Presynaptic G Protein-Coupled Receptors Differentially Modulate Spontaneous Glutamate Release in the Supraoptic Nucleus.

Spontaneous glutamate release in the supraoptic nucleus is modulated by a number of inhibitory G protein coupled receptors (GPCR), including GABAB , adenosine A1 and group III metabotropic glutamate receptors (mGluR). It remains unclear whether they have distinct roles or are redundant mechanisms that protect from hyperexcitation. To address this question, we facilitated spontaneous glutamate release using nifedipine or forskolin, which act in a protein kinase A (PKA)-independent and -dependent manner, respectively, and tested the effects of inhibitory GPCR agonists. We found that a GABAB receptor (GABAB R) agonist specifically inhibited forskolin-induced miniature excitatory postsynaptic currents (mEPSC), in contrast to an adenosine A1 receptor (A1R) agonist, which specifically inhibited nifedipine-induced mEPSCs. This suggests that GABAB Rs and A1 Rs modulate independent mechanisms activated by forskolin and nifedipine, respectively. However, the inhibitory effects of GABAB R and A1 R agonists on basal mEPSCs occluded each other, suggesting that these receptors also have an overlapping role. Group III mGluRs appear to have a greater control over glutamate release because agonists to these receptors inhibited both nifedipine- and forskolin-induced mEPSCs. mEPSCs induced by norepinephrine had the same characteristics as those induced by forskolin [i.e. PKA-dependence and sensitivity to GABAB R and group III mGluR agonists, but not an A1 R agonist]. In summary, the present study highlights the differential effects of GABAB R, A1 R and mGluR agonists on glutamate release stimulated by different secretagogues, including the endogenous neuromodulator norepinephrine. These results suggest that the roles of these inhibitory GPCRs are not completely redundant, and also indicate the physiological implications of having different excitatory and inhibitory GPCRs on the same synapse.

Four-dimensional quantitative analysis of the gait of mutant mice using coarse-grained motion capture.

To analyze an abnormal gait pattern in mutant mice (Hugger), we conducted coarse-grained motion capture. Using a simple retroreflective marker-based approach, we could detect high-resolution mutant-specific gait patterns. The phenotypic gait patterns are caused by extreme vertical motion of limbs, revealing inefficient motor functions. To elucidate the inefficiency, we developed a musculoskeletal computer model of the mouse hindlimb based on X-ray CT data. By integrating motion data with the model, we determined mutant-specific musculotendon lengths, suggesting that three major muscles were involved in the abnormal gait. This approach worked well on laboratory mice, which were putatively too small to be motion capture subjects. Motion capture technology was originally developed for human study, and our approach may help fill neuroscience gaps between mouse and human behavioral phenotypes.

Survival of Listeria innocua on hot and cold beef carcass surfaces.

AIMS: This study aimed to determine the survival and growth of Listeria innocua on hot and cold beef carcass surfaces. METHODS AND RESULTS: Four sites, the neck, outside round, brisket and foreshank/brisket, were inoculated with L. innocua (i) immediately after dressing while hot and (ii) when cold after chilling. After inoculation, all carcasses were stored at 4 degrees C for 72 h. Survival of L. innocua on cold surfaces declined during storage and was less than on hot carcasses at all times. Data on the survival of L. innocua in broth (maximum recovery diluent) indicated that counts could not be compared with those on carcasses, in particular on cold carcasses. CONCLUSIONS: The results indicate that L. innocua survives on hot carcass surfaces during chilling, but declines over time on cold surfaces. The decrease in L. innocua counts on cold surfaces may be related to a synergy between the combined stresses of low available water (a(w)) and low temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first to determine the effect of chilling on the survival and growth of Listeria on beef carcass surfaces. The information can potentially be used to determine the survival and growth of the pathogen, L. monocytogenes on beef surfaces.

The influence of attachment to beef surfaces on the survival of cells of Salmonella enterica serovar Typhimurium DT104, at different a(w) values and at low storage temperatures.

