Light-scattering Changes Research Articles

Morphological polarization of human polymorphonuclear leucocytes in response to three different chemoattractants: an effector response independent of calcium rise and tyrosine kinases.

Chemoattractants such as interleukin-8, C5a and N-formylmethionyl-leucyl-phenylalanine induce a cytosolic calcium rise involved in triggering the secretory functions of human polymorphonuclear leucocytes. We studied the possible role of calcium rise in membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization, which are all events contributing to chemotaxis. Membrane ruffling was assessed by right-angle light-scatter changes, the cellular content of polymerized actin by fluorescence of bodipy phallacidin, the intracellular distribution of filamentous actin by fluorescence microscopy and image digitization, and morphological polarization by scanning electron microscopy. Pretreatment of polymorphonuclear leucocytes with 50 microM BAPTA/AM, an intracellular calcium chelator, lowered the basal level in cell calcium and inhibited the transient calcium rise stimulated by 2 nM interleukin-8, 2 nM C5a, and 10 nM N-formylmethionyl-leucyl-phenylalanine. However, BAPTA pretreatment of polymorphonuclear leucocytes did not modify membrane ruffling, actin polymerization, filamentous actin distribution, and morphological polarization stimulated by chemoattractants. Downstream effectors may be protein tyrosine kinases. However, the tyrosine kinase inhibitor tyrphostin did not affect the cytoskeletal characteristics elicited by chemoattractants. Taken together, our results suggest that the transductional pathway leading to cytoskeleton organization and morphological polarization of polymorphonuclear leucocytes is different from that leading to secretion.

Forward light scatter at one month after photorefractive keratectomy.

Although it is known that backward light scatter increases transiently following most excimer laser photorefractive keratectomies (PRKs), it is not clear that there is a significant increase in forward light scatter, which is of primary concern for the patient. The object of this study was to determine if there is a significant change of forward light scatter at 1 month after (PRK) with an ablation zone diameter of 6 mm. Overlapping subsets of 24 normal myopic eyes were tested before (on the day of surgery) and 1 month after PRK, using three instruments: a Stray Light Meter (16 eyes); a Computerized Stray Light Meter (14 eyes); and a mesopic Increment Threshold-Glare Paradigm (six eyes). Differences between the two eyes before PRK were compared with the differences between the same eye before and after PRK, using repeated measured analysis of variance. In addition, increment threshold data obtained from 22 eyes after PRK were compared with those of 60 controls of the same age range and distribution by a t test. None of the statistical comparisons approached significance at the alpha = 0.05 level. Changes in light scatter as small as a factor of 1.95 (Stray Light Meter) and 1.55 (Increment Threshold-Glare Paradigm) could be detected as significant with a high power (0.8). Changes larger than a factor of 21 could be detected with a power of 0.8 for the Computerized Stray Light Meter. In these data, there is no support for the hypothesis that forward light scatter increases significantly 1 month after PRK with an ablation zone of 6 mm. Any increases in forward light scatter are unlikely to be greater than a factor of 1.5 to 2 under daytime or nighttime illumination conditions.

Development and validation of an automated particle-enhanced nephelometric immunoassay method for the measurement of human plasma C1q.

We have developed a sensitive immunoassay based on latex particle agglutination for measuring C1q concentrations in human plasma. In this simple and fast particle-enhanced immunoassay, we used carboxylated latex particles (diameter 210 nm) covalently coated with F(ab')2 fragments of anti-C1q antibodies. These particles are incubated with diluted sample (400-fold) for 6 min at room temperature, with the resulting agglutination quantified by measuring the change of light-scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hr. This assay generates a standard curve in the range of 24-775 mg/L, showing intraassay and interassay precision of < 8% and < 10%, respectively. Dilution linearity was validated throughout the dynamic range of the assay. There were no interferences from bilirubin, Intralipid, haemoglobin, and rheumatoid factor. Results obtained in 45 clinical samples correlated well with those obtained by a commercial radial immunodiffusion method (r = 0.936), and with those obtained by the Behring immunoprecipitation nephelometric test (r = 0.950). The mean concentration in plasma from healthy subjects was 180 mg/L and the reference interval was from 128 to 237 mg/L. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human C1q.

