Recovery from experimental parkinsonism in primates with GM1 ganglioside treatment.

A parkinsonian syndrome can be produced in nonhuman primates by administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Parkinsonian-like symptoms induced acutely by MPTP were ameliorated after treatment with GM1 ganglioside, a substance shown to have neurotrophic effects on the damaged dopamine system in rodents. Treatment with GM1 ganglioside also increased striatal dopamine and metabolite levels and enhanced the dopaminergic innervation of the striatum as demonstrated by tyrosine hydroxylase immunohistochemistry. These results suggest that GM1 ganglioside may hold promise as a therapeutic agent for the treatment of Parkinson's disease.

Antimicrobial use in the Australian pig industry: results of a national survey.

OBJECTIVE: To describe how various antimicrobials are used in commercial pig herds in Australia and for what disease conditions. PROCEDURE: Managers of large pig herds (> 200 sows) across Australia and their veterinarians participated in an internet-based survey in 2006. Questions were asked about herd management, the occurrence of bacterial diseases and the type and frequency of antimicrobial use. An antimicrobial usage index for each herd was derived as a summary of the risk of selection for antimicrobial resistance. Relationships between responses were explored with univariate and multivariate analysis. RESULTS: Responses were received for 197 herds estimated to represent at least 51% of all large pig herds in Australia. Most piggeries relied on drugs of low importance in human medicine (e.g. tetracyclines, penicillins and sulfonamides). For the two drugs of high importance in human medicine that can be legally prescribed to pigs in Australia, ceftiofur use was reported in 25% of herds and virginiamycin in none. Infections attributed to Lawsonia, Mycoplasma and Escherichia coli motivated the most use of antimicrobials. No useful association was found between management factors and the antimicrobial use index. CONCLUSION: Most antimicrobial use in the Australian pig industry is based on drugs of low importance to public health. Enhanced control of E. coli infections without reliance on antimicrobials would further reduce the risk of selecting for antimicrobial resistance relevant to public health. The amount of variation in the usage index between herds suggests that antimicrobial use should be constantly reviewed on a herd by herd basis.

Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells.

We describe the construction of an adeno-associated virus (AAV) vector in which the coding sequence of the procaryotic gene neo is expressed under the control of the major AAV promoter p40. This AAV-neo vector allowed stable expression of neo as a dominant selective marker in mammalian cells by selection of cells which were resistant to the antibiotic geneticin (G418). When the vector was introduced into human (293 or HeLa) cell lines by a DNA transfection procedure, stable geneticin-resistant colonies were obtained. When the vector was first packaged into AAV particles and then introduced into cells via particle infection, geneticin-resistant cells were obtained at higher frequencies than those obtained by DNA transfection. In geneticin-resistant cells the AAV-neo vector was integrated at low copy number and could be rescued by subsequent infection with wild-type AAV and the helper adenovirus or, in some cases, by infection with adenovirus alone. The rescued AAV-neo vector could then be recovered as amplified unintegrated DNA from a Hirt lysate. These results demonstrate that AAV can be used as a transducing viral vector for stable integration and expression of a foreign gene in mammalian cells. The high frequency of integration and the ability to rescue the integrated vector suggest that this vector system may be useful for selecting genes from cDNA libraries. This vector may also be useful for introduction of genes into cells which are refractory to transfection in procedures such as those involving the use of CaPO4 or DEAE-dextran.

Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae.

During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast.

Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis.

Phenotypic and genotypic profiling of antimicrobial resistance in enteric Escherichia coli communities isolated from finisher pigs in Australia.

