Superoxide dismutases of muscle in mitochondrial encephalomyopathies

Lmmunohistochemical analyses were made of the superoxide dismutases (Mn-SOD and CuiZn-SOD) in biopsied muscles from 7 patients with mitochondrial encephalomyopathies that included mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), and chronic progressive external ophthalmoplegia (CPEO). Mn-SOD mainly was present in the subsarcolemmal region, but it also was found in a coarsely granular, reticular, or diffuse pattern of staining within the muscle fibers. These Mn-SOD-positive fibers corresponded almost completely to the ragged-red fibers. The immunoreaction for CuiZn-SOD was weakly positive in some of the muscle fibers positive for Mn-SOD. In CPEO, Mn-SOD-positive fibers predominantly showed decreased cytochrome c oxidase (COX) activity. In MELAS, Mn-SOD-positive fibers tended to be stained deeply for COX although a few were COX-negative. These findings suggest that Mn-SOD-positive fibers can be used to make a differential diagnosis between CPEO and MELAS and that in mitochondrial encephalomyopathies Mn-SOD in the raggedred fibers may protect against oxidative stress. © John Wiley & Sons, Inc.     

PET examination of [11C]NNC 687 and [11C]NNC 756 as new radioligands for the D1-dopamine receptor

The benzazepines NNC 687 and NNC 756 have in animal studies been described as selective D₁-dopamine receptor antagonists. Both compounds have been labeled with¹¹C for examination by positron emission tomography (PET). In the present study central receptor binding was studied in monkeys and healthy men. After IV injection of both radioligands in Cynomolgus monkeys radioactivity accumulated markedly in the striatum, a region with a high density of D₁-dopamine receptors. This striatal uptake was displaced by high doses of the selective D₁-antagonist SCH 23390 (2 mg/kg) but not by the 5HT₂-antagonist ketanserin (1.5 mg/kg) or the selective D₂-antagonist raclopride (3 mg/kg). The cortical uptake after injection of [¹¹C]NNC 687 was not reduced in displacement experiments with ketanserin. The cortical uptake of [¹¹C]NNC 756 was reduced in displacement and protection experiments with ketanserin by 24–28% (1.5 mg/kg), whereas no reduction could be demonstrated on striatal uptake. In healthy males both compounds accumulated markedly in the striatum. For [¹¹C]NNC 687 the ratio of radioactivity in the putamen to cerebellum was about 1.5. For [¹¹C]NNC 756 the ratio was about 5. This ratio of 5 for [¹¹C]NNC 756 is the highest obtained so far for PET radioligands for the D₁-dopamine receptor.     

Proteolytic Processing Mechanisms in the Biosynthesis of Neuroendocrine Peptides: The Subtilisin-like Proprotein Convertases

The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2(SPC2) and PC1/PC3(SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member—furin/PACE (SPC1)—is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation or the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps.     

Are cytokines possible mediators of cancer cachexia?

The possible role of cytokines in the development of cancer cachexia was reviewed from the literature. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, IL-6, interferon (IFN)-gamma and leukemia inhibitory factor (LIF) can elicit many but not all host changes seen in cancer cachexia, including loss of appetite, loss of body weight, and the induction of acute-phase protein synthesis. However, these cytokines are not always demonstrated in the circulation of the cancer patients. The inability to detect circulating cytokines may be due to their low rate of production, their short half-life and rapid clearance from plasma, or their mode of action (autocrine or paracrine). Different cytokines are induced to stimulate the same response. This is very different from hormonal regulation, where a hormone acts on a cell directly through a specific receptor without depending on other mediators. Specific antibodies including anti-IFN-gamma, anti-TNF and anti-IL-6 antibodies, as well as the cyclooxygenase inhibitor indomethacin, have been used to reverse cancer cachexia. Overlapping physiologic activities make it unlikely that a single substance is the sole cause of cancer cachexia. It is hoped that further investigation on other cytokines and their possible relationships with hormones will help to clarify the mechanisms of cancer cachexia in the near future.This work was supported by a grant from the Japan-Sweden Foundation in 1991.     

