Ageing-related changes in GABAergic inhibition in mouse auditory cortex, measured using in vitro flavoprotein autofluorescence imaging.

KEY POINTS: Ageing is associated with hearing loss and changes in GABAergic signalling in the auditory system. We tested whether GABAergic signalling in an isolated forebrain preparation also showed ageing-related changes. A novel approach was used, whereby population imaging was coupled to quantitative pharmacological sensitivity. Sensitivity to GABAA blockade was inversely associated with age and cortical thickness, but hearing loss did not independently contribute to the change in GABAA ergic sensitivity. Redox states in the auditory cortex of young and aged animals were similar, suggesting that the differences in GABAA ergic sensitivity are unlikely to be due to differences in slice health. To examine ageing-related changes in the earliest stages of auditory cortical processing, population auditory cortical responses to thalamic afferent stimulation were studied in brain slices obtained from young and aged CBA/CAj mice (up to 28 months of age). Cortical responses were measured using flavoprotein autofluorescence imaging, and ageing-related changes in inhibition were assessed by measuring the sensitivity of these responses to blockade of GABAA receptors using bath-applied SR95531. The maximum auditory cortical response to afferent stimulation was not different between young and aged animals under control conditions, but responses to afferent stimulation in aged animals showed a significantly lower sensitivity to GABA blockade with SR95531. Cortical thickness, but not hearing loss, improved the prediction of all imaging variables when combined with age, particularly sensitivity to GABA blockade for the maximum response. To determine if the observed differences between slices from young and aged animals were due to differences in slice health, the redox state in the auditory cortex was assessed by measuring the FAD+/NADH ratio using fluorescence imaging. We found that this ratio is highly sensitive to known redox stressors such as H2 O2 and NaCN; however, no difference was found between young and aged animals. By using a new approach to quantitatively assess pharmacological sensitivity of population-level cortical responses to afferent stimulation, these data demonstrate that auditory cortical inhibition diminishes with ageing. Furthermore, these data establish a significant relationship between cortical thickness and GABAergic sensitivity, which had not previously been observed in an animal model of ageing.

COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility.

The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility. The wild-type allele, COI1, was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants. The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif. It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.

Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8.

Plant disease resistance (R) genes control the recognition of specific pathogens and activate subsequent defense responses. We show that the Arabidopsis thaliana locus RESISTANCE TO POWDERY MILDEW8 (RPW8) contains two naturally polymorphic, dominant R genes, RPW8.1 and RPW8.2, which individually control resistance to a broad range of powdery mildew pathogens. Although the predicted RPW8.1 and RPW8.2 proteins are different from the previously characterized R proteins, they induce localized, salicylic acid-dependent defenses similar to those induced by R genes that control specific resistance. Apparently, broad-spectrum resistance mediated by RPW8 uses the same mechanisms as specific resistance.

Arabidopsis Mutants Selected for Resistance to the Phytotoxin Coronatine Are Male Sterile, Insensitive to Methyl Jasmonate, and Resistant to a Bacterial Pathogen.

The phytotoxin coronatine and the plant growth regulator methyl jasmonate (MeJA) caused similar growth-inhibitory effects on Arabidopsis seedlings. To test whether these two compounds have similar action, 14 independent coi1 (coronatine-insensitive) mutants of Arabidopsis were selected. The mutants segregated as single recessive Mendelian markers, and all were alleles at the coi1 locus. All coi1 mutants were also insensitive to MeJA and were male sterile. Both coronatine and MeJA inhibited root growth, stimulated anthocyanin accumulation, and increased the level of two proteins of ~31 and ~29 kD detected in SDS-polyacrylamide gels of wild-type Arabidopsis but caused none of these effects in the coi1 mutant. Coronatine and MeJA also induced the systemic appearance of proteinase inhibitor activity in tomato. The male-sterile flowers of the coi1 mutant produced abnormal pollen and had reduced level of an ~31-kD protein, which was abundant in the wild-type flowers. A coronatine-producing strain of Pseudomonas syringae grew in leaves of wild-type Arabidopsis to a population more than 100 times greater than it reached in the coi1 mutant. We conclude that coronatine mimics the action of MeJA and that coi1 controls a step in MeJA perception/response and in flower development.