OBJECTIVE: To assess herd-to-herd variation in antimicrobial resistance phenotypes and associated antimicrobial resistance genes (ARGs) in faecal commensal Escherichia coli communities isolated from Australian slaughter-age pigs. METHODS: Hydrophobic grid-membrane filtration (HGMF) was used to screen populations of E. coli isolated from faecal samples obtained from pigs prior to or at slaughter. Multiplex PCRs were applied to the pooled DNA extracted from the samples to identify specific ARGs. METHODS: Pooled faecal samples from 30 finishers, from 72 different Australian pig farms, produced 5003 isolates for screening. HGMF techniques and image analysis were used to confirm E. coli resistance phenotypes to four antimicrobial agents (ampicillin, gentamicin, florfenicol and ceftiofur) using selective agars. Multiplex PCRs were performed on DNA from pooled samples for 35 ARGs associated with seven chemical classes. RESULTS: The prevalence of E. coli isolates showing no resistance to any of the drugs was 50.2% (95% confidence interval (CI) 41.8-58.6%). Ceftiofur resistance was very low (1.8%; CI 0.8-3.9%) and no ARGs associated with 3rd-generation cephalosporin resistance were detected. By contrast, ampicillin (29.4%, CI 22.8-37.0%), florfenicol (24.3%, CI 17.8-32.3%) and gentamicin (CI 17.5%, 10.7-27.2%) resistance prevalence varied greatly between farms and associated ARGs were common. The most common combined resistance phenotype was ampicillin-florfenicol. CONCLUSION: The use of registered antimicrobials in Australian pigs leads to the enteric commensal populations acquiring associated ARGs. However, despite a high intensity of sampling, ARGs imparting resistance to the critically important 3rd-generation cephalosporins were not detected.

Separation and characterization of low-molecular-weight nuclear RNA species that inhibit cell-free protein synthesis.

Various RNA fractions were isolated from nuclei of 12-day lactating rat mammary glands and examined for their ability to inhibit cell-free protein synthesis. Although total nuclear RNA was generally inactive, material contained in the poly(A)+ nuclear RNA fraction and the low-molecular-weight RNA derived from total nuclear RNA by sucrose gradient centrifugation, inhibited the translation of several mRNAs but not poly(U) or poly(A). Separation of the small nuclear RNAs by preparative polyacrylamide-urea gel electrophoresis allowed the identification of at least three active inhibitor RNA species. These differed both with respect to their ability to inhibit protein synthesis, and in their mechanism of action. While two of the RNA species inhibited elongation the other inhibited initiation of protein synthesis.

Isolation of intact larval haemoglobin from the brine shrimp Artemia salina. Prevention of degradation in vitro by proteases induced during larval development.

Haemoglobin induced in the larval stage of the brine shrimp, Artemia salina is extensively degraded when isolated from the later developmental stages of the larvae. Alkaline proteases appear in the organism a few hours after the induction of haemoglobin and cause the observed degradation. Addition of 2.6 mM phenylmethylsulphonyl fluoride or 20 micrograms/ml soybean trypsin inhibitor to the extraction buffer used for haemoglobin isolation prevents most of this degradation. Discrete haem proteins are found in extracts of the brine shrimp larvae isolated before induction of the proteases, and the major species has a molecular weight of over 200,000. This is believed to be the native haemoglobin. A spread of lower molecular weight haem-containing polypeptides is found in extracts of larvae isolated after induction of the proteases. These products are believed to result from degradation of the discrete haem proteins present in protease-free extracts.

Diabetes mellitus in Hopi and Navajo indians. Prevalence of microvascular complications.

In a cross-sectional study of Hopi and Navajo Indians with non-insulin-dependent diabetes mellitus, we found vascular complications to be strongly related to the duration of diabetes. In patients with diabetes of at least 10 yr duration, retinopathy was found in 57%, nephropathy in 40%, peripheral neuropathy in 21%, and peripheral vascular disease in 28%. For the Hopi and Navajo, the duration-specific prevalence rates of microvascular disease were very similar to prevalence rates found in many other populations. Thus we question the concept, based on reports in the late 1960s, that the Hopi and Navajo Indians have hyperglycemia as an isolated chemical abnormality unaccompanied by other manifestations of diabetes mellitus.

Some properties of DNA from phage-infected bacteria.

Replicating T5 or lambda phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and lambda replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with lambda the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of lambda-phage particles. The constituent polynucleotide chains of lambda but not T5 replicating DNA also appear to be longer than those of phage DNA.

Inhibition of progenitor cell proliferation in the dentate gyrus of rats following post-weaning lead exposure.

Although lead is a potent developmental neurotoxin, the effects of postnatal lead exposure on progenitor cell proliferation in the hippocampus has not been examined. Postnatal day 25 rats were fed a lead containing diet (1500 ppm lead acetate) for 30-35 days and administered bromodeoxyuridine (BrdU, 50 mg/kg, i.p.) during the last 5 days of lead exposure. Animals were killed 24 h after the last BrdU injection. Proliferation of new cells in the subgranular zone and dentate gyrus was significantly decreased in lead-exposed rats compared to control animals that ate a similar diet devoid of lead. These results suggest that postnatal lead exposure can have significant deleterious effects on progenitor cell proliferation and thus the structure and function of the hippocampus.

