A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma

Apicomplexan parasites cause serious human and animal diseases, the treatment of which requires identification of new therapeutic targets. Host-cell invasion culminates in the essential cleavage of parasite adhesins, and although the cleavage site for several adhesins maps within their transmembrane domains, the protease responsible for this processing has not been discovered. We have identified, cloned, and characterized the five nonmitochondrial rhomboid intramembrane proteases encoded in the recently completed genome of Toxoplasma gondii. Four T. gondii rhomboids (TgROMs) were active proteases with similar substrate specificity. TgROM1, TgROM4, and TgROM5 were expressed in the tachyzoite stage responsible for the disease, whereas TgROM2 and TgROM3 were expressed in the oocyst stage involved in transmission. Although both TgROM5 and TgROM4 localized to the cell surface in tachyzoites, TgROM5 was primarily at the posterior of the parasite, whereas adhesins were sequestered in internal micronemes. Upon microneme secretion, as occurs during invasion, the MIC2 adhesin was secreted to the apical end and translocated to the posterior, the site of cleavage, where it colocalized only with TgROM5. Moreover, only TgROM5 was able to cleave MIC adhesins in a cell-based assay, indicating that it likely provides the key protease activity necessary for invasion. T. gondii rhomboids have clear homologues in other apicomplexans including malaria; thus, our findings provide a model for studying invasion by this deadly pathogen and offer a target for therapeutic intervention.     

Clinical significance of high c-MYC and low MYCBP2 expression and their association with Ikaros dysfunction in adult acute lymphoblastic leukemia

Increased expression of c-MYC is observed in both Acute Myeloid Leukemia (AML) and T- cell Acute Lymphoblastic Leukemia (T-ALL). MYC binding protein 2 (MYCBP2) is a probable E3 ubiquitin ligase and its function in leukemia is unknown. IKZF1 deletion is associated with the development and poor outcome of ALL. Here, we observed significant high c-MYC expression and low MYCBP2 expression in adult ALL patients. Patients with high c-MYC expression and/or low MYCBP2 expression had higher WBC counts and a higher percentage of CD34+ or CD33+ cells, as well as splenomegaly, liver infiltration, higher BM blasts, and lower CR rate. Ikaros bound to the regulatory regions of c-MYC and MYCBP2, suppressed c-MYC and increased MYCBP2 expression in ALL cells. Expression of c-MYC mRNA was significantly higher in patients with IKZF1 deletion; conversely MYCBP2 mRNA expression was significantly lower in those patients. A CK2 inhibitor, which acts as an Ikaros activator, also suppressed c-MYC and increased MYCBP2 expression in an Ikaros (IKZF1) dependent manner in the ALL cells. In summary, our data indicated the correlation of high c-MYC expression, low MYCBP2 expression and high c-MYC plus low MYCBP2 expression with high-risk factors and proliferation markers in adult ALL patients. Our data also revealed an oncogenic role for an Ikaros/MYCBP2/c-MYC axis in adult ALL, providing a mechanism of target therapies that activate Ikaros in adult ALL.     

Randomized trial of rate-control versus rhythm-control in persistent atrial fibrillation: the Strategies of Treatment of Atrial Fibrillation (STAF) study

OBJECTIVES: This study was designed to compare two treatment strategies in patients with atrial fibrillation(AF): rhythm-control (restoration and maintenance of sinus rhythm) and rate-control (pharmacologic or invasive rate-control and anticoagulation). BACKGROUND: Atrial fibrillation is the most common arrhythmia. It is unclear whether a strategy of rhythm- or rate-control is better in terms of mortality, morbidity, and quality of life. METHODS: The Strategies of Treatment of Atrial Fibrillation (STAF) multicenter pilot trial randomized 200 patients (100 per group) with persistent AF to rhythm- or rate-control. The combined primary end point was a combination of death, cardiopulmonary resuscitation, cerebrovascular event, and systemic embolism. RESULTS: After 19.6 +/- 8.9 months (range 0 to 36 months) there was no difference in the primary end point between rhythm-control (9/100; 5.54%/year) and rate-control (10/100; 6.09%/year; p = 0.99). The percentage of patients in sinus rhythm in the rhythm-control group after up to four cardioversions during the follow-up period (rate-control group) was 23% (0%) at 36 months. Eighteen primary end points occurred in atrial fibrillation; only one occurred in sinus rhythm (p = 0.049). CONCLUSIONS: The STAF pilot study showed no differences between the two treatment strategies in all end points except hospitalizations. These data suggest that there was no benefit in attempting rhythm-control in these patients with a high risk of arrhythmia recurrence. It remains unclear whether the results in the rhythm-control group would have been better if sinus rhythm had been maintained in a higher proportion of patients, as all but one end point occurred during AF.     

