31P magnetic resonance spectroscopy in the frontal lobe of major depressed patients

Most research with 31P-magnetic resonance spectroscopy (31P-MRS) in affective disorders has been done in the field of bipolar disturbances. Reduced frontal and temporal lobe phosphomonoester (PME) concentrations were measured in the euthymic state, whereas increased values were found in the depressed state. In bipolar-II patients reduced phosphocreatine (PCr) concentrations were reported in the euthymic, depressed, and manic state. The aim of the present study was to explore whether PME and PCr were also altered in the frontal lobe of major depressed, unipolar patients. Therefore, we used 31P-MRS to investigate the relative phospholipid and high-energy phosphate concentrations in the frontal lobe of 14 unipolar patients, mostly medicated, and 8 age-matched controls. We found increased PME and decreased ATP values. Other 31P-MRS parameters were not different in both groups. Phosphomonoester percentages correlated negatively with the degree of depression. Thus, the main alterations found in bipolar depressed patients could also be demonstrated in unipolar depressed patients. The results are discussed with regard to disturbed phospholipid and intracellular high-energy phosphate metabolism in depressed patients.     

Crystal growth in dental enamel: the role of amelogenins and albumin

Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (< 21 KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However, synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage, permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.     

Identification of benz(othi)azepine-binding regions within L-type calcium channel alpha1 subunits

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.     

The role of angiotensin AT1 receptors in the diuretic, natriuretic, kaliuretic and blood pressure responses induced by angiotensin II activation of the median preoptic nucleus in conscious rats

We determined the effects of two classical angiotensin II (ANG II) antagonists, [Sar1, Ala8]-ANG II and [Sar1, Thr8]-ANG II, and losartan (a nonpeptide and selective antagonist for the AT1 angiotensin receptors) on diuresis, natriuresis, kaliuresis and arterial blood pressure induced by ANG II administration into the median preoptic nucleus (MnPO) of male Holtzman rats weighing 250-300 g. Urine was collected in rats submitted to a water load (5% body weight) 1 h later. The volume of the drug solutions injected was 0.5 microliters over 10-15 s. Pre-treatment with [Sar1, Ala8]-ANG II (12 rats) and [Sar1, Thr8]-ANG II (9 rats), at the dose of 60 ng reduced (13.7 +/- 1.0 vs 11.0 +/0 1.0 and 10.7 +/0 1.2, respectively), whereas losartan (14 rats) at the dose of 160 ng totally blocked (13.7 +/- 1.0 vs 7.6 +/- 1.5) the urine excretion induced by injection o 12 ng of ANG II (14 rats). [Sar1, Ala8]-ANG II impaired Na+ excretion (193 +/- 16 vs 120 +/- 19), whereas [Sar1, Thr8]-ANG II and losartan block Na+ excretion (193 +/- 16 vs 77 +/- 15 and 100 +/- 12, respectively) induced by ANG II. Similar effects induced by ANG II on K+ excretion were observed with [Sar1, Ala8]-ANG II, [Sar1, Thr8]- ANG II, and losartan pretreatment (133 +/- 18 vs 108 +/- 11, 80 +/- 12, and 82 +/- 15, respectively). The same doses as above of [Sar1, Ala8]-ANG II (8 rats), [Sar1, Thr8]-ANG II (8 rats), and losartan (9 rats) blocked the increase in the arterial blood pressure induced by 12 ng of ANG II (12 rats) (32 +/- 4 vs 4 +/- 2, 3.5 +/- 1, and 2 +/- 1, respectively. The results indicate that the AT1 receptor subtype participates in the increases of diuresis, natriuresis, kaliuresis and arterial blood pressure induced by the administration of ANG II into the MnPO.     

Accidental hypothermia: incidence, risk factors and clinical course of patients admitted to hospital

This study was initiated to identify the incidence, risk factors and outcome predictors of patients admitted to hospital in the Netherlands because of accidental hypothermia. Information about these patients was available for study through the National Health Care Data Bank. Between 1987 and 1990, 612 accidental hypothermic patients were admitted: 185 hypothermic patients also suffered from submersion (HYPSUBS), but this was not the case in the remaining 427 patients (HYPNOTSUBS). Patients in the HYPNOTSUBS group were older (average age 55.2 years versus 38.9 years; p < 0.001), remained longer in hospital (average 20.8 days versus 9.2 days; p < 0.001) and had a higher death rate than those in the HYPSUBS group (16.9% versus 5.9%; p < 0.001). In HYPNOTSUBS, increasing age correlated with increases in the length of hospital stay and death rate. This relationship was not found in HYPSUBS. Trauma was the major associated problem in both groups; these patients had the highest death rate (22.8% versus 16.7%; not significant). Death occurred within 2 days in 54% of HYPNOTSUBS non-survivors and 73% of HYPSUB non-survivors. HYPNOTSUBS admitted to university hospitals showed a lower death rate (5.9%) compared with HYPNOTSUBS admitted to non-university hospitals with less than 400 beds (13.4%) or more than 400 beds (21.7%). In contrast, the death rate in HYPSUB was higher in university hospitals (14.3%) than in non-university hospitals with less than 400 beds (5.2%) or more than 400 beds (3.6%). We observed that the incidence of accidental hypothermia is low at 1.1 per 100,000 inhabitants per year. We concluded that HYPNOTSUBS and HYPSUB are different groups of patients with respect to demographic data, risk factors and prognostic factors. Old age is an important unfavourable prognostic factor in HYPNOTSUB but not in HYPSUB. Hypothermia with trauma is an unfavourable combination in both groups. Almost half of the HYPNOTSUBS non-survivors died after more than 2 days. Because body temperature will have returned to normal by then, this must be the result of late complications. Most HYPSUB non-survivors died during the first 2 days, probably as a direct result of the submersion injury.     