This study investigated the influence of attachment to beef surfaces on the survival, injury and death of stationary phase cells of Salmonella enterica serovar Typhimurium DT104, compared to cells free in solution. The effects on cells are considered at different a(w) values and low temperatures in relation to osmotic and cold temperature shock effects. Attachment of cells to meat surfaces prevented cell injury and death from hyperosmosis and low temperatures, compared to meat solutions. Storage of cells for 72h resulted in higher levels of cell death on cells attached to meat surfaces. The improved survival of cells in solutions was considered to be related to adaptation to osmotic stress as a result of exposure to a previous hyperosmotic shock and the ability of the cells to produce cold shock proteins. Pathogen cell growth at low temperatures is discussed in relation to the presence of low levels of NaCl. Finally the data is discussed in relation to pathogen survival on beef carcass surfaces during refrigeration.

More or less CHAOS: case report and literature review suggesting the existence of a distinct subtype of congenital high airway obstruction syndrome.

Congenital obstruction of the upper airway (CHAOS) is a rare, usually lethal abnormality. A literature review of 36 prenatally diagnosed cases of CHAOS and the analysis of our own case suggest the existence of a distinct subtype of CHAOS, raising important implications for diagnosis and management. Serial fetal ultrasound examinations at 17-23 weeks' gestation showed hyperechoic and enlarged lungs, mediastinal shift, flattened diaphragm, polyhydramnios and apparently fluid-filled esophagus, findings interpreted as bilateral cystic adenomatoid malformation Type III. Ultrasound findings normalized around 32 weeks. The diagnosis of CHAOS was made after birth at term by direct laryngoscopy prompted by ventilatory difficulties and failed attempts at intubation. A pinhole opening posterior to the cricoid cartilage allowed the passage of an endotracheal tube. Based on observations in our case and those of five similar cases in the literature, we describe for the first time a subtype of CHAOS that is characterized by minor pharyngotracheal or laryngotracheal communications and associated with a less severe natural history and even resolution of ultrasound findings. In spite of this, a high index of awareness should be maintained because resolution of ultrasound findings does not necessarily indicate resolution of underlying pathology.

Impact of a novel spray-chilling system on surface microflora, water activity and weight loss during beef carcass chilling.

Commercially slaughtered and dressed beef carcass sides (n=30) were followed through a standard commercial chill unit fitted with a new "Jasca" air humidification system adjusted to provide intermittent water spraying of carcass sides (spray cycle 2 min on, 1 min off) for 15 h. Immediately after dressing, and after 24h in the chill unit, the surface water activity, and the weight of each side was measured, and 5 cm2 samples were recovered from four locations, i.e. rump, flank, brisket and neck on the surface of each side. These samples, and similar samples from control sides (n=30) processed in a standard commercial chill unit, were subjected to microbiological examination by direct and resuscitation counts on plate count agar (PCA), MacConkey agar (MAC) and violet red bile glucose agar (VRBGA). No significant differences were observed between bacterial numbers on test and control samples on each of the above agars, at each sample point/occasion. Comparison of direct and resuscitation counts suggested the presence of substantial numbers of injured cells, at both stages (pre- and post-chill), on test and control sides. After 24 h in chill units, test sides exhibited an average weight loss of 1.36% (+/-0.36%), which is significantly less (P<0.001) than the average weight loss (1.55%+/-0.24%) from control sides. These results suggest that the Jasca spray-chilling system can limit carcass shrinkage (on average by 0.19%) without significantly increasing the surface populations of selected bacterial groups.

Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays

It presents information on cells and viruses, laboratory facilities, serum samples, microneutralization assay, H5N1 Western blotting, ELISA with HA, HI assays, and results obtained.

Neurological manifestations of avian influenza viruses in mammals.

