Efficiency of the inbreeding coefficient f and other estimators in detecting null alleles, as revealed by empirical data of locus oke3 across 65 populations of chum salmon Oncorhynchus keta.

A survey of 65 populations of chum salmon Oncorhynchus keta across the species range revealed homozygote excess (947 homozygotes in 2954 fish) at a polymerase chain reaction (PCR)-based simple sequence repeat (SSR) locus oke3 with multiple alleles, whereas re-designed PCR primers indicated that 328 of these homozygotes were actually heterozygotes. Statistically significant high positive values of inbreeding coefficients, f, in multiple populations appeared to be a reliable predictor of null alleles. Based on these data, three methods were checked for their ability to estimate null-allele frequencies.

Immunoblotting and immunodetection.

This unit provides protocols for immunoblotting, which is used to identify specific protein sequences separated by electrophoresis and transferred to an appropriate membrane and recognized by a polyclonal or monoclonal antibody. After the proteins are separated on a gel, they are transferred to a membrane by electroblotting or a semidry transfer system. Proteins on the membrane can be visualized with the reversible stain Ponceau S to assess the completeness of transfer. Then the blot is analyzed with antibodies. The primary antibody is specific for the protein(s) of interest; the secondary antibody (an anti-Ig) is conjugated to horseradish peroxidase or alkaline phosphatase and detected colorimetrically or by chemiluminescence. The membrane can be stripped and reused for other probes.

Conjugation of enzymes to antibodies.

Conjugation of enzymes to antibodies involves the formation of a stable, covalent linkage between an enzyme [e.g., horseradish peroxidase (HRPO), urease, or alkaline phosphatase] and an antigen-specific monoclonal or polyclonal antibody in which neither the antigen-combining site of the antibody nor the active site of the enzyme is functionally altered. This unit describes procedures for cross-linking HRPO, urease or alkaline phosphatase to immunoaffinity-purified monoclonal or polyclonal antibodies (IgG).

Purification of monoclonal antibodies.

The uses of monoclonal antibodies as enzyme conjugates and as immunoaffinity reagents require their purification from the crude ascites fluid. This unit provides protocols for purification using protein A-Sepharose chromatography and affinity chromatography, which are superior to ammonium sulfate precipitation, gel filtration, or ion-exchange chromatography for the preparation of contaminant-free antibody.

Enzyme-linked immunosorbent assays (ELISA).

This unit describes six different ELISA systems for the detection of specific antibodies, soluble antigens, or cell-surface antigens. In all six systems, soluble reactants are removed from solution after specifically binding to solid-phase reactants. In the first four protocols, solid-phase reactants are prepared by adsorbing an antigen or antibody onto plastic microtiter plates; in the next two protocols, the solid-phase reactants are cell-associated molecules. In all protocols, the solid-phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or with a microtiter plate reader. The amount of product generated is proportional to the amount of analysate in the test mixture. Support protocols are provided for optimizing the different ELISAs and preparing lysates for use as test antigen from bacterial cultures containing expressed protein.

Immunization of mice.

In the protocols in this unit, antigen is prepared for injection either by emulsifying an antigen solution with Freunds adjuvant or by homogenizing a polyacrylamide gel slice containing the protein antigen. Mice are immunized at 2- to 3-week intervals. Test bleeds are collected 7 days after each booster immunization to monitor serum antibody levels. Mice are chosen for hybridoma fusions when a sufficient antibody titer is reached.

Preparation of myeloma cells.

In this unit, myeloma cells are cultured to ensure their sensitivity to the HAT selection medium used after cell fusion. Cell culture conditions are adjusted such that the Sp2/0 cells are in the log phase of growth and exhibit high viability at the time of collection for fusion. A support protocol is provided to determine the number of viable cells present in the cell culture.

Preparation of mouse feeder cells for fusion and cloning.

To maximize the yield of hybrids from fusion and cloning procedures, feeder cells are required to be cocultured with the hybrids, while hybrid cell density is low. Mouse peritoneal cells, most of which are macrophages, have been found to be convenient and effective feeder cells which are a source of soluble growth factors for hybridoma cells.

Fusion of myeloma cells with immune spleen cells.

Liquid nitrogen storage is the method of choice for long-term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.

Cloning of hybridoma cell lines by limiting dilution.

Cloning by limiting dilution is a method based on the Poisson distribution. Dilution of cells to an appropriate number per well can maximize the proportion of wells that contain one single clone. Two or more cloning procedures are carried out until >90% of the wells containing single clones are positive for antibody production.

Freezing and recovery of hybridoma cell lines.

Liquid nitrogen storage is the method of choice for long-term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.

Increasing rates of suicide in Western Australian psychiatric patients: a record linkage study.

