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PubMed1998

II. Steady-State Rheological Investigations.

Depletion flocculation of an alkane-in-water emulsion has previously been found to induce phase separation and, above certain polymer and oil concentrations, a delay period prior to the onset of creaming. In this paper the steady-state viscometry of an emulsion of 1-bromohexadecane-in-water has been investigated in the presence of hydroxyethylcellulose as the depletion flocculant. We have sought to characterize the flow behavior of this emulsion system and determine the characteristic rheology during the delay period. The use of this near-density-matched system eliminates the effect of creaming, and we have, therefore, characterized the delay period by reference to the behavior of a related system, differing only in oil composition. At oil and polymer concentrations conducive to the presence of a delay period, the emulsions were found to be shear thinning, whereas the emulsion system in the absence of polymer and the isolated continuous phases were Newtonian. The structure formation influencing the rheology of the emulsions at high polymer concentrations was attributed entirely to interdroplet depletion interactions. The concept of dynamic yield stress for such a system is discussed and the consequences for the structure of a buoyancy-resisting space-spanning phase or "transient gel." Copyright 1998 Academic Press.

Journal Article↗

PubMed1999

Probing Static Structure of Colloid-Polymer Suspensions with Multiply Scattered Light.

Time-dependent measurements of light propagation were conducted in aqueous dispersions of 523 nm diameter polystyrene at concentrations between 0.1 and 0.4 solids volume fraction in order to assess how particle correlation is influenced by depletion interactions arising from the addition of soluble polyethyleneoxide (PEO). In the absence of polymer, the transport scattering length can be predicted from Mie scattering theory and the Percus-Yevick (P-Y) model for static structure of a dense hard-sphere colloidal solution. Depletion forces arising from the addition of PEO of varying molecular weights influenced the spatial ordering of the dispersion and caused a further increase in the transport scattering length beyond that predicted by hard-sphere static structure factor but similar to that predicted by the mean sphere approximation (MSA) to the P-Y model described by Ye et al. (1996). Onset of flocculation occurred with increased PEO addition and correlated with PEO molecular weight. Phase separation was noted by no further change in the transport scattering length, except when flocculation was induced by the highest molecular weight PEO. The use of time-dependent measurements of light propagation in dense systems provides an alternative to small-angle light, neutron, and X-ray scattering characterization of interaction potentials in dense, multiply scattering samples and promises further fruitful investigation of colloidal particle interactions in suspensions. Copyright 1999 Academic Press.

Journal Article↗

PubMed2000

Microgel Particles as a Matrix for Polymerization: A Study of Poly(N-isopropylacrylamide)-Poly(N-methylpyrrole) Dispersions.

Poly(N-isopropylacrylamide) (PNIPAM) microgel particles dispersed in water have been used as a matrix for the polymerization of a hydrophobic monomer, N-methylpyrrole (MPy). The presence of poly(MPy) (PMPy) within the dried composite particles has been confirmed using electron paramagnetic resonance (EPR) measurements which show a characteristic free-radical signal at g=2.007. Electron microscopy data (TEM) show that the composite PNIPAM-xPMPy particles have a "raspberry-like" morphology. (The value for x represents the volume percent of MPy added during synthesis with respect to the total microgel volume.) PCS data indicate that PMPy occupies the majority of the collapsed composite particle volume. The maximum value of x consistent with colloid stability for PNIPAM-xPMPy dispersions is 4.5%. Higher values of x result in coagulation due to interparticle bridging by PMPy. Variable temperature PCS measurements of the PNIPAM-xPMPy dispersions have been used to study the thermally induced collapse of the composite particles. The extent of collapse becomes less with increasing values for x. The embedded PMPy particles restrict the extent of PNIPAM network contraction. The stability of the PNIPAM-4.5PMPy dispersions was investigated by means of turbidity measurements using aqueous 0.1 M NaCl solution. The upper critical flocculation temperatures (UCFT) for PNIPAM and PNIPAM-4.5PMPy dispersions were identical (38-39 degrees C). The flocculation observed was found to be fully reversible. The composite dispersion stability in the absence of salt was attributed to electrosteric stabilization afforded by the PNIPAM matrix. These results indicate that PNIPAM microgel particles may have application as a matrix for the polymerization of hydrophobic monomers in water. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Creaming and Rheology of Oil-in-Water Emulsions Containing Sodium Dodecyl Sulfate and Sodium Caseinate.

