Genomic structure of proton-coupled oligopeptide transporter hPEPT1 and pH-sensing regulatory splice variant

Proton-coupled oligopeptide transporter PEPT1 facilitates the transport of dipeptides and peptoid drugs (including antibiotics) across the cell membranes of endothelial and epithelial cells. Substrate transport by the proton symport is driven by pH gradients, while the profile of pH sensitivity is regulated by a closely related protein. hPEPT1-RF. We investigated the genomic structure of hPEPT1 and hPEPT1-RF. Analysis of the high-throughput genomic sequence (HTGS) database revealed that hPEPT1 and hPEPT1-RF are splice variants encoded by the same gene located in chromosome 13, consisting of 24 exons. hPEPT1 is encoded by 23 exons and hPEPT1-RF by 6 exons. Coding sequences of hPEPT1-RF share 3 exons completely and 2 exons partially with hPEPT1. The genomic organization of hPEPT1 shows high similarity with its mouse orthologue. Exon-intron boundaries occur mostly in the loops connecting transmembrane segments (TMSs), suggesting a modular gene structure reflecting the TMS-loop repeat units in hPEPT1. The putative promoter region of hPEPT1 contains TATA boxes and GC-rich regions and a potential insulin responsive element.     

A new triphenylethylene compound,Fc-1157a

Summary The antitumor effects of a new antiestrogen, Fc-1157a¹ have been studied in vitro and in vivo. In vitro the effect of Fc-1157a was comparable to that of tamoxifen. The effect was dose-dependent, and at concentrations higher than 10^-6 mol/l Fc-1157a induced real cell death of the MCF-7 cells. In DMBA-induced mammary cancer in rats Fc-1157a decreased the number of new tumors and inhibited the growth of existing tumors, these effects being statistically highly significant. The ratio of growing tumors to stable and regressing tumors was significantly decreased. Although these effects were slightly stronger with Fc-1157a than with tamoxifen, the difference between these two compounds was not statistically significant.Murine uterine sarcoma, an estrogen receptor-negative tumor, was resistant to tamoxifen, but was statistically significantly inhibited by high doses (100 and 200 mg/kg^-1 day^-1 for 5 days) of Fc-1157a.The antitumor effects of Fc-1157a are due mainly to the antiestrogenic activity. At high concentrations in vitro and at high doses in vivo Fc-1157a exerts antitumor effects some of which are different from those of tamoxifen and are directed even against estrogen receptor-negative tumors. The exact mechanism of the observed cytolytic effect at high doses is unknown.     

Different synergistic roles of small polyethylenimine and Dosper in gene delivery.

Low-molecular-weight PEIs and cationic liposomes can be combined resulting in a synergistic increase in transfection efficiency as we have reported earlier. Here, we have further investigated the potential mechanisms of this synergy. Complex morphology, complex sizes and DNA condensation were studied using transmission electron microscopy, light scattering methods and ethidium bromide exclusion, respectively. Cellular uptake, transfection efficiency, and effect of proton pump inhibitor bafilomycin A1 were examined in cell cultures. The cellular uptake of DNA was negligible with PEI2K-DNA complexes, whereas the uptake of the PEI2K-DNA-Dosper or the Dosper-DNA complexes was maximally about 40%. The number of transfected cells was two times higher with PEI2K-DNA-Dosper complexes than with Dosper-DNA complexes. The PEI2K-DNA-Dosper combination was slightly less sensitive to bafilomycin A1 than the PEI25K-DNA or Dosper-DNA complexes. There were no differences between PEI2K and PEI25K in DNA condensation. Dosper condensed DNA slightly more in PEI2K complexes. The PEI25K-DNA complexes were much smaller (<250 nm) than the PEI2K-DNA complexes (0.5-12 micro m) which were also rather polydisperse. It is suggested that two independent mechanisms would lead to synergistic transfection efficiency: (1) Dosper improves the cellular uptake of PEI2K-DNA complexes, and (2) PEI2K improves a transfer of the complexes from lysosomes to nucleus.     

