Curvilinear Three-Dimensional Modeling of Spinal Curves with Dual Kriging

In spinal deformation studies, three-dimensional reconstruction of the spine is frequently represented as a curve in space fitted to the vertebral centroids. Conventional interpolation techniques such as splines, Bezier and the least squares method are limited since they cannot describe precisely the great variety of spinal morphologies. This article presents a more general technique called dual kriging, which includes two mathematical constituents (drift and covariance) to adjust the interpolated functions to spinal deformity better. The cross-validation technique was used to compare the parametric representations of spinal curves with different combinations of drift and covariance functions. Model validation was performed from a series of analytic curves reflecting typical scoliotic spines. Calculation of geometric torsion, a sensitive parameter, was done to evaluate the accuracy of the kriging models. The best model showed an absolute mean difference of 1.2 x 10(-5) (+/- 7.1 x 10(-5) ) mm(-1) between the analytical and estimated geometric torsions compared to 5.25 x 10(-3) (+/- 3.7 x 10(-2) ) mm(-1) for the commonly used least-squares Fourier series method, a significant improvement in spinal torsion evaluation.     

In vitro lung delivery of bacteriophages KS4‐M and ΦKZ using dry powder inhalers for treatment of Burkholderia cepacia complex and Pseudomonas aeruginosa infections in cystic fibrosis

Aims: To determine the feasibility of formulating and aerosolizing powders containing bacteriophages KS4‐M and ΦKZ for lung delivery and treatment of pulmonary Burkholderia cepacia complex and Pseudomonas aeruginosa infections. Methods and Results: Endotoxin‐removed bacteriophages KS4‐M and ΦKZ were lyophilized in lactose/lactoferrin 60 : 40 w/w matrix and deagglomerated in a mixer mill (without beads) to formulate respirable powders. The powders were then aerosolized using an Aerolizer^® capsule inhaler. Mass median aerodynamic diameter (MMAD) of this inhalable aerosol was determined using Andersen cascade impactor at 60 l min^?1. Measured MMAD for both types of powders was 3·4 μm, and geometric standard deviation was 1·9–2·0. Viability of bacteriophages delivered distal to an idealized mouth‐throat replica was determined from bioassays of samples collected on filters placed after the idealized replica. As a percentage of inhaler load, amount of powder delivered distal to the mouth‐throat replica, which is a measure of lung delivery, was 33·7 ± 0·3% for KS4‐M and 32·7 ± 0·9% for ΦKZ. Titres collected downstream of the mouth throat were (3·4 ± 2·5) × 10⁶ PFU for KS4‐M with an Aerolizer capsule load of (9·8 ± 4·8) × 10⁶ and (1·9 ± 0·6) × 10⁷ for ΦKZ with an Aerolizer capsule load of (6·5 ± 1·9) × 10⁷. Conclusions: Bacteriophages KS4‐M and ΦKZ can be lyophilized without significant loss of viability in a lactose/lactoferrin 60 : 40 w/w matrix. The resulting powders can be aerosolized to deliver viable bacteriophages to the lungs. Significance and Impact of the Study: Development of lactoferrin‐based bacteriophage aerosol powders solidifies the ground for future research on developing novel formulations as an alternative to inhaled antibiotic therapy in patients with cystic fibrosis.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Myotube production in fast myotomal muscle is switched-off at shorter body lengths in male than female Atlantic halibut Hippoglossus hippoglossus (L.) resulting in a lower final fibre number

A sampling method is described to determine accurately the number of fast myotomal muscle fibres (N_F) in a large flatfish species, the Atlantic halibut Hippoglossus hippoglossus. An unusual feature of the fast myotomal muscle is the presence of internalized strips of slow muscle fibres. In fish of 1·5–3·5 kg (n = 24), the total cross‐sectional area (A_TC) of fast muscle was 18% greater in the dorsal than ventral myotomal compartments (P < 0·05), whereas there was no significant difference between left‐ and right‐hand sides of the body. Due the bilateral asymmetry, muscle blocks (5 × 5 × 5 mm) were prepared to systematically sample each myotomal quadrant (dorsal, ventral, left‐ and right‐side) and the diameters of 150 fast fibres measured per block. Smooth non‐parametric probability functions were fitted to a minimum of 800 measurements of fibre diameter per quadrant (n = 5). There were no significant differences in the distribution of muscle fibre diameters between myotomal compartments and therefore N_F could be estimated from a single quadrant. The number of blocks required to estimate N_F with a repeatability of ±2·5% increased from six at 300 g body mass to 17 at 96·5 kg, caused by variation within and between blocks. Gompertz curves were fitted to measurements of fibre number and fork length (L_F). The estimated final fibre number was 8·96 × 10⁵ (7·99–9·94 × 10⁵, 95% CI) for males and 1·73 × 10⁶ (1·56–1·90 × 10⁶, 95% CI) for female fish. The estimated L_F for cessation of fibre recruitment in the fast muscle of female fish (1775 mm) was almost twice that in males (810 mm), reflecting their greater ultimate body size.     

