Enterococcus species composition determined by capillary electrophoresis of the groESL gene spacer region DNA.

Marine recreational beaches are monitored for fecal contamination by Enterococcus spp. (ENT) counts. Although different ENT species in the environment tend to thrive in and originate from distinct hosts, the current monitoring method does not differentiate among species. Time-consuming isolation-based species identification precludes routine analysis of environmental ENT communities. Therefore, an isolation-independent DNA fingerprinting method was developed to characterize environmental ENT communities using DNA length polymorphism of the spacer region between the groES and groEL genes common to most ENT species. Capillary electrophoresis resulted in distinct peak sizes of PCR products that carried polymorphic groESL spacers (300-335 bp in length) among 8 different ENT species (Enterococcus avium, Enterococcus gallinarum, Enterococcus casseliflavus, Enterococcus mundtii, Enterococcus hirae, Enterococcus faecium, Enterococcus durans, and Enterococcus faecalis). Distortions in true species ratios observed in electropherograms were caused by PCR biases arising in a mixed ENT community DNA template. E. faecalis was overestimated and E. avium and E. faecium were underestimated compared to the original species ratios in the mixed community. The PCR product bias was constant between species, so good approximation of the species ratio in ENT communities is possible. In environmental samples, a high percentage of E. faecalis (96%) together with high total ENT counts were observed in samples collected from a sewer line and from several sites in a storm drain system where sewage leaks were suspected. In contrast, samples with <400 CFU 100 ml-1 ENT were either dominated by E. mundtii or had 4 or more ENT species. The latter ENT community profiles are considered to be signatures of enterococci rarely associated with animals with low or of non-fecal origin.

Bound on Lorentz and CPT violating boost effects for the neutron.

A search for an annual variation of a daily sidereal modulation of the frequency difference between colocated 129Xe and 3He Zeeman masers sets a stringent limit on boost-dependent Lorentz and CPT violation involving the neutron, consistent with no effect at the level of 150 nHz. In the framework of the general standard-model extension, the present result provides the first clean test for the fermion sector of the symmetry of spacetime under boost transformations at a level of 10(-27) GeV.

A system for low field imaging of laser-polarized noble gas.

We describe a device for performing MRI with laser-polarized noble gas at low magnetic fields (<50 G). The system is robust, portable, inexpensive, and provides gas-phase imaging resolution comparable to that of high field clinical instruments. At 20.6 G, we have imaged laser-polarized (3)He (Larmor frequency of 67 kHz) in both sealed glass cells and excised rat lungs, using approximately 0.1 G/cm gradients to achieve approximately 1 mm(2) resolution. In addition, we measured (3)He T(2)(*) times greater than 100 ms in excised rat lungs, which is roughly 20 times longer than typical values observed at high ( approximately 2 T) fields. We include a discussion of the practical considerations for working at low magnetic fields and conclude with evidence of radiation damping in this system.

Low-field MRI of laser polarized noble gas.

NMR images of laser polarized 3He gas were obtained at 21 G using a simple, homebuilt instrument. At such low fields magnetic resonance imaging (MRI) of thermally polarized samples (e.g., water) is not practical. Low-field noble gas MRI has novel scientific, engineering, and medical applications. Examples include portable systems for diagnosis of lung disease, as well as imaging of voids in porous media and within metallic systems.

Testing for isoniazid. An evaluation of the Arkansas method.

To better treat and eliminate tuberculosis, patient compliance must be improved. Compliance can be evaluated by measuring a drug or its metabolite in the urine. In Arkansas, a simple colorimetric method of checking the urine for isoniazid (the Potts-Cozart test) has been used for many years, but it is relatively unknown outside that state and its reliability has not been confirmed. To evaluate this test, urine was blindly tested from patients from a tuberculosis clinic. Controls included urine from patients from a substance abuse clinic and Veterans Medical Center. In more than 200 urine samples tested, no false-positives were found. Urinalysis showed normal values for three patients who were supposed to be receiving antituberculosis medication, but it is likely that these patients were noncompliant. A peculiarity of the test was that the color change with positive tests varied. To investigate this variation, absorption spectroscopy of many substances was performed. Nicotine accounted for the different shade of blue associated with the positive test, but the color produced and the absorption spectroscopy were different from isoniazid, so it did not confuse the interpretation of the results. This test for isoniazid in the urine is simple, quick, inexpensive, easy to interpret, and reliable. It also can be used to detect nicotine and its metabolites.

Cyclosporine A inhibition of microcystin toxins.

Cyclosporine A (CyA) given i.v. at a dose of 1.25 mg/mouse blocks a subsequent i.v. lethal dose (1.7-1.8 x LD50) of microcystin-LR for 24 hr, and is about 50% protective at 48 hr. Conversely, the fraction of mice that can be rescued by CyA (0.2 mg/mouse) after a lethal dose of microcystin-LR decreases rapidly with a pharmacodynamic half-time of only about 100 sec. The prophylactic action of CyA was tested against lethal doses of four microcystins. The acute lethality of 1.7-1.8 x LD50 dose of microcystin-LR, -RR, -LY, or -LA given 1 hr after administration of 0.2 mg of CyA is 0%, 0%, 58%, or 100%, respectively. Even a 0.6 mg/mouse dose of CyA is ineffective prophylaxis against a lethal dose of microcystin-LA. The inhibitory potency of CyA on microcystin toxicity can be completely reversed by the single L-amino acid substitution of alanine for arginine in the microcystin.

