The function of the adrenocortical axis in permanent middle cerebral artery occlusion: effect of glucocorticoids on the neurological outcome.

We characterized the effect of acute ischemic stroke on the activation of the hypothalamic-pituitary-adrenal (HPA) axis and evaluated the role of glucocorticoids (GC) in the clinical outcome following ischemic stroke. Male spontaneous hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO) and developed a cortical infarct. At 4h post-PMCAO or sham operation, serum levels of ACTH and corticosterone (CS) were elevated 5 and 4 fold respectively as compared to controls and then returned to basal levels at 24h post surgery. In these experimental groups we found also a significant depletion of median eminence (ME)-CRH(41). In adrenalectomized (Adx) rats that underwent PMCAO the degree of motor disability and infarct volume was similar to that of intact rats. Administration of dexamethasone (Dex) to Adx-PMCAO rats significantly improved the motor disability and decreased the infarct volume. However, in sham-Adx with PMCAO, Dex had no effect on these two parameters. In rats with PMCAO or sham-PMCAO, brain production of PGE(2) was significantly increased. This effect was further enhanced in Adx-PMCAO rats and significantly inhibited by Dex. In conclusion, activation of the HPA axis following PMCAO is due to stress induced by surgery. This activation is mediated by hypothalamic CRH(41). Absence of endogenous GC or administration of Dex in naïve rats does not alter motor and pathological parameters in the acute stage following PMCAO. In contrast, administration of Dex significantly improved the outcome following cerebral ischemia in Adx rats which may be due to increased glucocorticoid receptors. Brain production of PGE(2) does not play an important role in the pathophysiology of the acute phase of cerebral ischemia.

Combination of dexanabinol and tempol in focal cerebral ischemia: is there a ceiling effect?

Because ischemic neuronal death is triggered by several parallel mechanisms, a combination of drugs active against individual death-promoting mechanisms may have synergistic effects. Dexanabinol is a noncompetitive NMDA antagonist with anti-inflammatory effects and tempol is a nitroxide antioxidant. Therefore, we explored whether their combined use results in smaller infarct volumes as compared with their individual administration. Rats underwent permanent middle cerebral artery occlusion (PMCAO) and were given vehicle, dexanabinol alone, tempol alone, or a combination of dexanabinol and tempol (n = 13 per group) 1 h later. Five animals in each group were evaluated with a motor rating scale 24 h after PMCAO and the infarct volumes were then measured. The remaining animals were examined with motor and behavioral scales up to 30 days after PMCAO and their infarct volumes were then determined. Motor disability and water maze latencies at all time points examined and infarct volumes at days 1 and 30 were significantly reduced in all active treatment groups when compared with vehicle. However, no significant differences were observed between the active treatment groups. In conclusions, combination therapy with dexanabinol and tempol does not appear to have additional neuroprotective effects compared to those conferred by each agent alone even when administered at optimal timing and dosing. Therefore, a ceiling neuroprotective effect that is impossible to overcome may exist.

The nitroxide antioxidant tempol is cerebroprotective against focal cerebral ischemia in spontaneously hypertensive rats.

Free radicals appear to participate in the final common pathway of neuronal death in ischemia and may therefore be an adequate target for therapy. Tempol is a nitroxide antioxidant with proven protective efficacy in several animal models, including myocardial ischemia, that has not been previously tested in models of permanent cerebral ischemia. Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO). Following dose-response and time-window-finding experiments rats were given vehicle or tempol (50 mg/kg) subcutaneously 1 h after PMCAO (n = 10/group). Five animals in each group were evaluated with a motor scale 24 h after the infarct and were then sacrificed and the injury volume was measured. The remaining animals were examined daily with the motor scale and also with a Morris water maze test on days 26-30 after PMCAO and sacrificed on day 30. Motor scores at all time points examined were significantly better in the tempol-treated animals (P < 0.05 for all). Significantly better performance in the water maze test for performance on days 26-30 was noted in the tempol group compared with the vehicle-treated group (P < 0.05). Injury volumes at days 1 and 30 were significantly reduced in the tempol group (9.83 +/- 1.05 vs 19.94 +/- 1.43% hemispheric volume, P = 0.0009, and 13.2 +/- 2.97 vs 24.4 +/- 2.38% hemispheric volume, P = 0.02, respectively). In conclusion, treatment with tempol led to significant motor and behavioral improvement and reduced injured tissue volumes both in the short and in the long term after stroke.

