Sub-ppm level high energy resolution fluorescence detected X-ray absorption spectroscopy of selenium in articular cartilage.

The speciation of highly-diluted elements by X-ray absorption spectroscopy in a diverse range of materials is extremely challenging, especially in biological matrices such as articular cartilage. Here we show that using a high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) technique coupled to an array of crystal analyzers, selenium speciation down to 400 ppb (µg kg-1) within articular cartilage can be demonstrated. This is a major advance in the speciation of highly-diluted elements through X-ray absorption spectroscopy and opens new possibilities to study the metabolic role of selenium and other elements in biological samples.

Satellite cell-mediated breast muscle regeneration decreases with broiler size.

Satellite cells (SCs) reside between the sarcolemma and basal lamina of muscle fibers and are the primary contributor of DNA for post-hatch muscle growth and repair. Alterations in SC content or properties by intrinsic and extrinsic factors can have detrimental effects on muscle health and function, and ultimately meat quality. We hypothesized that disrupted SC homeostasis may account in part for the increased breast myopathies observed in growing broilers. To test this hypothesis, we selected broilers with different body weights at comparable ages and studied SC characteristics in vitro and in vivo. Data shows that SC numbers in the breast muscles decrease (P < 0.001) and their inherent abilities to proliferate and differentiate diminish (P < 0.001) with age and size. Further, when breast muscle is presented with an insult, muscle of larger broilers regenerates more slowly than their smaller, age-matched counterparts arguing that SC quality changes with size and age. Together, our studies show that birds with greater muscle hypertrophy have less SCs with diminished ability to function, and suggest that aggressive selection for breast growth in broilers may exhaust SC pools when birds are grown to heavier processing weights. These findings provide new insights into a possible mechanism leading to breast myopathies in the poultry industry and provide targets for mitigating adverse fresh breast quality.

Anaerobic Chemolithotrophic Growth of the Haloalkaliphilic Bacterium Strain MLMS-1 by Disproportionation of Monothioarsenate.

A novel chemolithotrophic metabolism based on a mixed arsenic-sulfur species has been discovered for the anaerobic deltaproteobacterium, strain MLMS-1, a haloalkaliphile isolated from Mono Lake, California, U.S. Strain MLMS-1 is the first reported obligate arsenate-respiring chemoautotroph which grows by coupling arsenate reduction to arsenite with the oxidation of sulfide to sulfate. In that pathway the formation of a mixed arsenic-sulfur species was reported. That species was assumed to be monothioarsenite ([H2As(III)S(-II)O2](-)), formed as an intermediate by abiotic reaction of arsenite with sulfide. We now report that this species is monothioarsenate ([HAs(V)S(-II)O3](2-)) as revealed by X-ray absorption spectroscopy. Monothioarsenate forms by abiotic reaction of arsenite with zerovalent sulfur. Monothioarsenate is kinetically stable under a wide range of pH and redox conditions. However, it was metabolized rapidly by strain MLMS-1 when incubated with arsenate. Incubations using monothioarsenate confirmed that strain MLMS-1 was able to grow (µ = 0.017 h(-1)) on this substrate via a disproportionation reaction by oxidizing the thio-group-sulfur (S(-II)) to zerovalent sulfur or sulfate while concurrently reducing the central arsenic atom (As(V)) to arsenite. Monothioarsenate disproportionation could be widespread in nature beyond the already studied arsenic and sulfide rich hot springs and soda lakes where it was discovered.

Circulating levels of pro-atrial natriuretic peptide in lower respiratory tract infections.

OBJECTIVE: To analyse the mid region of plasma N-terminal pro-atrial natriuretic peptide (MR-proANP) levels in patients with lower respiratory tract infections to evaluate its prognostic use for the severity of disease and outcome. DESIGN: Prospective observational study. Setting. Emergency department of a university hospital. SUBJECTS: A total of 545 consecutive patients with lower respiratory tract infections and 50 healthy controls. Interventions. MR-proANP was measured in serum from all patients using a new sandwich immunoassay. RESULTS: MR-proANP levels (median [IQR], in pmol L(-1)) were significantly higher in patients with lower respiratory tract infections when compared with controls (138.0 [74.1-279.0] vs. 72.7 [62.5-89.5], P < 0.001), with highest levels in patients with community-acquired pneumonia (CAP). MR-proANP, but not C-reactive protein (CRP) levels, gradually increased with increasing severity of CAP, classified according to the pneumonia severity index (PSI) score (P < 0.001). On admission, MR-proANP levels were significantly higher in nonsurvivors when compared with survivors (293.0 [154.0-633.0] vs. 129.0 [71.4-255.0], P < 0.001). In a receiver operating characteristic (ROC) analysis for the prediction of survival of patients with CAP the area under the ROC curve (AUC) for MR-proANP was 0.69, similar when compared with the PSI (AUC 0.74, P = 0.31), and better when compared with other biomarkers, i.e. procalcitonin (AUC 0.57, P = 0.08), CRP (AUC 0.52, P = 0.02), and leucocyte count (AUC 0.56, P = 0.07). CONCLUSIONS: MR-proANP levels are increased in lower respiratory tract infections, especially in CAP. Together with other clinical, radiographic and laboratory findings, MR-proANP levels might be helpful for the risk stratification in CAP.

