Tooth Size Variation in Pinniped Dentitions

It is contentious whether size variation among mammalian teeth is heterogeneous or homogeneous, whether the coefficient of variation is reliable, and whether the standard deviation of log-transformed data and the residual of standard deviation on mean variable size are useful replacements for the coefficient of variation. Most studies of tooth size variation have been on mammals with complex-crowned teeth, with relatively little attention paid to taxa with simple-crowned teeth, such as Pinnipedia. To fill this gap in knowledge and to resolve the existing controversies, we explored the variation of linear size variables (length and width) for all teeth from complete permanent dentitions of four pinniped species, two phocids (Histriophoca fasciata, Phoca largha) and two otariids (Callorhinus ursinus, Eumetopias jubatus). Size variation among these teeth was mostly heterogeneous both along the toothrow and among species. The incisors, canines, and mesial and distal postcanines were often relatively highly variable. The levels of overall dental size variation ranged from relatively low as in land carnivorans (Phoca largha and both otariids) to high (Histriophoca fasciata). Sexual size dimorphism varied among teeth and among species, with teeth being, on average, larger in males than in females. This dimorphism was more pronounced, and the canines were larger and more dimorphic relative to other teeth in the otariids than in the phocids. The coefficient of variation quantified variation reliably in most cases. The standard deviation of log-transformed data was redundant with the coefficient of variation. The residual of standard deviation on mean variable size was inaccurate when size variation was considerably heterogeneous among the compared variables, and was incomparable between species and between sexes. The existing hypotheses invoking developmental fields, occlusal complexity, and the relative timing of tooth formation and sexually dimorphic hormonal activity do not adequately explain the differential size variation along the pinniped toothrow.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Detection of Airborne Stachybotrys chartarum Macrocyclic Trichothecene Mycotoxins on Particulates Smaller than Conidia

Highly respirable particles (diameter, <1 μm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.     

A test of a model for planktivorous filter feeding by bream Abramis brama

Synopsis The planktivorous feeding of bream, Abramis brama on Daphnia hyalina and Bosmina coregoni was analyzed in a stepwise regression analysis with the average size (and standard deviation) of consumed organisms as dependent variable and the size of the fish, the average size (and standard deviation) of the organisms and their density in the environment as independent variables. Three basic predictions on filter feeding were formulated and tested. It was predicted that the (average) prey size should increase with fish size, but that the standard deviation should decline. Secondly the prey size should be strongly correlated with the prey size available and thirdly the prey density should have little effect on the size selection. These hypotheses could not be rejected for bream>10 cm feeding on B. coregoni and for bream>20 cm feeding on D. hyalina. The hypotheses were not valid for smaller bream as these acted as particulate or combined filter- and particulate feeders.     

Use of Centrifugal Filter Devices to Concentrate Dengue Virus in Mosquito per os Infection Experiments

Dengue virus (DENV) is an arbovirus transmitted to humans by the bite of infected Aedes mosquitoes. Experimental per os infection of mosquitoes with DENV is usually a preliminary step in virus/vector studies but it requires being able to prepare artificial blood-meals with high virus titers. We report here the convenient use of centrifugal filter devices to quickly concentrate DENV particles in cell-culture supernatants. The median viral titer in concentrated-supernatants was 8.50 log₁₀ TCID₅₀/mL. By using these DENV concentrated-supernatants to prepare infectious blood-meals in Aedes aegypti per os infection experiments, we obtained a mean mosquito-infection rate of 94%. We also evaluated the use of centrifugal filter devices to recover DENV particles from non-infectious blood-meals presented to infected mosquitoes through a feeding membrane to collect their saliva.     

Evaluation of Five Membrane Filtration Methods for Recovery of Cryptosporidium and Giardia Isolates from Water Samples

We evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n = 53 ± 15.4 per 10 liters) and cysts (n = 59 ± 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 ± 8, P = 0.015; cysts, 49.8 ± 12.2, P = 0.4260), and SMF (oocysts, 16.2 ± 2.8, P = 0.0079; cysts, 35.2 ± 3, P = 0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.     

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

A new manual dispensing system for in meso membrane protein crystallization with using a stepping motor-based dispenser

A reliable and easy to use manual dispensing system has been developed for the in meso membrane protein crystallization method. The system consists of a stepping motor-based dispenser with a new microsyringe system for dispensing, which allows us to deliver any desired volume of highly viscous lipidic mesophase in the range from ~50 to at least ~200 nl. The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing. Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well. The system was useful for crystallizing membrane and soluble proteins in meso.     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Analysis of local order in the spatial distribution of cell surface molecular assemblies.

