Death of an apprentice bodybuilder following 2,4-dinitrophenol and clenbuterol intake

International Journal of Legal Medicine - We present the case of a 17-year-old man, who died after 2,4-dinitrophenol (DNP) and clenbuterol consumption, which he likely took for physical...     

Infant mortality and prenatal care: contributions of the clinic in the light of Canguilhem and Foucault

This review study aimed to verify how studies conducted in Brazil have related infant mortality to prenatal care and to present contributions of the clinic in the light of Canguilhem and Foucault for qualification of the care. An integrative literature review was conducted from searches in the databases SciELO, LILACS, MEDLINE and BDENF for the period 2000 to 2009. The relationship between infant mortality and prenatal care is related to the insufficient number of consultations or to the quality of the care provided. Even when the number of and routine consultations in the prenatal care were adequate, avoidable deaths were present. For the qualification of prenatal care, it is suggested that the clinical knowledge and other elements that comprise the process of human living are considered, in order that the clinical view is enlarged and articulated to the technologies available in the health system and, together, they are able to contribute to the reduction of infant mortality in Brazil.     

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306–E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron–electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111–114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2–3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.Proteins in solution exist in conformational equilibria that cannot be appreciated from structures observed in crystal lattices (1–5). The members of a folded conformational ensemble may have distinct functions and hence are of interest in elucidating mechanisms of protein action (5–7). The free-energy differences between the conformations can range from zero to a few kilocalories per mole; the higher free-energy states are referred to as “invisible” or “excited” (E) states owing to their low equilibrium populations. The structural transition between the native ground state (G) and the E state may involve rigid body motion of the peptide backbone (5, 8) or local unfolding (9).For a complete understanding of molecular mechanisms underlying function, characterization of functionally relevant conformational substates is required. However, in the case of E states, low populations and short lifetimes present a challenge for biophysical characterization. The elegant high-resolution NMR studies from Akasaka (10, 11) and coworkers suggest that application of hydrostatic pressure on the order of a few kilobars may solve this problem by reversibly populating functional E states, making them amenable for study by spectroscopic methods. For example, high-pressure NMR has been used to identify and characterize E states crucial to ligand binding in ubiquitin (12) and dihydrofolate reductase (13). As a result, high-pressure biophysics is currently of mainstream interest in protein science.Application of sufficiently high pressure leads to population of a “pressure-denatured” state. For many proteins this state retains a relatively compact fold, at least near neutral pH and in the absence of denaturants, and is thus distinct from the unfolded states produced thermally or by chemical denaturants wherein both secondary and tertiary structures are largely lost (14–20). The pressure-denatured state is often labeled as the “unfolded state” (10), but here we reserve the term “unfolded” to describe a state with little tertiary or secondary structure. In this paper attention is focused on moderate pressures (<4 kbar) that shift conformational equilibria rather than leading to a pressure-denatured state.Pressure shifts equilibria in a direction to reduce the total volume of the system. The current view is that the volume reduction that accompanies pressure-modulated transitions in proteins, including formation of the denatured state, is dominated by the elimination of voids or cavities in the protein’s interior (14, 21–24) via hydration, although other factors contribute (15, 25–28). In the equilibrium between two folded conformations, G ↔ E for example, an alternative “structure-relaxation” mechanism may play a role in the pressure response. In this model, voids are eliminated by pressure owing to an increase in the population of an alternative packing arrangement of the core in which cavities are filled with native side chains rather than solvent. This model has been suggested to play a role in certain proteins at high pressure (1, 29–31), although to our knowledge direct observation of structure relaxation