BENDING STIFFNESS OF CYLINDRICAL PLANT ORGANS WITH A ‘CORE-RIND’ CONSTRUCTION: EVIDENCE FROM JUNCUS EFFUSUS LEAVES

The mechanical behavior of leaves of Juncus effusus L. in bending was investigated in terms of a closed-form analytical solution derived to predict the bending stiffness of a cylindrical sandwich beam consisting of an outer ‘rind’ (sclerenchyma and chlorenchyma) and an inner ‘core’ (aerenchyma). The elastic moduli (E_TOTAL) of intact leaves was measured by means of multiple resonance frequency spectra and compared to that of leaves for which the aerenchymatous core was surgically destroyed. Based on ten leaves, E_TOTAL = 22.33 × 10⁴ ± 5.37 ± 10⁴ kg · cm^–2 while the elastic modulus of the ‘rind’ was 22.29 × 10⁴ ± 5.69 × 10⁴ kg · cm^–2. The elastic modulus of the ‘core’ was estimated at 3.12 × 10⁴ ± 1.42 × 10⁴ kg · cm^–2. Load-deflection curves for three leaf segments indicated leaves were linearly elastic within the range of loading and could be predicted with considerable accuracy based on the closed-form solution. The aerenchymatous core was found to contribute very little to the bending stiffness of leaves, although its contribution appeared to increase as leaf diameter decreased. Leaves mechanically failed by Brazier buckling when excessively loaded and were best considered to mechanically operate as hollow tubes. Nonetheless, the analytical solution for bending stiffness could be applied and, in theory, can be used to predict the behavior of other plant organs with a ‘corerind’ construction.     

Semi‐permeable membrane retention of synovial fluid lubricants hyaluronan and proteoglycan 4 for a biomimetic bioreactor

Synovial fluid (SF) contains lubricant macromolecules, hyaluronan (HA), and proteoglycan 4 (PRG4). The synovium not only contributes lubricants to SF through secretion by synoviocyte lining cells, but also concentrates lubricants in SF due to its semi‐permeable nature. A membrane that recapitulates these synovium functions may be useful in a bioreactor system for generating a bioengineered fluid (BF) similar to native SF. The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 µm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes ± IL‐1β + TGF‐β1 + TNF‐α, a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes ± adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 µg/mL) was added to the BF compartment, and HA and PRG4 loss into the NF compartment after 2, 8, and 24 h was determined. Lubricant loss kinetics were analyzed to estimate membrane permeability. Experiment 1: Cytokine‐regulated HA and PRG4 secretion rates on membranes were comparable to those on TC plastic. Experiment 2: Transport of HA and PRG4 across membranes was lowest with 50 nm membranes and highest with 3 µm membranes, and transport of high MW HA was decreased by adherent synoviocytes (for 50 and 90 nm membranes). The permeability to HA mixtures for 50 nm membranes was ～20 × 10^?8 cm/s (? cells) and ～5 × 10^?8 cm/s (+ cells), for 90 nm membranes was ～35 × 10^?8 cm/s (? cells) and ～19 × 10^?8 cm/s (+ cells), for 170 nm membranes was ～74 × 10^?8 cm/s (± cells), and for 3 µm membranes was ～139 × 10^?8 cm/s (± cells). The permeability of 450 kDa HA was ～40× lower than that of 30 kDa HA for 50 nm membranes, but only ～2.5× lower for 3 µm membranes. The permeability of 4,000 kDa HA was ～250× lower than that of 30 kDa HA for 50 nm membranes, but only ～4× lower for 3 µm membranes. The permeability for PRG4 was ～4 × 10^?8 cm/s for 50 nm membranes, ～48 × 10^?8 cm/s for 90 nm membranes, ～144 × 10^?8 cm/s for 170 nm membranes, and ～336 × 10^?8 cm/s for 3 µm membranes. The associated loss across membranes after 24 h ranged from 3% to 92% for HA, and from 3% to 93% for PRG4. These results suggest that semi‐permeable membranes may be used in a bioreactor system to modulate lubricant retention in a bioengineered SF, and that synoviocytes adherent on the membranes may serve as both a lubricant source and a barrier for lubricant transport. Biotechnol. Bioeng. 2010; 106: 149–160. © 2009 Wiley Periodicals, Inc.     

