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PubMed1989

High-resolution localization of lactoferrin in human neutrophils: labeling of secondary granules and cell heterogeneity.

We studied by electron microscopy the topography of the iron-binding protein lactoferrin in human peripheral blood neutrophils by immunogold labeling of thin-sectioned cells. We show that fixation in 0.3% glutaraldehyde, supplemented by 2% formaldehyde, and combined with embedding in the hydrophilic resin LR White results in excellent preservation of the lactoferrin antigenicity and of the neutrophil microanatomy. Most of the immunogold marking was seen on the specific (secondary) granules of polymorphonuclear leukocytes, thus strengthening prevailing views on the subcellular distribution of lactoferrin in neutrophils. We document that a minority (8-10%) of neutrophils showed significant amounts of lactoferrin associated with a flocculent, protein-like material seen inside large cytoplasmic vacuoles. We show that circulating neutrophils comprise 6-10% of cells that are virtually devoid of lactoferrin labeling. This finding provides specific cytochemical support for the concept that human polymorphonuclear leukocytes are made up of a heterogeneous population of cells.

Adult↗

PubMed1990

Secretion of mucoid material by lymphokine-activated killer cells: study by light and electron microscopy.

When lymph node cells from nude mice were grown on embryonic fibroblast monolayers together with rat interleukin-2, only one type of colonies developed. These colonies were composed of cytotoxic cells termed "granular/lymphokine-activated killer/mucus-secreting cells" (LAK-GM). An extensive differentiation course, in which all the cellular components were involved, ended with a population of short-lived, mature, nondividing large cells that apparently synthesized and deposited a flowing mucoid material (FMM) that stained distinctly blue with periodic acid-Schiff/alcian blue (PAS-Ab) at pH 1 and distinctly red by the naphthol AS-D-chloracetate method for specific esterase. So far, the best monolayers to trigger the FMM synthesis were those prepared from 16- to 18-day-old whole embryos. These cells were compared with LAK cells that developed on monolayers (such as embryonic skin or adult kidney) that did not trigger FMM synthesis. They were also compared with other cell types that differentiated in colonies on the fibroblast monolayers: histiocytes (fixed macrophages), mixed granulocytes/monocytes, mucosal mast cells; and with populations of mature rat T-killer cells developed on same mouse monolayers. Features distinctive to the secreting LAK-GM cells were presence of masses of membrane-limited vesicles that were strictly confined to the surface of the cells in FMM-containing colonies. All transitional forms of budding activity could be seen on the cell surface facing the masses. Within the same cells, many granules displayed varying degrees of degradation, the granular material being transformed into flocculent material that formed small pools facing each degraded surface. Other characteristics of the LAK-GM lineage were the accumulation of glycogen prior to the appearance of the FMM, the presence of several structures of a ribosome-lamella complex in the LAK-GM in colonies that did not accumulate FMM, and filopodia commonly emerging from the pole proximal to the nucleus. Of various fixation methods tried, only after treatment with absolute alcohol and subsequent drying was the FMM stained with PAS-Ab. By subsequent wetting, the capacity to be stained was irreversibly lost. After incubation of the living cultures with the enzymes hyaluronidase or chondroitinases AC or ABC, the FMM disappeared. These observations suggest a triggering mechanism by the embryonic mesenchymal fibroblastoid cells for synthesis and secretion of mucous material that is a proteoglycan of the chondroitin sulfate group.

Animals↗

PubMed1977

Ultrastructure and histochemistry of the supportive structures associated with the radula of the slug, Limax maximus.