OBJECTIVE: To investigate the risk of suicide following contact with mental health services and whether it has changed over time. METHOD: Record linkage was used to obtain the records of previous hospital admissions and mental health service contacts for deaths due to suicide in the period 1980-98. Standardized incidence rates were calculated for the general population and for users of mental health services. Proportional hazards regression was used to assess risk factors within the cohort of people with mental health service contact. RESULTS: Suicide risk was significantly increased in users of mental health services (rate ratio 6.66 in males and 7.52 in females). Suicide risk was highest in the first 7 days after discharge from in-patient care, and decreased exponentially with time since discharge. Suicide risk in users of mental health services has increased over the study period. CONCLUSION: These results highlight the importance of adequate follow-up of patients discharged from in-patient services, and the need for adequate resources for community-based services.

Excess cancer mortality in Western Australian psychiatric patients due to higher case fatality rates.

OBJECTIVE: To investigate the association between mental illness and cancer incidence, mortality and case fatality. METHOD: A population-based record linkage study was undertaken based on 172,932 patients of mental health services in Western Australia. Records of mental health service contacts were linked with cancer registrations and death records. RESULTS: While there was little difference in overall cancer incidence rates between psychiatric patients and the general community (RR in males 1.05, 95%, CI 1.02-1.09, RR in females 1.02, 0.98-1.05), cancer mortality was 39%, higher in males (95%) CI: 32-46%) and 24% higher in females (17-32%). CONCLUSION: People with mental illness in Western Australia do not show an increased incidence rate of cancer, but do have higher cancer mortality. This was attributed to a higher cancer case fatality rate among people with mental illness.

Vaccination of seropositive subjects with CHIRON CMV gB subunit vaccine combined with MF59 adjuvant for production of CMV immune globulin.

The safety and immunogenicity of four different regimens of CHIRON cytomegalovirus (CMV) gB subunit vaccine combined with MF59 adjuvant and administered to seropositive plasma donors were evaluated to ascertain whether vaccination of seropositive subjects would significantly increase antibody titer to gB glycoprotein. This was done to select the best vaccination regimen for generating high-titered plasma for manufacture of CMV immune globulin. No serious adverse events were attributed to this vaccine, and the vaccine was well tolerated. Only the first dose of vaccine in each regimen stimulated a four-fold or greater antibody response to gB glycoprotein and each regimen induced similar antibody titers. However, initial vaccination followed by a 1 week rest from plasmapheresis and two booster vaccinations at 8 and 24 weeks, each followed with another 1 week rest from plasmapheresis, maintained the highest geometric mean gB ELISA titer of the four regimens over the 34-week post-vaccination period. CMVIG manufactured from a pool of high titered plasma units from two of four subject groups had gB ELISA and neutralizing antibody titers nine and six times higher, respectively, compared to Cytogam, indicating that vaccination of seropositive subjects with CHIRON gB vaccine combined with MF59 adjuvant prior to harvesting plasma can enhance functional antibody in a CMVIG product.

Suicide rates in psychiatric in-patients: an application of record linkage to mental health research.

OBJECTIVE: To study trends in the rate of suicide in psychiatric patients in Western Australia. To examine the associations of suicide with demographic and clinical factors. METHODS: A population-based cohort of 52,010 individuals whose first psychiatric admission occurred in 1980-95 was identified from the Health Services Research Linked Database. There were 471 deaths by suicide by 31 December 1995. Age standardised suicide rates per 1,000 person-years at risk were calculated. Suicide rates in the first year after a patient's first admission were also examined and a proportional hazards regression analysis was performed to examine risk factors for suicide. RESULTS: Male psychiatric patients were 3.4 times more likely to commit suicide than female patients (95% CI 2.76-4.24). Younger patients were at higher risk than older patients, and patients with extended periods of in-patient treatment were at more than double the risk of short-stay patients. Over the 16-year period, the rate of suicide in the first year after first psychiatric admission was found to increase by 3.4% a year (95% CI -0.7-7.6%). CONCLUSIONS: The findings confirm that psychiatric patients are at high risk of suicide. Patient outcomes in terms of risk of suicide after hospital discharge have deteriorated. IMPLICATIONS: Improvements are needed in the provision of community support to high risk psychiatric patients. Further work should be done to identify patients at highest risk of suicide.

Mapping of semantic, phonological, and orthographic verbal working memory in normal adults with functional magnetic resonance imaging.

Twelve neurologically normal participants (4 men and 8 women) performed semantic, phonological, and orthographic working memory tasks and a control task during functional magnetic resonance imaging. Divergent regions of the posterior left hemisphere used for decoding and storage of information emerged in each working memory versus control task comparison. These regions were consistent with previous literature on processing mechanisms for semantic, phonological, and orthographic information. Further, working memory versus control task differences extended into the left frontal lobe, including premotor cortex, and even into subcortical structures. Findings were consistent with R. C. Martin and C. Romani's (1994) contention that different forms of verbal working memory exist and further suggest that a reconceptualization of premotor cortex functions is needed.

Determination of drug-induced changes in functional MRI signal using a pharmacokinetic model.