Broadened clinical utility of gene gun-mediated, granulocyte-macrophage colony-stimulating factor cDNA-based tumor cell vaccines as demonstrated with a mouse myeloma model.

Effective immunization against the murine B16 melanoma by a nonviral approach in which a gene gun is used to transfer GM-CSF cDNA into tumor cells has been described. We have extended this nonviral approach by using the poorly immunogenic murine myeloma MPC11 model. Vaccination with the transfected, GM-CSF-expressing MPC11 cells induced a potent antitumor cytotoxic T lymphocyte response associated with tumor rejection in the majority of the test mice. Furthermore, nearly 100% (27 of 28) of the tumor-free mice were able to reject a tumor rechallenge. While this approach is clinically attractive because of minimal tissue manipulation/culturing and the absence of infectious agents, a number of tested human primary tumors, including myeloma cells, have failed to produce high levels of GM-CSF after gene gun transfection. To circumvent the low transfection efficiency in certain human tumor cells, we showed that combining irradiated tumor cells to provide tumor antigens together with gene gun-transfected fibroblasts to provide GM-CSF induced effective tumor rejection. We also report that normal human skin fibroblasts transfected by the gene gun produce high levels of human GM-CSF (250 ng/10(6) cells/24 hr). These results suggest that combining irradiated tumor cells with gene gun-transfected fibroblasts results in antitumor immune responses and may allow for a wider application of this approach to cancer immunotherapy.

Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens.

In previous work, UEA1 and UCSC1, two geographically distinct, powdery mildew isolates, were recognized for their ability to infect Arabidopsis thaliana. We have clarified the identity of these isolates by determining their host ranges, reexamining their morphology, and comparing their DNA sequences for the 5.8S ribosomal RNA and two flanking internal transcribed spacer sequences. These experiments confirm that UEA1 is a member of Erysiphe cruciferarum and that UCSC1 belongs to E. cichoracearum. Interactions of the two Erysiphe isolates with 360 A. thaliana accessions were examined to provide a comprehensive profile of naturally occurring powdery mildew resistance in this weedy species. The majority of A. thaliana accessions (213) were susceptible to both isolates. Among the accessions exhibiting some degree of resistance, most (84) responded differentially to UEA1 and UCSC1 and the remainder were resistant to both isolates. Notably, resistance to UCSC1 cosegregated with RPW7, a locus previously demonstrated to confer resistance to UEA1 in Ms-0 x Landsberg (erecta) crosses. With this large collection of resistant accessions, questions about species specificity, genetic diversity and the evolution of resistance to powdery mildews can be addressed.

Effects of beta-carboline on fear-related behavioral and neurohormonal responses in infant rhesus monkeys.

We examined the effects of the inverse benzodiazepine agonist ethyl-beta-carboline-3-carboxylate (beta-CCE) on behavioral, hormonal, and neurochemical responses in infant rhesus monkeys exposed to fearful situations. Our paradigm elicits three distinct adaptive patterns of defensive behavior. From previous work, we hypothesized that behaviors induced by attachment bond disruption are predominantly mediated by opiate systems, whereas behaviors induced by the threat of attack are mediated by benzodiazepine systems. When beta-CCE (0, 125, 250, and 500 micrograms/kg) was administered immediately after maternal separation, the 500 micrograms/kg dose increased freezing and the 250 and 500 micrograms/kg doses reduced environmental exploration. Test conditions produced increased plasma ACTH and cortisol concentrations and increased cerebrospinal fluid (CSF) concentrations of MHPG and DOPAC; beta-CCE did not further affect these metabolites. A dose of 1000 micrograms/kg of beta-CCE increased CSF concentrations of DOPAC and MHPG in infants left with their mothers. During test conditions, it further increased CSF MHPG (but not DOPAC) concentrations, and reduced cooing while increasing freezing and barking and other hostile behaviors. Our results thus confirm that benzodiazepine systems mediate threat-related behaviors and suggest that coos, which were thought to predominantly reflect the degree of distress during separation, can be modulated by the infant's level of fear. beta-CCE also activated stress-related pituitary-adrenal hormonal systems and brain norepinephrine (NE) and dopamine (DA) systems. These effects occurred when animals remained undisturbed in their home cages with their mothers, suggesting that benzodiazepine receptors directly modulate brain NE and DA systems.

Characterisation of genes involved in biosynthesis of coronafacic acid, the polyketide component of the phytotoxin coronatine.

