Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy

Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.     

Retrobulbar anesthesia for repair of ruptured globes

PURPOSE: To describe two cases involving patients with ruptured globes in whom retrobulbar anesthesia was used successfully. METHOD: A modified retrobulbar anesthesia technique was used to repair two ruptured globes in two patients in whom general anesthesia was contraindicated. RESULT: Using a modified technique, retrobulbar anesthesia did not cause wound gape or prolapse of intraocular contents. CONCLUSIONS: Most open globes should be repaired after administration of general anesthesia. Modified retrobulbar anesthesia can provide a reasonable alternative in rare situations in which general anesthesia subjects the patient to an unacceptable risk.     

Popliteal aneurysm presenting as acute thrombosis and ischemia in a middle-aged man with a history of Kawasaki disease

Kawasaki disease is known to cause a vasculitis of small and medium-sized vessels, with subsequent aneurysm formation. Most of the severe manifestations of the disease occur as a result of coronary aneurysm formation. However, many other arteries have been documented to be involved. A case is presented of a middle-aged man with a history of Kawasaki disease who had an acute ischemic limb from a thrombosed popliteal aneurysm that formed as a result of the disease. This is the first known case report of Kawasaki disease resulting in delayed lower extremity ischemia. Typical findings of patients with Kawasaki disease are presented, along with a case report and review of the literature. A history of Kawasaki disease is an extremely rare but possible cause of peripheral aneurysms, even in middle-aged patients.     

Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.     

Efficacy of radical hysterectomy as treatment for patients with small cell carcinoma of the cervix

BACKGROUND: This study was performed to identify pathologic and clinical features that best predict disease free survival of patients with early stage small cell carcinoma of the cervix treated by radical hysterectomy. METHODS: Three hundreds and seventy patients with cervical carcinoma were analyzed retrospectively to define those variable that best predict disease free survival (DFS). Variables included age, weight, race, marital status, economic status, tumor size, depth of invasion (DI), lymph-vascular space involvement (LVSI), cell type, tumor grade, lymph node metastasis (LNM), and total number of lymph nodes removed. Patients with lymph node metastasis, parametrial involvement, and positive or close surgical margins were offered postoperative radiation. RESULTS: Twelve patients were found to have small cell carcinoma (3.2%). One patient had microinvasive carcinoma of the cervix (MIC) as defined by the Society of Gynecologic Oncologists with a depth of invasion of 3 mm or less and no lymph-vascular space invasion, and has been reported previously. A detailed analysis of the other patients with nonsmall cell carcinoma is presented separately. Five patients achieved a DFS of at least 5 years, whereas 7 patients died with disease. Excluding the patient with MIC, the 5-year DFS rate was 36.4%. CONCLUSIONS: Relative to other cell types, small cell carcinomas of the cervix is an aggressive neoplasm with a higher rate of LVSI and LNM despite smaller DI and tumor size. These data suggest that multimodality therapy, combining radical surgery and radiation with cytotoxic chemotherapy, may provide these patients with the best chance for cure.     

The population risk as an explanatory variable in research synthesis of clinical trials

The population risk, for example the control group mortality rate, is an aggregate measurement of many important attributes of a clinical trial, such as the general health of the patients treated and the experience of the staff performing the trial. Plotting measurements of the population risk against the treatment effect estimates for a group of clinical trials may reveal an apparent association, suggesting that differences in the population risk might explain heterogeneity in the results of clinical trials. In this paper we consider using estimates of population risk to explain treatment effect heterogeneity, and show that using these estimates as fixed covariates will result in bias. This bias depends on the treatment effect and population risk definitions chosen, and the magnitude of measurement errors. To account for the effect of measurement error, we represent clinical trials in a bivariate two-level hierarchical model, and show how to estimate the parameters of the model by both maximum likelihood and Bayes procedures. We use two examples to demonstrate the method.     

Distinguishing clinically important from unimportant prostate cancers before treatment: value of systematic biopsies

PURPOSE: We assessed the ability of diagnostic tests to distinguish clinically unimportant cancers. MATERIALS AND METHODS: We correlated T stage (based on digital examination and ultrasound), prostate specific antigen (PSA), PSA density and pathological features of cancer in systematic biopsy specimens with features of cancer in 170 radical prostatectomy specimens. Clinically unimportant cancers were defined as small (0.5 cm.3 or less), well or moderately differentiated and confined to the prostate. RESULTS: Of the patients 10% had an unimportant cancer. On logistic regression analysis the 2 significant predictors were maximum length of cancer in any core and PSA density. Of 12 patients with maximum cancer length 2 mm. or less and PSA density less than 0.1., 75% had an unimportant cancer compared to 5% of the remaining 158 (p < 0.0005). CONCLUSIONS: Quantitative analysis of systematic biopsy specimens combined with PSA density provides valuable staging information and helps to identify cancers of low biological potential.     

Estrone formation from dehydroepiandrosterone in cultured human breast adipose stromal cells

OBJECTIVE: To describe the phenotype in a family with dominantly inherited cone-rod dystrophy with chromosome assignment to a 19q locus, and to correlate this with current classifications of this retinal dystrophy. DESIGN: A detailed clinical examination including Goldmann perimetry was undertaken in all family members. Six members under the age of 30 years underwent dark-adapted electroretinography, color contrast-sensitivity measurement, dark-adapted static perimetry, and dark adaptometry. PATIENTS: The study included 34 affected and 22 unaffected patients in four generations of a pedigree that manifested autosomal dominant cone-rod retinal dystrophy linked to a chromosome 19q locus by genetic linkage analysis. RESULTS: Loss of visual acuity occurred in the first decade of life, onset of night blindness occurred after 20 years of age, and little visual function remained after the age of 50 years. Central and, later, peripheral retinal fundus changes were associated with central scotoma, pseudoaltitudinal field defects, and finally global loss of function. Psychophysical and electrophysiologic testing before the age of 26 years showed more marked loss of cone than rod function. CONCLUSIONS: The phenotype associated with this mutation does not fit well into previous subtypes of cone-rod dystrophy. Further studies will be needed to correlate specific genetic mutations in this group of conditions with the various clinical phenotypes.     

Organic osmolyte channels: transport characteristics and regulation

Erythrocytes of the skate (Raja erinacea) exposed to hypotonic stress swell and then undergo a volume regulatory decrease by releasing taurine and other osmolytes. Previous studies showed that taurine release occurs via a volume-activated, Na(+)-independent, bi-directional transporter that has the properties of a size-limited channel. We now report on the transport characteristics of this channel and its regulation. Kinetic, competition and inhibitor studies indicate that polyols (myo-inositol) and trimethylamines (betaine) are transported by the same channel as taurine. Although the identity of the channel is still unknown a variety of evidence suggests that band 3 is involved in either channel formation or regulation. Hypotonicity causes phosphorylation and structural changes in band 3. Under isotonic conditions band 3 is predominantly in the dimeric form. Hypotonicity causes a shift to tetrameric band 3. We hypothesize that the band 3 tetramer either forms or regulates an osmolyte channel. The finding that expression of band 3 protein increases osmolyte channel activity in Xenopus oocytes supports this hypothesis.