Hierarchical Bayesian model for prevalence inferences and determination of a country's status for an animal pathogen.

Certification that a country, region or state is "free" from a pathogen or has a prevalence less than a threshold value has implications for trade in animals and animal products. We develop a Bayesian model for assessment of (i) the probability that a country is "free" of or has an animal pathogen, (ii) the proportion of infected herds in an infected country, and (iii) the within-herd prevalence in infected herds. The model uses test results from animals sampled in a two-stage cluster sample of herds within a country. Model parameters are estimated using modern Markov-chain Monte Carlo methods. We demonstrate our approach using published data from surveys of Newcastle disease and porcine reproductive and respiratory syndrome in Switzerland, and for three simulated data sets.

CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA.

Human immunodeficiency virus type 1 (HIV-1) Tat interacts with cyclin T1 (CycT1), a regulatory partner of CDK9 in the positive transcription elongation factor (P-TEFb) complex, and binds cooperatively with CycT1 to TAR RNA to recruit P-TEFb and promote transcription elongation. We show here that Tat also stimulates phosphorylation of affinity-purified core RNA polymerase II and glutathione S-transferase-C-terminal-domain substrates by CycT1-CDK9, but not CycH-CDK7, in vitro. Interestingly, incubation of recombinant Tat-P-TEFb complexes with ATP enhanced binding to TAR RNA dramatically, and the C-terminal half of CycT1 masked binding of Tat to TAR RNA in the absence of ATP. ATP incubation lead to autophosphorylation of CDK9 at multiple C-terminal Ser and Thr residues, and full-length CycT1 (amino acids 728) [CycT1(1-728)], but not truncated CycT1(1-303), was also phosphorylated by CDK9. P-TEFb complexes containing a catalytically inactive CDK9 mutant (D167N) bound TAR RNA weakly and independently of ATP, as did a C-terminal truncated CDK9 mutant that was catalytically active but unable to undergo autophosphorylation. Analysis of different Tat proteins revealed that the 101-amino-acid SF2 HIV-1 Tat was unable to bind TAR with CycT1(1-303) in the absence of phosphorylated CDK9, whereas unphosphorylated CDK9 strongly blocked binding of HIV-2 Tat to TAR RNA in a manner that was reversed upon autophosphorylation. Replacement of CDK9 phosphorylation sites with negatively charged residues restored binding of CycT1(1-303)-D167N-Tat, and rendered D167N a more potent inhibitor of transcription in vitro. Taken together, these results demonstrate that CDK9 phosphorylation is required for high-affinity binding of Tat-P-TEFb to TAR RNA and that the state of P-TEFb phosphorylation may regulate Tat transactivation in vivo.

Localization of GABA-like immunoreactivity in the central nervous system of Aplysia californica.

Gamma-aminobutyric acid (GABA) is present in the central nervous system of Aplysia californica (Gastropoda, Opisthobranchia) where its role as a neurotransmitter is supported by pharmacological, biochemical, and anatomical investigations. In this study, the distribution of GABA-immunoreactive (GABAi) neurons and fiber systems in Aplysia was examined by using wholemount immunohistochemistry and nerve backfill methods. GABAi neurons were located in the buccal, cerebral, and pedal ganglia. Major commissural fiber systems were present in each of these ganglia, whereas more limited fiber systems were observed in the ganglionic connectives. Some of the interganglionic fibers were found to originate from two unpaired GABAi neurons, one in the buccal ganglion and one in the right pedal ganglion, each of which exhibited bilateral projections. No GABAi fibers were found in the nerves that innervate peripheral sensory, motor, or visceral organs. Although GABAi cells were not observed in the pleural or abdominal ganglia, these ganglia did receive limited projections of GABAi fibers originating from neurons in the pedal ganglia. The distribution of GABAi neurons suggests that this transmitter system may be primarily involved in coordinating certain bilateral central pattern generator (CPG) systems related to feeding and locomotion. In addition, the presence of specific interganglionic GABAi projections also suggests a role in the regulation or coordination of circuits that produce components of complex behaviors.