Mechanism of Non-Photochemical Chlorophyll Fluorescence Quenching. II. Resolution of Rapidly Reversible Absorbance Changes at 530 Nm and Fluorescence Quenching by the Effects of Antimycin, Dibucaine and Cation Exchanger, A23187

The putative relationship between the light-induced absorbance increase at 530 nm (ΔA530), the so-called light-scattering change, and non-photochemical chlorophyll fluorescence quenching (NPQ) was examined by the effect of inhibitors. Antimycin at a low concentration (350 nM) completely inhibited fluorescence quenching while only partially inhibiting A530. This effect was independent of the mode of thylakoid energisation and preinduction of violaxanthin de-epoxidation. Dibucaine at 20 FM abolished NPQ but had little effect on ΔA530. Moreover, the light-induced ΔA530 signal was present even in the absence of de-epoxidised xanthophylls. The cation exchanger A23187 blocked the development of NPQ as well as relaxed fluorescence quenching at steady state without involving a major portion of ΔA530. Thus, the relationship between energy-dependent A530 changes and fluorescence quenching was non-linear under all conditions tested. The light-induced absorbance increase at 530 nm, therefore, is insufficient for NPQ. The differential effects of inhibitors are explained schematically, depicting three phases for NPQ: (a) formation of zeaxanthin and antheraxanthin by the xanthophyll cycle; (b) formation of a state reflected by A530 that is induced by the transthylakoid ApH, possibly involving aggregation of LHCII; and (c) fluorescence quenching by the combined effect of both steps and by the H+-cation exchange properties of thylakoid membranes.

Polyethylene Glycol Modified Phospholipids Stabilize Emulsions Prepared from Triacylglycerol

A stable lipid‐based carrier system containing a triacylglycerol core has been developed. This has been achieved by homogenization of com oil (primarily triacylglycerol) in the presence of phosphatidylcholine (PC), preformed 100 nm liposomes prepared from PC/cholesterol (55: 45; mol:mol) and polyethylene glycol modified phosphatidylethanolamine (PEG2000‐PE). The lipid/liposome mixtures were emulsified using a microfluidizer, and the resulting particles could, depending on the phospholipids used and the addition of cholesterol, be designed to exhibit a uniform mean particle size of less than 100 nm (as measured by quasielastic light scattering). The presence of an oil core within the emulsified lipid preparation was confirmed by freeze–fracture and cryoelectron microscopy. Stability of the resulting PEG2000‐PE‐coated triacylglycerol emulsion was determined by several techniques including (1) time dependent changes in light scatter determined by measuring changes in absorbance at 600 nm (decreases in absorbance are indicative of unstable oil emulsions), (2) column chromatography procedures evaluating the migration of incorporated lipids, specifically PEG2000‐PE and triolein, after emulsions were incubated at 37 °C in the presence and absence of serum, and (3) in vivo plasma clearance data demonstrating that the lipid mixtures were maintained at ratios specified prior to iv administration. This emulsion technology has been used to prepare formulations of several water insoluble compounds, such as the drugs taxol and dibucaine and the lipophilic dye sudan IV.

Rapid estimation of bacterial antibiotic susceptibility with flow cytometry.

Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15 degrees). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2-5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.

Detection and Characterization of Apoptotic Peripheral Blood Lymphocytes in Human Immunodeficiency Virus Infection and Cancer Chemotherapy by a Novel Flow Immunocytometric Method

AbstractWe have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (< 400/µL) than in patients at earlier stages (> 400 CD4 cells/µL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

Detection and characterization of apoptotic peripheral blood lymphocytes in human immunodeficiency virus infection and cancer chemotherapy by a novel flow immunocytometric method