Panic in otolaryngology patients presenting with dizziness or hearing loss.

This study compared 50 patients presenting to an otolaryngology clinic with a complaint of dizziness and 50 patients presenting with hearing loss on questionnaire measures of panic, phobic avoidance, generalized anxiety, and depression. Clinical and laboratory evaluations of vestibular and audiological complaints were also completed. Twenty percent of the group with dizziness and none of the group with hearing loss reported symptoms that met DSM-III-R criteria for panic disorder. Patients with dizziness and peripheral vestibulopathy had more symptoms of phobic avoidance, generalized anxiety, and depression than patients with confirmed hearing loss.

Transdermal clonidine compared to placebo in painful diabetic neuropathy using a two-stage 'enriched enrollment' design.

Because a variety of mechanisms may generate pain in neuropathic pain syndromes, conventional clinical trial methods may fail to identify some potentially useful drugs; a drug affecting just a single mechanism may work in too few patients to yield a statistically significant result for the trial. To test a previous clinical observation that approximately one-quarter of patients with painful diabetic neuropathy appear responsive to clonidine, we conducted a formal clinical trial of transdermal clonidine in painful diabetic neuropathy patients using a 2-stage enriched enrollment design. In the first stage (study 1), 41 patients with painful diabetic neuropathy completed a randomized, 3-period crossover comparison of transdermal clonidine (titrated from 0.1 to 0.3 mg/day) to placebo patches. Twelve apparent responders from study I were entered into the 'enriched enrollment' second stage (study II), consisting of an additional 4 double-blind, randomized, 1-week treatment periods with transdermal clonidine and placebo. Study I showed that in the overall group of 41 patients, pain intensity differed little during clonidine and placebo treatment. In study II, however, the 12 apparent responders from study I had 20% less pain with clonidine than placebo (95% confidence interval (CI): 4-35% pain reduction; P = 0.015), confirming that their pain was responsive to clonidine. None of the 3 consistent clonidine responders who were tested with the alpha-adrenergic blocker phentolamine had relief of pain, suggesting that clonidine's pain relief is not mediated by a decrease in sympathetic outflow. A post-hoc analysis of many variables suggested that patients who described their pain as sharp and shooting may have a greater likelihood of responding to clonidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid mediators of tumor necrosis factor-alpha-induced uveitis.

The authors tested the hypothesis that platelet-activating factor (PAF) and cyclooxygenase metabolites of arachidonic acid mediate the ocular inflammatory response to intravitreally injected tumor necrosis factor-alpha (TNF alpha). Rabbits were treated with the PAF receptor antagonist SRI 63-441, the cyclooxygenase inhibitors indomethacin and naproxen, or SRI 63-441 and indomethacin. At 24 hr after intravitreal injection of TNF (20,000 U), the severity of inflammation was assessed based on iridal hypermia, aqueous humor leukocyte number and aqueous humor protein, immunoreactive-prostaglandin E (I-PGE), and leukotriene B4 (LTB4) concentrations. Although all of the treatments significantly reduced the severity of anterior uveitis, SRI 63-441 plus indomethacin was the most effective, indomethacin and naproxen were intermediately effective, and SRI 63-441 was the least effective. The results of this study are consistent with an important role for cyclooxygenase metabolites of arachidonic acid in the inflammatory response to TNF alpha, particularly with respect to dilation of iridal blood vessels. Although naproxen was nearly as effective as indomethacin in reducing aqueous humor I-PGE levels, indomethacin conferred significantly more protection to the blood-aqueous barrier as shown by lower protein levels in the aqueous humor. Thus, although cyclooxygenase inhibition may partially explain the protection afforded the blood-aqueous barrier by these nonsteroidal anti-inflammatory agents, the data suggest that indomethacin may also exert anti-inflammatory effects that are independent of cyclooxygenase inhibition. Furthermore, the data are consistent with TNF alpha releasing PAF in the eye and PAF acting both directly, by increasing vascular permeability, and indirectly, by promoting release of cyclooxygenase and lipoxygenase metabolites of arachidonic acid.

Some comments on potency measures in mutagenicity research.

In this article, the measurement of the potency of a chemical or mixture from its dose response in a particular assay is addressed. Attention is focused on data from the Ames Salmonella assay. Three measures of potency are explored and shown to be highly correlated. The presentation then discusses specific areas of research that might benefit from a study of potency.