The H5N1 viruses isolated from humans in Hong Kong directly infected both mice and ferrets without prior adaptation to either host. Two representative viruses, A/Hong Kong/483/97 (HK/483) and A/Hong Kong/486/97 (HK/486) were equally virulent in outbred ferrets but differed in their virulence in inbred mice. Both HK/483 and HK/486 replicated systemically in ferrets and showed neurologic manifestations. In contrast, intranasal infection of mice with HK/483, but not HK/486, resulted in viral spread to the brain, neurologic signs, and death. However, HK/486 was able to replicate in the brain and induce lethal disease following direct intracerebral inoculation.

Pathogenesis of and immunity to a new influenza A (H5N1) virus isolated from duck meat.

The outbreak of avian influenza H5N1 in Hong Kong in 1997 raised concerns about the potential for the H5 subtype to cause a human pandemic. In 2001 a new H5N1 virus, A/Duck Meat/Anyang/AVL-1/2001 (A/Dkmt), was isolated from imported duck meat in Korea. The pathogenesis of this virus was investigated in mice. A/Dkmt virus had low infectivity but was lethal for mice at high doses, and at lethal doses, the virus replicated in the brains of infected mice. A/Dkmt virus cross-reacted poorly with ferret antisera raised against human H5N1 viruses, but prior infection with A/Dkmt virus protected mice from death after secondary infection with human H5N1 virus.

Mechanisms of pathogenicity of influenza A (H5N1) viruses in mice.

Avian-like H5N1 influenza viruses isolated from humans in 1997 were shown to have two distinct pathogenic phenotypes in BALB/c mice, after intranasal inoculation and without prior adaptation to this host. To further understand the mechanisms of H5N1 pathogenicity, we investigated the consequences of the mute of viral inoculation on morbidity and mortality, viral replication in pulmonary and systemic organs, and lymphocyte depletion. This study demonstrates the importance of extrapulmonary spread and replication, particularly in the brain, for the lethality of H5N1 viruses.

Salmonella serotypes present on a sample of Irish pig farms.

A survey of the prevalence of Salmonella species infection was conducted on 59 Irish farrow-to-finish pig herds. Faecal samples were collected from the pens of first-stage weaners (growing pigs approximately three to 10 weeks of age), second-stage weaners (approximately 10 to 17 weeks of age) and fatteners, and from the dry sow and farrowing sow houses. The prevalence of infection was estimated to within 5 per cent with a 95 per cent confidence interval. Thirty of the 59 herds were infected, 12 with Salmonella Typhimurium only, eight with Salmonella Derby only and seven with both S Typhimurium and S Derby; serotypes London, Livingstone and Infantis were each isolated from a single herd. Farms in Ireland are assigned to one of three infection categories on the basis of the antibody levels in samples of meat juice taken at slaughter. When a herd was classified as either positive or negative on the basis of the isolation of Salmonella from at least one faecal sample there was no association between the herd's category as determined by meat juice serology and the probability of the isolation of Salmonella from the faecal samples. However, there were differences in prevalence between pigs at different stages of production in herds of different categories. Farrowing sow houses in moderately infected (category 2) herds had significantly lower infection rates (P < or = 0.05) than other herd categories and other stages of production. Pigs from first-stage weaner pens in slightly infected (category 1) herds were more likely to be infected with Salmonella than pigs at any other stage of production or category of herd.

Pathogenicity and antigenicity of a new influenza A (H5N1) virus isolated from duck meat.