Germinal center kinetics in lymph nodes of primed mice stimulated with complexed as opposed to free antigen.

Primed mice with low titers of circulating tetanus antitoxin (AB) were stimulated via the hind footpads with either fluid tetanus toxoid alone (AG) to create in vivo complexes in AG excess, or the same dose of toxoid complexed at equivalence with isologous antibody (AB-AG CPX), to have in vivo complexes in AB excess. All experimental animals reacted with three topically distinct consecutive waves of enhanced proliferative activity in popliteal lymph nodes, i.e., in the T-zone (peak on day 2), in the medullary area, the main site of plasmocytopoiesis (day 3), and in lymphoid follicles (day 5-6). Maximum serum AB titers following injection of AG-AB CPX were only about 25% of those found in animals boosted with AG alone. This suppressive effect was best reflected in a comparable reduction in plasmocytopoiesis, and to an lesser extent in the proliferative activity within the T-zone, and not at all in the overall magnitude of germinal center formation and/or expansion. However, the patterns of germinal center kinetics differed markedly between the two groups: a high sharp peak of development on day 5, followed by a marked drop on day 6 characterized the response in mice given AG alone, and a broad peak around day 6 that of those receiving AG-AB CPX. These differences could not adequately be accounted for by variations in centroblast/centrocyte proliferation rate vs. pycnotic indices, so that different patterns of lymphoid cell emigration from the centers may be considered. The results suggest that immune complexes, fixed on follicular dendritic cells, with different antigen-to-antibody ratios have divergent effects on the development and kinetics of germinal centers, the principal sites of memory B cell generation.

Prophylaxis of cyanobacterial and mushroom cyclic peptide toxins.

The pathogenicity of virulent cyclic peptide toxins of the cyanobacterium, Microcystis aeruginosa, and the mushroom, Amanita phalloides, was prevented in mice by pretreatment with a variety of chemically unrelated agents including hydrocortisone, shellac, certain diazo and triazine dyes and cyclosporine. A. Despite the diverse nature of the protective agents, a feature commonly associated with protection was the ability to impair hepatic uptake of 51Cr-labeled sheep erythrocytes, a function of hepatic macrophages (Kupffer cells). In addition, several of the protective agents are known to affect other aspects of reticuloendothelial cell function. Therefore it seems likely that the hepatic macrophage is involved in the observed protection, although by what mechanism(s) is unknown. The most remarkable prophylaxis was seen with a single injection of Trypan red, which provided nonimmunologic protection against a lethal dose of a cyanobacterial toxin, cyanoginosin-LR, for periods up to 3 months.

Tetanus toxoid complexed with heterologous antibody can induce germinal centre formation and B cell memory in mice without evoking a detectable anti-toxin response.

Fluid free tetanus toxoid (FTT) alone or FTT complexed in vitro at equivalence (EQ) or in antibody excess (ABEX) with anti-toxin contained in a human gammaglobulin preparation (HGG), or HGG alone, were injected into the hind leg footpads of mice. Anti-toxin titres of mouse serum were measured and compared with proliferative reactions in popliteal lymph nodes, based on combined 3H-thymidine autoradiography and planimetry, as a function of time. FTT in complex with HGG in ABEX failed to elicit a measurable anti-toxin response but caused, of all the materials tested, the most marked numerical increase of germinal centres. This finding is in accord with results of earlier studies indicating that the same heterologous antigen-antibody complexes at EQ or in ABEX can prime the animals, usually without eliciting antibody production detectable by serum titration. The model system used in the present experiments is thus well suited for a separation of the two principal arms of the dichotomous humoral immune response, i.e. by inducing germinal centre and B cell memory development but not specific antibody formation.

The effects of single L-amino acid substitutions on the lethal potencies of the microcystins.

Microcystin-LR and -LA are more toxic than microcystin-LY and -RR in adult mice. They induce different degrees of thrombocytopenia and leukopenia, and the lethalities of their binary and ternary mixtures are addictive. Postnatal mice are resistant to doses of microcystin-LR that are lethal to adults but they are susceptible to higher doses. Substitution of a single L-amino acid for another in a microcystin markedly affects the dosimetric potency, but not the pathophysiology of its toxicity.

Ventricular fibrillation during rectal examination.

The case of a 74-year-old man who developed ventricular fibrillation during a digital rectal examination is presented. The patient was subsequently resuscitated and developed cardiac enzyme elevation without ECG changes, indicating a nontransmural myocardial infarction. Although controlled studies have not shown any ill effects of rectal examination in patients with acute myocardial infarction, there have been multiple case reports of bradycardia, ectopy, and ventricular arrhythmias resulting from rectal examination. The postulated etiology of the ectopy is twofold; increased vagal tone from rectal parasympathetic innervation or increased sympathetic tone from anxiety-stimulated catecholamine release. Rectal examination is definitely indicated in a subset of patients including those with gastrointestinal or genitourinary complaints, unexplained hypotension or anemia, trauma, and neurological deficits, and those who will receive anticoagulation or thrombolytic therapy. In the remaining patients, the decision must be made on a case-by-case basis. Awareness of and precautions for possible ill effects of the examination are prudent.