High Dose Adenosine Stress Echocardiography for Noninvasive Detection of Coronary Artery Disease

Objectives. The aim of this study was to assess the tolerability and incremental diagnostic value of high adenosine doses in stress echocardiography testing in patients with coronary artery disease (CAD).

Background. In comparison with other pharmacologic stress echocardiography tests, standard dose adenosine stress has suboptimal sensitivity for detecting milder forms of CAD.

Methods. Adenosine stress echocardiography was performed in 58 patients using a starting dose of 100 μg/kg body weight per min over 3 min followed by 140 μg/kg per min over 4 min (standard dose). If no new wall motion abnormality appeared, the dose was increased to 200 μg/kg per min over 4 min (high dose). All patients underwent coronary angiography. Significant CAD was defined as ≥50% diameter stenosis in at least one major coronary artery. Thirty-three patients had one-vessel and seven had multivessel CAD. Coronary angiographic findings were normal in 18 patients.

Results. The high adenosine dose caused a slight but significant increase over baseline values in rate-pressure product. Limiting side effects occurred in two patients during the standard dose protocol and in one patient receiving the high dose regimen. The test was stopped in 30 patients after the standard adenosine dose regimen because of a provoked new wall motion abnormality. The sensitivity of adenosine echocardiography with the standard dose was 75% (95% confidence interval [CI] 63% to 87%). After completion of the standard dose protocol, 28 patients continued testing with the high dose adenosine protocol. The overall sensitivity of adenosine echocardiography, calculated as cumulative, increased to 92% (95% CI 84% to 100%) with the high dose (p < 0.05). The specificity of adenosine testing was 100% and 88%, respectively, with the standard and high dose regimen (p = 0.617).

Conclusions. We believe that use of a higher than usual adenosine dose protocol for stress testing may improve the diagnostic value of adenosine echocardiography, mainly by increasing sensitivity in patients with single-vessel disease without deterioration of the safety profile and with only a mild reduction in specificity.

(J Am Coll Cardiol 1996;28:1689–95)>     

Dipyridamole-Atropine-Induced Myocardial Infarction in a Patient with Patent Epicardial Coronary Arteries

BACKGROUND: The diagnostic accuracy of the physical and pharmacological stress echocardiography tests is higher than routine exercise electrocardiography. They have an acceptable safety profile and have been rarely associated with severe adverse effects. CASE REPORT: We present a case of acute anterior myocardial reinfarction immediately after exercise and pharmacological (dipyridamole-atropine) stress echocardiography testing 1 month after successful stent implantation in LAD. Our patient was a 43-year-old man with a history of heavy smoking and hypertension. Remarkably, the stress echocardiogram was non-diagnostic few hours before the infarction occurred. Angiography performed 4 months after the reinfarction revealed neither a culprit lesion nor stent thrombosis. CONCLUSION: Aggressive "last generation" pharmacological stress testing may provide optimal diagnostic accuracy, but as in our case, complications may occur, even after negative stress testing. To our knowledge, this is the first reported case of an acute myocardial infarction as a severe complication of stress testing, which developed in a patient after stent implantation.     