Electron micrographs obtained from platinum-carbon replicas of freeze-cleaved or freeze-dried cells and exhibiting intramembranous or externally-disposed particles were analysed statistically for degree of non-random particle association. Analysis was based on use of an analogy to the radial distribution function, g(r), which provided the average density of neighbor particles around a particle in the micrograph at 30 Å increments from particle center. The ratio of average density of neighbors around a particle to overall particle density in the micrograph was then determined as a function of distance from particle center. For a random plot of 90 Å particles, the computed ratio deviated from 1 by less than 10% for a distance up to 900 Å from particle center. In a field of similarly-sized intramembranous particles within the freeze-cleaved surface membrane of a mouse fibroblast, there were indications of slight non-random particle associations at distances of 120–240 Å and 300–330 Å from particle center, while a significant association of particles comprising an intercellular gap junction was obtained at 90–120 Å. Virus-related molecular assemblies on the surfaces of fibroblasts infected with a temperature-sensitive mutant of murine luekemia virus and grown under conditions that did not permit virus assembly to occur were analysed similarly. Significant deviation from randomness was found at several distances from particle center, both on areas of membrane that were populated by large numbers of particles and on low-density areas.     

The effect of the transmission of the beet mosaic virus on the variability of its incubation period

The variability of the incubation period of the beet mosaic virus [Beta virus 2(Lind) Smith] was investigated in a three year experiment. The virus was transmitted by the black bean aphid (Aphis fabae Scop.) or by a mechanical inoculation onAmaranthus caudatus L. Variability characterized by the standard deviation indicates a closer distribution of measurements when the virus was transmitted by mechanical inoculation. The differences in variability of the incubation period between the two methods of virus tramission are statistically significant; dispersion and standard deviation are lower after mechanical inoculation. The average length of the incubation period after virus transmission by aphids and by mechanical inoculation was 10.8 and 7.7 days, respectively. The difference was found statistically significant and may be explained by the higher number of viral particles transmitted by mechanical inoculation which may accelerate the process of infection. A significant correlation between the temperature of the environment and the length of the incubation period was also found in some experiments.     

Two swinging-bucket ultracentrifugal filters for receptor-binding assays

Centrifugal filters for SW 25.1 and 50.1 swinging-bucket ultracentrifuge rotors have been tested up to the maximum speeds allowed, 90,000 and 300,000g, respectively. The filters are 1 and 0.5 in. in diameter and accept standard 25-mm polycarbonate filter membranes. The filter membranes are both cup-shaped to prevent loss of particulates to the support materials of the filters. The filter for the SW 25.1 rotor can take 0.75 ml and that of the 50.1 head 0.5 ml. The fluid retained after centrifuging consists of the fluid on the filter membrane and in its pores and that retained by the material filtered. The calculated volume of the pores of the 0.2-μm filter was 0.46 μl. Total liquid retentions of about 0.8 μl have been achieved with both filters using a particulate concentration of 0.5 mg/ml.     

PDADMAC flocculation of Chinese hamster ovary cells: Enabling a centrifuge-less harvest process for monoclonal antibodies

High titer (>10 g/L) monoclonal antibody (mAb) cell culture processes are typically achieved by maintaining high viable cell densities over longer culture durations. A corresponding increase in the solids and sub-micron cellular debris particle levels are also observed. This higher burden of solids (≥15%) and sub-micron particles typically exceeds the capabilities of a continuous centrifuge to effectively remove the solids without a substantial loss of product and/or the capacity of the harvest filtration train (depth filter followed by membrane filter) used to clarify the centrate. We discuss here the use of a novel and simple two-polymer flocculation method used to harvest mAb from high cell mass cell culture processes. The addition of the polycationic polymer, poly diallyldimethylammonium chloride (PDADMAC) to the cell culture broth flocculates negatively-charged cells and cellular debris via an ionic interaction mechanism. Incorporation of a non-ionic polymer such as polyethylene glycol (PEG) into the PDADMAC flocculation results in larger flocculated particles with faster settling rate compared to PDADMAC-only flocculation. PDADMAC also flocculates the negatively-charged sub-micron particles to produce a feed stream with a significantly higher harvest filter train throughput compared to a typical centrifuged harvest feed stream. Cell culture process variability such as lactate production, cellular debris and cellular densities were investigated to determine the effect on flocculation. Since PDADMAC is cytotoxic, purification process clearance and toxicity assessment were performed.     

On the polydispersity of mitochondria in tissue homogenates and the determination of the average sedimentation coefficients of mixed populations of mitochondria

The sedimentation profile (sediterm) of subcellular particles in homogenous media depends on the average sedimentation coefficient ( value) and the size distribution. The present study has focused on the two common types of polydispersity, i.e., (i) a variable standard deviation in a normal (Gaussian) size distribution, and (ii) two populations of partieles with defined values and size distributions. Theoretical considerations and experimental data indicate that rat liver mitochondria have a normal size distribution, ( ) with much smaller standard deviation than previously assumed (σ = 0.118 μm) based on isokinetic gradient centrifugation and electron microscopy. Sedimentation of a mixture of rat liver and guinea pig ileal mitochondria having the values 17,040 S and 5640 S, respectively, gave the expected profile (sediterm) of two populations of particles. Their values were estimated to be identical to those obtained when the individual mitochondrial populations were sedimented. The ratio between the populations (based on the assay of marker enzyme) was found to be identical to the expected value.     