Extracellular and intracellular factors influencing gene transfection mediated by 1,4-dihydropyridine amphiphiles

Double-charged 1,4-dihydropyridine (1,4-DHP) amphiphiles have been shown to condense DNA and efficiently transfect it into cells in vitro [Hyvönen et al., Biochim. Biophys. Acta 1509 (2000) 451]. Alkyl chain length and buffering capacity at endosomal pH range (5.0–7.4) affected complexation and transfection activity. In this study we examined how those chemical modifications of amphiphile–DNA complexes (amphiplexes) affect their interactions with extracellular polyanions (glycosaminoglycans, albumin) and lipid bilayers, their cellular uptake and intracellular distribution. To evaluate cellular uptake, CV1-P cells were incubated with labeled DNA–amphiphile complexes and analyzed by flow cytometry. Confocal laser fluorescence microscopy was used to investigate the intracellular distribution of amphiplexes. The results showed that biophysical properties of compounds can be changed by slight structural modifications. These factors determine the intracellular kinetics and transfection efficacy of the compounds. Some extracellular glycosaminoglycans and serum interfere with 1,4-DHP-amphiphile-mediated transfection by destabilizing the amphiplexes. Neither high cellular uptake, membrane destabilizing activity nor buffering capacity alone is adequate for high transfection efficacy. The activity results from complex interplay of various factors that determine intracellular kinetics and, consequently, transfection.     

Lumbar spinal stenosis patients are satisfied with short-term results of surgery - younger age, symptom severity, disability and depression decrease satisfaction

Purpose. To study the lumbar spinal stenosis (LSS) patients' (n = 98) satisfaction with surgery outcome and associated factors at three months post-operative stage.

Method. LSS-related physical functioning and pain were assessed with Oswestry disability index, Stucki questionnaire, Visual Analogue Scale and pain drawings. Depression was assessed with 21-item Beck Depression Inventory. Psychological well-being was assessed with the Life satisfaction scale, Toronto Alexithymia Scale and Sense of Coherence Scale. All questionnaires were administered before and 3 months after surgical treatment of LSS. Satisfaction with surgery outcome was assessed with a separate scale.

Results. Considerable improvement was evident in all the functional and pain-related variables. Two-thirds (66%) of the patients were at least clearly satisfied with the surgery outcome. Younger age, symptom severity, disability and depression were independently associated with dissatisfaction with surgery outcome.

Conclusion. The lack of physical, functional and emotional well-being is associated with the patients' dissatisfaction with the surgery outcome. Patient satisfaction is a valid outcome to be measured in LSS patients undergoing surgery. It is recommended that patients should be provided with realistic pre-operative patient information and that depression be assessed pre-operatively.     

The clinical utility of serum anti‐Müllerian hormone in the follow‐up of ovarian adult‐type granulosa cell tumors—A comparative study with inhibin B

Ovarian adult‐type granulosa cell tumors (AGCTs) require prolonged follow‐up, but evidence regarding the optimal follow‐up marker is lacking. The objective of our study was to validate the clinical usefulness of serum anti‐Müllerian hormone (AMH) and the current marker inhibin B as single and combined markers of AGCTs. We conducted a longitudinal, partially prospective cohort study of 123 premenopausal and postmenopausal AGCT patients with a median follow‐up time of 10.5 years (range 0.3–50.0 years). Serum AMH and inhibin B levels were measured from 560 pretreatment and follow‐up serum samples by using immunoenzymometric assays. We found that serum AMH and inhibin B levels were significantly elevated in patients with primary or recurrent AGCTs. The levels of both markers positively correlated to tumor size (p < 0.05). AMH and inhibin B performed similarly in receiving operator characteristic analyses; area under the curve (AUC) values were 0.92 [95% confidence interval (CI) 0.88–0.95] for AMH, and 0.94 (95% CI 0.90–0.96) for inhibin B. AMH was highly sensitive (92%) and specific (81%) in detecting a macroscopic AGCT. However, in AUC comparison analyses, the combination of the markers was superior to inhibin B alone. In conclusion, serum AMH is a sensitive and specific marker of AGCT, and either AMH or inhibin B can be monitored during follow‐up. However, combining AMH and inhibin B in AGCT patient follow‐up improves the detection of recurrent disease.     

Feasibility of abdominal plain film images in evaluation suspected drug smuggler

Objective

Drug smuggling in the gastrointestinal tract has soared within the last 20 years. Though illegal substances in the gastrointestinal tract can be visualized with ultrasound, MRI and CT, the abdominal radiograph has by far remained the most frequently used way of detecting smuggled drugs. The purpose of the study was to evaluate the inter-radiologist interpretation error and the reliability of the abdominal radiograph in detecting smuggled drugs.