Coronafacic acid (CFA) is the polyketide component of coronatine (COR), a phytotoxin produced by the plant pathogen, Pseudomonas syringae. In the present study we have determined the nucleotide sequence of a 3.92-kb DNA fragment involved in CFA biosynthesis. Analysis of the sequence revealed four complete open reading frames (ORFs) designated cfa1 to cfa4 and one incomplete ORF (cfa5), all transcribed in the same direction. The predicted translation products of cfa1, cfa2 and cfa3 showed relatedness to acyl carrier proteins, fatty acid dehydrases and beta-ketoacylsynthases, respectively, which are required for polyketide synthesis. cfa1 was subcloned, its sequence was confirmed, and it was overexpressed in E. coli to yield a peptide with an apparent molecular mass of 6 kDa.

Interleukin-12 cDNA skin transfection potentiates human papillomavirus E6 DNA vaccine-induced antitumor immune response.

Human papillomaviruses are associated with >90% of all cases of uterine cervical tumors. The E6 and E7 oncoproteins of human papillomavirus are potentially ideal targets of immune therapy for cervical cancer, because their expression is necessary for cellular transformation. Although both E6 and E7 proteins contain numerous predicted cytotoxic T lymphocyte (CTL) epitopes that are capable of binding to human leukocyte antigens, the majority of earlier in vivo tumor rejection studies have focused on E7. We show here that gene gun-mediated skin transfection of plasmid vector encoding the nontransforming, amino-terminal half of E6 resulted in the induction of E6-specific CTL activity and tumor rejection in a murine model. The use of recombinant murine interleukin-12 (rmIL-12) as a vaccine adjuvant has been shown to result in both an enhancement and suppression of immune responses, depending upon the doses of rmIL-12 and the experimental systems used. We demonstrate here that local expression of transgenic mIL-12 at the E6 DNA vaccination site potentiated E6-specific CTL responses and increased vaccine-induced antitumor therapeutic efficacy. Our results indicate that transfection of the mIL-12 gene at the vaccination site may represent an attractive adjuvant for cancer gene immunotherapy.

A clinical comparison of Tc-99m HEDP and Tc-99m MDP in the detection of bone metastases: concise communication.

We have compared bone scintigrams made with Tc-99m-tagged HEDP (1-hydroxyethylidene diphosphonate)and MDP (methylene diphosphonate), the former at 4 hr after injection, the latter at both 2 and 4 hr. In 17 patients with skeletal metastases, there was no significant difference in lesion count or scan quality between the 4-hr images. The tumor-to-bone ratio (T/B) was significantly higher with Tc-HEDP (p less than 0.02). Lesion detection rate and T/B ratios were both lower with Tc-MDP at 2 hr when compared with the 4-hr values for both Tc-HEDP (p less than 0.02, p less than 0.005) and Tc-MDP (p less than 0.02, p less than 0.01). The 4-hr Tc-MDP scan was of significantly higher quality than the 2 hr Tc-MDP scan (p less than 0.01). Although Tc-HEDP produces a higher T/B ratio at 4 hr, the present study does not suggest that either agent is superior in clinical practice.

The role of bone scanning in osteomalacia.

The presence of eight "metabolic features" was assessed on the bone scintigrams of ten patients with osteomalacia. In all of these bone images, sufficient features were present to strongly suggest a metabolic disorder. There scintiphotos were included in a controlled blind study using 30 normal bone scans and 20 scans of metastatic disease. Nine of the ten metabolic bone images were correctly identified by two independent observers. Skeletal uptake of radiotracer, expressed as bone-to-soft tissue ratio, was significantly higher in the osteomalacic patients than in a group of 80 controls.

The use of whole-body retention of Tc-99m diphosphonate in the diagnosis of metabolic bone disease.