A simple disc technique for the presumptive identification of Legionella pneumophila.

A method is described for the presumptive identification of Legionella pneumophila by the formation of satellite colonies around filter paper discs impregnated with ferric pyrophosphate and L-cysteine hydrochloride on a deficient basal medium. This technique simplifies the differentiation of picked colonies of L pneumophila from other organisms in mixed cultures from environmental and contaminated clinical samples.

Transfer of R factors from Escherichia coli to salmonellas in the rumen of sheep.

Adult sheep were given into the rumen c. 10(8) cells each of donor strains of E. coli containing an R factor and prospective salmonella-recipient organisms and were maintained on a diet of lucerne chaff; the animals excreted the organisms, remained healthy, and no transfer of the R factor was detected. When the animals were starved for 48 h before inoculation, the ruminal environment was altered so that, on resumption of feeding, small numbers (c. 10(2)-10(4) cells--less than one cell per ml of rumen fluid) of the introduced organisms were able to multiply and reach sufficient numbers for the transfer of R factors to occur within the rumen. One animal, given 7-8 X 10(3) cells of recipient S. lomita after starvation for 48 h, became a carrier of this organism. A second animal, given 4-4 X 10(2) cells of S. typhimurium after starvation for 48 h, developed acute, fatal salmonellosis 5 days later; at the time of death, large numbers of salmonella organisms (c. 10(9) cells per g) were present in the faeces; these included many (c. 10(6) cells per g) that had received the R factor by transfer in vivo. These results indicate that short periods of starvation may enhance the transfer of R factors and possibly other plasmids between suitable micro-organisms in vivo, and may increase the susceptibility of animals to pathogenic micro-organisms.

GM1 ganglioside treatment partially reverses the nigrostriatal dopamine defect in the weaver mutant mouse.

The weaver mutation in the mouse is a developmental disorder characterized by cerebellar atrophy as well as decreased numbers of substantia nigra dopaminergic neurons and a striatal dopamine loss. Since the nigrostriatal dopamine loss occurs postnatally, the present study was performed to determine whether early intervention with GM1 ganglioside could alter the extent of this dopamine loss. Weaver mice that received injections of GM1 ganglioside (30 mg/kg) daily, beginning at 7-10 days of age, had significantly higher striatal dopamine levels and significantly more tyrosine hydroxylase-positive substantia nigra pars compacta neurons than weaver mice that received only daily saline injections. These results show that GM1 treatment can alter at least some aspects of this inherited developmental disorder. If the weaver defect is related to a deprivation of trophic support for certain midbrain dopaminergic neurons, the presence of GM1 may be able to enhance the survival of these neurons.

Predicting rodent carcinogenicity from mutagenic potency measured in the Ames Salmonella assay.

Many in vitro tests have been developed to identify chemicals that can damage cellular DNA or cause mutations, and secondarily to identify potential carcinogens. The test receiving by far the most use and attention has been the Salmonella (SAL) mutagenesis test developed by Ames and colleagues [(1973): Proc Natl Acad Sci USA 70:2281-2285; (1975): Mutat Res 31:347-364], because of its initial promise of high qualitative (YES/NO) predictivity for cancer in rodents and, by extension, in humans. In addition to the initial reports of high qualitative predictivity, there was also an early report by Meselson and Russell [in Hiatt HH et al (1977): "Origins of Human Cancer, Book C: Human Risk Assessment," pp 1473-1481] of a quantitative relationship between mutagenic potency measured in SAL and carcinogenic potency measured in rodents, for a small number of chemicals. However, other reports using larger numbers of chemicals have found only very weak correlations. The primary purpose of this study was to determine whether mutagenic potency, as measured in a number of different ways, could be used to improve predictivity of carcinogenicity, either qualitatively or quantitatively. To this end, eight measures of SAL mutagenic potency were used. This study firmly establishes that the predictive relationship between mutagenic potency in SAL and rodent carcinogenicity is, at best, weak. When predicting qualitative carcinogenicity, only qualitative mutagenicity is useful; none of the quantitative measures of potency considered improves the carcinogenicity prediction. In fact, when qualitative mutagenicity is forced out of the model, the quantitative measures are still not predictive of carcinogenicity. When predicting quantitative carcinogenicity, several possible methods were considered for summarizing potency over all experiments; however, in all cases, the relationship between mutagenic potency predictors and quantitative carcinogenicity is very weak.