CYP1A2 activity, gender and smoking, as variables influencing the toxicity of caffeine

We have investigated several factors that might be related to the occurrence of toxic effects during the performance of a urinary test with caffeine (300  mg p.o), in 120 healthy volunteers. A total of 218 toxic effects were self-reported by eighty-two (68%) subjects. Females and nonsmokers were at the highest risk (chi-square test, P =0.01). Furthermore, two nonsmoking females experienced a symptomatology with delirium, restlessness, muscle tremor, vomiting and wakefulness. Among females and nonsmokers, those subjects who experienced toxic effects had lower caffeine N3-demethylation index (CYP1A2 activity) compared with unaffected females (1.87±0.51 vs 1.47±0.27, P <0.0005) and nonsmokers (1.69±0.23 vs 1.49±0.31, P <0.02). Caffeine N1- and N7-demethylations indices were also lower among females ( P <0.0005) and nonsmokers ( P <0.02) who reported toxic symptoms. We conclude that CYP1A2 activity, gender and smoking are variables to be considered as influencing the toxicity of caffeine.     

Immediate ultrastructural effects of endothelin-1 on rabbit basilar artery

Summary In this study intra-arterial Endothelin-1 was applied to rabbit basilar arteries and morphological findings were compared between two groups who were either perfusion fixed or immersion fixed. We planned to establish the quantitative dimension of the drug-induced morphological alterations, independent of the fixation technique's effect.There was an abundance of collagenous fibres deposited among the smooth muscle cells which was not observed in control arteries and after immersion fixation. These degenerative changes are similar to the finding following subarachnoid haemorrhage. The only fixation-related difference was the fact that lamina elastica interna was not corrugated in the perfusion fixation group.It is concluded that, the observed changes in the connective tissue of the arterial wall alter the passive elastic properties and so affect the degree of the response to the vasoactive messengers.     

Influence of preformed α-helix and α-helix induction on the activity of cationic antimicrobial peptides

One prominent class of cationic antibacterial peptides comprises the α-helical class, which is unstructured in free solution but folds into an amphipathic α-helix upon insertion into the membranes of target cells. To investigate the importance of α-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form α-helices or to have preformed α-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i+ 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized α-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of α-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase α-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r²= 0.88) with a helican parameter which was calculated as the induction of α-helix in α membrane-mimicking environment divided by the α-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.     

Effect of nilvadipine on the voltage-dependent Ca channels in rat hippocampal CA1 pyramidal neurons

Effects of nilvadipine on the low- and high-voltage activated Ca²⁺ currents (LVA and HVA I_Ca, respectively) were compared with other organic Ca²⁺ antagonists in acutely dissociated rat hippocampal CA1 pyramidal neurons. The inhibitory effects of nilvadipine, amlodipine and flunarizine on LVA I_Ca were concentration- and use-dependent. The apparent half-maximum inhibitory concentrations (IC₅₀s) at every 1- and 30-s stimulation were 6.3×10⁻⁷ M and 1.8×10⁻⁶ M for flunarizine, 1.9×10⁻⁶ M and 7.6×10⁻⁶ M for nilvadipine, and 4.0×10⁻⁶ M and 8.0×10⁻⁶ M for amlodipine, respectively. Thus, the strength of the use-dependence was in the sequence of nilvadipine>flunarizine>amlodipine. Nilvadipine also inhibited the HVA I_Ca in a concentration-dependent manner with an IC₅₀ of 1.5×10⁻⁷ M. The hippocampal CA1 neurons were observed to have five pharmacologically distinct HVA Ca²⁺ channel subtypes consisting of L-, N-, P-, Q- and R-types. Nilvadipine selectively inhibited the L-type Ca²⁺ channel current which comprised 34% of the total HVA I_Ca. On the other hand, amlodipine non-selectively inhibited the HVA Ca²⁺ channel subtypes. These results suggest that the inhibitory effect of nilvadipine on the neuronal Ca²⁺ influx through both LVA and HVA L-type Ca²⁺ channels, in combination with the cerebral vasodilatory action, may prevent neuronal damage during ischemia.     

Abdominal vagotomy attenuates interleukin-1β-induced nitric oxide release in the paraventricular nucleus region in conscious rats

Nitric oxide (NO) has recently been shown to modulate the hypothalamic–pituitary–adrenal axis response to interleukin-1β (IL-1β). We measured levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the hypothalamic paraventricular nucleus (PVN) region using an in vivo brain microdialysis technique in conscious rats. Intraperitoneally administered IL-1β produced a significant increase in both NO₂⁻ and NO₃⁻ levels in the PVN region. We also examined the possible involvement of the abdominal vagal afferent nerves in this effect. In abdominal-vagotomized rats, the increase was significantly attenuated compared to that in sham-operated rats. Our results suggest that the abdominal vagal afferent nerves are involved in intraperitoneally administered IL-1β-induced NO release in the PVN region.