Avian influenza A viruses are the ancestral origin of all human influenza viruses. The outbreak of highly pathogenic (HP) avian H5N1 in Hong Kong in 1997 highlighted the potential of these viruses to infect and cause severe disease in humans. Since 1999, HP H5N1 viruses were isolated several times from domestic poultry in Asia. In 2001, a HP H5N1 virus, A/Duck/Anyang/AVL-1/2001 (Dk/Anyang), was isolated from imported frozen duck meat in Korea. Because of this novel source of HP H5N1 virus isolation, concerns were raised about the potential for human exposure and infection; we therefore compared the Dk/Anyang virus with HP H5N1 viruses isolated from humans in 1997 in terms of antigenicity and pathogenicity for mammals. At high doses, Dk/Anyang virus caused up to 50% mortality in BALB/c mice, was isolated from the brains and lymphoid organs of mice, and caused lymphopenia. Overall Dk/Anyang virus was substantially less pathogenic for mice than the H5N1 virus isolated from a fatal human case in 1997. Likewise, Dk/Anyang virus was apathogenic for ferrets. Dk/Anyang virus was antigenically distinguishable by hemagglutination-inhibition (HI) assay from human H5N1 viruses isolated in 1997 and avian H5N1 viruses isolated in 2001 in Hong Kong. Nevertheless, prior infection with Dk/Anyang virus protected mice from death after secondary infection with HP human H5N1 viruses. These results indicate that compared with HP human H5N1 viruses, Dk/Anyang virus is substantially less pathogenic for mammalian species. Nevertheless, the novel source of isolation of this avian H5N1 virus must be considered when evaluating the potential risk to public health.

Treatment of refractory acute leukemia with timed sequential chemotherapy using topotecan followed by etoposide + mitoxantrone (T-EM) and correlation with topoisomerase II levels.

A phase I/II clinical study evaluated 17 patients with refractory/recurrent acute leukemia treated with 1.5 mg/m2/day topotecan on days 1-3 followed by etoposide (100 mg/m2/day)+mitoxantrone (10 mg/m2/day) on days 4, 5 and 9, 10. Timed sequential chemotherapy using the topoisomerase I-inhibitor topotecan before the topoisomerase II-inhibitors, etoposide+mitoxantrone (T-EM) treatment is proposed to induce topoisomerase II protein levels and potentiate the cytotoxic activity of the topoisomerase II-directed drugs. Fourteen patients had refractory and three had recurrent acute leukemia. The majority of patients were heavily pre-treated with greater than three re-induction chemotherapy regimens. Ten patients responded to T-EM treatment (59%). Four of seventeen (24%) had a complete remission and one had a partial remission. Four additional patients (24%) who scored complete leukemia clearance had no evidence of disease with complete white and red blood cell recovery but with platelet counts less than 100,000. The lack of platelet recovery in one patient having a partial response was scored as a partial leukemia clearance. The toxicity profile included major non-hematological toxicity including grade 3 mucositis (29%) and neutropenic fever (65%). Paired measurements of intracellular levels of topoisomerase II isoforms alpha and beta in leukemia blast cells (bone marrow) collected before (day 0) and after topotecan treatment (day 4) showed that a relative increase of topoisomerase IIalpha (Topo IIalpha) > or = 40% strongly correlated with response after T-EM treatment. Increased Topo IIalpha levels also corresponded to increased DNA fragmentation. Two patients who had an increase of Topo IIalpha of 20-25% had either a PR or PLC while patients with a < 10% increase showed no response to T-EM treatment. We conclude that timed sequential chemotherapy using topotecan followed by etoposide+mitoxantrone is an effective regimen for patients with refractory acute leukemia, and demonstrate Topo IIalpha protein level increases after topotecan treatment.

Organizations and promoter analyses of the human and the mouse genes for PACT, the protein-activator of the interferon-induced protein kinase, PKR.

PACT is an activator of the protein kinase, PKR. Here we report the isolation and the characterization of the mouse Pact gene. It contains eight exons ranging in size from 79 to 630 bp spanning a region of 18 kb with the largest and smallest introns being 3700 and 500, respectively. The human PACT gene, as analyzed from sequence available in the GenBank database, has a very similar organization. The 5' flanking regions of both mouse and human PACT genes are devoid of TATA boxes but are rich in GC boxes. Although there are putative binding sites of numerous transcription factors on both promoters, their organizations and identities are different. For examining promoter activities, about 2 kb of DNA 5' to the transcription start sites of both genes was cloned upstream of a reporter luciferase gene. Transient transfection assays demonstrated that both promoters are strong. Deletion analyses revealed that most of the positive cis-elements lie within 400 bp upstream of the transcription start sites of both mouse and human PACT genes.

