The Sertoli cell of the water buffalo (Bubalus bubalis) during the spermatogenic cycle

Summary Ultrastructural features and morphometric evaluations of buffalo Sertoli cells are reported for the six phases of the spermatogenic cycle. The phases of the tubular seminiferous epithelium are identified according to characteristic cellular associations with completed spermiation as demarcation between two cycles. Average tubular diameter (245 m) and epithelial height (61 m) do not vary significantly during the cycle. The relative Sertoli cell volume in the seminiferous epithelium varies between 30% (phase 4) and 39% (phase 8). The calculated volume of a single Sertoli cell increases from a nadir of 7118 m³ in phase 3 abruptly to a maximum of 8968 m³ in phase 4 and is then gradually reduced during the following phases. The Sertoli cell surface area shows a similar trend: it amounts to 11105 m² in phase 3 and to 14260 m² in phase 4. The contact area of the Sertoli cell with adjacent cells and structures is subject to characteristic changes; from the expansion of basal Sertoli-Sertoli contacts it is concluded that the blood-testis barrier in the buffalo is particularly tight during phases 8, 1 and 2. The irregularly contoured nucleus contains a vesicular nucleolus, has a calculated volume from 465 m³ to 543 m³ and occupies 5 to 7% of the cell. Volume percentages of mitochondria (4%), Golgi apparatus and lysosomal bodies are rather constant during the cycle. Whorls and orderly arranged aggregates of the smooth endoplasmic reticulum occur in basal location as well as in close association with elongating spermatids. Smooth ER is the organelle that exhibits the most prominent changes during the Sertoli cell cycle: it occupies 5.79% in phase 3 and 20.9% in phase 4 of the total cellular volume. Phagocytosis of residual bodies is insignificant in this species and a lipid cycle is absent in buffalo Sertoli cells.     

Basal lamina of human seminiferous tubule—Its role in material transport

Summary Electron microscopy of testicular biopsies obtained from two adult males with tunica vaginal hydrocele revealed some protrusions from the basal lamina to the germinal epithelium in the seminiferous tubule. The protrusions were of three types: some between the spermatogonia and Sertoli cells, some directly within the Sertoli cell cytoplasm and others inside the spermatogonia. The protrusions inside the spermatogonia were only 0.5 m deep whereas the other types were from 1–11 m deep. Occasionally some cut off portions of these protrusions were seen inside the ground cytoplasm of the Sertoli cell without an apparent connection with the original stalk. The matrix of the protrusions contained a homogenous component (composed of a fine filamentous element) and granular and membranous components. These components closely resemble the materials found in the basal lamina of the seminiferous tubule. It has been suggested that under mild pathological conditions, i.e., hydrocele, the junctions between the seminiferous tubule epithelium and the basal lamina become somewhat more flexible. As a result, the protrusions become longer and a passage might be formed to allow the flow of raw materials in or out of the seminiferous tubule.This work was supported by USPHS Research Grant HD-03266 and GRS 94802     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Endocrine cells in the human fetal small intestine

Summary In this report we describe the time of appearance and ultrastructural features of enteroendocrine cells (EECs) in the human fetal small intestine (SB) between 9 and 22 weeks gestation. Thirteen distinctive EECs were identified in fetal SB. Two of these, not found in normal adult SB, appeared within the stratified epithelium of the proximal SB at 9–10 weeks. They were arbitrarily termed primitive and precursor cells. As in all fetal EECs, the pale cytoplasm of the primitive cell contains a distinctive population of secretory granules (SGs). Primitive cell SGs average 200–330 nm; some have dense cores with lucent halos while others are filled with a homogeneous dense or flocculent material. The SGs of the precursor cells are larger, averaging up to 1 m in diameter and their contents vary in electron density. A third group of cells not described in normal adult SB was arbitrarily termed transitional cells. These have two populations of SGs; one resembles the SGs of the precursor cells, and the other resembles the SGs of some of the specific adult type EECs. Transitional EC, S, I and G cells are seen. In addition, mature appearing EC, S, G, I, L, D, and D₁ cells were identified by 12 weeks of gestation. The primitive, precursor, and transitional cells may represent sequential developmental precursors of adult type EECs.Supported by Research Grant AM-17537 from the National Institutes of Health, Besthesda, MarylandThe authors would like to thank Ms. Linda Barstein for her excellent technical assistance     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Ultrastructure of light cells in the dolphin thyroid

Summary The ultrastructural appearance of the light cells is described in the thyroid gland of the common (Pacific) dolphinDelphinus bairdi. The appearances of active light cells are discussed in relation to the production of thyrocalcitonin and the special osteological characteristics of marine mammals. The presence of nerve processes possibly associated with light cells is discussed.Also known as parafollicular cells (Nonidez, 1932) and C cells (Pearse, 1966).     