Zinc and salinity effects on membrane transport in Chara connivens

Pressure-probe measurements showed that the pressure relaxation of internodal cells of the freshwater alga Chara connivens slowed considerably when 1–5 mol m^?3 Zn²⁺, or more especially Zn²⁺ and 75 mol m^?3 NaCl, were present in the medium for periods of 1 h or longer. These results indicate that the water permeability of the Chara membrane is decreased by Zn²⁺, and that this effect is enhanced by 75 mol m^?3 NaCl. Specific values taken after 375 min exposure were: 5 mol m^?3 Zn²⁺ and 75 mol m^?3 NaCl caused the half-time for bulk water movement to increase from 7·8±2·3 to 79·5±5·4s, corresponding to a decrease in the hydraulic conductivity (Lp) from (13·0±3·3) × 10^?7 m s^?1 mPa^?1 to (1·25±0·23) × 10^?7 m s^?1 MPa^?1 (mean±S.D., n= 10). These changes are not seen in the presence of NaCl alone, and to a reduced extent in the presence of 5 mol m^?3Zn²⁺ alone (after 375 min, Lp was (2·4±0·1) × 10^?7 m s^?1 MPa^?1, mean±S.D., n = 6). Ca²⁺ cannot substitute for Zn²⁺, but seems to competitively inhibit Zn²⁺. There was another, kinetically distinct effect of Zn²⁺: the ingress of Na⁺ within 15 min of exposure to 75 mol m^?3 NaCl is halved by the presence of 1–5 mol m^?3 Zn²⁺, although internal osmolality is little changed by Zn²⁺. In spite of this, Zn²⁺ does not exert the long-term protection against NaCl that has been reported for Ca²⁺. Depending on the concentration of Zn²⁺ and the duration of the exposure, the effects on water permeability were fully or partly reversible within 24–48 h. The mechanism of these changes is difficult to identify. One possibility is a zinc-induced restriction of trans-membrane channels to give single-file channels which can be blocked by salt.     

Residence time and drift patterns of larval June sucker Chasmistes liorus in the lower Provo River as determined by otolith microstructure

Estimates of age derived from daily ring counts from otoliths and capture rates of larval June sucker Chasmistes liorus were used to determine the relationship between discharge rates of the Provo River and residence time and patterns of larval drift. During 1997, larval drift occurred over a 22 day period when discharge rates were low (mean ±s.d. 3·2 ± 0·0 m³ s^?1). In 1998, larval drift occurred in two separate events over a 40 day period. Discharge was higher during the first larval drift period (19 days; 24·8 ± 1·3 m³ s^?1) and lower during the second larval drift period (17 days; 7·0 ± 0·9 m³ s^?1). In 1997, no larval fish were collected at the lowermost transect on the Provo River (nearest Utah Lake), and few larvae >21 days of age were found. During the first drift period of 1998, larval C. liorus were collected at all transects, and mean age of larvae collected between upstream and downstream transects increased by c. 7 days. During the second drift period of 1998, only a few were collected in the lowermost transects, and age did not increase with proximity to the lake. Patterns in catch and age distribution of larval C. liorus in the lower Provo River suggest that recruitment failure occurs during the larval drift period in years with insufficient discharge to transport larvae into the lake.     

