An alternative method for the quantitation of neuronal damage after experimental middle cerebral artery occlusion in rats: analysis of behavioral deficit

We tested the hypothesis that increasing durations of focal ischemia that have been shown to result in enlargement of cortical infarct will be associated with progression of behavioral dysfunction that can be measured by a battery of tests sufficiently sensitive and reproducible to detect a positive effect of pharmacotherapy. Untreated or N-methyl-D-aspartate receptor antagonist (CNS-1102)-treated spontaneously hypertensive rats underwent 45, 60, 90, or 120 min of tandem middle cerebral and common carotid artery occlusion followed by reperfusion. We then evaluated the extent of damage and its recovery for up to 21 days using nine behavioral tests aimed at analyzing strength, coordination, and bilateral asymmetry. Also using a graded bioassay that employs a curve-fitting computer program (ALLFIT) to correlate duration of ischemia with degree of behavioral dysfunction, we calculated the average of maximal behavioral dysfunction and duration of ischemia required to produce half-maximal behavioral dysfunction and compared these values in untreated controls with analogous values obtained from animals treated with CNS-1102. Three behavioral tests, forearm flex, tape (somatosensory neutralization), and foot-fault placing, were each separately and combined able to distinguish between the degrees of damage produced by increasing durations of ischemia. The behavioral abnormalities assessed using the tape test were reversible within a week, whereas those using forearm flex or foot-fault tests persisted for at least 21 days. CNS-1102 significantly reduced behavioral dysfunction measured by all three tests. This analysis of behavioral dysfunction represents a useful experimental model to grade efficacy of therapies aimed at protecting the brain from damage produced by acute stroke and might also be used to assess recovery from preexisting ischemic damage.     

Pneumococcal polysaccharide vaccine in children with chronic renal disease: a prospective study of antibody response and duration

We studied the antibody response to pneumococcal serotypes 3 and 14 after pneumococcal polysaccharide vaccine was administered to 41 children with renal disease. One month after vaccination, 76% and 61% of patients achieved at least a twofold titer rise to serotypes 3 and 14, respectively; this finding was comparable to historic control values. One year after vaccination, the majority of patients retained protective antibody levels. Achieving a titer > or = 1.0 microgram/ml IgG at 1 month was highly predictive of retaining a protective antibody level > or = 0.15 microgram/ml at 1 year.     

Frequent clones of p53-mutated keratinocytes in normal human skin

The multiple genetic hit model of cancer predicts that normal individuals should have stable populations of cancer-prone, but noncancerous, mutant cells awaiting further genetic hits. We report that whole-mount preparations of human skin contain clonal patches of p53-mutated keratinocytes, arising from the dermal-epidermal junction and from hair follicles. These clones, 60-3000 cells in size, are present at frequencies exceeding 40 cells per cm2 and together involve as much as 4% of the epidermis. In sun-exposed skin, clones are both more frequent and larger than in sun-shielded skin. We conclude that, in addition to being a tumorigenic mutagen, sunlight acts as a tumor promoter by favoring the clonal expansion of p53-mutated cells. These combined actions of sunlight result in normal individuals carrying a substantial burden of keratinocytes predisposed to cancer.     

Lymphocytopenia and thrombocytopenia during treatment with risperidone or clozapine

The atypical antipsychotic clozapine is associated with several well-known abnormalities of blood cell count, whereas only rare reports are associated with the neuroleptic risperidone. This report describes lymphocytopenia and thrombocytopenia under treatment with risperidone which continued after changing to clozapine without other clinically significant abnormal hematological parameters. Within one week after discontinuation of both neuroleptics abnormalities of blood cell count reversed to the initial values. Abnormalities of lymphocytes or thrombocytes are rare side-effects under treatment with risperidone or clozapine.     

