Confirmation of the single-step membrane filtration procedure for estimating Pseudomonas aeruginosa densities in water.

The efficiency of two procedures, membrane filtration and most probable number, to resuscitate and enumerate Pseudomonas aeruginosa have been compared at two temperatures and varying incubation periods. Data indicate that the membrane filtration procedure using mPA or mPA medium B is more efficient than the most-probable-number procedure in estimating P. aeruginosa populations. It was also found that the specificity of the membrane filtration procedure was such that 92 to 99% of the colonies counted as P. aeruginosa were confirmed, whereas only 2.7 to 10% of the nontypical colonies were confirmed as P. aeruginosa. Furthermore, the data indicate that mPA medium B combined with a 3- to 4-day incubation period at 4.15 degrees C is slightly more specific than mPA medium and is a valid single-step procedure for the resuscitation and enumeration of P. aeruginosa from water or sewage effluent.     

Platelet activating factor receptor blockade enhances recovery after multifocal brain ischemia

We treated four anesthetized dogs (Canis familiaris) with the platelet activating factor (PAF) receptor antagonist kadsurenone prior to 60 min of multifocal ischemia induced by air embolism, and measured neuronal recovery, blood flow and autologous 111In-labeled platelet accumulation for 4 h after ischemia. Four anesthetized animals with identical ischemia served as controls. Kadsurenone (3 mg/kg) administered 5 min prior to ischemia and continuously (1 mg/kg/hr) throughout ischemia and recovery significantly enhanced recovery of cortical somatosensory evoked response (CSER) amplitude (% of baseline) when compared to controls (27-36% vs 9-14%, p less than 0.05). We estimated platelet accumulation as 111In activity (cmp/g tissue) in the injured hemisphere minus that in the non-injured hemisphere. Kadsurenone treated animals did not exhibit significantly altered 111In-labeled platelet accumulation when compared to controls (6158 +/- 2386 vs 9979 +/- 3852, mean +/- SEM). Beneficial effects of PAF receptor blockade other than those on platelet accumulation may be involved.     

AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.     

Enumeration of Klebsiella spp. in cold water by using MacConkey-inositol-potassium tellurite medium

MacConkey-inositol-potassium tellurite agar was field tested for its ability to selectively enumerate Klebsiella species from the waters of the Saint John River Basin, which include fresh and marine waters. Water temperature varied from 1 to 6 degrees C during the survey period. Results of the study indicated that 77% of the typical colonies on MacConkey-inositol-potassium tellurite medium were Klebsiella species, but the total Klebsiella population enumerated was greatly underestimated.     

Effect of Pressure on the Release of Radioactive Glycine and γ-Aminobutyric Acid from Spinal Cord Synaptosomes

Exposure to high hydrostatic pressure produces neurological changes referred to as the high-pressure nervous syndrome (HPNS). Manifestations of HPNS include tremor, EEG changes, and convulsions. These symptoms suggest an alteration in synaptic transmission, particularly with inhibitory neural pathways. Because spinal cord transmission has been implicated in HPNS, this study investigated inhibitory neurotransmitter function in the cord at high pressure. Guinea pig spinal cord synaptosome preparations were used to study the effect of compression to 67.7 atmospheres absolute on [3H]glycine and [3H]gamma-aminobutyric acid ([3H]GABA) release. Pressure was found to exert a significant suppressive effect on the depolarization-induced calcium-dependent release of glycine and GABA by these spinal cord presynaptic nerve terminals. This study suggests that decreased tonic inhibitory regulation at the level of the spinal cord contributes to the hyperexcitability observed in animals with compression to high pressure.     

Bacterial Density in Water Determined by Poisson or Negative Binomial Distributions

The question of how to characterize the bacterial density in a body of water when data are available as counts from a number of small-volume samples was examined for cases where either the Poisson or negative binomial probability distributions could be used to describe the bacteriological data. The suitability of the Poisson distribution when replicate analyses were performed under carefully controlled conditions and of the negative binomial distribution for samples collected from different locations and over time were illustrated by two examples. In cases where the negative binomial distribution was appropriate, a procedure was given for characterizing the variability by dividing the bacterial counts into homogeneous groups. The usefulness of this procedure was illustrated for the second example based on survey data for Lake Erie. A further illustration of the difference between results based on the Poisson and negative binomial distributions was given by calculating the probability of obtaining all samples sterile, assuming various bacterial densities and sample sizes.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Bubble-induced dysfunction in acute spinal cord decompression sickness

Five anesthetized dogs undertook a chamber dive, on air, to 300 feet of seawater for 15 min. After the dive, spinal cord decompression sickness was detected by recording a reduced amplitude of the somatosensory evoked potential compared with predive base-line values. After the diagnosis of decompression sickness and rapid perfusion fixation of the animal, the spinal cord was removed and examined histologically. Numerous space-occupying lesions (SOL) that disrupted the tissue architecture were found in each cord, mainly in the white matter. The size and distribution of the SOL were determined using computerized morphometry. Although SOL occupied less than 0.5% of the white matter volume, we tested a number of algorithms to assess whether the SOL may have been directly involved in the loss of spinal cord function that followed the dive. We determined that the loss of somatosensory evoked potential amplitude may be attributed to the SOL if 30-100% of the spinal cord fibers that they displaced were rendered nonconducting. A number of possible mechanisms by which SOL may interfere with spinal nerve conduction are discussed.