Stunning and myocardial contractile autoregulation studied on the isolated isovolumic blood-perfused dog heart

AIM: To study, for the first time, the effects of stunning on homeometric and heterometric autoregulation. METHODS AND RESULTS: Ischaemia (15 min)/reperfusion (30 min) was induced in the isovolumic blood-perfused dog heart preparation. Heart rate elevations (n = 9) from 60 to 200 beats min-1, in steps of 20 beats min-1, promoted the same inotropic stimulation in control (C) and stunning (S), indicating that ischaemia/reperfusion does not affect the changes in calcium kinetics elicited by the Bowditch effect. Sudden ventricular dilation (VD) (n = 10) evoked an instantaneous increase in developed pressure (Delta1DP) followed by a continuous slow performance increase (Delta2DP) in C and S. Delta1DP (C: 35 +/- 2.2 mmHg; S: 27 +/- 2.1 mmHg; P = 0.002) and Delta2DP (C: 20 +/- 1.6 mmHg; S: 14 +/- 1.3 mmHg; P = 0.002) decreased proportionally, while Delta2/Delta1DP (C: 0.57 +/- 0.13; S: 0.58 +/- 0.14) and slow response time course (T/2) were unchanged (C: 55 +/- 6.6 s; S: 57 +/- 7.7 s) after ischaemia/reperfusion. The reduction of Delta1DP can be understood as a decline of the myofilaments calcium responsiveness, the main pathophysiological effect of stunning. The reason for the weakening of Delta2DP, due to intracellular calcium gain, was not determined but it was supposed that its complete manifestation could be restricted by cyclic adenosine monophosphate (cAMP) myocardial content reduction. As reported by others, Delta2DP depends on myocardial cAMP, and it has been shown that myocardial cAMP is decreased after ischaemia/reperfusion. CONCLUSIONS: Contractile depression due to stunning has no effect on the inotropic stimulation generated by the Bowditch phenomenon. Immediate and time-dependent enhancements of contraction evoked by sudden VD are proportionally reduced and the slow response time course is unaffected in the stunned myocardium.     

Predisposing effect of spontaneous mesenchymal intimal thickenings of rabbit aorta to early lipid deposition

Summary Ten rabbits received by gastric tube 0.3 to 0.8 g pure cholesterol every second day for 4 to 8 weeks. Ten animals were used as control. Total serum cholesterol was determined weekly in experimental and control groups. The mean value of the total serum cholesterol in the experimental group was 482±79.6 mg/100 ml, about 2.5 times more than the cholesterol value of the control group (206±51.0 mg/100 ml).Early and selective lipid deposition was present in spontaneous mesenchymal thickenings of all aortas of the animals receiving cholesterol. The deposition failed to be observed in the aortic intima without that thickening. The intimal thickening is a change of the arterial wall that predisposes to lipid deposition.This observation supports the view that the proliferative changes seen in human atherosclerosis precede the lipid deposition and are a predisposing factor to this deposition.     

Inhibition of cysteine proteinases by autolytic digestion is mediated by CBP2/Hsp47

CBP2/Hsp47 is a glycoprotein normally limited to the ER-Golgi where it is first associated with procollagen chains at a very early point during translation of nascent chains and later with properly folded procollagen. Although CBP2/Hsp47 is regarded as a molecular chaperone belonging to the serpin superfamily, this protein does not appear to inhibit serine proteinases. Here we demonstrate that CBP2/Hsp47 functions in a manner similar to other serpin superfamily members by cross class inhibiting cysteine proteinases. A CBP2/Hsp47 to cathepsin L inactivation stoichiometery of approximately 1.5 revealed concurrent cleavage of CBP2/Hsp47 with proteinase inactivation. Cleavage of the CBP2/Hsp47 was shown to occur outside the P1-P1' at the P16-P15 and P2'-P3' bonds. In addition, the proteinase bands in SDS/PAGE diminished on reaction of the enzyme with CBP2/Hsp47. These results sustain a mechanism advocated by Bjork et al. (1998), in which cysteine proteinases assault a peptide bond in the reactive site loop of serpins, (CBP2/Hsp47) adjacent to the P1-P1' bonds involved in serine proteinase inhibition. The reaction proceeds with the substrate pathway dominating in the cysteine proteinase reaction. In these complexes the cysteine proteinases, papain and cathepsin L, are rendered more susceptible to proteolysis and are degraded by active enzyme. These properties help explain the mechanism by which CBP2/Hsp47 increases the fidelity of collagen production. Moreover, if CBP2/Hsp47 is shown to involve the multiplexin subclass of collagens, it may further provide a mechanism by which the motogen and angiogenic properties during development and/or neoplasia are regulated.     