As the applications of functional magnetic resonance imaging (fMRI) expand, there is a need for the development of new strategies for data extraction and analysis that do not require the presentation of stimuli in a repeated on/off pattern. A description and evaluation of a method and computer algorithm for the detection and analysis of brain activation patterns following acute drug administration using fMRI are presented. A waveform analysis protocol (WAP) input function has been developed that is based upon the single-dose pharmacokinetics of a drug of interest. As a result of this analysis, regional brain activation can be characterized by its localization and intensity of activation, onset of action, time to peak effect, and duration of action. A global statistical test for significant drug effects based upon the probability of a voxel being activated by a saline vehicle injection is applied to grouped data on a voxel by voxel basis. Representative data are presented using nicotine as a prototypical agent. Using this method, statistically significant drug-induced brain activation has been identified in several key cortical and subcortical brain regions.

Selective effects of the endogenous cannabinoid arachidonylethanolamide (anandamide) on regional cerebral blood flow in the rat.

Recent biochemical data suggest that arachidonylethanolamide (AEA; anandamide) may be an endogenous ligand for brain cannabinoid receptors. The functional neuronal consequences of AEA binding to cannabinoid receptors are only poorly understood. Using regional cerebral blood flow (rCBF) as an indirect marker of neuronal activity, acute AEA administration dose-dependently depressed rCBF in unanesthetized rats. Although 3.0 mg/kg was ineffective in altering rCBF, 10 mg/kg led to a decrease in rCBF in seven brain areas including the amygdala, cingulate, frontal, prepyriform, sensorimotor, and claustrocortex. An additional 16 areas responded in a similar manner to AEA, but only after 30 mg/kg, including the CA1 and CA3 regions of the hippocampus, the rostral core portion of the nucleus accumbens, and rostral caudate nucleus. Most of these rCBF effects dissipated between 15 and 20 min after drug administration, with only 4 regions, the basomedial and lateral amygdala, CA3 hippocampus and claustrocortex still depressed 60 min after an acute drug injection. No significant changes in heart rate, blood pressure, or blood gases were seen at the time of rCBF measurement, suggesting that the observed drug effects were neuronally mediated. Taken together with existing behavioral data, these data support the hypothesis that an endogenous cannabinoid neural system exists in mammalian brain and may help to explain the unique behavioral profile seen after cannabinoid administration.

Nicotine-induced limbic cortical activation in the human brain: a functional MRI study.

OBJECTIVE: Nicotine is a highly addictive substance, and cigarette smoking is a major cause of premature death among humans. Little is known about the neuropharmacology and sites of action of nicotine in the human brain. Such knowledge might help in the development of new behavioral and pharmacological therapies to aid in treating nicotine dependence and to improve smoking cessation success rates. METHOD: Functional magnetic resonance imaging, a real-time imaging technique, was used to determine the acute CNS effects of intravenous nicotine in 16 active cigarette smokers. An injection of saline followed by injections of three doses of nicotine (0.75, 1.50, and 2.25 mg/70 kg of weight) were each administered intravenously over 1-minute periods in an ascending, cumulative-dosing paradigm while whole brain gradient-echo, echo-planar images were acquired every 6 seconds during consecutive 20-minute trials. RESULTS: Nicotine induced a dose-dependent increase in several behavioral parameters, including feelings of "rush" and "high" and drug liking. Nicotine also induced a dose-dependent increase in neuronal activity in a distributed system of brain regions, including the nucleus accumbens, amygdala, cingulate, and frontal lobes. Activation in these structures is consistent with nicotine's behavior-arousing and behavior-reinforcing properties in humans. CONCLUSIONS: The identified brain regions have been previously shown to participate in the reinforcing, mood-elevating, and cognitive properties of other abused drugs such as cocaine, amphetamine, and opiates, suggesting that nicotine acts similarly in the human brain to produce its reinforcing and dependence properties.

Dopamine release in the nucleus accumbens during heroin self-administration is modulated by kappa opioid receptors: an in vivo fast-cyclic voltammetry study.

Mu and kappa opioid agonists are known to produce different, and sometimes opposite, effects on several pharmacological and behavioral measures. However, whether kappa agonists can be used to antagonize the reinforcing and putative dopamine (DA)-releasing properties of a mu agonist such as heroin is unclear. With the use of the high temporal and spatial resolution of in vivo fast-cyclic voltammetry to measure changes in extracellular DA in the nucleus accumbens (NAcc), we observed (1) dose-dependent increases in DA in the NAcc during heroin self-administration (SA), (2) that coadministration of the kappa agonist U50,488H with heroin or intracerebroventricular dynorphin A pretreatment significantly depressed the heroin-stimulated DA release during SA, where U50,488H alone inhibited the basal DA release in the NAcc, (3) that coadministration of low-dose U50,488H or dynorphin A significantly increased heroin SA behavior, whereas high-dose U50,488H, which alone did not support SA behavior, reduced or completely blocked heroin SA and (4) that nor-binaltorphimine dihydrochloride (a selective kappa receptor antagonist) potentiated DA release in the NAcc and modestly decreased heroin SA. Taken together, these data suggest that endogenous kappa receptor activation can inhibit mu agonist-induced activation of the mesolimbic DA pathway, which may in turn depress heroin-induced reinforcement.