The odontophore and connective tissue-filled portion of the radular sac (called the "collostyle") of the slug, Limax maximus, were examined by light and electron microscopy. While both of these structures grossly resemble vertebrate cartilage, neither is composed of a type of tissue with the microscopic appearance and histochemical properties of cartilage. The roughly U-shaped odontophore possesses a thin capsule composed of connective tissue. The parenchyma of the odontophore consists of modified muscle cells which are organized into irregular groups by incomplete trabeculae composed of conventional muscle cells. The odontophoral cells are variable in size; they contain glycogen-filled "cores" as well as bundles of peripherally located filaments resembling myofilaments; and they are innervated like muscle cells. The nuclei of the cells are located eccentrically in the glycogen-filled portions of the cells and typically contain prominent nucleoli. The nuclei are surrounded by multiple small Golgi complexes and pleomorphic dense bodies resembling lysosomes. The extracellular matrix of the odontophore is very sparse and contains glycogen and fibrillar material but no histochemically demonstrable acidic mucosubstances. The collostyle consists of a gelatinous type of tissue somewhat like vertebrate mucoid connective tissue. The abundant extracellular matrix contains cross-banded filaments, a flocculent material disposed in wavy indefinite strands, and small electron-dense particles. The matrix contains histochemically demonstrable neutral and weakly acidic mucosubstances. The cell population of the collostyle includes solitary muscle cells and fibrocytes containing large quantities of glycogen.

Animals↗

PubMed1983

Ultrastructure of the adenohypophysis in the larval anadromous sea lamprey, Petromyzon marinus L.

The ultrastructure of the adenohypophysis (AH) in the larval anadromous sea lamprey, Petromyzon marinus L., was examined. The AH is subdivided into three regions, the pro-, meso-, and meta-AH. Cells of the nasopharyngeal stalk extend directly beneath the pro- and meso-AH to form the ventral surface of the gland. Some cells in the pro- and meso-AH are arranged into small follicles. Each region of the AH is characterized by a single granulated (secretory) cell type. Granulated cells constitute 80-90% of the pro-AH and contain secretory granules that range from 800 to 2400 A in diameter. Only 10-20% of the cells in the meso-AH are granulated and they contain much smaller secretory granules (400 to 1250 A diameter) than those in the pro-AH. Granulated cells constitute 80-90% of the meta-AH and contain only a few secretory granules, ranging from 1000 to 2500 A in diameter, and many vesicles containing either a loose flocculent or dense granular material. Non-granulated (stellate) cells are found in all regions. They are characterized by their long cell processes, abundant cytoplasmic filaments, and variable electron density. The appearance of organelles in these cells suggests they are nonsecretory. They may play a role in maintaining the structural integrity of the gland and the regulation of granule release in the pro-AH. Two types of nongranulated cells make up 80-90% of the meso-AH. Type I are stellate cells, type II may be undifferentiated cells. The functional significance of the secretory cells in the larval AH is discussed.

Animals↗

PubMed1989

Ultrastructural features of the developing eosinophils in bone marrow and spleen of the musk shrew, Suncus murinus.

Eosinophilopoiesis in the musk shrew, Suncus murinus, a representative of the order Insectivora, was studied by light and electron microscopy. To examine biochemical features of cytoplasmic granules, extraction with proteolytic enzymes was carried out on ultrathin sections of bone marrow. In this species, eosinophils are produced in the same manner in both spleen and bone marrow. Developing eosinophils were distinguished as belonging to four stages, recognized by ultrastructural changes in cytoplasmic organelles as well as the eosinophilic granules during maturation. Granulogenesis began by budding of vacuoles containing flocculent material from the concave face of the Golgi apparatus, in the promyelocyte to myelocyte stage. The matrix of developing granules transformed into a finely granular structure, and the large spherical granules of mature eosinophils were homogeneous without crystalline cores. It was shown by proteolytic enzyme extraction that the proteinaceous cores of mature granules were uniformly removed; there was no evidence that they contained crystalloid inclusions. These results indicate that shrew eosinophils can be regarded as cells that retain a prototype of eosinophil granules, probably like those of ancestral mammals rather than those of higher living Mammalia.

Animals↗

PubMed2003

Preparation of disodium fluorescein powders in association with lauric and capric acids.

In previous studies micronized disodium fluorescein (DF) powders were coated with lauric or capric acid. This was a precursor to evaluating the effects of the fatty acids on the hygroscopic growth of the DF aerosols. Studies of the association of fatty acids with DF reflected a limited interpretation of the data obtained. The present study examines more closely the phenomena involved in coating the DF. It is concluded that lateral interactions due to the hydrophobic aliphatic tails of the fatty acids give rise to adsorption, aggregation, and flocculation, all of which cause inflections in the association isotherm rendering it nonlinear.

