submandibular gland), intercalated duct cells (parotid gland), acinar cells (sublingual gland), striated and interlobular duct cells (all glands). They were also localized in superficial lining epithelial cells of the digestive system, in liver cells, and acinar cells of the pancreas.Enzymes with kallikrein-like activity were found only in the striated and interlobular duct cells of salivary glands, in acinar cells of the pancreas, and in proximal tubular cells of the kidney. Free cells (including mast cells) normally distributed in the connective tissue of various organs showed reactivity towards N-O-met. Some of these cells were also reactive against Val-Leu-Arg-4-MNA.     

Isolation of protoplasts by means of a “species-specific” autolysine inChlamydomonas

Summary Prior to fusion, gametes ofChlamydomonas reinhardii discard their cell walls. This naturally occuring phenomenon has provided the basis for a method of protoplast isolation from both gametes and vegetative cells within the genusChlamydomonas. When synchronized cultures of compatibleChlamydomonas gametes are mixed it is possible, after removal of the cells, to obtain a solution having a high cell wall lytic activity. That vegetative and gamete cells after treatment with this gamete-autolysine are indeed protoplasts has been proved by various light and electron microscopical methods.The species specifity of this autolysine, its difference to the previously described sporangialautolysine (Schlösser 1966) and furthermore its use in the large scale production of protoplasts is also described. Since this wall autolysine is a factor produced by the cells themselves at a particular stage in their life cycle it represents a non-foreign agent in contrast to all other enzymic methods previously employed for protoplast isolation.     

Folding versatility of the C-terminal tetrapeptide amide sequence of the lipopeptaibol antibiotics trichodecenin and trichogin

Summary An X-ray diffraction analysis ofZ-l-Leu-Aib-Gly-l-Ile-l-Leu-OMe, containing the N-acylated tetrapeptide amide sequence-l-Leu-Aib-Gly-l-Ile-, showed that in the crystal state the carbonyl group preceding thel-Leu¹ residue acts as the acceptor of two C=OH–N intramolecular H-bonds, which give rise to an-l-Leu¹-Aib²-type-III' -turn and an-l-Leu¹-Aib²-Gly³-l-Ile⁴--turn, respectively. A second (type-I') -turn encompasses the-Aib²-Gly³-sequence. This is the third type of folding motif known for that tetrapeptide sequence, considering also those already published for the C-terminal segment of the lipopeptaibol antibiotics trichodecenin I and trichogin A IV.     

Abscisic acid metabolism —vacuolar/extravacuolar distribution of metabolites

The biotransformation of abscisic acid (ABA) was studied in cell suspension cultures of Lycopersicon esculentum. The ABA was converted by the cells to phaseic acid, nigellic acid, dihydrophaseic acid, abscisic acid--D-glucopyranosyl ester (ABA-Glc) and other ABA and phaseic acid conjugates. Investigation of their cellular distribution showed that the conjugated forms were located only in the vacuoles whereas ABA and its acidic metabolites were found mainly in the extravacuolar fractions. Our results, together with a number of studies on the increase of ABA-Glc as a response to stress, allow us to propose that ABA-Glc is irreversibly compartmented in the vacuoles of plant cells.Abbreviations ABA abscisic acid - ABA-Glc -D-glucopyranosyl ester of ABA - DPA 4-dihydrophaseic acid; nigellic acid=3-methyl-5-(1-hydroxy-2-hydroxymethyl-6-dimethyl-4-oxo-cyclohex-2-enyl)-penta-2Z, 4E-dienoic acid - PA phaseic acid     

Critical survey of the present stand of the production of perfect stage of organs of fructification in dermatophytes

Summary Very little progress had been made on the question whether organs of higher fructification occur in dermatophytes. Only two significant studies can be noted. One was the elaboration of the hypothesis byMatruchot &Dassonville that dermatophytes because of morphological similarities with certain fungi (Myxotrichum, Gymnoascus, Arachniotus) belong with them to the Gymnoascaceae. However, this generalization was unwarranted, since their hypothesis was based on a single strain of a dermatophyte, designated by them as Trichophyton, sp. without the species having been more closely determined. Moreover, they did not produce any evidence for their generalization that all dermatophytes should, or necessarily must belong to the Gymnoascaceae. Finally,Matruchot &Dassonville had never seen either pycnidia or perithecia in their cultures. To say the least, their hypothesis, whether right or wrong, must await further demonstrations that pycnidia and/or perithecia do exist in the dermatophytes.The only apparently successful attempt at solving the question of the existence of higher organs of fructification in dermatophytes has been made byNannizzi. His findings must be considered valid, unless duplications of his experiments would prove to the contrary. However, the same objection has to be made against his generalization as was raised against the generalization ofMatruchot &Dassonville. This is that ALL dermatophytes must necessarily belong to the Gymnoascaceae.Nannizzi succeeded in producing pycnidia only in the form-genusTrichophyton, in the speciesTr. mentagrophytes (Ch. Robin, 1853) andTr. equinum Matruchot. He also was successful in his endeavor in the form—genusAchorion, in the speciesAchorion gypseum Bodin. However, his attempts to produce higher organs of fructification inMicrosporon lanosum Sabouraud and inAchorion Quinckeanum Zopf failed.