Expression of endothelial nitric oxide synthase in the ischemic penumbra: relationship to expression of neuronal nitric oxide synthase and vascular endothelial growth factor.

Expressional patterns of the endothelial and neuronal forms of nitric oxide synthase (NOS) in cerebral ischemia were studied utilizing a permanent middle cerebral artery occlusion (PMCAO) model. Motor performance and infarct volumes were determined in the rats. Immunohistochemical staining for eNOS, nNOS and neurofilament were performed at 1, 2, 3, 5, 7 and 14 days after PMCAO. Vascular endothelial growth factor (VEGF) expression was determined by in-situ hybridization. PMCAO caused a reproducible cortical infarct with motor deficits in the rats. Double immunohistochemical stainings indicated that eNOS and nNOS were induced in ischemic neurons. Most stained neurons were positive for both NOS forms but some reacted with only one NOS antibody. nNOS expression peaked at 24-48 h after PMCAO, stained mainly the cytoplasm of core neurons, and disappeared after the 3rd day. eNOS expression increased until the 7th day, stained mainly the cytoplasm and membrane of penumbral cells and disappeared by the 14th day after PMCAO. VEGF expression was significantly induced in the penumbral zone in a similar distribution to eNOS. The anatomical and temporal pattern of VEGF and eNOS induction in the brain after permanent ischemia suggest that these mediators may play a role in protecting penumbral tissue from additional ischemic damage.

Long term cerebroprotective effects of dexanabinol in a model of focal cerebral ischemia.

In order to test the long-term cerebroprotective effects of dexanabinol, a synthetic non-competitive NMDA antagonist that also has anti-TNFalpha effects, spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO). Rats were given vehicle or dexanabinol (4.5 mg/kg) 1, 3 or 6 h after PMCAO. The research consisted of 2 stages. In the short-term set of experiments animals (n=5/group), were tested with a motor disability scale 24 h post PMCAO, then sacrificed and the infarct volume was measured using 2,3,5-Triphenyltetrazolium chloride (TTC) staining. In the long-term set of experiments the rats (n=7/group) were examined daily with a motor disability scale up to 30 days after PMCAO and then sacrificed and infarct volumes were determined using TTC staining. Motor scores were significantly improved in the dexanabinol treated rats (P<0.05 for all groups) at all the time points examined. Infarct volumes were significantly reduced 24 h after PMCAO in the groups treated 1 or 3 h, but not 6 h after PMCAO compared with vehicle (Mean+/-S.D., 11.5+/-2.02, 12+/-3.2 and 14.4+/-2.4% vs. 20.8+/-1.3% hemispheric volume respectively). The lesions remained significantly smaller in the dexanabinol groups 30 days after PMCAO (Mean+/-S.D., 24.49+/-1.9% vs. 8.1+/-0.6, 11.1+/-2.3 and 13.8+/-2.5% hemispheric volume in animals treated with vehicle vs. dexanabinol 1, 3 or 6 h after PMCAO respectively; P<0.05 for all). In conclusion, the extended therapeutic window and the multi-mechanistic durable neuroprotective effects of dexanabinol make it a promising candidate for future stroke therapy.

Heat-shock-proteins-antibodies in patients with Helicobacter pylori associated chronic gastritis.

Twenty eight patients (15 F, 13 M mean age 37.7 SD +/- 9.93 range 22-55) affected by Helicobacter Pylori infection associated gastritis were studied. HSP 70 Antibodies were found in 21.4% of patients and their mean values were significantly higher in the patients than in the subjects affected by gastritis HP negative used as controls (p = 0.05). This datum was confirmed by Western blotting. The presence of HSP 70 antibodies in the sera of those patients may support the link between the protein and the development and persistence of chronic inflammation in the gastric mucosa.

Treatment with cyclosporine A promotes axonal regeneration in rats submitted to transverse section of the spinal cord--II--Recovery of function.

Previous reports have suggested a correlation between autoimmunity and abortive axonal regeneration in mammalian CNS. In this study we investigated the effects of immunosuppressive treatment with Cyclosporine A (CyA) (2.5-5 mg/kg/day) on axonal regeneration after complete spinal transection in rats. Partial recovery of function was observed 30 days after surgery in rats treated with CyA, with the presence of incomplete spontaneous locomotion and a positive contact placing reaction. Restoration of somatosensory evoked potentials and positive retrograde fluorescent tracing were also observed. CyA reduced the autoimmune reaction which targeted components of the axons. These results provide further evidence of the role played by autoimmunity in blocking regeneration of fibre tracts in mammalian CNS.