Materials and methods

A total of 279 abdominal radiographs of suspected smugglers were classified by three radiologists as clearly positive or negative for drug smuggling. All available information about the cases was collected from the customs officers and police.

Results

Out of these cases 203 (73%) were interpreted as negative and 35 (13%) as positive by all three radiologists. In 86% of the cases there was, therefore, an inter-radiological agreement in interpreting the images. In 41 (14%) cases, however, there was an inter-radiologist disagreement. Kappa-value for inter-observer variability was 0.70.

Conclusions

In up to a seventh of the abdominal radiographs the interpretation can be challenging even for an experienced radiologist. False positive interpretation can lead to innocent passengers being detained in vain. As negatively interpreted images usually result in releasing of the suspect, there is no way of knowing how many false negative occur. This makes the abdominal radiograph a suboptimal examination, and low dose CT should be considered as the screening modality for gastrointestinal drug smugglers.     

Erratum to: Long-term results of surgery for lumbar spinal stenosis: a randomised controlled trial

European Spine Journal -     

The clot burden score, the Boston Acute Stroke Imaging Scale, the cerebral blood volume ASPECTS, and two novel imaging parameters in the prediction of clinical outcome of ischemic stroke patients receiving intravenous thrombolytic therapy

Introduction

Recently two classification methods based on the location and the extent of thrombosis detected with CT angiography have been introduced: the Boston Acute Stroke Imaging Scale (BASIS) and the clot burden score (CBS). We studied the performance of BASIS and CBS in predicting good clinical outcome (mRS ≤2 at 90?days) in an acute (<3?h) stroke cohort treated with intravenous thrombolytic therapy.

Methods

Eighty-three consecutive patients who underwent multimodal CT were analyzed. Binary logistic regression model was used to assess how BASIS, CBS, and cerebral blood volume (CBV) ASPECTS predict favorable clinical outcome. Diagnostic sensitivities and specificities were calculated and compared.

Results

Patients with low CBS and CBV ASPECTS scores and major strokes according to BASIS had significantly higher admission NIHSS scores, larger perfusion defects, and more often poor clinical outcome. In logistic regression analysis, CBV ASPECTS, CBS and BASIS were significantly associated with the clinical outcome. The performance of BASIS improved when patients with thrombosis of the M2 segment of the middle cerebral artery were classified as having minor stroke (M1-BASIS). In the anterior circulation, the sum of CBS and CBV ASPECTS (CBSV) proved to be the most robust predictor of favorable outcome. CBV ASPECTS and CBS had high sensitivity but moderate to poor specificity while BASIS was only moderately sensitive and specific.

Conclusion

CBS, BASIS, and CBV ASPECTS are statistically robust and sensitive but unspecific predictors of good clinical outcome. Two new derived imaging parameters, CBSV and M1-BASIS, share these properties and may have increased prognostic value.     

CD14 and TNfa promoter polymorphisms in patients with acute arthritis. Special reference to development of chronic spondyloarthropathy

OBJECTIVE: To examine CD14 and TNFalpha gene polymorphisms in early arthritis in relation to clinical outcome. METHODS: We studied 141 Caucasians who had had early arthritis 10 to 38 years earlier. We analysed CD14 (-159) and TNFalpha (-238, -308, -376) polymorphisms using a novel cycle minisequencing method. DNA pools from 370 Caucasian blood donors served as controls. RESULTS: CD14 (-159)C-->T allele frequencies were comparable among patients and controls (39% vs 40%). Fifty men and 42 women had recovered while 24 men and six women had chronic spondyloarthropathy (SpA). Mutant T allele frequency was higher in the chronic SpA group than in the recovered group in women (75% vs 32%, relative risk 1.3, 95% confidence limit 1.1 to 1.6, P = 0.011), but not in men (38% vs 44%). All female patients with chronic SpA had CD14 (-159)T allele and none had a possibly protective TNFalpha (-308)G-->A allele. CONCLUSIONS: Possession of CD14 (-159)T allele does not increase risk of ReA but may increase susceptibility of female patients to development of chronic SpA.