---

Zusammenfassung Versuche, betreffs des gegenwärtigen Standes der Fruchtkörperproduktion in Dermatophyten, die sich nun auf sechs Jahrzehnte erstrecken, sind kritisch beleuchtet worden.Es konnte nur ein einziger, anscheinend erfolgreicher Versuch verzeichnet werden, der vonNannizzi, 1926. Mit seiner speziellen Technik, indem er Dermatophyten auf natürlichen Nährböden (Federn, Leder, Knochen, Haare) züchtete, war es ihm gelungen, Pykniden in der Form-GattungTrichophyton, in den ArtenTr. mentagrophytes (Ch. Robin, 1853) undTr. equinum Matruchot hervorzurufen. In der Form-GattungAchorion sind Pykniden in der ArtAchorion gypseum Bodin produziert worden in einem Nährboden von Waldboden gemischt mit Stückchen von altem Leder. Jedoch sind seine Versuche, Pykniden inMicrosporon lanosum Sabouraud und inAchorion Quinckeanum Zopf hervorzurufen, fehlgeschlagen.

     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Heterogeneity of calcium compartmentation: Electron probe analysis of renal tubules

Summary The objective of this study has been to determine the intracellular localization of calcium in cryofixed, cryosectioned suspensions of kidney proximal tubules using quantitative electron probe X-ray microanalysis. Two populations of cells have been identified: 1) Viable cells, representing the majority of cells probed, are defined by their relatively normal K/Na concentration ratio of 41. Their measured Ca content is 4.1±1.4 (sem) mmol/kg dry wt in the cytoplasm and 3.1 ± 1.1 mmol/kg dry wt in the mitochondria, or an average cell calcium content of 3.8 mmol/kg dry wt. 2) Nonviable cells, defined by the presence of dense inclusions in their mitochondria and a K/Na concentration ratio of 1. The Ca content is 15±2 mmol/kg dry wt in the cytoplasm and 685±139 mmol/kg dry wt in the mitochondria of such cells. Assuming 25 to 30% of the cell volume is mitochondrial, the overall calcium content of such nonviable cells is 210 mmol/kg dry wt. The presence of these inclusions in 4 to 5% of the cells would account for the average total Ca content measured in perchloric acid extracts of isolated proximal tubule suspensions ( 18 nmol/mg protein or 12.6 mmol/kg dry wt). Whole kidney tissues display a large variability in toal Ca content (4.5 to 18 nmol/mg protein, or 3.4 to 13.5 mmol/kg dry wt), which could be accounted for by inclusion in 0 to 4% of the cells. The electron probe X-ray microanalysis (EPXMA) data conclusively demonstrate that thein situ mitochondrial Ca content of viable cells from the kidney, proximal tubule is low and support the idea that mitochondrial Ca may regulate dehydrogenase activity but probably does not normally control cytosolic free Ca.     

Metabolic variants of intestinal alkaline phosphatase in relation to fat absorption: in situ demonstration with the organ-specific inhibitors l-phenylalanine and l-homoarginine

Summary The localization of rat intestinal alkaline phosphatase has been studied in relation to fat absorption. The observations support a theory of conversion, within the intestinal mucosa, of intestinal type to liver type alkaline phosphatase when the criterion of differential sensitivity to two amino acid inhibitors, l-phenylalanine, and l-homoarginine, is applied.Following a three hour in vivo exposure to mixtures of oleic acid, sodium taurocholate, and lauric acid, the epithelium becomes depleted of its l-phenylalanine-sensitive, intestinal type alkaline phosphatase. At the same time, enriched activity is seen in the lamina propria; this activity is both particulate and diffuse, and is present both in the connective tissue matrix and in cells, including macrophages, eosinophils and lymphocytes. Most of this enzyme is inhibited by l-homoarginine, a property characteristic of liver type alkaline phosphatase.The localization of enzyme-positive particles 0.5 to 1.0 in diameter in both epithelium and lamina propria appears identical to that of particulate fat. A physical association between transport of absorbed fat and metabolic conversion of intestinal type alkaline phosphatase is postulated.This work was aided in part by grants-in-aid [CA-3332-01, K6-CA-18,453] from the National Cancer Institute, National Institutes of Health, U.S.P.H.S. and the John A. Hartford Foundation, Inc.Pre-doctoral trainee, U.S.P.H.S. grant GM01451.Holder of a Juan Marsh Foundation Fellowship, Lemuel Shattuck Hospital.     

The fine structure of the capillaries and their surroundings in the cerebral hemispheres of Myxine glutinosa (L.)

Summary The fine structure of blood capillaries and their surroundings in the hemispheres of the hagfish, Myxine glutinosa, is described. As in other species, one or more endothelial cells delinate the lumen. Where such cells meet, they show complicated interdigitations. At some places tight junctions (zonulae occludentes) are found.The cytoplasm pressents at places many glycogen particles, ergastoplasmic membranes and free ribosomes in addition to the usual organelles. In addition, the luminal surface gives origin to deep invaginations from which channels penetrate into the cytoplasm. Numerous, presumably pinocytotic vesicles ranging from 450–1000 Å in diameter are present in the endothelial cell cytoplasm. A special type of cytoplasmic tubules is also present. The basement membrane is very thick, and measures 1000 Å at the narrowest place.Only one type of glial cell is present. This cell has certain characteristics in common with astrocytes of mammals but is for certain reasons considered to be a primitive glial cell. All capillaries have a complete lining of glial elements. The glial processes in contact with the vessels vary in shape and size. Glia cell bodies also participate. Closed contacts are present between the glial cells, but also desmosomal attachments are observed.The finding that capillaries in the hemispheres of Myxine are entirely surrounded by processes or bodies of glial cells shows that even at the most primitive vertebrate level glial cells are essential for the transport of material from the blood into the nervous tissue. Addendum. After the paper was submitted for publication, Dewey and Barr have considered the possible role of the zonulae occludentes in electronic coupling between cells [J. Cell Biol. 23, 553–585 (1964)]. The reader is referred to this paper.