A glass-membrane pH microelectrode.

By miniaturizing the original MacInnes and Dole glass-membrane pH electrode a new pH microelectrode has been developed. The technique developed utilizes the tip of a high electrical resistance glass pipet that can be sealed with a thin membrane of H⁺-sensitive glass. Single-barreled electrodes have been made with tip diameters ranging from 1.5 to 100 μm and double-barreled electrodes with tip diameters from 2 to 28 μm. The glass-membrane pH microelectrode provided a means for sensing the pH of biological solutions with an electrode having theoretical slope and tip configurational control. The most unique characteristics of the electrode were: the pH sensing surface was quite small, the tip diameter could be controlled, and the problem of electrode insulation was eliminated.     

Regular superstructures of purified DNA in ethanolic solutions

Aqueous solutions of purified DNA from bacteriophage T7 were subjected to various concentrations of ethanol and visualized by electron microscopy. Compact, linear, unbranched particles with uniform diameters were found which have three distinct lengths, 3.04±0.04 μm, 0.69±0.01 μm and 0.159±0.014μm, with increasing diameters. An analysis of the observations revealed supercoils of first, second and third order for the above lengths. DNA supercoiling may be the consequence of dehydration by ethanol and drying and, in native chromatin, of dehydration by histone.     

A model for discrete spindle elongation

It is not understood how spindles elongate by discrete lengths of approximately 0.7 µm or multiples of 0.7 µm during anaphase B of the cell cycle. Here, we report that GTP-tubulin segments (newly synthesized segments) on microtubules have discrete lengths of approximately 0.35 µm or multiples of 0.35 µm. Because two sets of microtubules, namely anti-parallel interpolar microtubules, contribute to spindle elongation, the total lengths of the GTP-tubulin segments on their plus ends should be 0.7 µm or multiples of 0.7 µm. Microtubule synthesis in such discrete lengths is thus hypothesized to underlie the discrete lengths in spindle elongation.     

Xylem of rattans: vessel dimensions in climbing palms

We examined 11 species in four genera of rattans (Calamus, Daemonorops, Korthalsia, Plectocomia) growing in their native rainforest habitat in Singapore. Using aqueous safranin dye, we found that >95% of all vascular bundles at the base of a mature stem were functioning to transport water. We determined the frequency of vessel lengths in the long stems of these climbing palms by infiltration with dilute latex paint. Separate length distributions were made for metaxylem and protoxylem vessels; in both, there were many short and a few long vessels. The longest protoxylem vessels ranged from 7.5 to 62 cm in length, but one stem had an exceptional protoxylem vessel measuring 3.0 m. Maximum metaxylem vessel diameters were positively correlated to maximum vessel lengths in these species. The longest metaxylem vessel was found in K. rigida and was 3.96 m in length and was constructed from ～1200 vessel elements (cells). The widest vessel in that same stem was 532 μm in diameter. Long, wide vessels decrease resistance and increase water transport efficiency. In addition, we suggest that wide metaxylem vessels may have an important function in water storage.     

Single acetylcholine-activated channel currents in developing muscle cells

The properties of single acetylcholine-activated ion channels in developing rat myoblasts and myotubes in tissue culture have been investigated using the gigaohm seal patch clamp technique. Two classes of ACh-activated channels were identified. The major class of channels (accounting for >95% of all channel openings) has a conductance of 35 pS and a mean open time of 15 msec (at room temperature and ?80 mV). The minor class of channels has a larger conductance (55 pS) and a briefer mean open time (2–3 msec). Functional ACh-activated channels are present in undifferentiated mononucleated myoblasts 1–2 days in culture, although the channel density on such cells is low. Over the next week in culture, as the myoblasts fuse to form multinucleate myotubes, there is a marked increase in channel density and an increase in the proportion of large conductance channels. No significant change, however, occurs in channel conductance or open time (within a given class of channels) during this period. At high concentrations of ACh, channels desensitize and channel openings occur in groups, similar to what has been previously described in adult muscle. The rate of channel opening within a group of openings increases with increasing agonist concentration while mean open time is independent of agonist concentration, as expected from simple models of drug action. During a group of openings, the channel is open for half the time (i.e., channel opening rate is equal to channel closing rate) at a concentration of approximately 6 μm ACh.     