Heterosubtypic immunity against human influenza A viruses, including recently emerged avian H5 and H9 viruses, induced by FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage function.

Induction of heterosubtypic immunity to influenza viral antigens is of paramount importance to the prevention of epidemics and potential pandemics. The 1997 incidence of avian influenza infections in humans in Hong Kong heightened the need for pandemic preparedness and a search for vaccines and vaccine delivery systems that can confer broad protection. In this report, we demonstrate that the delivery of H1N1 subtype influenza viral antigens as immunostimulating complexes (ISCOM) induces broad cross-protection in mice against challenge with various influenza virus subtypes, including the avian H9 and the H5 strains that were recently responsible for deaths in humans. The ISCOM delivery system induced high and long-lived serum antiviral antibodies and class I-restricted cytotoxic T-lymphocytes (CTL). Studies with perforin, IFN-gamma, and mu-chain gene knock-out mice demonstrated that the heterosubtypic protection required cross-reactive, functional cytotoxic T cells and nonhemagglutination inhibiting serum antibodies. Interferon-gamma, a major player in viral clearance by nonlytic mechanisms, did not appear to play a role in heterosubtypic immunity. Nonformulated H1N1 influenza antigens failed to induce significant CTL or long-lasting antibody responses or to protect mice against challenge with heterosubtypic viruses. Furthermore, while influenza virus infection induced a dominant nucleoprotein (NP)-specific CTL response in H2 mice, the ISCOM delivery system induced a dominant hemagglutinin-specific CTL response. Moreover, non-neutralizing but cross-reactive antibodies played a role in reducing viral titers by macrophages. These results suggest that exogenous delivery of influenza antigens as ISCOM can influence their antigen processing and presentation, their ability to induce/recall CTL specificities, and their capacity to mediate broad cross-protection against influenza virus variants.

Prevalence of Salmonella serotypes on pig carcasses from high- and low-risk herds slaughtered in three abattoirs.

The aim of this study was to compare the prevalence of Salmonella serotypes at two different sites on pig carcasses from herds classified as high-risk or low-risk and to elucidate the relationship between carcass contamination levels and serological status. Caecal samples and carcass surface swabs were cultured for Salmonella from a total of 210 pigs from low risk herds (< 19% of pigs in herd Salmonella seropositive) and 209 pigs from high risk herds (> 32% of pigs in herd Salmonella seropositive) in three abattoirs. Meat juice samples were collected for analysis by ELISA. The prevalence of Salmonella in the caecal contents of "low-risk" pigs was 10%, which was significantly lower than the 19% prevalence in "high-risk" pigs (p < 0.01). The corresponding figures for skin samples collected immediately post-evisceration were 2% and 12%. The predominant Salmonella serotype in the caecal contents of both the low-risk and high-risk pigs was Salmonella Typhimurium. Salmonella Kentucky and Salmonella Derby were the most frequent isolates from the carcass surface swabs of low- and high-risk pigs respectively. There was a positive association between seropositivity of pigs from high-risk herds and caecal carriage (p < 0.05). Results showed that herd categorisation based on serological results was useful in predicting Salmonella isolation rates from caecal samples and surface swabs of slaughtered pigs.

Diagnosing pediatric asthma: validating the Easy Breathing Survey.

OBJECTIVE: To determine the sensitivity, specificity, and predictive value of a simple, self-administered questionnaire for the diagnosis of asthma in children. STUDY DESIGN: A questionnaire specifically designed to assist primary care providers in making a diagnosis of asthma in children was developed and administered in 4 different primary care and subspecialty clinics, validated, and then used as part of an asthma management program called Easy Breathing. Asthma diagnoses were made according to recommended National Asthma Expert Panel Guidelines. RESULTS: Four questions on the survey were shown to be sensitive and specific for asthma. The sensitivity was greater for all levels (mild, moderate, and severe) of persistent asthma than for mild, intermittent asthma. A positive response to any 1 of the 4 questions was over 94% sensitive for asthma; a negative response to all 4 questions was 55% specific for ruling out asthma. CONCLUSIONS: Patient responses to 4 specific respiratory symptom questions can assist primary care providers in diagnosing asthma in children. Primary care providers serving pediatric populations at high risk for asthma should consider asking patients or their parents these 4 questions regarding asthma symptoms on a regular basis.