under pressure has not been reported.That a structure-relaxation mechanism may play a role in the pressure response is suggested by recent studies of cavity-creating mutants in T4 lysozyme (T4L) at atmospheric pressure (1). Although the crystal structures of the cavity mutants are nearly identical to those of the WT protein (32–35), in solution there are multiple conformations in equilibrium (1, 4). For example, in the T4L cavity mutants L121A/L133A, L133G, and W138A, two conformations of similar free energy were identified in solution (1). Perhaps the most extensively investigated cavity-forming mutation is T4L L99A that enlarges a preexisting cavity in the rigid four-helix bundle of the protein. In this mutant, a minor conformation (E) in equilibrium with the ground state (G) was detected that accounted for ∼3% of the population (4, 36). Remarkably, in each of the above cavity-creating mutants, one member of the conformational ensemble corresponds to a structural rearrangement that fills the cavity with a side chain; for L99A it is Phe114 that occupies the engineered cavity (5). Such conformations in which cavities are absent or reduced are expected to have a lower molar volume, and thus may be populated by pressure.The pressure dependence of the L99A mutant was recently studied with NMR methods by Nucci et al. (27) and independently by Maeno et al. (31). In the study of Maeno et al. (31), the disappearance of cross-peaks in a ¹H-¹³C heteronuclear single-quantum coherence (HSQC) NMR experiment at pressures up to 3 kbar was interpreted to reflect an increase in population of the E conformation with Phe114 occupying the cavity, consistent with a structure-relaxation mechanism rather than cavity hydration or unfolding. However, based on ¹H-¹⁵N HSQC NMR studies on the same protein, Nucci et al. (27) concluded that cavity hydration and unfolding occurred at pressures less than 2.5 kbar rather than populating the E conformation. Commentaries on these conflicting interpretations were recently published (37, 38).In the present study, we investigate the pressure dependence of T4L WT*, L99A, and L99A containing the additional mutations G113A and G113A/R119P, both of which have been shown to lower the free energy of the E conformation and hence increase the equilibrium population (5). The experimental approach is based on site-directed spin labeling EPR (SDSL-EPR) and the recently developed technologies of high-pressure continuous wave (CW) EPR spectroscopy for SDSL (30, 39), pressure-resolved double electron–electron resonance (PR DEER) spectroscopy (40), and high-pressure circular dichroism (HP-CD) (30). HP-CD reveals the global secondary structure of the protein at pressures up to 2.4 kbar. CW EPR line shapes of spin-labeled proteins are sensitive to backbone fluctuations on the nanosecond time scale (41, 42) and can unambiguously identify site-specific unfolding under pressure to 4 kbar, as well as identify sequences in slow conformational exchange on the microsecond–millisecond time scale (30, 39). The intrinsic time scale of SDSL-EPR is much shorter than NMR, such that spectral averaging of microsecond–millisecond protein conformational exchange does not occur. Hence, EPR spectra of a spin-labeled protein provide a “snapshot” of conformational equilibria frozen in time. Although the CW spectra can reveal conformational equilibria with exquisite sensitivity, they do not provide quantitative information on the structures involved or resolve the true heterogeneity of a conformational manifold. This information is uniquely provided by distance mapping using pairs of spin labels and DEER spectroscopy. At the present state of development, PR DEER allows direct structure mapping of the states populated by pressure to 6 kbar.The results reported below indicate that at pressures up to 2.4 kbar the secondary structure content of T4L L99A is unchanged, eliminating the possibility of global or subglobal unfolding of the helical C domain, in agreement with the results of Maeno et al. (31). Collectively, the data do not support a large-scale shift to the E conformation of T4L L99A under high pressure, but rather cavity hydration and finally transition to a partially disordered state at pressures of 4 kbar. However, pressure strongly shifts the G ↔ E equilibrium toward E in the presence of the additional G113A and R119P mutations that lower the energy of the E conformation, thus demonstrating a structure-relaxation mechanism for the pressure response. Moreover, ligand binding to the engineered cavity strongly stabilizes the G conformation. Taken together, the results show that both cavity hydration and structure relaxation are valid models for the pressure dependence in proteins, and which prevails depends on the details of the energy landscape.     