Analysis of circular bordered pit function II. Gymnosperm tracheids with torus-margo pit membranes

A model of xylem conduit function was applied to gymnosperm tracheids with torus-margo pit membranes for comparison with angiosperm vessels. Tracheids from 17 gymnosperm tree species with circular bordered pits and air-seed pressures from 0.8 to 11.8 MPa were analyzed. Tracheids were more reinforced against implosion than vessels, consistent with their double function in transport and support. Tracheid pits were 3.3 to 44 times higher in hydraulic conductivity than vessel pits because of greater membrane conductivity of the torus-margo configuration. Tight scaling between torus and pit size maximized pit conductivity. Higher pit conductivity allowed tracheids to be 1.7-3.4 times shorter than vessels and still achieve 95% of their lumen-limited maximum conductivity. Predicted tracheid lengths were consistent with measured lengths. The torus-margo structure is important for maximizing the conductivity of the inherently length-limited tracheid: replacing the torus-margo membrane with a vessel membrane caused stem tracheid conductivity to drop by 41%. Tracheids were no less hydraulically efficient than vessels if they were long enough to reach their lumen-limiting conductivity. However, this may only be possible for lumen diameters below approximately 60-70 μm.     

Imaging of chemiluminescent reactions in mesoscale silicon-glass microstructures

Chemiluminescent reactions in mesoscale analytical structures (chips) containing micrometer-sized interconnecting channels and chambers (pL-nL total volume) were imaged. The chips were fabricated by bonding Pyrex glass to etched pieces of silicon using a high-temperature diffusive bonding technique. In initial experiments light emission from an enhanced chemiluminescent horseradish peroxidase reaction and from a peroxyoxalate reaction contained in straight channels (300 μm wide × 20μ deep; volume 70.2 nL) and open chambers (812 μm wide, 400 μm deep, 5.2 mm long) linked by channels (100μm wide, 20 μm deep) to an exit and entry port were studied using a specially modified microplate holder and an Amerlite microplate luminometer. Light emission from more complex structures (two chambers interconnected by a branching channel 100 μm wide, 20 μm deep) filled with a solution containing alkaline phosphatase, Emerald, and CSPD^TM was imaged using a Photometrics Star 1 CCD camera. Detailed investigation of the detection and spatial resolution of the signal was performed on a Berthold Luminograph LB 980 using both the enhanced chemiluminescent horseradish peroxidase reaction and a peroxyoxalate reaction. We successfully resolved light emission from silicon structures with dimensions 100 μm wide and 20 μm deep. These simple silicon structures served as models for more complex designs that will be used for simultaneous multi-analyte assays in which an imaging system resolves and quantitates light emission from different locations on a silicon-glass analytical device.     

The long activations of α2 glycine channels can be described by a mechanism with reaction intermediates ("flip")

The α2 glycine receptor (GlyR) subunit, abundant in embryonic neurons, is replaced by α1 in the adult nervous system. The single-channel activity of homomeric α2 channels differs from that of α1-containing GlyRs, as even at the lowest glycine concentration (20 μM), openings occurred in long (>300-ms) groups with high open probability (P(open); 0.96; cell-attached recordings, HEK-expressed channels). Shut-time intervals within groups of openings were dominated by short shuttings of 5-10 μs. The lack of concentration dependence in the groups of openings suggests that they represent single activations, separated by very long shut times at low concentrations. Several putative mechanisms were fitted by maximizing the likelihood of the entire sequence of open and shut times, with exact missed-events allowance (program hjcfit). Records obtained at several glycine concentrations were fitted simultaneously. The adequacy of the different schemes was judged by the accuracy with which they predicted not only single-channel data but also the time course and concentration dependence of macroscopic responses elicited by rapid glycine applications to outside-out patches. The data were adequately described only with schemes incorporating a reaction intermediate in the activation, and the best was a flip mechanism with two binding sites and one open state. Fits with this mechanism showed that for α2 channels, the opening rate constant is very fast, ～130,000 s(-1), much as for α1β GlyRs (the receptor in mature synapses), but the estimated true mean open time is 20 times longer (around 3 ms). The efficacy for the flipping step and the binding affinity were lower for α2 than for α1β channels, but the overall efficacies were similar. As we previously showed for α1 homomeric receptors, in α2 glycine channels, maximum P(open) is achieved when fewer than all five of the putative binding sites in the pentamer are occupied by glycine.     