Stage-specific localization of cytoskeletal actin mRNA in murine seminiferous tubules and intestinal epithelia as demonstrated by in-situ hybridization

Summary In-situ hybridization experiments have been performed using isoactin ( and )-specific riboprobes in various tissues of the rat and mouse. Distribution of the grains of actin mRNAs for both and types was similar throughout sections of the rat testis. Although both mRNAs were evenly distributed in the seminiferous tubule, extremely heavy labeling was observed in about 10% of the seminiferous tubules that could be identified as stage XII of spermatogenesis. At high magnification, grains of the mRNA were found in the cytoplasm of elongating spermatids and in the Sertoli cell cytoplasm at the adluminal side. Much higher density of the grains of mRNA was observed in the neck region of the spermatids at stage XII. Thus, the dense distribution of cytoskeletal actin mRNAs is stage-specific in the tubule during spermatogenesis in the rat. The high expression of both and actin mRNAs was also observed in the epithelial cells of the intestinal crypts.     

Sertoli cells of adult rats “in vitro”

Summary A cell line obtained from isolated seminiferous tubules of adult rat testis has been studied in vitro over a period of 35 days.Light and electron microscopic studies performed from hour 2 to the end of culture have shown the presence of a monomorphic cell population. After 5–6 days of culture the cells formed a monolayer. The cytoplasm of the cells contained numerous lipid bodies and produced numerous projections. The nucleus showed several indentations and one or more nucleoli. From the 9th to the 15th day of culture the cells developed a large amount of endoplasmic reticulum, Golgi apparatus and aggregates of electron dense granules. From the 20th to 40th day the cell cultures progressively degenerated.Immunochemical analysis of the culture medium revealed the presence of estradiol-17, which reached its maximum production rate from the 8th day to the 18th day of culture. Corresponding to cell involution estradiol concentration underwent a rapid decrease.On the basis of morphological and biochemical data the cells could be considered Sertoli cells.This work was supported by Grants n. 74.00155.04 and n. 75.01224.04 from the Consiglio Nazionale delle Ricerche (C.N.R.), Rome, Italy, and by Istituto di Ricerca F. Angelini, Rome, ItalyPart of this work was presented at the 10th Italian Congress of Electron Microscopy. Ostuni 1–4 October 1975The excellent technical assistance of Miss Laura Vassallo, Daniela Venturini and Mr. Massimo Rosati and Mario Termine is deeply appreciated     

Polygonal inequalities as a key to neuronic equations

Neuronic or decision equations, first proposed as a mathematical model of neural activity, have shown, after their exact, compact solution was found, typical behaviours that make them natural tools for General Systems studies. It is shown here that their mathematical investigation is remarkably furthered by generalizing the triangular inequality to polygonal ones. These permit the immediate computation of the tensorial expansion of linearly separable boolean functions, and exhibit clearly the connection between their continuous and discontinuous aspects.     

Voltage dependence of the basolateral membrane conductance in theAmphiuma collecting tubule

Summary The basolateral potassium conductance of cells of most epithelial cells plays an important role in the transcellular sodium transport inasmuch as the large negative equilibrium potential of potassium across this membrane contributes to the electrical driving force for Na⁺ across the apical membrane. In the present study, we have attempted to establish, theI-V curve of the basolateral membrane of theAmphiuma collecting tubule, a membrane shown to be K⁺ selective. TransepithelialI-V curves were obtained in short, isolated perfused collecting tubule segments. The shunt conductance was determined using amiloride to block the apical membrane Na⁺ conductance. In symmetrical solutions, the shuntI-V curve was linear (conductance: 2.2±0.3 mS·cm^–2). Transcellular current was calculated by subtracting the shunt current from the transepithelial current in the absence of amiloride. Using intracellular microelectrodes, it was then possible to measure the basolateral membrane potential simultaneously with the transcellular current. The basolateral conductance was found to be voltage dependent, being activated by hyperpolarization: conductance values at –30 and –80 mV were 3.6±1.0 and 6.6±1.0 mS·cm^–2, respectively. BasolateralI-V curves were thus clearly different from that predicted by the constant field model. These results indicate that the K⁺-selective basolateral conductance of an amphibian collecting tubule shows inward (anomalous) rectification. Considering the electrogenic nature basolateral Na–K-pump, this may account for coupling between pump-generated potential and basolateral K⁺ conductance.     