Inverse association between skin response to aeroallergens and Schistosoma mansoni infection

BACKGROUND: Helminthic infections and allergic disease are highly prevalent in many areas of the world. It is known that IgE antibodies are involved in the pathogenesis of both helminthiasis and atopy. However, the consequences of the presence of helminthic infections in atopic patients are still not completely understood. METHODS: Subjects infected by Schistosoma mansoni with more than 200 eggs/g of feces (n = 42) and uninfected subjects (n = 133) were selected from an endemic area of schistosomiasis. The history of allergy and results of the immediate hypersensitivity prick tests with inhalant allergen extracts were registered. Total IgE and IgE specific to S. mansoni and aeroallergens were measured in serum by ELISA. RESULTS: The proportion of individuals with a positive skin test to allergens was higher in the uninfected group (24.3%) than in the group with more than 200 eggs/g of feces (4.8%). The odds of atopy (defined as a positive test for at least one of the antigens) were 5 times higher (odds ratio = 7.0; 95% confidence interval = 1.6-31.1%; p = 0.01) in the uninfected group, after taking into account the potential influence of gender and age. While there was a tendency for higher total and S. mansoni-specific IgE levels in infected patients, an opposite trend, that is higher aeroallergen-specific IgE, was observed in uninfected subjects. CONCLUSIONS: There was a strong and statistically significant inverse association between the immediate skin test response to common aeroallergens and infection by S. mansoni. The results indicate that immediate hypersensitivity reactions may be suppressed in S. mansoni-infected individuals.     

Aberrant cellular retinol binding protein 1 (CRBP1) gene expression and promoter methylation in prostate cancer

AIMS: Retinoids are involved in cell growth, differentiation, and carcinogenesis. Their effects depend on cytosolic transport and binding to nuclear receptors. CRBP1 encodes a protein involved in this process. Because altered CRBP1 expression and promoter hypermethylation occur in several tumours, these changes were investigated in prostate tumorigenesis. METHODS: The CRBP1 promoter was assessed by methylation specific polymerase chain reaction on tissue samples from 36 radical prostatectomy specimens (paired normal tissue, adenocarcinoma, and high grade prostatic intraepithelial neoplasia (HGPIN)), 32 benign prostatic hyperplasias (BPHs), and 13 normal prostate tissue samples from cystoprostatectomies. Methylation of DNA extracted from microdissected tissue was examined blindly. CRBP1 expression was assessed by immunohistochemistry on formalin fixed, paraffin wax embedded tissue. RESULTS: Loss of CRBP1 expression was seen in 15 of 36 adenocarcinomas and 18 of 36 HGPINs. Fifteen adenocarcinomas and nine HGPINs showed overexpression, whereas the remainder showed normal expression. BPH displayed normal expression. No significant associations were found between CRBP1 expression and Gleason score or stage. CRBP1 promoter hypermethylation was found in 17 of 36 adenocarcinomas, three of 35 HGPINs, one of 36 normal prostate tissues from the same patients, none of 32 BPHs, and none of 13 normal prostate tissues from cystoprostatectomies. Loss of expression and hypermethylation of CRBP1 were not significantly associated. CONCLUSIONS: Altered CRBP1 expression and hypermethylation are common in prostate carcinoma, although CRBP1 hypermethylation is not an early event in tumorigenesis. Moreover, both adenocarcinoma and HGPIN show frequent CRBP1 overexpression. The molecular mechanisms underlying altered CRBP1 expression in prostate cancer deserve further study.     

Adenosine A(2A) receptor facilitation of hippocampal synaptic transmission is dependent on tonic A(1) receptor inhibition

Adenosine tonically inhibits synaptic transmission through actions at A(1) receptors. It also facilitates synaptic transmission, but it is unclear if this facilitation results from pre- and/or postsynaptic A(2A) receptor activation or from indirect control of inhibitory GABAergic transmission. The A(2A) receptor agonist, CGS 21680 (10 nM), facilitated synaptic transmission in the CA1 area of rat hippocampal slices (by 14%), independent of whether or not GABAergic transmission was blocked by the GABA(A) and GABA(B) receptor antagonists, picrotoxin (50 microM) and CGP 55845 (1 microM), respectively. CGS 21680 (10 nM) also inhibited paired-pulse facilitation by 12%, an effect prevented by the A(2A) receptor antagonist, ZM 241385 (20 nM). These effects of CGS 21680 (10 nM) were occluded by adenosine deaminase (2 U/ml) and were made to reappear upon direct activation of A(1) receptors with N(6)-cyclopentyladenosine (CPA, 6 nM). CGS 21680 (10 nM) only facilitated (by 17%) the K(+)-evoked release of glutamate from superfused hippocampal synaptosomes in the presence of 100 nM CPA. This effect of CGS 21680 (10 nM), in contrast to the isoproterenol (30 microM) facilitation of glutamate release, was prevented by the protein kinase C inhibitors, chelerythrine (6 microM) and bisindolylmaleimide (1 microM), but not by the protein kinase A inhibitor, H-89 (1 microM). Isoproterenol (30 microM), but not CGS 21680 (10-300 nM), enhanced synaptosomal cAMP levels, indicating that the CGS 21680-induced facilitation of glutamate release involves a cAMP-independent protein kinase C activation. To discard any direct effect of CGS 21680 on adenosine A(1) receptor, we also show that in autoradiography experiments CGS 21680 only displaced the adenosine A(1) receptor antagonist, 1,3-dipropyl-8-cyclopentyladenosine ([(3)H]DPCPX, 0.5 nM) with an EC(50) of 1 microM in all brain areas studied and CGS 21680 (30 nM) failed to change the ability of CPA to displace DPCPX (1 nM) binding to CHO cells stably transfected with A(1) receptors.Our results suggest that A(2A) receptor agonists facilitate hippocampal synaptic transmission by attenuating the tonic effect of inhibitory presynaptic A(1) receptors located in glutamatergic nerve terminals. This might be a fine-tuning role for adenosine A(2A) receptors to allow frequency-dependent plasticity phenomena without compromising the A(1) receptor-mediated neuroprotective role of adenosine.     