Teaching the Laufe-Piper forceps technique at cesarean delivery.

OBJECTIVE: To present a method of teaching forceps technique during cesarean delivery of breech-presenting infants using Laufe-Piper forceps and to evaluate its usefulness. STUDY DESIGN: For several years, residents at the University of Texas Medical Branch, Galveston, have learned and practiced Piper forceps technique during cesarean delivery. To assess their experience with this method, we mailed questionnaires to third- and fourth-year residents and recent graduates of the Galveston program. The same surveys were mailed to a control group of residents and recent graduates of two other programs where this teaching exercise is not practiced routinely. RESULTS: Responses were received from 32 (74%) study subjects and 63 (71%) controls. Demographic characteristics and experience with vaginal breech delivery were similar between the two groups. Respondents from the Galveston program noted greater annual use of forceps for vaginal delivery of cephalic-presenting infants (P = .012). They also rated themselves as more comfortable (P = .023) and more skilled (P = .006) with Piper forceps than controls. Of 53 respondents who had had previous experience with this teaching method, 47 noted that it provided a great or moderate educational benefit, and 36 strongly or moderately believed it gave them more confidence in using Piper forceps during vaginal breech delivery. Using multiple regression analysis, sex, overall level of experience, Piper forceps experience during vaginal delivery and overall forceps use were stronger determinants of self-rated comfort and skill than was experience with Laufe-Piper forceps during cesarean. CONCLUSION: Laufe-Piper forceps can be used for cesarean delivery of breech-presenting infants. This practice promotes confidence and skill for their use at vaginal delivery.

Mitochondrial DNA metabolism targeting drugs.

Numerous drugs are known to deplete mitochondrial DNA (mtDNA) from mammalian cells. These include DNA polymerase gamma and type II topoisomerase inhibitors, lipophilic cationic compounds, and DNA intercalating and non intercalating agents. The effects of these drugs on mtDNA metabolism will be discussed and potential mechanisms underlying their depletion of mtDNA presented.

A specific isozyme of 2'-5' oligoadenylate synthetase is a dual function proapoptotic protein of the Bcl-2 family.

2-5(A) synthetases are a family of interferon-induced enzymes that polymerize ATP into 2'-5' linked oligoadenylates that activate RNase L and cause mRNA degradation. Because they all can synthesize 2-5(A), the reason for the existence of so many synthetase isozymes is unclear. Here we report that the 9-2 isozyme of 2-5(A) synthetase has an additional activity: it promotes apoptosis in mammalian cells. The proapoptotic activity of 9-2 was isozyme-specific and enzyme activity-independent. The 9-2-expressing cells exhibited many properties of cells undergoing apoptosis, such as DNA fragmentation, caspase activation, and poly ADP-ribose polymerase and lamin B cleavage. The isozyme-specific carboxyl-terminal tail of the 9-2 protein was shown, by molecular modeling, to contain a Bcl-2 homology 3 (BH3) domain, suggesting that it may be able to interact with members of the Bcl-2 family that contain BH1 and BH2 domains. Co-immunoprecipitate assays and confocal microscopy showed that 9-2 can indeed interact with the anti-apoptotic proteins Bcl-2 and Bclx(L) in vivo and in vitro. Mutations in the BH3 domain that eliminated the 9-2-Bcl-2 amd 9-2-Bclx(L) interactions also eliminated the apoptotic activity of 9-2. Thus, we have identified an interferon-induced dual function protein of the Bcl-2 family that can synthesize 2-5(A) and promote cellular apoptosis independently. Moreover, the cellular abundance of this protein is regulated by alternative splicing; the other isozymes encoded by the same gene are not proapoptotic.