Contribution of terrestrial invertebrates to the annual resource budget for salmonids in forest and grassland reaches of a headwater stream

1. The annual input, contribution to the diet of salmonids, and quantitative input of terrestrial invertebrates to four reaches with contrasting forest (n=2) and grassland riparian vegetation (n=2) were compared in a Japanese headwater stream.
2. The annual input of terrestrial invertebrates falling into the forest reaches (mean±1 SE=8.7×10³±0.3×10³ mg m^?2 year^?1) was 1.7 times greater than that in the grassland reaches (5.1×10³±0.8×10³ mg m^?2 year^?1), with clear seasonality in the daily input of invertebrates in both vegetation types. The daily input, however, differed between the vegetation types only in summer, when it rose to a maximum in both vegetation types.
3. Fish biomass also differed among the seasons in both vegetation types, being less in the grassland reaches. The contribution of terrestrial invertebrates to the salmonid diet in the forest and grassland reaches was 11 and 7% in spring, 68 and 77% in summer, 48 and 33% in autumn, and 1 and 1% in winter, respectively. The prey consumption rate of fish, which was similar between the vegetation types, increased with stream temperature and was highest in summer. Terrestrial invertebrates supported 49% (mean±1 SE=5.3×10³±0.4×10³ mg m^?2 year^?1) of the annual, total prey consumption (10.9×10³±1.7×10³ mg m^?2 year^?1) by salmonids in the forest and 53% (2.0×10³±0.3×10³ mg m^?2 year^?1) (3.8×10³±0.6×10³ mg m^?2 year^?1) in the grassland reaches.
4. Salmonids were estimated to consume 51 and 35% of the annual total (falling plus drift) input of terrestrial invertebrates in the forest and grassland reaches, respectively. The input of terrestrial invertebrates by drift, however, was almost equal to the output in both vegetation types, suggesting that the reach‐based, in‐stream retention of terrestrial invertebrates almost balanced these falling in.
5. Difference in the riparian vegetation, which caused spatial heterogeneity in the input of terrestrial invertebrates, could play an important role in determining the local distribution of salmonids.     

Ecophysiology of the hypotonic response in the salt-tolerant charophyte alga Lamprothamnium papulosum

The ecophysiology of the hypotonic response was studied in the charophyte alga, Lamprothamnium papulosum, which was grown in a marine (SW; 1072 mosmol kg^–1) and a brackish (1/2 SW; 536 mosmol kg^–1) environment. The cells produced an extracellular mucilage identified by histochemical staining as a mixture of sulphated and carboxylated polysaccharides. The thickness and chemical composition of the mucilage layer was a function of environmental salinity and cell age. Mucilage progressively increased in thickness from the apex (9 SW cells: 12·6 ± 1·8 μm; 15 1/2 SW cells: 4·8 ± 0·7 μm) to the base of the plants (15 SW cells: 44·8 ± 3·3 μm; nine 1/2 SW cells: 23·8 ± 2·5 μm); with a corresponding increase in the sulphated proportion. The mucilage was significantly thicker in SW plants. Hydraulic conductivity (Lp) at the apex of SW plants, measured by transcellular osmosis, was 8·3 × 10^–13 m s^–1 Pa^–1. This was close to Lp of freshwater Chara (8·5 × 10^–13 m s^–1 Pa^–1) which lacked mucilage. Basal SW cells with thicker mucilage had a smaller apparent Lp of 3·5 × 10^–13 m s^–1 Pa^–1. The electrophysiology of the resting state and hypotonic response was compared in cells from the two environments based on current/voltage (I/V) analysis. The resting potential difference (PD) and conductance differed (11 SW cells: – 102·4 ± 10·1 mV, eight SW cells: 18·6 ± 2·4 S m^–2; 19 1/2 SW cells: –125·7 ± 5·9 mV, 8·3 ± 0·8 S m^–2). The type of cellular response to a hypotonic shock (decrease of 268 mosmol kg^–1) also differed. In 1/2 SW plants, only the apical cells with thin mucilage responded classically with depolarization, conductance increase, Ca²⁺ influx, cessation of cytoplasmic streaming, and K⁺ and Cl^– effluxes. Older cells making up the bulk of the plants responded with depolarization, but continued cytoplasmic streaming, and had only a small increase in conductance; or depolarized transiently without altering the I/V profile, conductance or streaming speed. Most cells remained depolarized and in the K⁺ state 1 h post-shock. Cells treated with the K⁺ channel blocker tetraethylammonium chloride also depolarized and remained depolarized. The SW cells depolarized but otherwise responded minimally to a 268 mosmol kg^–1 drop in osmolarity and required a further 268 mosmol kg^–1 down-step to elicit a change in the conductance. A spectrum of responses was measured in successively older and more mucilaginous cells from the same marine plant. We discuss the ecophysiological significance of the mucilage layer which modulates the cellular response to osmotic shock and which can be secreted to different degrees by plants inhabiting environments of different salinity.     