Effect of treatment with LHRH analogs containing cytotoxic radicals on the binding characteristics of receptors for luteinizing-hormone-releasing hormone in MXT mouse mammary carcinoma

Binding capacities and apparent dissociation constants of receptors for luteinizing-hormone-releasing hormone (LHRH) were investigated in estrogen-independent MXT mammary cancers of untreated mice and after in vivo treatment with agonistic or antagonistic analogs of LHRH containing cytotoxic radicals: AJ-04 (agonist [d-Lys⁶]LHRH linked to methotrexate), T-98-{[d-Lys⁶]LHRH coupled to glutaryl-2-(hydroxmethyl)anthraquinone (G-HMAQ)} and T-121/B (LHRH antagonist T-147 containing two residues of G-HMAQ), which induced tumor growth inhibition. The effects were compared to LHRH agonist [d-Trp⁶]LHRH and carriers [d-Lys⁶]LHRH, LHRH antagonist T-147, as well as to methotrexate, G-HMAQ and surgical bilateral overiectomy. Analysis of the binding data revealed that in control tumors the interaction of¹²⁵I-[d-TRP⁶]LHRH was consistent with the presence of one class of saturable, specific, noncooperative, high-affinity and low-capacity binding sites. Chronic treatment of mice bearing MXT tumors with LHRH analogs AJ-04 and T-121/B carrying cytotoxic radicals, but not with T-98 produced significant down-regulation of membrane receptors for LHRH. The largest decrease in dissociation binding constant andB _max of receptors for LHRH was also found in animals treated with T-121/B. Specific, high affinity binding of¹²⁵I-labelled epidermal growth factor (EGF) was detected in the membranes from control and treated MXT tumors. Treatment with cytotoxic LHRH analogs, AJ-04, T-98 and especially with T-121/B, reduced maximal binding capacity of EGF receptors. Our results indicate that LHRH analogs carrying cytotoxic radicals retain their hormonal activity and inhibit tumor growth while inducing down-regulation of LHRH receptors. In addition, probably both components of the cytotoxic LHRH analog, peptide carriers and cytotoxic radicals, reduce the binding capacity of EGF receptors, which might be useful in the treatment of breast cancer.Abbreviations LHRH luteinizing-hormone-releasing hormone - EGF epidermal growth factor     

Inhibition of growth of experimental prostate cancer in rats by LH-RH analogs linked to cytotoxic radicals

The effects of hybrid cytotoxic LH-RH analogs, produced by linking anthraquinone or methotrexate to carrier LH-RH agonist [D-Lys⁶]LH-RH, were evaluated in Copenhagen-Fisher Fl rats bearing Dunning R-3327H prostate adenocarcinoma. The two cytotoxic LH-RH analogs T-98 [(D-Lys⁶)LH-RH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)], and AJ-04 [(D-Lys⁶)LH-RH linked to methotrexate (MTX)], carrier [D-Lys⁶]LH-RH, or the free cytotoxic compounds MTX and G-HMAQ were administered from Alzet Osmotic minipumps for 7–8 weeks. The cytotoxic LH-RH analogs caused somewhat greater tumor growth inhibition than the carrier peptide, while anthraquinone or methotrexate alone, administered in equimolar doses, were ineffective. The inhibition of androgen sensitive organs (testes, ventral prostates, and seminal vesicles) was pronounced with both carrier and cytotoxic analogs, showing the latter to be fully hormonally active in suppressing the pituitary-gonadal axis. Histological changes were also evaluated. The inhibition of mitosis and the frequency of apoptosis were higher in tumors treated with AJ-04, T-98, [D-Lys⁶]LH-RH, or by castration than in those of controls. Serum hormone levels were lowered by both carrier peptide and cytotoxic analogs, LH being substantially depressed, and testosterone not detectable. These results and other findings indicate that LH-RH analogs containing cytotoxic radicals anthraquinone or methotrexate retain their hormonal activity after administration in vivo, and can effectively inhibit tumor growth. Extensive further studies are required on this new class of compounds, but apparent binding of cytotoxic LH-RH analogs to tumors such as prostate cancer, which have receptors for LH-RH, could greatly reduce peripheral toxicity of chemotherapeutic agents. This approach, based on targeted chemotherapy, might be of practical therapeutic importance for the management of advanced prostate cancers, which eventually relapse after palliative hormonal therapy. © 1993 Wiley-Liss, Inc.     