Treatment with cyclosporine A promotes axonal regeneration in rats submitted to transverse section of the spinal cord.

The abortive axonal regeneration in the central nervous system (CNS) of mammals has been attributed to a series of inhibitory factors. Previous reports suggest the development of autoimmune reactions following CNS lesion in mammals. In this study we investigated whether immunosuppressive treatment with cyclosporine A is able to facilitate axonal regeneration in rats submitted to complete transverse section of the spinal cord at the level of T7-T8. Treated animals received daily subcutaneous injections of cyclosporine A (2.5 mg/kg), while control rats were given a similar treatment with saline. Immunosuppression was begun immediately after spinal cord transection. Strong evidence of morphological axonal regeneration was observed 15 days after surgery in all cyclosporine A treated animals. Furthermore, treatment with cyclosporine A markedly reduced the seric immune reaction elicited by the lesion. The results of this study, although preliminary, provide further evidence for the development of autoimmune processes following lesion in the CNS, and suggest that blocking of the immune reaction facilitates axonal regeneration in the rat.

Thermal behaviour of A4 lactate dehydrogenase purified from the heterothermic and sympatric vertebrate species Brook lamprey (Lampetra planeri), tench (Tenca tenca), smooth (Triturus vulgaris) and alpine newt (Triturus alpestris).

1. The A4 lactate dehydrogenase isozyme was purified to homogeneity from the tissues of Brook lamprey (Lampetra planeri), tench (Tenca tenca), smooth newt (Triturus vulgaris) and alpine newt (T. alpestris). 2. These four species share their geographical distribution in the same freshwater habitats, often live together in the same station and two of them are congeneric. Steady-state kinetic investigations have shown that: 3. Km (apparent) for pyruvate vs. temperature and (apparent) product Ki (Pyruvate) and Ki (Lactate) are fairly similar among species; 4. kcat/Km decreases with temperature in the case of the newts but increases in the case of both lamprey and tench; 5. Thermostability does not correlate to preferred ambient temperature and, in particular, tench LDH starts being inactivated up to 65 degrees C. 6. Thermostability does not correlate with activation energy either; 7. No clear relationships can be demonstrated either between activation energy and conformational transitions in the molecule (these latter indicated by breaks in the Arrhenius plots) nor between activation energy and molecular flexibility, investigated by melting experiments.

Lactate dehydrogenase from Lampetra planeri is composed of chains of unique type which show intermediate properties between the heart and the muscle isozymes of vertebrates.

1. Like other lamprey species, Lampetra planeri displays LDH chains of a single type. Since lampreys are more related to vertebrates than myxines, which do have usual A and B monomers, we suspect that either a gene inactivation or a gene loss occurred in the former group. 2. The characterization of the enzyme gave interesting results. From the standpoint of its affinity for ion exchangers, it behaves as if it is composed of A-type chains. 3. From the standpoint of substrate and product inhibition, it resembles much more closely the B containing isozyme. 4. Since literature reports that the other known single-chained LDH's from lampreys are definitely of the A type, we suggest the possibility that L. planeri enzyme underwent some orthologous evolution which brought it to resemble the heart isozyme.

Insensitivity of archaebacterial ribosomes to protein synthesis inhibitors. Evolutionary implications.

The effect on Sulfolobus solfataricus (an extremely thermoacidophilic archaebacterium) of selected inhibitors affecting reactions of the polypeptide elongation cycle has been tested by using poly(U) and poly(UG) directed cell-free systems. The results reveal a unique pattern of antibiotic sensitivity of Sulfolobus ribosomes with an inhibitory effect observed for only three of 60 compounds tested. Through comparison with suitable eubacterial and eukaryotic cell-free systems the insensitivity of Sulfolobus ribosomes to most inhibitors of protein synthesis appears to reflect a phylogenetic distinction of ribosome structure, rather than the high temperature conditions of the Sulfolobus assay system. In this respect ribosomes of thermoacidophilic archaebacteria differ not only from their eubacterial and eukaryotic counterparts, but also from ribosomes of archaebacteria belonging to the methanogenic-halophilic branch of the 'third' kingdom. The evolutionary implications of these findings are discussed.