Postprocessing microflora of commercial attieke (a fermented cassava product) produced in the south of Côte d'Ivoire

The distribution and presence of hygiene indicator and pathogenic micro‐organisms in 375 samples of attieke marketed in Côte d'Ivoire, and their roles in the food poisoning were evaluated. Microbiological analyses were carried out, which included the total viable bacteria, coliforms, Escherichia coli, Staphylococcus aureus, Salmonella, Bacillus spores, fungi and Clostridium perfringens. The results revealed that the viable bacteria counts ranged from 2·2 ± 1·2 × 10⁵ to 3·4 ± 1·4 × 10⁶ CFU g^?1, while the yeasts and the moulds counts ranged, respectively, from 2·4 ± 0·12 × 10⁴ to 9·8 ± 0·4 × 10⁵ CFU g^?1 and 1·3 ± 0·7 × 10¹ to 1·7 ± 0·7 × 10² CFU g^?1. Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Citrobacter freundi, Enterobacter amnigenus, Citrobacter youngae, Enterobacter aerogenes, Klebsiella pneumoniae, Serratia marcescens, Enterobacter agglomerans and Klebsiella oxytoca were the bacteria isolated, and Rhizopus spp., Mucor spp., Thamnidium spp., Fusarium spp., Moniliella spp. the fungi. Escherichia coli, Clostridium perfringens and Salmonella spp. were not found. The occurrence of some bacteria and fungi illustrate that attieke collected in Côte d'Ivoire markets may act as a reservoir of pathogenic micro‐organisms for human.

Significance and Impact of Study

This study demonstrates the great need to carry out microbiological tests frequently on attieke and even more the need to apply correct HACCP system during the production. Attieke is especially a well‐known product in West Africa; hence, it is extremely important to ensure an adequate microbiological quality to guarantee consumers health. Overall, the study highlighted the need for effective communication on microbiological food risks, proper instruction and supervision in food‐handling procedures, greater education on food safety risks.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

Light-scattering spectroscopic study of polysaccharide protein conjugate

By combining gel permeation chromatography (GPC) and light-scattering spectroscopy, including photon correlation and angular distribution of absolute scattered intensity, we were able to characterize immunologically active Haemophilus influenzae type b polysaccharide (HIB Ps) bovine serum albumin (BSA) conjugates in terms of equivalent hydrodynamic radius r_h ～ (6.2 ± 0.6) × 10² Å, apparent radius of gyration r_g ～ (5.4 ± 0.3) × 10² Å, apparent molecular weight M_w ～ (3.5 ± 0.4) × 10⁶ g/mol, and a second virial coefficient A₂ ～ (1.9 ± 0.3) × 10^?4 cm³ mol/g². We could study the effects of each of the processes in the conjugate formation according to the following procedure: BSA (dialysis, modification, fractionation) + HIB Ps → HIB Ps/BSA conjugate (conjugate formation, fractionation). Narrow distributions of HIB Ps BSA conjugate formation can be achieved using fractionated BSA.     

Laboratory studies on the fungus Verticillium lecanii, a larval pathogen of the large elm bark beetle (Scolytus scolytus)

From 1972 to 1974, estimates of the natural larval mortality (> second instar) of elm bark beetles caused by pathogenic organisms were always below 7'5 % of the beetle population. The pathogenic fungus Verticillium lecanii was frequently isolated from field-collected dead larvae, and in the laboratory all larvae were killed in 5 days when exposed to spore concentrations of 4·5 × 10⁶ spores/ml. V. lecanii begins to lose its pathogenicity after prolonged culture on artificial media. The time taken for V. lecanii to kill Scolytus scolytus larvae when exposed to a logarithmic series of spore dilutions from 9·1 × 10⁷/ml to 9·1 × 10³/ml increased with decreasing amounts of inoculum. Even at spore concentrations as low as 9·1 × 10³/ml the mortality of treated larvae was greater than that of untreated individuals. At 100% r.h. all treated larvae were killed over a temperature range of 5–30 °C; those maintained at 25 °C were killed most rapidly and those kept at 5 °C the slowest.     