Uptake mechanism of technetium-99m-d, 1-HMPAO in cell cultures of the dissociated postnatal rat cerebellum.

The accumulation and retention mechanisms of 99mTc-d, 1-hexamethylpropyleneamine oxime (99mTc-d, 1-HMPAO) were investigated in cultures of the dissociated rat cerebellum. Our experiments indicate a linear dependency of the uptake on incubation time and on the concentration of the radioligand. Upon chloroform extraction and distribution between the lipophilic and the hydrophilic phases, we located 69.1% of the retained radioactivity in the hydrophilic phase, 24.1% in a bound state and 6.8% in the lipophilic phase. The water-soluble, unbound radioactive contents of the cultures were identified as 99mTcO4- by HPLC analysis. Treatment of cultures with diethyl maleate (DEM) inhibited the accumulation of radioactivity along with a reduction of the GSH contents of the cultures. However, even in the absence of GSH, significant amounts of radioactivity were accumulated. DEM reduced the radioactive contents of cultures predominantly by diminishing the aqueous phase of the chloroform-extracted material. By contrast, the metabolic state, manipulated by treating the cultures with oligomycin B or 2,4-dinitrophenol, had no significant effect on the accumulation of radioactivity. Our experiments suggest two major mechanisms for the retention of radioactivity following the exposure of neuronal tissue to 99mTc-d, 1-HMPAO: Conversion of the lipophilic complex to the hydrophilic product, 99mTcO4-, and binding to non-diffusible cell components.

Technetium-99m-d,1-hexamethylpropyleneamine oxime (HMPAO) uptake and glutathione content in brain tumors.

Technetium-d, HMPAO SPECT was performed in 70 patients suffering from intracerebral tumors of various histologic types (glioma n = 30, meningioma n = 19, metastases n = 10, angioma n = 3, neuroma n = 2, lymphoma n = 2, neurocytoma n = 1, epidermoid n = 1, gliosis n = 1, cholesteatoma n = 1). Tumor classification was histologically verified in all subjects except in two cases with inoperable angiomas. SPECT was performed under resting state conditions with a dual-head rotating camera (SIEMENS ZLC 37) following intravenous injection of 18-25 mCi 99mTc-d, 1-HMPAO. Regional tracer deposit was expressed in terms of a cerebellar index (CBI). Significantly higher regional HMPAO uptake was found in meningiomas when compared with gliomas of different malignancy (ANOVA p less than 0.05). Within gliomas, regional uptake increased with malignancy (n.s.). In 23 patients, a total of 32 tumor specimens were obtained for histochemical analysis of glutathione (GSH) content using high-pressure liquid chromatography. A significant correlation (least square method, p less than 0.001) between CBIs and GSH values was found, supporting the hypothesis that GSH is the predominant factor for the conversion of the lipophilic complex to hydrophilic derivates.

A contribution to classification of hallucinations.

Proceeding from the hypothesis that auditory hallucinations in psychotic patients have another biological basis than hallucinations provoked by hypnotic suggestion in healthy persons, we performed a symptom-comparative study by means of 99mTc-HMPAO single photon emission computed tomography. The results confirm the importance of pathogenesis in symptom-oriented psychiatric research.

The cerebral localization of neuropsychological impairment in Alzheimer's disease: a SPECT study.

Twenty-three patients with a clinical diagnosis of Alzheimer's disease were examined with a set of neuropsychological tests and with 99mTc-hexamethyl-propyleneamineoxime (HMPAO) single photon emission computed tomography (SPECT). Correlations between test results and indices of regional HMPAO distribution were analysed by multidimensional scaling (MDS). Test results covaried positively with relative HMPAO uptake of frontal, inferior parietal and superior temporal regions but not, or in a negative way, with the remainder of the regions. When only positive correlations were analysed, MDS suggested two dimensions of organization: one was related to a dichotomy between frontal and temporo-parietal regions. The relationship of test results to this dimension was largely consistent with common neuropsychological knowledge. A second, less stringent dimension of organization opposed right and left hemisphere regions. The ordering of test results with respect to this dimension was only partly consistent with what is known about the lateralization of neuropsychological deficits from the study of localized brain lesions. The possibility is considered that these inconsistencies may reflect the effect of disproportionally severe involvement of extended cortical systems which modulates the sequels of localized brain damage.

Quantification of regional cerebral blood flow with IMP-SPECT. Reproducibility and clinical relevance of flow values.