We have developed a quantitative and sensitive flow cytometric method for the detection of human apoptotic lymphocytes that, unlike previously described assays, allows their identification in mixed populations of peripheral blood leukocytes as well as their immunophenotyping. Apoptotic lymphocytes are identified on the basis of peculiar light scatter changes, reflecting their smaller size and their modified nucleus/cytoplasm organization, and of the decreased expression of surface CD45 molecules. Based on these criteria, apoptotic lymphocytes generated by exposure to ionizing radiation can be easily distinguished from viable cells and from necrotic lymphocytes generated by treatment with antibody and complement. Using this assay, we reappraised the phenomenon of the in vitro apoptosis of lymphocytes from patients with human immunodeficiency virus (HIV) infection. Lymphocytes from HIV patients, unlike those from normal HIV-negative subjects, undergo apoptosis upon simple in vitro culture. We found that the percentages of lymphocytes undergoing apoptosis were significantly higher in patients with low CD4 cell counts (&lt; 400/microL) than in patients at earlier stages (&gt; 400 CD4 cells/microL). However, phenotypic analysis disclosed that apoptotic lymphocytes generated in these cultures were mostly CD8+ T cells and CD19+ B cells. Thus, in contrast to what has been previously suggested, the phenomenon of in vitro lymphocyte apoptosis might not be pathogenetically related to the depletion of CD4+ T cells in acquired immunodeficiency syndrome. Nevertheless, it might represent an useful marker of disease progression. Our assay allows the analysis of unfractionated peripheral blood leukocytes and thus the identification of apoptotic lymphocytes circulating in vivo. Apoptotic lymphocytes could indeed be detected in the circulation of a patient with cancer shortly after high-dose cytotoxic chemotherapy. By contrast, no apoptotic lymphocytes could be detected in vivo in patients with early or advanced HIV infection.

Relationships among violaxanthin deepoxidation, thylakoid membrane conformation, and nonphotochemical chlorophyll fluorescence quenching in leaves of cotton (Gossypium hirsutum L.)

The kinetics and temperature dependencies of development and relaxation of light-induced absorbance changes caused by deepoxidation of violaxanthin to antheraxanthin and zeaxanthin (ΔZ; peak at 506 nm) and by light scattering (ΔS; peak around 540 nm) as well as of nonphotochemical quenching of chlorophyll fluorescence (NPQ) were followed in cotton leaves. Measurements were made in the absence and the presence of dithiothreitol (DTT), an inhibitor of violaxanthin deepoxidase. The amount of NPQ was calculated from the Stern-Volmer equation. A procedure was developed to correct gross AS (ΔSg) for absorbance changes around 540 nm that are due to a spectral overlap with ΔZ. This protocol isolated the component which is caused by light-scattering changes alone (ΔSn). In control leaves, the kinetics and temperature dependence of the initial rate of rise in ΔSn that takes place upon illumination, closely matched that of ΔZ. Application of DTT to leaves, containing little zeaxanthin or antheraxanthin, strongly inhibited both ΔSn and NPQ, but DTT had no inhibitory effect in leaves in which these xanthophylls had already been preformed, showing that the effect of DTT on ΔAn and NPQ results solely from the inhibition of violaxanthin deepoxidation. The rates and maximum extents of ΔSn and NPQ therefore depend on the amount of zeaxanthin (and/or antheraxanthin) present in the leaf. In contrast to the situation during induction, relaxation of ΔZ upon darkening was much slower than the relaxation of ΔSn and NPQ. The relaxation of ΔSn and NPQ showed quantitatively similar kinetics and temperature dependencies (Q10=2.4). These results are consistent with the following hypotheses: The increase in lumen-proton concentration resulting from thylakoid membrane energization causes deepoxidation of violaxanthin to antheraxanthin and zeaxanthin. The presence of these xanthophylls is not sufficient to cause ΔSn or NPQ but, together with an increased lumen-proton concentration, these xanthophylls cause a conformational change, reflected by ΔSn. The conformational change facilititates nonradiative energy dissipation, thereby causing NPQ. Membrane energization is prerequisite to conformational changes in the thylakoid membrane and resultant nonradiative energy dissipation but the capacity for such changes in intact leaves is quite limited unless zeaxanthin (and/or antheraxanthin) is present in the membrane. The sustained ΔSn and NPQ levels that remain after darkening may be attributable to a sustained high lumen-proton concentration.

Cell membrane changes induced by the cytolytic peptide, melittin, are detectable by 90 degrees laser scatter.