Reduced HIV-1 infectability of CD4+ lymphocytes from exposed-uninfected individuals: association with low expression of CCR5 and high production of beta-chemokines

Escherichia coli leader peptidase, an integral membrane protein, is responsible for the cleavage of the signal sequence of many exported proteins. Recent studies suggest that it is a novel serine protease that utilizes a serine-lysine catalytic dyad. In an effort to further understand the mechanism of this enzyme, an internally quenched fluorescent peptide substrate incorporating the leader peptidase cleavage site of maltose binding protein signal peptide, Y(NO2)-F-S-A-S-A-L-A-K-I-K(Abz) (anthraniloyl), was designed and synthesized. In the intact peptide, the fluorescence of the anthraniloyl group is quenched by the 3-nitrotyrosine. This quenched fluorescence is liberated upon cleavage of the peptide by the leader peptidase, resulting in increased fluorescence that could then be monitored fluorometrically. The designed substrate can be cleaved effectively by E. coli leader peptidase as detected by both HPLC and fluorescent spectroscopy. Mass spectra of cleavage products demonstrated that the cleavage occurs at the predicted site (A-K). The cleavage of the peptide substrate has a linear dependence on the enzyme concentration (0.1 to 1.9 microM) and the kcat/K(m) was calculated to be 71.1 M-1 s-1. These data are comparable with the unmodified peptide substrate. This report represents the first direct continuous assay based on fluorescence resonance energy transfer for E. coli leader peptidase.     

Effect of oral beta-carotene supplementation on plasma human immunodeficiency virus (HIV) RNA levels and CD4+ cell counts in HIV-infected patients

We conducted a pilot, open-label study to assess the effect of short-term beta-carotene administration (180 mg/d with meals for 4 weeks) on the plasma human immunodeficiency virus (HIV) RNA levels and CD4+ lymphocyte counts in 21 HIV-infected patients. We found that plasma HIV RNA levels and CD4+ lymphocyte counts did not change following this short course of beta-carotene supplementation. Patients with lower serum concentrations of beta-carotene before supplementation were no more likely to have an increase in their CD4+ lymphocyte count or plasma HIV RNA copy number than were those with higher concentrations. No correlation was found between pre- or postsupplementation beta-carotene or vitamin A concentrations and pre- or postsupplementation CD4+ lymphocyte counts or plasma HIV RNA titers. This study provides no support for beta-carotene supplementation for HIV-infected subjects with normal baseline serum levels of beta-carotene and vitamin A.     

Effect of CYP2D1 inhibition on the behavioural effects of d-amphetamine

In rats, amphetamine (AMP) conversion to 4-OH-AMP is metabolized by CYP2D1, the rat equivalent of the human enzyme CYP2D6. To determine the impact of impaired AMP metabolism on its behavioural effects, AMP-induced hyperactivity, AMP discrimination and AMP self-administration were examined in male Wistar rats with or without pretreatment with the CYP2D1 inhibitors quinine and budipine. In vivo, quinine (20 mg/kg) and budipine (10 mg/kg) increased the plasma area under the curve of AMP 4-fold and 3.6-fold respectively, and decreased the plasma levels of 4-OH-AMP, 3-fold and 8.6-fold, confirming that the doses used suppressed CYP2D1 activity. Both inhibitors prolonged AMP-induced hyperactivity (0.3 mg/kg) and prolonged the duration of AMP-appropriate responding for periods of up to 90 min post-AMP administration in a drug discrimination procedure. In rats given a preload dose of AMP (0.8 mg/kg) 3 h prior to the self-administration test session, CYP2D1 inhibition resulted in fewer AMP infusions being taken compared with rats receiving the AMP preload dose alone. These studies indicate that AMP is responsible for the behavioural effects seen in rats and that a rat phenocopy model of the human CYP2D6 deficiency state can be produced by CYP2D1 inhibitors.     

Systemic release of soluble TNF receptors after high-dose TNF in isolated limb perfusion

Isolated limb perfusion (ILP) with high dose tumour necrosis factor (TNF), interferon gamma and melphalan (TIM) is an efficient treatment for patients with regionally advanced melanoma and sarcoma. In 44 patients, we determined the kinetics of soluble TNF receptors (sTNF-RI and RII) plasma concentrations, and correlated them with systemic TNF and interleukin 6 (IL-6) levels and shock. Seven patients treated conventionally by ILP without cytokine served as controls. Elevated levels of both sTNF-Rs were observed within 30 min after beginning of the TIM-ILP. A first peak of sTNF-Rs levels was observed 3 h after ILP and was followed by a rapid decrease reaching a nadir at 12-14 h post ILP. This first peak was followed by a second, long-lasting elevation of both sTNF-Rs levels persisting for 4 to 5 days after TIM-ILP. Patients treated by ILP without TNF/interferon gamma (IFN-gamma) had no detectable increase in either sTNF-Rs or in circulating TNF, demonstrating that the release of TNF-Rs was dependent upon the administration of TNF/IFN-gamma. High plasma levels of TNF and IL-6 were observed in patients that had more than 5% leakage during the TIM-ILP, but no significant correlation between TNF levels and the peak values of both sTNF-Rs was observed. The levels of TNF and IL-6 were, however, significantly related to each other. TNF systemic levels, but not sTNF-Rs concentrations, correlated significantly with the severity of the shock observed after TIM-ILP. Patients in which sTNF-RII concentration was in excess over circulating TNF, had no shock or grade I shock only, suggesting that sTNF-RII may play a protective, although limited, role in inhibiting activity of circulating TNF.