The creaming and rheology of fine n-tetradecane oil-in-water emulsions at pH 6.8 containing the commercial protein sodium caseinate and the ionic surfactant sodium dodecyl sulfate (SDS) have been studied, and an overview diagram relating surfactant composition and creaming stability has been constructed. The presence of both SDS and sodium caseinate in an emulsion system increases the overall stability with respect to creaming. Excess SDS promotes destabilization through fast creaming; this can be attributed to depletion flocculation brought about by unadsorbed surfactant micelles. Addition of sodium caseinate was found to reduce this effect, even at relatively high SDS concentrations. The behavior of the caseinate + SDS emulsions is thus different from the behavior of the previously reported caseinate + Tween 20 systems, where the combination of the two surface-active agents was found to reduce the emulsion stability, as indicated by fast creaming and shear-thinning rheology. Addition of sodium chloride was found to increase the extent of non-Newtonian behavior and to enhance the degree of creaming for SDS-containing emulsions. Increased caseinate levels in these systems seem to offer some stabilization through reduction of the shear-thinning character and improvement in creaming stability. These phenomena can be explained in terms of a considerable amount of SDS binding to the protein, which reduces the amount of SDS available to promote protein displacement and depletion flocculation. In contrast to the SDS systems, the properties of equivalent emulsions containing caseinate + nonionic surfactant Tween 20 are relatively insensitive to salt content. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Effect of Particle Association on 2,2'-Bipyridyl Adsorption onto Kaolinite.

The effect of particle concentration, in kaolin suspensions, on the adsorption of 2,2'-bipyridyl was studied. Adsorption expressed in units of micromoles per gram decreased as a result of the increase in particle concentration and also as a result of the presence of coagulant (0.25 M NaCl). Dispersion treatment with sodium hexametaphosphate increased the adsorption of bipyridyl. The decrease in adsorption with the increase in particle concentration suggests a possible relation between adsorption and flocculation phenomena. On the basis of classic flocculation theory a straight-line relation was obtained between the square root of the adsorption maximum (mmol/L) and particle concentration (g/L). It is concluded that particle association, which is a function of particle concentration, reduces the surface/aqueous interface and consequently the adsorption of bipyridyl. Copyright 2000 Academic Press.

Journal Article↗

PubMed2002

Two-dimensional colloidal aggregation: concentration effects.

Extensive numerical simulations of diffusion-limited (DLCA) and reaction-limited (RLCA) colloidal aggregation in two dimensions were performed to elucidate the concentration dependence of the cluster fractal dimension and of the different average cluster sizes. Both on-lattice and off-lattice simulations were used to check the independence of our results on the simulational algorithms and on the space structure. The range in concentration studied spanned 2.5 orders of magnitude. In the DLCA case and in the flocculation regime, it was found that the fractal dimension shows a linear-type increase with the concentration phi, following the law: d(f)=d(fo)+aphi(c). For the on-lattice simulations the fractal dimension in the zero concentration limit, d(fo), was 1.451+/-0.002, while for the off-lattice simulations the same quantity took the value 1.445+/-0.003. The prefactor a and exponent c were for the on-lattice simulations equal to 0.633+/-0.021 and 1.046+/-0.032, while for the off-lattice simulations they were 1.005+/-0.059 and 0.999+/-0.045, respectively. For the exponents z and z', defining the increase of the weight-average (S(w)(t)) and number-average (S(n)(t)) cluster sizes as a function of time, we obtained in the DLCA case the laws: z=z(o)+bphi(d) and z'=z'(o)+b'phi(d'). For the on-lattice simulations, z(o), b, and d were equal to 0.593+/-0.008, 0.696+/-0.068, and 0.485+/-0.048, respectively, while for the off-lattice simulations they were 0.595+/-0.005, 0.807+/-0.093, and 0.599+/-0.051. In the case of the exponent z', the quantities z'(o), b', and d' were, for the on-lattice simulations, equal to 0.615+/-0.004, 0.814+/-0.081, and 0.620+/-0.043, respectively, while for the off-lattice algorithm they took the values 0.598+/-0.002, 0.855+/-0.035, and 0.610+/-0.018. In RLCA we have found again that the fractal dimension, in the flocculation regime, shows a similar linear-type increase with the concentration d(f)=d(fo)+aphi(c), with d(fo)=1.560+/-0.004, a=0.342+/-0.039, and c=1.000+/-0.112. In this RLCA case it was not possible to find a straight line in the log-log plots of S(w)(t) and S(n)(t) in the aggregation regime considered, and no exponents z and z' were defined. We argue however that for sufficiently long periods of time the cluster averages should tend to those for DLCA and, therefore, their exponents should coincide with z and z' of the DLCA case. Finally, we present the bell-shaped master curves for the scaling of the cluster size distribution function and their evolution when the concentration increases, for both the DLCA and RLCA cases.