BATRACHOSPERMUM HETEROCORTICUM SP. NOV. AND POLYSIPHONIA SUBTILISSIMA (RHODOPHYTA) FROM FLORIDA SPRING-FED STREAMS1

Polysiphonia subtilissima Mont. Is reported for the first time from a freshwater environment. The presence of four pericentral cells, subdichotomous branching, apical trichoblasts and rhizoids arising from pericentral cells combined with a lack of cortication and reproductive cells is consistent with marine populations of this species. The range of filament length is 1.4–4.7 cm. Branch diameters are 38–76 μm and pericentral cell lengths are 58–125 μm. Batrachospermum heterocorticum sp. nov. is distinguished primarily by a developmental change in cortical filaments from typical cylindrical cells (5.0–7.9 μm diam in initial stages to enlarged, elliptical cells (12.9–24.1 μm diam) in mature axes. Another unique feature of this species is carpogonia with cylindrical, pedicellate trichogynes on stringht carpogonial branches in mid to outer portions of lateral whorls. Other characteristics of B. heterocorticum include the following: olive-green color, 2–6 cm length, dichotomous to trichotomous fascicles in 4–7 tiers, 385–647 μm whorl diameters, 109–198 μm carpospore diameters and relatively small “chantransia” filaments.     

Coccidian Parasites (Apicomplexa: Eimeriidae) from Insectivores: New Species from Shrew Moles (Talpidae) in the United States1

All of 18 shrew moles, Neurotrichus gibbsii, collected in Oregon and Washington were infected with one or more species of coccidia. Three eimerians and one isosporan were identified and described as new species. Sporulated oocysts of Eimeria heterocapita n. sp. were subspheroid to ellipsoid, 25.5 times 21.4 (23–27 times 18–23) μm. A membranous, cap-like structure was present at one pole of the oocyst, but a micropyle, oocyst residuum, and polar body were absent. Ovoid sporocysts were 13.6 times 10.0 (12–15 times 9–11) μm; a compact sporocyst residuum was present, but Stieda, sub-, and parastieda bodies were absent. This species was found in 2 of 18 (11%) hosts. Sporulated oocysts of Eimeria neurotrichi n. sp. were avoid, 17.6 times 13.6 (16–20 times 11–16) μm; micropyle and oocyst residuum were absent, but a polar body was present. Ovoid sporocysts were 10.7 times 5.5 (9–12 times 5–6) μm; Stieda body and sporocyst residuum were present, but sub- and parastieda bodies were absent. This species was found in 2 of 18 (11%) hosts. Sporulated oocysts of Eimeria parastiedica n. sp. were subspheroid, 27.4 times 25.5 (25–30 times 22–28) μm; micropyle, oocyst residuum, and polar body were absent. Ovoid sporocysts, pointed at both ends, were 18.3 times 10.4 (16–20 times 9–11) μm; Stieda, sub-, and parastieda bodies were present as was a sporocyst residuum. This species was found in 2 of 18 (11%) hosts. Sporulated oocysts of Isospora neurotrichi n. sp. were subspheroid, 13.9 times 12.0 (11–16 times 10–15) μm; micropyle and oocyst residuum were absent, but 1–3 polar bodies were present. Ellipsoid sporocysts were 9.2 times 6.1 (8–11 times 5–8) μm; sub- and parastieda bodies were absent, but a Stieda body and sporocyst residuum were present. This species was found in 17 of 18 (94%) hosts. Ten of 18 (56%) hosts were seen to be naturally infected with only one coccidium.     

Hair diameter distribution in sheep interspecies hybrids

Hair diameter distribution was studied in wild sheep species (argali—Ovis ammon, mouflon—O. Musimon; domestic sheep—O. aries) and their hybrids. In wild species and their hybrids, rather distinctly separated subpopulations of thin (underwool) hairs (10–22 μm diameter) and thick (kemp) hairs (85–302 μm) were detected, and intermediate diameters were rare. In domestic sheep, the kemp subpopulation was very poor, not distinctly separated from the underwool, while maximal hair diameter was <150 μm. In wild × domestic crosses, the kemp subpopulation was clearly expressed, shifted to lower diameter range, and maximal diameters was <200 μm. The same restriction was found in two-month-old wild species lambs compared with adults.     

CHROOCOCCOID CYANOBACTERIA IN LAKE ONTARIO: VERTICAL AND SEASONAL DISTRIBUTIONS DURING 19821

Chroococcoid cyanobacteria (0.7–1.3 μm in diameter) were discovered to be a significant component of the Lake Ontario plankton. Using epifluorescence microscopy, the densities of these microorganisms were found to vary by four orders of magnitude with a single large peak in abundance (6.5 × 10⁵ cells mL^?1) corresponding to the time of maximum water temperature. The morphology and abundance of these cyanobacteria were similar to those previously found in oceanic systems. They constituted 10% of the bacterial numbers in the epilimnion during this period, approximately 40% of the biomass of prokaryotes less than 2.0 μm, and 30% of the biomass of all microorganisms less than 20 μm in size. Size fractionation studies indicated that they were responsible for approximately 38% of the total primary production during times of peak abundance, and were important in phosphorus uptake. Cyanobacteria observed in the food vacuoles of heterotrophic microflagellates and in the guts of rotifers suggest that the latter organisms may be important consumers of this prokaryote population.     