Chromatography, Gas↗

PubMed1976

Filtration rates of pharmaceutical suspension systems.

This study was undertaken to find it a correlation exists between the filtration rate and the Hu/Ho ratio. For this purpose, flocculated and coagulated structures of sulfamerazine were used. Suspension settling profiles and filtration rates, using additives and additive-free sulfamerazine suspensions, were studied. A straight-line correlation was found. Effects of filter bed parameters on refiltration rates were studied, and a relationship was found by the use of the Kozeny-Carman equation. A straight-line relationship existed between the H1/Ho ratio and the filtration rate exhibited by the different suspension aggregates.

Aluminum↗

PubMed1976

Attainment of highly uniform solid drug dispersions employing molecular scale drug entrapment in polymeric latices.

The uniformity of distribution attainable for an amine drug in solid dispersions prepared using a molecular scale entrapment procedure was investigated. Excellent reproducibility of drug content throughout the entire entrapment product was demonstrated in both flocculated (high drug levels) and deflocculated (low drug levels) systems. Drug content and content uniformity were found to be predictable for deflocculated systems, even at high drug dilution ratios. Milling or particle-size fractionation appeared to have no effect on the distribution of drug throughout the solid dispersion entrapment products. Dry blending was inferior to molecular scale drug entrapment in distributing small quantities of drug uniformly.

Chemistry, Pharmaceutical↗

PubMed1977

Effect of chloroquine adsorption on acid reactivity of magnesium trisilicate.

Due to the adsorption of chloroquine by magnesium trisilicate, both the BP acid absorption test and the rate of hydrochloric acid uptake, as monitored by pH measurements, were significantly reduced. This reduction was dependent on the amount of chloroquine adsorbed, since multilayer adsorption produced relatively more suppressive effects than did monolayer adsorption. The presence of adsorbed chloroquine also decreased the amounts of magnesium released in an acid medium. The inhibition of the antacid property due to chloroquine adsorption may be attributed to the occupation of the reactive sites of the antacid surface by chloroquine and to a reduction of the surface of the antacid due to flocculation of the particles.

Adsorption↗

PubMed1980

Comparison of polyethylene glycol and polyoxyethylene stearate as excipients for solid dispersion systems of griseofulvin and tolbutamide II: dissolution and solubility studies.

The effects of joining a long-chain ester group with the polyethylene glycol molecule were studied in solid dispersion systems by comparing the dissolution and solution properties of such systems prepared from polyethylene glycol 2000 with those prepared from the nontoxic, water-soluble, solid excipient polyoxyethylene 40 stearate. Solid dispersion systems of griseofulvin and tolbutamide were prepared by physical mixing, fusion, or coprecipitation from ethanol. The compacted dispersion systems dissolved by progressive erosion, releasing floccules of microcrystals. The released microcrystals of tolbutamide (3--10 micrometer) were smaller than the original drug particles (approximately 20 micrometer), but those of griseofulvin were of similar size to the original particles. In general, the rate of and extent of release of each drug were greater from polyoxyethylene 40 stearate than from polyethylene glycol 2000 dispersions. The aqueous solubility and dissolution rate of nondisintegrating disks of each pure drug increased only slightly in the presence of polyethylene glycol 2000 but increased considerably with increasing concentration of polyoxyethylene 40 stearate due to micellar solubilization. Thus, polyoxyethylene 40 stearate generally is superior to polyethylene glycol 2000 in promoting the dispersion of the drugs in solids, disintegration of the compacted solids, and solubilization of the drug during dissolution.

Chemical Precipitation↗

PubMed1983

Effect of inorganic additives on solutions of nonionic surfactants V: Emulsion stability.