The 90 degrees light scatter parameter of the flow cytometer was used to observe changes in the membrane of human lymphoblastoid cells (HMy2) as a result of the action of the cytolytic peptide, melittin. There was a rapid and concentration-dependent increase in 90 degrees light scatter after incubation of the cells with melittin, with the level of 90 degrees light scatter reaching a maximum after 2 min. Even after all the cells were killed, as determined by ethidium bromide incorporation, the 90 degrees light scatter continued to increase. Further, the 90 degrees light scatter changes were temperature dependent. The data are consistent with the formation of lipid vesicles, either attached to the cell membrane or intracellular, as confirmed by electron microscopy of cells treated with melittin. The results demonstrate the use of the flow cytometer to detect changes in the integrity of the cell membrane.

Chapter 25 Functional Measurements Using HL-60 Cells

Publisher Summary This chapter discusses functional measurements using HL-60 cells. Exposure of HL-60 cells to a variety of agents induces terminal differentiation along either of two distinct pathways—one that bears many of the surface properties and functional characteristics of mature neutrophils and the other that resembles characteristics of monocytes/macrophages. HL-60 cells show some unique light scatter changes with differentiation. The chapter shows this change as there is a dramatic reduction in cell size and increase in 90° light scatter. As spontaneous differentiation is continually occurring, some differentiated cells are always present in so-called “undifferentiated” populations. A rapid method for checking the differentiation status of HL-60 cells is to evaluate the esterase content. Undifferentiated cells have reasonably high esterase content, which decreases in granulocytic differentiation and increases in monocytic differentiation. Differentiated HL-60 cells also express increased numbers of cell receptors such as CDllb.

Effect of polyamines on surface charge and light-scattering changes in thylakoid membranes

Effect of polyamines on surface charge and light-scattering changes in thylakoid membranes

Glare measurements before and after cataract surgery.

The efficacy of cataract surgery in improving visual performance was evaluated in 10 patients by measuring changes in intraocular light scatter, contrast sensitivity and glare induced visual loss. Two different methods were used to estimate these functions before and after cataract extraction with implantation of a monofocal posterior intraocular lens. By one method intraocular light scattering was assessed. By the other, letter contrast sensitivity and glare induced visual loss were determined. Preoperatively most patients had various glare problems, glare induced visual loss and increased intraocular light scatter. Following surgery the letter contrast sensitivity increased by a factor of 3 or more (mean 4.3). The glare problems diminished or disappeared after surgery, although several patients still had increased intraocular light scattering and glare induced visual loss compared to normals. Postoperatively the contrast sensitivity both with and without glare had increased to a level where the glare induced visual loss did not create any visual problems.

F-actin content and spatial distribution in resting and chemoattractantstimulated human polymorphonuclear leucocytes: Which role for intracellular free calcium?

Intracellular free calcium concentration ([Ca2+]i) plays a pivotal role for many responses in polymorphonuclear leucocytes (PMNs) stimulated by chemoattractants such as N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The importance of [Ca2+]i in the morphological polarization was investigated by using calcium-manipulated PMNs. We loaded human PMNs with BAPTA/AM to buffer or chelate [Ca2+]i in the presence or the absence of extracellular calcium by using fluo-3/AM as calcium indicator. The shape changes of PMNs were determined by microscopic examination, and membrane ruffling by right-angle light-scatter changes. Actin polymerization and F-actin distribution were recorded by staining PMNs with bodipy-phallacidin and quantified by quantitative fluorescence microscopy. We found that calcium-free incubation of PMNs loaded or not with 50 microM BAPTA/AM did not modify morphological polarization, membrane ruffling, actin assembly and F-actin distribution of PMNs stimulated with fMet-Leu-Phe, suggesting that these responses were probably functionally linked. It should be noted that incubation of PMNs in calcium-free conditions resulted in a radial distribution of F-actin and a moderate polymerization of actin, but not in morphological polarization of PMNs. Moreover, both calcium-sensitive and calcium-insensitive mechanisms of actin polymerization were additive, and inhibitable by 5 micrograms/ml cytochalasin B.

Uncoupling of early signal transduction events from effector function in human peripheral blood neutrophils in response to recombinant macrophage inflammatory proteins-1 alpha and -1 beta.