Influence of the glass packing on the contamination of pharmaceutical products by aluminium. Part III: Interaction container-chemicals during the heating for sterilisation

The interaction of chemicals with the container materials during heating for sterilisation was investigated, storing the components of parenteral nutrition solutions individually in sealed glass ampoules and in contact with a rubber stopper, and heating the system at 121 degrees C for 30 min. Subsequently, the aluminium content of the solutions was measured by atomic absorption spectrometry (AAS). The assay was also carried out with acids, alkalis and some complexing agents for Al. The containers were decomposed and also assayed for aluminium. 30 different commercial solutions for parenteral nutrition, stored either in glass or in plastic containers, were assayed measuring the aluminium present in the solutions and in the container materials. The results of all investigated container materials revealed an aluminium content of 1.57% Al in glass, 0.05% in plastic and 4.54% in rubber. The sterilisation procedure showed that even pure water was able to extract Al from glass and rubber, 22.5 +/- 13.3 microg/L and 79.4 +/- 22.7 microg/L respectively, while from plastic the aluminium leached was insignificant. The Al released from glass ampoules laid between 20 microg/L for leucine, ornithine and lysine solutions and 1500 microg/L for solutions of basic phosphates and bicarbonate; from rubber stoppers it reached levels over 500 microg/L for cysteine, aspartic acid, glutamic acid and cystine solutions. Ion-exchange properties and influence of pH can explain the interaction of glass with some chemicals (salts, acids and alkalis), but only an affinity for aluminium could explain the action of some amino acids and other chemicals, as albumin and heparin, on glass and rubber, considering the aluminium release. Experiments with complexing agents for Al allowed to conclude that the higher the stability constant of the complex, the higher the Al release from the container material.     

Fiber composition of the distal accessory flexor muscle in several decapod crustaceans

The fiber composition of the distal accessory flexor muscle (DAFM) and the branching pattern of its excitor axon were compared in several species of crabs, in the lobster and the crayfish. The muscle is composed exclusively of long sarcomere (> 6 μm) fibers and therefore of the slow type. In all the crab species, except one, there is a distal to proximal gradient of fibers with increasing sarcomere lengths; this gradient is reverse in lobsters and crayfish. A proximal to distal gradient of increasing fiber diameters occurs in the DAFM of all crab species but not in the lobster and crayfish, in which all the fibers are approximately equal in diameter. The single excitatory axon traverses the width of the DAFM and gives off primary branches on either side in the lobster and crayfish but on only one side in crabs. The hypothesis that the axonal branching pattern may govern the regional distribution of fibers with differing sarcomere lengths in proposed.     

A submicrometer glass-membrane pH microelectrode.

The glass-membrane pH microelectrode (GMpHME) described previously (Anal. Biochem.73, 501, 1976) had a limitation in the minimum size (tip diameter) that could be manufactured (about 1 μm). In addition, when made at this small size the electrical resistance was usually high (10¹¹ Ω) and the response time long (greater than 5 min). The inability to manufacture the GMpHME with tip diameters less than 1 μm was primarily due to the thickness of the pH glass used to form the H⁺-sensitive membrane. In this report we detail a method for thinning the pH glass in such a way that the manufacture of submicrometer glass-membrane pH microelectrodes is possible. These submicrometer pH electrodes have rapid response times (1 to 3 min) and maintain the desirable characteristics of all GMpHMEs, that is, near theoretic slope and a well-defined sensing surface area.     

Impact of tightly focused femtosecond laser pulses on nucleolus-like bodies of mouse GV oocyte and the ability of mouse oocytes to mature

Using femtosecond laser radiation, nucleolus-like bodies (NLBs) of mouse oocytes were locally dissected without damage to zona pellucida, cytoplasmic membrane, nuclear membrane, and nucleoplasm surrounding NLB. It was found that, after dissection of 2.7 × 10^–11 cm³ of NLB material, which is approximately 5.2% of 10 μm NLB volume, the probability of germinal vesicle oocyte development to metaphase II stage of meiosis decreased 3–7 times compared to the non-treated oocytes. This result indicates that NLB material organization is significant for mouse oocyte maturation.     