Thrombin enhancement of interleukin-1 and tumor necrosis factor-alpha induced polymorphonuclear leukocyte migration.

BACKGROUND: Cytokines such as IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) activate vascular endothelium to express leukocyte adhesion molecules that promote polymorphonuclear leukocyte (PMNL) migration and to synthesize tissue factor, thus making the endothelium a procoagulant surface. alpha-Thrombin, generated during coagulation, also activates endothelial cells. Since all these processes are likely involved in inflammation, the effect of alpha-thrombin on PMNL interaction with cytokine activated endothelium was investigated. EXPERIMENTAL DESIGN: Human umbilical vein endothelium was grown on polycarbonate filters to investigate the effects interleukin-1 alpha (IL-1 alpha), TNF-alpha, and alpha-thrombin on PMNL transendothelial migration quantitated with 51Cr-labeled PMNL, and on endothelial monolayer permeability, quantitated with 125I-labeled albumin (HSA). To evaluate the expression of endothelial-leukocyte adhesion molecules, enzyme-linked immunosorbent assay was performed on human umbilical vein endothelium monolayers. The effect of thrombin on PMNL accumulation and plasma exudation in inflammation was studied in a rabbit dermal model, using 51Cr-labeled blood leukocytes and [125I]HSA respectively. RESULTS: On resting human umbilical vein endothelium, alpha-thrombin induced a transient increase (2.5- to 4-fold) in monolayer permeability lasting 30 minutes. Slight but significant transendothelial migration of 51Cr-labeled PMNL was induced by alpha-thrombin (7.4 +/- 0.6% of cells added, unstimulated = 1.9 +/- 0.4%), although this response was less than that induced by f-norLeu-Leu-Phe (17%), IL-1 alpha (29%) or TNF-alpha (21%). alpha-Thrombin enhanced the initial rate of IL-1, TNF-alpha and f-norLeu-Leu-Phe induced PMNL transendothelial migration in an additive or supradditive manner (e.g., with IL-1 alpha+alpha-thrombin, migration was 58% greater than additive at 15 to 30 minutes, p < 0.001). Catalytically inactivated alpha-thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone and diisopropyl-fluorophosphate alpha-thrombin, did not enhance migration or permeability. In dermal inflammation in rabbits, alpha-thrombin (10 units/site) induced an increase in plasma protein exudation, with only a mild infiltration of PMNL. However, alpha-thrombin synergistically enhanced the PMNL infiltration induced by IL-1 alpha, TNF-alpha, but not that induced by zymosan activated plasma (C5a) or IL-8 (neutrophil-activating peptide-1). These measurements were confirmed histologically. Investigations into the mechanisms of the enhancement of PMNL migration indicated that individually vascular permeability changes, prostaglandins, platelet activating factor, and P-selectin expression did not account for the observation effects. CONCLUSIONS: Alpha-thrombin may have a role in synergistically enhancing PMNL infiltration at sites of inflammation, in part via enzymatic action on the cytokine activated endothelium. The mechanisms involved in this effect are likely a complex interaction.     

Ectopic T cell receptor expression causes B cell immunodeficiency in transgenic mice

Mice expressing transgenic T cell receptors (TCR) are used to explore important questions in immunity. However, transgene expression may have unexpected effects. We previously reported a B cell immunodeficiency, comprising decreased B cell numbers and diminished antibody responses, in mice that express a transgenic TCR specific for nicotinic acetylcholine receptor; the mice were generated using cassette vectors designed specifically for transgenic TCR expression [see Kouskoff et al. J. Immunol. Methods 1995. 180: 273-280]. We now show data suggesting that this defect is due to the expression and accumulation of TCR alpha and beta chains inside B cells and induction of an endoplasmic reticulum stress response, causing apoptosis at the pre B-I and later B cell stage. Thus, inappropriate transgene expression can profoundly affect B cells, leading to a previously undescribed mechanism of immunodeficiency.     

Glaucolide B, A Molluscicidal Sesquiterpene Lactone, and Other Constituents of Vernonia eremophila

The sesquiterpene lactone glaucolide B was isolated from the molluscicidal extract of VERNONIA EREMOPHILA Mart, and shown to be responsible for the molluscicidal activity. Hydrogenolysis of glaucolide B resulted in the disappearance of the activity. Other inactive constituents of the extract were 3,7-dimethoxy-5,3',4'-trihydroxyflavone and two transformation products, possibly artefacts, of glaucolide B. Glaucolide B exhibited strong antimicrobial activity against B, CEREUS and moderate activity against S. AURENS, but was inactive in several other systems.