Single-photon emission computed tomography with N-isopropyl[123I]-p-iodoamphetamine (IMP-SPECT) was performed in 14 normal volunteers (seven men and seven women aged 25.1 +/- 5.3 years) and 29 patients with cerebrovascular disease (18 men and 11 women aged 54.1 +/- 13.7 years). The fluid microsphere model was used to estimate cerebral blood flow (CBF). Normal subjects were scanned twice, 1 week apart, to determine the reproducibility of the CBF estimates. Hemispheric blood flow (hCBF) was calculated as the mean of regional cerebral blood flow (rCBF) values in 16 gray matter regions per hemisphere. In normal subjects mean hCBF was 68 ml/100 g/min. The highest rCBF was found in the occipital cortex, followed by the frontal, temporal, and parietal cortexes. CBF values were reproducible (p less than 0.001 except the right thalamic region, where p less than 0.01). Intraindividual variation ranged between 0.3% and 15%. Women exhibited significantly higher (16%, p less than 0.02) CBF than men. Patients were subdivided into groups with reversible (n = 19) and persistent (n = 10) symptoms. Significant hCBF differences between the affected and the contralateral hemispheres were recorded only in the group with reversible symptoms (p less than 0.005), whereas the group with persistent symptoms showed a significant bilateral decrease of hCBF compared with normal subjects and patients with reversible symptoms. Focal CBF was significantly lower in patients with completed stroke than in patients with transient symptoms (p less than 0.001). Our results indicate that IMP-SPECT can be used for the routine estimation of CBF in normal and pathologic states.

Regional cerebral blood flow patterns in visual imagery.

Regional cerebral blood flow patterns were investigated by means of single photon emission computerized tomography in subjects who solved cognitive tasks which either did or did not require the use of visual imagery. In a first experiment judging the correctness of visual imagery sentences like "a grapefruit is bigger than an orange" led to increases of regional activity in inferior temporal and in the left inferior-occipital region when compared to blood flow patterns elicited by judgements about low imagery sentences or by responding differentially to "yes" and "no". Motor imagery sentences did not cause such an increase. In a second experiment a condition in which visual images were used for counting the corners of letters was compared to a condition in which subjects internally rehearsed the alphabet. The only difference concerned the inferior frontal regions which showed higher activity in rehearsing the alphabet. However, activity in inferior temporal and inferior occipital regions showed a positive correlation to the self-rated vividness of the visual images in the corner counting condition. The results of both experiments yield converging evidence that visual imagery is selectively related to activity of inferior-temporal and occipital regions. They thus support the hypothesis that the cerebral correlate of visual imagery is different from that of non-imaginal thinking.

Regional brain function in hallucinations: a study of regional cerebral blood flow with 99m-Tc-HMPAO-SPECT in patients with auditory hallucinations, tactile hallucinations, and normal controls.

From the supposition that there exists a possible connection between certain psychopathological symptoms, and/or syndromes (e.g., hallucinations) and regional cerebral dysfunction, patients suffering from auditory and tactile hallucinations were investigated, in a symptom-oriented study, using the method of technetium-99m-Hexamethyl-propylenamine Oxime (99m-Tc-HMPAO)-Single Photon Emission Computed Tomography (SPECT) and compared with normal controls. The results support Jackson's hypothesis to the effect that hallucinatory phenomena will primarily occur when the normally inhibitive influence of the upper cortical centers over the lower brain structures diminishes, resulting in relative hyperactivity in the basal regions. In addition to the brain activity-changes generally observed in hallucinating patients, it was possible to identify regional cerebral blood flow (rCBF)-distribution patterns characteristic in certain forms of hallucinatory phenomena, i.e., a significant increase of activity in the hippocampal regions (including hippocampus, parahippocampus, and amygdala) only in patients with auditory hallucinations, and a significant reduction of rCBF in the inferior temporal regions in patients with tactile hallucinations.

Single photon emission computerized tomography during and between seizures.

In three patients, patterns of brain activity were measured by 99mTc-hexamethyl-propyleneamineoxime (99mTc-HM-PAO) brain SPECT (single photon emission computerized tomography) in ictal and interictal states. Increased relative blood flow indicated the focus of partial seizures, its spreading to adjacent cortical regions and to distant brain structures via neuronal pathways. Ictal patterns of regional cerebral blood flow (rCBF) were in agreement with clinical symptomatology. Successive SPECT studies were performed after 3-7 days in the absence of electroencephalographic and clinical signs of seizures but still revealed increased relative blood flow in the focus of the seizures. SPECT studies, performed 2-6 weeks after the last clinically observable seizures, demonstrated the transition from increased to decreased relative blood flow in the focus of the seizures. In one patient, the EEG was complementary to and corresponded with the rCBF patterns in the ictal state. However, the dynamics of interictal changes could only be assessed by brain SPECT.