The effects of environmental salinity on the growth and physiology of totoaba Totoaba macdonaldi and shortfin corvina Cynoscion parvipinnis

Totoaba Totoaba macdonaldi and shortfin corvina Cynoscion parvipinnis, were acclimated and reared together at salinities of 0, 2, 5, 10, 20 and 35 for 56 days. Initial overall mean ± s.d . body masses of 67·6 ± 7·1 g T. macdonaldi and 37·3 ± 3·1 g C. parvipinnis increased to final overall masses of 217·4 ± 30·3 and 96·5 ± 16·5 g, respectively, at the end of the study. Totoaba macdonaldi was not able to tolerate salinities of 0 and 2 and C. parvipinnis of 0. In contrast, both species had 100% survival at salinities ≥ 10. Somatic growth was highest not at natural seawater salinity of 35, but at 10. Plasma osmolality ranged from 172·5 to 417·0 mOsmol kg^?1 for T. macdonaldi and from 207·0 to 439·5 mOsmol kg^?1 for C. parvipinnis and varied in direct proportion to salinity. The estimated isosmotic salinities of T. macdonaldi and C. parvipinnis were 12·3 and 13·4, respectively. Cynoscion parvipinnis reared at two had significantly lower plasma lysozyme activity (95·0 Units ml^?1) than fish held at salinities from 5 to 35 (ranging from 215·0 to 355·0 Units ml^?1), but without clear trends over this range. Blood neutrophil oxidative radical production (NBT) (ranging from 3·9 to 6·7 mg ml^?1) had some significant differences among salinities, but these did not follow a clear pattern. For T. macdonaldi, neither lysozyme activity nor NBT was affected by salinity. Ash content of whole fishes varied directly and moisture content inversely, with salinity for both species.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Physicochemical studies on xylinan (acetan). III. Hydrodynamic characterization by analytical ultracentrifugation and dynamic light scattering

A laboratory-made sample of the polysaccharide xylinan (acetan) has been further characterized with respect to (i) purity, (ii) molar mass and polydispersity, and (iii) gross conformation by a combination of hydrodynamic measurements (sedimentation velocity and equilibrium analytical ultracentrifugation, viscometry, and dynamic light scattering) in aqueous NaCl (I = 0.10 mol·L⁻¹). Sedimentation velocity diagrams recorded using Schlieren optics revealed highly pure material sedimenting as a single boundary [s^o_20.w = 9.5 ± 0.7) S; k_s = (273 ± 112) mL/g]. The hypersharp nature of these boundaries is symptomatic of a polydisperse and highly nonideal (in the thermodynamic sense) system. Low speed sedimentation equilibrium in the analytical ultracentrifuge using Rayleigh interference optics and two different types of extrapolation procedure (involving point and whole-cell molar masses) gave a weight average molar mass M_w of (2.5 ± 0.5) × 10⁻⁶ g·mol⁻¹ and also a second virial coefficient, B = (2.8 ± 0.7) × 10⁻⁴ mL·mol·g⁻², both values in good agreement with those from light scattering-based procedures (Part II of this series). A dynamic Zimm plot from dynamic light scattering measurements gave a z-average translational diffusion coefficient D^o_20.w = (3.02 ± 0.05) × 10⁻⁸ cm²·s⁻¹ and the concentration-dependence parameter k_D = (370 ± 15) mL/g. Combination of s^o_20.w with D^o_20.w via the Svedberg equation gave another estimate for M_w of ≅ 2.4 × 10⁶ g/mol, again in good agreement. Both the Wales-van Holde ratio (k_s/[η]) ≅ 0.4 (with [η] = (760 ± 77) mL/g) and the ρ-parameter (ratio of the radius of gyration from static light scattering to the hydrodynamic radius from dynamic light scattering) as ρ > 2.0 all indicate an extended conformation for the macromolecules in solution. These findings, plus Rinde-type simulations of the sedimentation equilibrium data are all consistent with the interpretation in terms of a unimodal wormlike coil model performed earlier. © 1996 John Wiley & Sons, Inc.     