Evaluation of right atrial size in patients with atrial arrhythmias: comparison of 2D versus real time 3D echocardiography

Aim: This study compares different parameters of right atrial size evaluated by two-dimensional (2D) echocardiography with right atrial volume measured using three-dimensional echocardiography (3DE). Methods and Results: One hundred sixty-three consecutive patients with a history of atrial arrhythmias were studied by standard two-dimensional and by real time 3DE. Of these 142 (87%) recordings were of sufficient quality for interpretation of the right atrium by both imaging techniques. The following parameters of right atrial size were measured: apical four-chamber short-axis diameter (4CH short axis), apical four-chamber long axis diameter (4CH long axis), and apical four-chamber planimetry area. The 2D-derived right atrial volume was calculated by using the single plane area-length method (4CH area-length). The 2D parameters were then correlated with right atrial volume measured by real time 3DE. Linear regression analysis showed moderate correlation for four-chamber planimetry area (r = 0.72, P < 0.001) and 2D-derived volume calculation (r of 4CH single plane area-length RA volume = 0.70, P < 0.001). Diameters correlated clearly less well with 3DE volume (r of 4CH short axis = 0.61, 4CH long axis = 0.59, P < 0.001 respectively). Conclusion: Real time 3DE is highly feasible for right atrial volume determination. The results demonstrate that measurements of dimensions using 2D echocardiography may not accurately assess right atrial size. If 3DE is not available, apical 4CH planimetry area is a simple alternative that may be used for evaluating right atrial size in clinical practice. The 2D-derived right atrial volume by single plane area-length method was not better correlated with 3DE volume than four-chamber planimetry area.     

N-Cadherin cleavage during activated hepatic stellate cell apoptosis is inhibited by tissue inhibitor of metalloproteinase-1

Apoptosis of hepatic stellate cells (HSC) has previously been shown to occur during spontaneous resolution of experimental liver fibrosis. TIMP-1 has also been shown to have a key role because of its ability to inhibit apoptosis of HSC via matrix metalloproteinase (MMP) inhibition. This has led to further study of novel substrates for MMPs that might impact on HSC survival. N-Cadherin is known to mediate cell-cell contacts in fibroblasts. In this study we demonstrate that N-Cadherin is expressed by activated rat HSC. Furthermore, during apoptosis of HSC, the N-Cadherin is cleaved into smaller fragments. Apoptosis of HSC may be inhibited by TIMP-1. This is associated with reduced fragmentation of N-Cadherin. N-Cadherin may have an important role in supporting HSC survival while N-Cadherin cleavage may play a part in promoting HSC apoptosis in recovery from liver fibrosis.     

Alloimmunization prevents the migration of transfused indium-111- labeled granulocytes to sites of infection

111In-labeled granulocytes were used to study the effects of histocompatibility factors on the migration of transfused granulocytes to infected sites. Fourteen alloimmunized and 20 nonalloimmunized patients received approximately 10(8) 111In-labeled granulocytes from ABO-compatible, non-HLA-matched donors, and scans were performed over known infected sites. All 14 alloimmunized patients had lymphocytotoxic antibody (LCTAb) and required HLA-matched platelet transfusions. Of the nonalloimmunized patients, 20/20 had positive scans at sites of infection. None of the 20 had LCTAb, 0/17 had a positive lymphocytotoxic crossmatch (LCTXM) with the donor, and 3/18 had a positive leukoagglutinin crossmatch (LAXM). Thus, histocompatibility testing was not found to be important in nonalloimmunized patients. In contrast, only 3/14 alloimmunized patients had positive scans at sites of infection (p = 0.00001 compared to nonalloimmunized patients). One of 3 had a positive LCTXM and 2/3 had a positive LAXM. Of the alloimmunized patients, 10/11 with negative scans had a positive LCTXM and 8/11 had a positive LAXM. Labeled granulocytes failed to reach sites of infection in 11/14 (78%) alloimmunized patients, demonstrating that histocompatibility factors can be of major importance in affecting the outcome of granulocyte transfusions. Granulocytes from random donors are unlikely to be effective in alloimmunized patients. The lack of an adequate crossmatching technique is a major problem limiting the ability to provide granulocyte transfusions for alloimmunized patients.     