The limited role of bone scanning in the diagnosis of metabolic bone disease might be considerably improved by accurate quantification of skeletal uptake of the radiopharmaceutical. Using a standard shadow-shield whole-body monitor, we have measured whole-body retention (WBR) of Tc-99m HEDP up to 24 hr in 11 patients with renal osteodystrophy (mean WBR 88.6% at 24 hr); in ten patients with Paget's disease (mean 56.9%); in seven patients with osteomalacia (mean 40.7%); in five patients with primary hyperparathyroidism (mean 50.7%); in four patients with osteoporosis (mean 21.2%); and in 12 normals (mean 19.2%). The osteoporotic group could not be differentiated from the normal group, but the other groups were significantly different from the normal group at 24 hr (p less than 0.002), and each individual rest for the 24-hr WBR of Tc-99m HEDP in these groups lay outside our normal range. This test may, therefore, provide a sensitive means of detecting conditions with increased bone turnover. We obtained measurements of plasma activity of Tc-99m HEDP in these patients up to 24 hr, and 4-hr bone to soft-tissue ratios from bonescan images, but little additional information resulted.

Can the extent of coronary artery disease be predicted from thallium-201 myocardial images?

The accuracy with which the extent of coronary artery disease can be predicted from stress thallium-201 myocardial images has been assessed in 81 patients with chest pain. Whereas the appearance of the myocardial images was both a sensitive means of detecting coronary artery disease (images abnormal in 43 of 47 patients with abnormal coronary arteriograms) and specific in excluding it (images normal in 31 of 34 patients with normal arteriograms), there was poor correlation between the extent of disease predicted from the Tl-201 images and the findings at arteriography. It is concluded that although stress Tl-201 myocardial imaging is a useful method for the noninvasive diagnosis of coronary artery disease, it cannot be relied upon to predict the number of abnormal vessels.

Comparison of enalapril versus captopril on left ventricular function and survival three months after acute myocardial infarction (the "PRACTICAL" study).

Left ventricular (LV) function and survival can be improved with captopril when initiated later than 24 hours after acute myocardial infarction. Animal studies suggest additional benefits may be obtained with earlier initiation of angiotensin-converting enzyme (ACE) inhibitors. The effects on LV function of captopril and enalapril initiated within 24 hours of myocardial infarction were studied. Two hundred twenty-five patients with acute myocardial infarction were enrolled within 24 hours of the onset of chest pain. They were randomized to receive either captopril 25 mg three times daily, enalapril 5 mg three times daily, or placebo. LV ejection fraction (EF) and volumes were measured by radionuclide ventriculography at baseline during treatment and at 3 months after a 3-day withdrawal from therapy. The ACE inhibitor group had a significant increase in EF (45 +/- 1 to 47 +/- 1%; p = 0.005) and significantly attenuated LV dilatation compared with results in the placebo group (175 +/- 6 to 189 +/- 7 ml in the placebo group vs 168 +/- 4 to 172 +/- 4 ml in the ACE inhibitor group; p = 0.051 for LV end-diastolic volume; and 99 +/- 6 to 108 +/- 7 ml in the placebo group vs 94 +/- 3 to 94 +/- 4 ml; p = 0.026 for LV end-systolic volume). The beneficial effects of ACE inhibitor therapy on LV function were observed irrespective of the degree of initial LV dysfunction and were comparable in both the captopril and enalapril groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged stress-induced elevation in plasma corticosterone during pregnancy in the rat: implications for prenatal stress studies.

Experiments were conducted to test the hypothesis that exposure to uncontrollable stress during pregnancy results in a heightened elevation of plasma glucocorticoids. Rats were exposed to uncontrollable electric tail shocks every other day during the 3 weeks of pregnancy. Plasma corticosterone concentrations in stressed dams increased significantly from gestation days 4 to 20. Importantly, this increase in plasma corticosterone occurred 24- and 48-h after exposure to stress suggesting a prolonged elevation in stress-induced glucocorticoid secretion. In addition, the stress-induced rise in plasma corticosterone was accompanied by a significant decrease in maternal levels of corticosteroid binding globulin which suggests increased circulating levels of free corticosterone. Significant stress-induced elevations in plasma corticosterone also occurred in fetuses that were examined on gestation day 20. Furthermore, a significant positive correlation was found between maternal and fetal plasma corticosterone. Results demonstrate that repeated exposure to uncontrollable stress increases plasma concentrations of glucocorticoids throughout pregnancy. In the unbound state, corticosterone may be highly effective in producing alterations in brain development of offspring. These data have important implications for understanding the process underlying the effects of prenatal stress.

Monitoring universal precautions: a new assessment tool.