Further studies on double-stranded RNA species from raspberry plants infected with aphid-borne viruses

Analysis by electrophoresis in polyacrylamide gels, followed by silver staining, of dsRNA extracted from many samples of raspberry leaves infected with raspberry leaf mottle virus (RLMV) and/or raspberry leaf spot virus (RLSV) failed to detect reliably any significant quantities of dsRNA species in excess of 1·0 × 10⁶mol. wt. This contrasts with results reported from Canada where three dsRNA species of estimated mol. wt 2·6 × 10⁶1·6 × 10⁶and 1·1 × 10⁶were consistently associated with infection with RLSV but none were associated with RLMV. However, in Scotland, four dsRNA species of estimated mol. wt 2·4 × 10⁶1·6 × 10⁶0·7 × 10⁶and 0·3 × 10⁶were detected in raspberry infected with apple mosaic ilarvirus. These results suggest that the dsRNA species reported from Canada are not those of RLSV but are probably those of a second virus, possibly an ilarvirus, which occurs together with RLSV and/or induces similar symptoms. A few samples from plants infected with RLMV and RLSV contained very small amounts of two dsRNA species of estimated mol. wt 4·7 × 10⁶and 4·5 × 10⁶. It is not known whether these species are those of RLMV and RLSV.     

INORGANIC CARBON ACQUISITION BY CHRYSOPHYTES1

Twelve species, representing 12 families of the chrysophytes sensu lato, were tested for their ability to take up inorganic carbon. Using the pH‐drift technique, CO₂ compensation points generally varied between 1 and 20 μmol · L^?1 with a mean concentration of 5 μmol · L^?1. Neither pH nor alkalinity affected the CO₂ compensation point. The concentration of oxygen had a relatively minor effect on CO₂‐uptake kinetics, and the mean CO₂ compensation point calculated from the kinetic curves was 3.6 μmol · L^?1 at 10–15 kPa starting oxygen partial pressure and 3.8 μmol · L^?1 at atmospheric starting oxygen partial pressure (21 kPa). Similarly, uptake kinetics were not affected by alkalinity, and hence concentration of bicarbonate. Membrane inlet mass spectrometry (MIMS) in the presence and absence of acetazolamide suggested that external carbonic anhydrase in Dinobryon sertularia Ehrenb. and Synura petersenii Korschikov was either very low or absent. Rates of net HCO₃^? uptake were very low (～5% of oxygen evolution) using MIMS and decreased rather than increased with increasing HCO₃^? concentration, suggesting that it was not a real uptake. The CO₂ compensation points determined by MIMS for CO₂ uptake and oxygen evolution were similar to those determined in pH‐drift and were >1 μmol · L^?1. Overall, the results suggest that chrysophytes as a group lack a carbon‐concentrating mechanism (CCM), or an ability to make use of bicarbonate as an alternative source of inorganic carbon. The possible evolutionary and ecological consequences of this are briefly discussed.     

Molecular size and shape of the native and reassociated forms of the extracellular haemoglobin of Tubifex tubifex

The extracellular haemoglobin of Tubifex tubifex and the product of its reassociation at neutral pH subsequent to dissociation at alkaline pH, were examined by small-angle X-ray scattering. The following molecular parameters were determined for the native and reassociated molecules, respectively: maximum diameter 30.0±1.0 and 32.0±1.0nm; radius of gyration 10.66±0.15 and 11.07±0.15 nm; molecular weight (3.09±0.15) × 10⁶ and (2.99±0.15) × 10⁶ dalton. Although the scattering curves of the native and reassociated haemoglobin possess similar shapes the distance distribution functions exhibit slight differences in their shape as well as in the position of their maximum. The best fit with the experimental distribution functions was obtained with models consisting of 12 spheres arranged in two hexagonal layers. In the case of the native haemoglobin each of the 12 spheres has a diameter of 9.3 nm while for the reassociated haemoglobin each of the 12 spheres has a diameter of 11.5 nm. The results suggest that although their molecular weights are the same, the reassociated molecule is slightly larger than the native molecule