The 1.6 angstroms resolution crystal structure of nuclear transport factor 2 (NTF2)

Nuclear transport factor 2 (NTF2) facilitates protein transport into the nucleus and interacts with both the small Ras-like GTPase Ran and nucleoporin p62. We have determined the structure of bacterially expressed rat NTF2 at 1.6 angstroms resolution using X-ray crystallography. The NTF2 polypeptide chain forms an alpha + beta barrel that opens at one end to form a distinctive hydrophobic cavity and its fold is homologous to that of scytalone dehydratase. The NTF2 hydrophobic cavity is a candidate for a potential binding site for other proteins involved in nuclear import such as Ran and nucleoporin p62. In addition, the hydrophobic cavity contains a putative catalytic Asp-His pair, which raises the possibility of an unanticipated enzymatic activity of the molecule that may have implications for the molecular mechanism of nuclear protein import.     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988), Adv. Enzymol. Related Areas Mol. Biol. 61, 381-436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn2+ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

Kinetics of modification of the mitochondrial succinate-ubiquinone reductase by 5,5''-dithiobis-(2-nitro-benzoic acid)

In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-beta (TGF-beta) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-beta in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-beta was supported by the following observations: (1) exogenous human TGF-beta 1 increased protein synthesis; (2) antibody to TGF-beta blocked both TGF-beta- and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-beta bioactivities; and (4) the actions of U46619 on protein content and TGF-beta bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-beta and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-beta (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-beta that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-beta bioactivity and stimulated protein synthesis. The hyper-trophic response to bFGF was blocked by anti-TGF-beta. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-beta-induced suppression of proliferation, as evidenced by the ability of antibody to TGF-beta to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis as well as active and total TGF-beta bioactivities. By contrast, the protein kinase C inhibitors did not prevent the increases in protein synthesis induced by exogenous TGF-beta. The results demonstrate that thromboxane/prostaglandin endoperoxide signals increased TGF-beta bioactivity via protein kinase C. Increases in both bFGF and TGF-beta are required for an optimal hypertrophic response to U46619. The hypertrophic response to TGF-beta occurs through a protein kinase C-independent pathway.     

CT and MRI of experimentally induced mesenteric ischemia in a porcine model

OBJECTIVE: Our goal was to assess the value of CT and MRI for the detection of bowel wall changes in experimentally induced mesenteric ischemia. METHODS: in 18 female pigs, a percutaneous embolization of the superior mesenteric artery was performed with buthyl-2-cyanoacrylate and Lipiodol (1:1) (experimental group). In six animals, only diagnostic imaging and histologic evaluation were performed (control group). CT was carried out 3, 6, and 12 h after occlusion. Incremental CT (1 s scan time, 5 mm slice thickness, 7 mm increment, 120 kV/290 mAs) and spiral CT (slice thickness 5 mm, pitch 1.5, 120 kV/165 mA) were performed pre and post contrast injection (Somatom Plus/Siemens). Serial CT was carried out after intravenous contrast injection (1 ml/kg, 2 ml/s). MRI (Magnetom 1.5 T; Siemens) was performed with T1 (pre and post 0.01 mmol/kg Gd-DTPA; Magnevist; Schering, Germany), T2, and proton density images in axial orientation. Slice thickness was 3 mm and slice gap 1 mm. Additionally, a T1-weighted GE sequence (multislice FLASH 2D) was obtained in dynamic technique (before and 30, 60, and 90 s after contrast agent injection) with a slice thickness of 5 mm. Biometrical monitoring included blood pressure, heart frequency, blood cell count, electrolyte status, blood gas analysis, and determination of serum lactate. Image evaluation included morphological analysis and determination of the enhancement pattern. Histological specimens were obtained and analyzed according to the Chiu classification. RESULTS: The histologic workup of the specimen 3, 6, and 12 h after vascular occlusion revealed an average Chiu state 3, 4, and 5. On CT, the bowel wall had a thickness of 4.7 mm on average in the ischemic segments. There was a significant difference from the control group (average 3 mm). Free intraperitoneal fluid and intramural gas were seen after 12 h of ischemia in 80%. In ischemic bowel segments, no mural enhancement was seen. Normal segments and the bowel of the control animals showed an enhancement of 34 HU on average (SD = 3.1 HU; p.<0.01). In MRI, S/N and C/N differed significantly between experimental and control groups in T1 and proton density images. In ischemic segments of all phases, the bowel wall did not show contrast enhancement. Healthy segments and bowel of control animals showed a significant enhancement (p<0.01). CONCLUSION: Cross-sectional imaging has a high sensitivity for delineation of ischemic bowel wall segments. The enhancement pattern of the bowel wall enables detection of location, extent, and cause of a acute arterial mesenteric ischemia with high accuracy in an early phase.