A Monitor for Airborne Bacteria

A unique agar drum sampler is described which indicates, continuously, the number of viable, bacterial particles per unit volume of air at the time and point of sampling. By selection of the timer and the sampling rate the sampler is suitable for quite a wide range of concentration and time. An impaction line of 484 in. greatly increases the capacity of this device over slit samplers and other instruments designed to give time-concentration data for viable airborne particles. This sampler should prove useful for: (i) monitoring airborne bacteria in hospitals, public places, and food and industrial plants; (ii) decay rate studies of bacterial aerosols; (iii) evaluation of aerial germicides; (iv) determination of effectiveness of air conditioning systems in removing airborne bacteria; and (v) many other studies in aerobiology.     

Division competence in Tetrahymena: determination of minimum cell volume and rate of nutrient uptake

Cell volume and doubling time have been determined for exponentially growing Tetrahymena pyriformis cells in broth medium with and without glucose and in media made from these media by dilution with water. The cells tolerate media with dry weights from 105 down to 0.06 g/L. In the diluted media the cells have small volumes and the doubling time is increased. When the cell volume increase per time per cell in a given medium is expressed as a function of the cell volume in this same medium, a direct proportionality is found. From this equation the minimum cell volume of division competence (MVDC) can be found. It is 2,100 microns 3 for T. pyriformis at 28 degrees C. The lag period resulting from an upshift of exponentially growing cells from diluted media to more concentrated media is a function of the initial and resulting cell volumes and MVDC. The increase in cell volume per unit of time for a given cell depends on the dry weight of the medium. This parameter can be transformed to mass increase per cell surface area per time, which represents rate of nutrient uptake. When plotted against the dry weight of the media, a Michaelis-Menten-like curve is obtained with two Km values of 3.8 and 0.08 g/L with corresponding Vmax values of 20 and 4 ng/cm2.s. The low Km value (0.08 g/L) indicates that Tetrahymena is able to take up nutrients from highly diluted media. The high value of Vmax (20 ng/cm2.s) increases the ability of growth in more concentrated media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence.

To determine whether amino acid side-chain substitutions in linear gramicidins after the structure of membrane-spanning channels formed by the modified peptides, we have developed a quantitative measure of structural equivalence of the peptide backbone among gramicidin channels based on functional (single-channel) measurements. The experiments exploit the fact that gramicidin channels are symmetrical dimers, and that channels formed by different gramicidin analogues can be distinguished on the basis of their single-channel current amplitudes or durations. It is thereby possible to determine whether hybrid channels can form between chemically dissimilar peptides, i.e. whether the peptides can adapt to each other. Further, since the relative rates of channel formation as well as the relative concentrations of pure and hybrid channel types can be measured in the same membrane, these experiments provide a quantitative measure of the energetic cost of hybrid channel formation relative to the formation of the pure channels. For a wide variety of different side-chains, we find that substitutions as extreme as glycine to phenylalanine at position 1, at the join between the two monomers in a membrane-spanning dimer, incur no energetic cost for channel formation, which implies that channels formed by each of the modified peptides are structurally equivalent. In addition, the average durations of the hybrid channels (except those having tyrosine or hexafluorovaline at position 1) are intermediate to the average durations of the respective pure channel types, thus providing further evidence for structural equivalence among channels formed by sequence-substituted gramicidins.     

Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

Analysis of extracellular calcium and volume changes in the compound eye of the honeybee drone,Apis mellifera

Summary Superfused slices of drone retina were used for a quantitative analysis of light-induced changes in extracellular Ca²⁺ concentration ([Ca²⁺]_o) and extracellular space (ECS) volume. 20-ms light flashes elicited biphasic changes in [Ca²⁺]_o. For a saturating flash a brief, initial decrease was followed by a transient increase of 120±34 M. Long, dim steps of light (5 min) produced either a decrease or an increase in [Ca²⁺]_o depending strongly on the previous illumination. Brighter continuous lights caused the [Ca²⁺]_o to increase transiently by 1.4 mM to a peak from which it decayed to a plateau, up to 0.6 mM above the dark concentration.Light flashes (20 ms) caused a shrinkage in ECS volume not exceeding 4%. Thus, changes in [Ca²⁺]_o were almost completely due to Ca²⁺ fluxes between the ECS and adjacent cells. Continuous lights caused a shrinkage in ECS volume rarely exceeding 16%–20%. Thus, less than 15% of the measured Ca²⁺ changes could be attributed to shrinkage of the ECS. These data confirm that the ECS functions as a source and a sink for Ca²⁺ mobilized by light. For comparison, we also made a few measurements of changes in [Ca²⁺]_o in the retina ofCalliphora.Abbreviations [Ca ²⁺]_i intracellular free Ca²⁺ concentration - [Ca ²⁺]_o extracellular free Ca²⁺ concentration - ECS extracellular space - ER endoplasmic reticulum - TMA ⁺ tetramethylammonium ion     

Functional consequences of proline mutations in the cytoplasmic and transmembrane sectors of the Ca2(+)-ATPase of sarcoplasmic reticulum

Site-specific mutagenesis was used to investigate whether Pro160, Pro195, Pro308, Pro312, Pro803, and Pro812 play essential roles in the function of the sarcoplasmic reticulum Ca2(+)-ATPase. All six prolines were substituted with alanine; and in addition, Pro308 was replaced by glycine and Pro312 by glycine as well as by leucine. Mutant cDNAs were expressed in COS-1 cells, and mutant Ca2(+)-ATPases located in the isolated microsomal fraction were examined with respect to Ca2+ uptake activity, Ca2+ dependence of phosphorylation from ATP, and the kinetic properties of the phosphoenzyme intermediates formed from both ATP and Pi. The enzymatic cycle was little affected by substitution of Pro160, Pro195, and Pro812, which are located in the cytoplasmic domain; but replacement of Pro308, Pro312, and Pro803, in the putative transmembrane helices, had a profound impact on the function of the enzyme. All mutations of Pro308 and Pro803 led to ATPases which were characterized by a reduced affinity for Ca2+. These prolines may therefore be involved in the structure of the high affinity Ca2(+)-binding sites in the enzyme. Substitution of Pro312 with alanine or glycine gave rise to mutants unable to transport Ca2+ even though their apparent affinities for Ca2+ in the phosphorylation reaction with ATP were increased. In these enzymes, the ADP-sensitive phosphoenzyme intermediate was stable for at least 5 min at 0 degrees C, whereas the ADP-insensitive phosphoenzyme intermediate decay at a rate similar to that of the wild type. Thus, the inability to transport Ca2+ could be accounted for by a block of ADP-sensitive to ADP-insensitive phosphoenzyme intermediate conformational transition. In contrast, substitution of Pro312 with leucine gave rise to a mutant enzyme that retained about 7% of the normal Ca2+ transport rate. Phosphoenzyme turnover in this mutant also occurred at a low but significant rate, suggesting that the leucine side chain can substitute to some extent for proline.