Pathophysiology of cyanoginosin-LR: in vivo and in vitro studies.

Cyanoginosin-LR, one of the group of virulent cyclic heptapeptide toxins (cyanoginosins) isolated from some strains of the cyanobacterium, Microcystis aeruginosa, kills mice within 1-2 hr after iv or ip injection. Although the liver is a target organ of the toxin, the rapidity of lethality is incompatible with metabolic death from failure of hepatocellular function. However, disintegration of sinusoidal endothelium causes massive intrahepatic hemorrhage. The loss of the structural integrity of hepatic sinusoids provides a previously undescribed mechanism for embolization of disintegrating cells from the liver to the lung. No injury to either cultured bovine pulmonary artery endothelial cells or mouse peritoneal macrophages was observed following prolonged incubation with high concentrations of the toxin, and there was no increase in vascular permeability to 125I-labeled albumin detected before intrahepatic hemorrhage. However, plasma fibronectin increased transiently after toxin injection. Acute, severe thrombocytopenia, a characteristic of cyanoginosin-LR toxicity, remains unexplained since platelets did not concentrate in the lungs, liver, or spleen. There are similarities between the effects of cyanoginosin-LR and of the lipopolysaccharide endotoxins, such as elevations of plasma levels of thromboxane B2 and 6-keto-prostaglandin F1 alpha.

Central nervous system radiation syndrome in mice from preferential 10B(n, alpha)7Li irradiation of brain vasculature.

Ionizing radiations were directed at the heads of anesthetized mice in doses that evoked the acute central nervous system (CNS) radiation syndrome. Irradiations were done using either a predominantly thermal neutron field at a nuclear reactor after intraperitoneal injection of 10B-enriched boric acid or 250-kilovolt-peak x-rays with and without previous intraperitoneal injection of equivalent unenriched boric acid. Since 10B concentrations were approximately equal to 3-fold higher in blood than in cerebral parenchyma during the reactor irradiations, more radiation from alpha and 7Li particles was absorbed by brain endothelial cells than by brain parenchymal cells. Comparison of the LD50 dose for CNS radiation lethality from the reactor experiments with the LD50 dose from the x-ray experiments gives results compatible with morphologic evidence that endothelial cell damage is a major determinant of acute lethality from the CNS radiation syndrome. It was also observed that boric acid is a low linear energy transfer radiation-enhancement agent in vivo.

Satisfactory primary tetanus antitoxin responses but markedly reduced germinal centre formation in first draining lymph nodes of ageing mice.

The present report deals with primary antibody responses to tetanus toxoid in 50-54-week-old ('ageing') as compared to 8-9-week-old ('young adult') mice. Antitoxin in the serum appeared 6 days earlier in the older than in the young animals, but in the latter reached 5 times higher titres on day 20. The magnitude of the proliferative response in the paracortex and the medulla of popliteal lymph nodes, as estimated by combined 3H-thymidine autoradiography and planimetry, was 3-7 times greater in the younger than in the older age group, thus approximately reflecting the difference in antibody titres on day 20. In contrast, germinal centre formation in response to the stimulus proved to be about 14 times less in ageing than in young adult mice. The findings demonstrate that, in the model system used, the age-related slopes of decline in humoral antibody responsiveness and proliferative reactivity in paracortex and medulla of first regional lymph nodes tend to be in parallel, while the ability of the immune apparatus to form germinal centres at this site deteriorates at a considerably faster pace. Results are also in line with the notion that centroblasts/centrocytes contribute little, if anything, to the ongoing antibody production elicited by the same stimulus which had triggered germinal centre formation. Finally, the observations made disprove the general validity of the suggestion that immune reactivity is maintained on the same level throughout life if tested with a novel antigen.

Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa.

Toxin-LR, a hexapeptide produced by Microcystis aeruginosa, causes marked hepatic vascular congestion, thrombocytopenia, microscopic pulmonary thrombi and death in 50-70 min when injected into mice. Although it is considered an hepatotoxin, we report that sublethal hepatocellular damage produced by CCl4 given 24 hr prior to toxin-LR administration prevents the acute deaths. However, CCl4-treated mice surviving toxin-LR acute effects often died during the subsequent three days. Pretreatment of mice with the microsomal enzyme inhibitors SKF 525A or cobaltous chloride did not alter the acute lethality of toxin-LR, but pharmacologic doses of hydrocortisone prevented both the acute and delayed deaths. X irradiation-induced thrombocytopenia or thrombocytopenia and leukopenia did not significantly affect the toxin's lethality. In vitro platelet aggregation or lysis did not occur during incubation with toxin-LR, nor was a humoral aggregating factor detected in plasma from toxin-injected mice.