Journal Article↗

PubMed2002

A small reservoir of disabled ORFs in the yeast genome and its implications for the dynamics of proteome evolution.

We surveyed the sequenced Saccharomyces cerevisiae genome (strain S288C) comprehensively for open reading frames (ORFs) that could encode full-length proteins but contain obvious mid-sequence disablements (frameshifts or premature stop codons). These pseudogenic features are termed disabled ORFs (dORFs). Using homology to annotated yeast ORFs and non-yeast proteins plus a simple region extension procedure, we have found 183 dORFs. Combined with the 38 existing annotations for potential dORFs, we have a total pool of up to 221 dORFs, corresponding to less than approximately 3% of the proteome. Additionally, we found 20 pairs of annotated ORFs for yeast that could be merged into a single ORF (termed a mORF) by read-through of the intervening stop codon, and may comprise a complete ORF in other yeast strains. Focussing on a core pool of 98 dORFs with a verifying protein homology, we find that most dORFs are substantially decayed, with approximately 90% having two or more disablements, and approximately 60% having four or more. dORFs are much more yeast-proteome specific than live yeast genes (having about half the chance that they are related to a non-yeast protein). They show a dramatically increased density at the telomeres of chromosomes, relative to genes. A microarray study shows that some dORFs are expressed even though they carry multiple disablements, and thus may be more resistant to nonsense-mediated decay. Many of the dORFs may be involved in responding to environmental stresses, as the largest functional groups include growth inhibition, flocculation, and the SRP/TIP1 family. Our results have important implications for proteome evolution. The characteristics of the dORF population suggest the sorts of genes that are likely to fall in and out of usage (and vary in copy number) in a strain-specific way and highlight the role of subtelomeric regions in engendering this diversity. Our results also have important implications for the effects of the [PSI+] prion. The dORFs disabled by only a single stop and the mORFs (together totalling 35) provide an estimate for the extent of the sequence population that can be resurrected readily through the demonstrated ability of the [PSI+] prion to cause nonsense-codon read-through. Also, the dORFs and mORFs that we find have properties (e.g. growth inhibition, flocculation, vanadate resistance, stress response) that are potentially related to the ability of [PSI+] to engender substantial phenotypic variation in yeast strains under different environmental conditions. (See genecensus.org/pseudogene for further information.)

Chromosomes, Fungal↗

PubMed1983

Differences in the ultrastructure of the submandibular glands of baboon and Rhesus monkey revealed by the use of different fixatives.

Submandibular glands of adult baboon and Rhesus monkeys were compared after different methods of fixation. In both species, serous acinar cells outnumber mucous acinar cells. In the baboon, serous cells contain secretory granules showing dense cores, moderately dense crescents, and flocculent material. In mucous cells, secretory granules vary in appearance from amorphous to highly ordered depending on fixation. In the Rhesus monkey, serous cells contain the same 3 components of secretory granules as in the baboon. Additionally, a fourth component is represented by a layer of electron-dense material between the crescent and flocculent material. Mucous cells contain electron-dense granules when fixed in Millonig's buffered fixatives, but when Clarke's or Sorensens' buffer is used the granules resemble more typical mucous granules. Duct systems of the two species are similar, but differ mainly in that large foci of glycogen are present in the striated duct cells of the Rhesus monkey.