Electrolytes often break emulsions to which they were added as active ingredients, adjuvants, or impurities. The stability of oil-in-water emulsions containing octoxynol 9 NF as the emulsifier and various added electrolytes was investigated by measuring droplet size, turbidity, and oil separation on storage at various temperatures and in a centrifugal field at 25 degrees. Electrolytes were added to hexadecane emulsions after emulsification (direct addition); alternatively, hexadecane was emulsified in octoxynol 9-electrolyte mixtures (reverse addition). Xylene emulsions were prepared by direct addition only. Hexadecane emulsions containing 0.10% octoxynol 9 were considerably more stable than xylene emulsions containing 0.60% because the surfactant is practically insoluble in hexadecane, but miscible in all proportions with xylene. An emulsifier soluble in the disperse phase as well as the continuous phase evidently forms less stable interfacial films. The electrolytes investigated were sulfuric and hydrochloric acids, magnesium nitrate, and aluminum nitrate, which salt octoxynol 9 in by complexation between its ether groups and their cations; sodium thiocyanate, which salts the surfactant in by destructuring water; and sodium chloride and sodium sulfate, which salt octoxynol 9 out. The addition of these electrolytes at concentrations up to 2 or 3 m to hexadecane emulsions produced fast and extensive creaming, little or no flocculation, no coalescence, and only minor changes in droplet size or turbidity on storage at room temperature. The extent of coalescence during centrifugation was actually reduced by the additives. Such stability is unusual. Droplet size and turbidity depended mainly on octoxynol 9 concentration. The greatest decrease in the former and increase in the latter occurred when the concentration was increased from 0.10 to approximately 0.4%. All emulsions became slightly coarser on storage at 25 degrees. Stability at 50 degrees was impaired by aluminum nitrate and magnesium nitrate and to a lesser extent by sodium sulfate and sodium chloride. Reverse-addition emulsions differentiated better between the electrolytes than direct-addition emulsions. Electrolytes salting octoxynol 9 in, especially by complexation, generally produced the finest and most stable emulsions. Similarly, xylene emulsions were destabilized more by the electrolytes which salted the emulsifier out than by those salting it in. Centrifugation of hexadecane emulsions at 7800 X g compressed the creamed emulsion layer into a plug of clear, transparent, isotropic gel from which coalesced hexadecane separated slowly on further centrifugation.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Phenomena↗

PubMed1984

Nonaqueous cephalosporin suspension for parenteral administration: cefazolin sodium.

The flocculation-deflocculation behavior of cefazolin sodium (I) in nonaqueous media and the effect of surfactants as measured by zeta potential, sedimentation, and porosity were studied. A significant difference in zeta potential was observed when the particles were suspended in different nonaqueous media. The addition of surfactant produced a deflocculated state. The surfactant deflocculated the particles by a process of supersaturation and crystallization involving a surfactant-cefazolin complex. The shielding effect of the surfactant on the surface of the particles also apparently affected their electrophoretic properties. Kinetic studies on the stability of the drug as a function of temperature were conducted; it appears that the chemical stability in ethyl oleate at room temperature is adequate for a reasonable shelf life. The efficiency of absorption of the drug from the ethyl oleate suspension was evaluated after intramuscular administration in dogs. The area under the plasma concentration versus time curve and urinary recovery indicated that cefazolin was 100% bioavailable from this nonaqueous preparation.

Absorption↗

PubMed1985

Effect of nonionic surfactants on aqueous primidone suspensions.

The following interactions between the soluble surfactant, octoxynol 9, and the very slightly soluble, finely powdered drug, primidone, in aqueous suspension were investigated: adsorption/desorption of the surfactant, micellar solubilization of the drug, and deflocculation of its particles. The last effect, measured by the sedimentation volume of the suspensions, was also investigated for other octoxynols. The adsorption of octoxynol 9 on solid primidone was proportional to the equilibrium surfactant concentration up to the critical micelle concentration. It leveled off at higher concentrations, reaching saturation at completion of a close-packed surfactant monolayer. The adsorption was essentially completely reversible. The solubility of primidone in water was very slight; its micellar solubilization was even less extensive. The sedimentation volume of primidone suspensions decreased with increasing equilibrium concentration of octoxynol 9 and began to level off at the critical micelle concentration of 0.018%. At about twice that concentration, the sedimentation volume became almost constant, but reached its lowest value only at less than or equal to 0.5%. Slow rotation of suspensions prior to sedimentation promoted flocculation and higher sedimentation volumes between 0.01 and 0.03% octoxynol 9. Octoxynols with higher hydrophilic-lipophilic balance than octoxynol 9 produced considerably larger sedimentation volumes at comparable concentrations, due to a lower surface activity and a lesser tendency to adsorb on primidone.