Alpha-to-beta structural transformation of ovalbumin: heat and pH effects

Ovalbumin is an important member of the serpin superfamily without inhibitory activity. The heat- and pH-induced -to- structural transformations of ovalbumin were investigated by means of circular dichroism and binding of ANS and Congo red dyes. The native ovalbumin shows a mixture of -helix and -sheet, while both the heat and alkali treatments are able to transform the native protein into a predominance of -sheet secondary structure. The free energy changes during transitions to the unfolded state are 5.19 kcal/mol from the native state and 4.00 kcal/mol from the heat-treated one. The binding abilities of the heat-treated and the alkali-treated forms to ANS and Congo red suggest that the altered forms exhibit hydrophobic exposure and intermolecular interaction. The results substantiate that the altered protein forms bearing increased -sheet structures are prone to aggregation, which is implicated in the pathogenesis of some conformational diseases.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Design of superactive and selective integrin receptor antagonists containing the RGD sequence

Summary Integrins play a major role in cell-cell and cell-matrix interactions. The majority of the different types of integrins recognize the tripeptide sequence arginine-glycine-aspartic acid (RGD). To explore the spatial requirements of the pharmacophore for receptor selectivity and high activity, a new procedure, spatial screening, was used. The procedure is based on the experience that the conformation of small cyclic peptides is mainly determined by the chirality of the amino acids (and glycine or proline). For example, cyclic pentapeptides with one d and four l amino acids prefer a II'/ conformation. The sequence RGDFV was shifted around this spatial II'/ template by synthesis of five peptides in which one of the amino acids was used in d-configuration. It turned out that cyclo(-RGDfV-) is a selective inhibitor for the _v3 integrin, which is strongly expressed in cancer cells. Systematic variations with different turn mimetics, retro-inverso structures, modified peptide bonds and sugar amino acids are discussed.     

Integrins: cell adhesives and modulators of cell function

Summary Integrins encompass a family of cell-surface molecules which play a crucial role in cell-cell and cell-extracellular matrix interaction. Of these heterodimeric transmembrane glycoproteins (consisting of an and chain) as yet at least 20 different types have been described, all with a different pattern of reactivity with extracellular matrix components. In this review the cell and tissue distribution of the integrins is discussed, with special emphasis on immunohistochemical localization of the ₁ integrins and the ₆₄ integrin. The ₁ integrins comprise a subfamily in which eight chains combine with one (the ₁) chain. The ₂₁, ₃₁ and ₆₁ and the ₆₄ integrins are expressed on a wide variety of epithelia on the basolateral surface or exclusively on the basal surface facing the basement membrane (e.g. ₆₁ and ₆₄). Leucocyte integrins, which share a common ₂ chain, occur almost exclusively on white blood cells and their precursors. The vitronectin receptors, which share a common _v chain, occur in a wide variety of cell types. Integrins play a major role in the interaction of the cell with the extracellular matrix in order to create and maintain tissue architecture. It has become clear, however, that through integrin-ligand interaction cell function is also modulated. Furthermore, in pathological conditions integrins play a role of some significance. Integrins mediate leucocyte traffic in developing inflammatory processes and function in neoplastic growth when it comes to invasion and metastasis.     

Histochemical demonstration of peptidases in the human kidney

Summary The localization of several peptidases in the human kidney was investigated histochemically. The membrane-bound peptidases, aminopeptidase A (APA), aminopeptidase M, -glutamyltransferase (-GT) and dipeptidylpeptidase IV, were mainly demonstrable in the brush border of the proximal tubule. In addition, APA was found in the glomeruli, while -GT was found in the basal labyrinth of the proximal tubule. The lysosomal peptidases, dipeptidylpeptidase I and cathepsin B, were most strongly concentrated in the different-sized lysosomes of the proximal tubule, but they were also found in the small lysosomes of the distal tubule. Dipeptidylpeptidase II showed only a weak reaction in lysosomes of the proximal tubule. It is concluded that, in comparison with other previously studied species, the human kidney has a well-developed equipment with membrane-bound and lysosomal peptidases.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

The mature mesonephric nephron of the rabbit embryo

Summary In freeze-fracture replicas of the entire cross-fractured mesonephros of 18 day rabbit embryos the basolateral and luminal cell faces of the different nephron segments were studied and compared with their metanephric counterparts. In the proximal tubule, the shallow zonula occludens exhibited only 1–2 strands and resembled the corresponding metanephric zonula, a very leaky type, which was found with a considerable paracellular flow component in sites of isotonic reabsorption. Gap junctions were restricted to the proximal tubule and were seen more frequently in its terminal segment. The distal tubule harboured two types of tight junctions. The most common type, a band of 5–8 closely parallel strands, matched the zonula occludens of the metanephric straight distal tubule. The observed particle density of the basolateral membrane (2,500±328/m²) was less than that of the proximal tubule (2,642±306). In addition, the collecting tubule exhibited a zonula occludens of the tight variety similar to that which occurred in the metanephric collecting duct. Rod-shaped particles of the luminal membrane were mainly concentrated in some of the intercalated cells but also had developed on principal cells, and occasionally, in the distal tubule. The Wolffian duct, with a deep tight zonula occludens, had an obviously rather inactive epithelium with no conspicuous transport-linked membrane specializations.     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.