Cardiopulmonary Interactions Following REM Sleep Deprivation in Sprague–Dawley Rats

We characterized the effects of 48 h of rapid-eye-movement (REM) sleep deprivation on cardiovascular and respiratory variables and on sleep-related cardiopulmonary interactions in adult male Sprague–Dawley rats. Rats were instrumented for monitoring EEG, EMG, and aortic blood pressure. Respiratory rate and minute ventilation were measured by unrestrained single-chamber plethysmography. By using radiotelemetry to monitor blood pressure we clearly demonstrated progressive decreases in mean blood pressure with transitions from wakefulness to non-rapid-eye-movement and REM sleep which were unaffected by REM sleep deprivation. Mirror-image state-dependent increases in heart period suggest that baroreflexes were augmented during sleep with respect to wakefulness. REM sleep deprivation was also associated with lower blood pressure and longer heart period over all sleep/wake states, although this achieved statistical significance only during REM sleep and only during the first hour of recovery sleep. These cardiovascular changes coupled with the observed decreases in respiratory rate and minute ventilation suggest a further augmentation of baroreflexes following REM sleep deprivation.     

Neuroglobin and cytoglobin: oxygen-binding proteins in retinal neurons

PURPOSE: The goal of this study was to describe the detailed localization of the novel oxygen-binding molecules, neuroglobin (Ngb) and cytoglobin (Cygb), in mammalian retinas and to determine whether Ngb and Cygb are neuronal or glial proteins in the retina. METHODS: Antibodies directed against Ngb and Cygb were used to examine their patterns of distribution in normal canine retinas. Immunoblot analysis was performed to verify antibody specificity and the presence of Ngb and Cygb in canine tissues. Double-labeling immunohistochemistry was performed with the Ngb and Cygb antibodies along with antibodies against neuronal (MAP-2, class III beta-tubulin (TUJ1), PKCalpha, and calretinin) and glial antigens (vimentin and CRALBP). Tissue sections were analyzed with light and confocal microscopy. RESULTS: Ngb and Cygb proteins were observed in different retinal cells. Cygb (but not Ngb) was also present in canine kidney, liver, lung, and heart tissue. Immunohistochemical analysis of canine retinas demonstrated Ngb immunoreactivity (IR) in the ganglion cell layer (GCL), inner (INL) and outer (ONL) nuclear layers, inner (IPL) and outer plexiform (OPL) layers, photoreceptor inner segments (IS), and retinal pigment epithelium (RPE). Ngb IR was localized within retinal neurons, but not in glia. Cygb IR was found in neurons and their processes in the GCL, IPL, INL, and OPL and within the RPE, but not in glia. CONCLUSIONS: Ngb and Cygb are widely distributed in retinal neurons and RPE, but not in glial cells of the canine retina. Their structure and distribution is suggestive of a possible role in oxygen transport in the mammalian retina.     

Approach to patients with implanted pacemaker and scheduled surgical or diagnostic procedure

Application of cardiac electrostimulation in strictly defined indications has been on the increase over the last few decades. Frequent use of this therapy as well as the fact that it is applied predominantly in patients in the seventh decade of life, implies possible signifficant comorbidities and need for various diagnostic and surgical procedures. These are the reasons we decided to point out certain specific features in approaching this patient group in preparation and implementation of these procedures. Preoperative approach starts with usual patient history, with additional information on the type of pacemaker, last pacemaker check and electrocardiogram. This general approach is not substantialy different for pacemaker or ICD patients. What is specific is the possible interferrence caused by devices used in diagnostic or therapeutic procedures (diathermy, lithotripsy). Complications that may arise are usually related to the underlying disease rather than the pacemaker malfunction, but still, careful approach and pacemaker check are warranted, especially in the group marked as "pacemaker dependent". Adequate preoperative assessment, only slightly different from the usual, represents a sufficient guarantee for safe procedures diagnostic, therapeutic or surgical.