Micelles↗

PubMed2006

Monomodal polyelectrolyte complex nanoparticles of PDADMAC/Poly(styrenesulfonate): preparation and protein interaction.

The binding of the model proteins HSA, LYZ and MYO to PEC nanoparticles is reported. PEC particles were prepared by mixing solutions of PDADMAC either with PSS or PMA-MS, followed by consecutive centrifugation. Monomodal anionic (PEC-1.50) and cationic (PEC-0.66) PEC particles were obtained using non-stoichiometric mixing ratios. PEC/protein conjugates were prepared by adding charged protein solutions to dispersions of respective like charged PEC particles, followed by one centrifugation step. Mixing proteins and PEC particles under attractive conditions led to flocculation of the dispersion. From CD, DLS and AFM the following trend for protein binding at PEC particles under repulsive conditions was obtained: HSA/PEC-1.50 > MYO/PEC-1.50 > LYZ/PEC-0.66. Protein uptakes up to 0.33 g x g(-1) (protein/PEC) (CD) and particle diameter enlargements up to 13 nm (AFM) were obtained at c(PROT) = 0.091 mg . mL(-1). Furthermore, novel spin coated films of PEC particles were interacted with proteins under both repulsive and attractive conditions. In-situ ATR FT-IR spectroscopy revealed that the adsorbed amount of HSA and LYZ under attractive conditions was significantly higher than under repulsive ones, which is analogous to protein adsorption at polyelectrolyte multilayers terminated either by polycation or polyanion. Similarly to the dispersed PEC/protein conjugates, under repulsive conditions the uptake of HSA was higher compared to LYZ. The shown protein uptake under repulsive conditions is related to concepts of mild enzyme or protein binding at nonbiogenic substrates.

Circular Dichroism↗

PubMed1983

An ultrastructural study of the macrophages of the carrageenan-induced granuloma in the rat lung.

The intralobular injection of 0.17 ml of 2 per cent. carrageenan into the rat lung induced an inflammatory granulomatous response. This inflammation was characterized in the early stages by immediate polymorphonuclear (PMN) leucocyte infiltration into the alveoli. Within 2 days the PMN's began to disappear and were replaced by carrageenan-containing macrophages. Alveolar macrophages actively phagocytised the injected carrageenan and were the dominant cell type in he alveoli for the length of this study (365 days). These low-turnover alveolar macrophages, which with light microscopy stained pinkish-red metachromatically with toludine blue due to their carrageenan content, underwent changes in vacuole morphology as well as changes in size and shape. Throughout the course of this inflammation, these macrophages had carrageenan-containing vacuoles which could be seen undergoing fusion to form larger vacuoles which at times constituted half the size of the entire macrophage cytoplasm. The carrageenan in the vacuoles was an amorphous flocculent appearing material for the initial 14 days but changes to a more fine filamentous form for the remainder of the study. This was probably due to its partial digestion by lysosomal enzymes. The general composition of the cytoplasm remained fairly constant during the 365 days. Pinocytotic vesicles and free ribosomes were abundant and the many mitochondria were small and rounded. Smooth endoplasmic reticulum lysosomes, and Golgi apparati, although not prominent, were present but the most striking feature of the cytoplasm was the presence of numerous lamellar bodies (phagocytosised surfactant). Some of the macrophages increased greatly in overall size (five to seven times) compared to their initial size. A few isolated macrophages could be seen degenerating but no general necrosis was seen. Except for one isolated case, no epithelioid cells were observed in this carrageenan inflammation. Fibrosis, if present at all, was very localised and was only evident at day 340 post-injection. This fibrosis generally involved one or two carrageenan-containing macrophages encapsulated by a few collagen fibres. No widespread fibrosis was ever observed in this study which confirmed earlier histological and biochemical investigations.