Evaluating the Membrane Fecal Coliform Test by Using Escherichia coli as the Indicator Organism

The fecal coliform membrane filter method (MFC) currently used in water pollution analysis was evaluated by using two strains of Escherichia coli, a known fecal coliform, as the indicator organism. A large relative error in the results obtained with this method was found to be dependent upon the brand of membrane filter employed, the medium, and the temperature of incubation. MFC densities varied between 10 and 60% of the densities determined by means of total bacteria counts and total coliform counts performed at 35 C. Due to the large relative error encountered, the MFC method cannot be recommended as an analytical tool for the laboratory enumeration of E. coli. The results do show that the MFC method can be used at 35 C for enumeration of E. coli and for differential counts of E. coli and Enterobacter aerogenes.     

Baleen Hydrodynamics and Morphology of Cross-Flow Filtration in Balaenid Whale Suspension Feeding

The traditional view of mysticete feeding involves static baleen directly sieving particles from seawater using a simple, dead-end flow-through filtration mechanism. Flow tank experiments on bowhead (Balaena mysticetus) baleen indicate the long-standing model of dead-end filtration, at least in balaenid (bowhead and right) whales, is not merely simplistic but wrong. To recreate continuous intraoral flow, sections of baleen were tested in a flume through which water and buoyant particles circulated with variable flow velocity. Kinematic sequences were analyzed to investigate movement and capture of particles by baleen plates and fringes. Results indicate that very few particles flow directly through the baleen rack; instead much water flows anteroposteriorly along the interior (lingual) side of the rack, allowing items to be carried posteriorly and accumulate at the posterior of the mouth where they might readily be swallowed. Since water flows mainly parallel to rather than directly through the filter, the cross-flow mechanism significantly reduces entrapment and tangling of minute items in baleen fringes, obviating the need to clean the filter. The absence of copepods or other prey found trapped in the baleen of necropsied right and bowhead whales supports this hypothesis. Reduced through-baleen flow was observed with and without boundaries modeling the tongue and lips, indicating that baleen itself is the main if not sole agent of crossflow. Preliminary investigation of baleen from balaenopterid whales that use intermittent filter feeding suggests that although the biomechanics and hydrodynamics of oral flow differ, cross-flow filtration may occur to some degree in all mysticetes.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.     

AN INTERPRETATION OF LIVER CELL MEMBRANE AND JUNCTION STRUCTURE BASED ON OBSERVATION OF FREEZE-FRACTURE REPLICAS OF BOTH SIDES OF THE FRACTURE

A modification of the freeze-fracturing technique to permit observation of replicas of both sides of the fracture is described. It has been used to study mouse liver cell membrane structure. Membranes break to give two faces with three-dimensional complementarity, although there is some small-scale mismatching which is discussed. Since the two distinctive sets of membrane faces are complementary sets, they cannot be the two outside surfaces. In particular, structures (such as particles) seen on these faces are within the membrane. It is not possible from this work to say precisely where the fracture plane goes with respect to a plasma membrane, only that it must be close to the interface between membrane and cytoplasm, or at that interface. Models, consistent with the appearance of the matching replicas, are derived for three regions of the plasma membrane: (a) The nonjunctional plasma membrane, which contains many scattered particles. Except for these particles, the otherwise flat fracture face is not at variance with a bimolecular leaflet structure. (b) Gap junctions. Each of the two membranes comprising a gap junction contains a close-packed array of particles. (c) Tight junctions. Here membranes have ridges within them.     

Kinetic properties of aspartate transport in rat heart mitochondrial inner membranes.

The mitochondrial glutamate-aspartate exchange carrier catalyzes the electrogenic exchange of intramitochondrial aspartate for extramitochondrial glutamate. Protons are cotransported with glutamate in a 1:1 ratio. In the present study, the effects of pH and glutamate concentration on glutamate entry into intact mitochondria were determined. Hydrogen ions were found to decrease the K_m for glutamate entry. In addition, using glutamate-loaded submitochondrial particles, aspartate transport into the particles was measured as a function of internal and external glutamate concentrations, pH, and electrical potential across the membrane. Glutamate, was a competitive inhibitor of aspartate transport when both amino acids were present on the same side of the membrane, while H⁺ was a noncompetitive inhibitor of aspartate entry into the particles. A decrease in glutamate concentration on the inside of the particles brought about a parallel decrease in V and K_m for aspartate outside of the particles, thus suggesting a ping-pong mechanism for the carrier. The uncoupling agent, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), lowered both the K_m and V of aspartate transport, while the effect on V was somewhat larger. Data obtained in the presence of KSCN was similar to that obtained with FCCP, and therefore it is concluded that both K_m and V changes are dependent on a change of electrical potential across the membrane. A model for the carrier is proposed, which is consistent with the data presented. The model includes a single binding site specific for either glutamate or aspartate, and a separate binding site for the cotransported proton. The affinity of the binding site for protons is increased by simultaneous glutamate binding, but decreased by aspartate binding. The data suggest that an increase in the membrane potential increases the mobility of the charged carrier-aspartate complex, but also facilitates some additional step in the exchange cycle involving subsequent return of the carrier to the matrix side of the membrane. The additional membrane-potential-dependent step could be proton binding on the cytosolic side of the carrier.