Macrophage inflammatory proteins-1 (MIP-1) alpha and beta are members of the C-C branch of the platelet factor 4 superfamily of cytokines, recently designated the "chemokine" superfamily. It has been suggested that the major cellular targets for the biologic activities of the C-C chemokines are the mononuclear leukocytes. However, the original designation of murine MIP-1 proteins as inflammatory mediators was based on suggestions that they activated neutrophil functions such as chemotaxis, the respiratory burst, and degranulation. In this study, we have evaluated the ability of human (Hu) MIP-1 alpha and beta to affect purified human neutrophil function. Although both rHuMIP-1 alpha and -1 beta stimulated significant calcium mobilization in human monocytes, only HuMIP-1 alpha exerted a detectable effect on neutrophils. HuMIP-1 alpha stimulated a small, dose-dependent increase in intracellular calcium, which was accompanied by a simultaneous change in right-angle light scatter, the latter indicating induction of shape change. While the effect of HuMIP-1 alpha on calcium mobilization in neutrophils was small when compared with that elicited by IL-8 or Gro alpha, it had similar characteristics to that by other receptor-dependent neutrophil agonists in that it was dependent on pertussis toxin-sensitive G proteins and on both mobilization of calcium from intracellular sources as well as influx from the extracellular environment. In addition, stimulation of neutrophils with HuMIP-1 alpha led to desensitization to subsequent additions of HuMIP-1 alpha. The stimulatory effect of HuMIP-1 alpha on neutrophil calcium mobilization and shape change was not coupled to other standard measures of neutrophil effector function. For instance, neither HuMIP-1 alpha nor -1 beta had any detectable stimulatory effect on the Na+/H+ antiport, degranulation, actin polymerization, or chemotaxis. Moreover, although HuMIP-1 alpha binding could easily be measured on monocytes or monocytic cell lines, the number of sites were too few to characterize on neutrophils by the same technique. Taken together, these results show that neither HuMIP-1 alpha nor -1 beta stimulate significant neutrophil activation and support the concept that the biologic effects of members of the C-C branch of the platelet factor 4 superfamily are not primarily directed toward neutrophils.

Platelet activating factor modulates signal transduction in Dictyostelium

During development, Dictyostelium discoideum cells produce platelet activating factor (PAF). When cells are stimulated with external cAMP pulses, PAF is transiently synthesized. To determine whether PAF is involved in signal transduction, we have tested the effect of PAF on some cellular responses which are regulated by cAMP, such as spontaneous light-scattering oscillations of suspended cells, cAMP relay, transient increases of cGMP level, and extracellular calcium uptake. Our results show that PAF specifically interferes with spontaneous spike-shaped oscillations, without affecting sinusoidal ones. PAF increases the amplitude of a spike, but has no effects on its phase or frequency. When cells fail to oscillate spontaneously, PAF does not induce spikes; however, if administered together with cAMP, it amplifies the light-scattering response to cAMP. Amplification of light-scattering changes is accompanied by a threefold increase in the concentration levels of both cellular cAMP and cGMP. Extracellular Ca2+ uptake is also stimulated by PAF. This latter response is independent of endogenous or exogenously added cAMP. All these effects are specific for the naturally occurring R-enantiomer of PAF, the S-enantiomer and lyso-PAF being inactive. These results suggest that PAF modulates signal transduction in Dictyostelium, probably by interacting with an intracellular acceptor, which is involved in the pathways regulating membrane Ca2+ channels, adenylate and guanylate cyclase.

Automated latex nephelometric immunoassay for the measurement of serum lipoprotein (a)

A sensitive immunoassay based on latex particle agglutination has been developed for measuring lipoprotein Lp(a) concentrations in serum or plasma. Carboxylated latex particles (diameter 240 nm) covalently coated with F(ab')2 fragments of anti-lipoprotein Lp(a) antibodies are incubated with diluted sample (400-fold) for 12 min at room temperature, with the resulting agglutination quantified by measuring the change of light-scatter produced. The assay has been automated on the Behring nephelometer analyzer with a sampling rate of 150 samples/hour. This assay generates a standard curve in the range of 27 to 1750 mg/L, showing inter-assay precision of less than 8%. There were no interferences from plasminogen, bilirubin, Intralipid, haemoglobin, rheumatoid factor, and apolipoprotein B. No significant differences were observed when fresh and frozen samples were compared. Sample pretreatment with "Lipoclean" clearing agent and sample lyophilization decreased the agglutinating reaction. In two separate studies using 77 and 112 patient sera the Lp(a) values, determined by the latex nephelometric method, the Terumo Macra Lp(a) ELISA test, and the Pharmacia Apo(a) radioimmunoassay method, gave correlation coefficients of 0.948 and 0.974, respectively. Physiological lipoprotein (a) values were determined in a blood donor group, with the distribution of serum Lp(a) highly skewed, with a mean (SD) and median values of 213(236) mg/L and 116 mg/L, respectively. Concentrations of Lp(a) were found to be age- and sex-independent. This latex nephelometric procedure is a convenient method and an interesting alternative to other immunoassays for routine measurement of human lipoprotein (a).