Animals↗

PubMed1987

Metamorphic changes within the lateral-line system of Anura.

The metamorphic loss of lateral-line organs, lateral-line nerves and second order lateral-line neurons was examined in two Anuran species. At the onset of metamorphic climax, terminals within the lateral-line neuropil showed accumulation of glycogen-like granules. Neither the lateral-line nerve nor the organs or the nerve terminals inside the organs displayed any sign of degeneration at this stage. A few second order neurons exhibited accumulations of chromatin into conspicuous masses. These cells were partially or completely engulfed by phagocytes. At mid-metamorphosis all lateral-line organs were lost. The proximal parts of the lateral-line nerve fibers entering the rhombencephalic alar plate showed signs of degeneration. Within the lateral-line neuropil, pre- and some postsynaptic elements exhibited the flocculent type of degeneration or, to a lesser extent, the dark type of degeneration. Second order lateral-line neurons underwent an electron-dense or electron-lucent type of degeneration and were taken up by phagocytes. At the end of metamorphic climax the distal parts of the lateral-line nerves showed numerous dark degenerating fibers inside an intact myelin sheath. Within the lateral-line neuropil, numerous dark degenerating presynaptic elements were found next to some elements showing flocculent degeneration. Fewer degenerating second order neurons were found in the alar plate. They showed predominantly the dark type of degeneration. In contrast to earlier reports, our data suggest that the degenerative metamorphic changes observed in the present study are initiated in all parts of the lateral-line system simultaneously, and lead to the complete loss of all lateral-line organs and nerves and presumably all second order lateral-line neurons as well.

Animals↗

PubMed1977

Spontaneous atherosclerosis: an ultrastructural study in the white Carneau pigeon.

The atheroscleroit lesions, associated with the celiac intimal smooth muscle cushions, of four and five year old White Carneau pigeons were studied with the light and electron microscopes. Light microscopic examination of the spontaneous lesions demonstrated large intimal cushions composed of smooth muscle abundant collagen, clusters of foam cells and cholesterol crystal clefts. Ultrastructural examination of the intimal atheroma revealed dilatations between apposing endothelial cells which contained a flocculent material, similar to that seen in the subendothelial space. The subendothelial compartment contained abundant collagen, extracellular lipid, vesiculated material and cell processes which contained a flocculent matrix and tubular-like elements. In addition, fibroblast-like interlaminar cells were often observed. Numerous intimal smooth muscle cells were seen which displayed varied morphology. Abundant foam cells were also present within the intimal atheromas. The presence of atherosclerotic lesions in preexisting intimal smooth muscle cushions suggests that hemodynamic factors may be important in the progression of these spontaneous lesions. Endothelial cell dilatations may provide an important route of transport for circulating elements which may accumulate withing the subendothelial space. Morphologically, it appears that the smooth muscle cells undergo modification and may represent the precursors of foam cells in this species.

Animals↗

PubMed1988

The nature and timing of excitotoxic neuronal necrosis in the cerebral cortex, hippocampus and thalamus due to flurothyl-induced status epilepticus.

Flurothyl-induced status epilepticus was studied by light and electron microscopy (LM, EM) to determine the time course and structural features of neuronal necrosis in the vulnerable brain regions in epilepsy. The cerebral cortex, hippocampus and thalamus were examined after closely spaced recovery periods of up to 1 week. The results showed that acidophilic neurons appeared simultaneously in neurons of the neocortex, hippocampus and thalamus, and that this occurred within 1 h following the end of the epilepsy. The corresponding features of acidophilic neurons by EM were mitochondrial flocculent densities and large discontinuities in cell and nuclear membranes. Dark neurons were ubiquitous during the epilepsy, but recovered almost universally. A few dark neuronal forms persisted and underwent cytorrhexis after 12-h recovery or longer. Axon-sparing dendritic lesions characteristic of excitotoxic neuronal death were found in the neuropil of the neocortex, and in both vulnerable CA1 and resistant CA3 neurons of the hippocampus. Other than acute edema, glial changes were absent. The findings support an excitotoxic mechanism in epilepsy-induced selective neuronal necrosis also in brain regions outside the hippocampus, and contrast with previous reports in ischemia and hypoglycemia in that neuronal necrosis occurs virtually immediately after an epileptic insult. No "maturation" of cell damage, as described in ischemia, was seen. Furthermore, even exceedingly dark neuronal forms and massive dendritic swelling must be considered sub-lethal or prelethal cellular changes. Lethal cellular changes include acidophilia by LM, cell membrane breaks, and mitochondrial flocculent densities by EM.