Animals↗

PubMed2006

Molecular species of phosphatidylethanolamine from continuous cultures of Saccharomyces pastorianus syn. carlsbergensis strains.

Saccharomyces pastorianus syn. carlsbergensis strain 34/70 is well known to be the most used strain for lager beer production. The difference between this strain and very closely related strain 34/78 is the latter's greater flocculating character. This single physiological trait can cause technical difficulties in beer production. The aim of this study was to determine whether lipid analysis by a combination of thin layer chromatography (TLC) with electrospray ionization mass spectrometry (ESI-MS) could be used as a strain-typing technique in order to distinguish S. pastorianus syn. carlsbergensis strain 34/70 from strain 34/78. Both strains (34/70 and 34/78) were harvested after continuous culture under standard conditions. Polar lipids were then extracted from lyophilized cultures and analysed by TLC in order to separate phospholipid families. Phosphatidylethanolamine (PE) was extracted and investigated using ESI-MS, to gain further information on individual molecular species. Using TLC analysis, lipids were separated corresponding to standards for PE, phosphatidylcholine (PC), phosphatidylglycerol (PG), cardiolipin (CL), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA) and sphingomyelin (SM). ESI-MS of the PE band, separated by TLC, showed that electrospray mass spectra were highly reproducible for repeat cultures. Novel findings were that both brewing strains displayed major phospholipid peaks with m/z 714, PE (34 : 2) m/z 742, PE (36 : 2) and m/z 758, PE (37 : 1). However, strain 34/78 had additional peaks of m/z 700, PE (33 : 2) and m/z 728, PE (35 : 2). Strain 34/70 had an extra peak with m/z 686 PE (32 : 2). We conclude that combined TLC/ESI-MS can distinguish between S. pastorianus syn. carlsbergensis 34/70 and 34/78 and may be a useful typing technique for differentiation of closely related yeast strains. This novel approach may aid quality assurance and could be suitable for yeast collections and larger industrial companies.

Cardiolipins↗

PubMed1995

Characterization of the glyceraldehyde-3-phosphate dehydrogenase gene family from Kluyveromyces marxianus--polymerase chain reaction-single-strand conformation polymorphism as a tool for the study of multigenic families.

Three glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Kluyveromyces marxianus were identified and characterized. The coding region of two of them (GAP2 and GAP3) is very similar (99.6% homology). The other gene (GAP1) is only 86% homologous to GAP2 or GAP3 and is responsible for the expression of Gap1p. This protein is extremely homologous to the K. marxianus cell wall protein p37, presumably involved in flocculation. However, no leader sequence could be detected in this gene. The identification of the three genes was possible with the use of polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), as it permits us to overcome the difficulties caused by the high homology amongst the genes. Expression of the GAPDH genes under different carbon sources (glucose or ethanol) was assessed either by Northern blot or reverse transcription-PCR-SSCP analysis, revealing that genes GAP1 and GAP2, but not GAP3, are transcribed. The results also indicate that the transcription of the gene encoding the cell wall protein p37 (Gap1p) is not dependent on the carbon source, in contrast with the expression of the gene GAP2, which is affected in cells growing in a glucose-depleted medium.

Amino Acid Sequence↗

PubMed1995

Genetically-modified brewing yeasts for the 21st century. Progress to date.

Academic studies and traditional breeding of yeasts depend upon their sporulation lifestyle. The strains used have been specially selected to sporulate readily and to mate producing new yeast types. Unfortunately brewing yeast strains do not behave in this way. They sporulate poorly, any spores which are formed are usually non-viable and any haploid strains produced are invariably non-maters. Only in recent years, with the development of recombinant-DNA techniques, has the specific breeding of new brewing yeast strains become widespread. Strains have been produced with the ability to ferment a wider range of carbohydrates, with altered flocculation properties and which produce beers with modified flavours. Many have been tested on the pilot scale and one, an amylolytic brewing yeast, has received approval for commercial use.

Carbohydrate Metabolism↗

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