DC-potential shifts and regional cerebral blood flow reveal frontal cortex involvement in human visuomotor learning.

In the present study, two different physiological parameters were measured to describe brain activity related to visuomotor learning: performance-related DC-potential shifts and regional cerebral blood flow (rCBF) by Tc-99m HMPAO brain SPECT (Single Photon Emission Computerized Tomography). Visuomotor learning was required in a conflicting situation: a visual target moved on a screen and had to be tracked by moving the right hand in an inverted fashion (IT), e.g. movements of the target to the right side required hand movement to the left and vice versa. Compared to a normal, non-inverted control task (T), IT required the development of a novel motor program and the prevention of returning to routine direct pursuit. These additional demands in IT caused a relative hyperperfusion in regions including the middle frontal gyri, frontomedial cortex (including the supplementary motor area, SMA), right basal ganglia (caudate-putamen) and left cerebellum. Correlations of rCBF values between the middle frontal gyrus and basal ganglia may indicate a functional relation between these two brain structures. Visuomotor performance was accompanied by slow negative DC-potential shifts. In frontal and to a lesser degree in central recordings, amplitudes of DC-negativity were larger in IT than they were in T. This additional frontal negativity covaried with the success of learning. Results substantiate, now using a dual approach, previous suggestions that the frontal lobe plays an important role in visuomotor learning.

Neurophysiological aspects of auditory hallucinations. 99mTc-(HMPAO)-SPECT investigations in patients with auditory hallucinations and normal controls--a preliminary report.

In a symptom-oriented study 17 patients suffering from chronic auditory hallucinations were investigated by means of 99mTc-HMPAO-SPECT and compared with healthy controls. The results confirm the relative frontal hypoactivity in junction with a relative hyperactivity in the basal ganglia and mesial limbic structures in both hemispheres found in a previous pilot study in auditorily hallucinating patients. Our results should fortify the symptom-oriented approach in psychiatric research.

Hexa-methyl-propylene-amine-oxime (HMPAO) single photon emission computed tomography (SPECT) in epilepsy.

Twenty-eight normal volunteers and 74 seizure patients were investigated with hexa-methyl-propylene-amine-oxime (HMPAO) brain single photon emission computed tomography (SPECT). Fifty-four patients suffered from partial seizures and 20 patients had generalized seizures. Indices describing regional tracer distribution (RIs) were calculated in all investigated subjects. Regions whose RI exceeded the mean normal RI +/- 3 SD were defined as abnormally perfused. In normals a significant interhemispheric asymmetry of HMPAO deposition was found, with higher RI values in the right frontal, parietal, temporal and occipital regions. In 59.3% of partial seizure patients abnormal RIs were found. Low RIs were detected predominantly in the frontal and temporal cortex, while elevated RIs were observed predominantly in the anterior basal ganglia. Only in 20% of the cases with generalized seizures, abnormal RIs were found. In one patient an ictal and 3 follow-up SPECT studies were obtained. Here SPECT results indicated transient rCBF changes between the ictal and seizure free state. EEG and SPECT foci were ipsilaterally located in 69.2% of the partial seizure cases. The results indicate that HMPAO brain SPECT is valuable for the detection of rCBF abnormalities in seizure patients and that patients with partial seizures have mostly several abnormally perfused areas in their brains.

Initial experience with technetium-99m HM-PAO brain SPECT.

Technetium-99m hexamethylpropyleneamineoxime ([99mTc]HM-PAO) brain single photon emission computed tomography (SPECT) was performed with a dual head rotating scintillation camera. Normal tracer distribution and side/side differences of counting rates were obtained in 11 healthy volunteers. Almost stable gray/white matter ratios were found (1.97-2.1) in one normal subject during 2 hr after tracer administration. Eighty-three investigated patients had the following diagnoses (in parentheses is percent of positive findings in each group): cerebral vascular disease 18 (94.4%), epilepsy 23 (82.6%), extrapyramidal disorders 8 (100%), dementia 12 (100%), headache 11 (63.6%), psychiatric disorders 11 (27.3%). In addition, SPECT was performed in 28 male volunteers during motor or visual imagery tasks and a significant increase (p = 0.035) of relative tracer deposition was observed in the left inferior occipital region during visual imagery when compared with motor imagery. The results indicate that [99mTc]HM-PAO SPECT is valuable for demonstrating pathologic and physiologic changes of the brain.