OBJECTIVES: Two pilot studies were conducted to produce efficacy data on an observational tool designed to assess the use of Universal Precautions (UP) in patient care settings. The instrument addresses barrier precautions, hand-washing, handling of sharps, and avoidance of unprotected mouth to mouth resuscitation. DESIGN: The Universal Precautions Assessment Tool was submitted to a panel of 3 experts to establish consensual validity. It was pilot tested by 2 simultaneous observers to establish interrater reliability. SETTING: Pilot Study I was conducted in 3 different units within a 100-bed U.S. Army hospital. Pilot Study II was conducted in the emergency department of a large university-based hospital. PARTICIPANTS: Subjects observed were registered nurses providing acute patient care. RESULTS: Two simultaneous raters calculated UP compliance rates of 76.4% and 78.6%, respectively, for 9 nurses in Pilot Study I, and 62% and 65%, respectively, for 5 nurses in Pilot Study II. The intraclass correlation coefficient for the raters' scores in Pilot Study I was 0.992 with a 95% confidence interval (0.979, 0.997). Consensual validity was established. CONCLUSIONS: The instrument has acceptable interrater reliability under the conditions used. Limitations to use include the possibility of a Hawthorne effect and the fact that assessing proper implementation of UP occasionally relies on a "judgment call" by the observer. With test conditions adjusted to minimize these limitations and with proper consideration of sample size, the tool can be used by researchers and by monitors of hospital quality control to measure UP compliance of caregivers individually or collectively.

Fear-potentiated startle in two strains of inbred mice.

The fear-potentiated startle paradigm has been used with great success to examine conditioned fear in both rats and humans. The purpose of this study was to examine fear-potentiated startle in inbred mice. One-month-old C57BL/6J (C57) and DBA/2J (DBA) mice were given tone + foot shock training trials. The amplitude of the acoustic startle reflex was measured in the presence and absence of the tone both before and after training. Both strains showed fear-potentiated startle after training as evidenced by larger startle amplitudes in the presence of the tone than in its absence. However, the magnitude of fear-potentiated startle was greater in DBA mice than in C57 mice. These results not only demonstrate fear-potentiated startle in mice for the first time but also suggest that fear-potentiated startle can be influenced by characteristics of the mouse strain.

Fear-potentiated startle, but not prepulse inhibition of startle, is impaired in CREBalphadelta-/- mutant mice.

Fear-potentiated startle was assessed in mice with a targeted disruption of the alpha and delta isoforms of the transcription factor cAMP response element binding protein (CREB) 24 hr after 5 tone + shock training trials. Whereas wild-type mice showed fear-potentiated startle that persisted up to 45 days after training, CREBalphadelta-/- mice failed to show fear-potentiated startle. However, CREBalphadelta-/- and wild-type mice had similar startle amplitudes and similar magnitudes of prepulse inhibition of startle, suggesting that CREBalphadelta-/- mice have no obvious sensory or motor deficits. These results add to the literature indicating that CREB-activated transcription plays a critical role in the formation of long-term memory and illustrate the utility of the fear-potentiated startle paradigm for assessing cognition in genetically altered mice.

Bing de ling, a Chinese herbal formula, stimulates multifaceted immunologic responses in mice.

Bing de ling is a Chinese herbal formula most commonly used in complementary medical settings against viral disorders. We have found that bing de ling potentiates upregulation of immune activity when administered to mice in dosages proportional to those used clinically. These mice demonstrated significant elevation of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production in splenocytes and enhancement of macrophage, natural killer cell, and lymphokine-activated killer cell cytotoxicity. These data are consistent with bing de ling's clinically observed efficacy against viruses and identify the formula as a promising candidate for clinical trials against diverse diseases that may respond to increased immunologic activity.

Review of infection control literature 1973 through 1981.

A total of 446 manuscripts published between 1973 and 1981 were selected for analysis in this review of the literature. More than half of these articles used research designs, with presentations of epidemic outbreaks the single greatest category. The overall category of surveillance was addressed in some way in almost three fourths of all manuscripts reviewed. Some aspect of ICP role components was the topic of about 10% (n = 55) of the total sample. Physicians were credited as first authors in 56.7% of the articles. It was not possible to determine the exact extent of ICP authorship because credentials varied widely. Finally, a cursory effort was undertaken to quantify numbers of articles that might have met study criteria for inclusion in years after 1981. That effort confirmed the investigators' suspicion that the yearly total of published manuscripts continued to escalate, as did the number of specialty journals in infection control.