Animals↗

PubMed1985

The temporal evolution of hypoglycemic brain damage. I. Light- and electron-microscopic findings in the rat cerebral cortex.

In the course of a study on the pathogenesis of neuronal necrosis in severe hypoglycemia, the morphological characteristics reflecting reversible and irreversible neuronal lesions were examined as a function of time following normalization of blood glucose. To that end, closely spaced time intervals were studied in the rat cerebral cortex before, during, and up to 1 year after standardized pure hypoglycemic insults of 30 and 60 min of cerebral isoelectricity. Both the superficial and deep layers of the cerebral cortex showed dark and light neurons during and several hours after the insult. By electron microscopy (EM) the dark neurons were characterized by marked condensation of both karyoplasm and cytoplasm, with discernible, tightly packed cytoplasmic organelles. The light neurons displayed clustering of normal organelles around the nucleus with clearing of the peripheral cytoplasm. Some cells, both dark neurons and neurons of normal electron density, contained swollen mitochondria with fractured cristae. Light neurons disappeared from the cerebral cortex by 4 h of recovery. Some dark neurons in the superficial cortex and almost all in the deep cortex evolved through transitional forms into normal neurons by 6 h recovery. Another portion of the dark neurons in the superficial cortex became acidophilic between 4 and 12 h, and by EM they demonstrated karyorrhexis with stippled electron-dense chromatin. The plasma membrane was disrupted, the cytoplasm was composed of amorphous granular debris, and the mitochondria contained flocculent densities. These definitive indices of irreversible neuronal damage were seen as early as 4-8 h recovery. Subsequently, the acidophilic neurons were removed from the tissue, and gliosis ensued. Thus, even markedly hyperchromatic "dark" neurons are compatible with survival of the cell, as are neurons with conspicuous mitochondrial swelling. Definite nerve cell death is verified as the appearance of acidophilic neurons at which stage extensive damage to mitochondria is already seen in the form of flocculent densities, and cell membranes are ruptured. Our previous results have shown that hypoglycemic neocortical damage affects the superficial laminae, chiefly layer 2. The present results demonstrate that, following the primary insult, this damage evolves relatively rapidly within the first 4-12 h. We have obtained no evidence that additional necrotic neurons are recruited after longer recovery periods.

Animals↗

PubMed1976

Enzymatic synthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera.

The enzyme activity synthesizing poly-beta-hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction of Zoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction when the bacterium grew dispersedly in a glucose-starved medium. The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose. PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated with D(-)-beta-hydroxybutyryl CoA in vitro. Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity for D(-)-beta-hydroxybutyryl CoA, and showed the same pH optimum at 7.0.

Coenzyme A Ligases↗

PubMed1991

The response of the three brood Ceriodaphnia test to fifteen formulations and pure compounds in common use.

The three brood Ceriodaphnia dubia test was carried out on fifteen formulations and pure compounds presently in general use in a variety of treatment processes or as reference toxicants. The most toxic substances (LC50 (based on survival/EC50 (based on total progeny) less than 10 mg/L) were metallic Cu, tertiary dodecyl mercaptan, an antioxidant TIA-RCO T-424, Cu(NO3)2.3H2O, K2Cr2O7, NALCO 7139--a flocculent, DOWFAX 2EP--a surfactant, TB-66--an algicide and ammonia. Exposure to these compounds elicited the same response from C. dubia for the two endpoints examined--survival and reproductive success. Two antimicrobials, PROXEL GXL and DOWICIL 75, and the surfactant sodium lauryl sulfate, were slightly toxic (LC50/EC50 less than 100 mg/L) for both endpoints studied. Two coagulants, NALCO 7109 and NALCO 8120 and a flocculent PERCOL 757 were practically nontoxic (LC50 greater than 100 mg/L) to slightly toxic (LC50 less than 100 mg/L) when the survival endpoint was examined, but were moderately toxic (EC50 less than 10 mg/L) to slightly toxic (EC50 less than 100 mg/L) when the endpoint of reproductive success was examined. These formulations and pure compounds had generally a declining effect on mean brood size, though there were exceptions to this observation as the broods progressed in number.