Kinetics of P2X7 receptor-operated single channels currents

Human P2X7 receptors were expressed in Xenopus laevis oocytes and single channels were recorded using the patch-clamp technique in the outside-out configuration. ATP4- evoked two types of P2X7 receptor-mediated single channel currents characterized by short-lived and long-lived openings. The short- and long-lasting open states had mean open times of approximately 5 and approximately 20 ms and slope conductances near -60 mV of 9 and 13 pS, respectively. The open probabilities of the short and long openings were strongly [ATP4-]-dependent with EC50 values of approximately 0.3 mM and approximately 0.1 mM ATP4-, respectively. The channel kinetics did not change significantly during sustained P2X7 receptor activation for several minutes, as was also observed in recordings in the cell-attached patch-clamp configuration. Activation and deactivation of the short openings followed exponential time courses with time constants in the range of 20 ms, and displayed a shallow [ATP4-] dependence of the activation process. The kinetics of the short channel openings at negative membrane potentials fitted well to a linear C-C-C-O model with two ATP4- binding steps at equal binding sites with a dissociation constant Kd of 139 microM.     

Influence of glass microbeads on growth,activity and morphological changes of Bacillus megaterium

Cells of Bacillus megaterium growing in the presence of glass microbeads with average diameters of 29 and 53 μm were frequently filamentous and sometimes reached lengths of 600 μm. Some of the filaments were nonseptate. The formation of filaments was prevented by magnesium but not by several other cations. In media with supplemental magnesium, the time required before active proliferation commenced was inversely related to the diameter of the particles. B. megaterium growing in media with the smaller size beads consumed oxygen and utilized glucose at greater rates than bacteria in media with the larger spheres or in bead-free solutions, and the uptake of oxygen was maintained for a longer period.     

Specialization for underwater hearing by the tympanic middle ear of the turtle, Trachemys scripta elegans

Turtles, like other amphibious animals, face a trade-off between terrestrial and aquatic hearing. We used laser vibrometry and auditory brainstem responses to measure their sensitivity to vibration stimuli and to airborne versus underwater sound. Turtles are most sensitive to sound underwater, and their sensitivity depends on the large middle ear, which has a compliant tympanic disc attached to the columella. Behind the disc, the middle ear is a large air-filled cavity with a volume of approximately 0.5 ml and a resonance frequency of approximately 500 Hz underwater. Laser vibrometry measurements underwater showed peak vibrations at 500-600 Hz with a maximum of 300 μm s(-1) Pa(-1), approximately 100 times more than the surrounding water. In air, the auditory brainstem response audiogram showed a best sensitivity to sound of 300-500 Hz. Audiograms before and after removing the skin covering reveal that the cartilaginous tympanic disc shows unchanged sensitivity, indicating that the tympanic disc, and not the overlying skin, is the key sound receiver. If air and water thresholds are compared in terms of sound intensity, thresholds in water are approximately 20-30 dB lower than in air. Therefore, this tympanic ear is specialized for underwater hearing, most probably because sound-induced pulsations of the air in the middle ear cavity drive the tympanic disc.     

Action of shear on enzymes: studies with catalase and urease

Intermittent shear was applied to approximately 1 mg/ml solutions of bovine liver catalase in a coaxial cylindrical viscometer at temperatures from 20 to 60°C and shear rates up to 683 sec^?1. The viscometer was sealed to prevent evaporation. Up to 40°C there were no activity losses during 3 hr total shearing. Above 40°C shearing reduced losses due to thermal inactivation, possibly by interfering with precipitation. At 3440 sec^?1 and 40°C fine precipitates formed but little activity was lost. No activity losses were found with experimental conditions under which Taylor vortexing occurred, nor when shear stresses were increased up 57 times by adding glycerol to raise the, viscosity. There were no significant losses in a capillary rheometer at shear rates up to 10⁶ sec^?1. When low concentration (6 μg/ml) catalase solutions were sheared there was little loss in sealed systems, but there were losses in “open” systems even in low-temperature nonshear experiments. Although there were no losses with 1 mg/ml solutions, 6 μg/ml catalase solutions from an alternative source did lose activity in sealed systems but much less than expected from previously published work. Approximately 1 mg/ml jack bean urease solutions were sheared in the sealed system at 23°C and 683 sec^?1 for 3 hr. No losses were found. No evidence of temporary or permanent inactivation was found with 28°g/ml solutions sheared in the presence of urea. Shear forces alone were not found to be as effective in causing enzyme inactivation as is generally believed and alternative mechanisms for damage are discussed.