Reevaluation, using intact cells, of the exclusion limit and role of porin OprF in Pseudomonas aeruginosa outer membrane permeability.

Earlier studies that used model membrane reconstitution methods have come to different conclusions regarding the exclusion limit of the outer membrane of Pseudomonas aeruginosa and whether OprF is the major channel-forming protein in the outer membrane. In this study, a 6.2-kbp SalI fragment, encoding only two cytoplasmic enzymes, alpha-galactosidase and sucrose hydrolase, and the inner membrane raffinose permease, was cloned behind the m-toluate-inducible tol promoter of vector pNM185 to create plasmid pFB71. P. aeruginosa strains harboring pFB71, when grown with inducer, produced both enzymes encoded by the insert and had acquired the ability to grow on the disaccharide melibiose and the trisaccharide raffinose. The rate of growth was dependent on the concentration and size of the saccharide and was decreased three- to fivefold by the absence of OprF, as examined by measuring the growth on melibiose and raffinose of an isogenic OprF-deficient omega insertion derivative, H636(pFB71). At high concentrations, di-, tri-, and tetrasaccharides could pass across the outer membrane to plasmolyze P. aeruginosa, as measured by light scattering and confirmed by electron microscopy. The initial rate kinetics of light-scattering changes were dependent on the size of the saccharide being used. Furthermore, the rates of change in light scattering due to raffinose and stachyose uptake across the outer membrane for strain H636 were fivefold or more lower than for its OprF-sufficient parent H103. These data are consistent with model membrane studies showing that OprF is the most predominant porin for compounds larger than disaccharides in P. aeruginosa and suggest that the exclusion limit for this porin and the outer membrane is greater than the size of a tetrasaccharide. In addition, these data confirmed the existence of other porins with a predominant function in monosaccharide uptake and a more minor function in the uptake of larger saccharides.

Flow cytometric characterization of microspore development in Brassica napus

Flow cytometry was found to be sensitive to change in the physical characteristics of developing Brassica napus microspores. By measuring forward angle (10° – 19°) light scatter (FALS) and log 90° light scatter (L90LS) several stages of microspore development were characterized in small (1.5 mm to 2.5 mm), medium (2.5 mm to 3.5 mm), and large (3.5 mm to 4.5 mm) buds. Cell sorting was used to identify the types of cells that made up the subpopulations in two parameter plots of FALS versus L90LS including tetrad, translucent, trilobate, round, and oval microspores. Microspore development was found to be synchronous in the variety that was studied (‘Topas’) since only a few stages were found in each bud-size category. The study defined the normal process of pollen ontogeny in terms of the changes in light scatter that the cells underwent and demonstrated that flow cytometry is a useful method for analyzing this process. Key words: flow cytometry, Brassica napus, microspores, pollen, light scatter.

Changes in light scatter and width measurements from the human lens cortex with age.

Light scatter and width measurements of the anterior cortical layers of the human lens were made in 50 eyes of 50 subjects using computerised linear scanning densitometry of Scheimpflug images. It was demonstrated that the amount of light scatter increased with age in all of the three major zones and that zone C3 showed the most marked increase. Most lens growth occurs in zone C2 with C3 showing little increase in width once it has become established. Zone C1 showed a tendency to decrease in width with age. In addition it was shown that the C3 zone, which is not present at birth and during early childhood, appears as a distinct layer during the second decade of life. Its scattering properties continue to increase throughout life, exceeding all other zones after approximately 30 years of age, in the absence of cataract. Possible explanations for the lens zone pattern are discussed.