IN VIVO LABELLING OF ACETYL-ASPARTYL PEPTIDES IN MOUSE BRAIN FROM INTRACRANIALLY AND INTRAPERITONEALLY ADMINISTERED ACETYL-l-[U-14C] ASPARTATE

Abstract— Following intracranial and intraperitoneal injection of acetyl- l -[U-¹⁴C]aspartate into mice about 5% and 0.7% of the radioactivity, respectively, was recovered from the brain after 30 min.
On chromatographic separation of the cationic and anionic compounds on a Dowex 50 column, the former fraction contained about 60% of the radioactivity, predominantly as labelled aspartate and glutamate. The anionic compounds, containing 20% of the labelled compounds, were fractionated in several chromatographic systems and resolved into a great variety of labelled peptidic compounds of which five acetyl-[U¹⁴-C]aspartyl peptides, containing two to four amino acids, were purified. One of these, acetyl-aspartyl glutamine, has not previously been found in brain.     

Phosphate-Activated Glutaminase and Mitochondrial Glutamine Transport in the Brain

A review of the properties of purified and tissue bound phosphate activated glutaminase (PAG) in brain and kidney (pig and rat) is presented, based on kinetic, electron microscopic and immunocytochemical studies. PAG is a mitochondrial enzyme and two pools can be separated, a soluble and membrane associated one. Intact mitochondria appear to express PAG accessible only to the outer phase of the inner mitochondrial membrane. This PAG has properties similar to that of the membrane fraction and polymeric form of purified enzyme. PAG in the soluble fraction has properties similar to that of the monomeric form of purified enzyme and is assumed to be dormant due to the high matrix concentration of the inhibitor glutamate. A hypothetical model for the localization of PAG in the mitochondria is presented. The activity of PAG in vivo is assumed to be regulated by cytosolic glutamate and other compounds, that affect the activation by phosphate. Glutamine is transported into brain and kidney mitochondria by a protein catalyzed energy requiring process, which may be mediated by more than one protein. There is no correlation between glutamine hydrolysis and transport.     

Seasonal Dynamics of Atlantic Herring (Clupea harengus L.) Populations Spawning in the Vicinity of Marginal Habitats

Gillnet sampling and analyses of otolith shape, vertebral count and growth indicated the presence of three putative Atlantic herring (Clupea harengus L.) populations mixing together over the spawning season February–June inside and outside an inland brackish water lake (Landvikvannet) in southern Norway. Peak spawning of oceanic Norwegian spring spawners and coastal Skagerrak spring spawners occurred in March–April with small proportions of spawners entering the lake. In comparison, spawning of Landvik herring peaked in May–June with high proportions found inside the lake, which could be explained by local adaptations to the environmental conditions and seasonal changes of this marginal habitat. The 1.85 km² lake was characterized by oxygen depletion occurring between 2.5 and 5 m depth between March and June. This was followed by changes in salinity from 1–7‰ in the 0–1 m surface layer to levels of 20–25‰ deeper than 10 m. In comparison, outside the 3 km long narrow channel connecting the lake with the neighboring fjord, no anoxic conditions were found. Here salinity in the surface layer increased over the season from 10 to 25‰, whereas deeper than 5 m it was stable at around 35‰. Temperature at 0–5 m depth increased significantly over the season in both habitats, from 7 to 14°C outside and 5 to 17°C inside the lake. Despite differences in peak spawning and utilization of the lake habitat between the three putative populations, there was an apparent temporal and spatial overlap in spawning stages suggesting potential interbreeding in accordance with the metapopulation concept.     