[Long-term experiences with the "St. Georg" sliding endoprosthesis]. / Längerfristige Erfahrungen mit Schlittenendoprothesen "St. Georg".

Primary healing, secondary operations necessary and--by means of follow-up examinations-local sequelae were analyzed after implantation of 65 "St. Georg" sliding prostheses; the operations had been performed at least 5 years and on average 77 months previously, in 64 patients. The overall incidence of early complications was 29.2%, the re-arthrotomy quota, leaving the prosthesis in situ 7.7%, and the incidence of loosening of the prostheses 5.5%. Complications and unsatisfactory results were due less to errors during implantation than to faulty indication. Apart from the loosening, it has been demonstrated that sliding surface prostheses provide satisfactory function, even over many years.

Management of tuberculin reactors in children and adolescents previously vaccinated with BCG.

Between 1960 and 1981, 142 Bacillus Calmette-Guérin (BCG)-vaccinated children and adolescents were evaluated in the Children's Chest Clinic, Bellevue Hospital. These patients came from 28 countries and had positive tuberculin reactions and/or exposure to active tuberculosis. Antituberculous therapy was given to 8 with active tuberculosis, to 8 with calcified pulmonary primary lesions and to 105 positive tuberculin reactors without disease. In a 1 1/2- to 16-year follow-up no tuberculous disease occurred, and two patients with previously negative roentgenograms developed calcified pulmonary lesions. Guidelines are given for the management of patients previously vaccinated with BCG with special reference to the value of prophylactic isoniazid therapy for those considered infected by Mycobacterium tuberculosis but without evidence of disease.

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.

Particle weights and protein composition of the ribosomal subunits of the extremely thermoacidophilic archaebacterium Caldariella acidophila.

1. The ribosomal subunits of one thermoacidophilic archaebacterium (Caldariella acidophila) and of two reference eubacterial species (Bacillus acidocaldarius, Escherichia coli) were compared with respect to ribosome mass and protein composition by (i) equilibrium-density sedimentation of the particles in CsCl and (ii) gel-electrophoretic estimations of the molecular weights of the protein and the rRNA. 2. By either procedure, it is estimated that synthetically active archaebacterial 30S subunits (52% protein by wt.) are appreciably richer in protein than the corresponding eubacterial particles (31% protein by wt.) 3. The greater protein content of the archaebacterial 30S subunits is accounted for by both a larger number and a greater average molecular weight of the subunit proteins; specifically, C. acidophila 30S subunits yield 28 proteins whose combined mass is 0.6 X 10(6) Da, compared with 20 proteins totalling 0.35 X 10(6) Da mass for eubacterial 30S subunits. 4. No differences in protein number are detected among the large subunits, but C. acidophila 50S subunits exhibit a greater number-average molecular weight of their protein components than do eubacterial 50S particles. 5. Particle weights estimated by either buoyant-density data, or molecular weights of rRNA plus protein, agree to within less than 2%. By either procedure C. acidophila 30S subunits 1.15 X 10(6) Da mass) are estimated to be about 300 000 Da heavier than their eubacterial counterparts (0.87 X 10(6) Da mass); a smaller difference. 0.15 X 10(6) Da, exists between the archaebacterial and the eubacterial 50S subunits (respectively 1.8 X 10(6) and 1.65 X 10(6) Da). It is concluded that the heavier-than-eubacterial mass of the C. acidophila ribosomes resides principally in their smaller subunits.

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin.

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.

A new agglutinating activity from wheat flour inhibited by tryptophan.

A new compound endowed with agglutinating activity, designated the flour agglutinin, was extracted from wheat flour with water and purified by gel filtration and ion-exchange chromatography. The haptenic inhibitors of the plant agglutinins do not affect flour agglutinin activity which, on the other hand, is inhibited by D- and L-tryptophan. Flour agglutinin has a molecular weight of about 5 - 10(4) as determined by gel filtration. It consists of a neutral heteropolysaccharide constituted of D-xylose and L-arabinose, and is homogeneous as judged by sedimentation analysis. Flour agglutinin activity is destroyed by treatment with Cellulase 2000 and periodate, but is not affected by alpha-amylase and proteolytic enzymes. Compared to germ agglutinin, flour agglutinin exhibits a peculiar range of cell specificity. It agglutinates several normal cell types, but has no effects on some neoplastic cells tested. Tryptic digestion of erythrocytes does not affect their susceptibility to flour agglutinin-induced agglutination.