Animals↗

PubMed1979

Radiologic abnormalities and autonomic neuropathology in the digestive tract of the ketonuric diabetic Chinese hamster.

Barium x-ray patterns of ketonuric diabetic Chinese hamsters displayed marked dilatation of the stomach, small and large intestine. Hypomotility was manifested by flocculation of barium in the small and large bowel. Impaired transit time was further characterized by prolonged emptying of the stomach (mean 570 min diabetics; 200 min controls) and delayed stool formation (mean 230 min diabetics; and 100 min controls) and passage (mean 457 min diabetics; 210 min controls). Ultrastructural analysis of Auerbach's myenteric plexuses of the small intestine indicated acute degeneration in certain distal, unmyelinated axons. Swelling, deposition of glycogen, aggregation of neurofilaments and dense accumulation of lamellar bodies were observed. The severity and frequency of barium flocculation, glycogen deposition, aggregation of neurofilaments and lamellar inclusion bodies in axons were directly related to duration of ketonuria. The data strongly suggest that autonomic neuropathology in the plexuses of Auerbach may be a critical factor underlying gastrointestinal dysfunction in the ketonuric diabetic Chinese hamster.

Animals↗

PubMed1984

Latter reorganization of membrane-glycogen complexes in rabbit extraocular muscle.

Characteristic modes of secondary reorganization were observed in membrane-glycogen complexes of rabbit extraocular muscle. These included (a) an irregular widening or narrowing of the intracisternal space, (b) a loss of the typical intracisternal flocculent densities, and (c) the acquisition of intercisternal flocculent densities. In irregularly widened cisternae, the membranes tended to remain closely adjacent to the intervening glycogen layer, thereby forming triads composed of a glycogen layer enclosed within the apposing membranes of adjacent cisternae. In the absence of glycogen particles from contiguous portions of several lamellae, the membranes became compacted to form myeloidlike figures. Degenerating complexes sometimes displayed distention of intercisternal spaces and layers of atypically small particles. The above modifications of membrane-glycogen complexes would be compatible with the notion that these structures are involved in the process of glycogen metabolism, as opposed to the previously suggested notion that these structures are transient vehicles for the accumulation of glycogen masses.

Animals↗

PubMed1988

Structure of the vitread face of the monkey optic disc (Macaca mulatta). SEM on frozen resin-cracked optic nerveheads supplemented by TEM and immunohistochemistry.

The authors applied frozen resin cracking after hexamethyldisilazane (HMDS) desiccation on the monkey optic disc region. The cracked face through the central part of the optic disc showed that the inner limiting membrane of the retina continued into the limiting membrane of Elschnig, and this, in turn, continued into the central meniscus of Kuhnt. At the disc margin the membrane was about 70 nm in thickness, due to a large fibrillar component. Elschnig's membrane was about 50 nm in thickness and was composed of both fibrils and flocculent material. The membrane covering the central meniscus of Kuhnt was about 20 nm in thickness. The number of fibrils here was very low, and the membrane consisted of flocculent material. The positive immunohistochemical stainings for GFA and vimentin of Elschnig's membrane and Kuhnt's meniscus were noteworthy. The positive staining disappeared when the membrane continued into the inner limiting membrane of the retina, supporting the different structural composition.

Animals↗

PubMed1982

The adverse effects of HEPES, TES, and BES zwitterion buffers on the ultrastructure of cultured chick embryo epiphyseal chondrocytes.

Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 micron diam) formed by the fusion and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red. Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium deposits seemed much larger and more numerous in the presence of zwitterion buffers. It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems, and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.

Alkanesulfonates↗