Molecular simulations as a tool for selection of kinetic hydrate inhibitors

Natural gas hydrates are ice-like structures in which water molecules form a cage around gas molecules. They have been a problem in the petroleum industry. The heavy cost of alcohol and glycol injections needed to suppress the formation of hydrates has spurred an interest in so-called “kinetic inhibitors”, able to slow down the hydrate formation rather than prevent it. An earlier work (Kvamme, B. et al. 1997, Mol. Phys., 90, p. 979) proposed a simulation-based scheme to assess the comparative performance of prospective inhibitors and select the best candidates for experimental testing. In this work, we employed molecular dynamics simulations to test several kinetic inhibitors in a multiphase water–hydrate system with rigid hydrate interface. In addition, a long-scale run was implemented for a system where the hydrate was free to melt and reform. Our conclusion that PVCap inhibitor will outperform PVP as a kinetic hydrate inhibitor is supported by experimental data. We demonstrate that numerical experiments can be a valuable tool for selecting kinetic inhibitors as well as provide insight into mechanisms of kinetic inhibition and hydrate melting and reformation.     

Zinc Deficiency Is Common in Several Psychiatric Disorders

Background

Mounting evidence suggests a link between low zinc levels and depression. There is, however, little knowledge about zinc levels in older persons with other psychiatric diagnoses. Therefore, we explore the zinc status of elderly patients suffering from a wide range of psychiatric disorders.

Methods

Clinical data and blood samples for zinc analyzes were collected from 100 psychogeriatric patients over 64 of age. Psychiatric and cognitive symptoms were assessed using the Montgomery and Aasberg Depression Rating Scale, the Cornell Scale for Depression in Dementia, the Mini-Mental State Examination, the Clockdrawing Test, clinical interviews and a review of medical records. In addition, a diagnostic interview was conducted using the Mini International Neuropsychiatric Interview instrument. The prevalence of zinc deficiency in patients with depression was compared with the prevalence in patients without depression, and the prevalence in a control group of 882 older persons sampled from a population study.

Results

There was a significant difference in zinc deficiency prevalence between the control group (14.4%) and the patient group (41.0%) (χ² = 44.81, df = 1, p<0.001). In a logistic model with relevant predictors, zinc deficiency was positively associated with gender and with serum albumin level. The prevalence of zinc deficiency in the patient group was significantly higher in patients without depression (i.e. with other diagnoses) than in patients with depression as a main diagnosis or comorbid depression (χ² = 4.36, df = 1, p = 0.037).

Conclusions

Zinc deficiency is quite common among psychogeriatric patients and appears to be even more prominent in patients suffering from other psychiatric disorders than depression.

Limitations

This study does not provide a clear answer as to whether the observed differences represent a causal relationship between zinc deficiency and psychiatric symptoms. The blood sample collection time points varied in both the control group and the patient group. No data regarding zinc supplementation were collected.     

Vegetational changes and human presence in the low-alpine and subalpine zone in Val Febbraro,upper Valle di Spluga (Italian central Alps), from the Neolithic to the Roman period

An interdisciplinary palaeoecological study in the low-alpine and subalpine zones of Val Febbraro, upper Valle di Spluga (Italy), between 1830 and 2304 m a.s.l., suggests the temporary presence of early Neolithic groups at about 6000 uncal b.p. Evidence for local woodland clearance and charcoal dust were found. Phases of woodland and treeline disturbances, and indications of increased human presence are evident at about 5500, 5100, and 4000 b.p. A marked increase in disturbance, mainly related to pasturing, is dated to the beginning of the Bronze Age. The last major stage of human impact on the vegetation coincides with the Final Bronze phase and the beginning of the Iron Age, with a small temporary reduction during the Roman period. ¹⁴C dated archaeological sites and finds are broadly concordant with the phases of human impact on the vegetation. A summary figure is presented. No locally significant climatic changes have been traced during the last 6000 years, and if present, they are probably overshadowed by the vegetational changes caused by human activity. Communicated by F. Bittmann     

Comparative responses to endothelin-2 and sarafotoxin 6b in the pulmonary vascular bed of the cat

Pulmonary vascular responses to endothelin-2 and sarafotoxin 6b were investigated in the feline pulmonary vascular bed under natural flow and constant flow conditions. Injections of endothelin-2 and sarafotoxin 6b in a dose of 0.3 nmol/kg iv increased pulmonary arterial and left atrial pressures and cardiac output, and caused a biphasic change in calculated pulmonary vascular resistance. Endothelin-2 caused a biphasic change in systemic arterial pressure, while sarafotoxin 6b only decreased arterial pressure. Under constant flow conditions in the intact-chest cat, injections of endothelin-2 and sarafotoxin 6b in doses of 0.1-1 nmol into the perfused lobar artery increased lobar arterial pressure in a dose-related manner but were less potent than the thromboxane A2 mimic, U46619. An ET analog with only the Cys1-Cys15 disulfide bond and an amidated carboxy terminus had no significant activity in the pulmonary vascular bed. The present data show that endothelin-2 and sarafotoxin 6b have significant vasoconstrictor activity in the pulmonary vascular bed of the cat.     

Evidence for Compartmentation of Synaptosomal Phosphate-Activated Glutaminase

Abstract: Phosphate-activated glutaminase (EC 3.5.1.2) in synaptosomal preparations is inhibited 40–60% by the sulphydryl group reagent N -ethylmaleimide (NEM), forming the basis for distinction between NEM-sensitive and NEM-insensitive glutaminases. The NEM effect cannot be explained by differential effects on distinct glutaminases because other glutaminases have not been detected, and the synaptosomal glutaminase activity can be fully accounted for by the activity of phosphate-activated glutaminase. By fractionation of mitochondria isolated from synaptosomal preparations, which are preincubated with and without NEM, both NEM-sensitive and NEM-insensitive glutaminases are found to be localized to the inner mitochondrial membrane. Variations in pH (7.0–7.6) and the phosphate concentration (5–10 mM) affect chiefly NEM-sensitive glutaminase, demonstrating that this glutaminase may be subject to regulation by compounds in the cytosol having restricted permeability to the inner mitochondrial membrane. Since p -hydroxymercuribenzoate, which is known to be impermeable to the inner mitochondrial membrane, inhibits glutaminase similarly to NEM, phosphate-activated glutaminase is assumed to be compartmentalized within the inner mitochondrial membrane. Thus, NEM-sensitive glutaminase is localized to the outer face and NEM-insensitive glutaminase to the inner region of this membrane and probably also to the matrix region.     

PURIFICATION OF PHOSPHATE-DEPENDENT PIG BRAIN GLUTAMINASE

Abstract— A procedure for preparing highly purified phosphate-activated glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris-HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate-borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.     

Uptake and release for glutamine and glutamate in a crude synaptosomal fraction from rat brain

[14C]Glutamine uptake in a crude synaptosomal (P2) fraction, (representing the sum of [¹⁴C]glutamine accumulated and [¹⁴C]glutamate formed by hydrolysis), is distinct from glutamate uptake. Glutamine uptake is Na⁺-independent and unaffected by the Na⁺–K⁺-ATPase inhibitor ouabain, whereas glutamate uptake is Na⁺-dependent and inhibited by ouabain. The uptake of both glutamine and glutamate is unaffected by the gamma-glutamyltransferase inhibitor, Acivicin. This indicates that glutamine uptake is not mediated by a carrier, as distinct from that of glutamate, and also not linked to gamma-glutamyl-transferase. Na⁺ affects the distribution of glutamine-derived glutamate by increasing the synaptosomal content and reducing that of the medium. When glutamate release from synaptosomes preloaded with [¹⁴C]glutamate is measured by superfusion technique in order to prevent reuptake, Na⁺ has been found to inhibit release in a non-depolarizing medium (Ringer buffer with no Ca²⁺) of the [¹⁴C]glutamate as well as of endogenous glutamate. The specific activity of the [¹⁴C]glutamine-derived glutamate in the incubation medium is much higher than that in the synaptosomes, indicating that there exists a readily releasable pool of newly formed glutamate in addition to another pool. The latter glutamate pool is partially reduced by Na⁺.Special Issue Dedicated to Dr. Abel Lajtha.