Up-regulation,nuclear import,and tumor growth stimulation of the adhesion protein p120 in pancreatic cancer

BACKGROUND & AIMS: Cell adhesion proteins have been implicated as tumor suppressors because they prevent malignant cells from dissociating their cell contacts. We have studied the role of p120(ctn), a recently discovered member of the cadherin/catenin family, in human pancreatic cancer. METHODS: In 32 resection specimens of pancreatic adenocarcinoma and 10 control samples the expression of p120(ctn) was studied by Northern blot, immunocytochemistry, and immunogold electron microscopy. Patient survival data, tumor grading, and staging were correlated to the experimental results. In PaTu 8889 T pancreatic cancer cells, p120(ctn) expression was suppressed with 21-nucleotide small interfering RNA (siRNA) duplexes and proliferation was determined by bromodeoxyuridine (BrdU) incorporation. RESULTS: In pancreatic cancer p120(ctn) messenger RNA (mRNA) was increased 3- to 4-fold. Although p120(ctn) was localized exclusively at cell contacts in controls it was found in the cytosol and nucleus of pancreatic cancer cells. This redistribution correlated to the degree of tumor dedifferentiation but was independent of tumor stage. The mean survival of patients with predominant membrane localization of p120(ctn) was 24 +/- 7 (SEM) months vs. 9 +/- 2 months for patients with predominant cytoplasmic p120(ctn) expression (P < 0.05). Silencing of p120(ctn) with siRNA duplexes reduced pancreatic cancer cell growth by 40%. CONCLUSIONS: Up-regulation, cytoplasmic redistribution, and nuclear import of p120(ctn) are associated with a more malignant phenotype of pancreatic cancer. This study further represents conclusive evidence for a direct involvement of p120(ctn) in malignant tumor cell proliferation. Both p120(ctn)-defective tumor cell contacts and p120(ctn)-mediated growth signals appear to contribute to the aggressive spread of pancreatic cancer.     

Papillary mesothelioma of the peritoneum in the absence of asbestos exposure

BACKGROUND: Malignant mesothelioma of the peritoneum is a very rare neoplasm, commonly associated with asbestos exposure and often rapidly fatal. Well Differentiated Papillary Mesothelioma of the Peritoneum (WDPMP) is regarded as a less aggressive variety of the tumor. Progressive ascites is often the only clinical manifestation of the disease and differentiation of WDPMP from benign mesothelial hyperplasia or adenocarcinoma is difficult. PATIENTS AND METHODS: Here we report the case of a 45-year-old patient who presented with ascites but without evidence of portal hypertension, liver disease or abdominal malignancy. On diagnostic laparoscopy small tumor nodules were found to cover the parietal peritoneum and the greater omentum and histopathologically corresponded to papillary mesothelial hyperplasia with minimal nuclear atypia. Histochemically biopsies were positive for Calretinin, Cytokeratins and Epithelial Membrane Antigen (EMA). Based on these findings the diagnosis of WDPMP was made and the patient was closely followed without primary cytostatic therapy. CONCLUSIONS: Progressive ascites was the only clinical symptom in this patient, while liver disease, portal hypertension and gastrointestinal malignancies were ruled out by clinical, laboratory and imaging techniques. Laparoscopic biopsy revealed WDPMP to be the underlying disease. Immunocytochemistry is required to establish the diagnosis of this rare malignant disorder which is even more uncommon in the absence of a history of asbestos exposure. Due to the indolent course of WDPMP therapy should only be initiated when signs of rapid tumor progression become apparent.     

起搏器心内膜炎的外科治疗

目的　介绍 15例起搏器心内膜炎外科治疗的临床经验 ,讨论手术指征和时机。方法　1993年至 2 0 0 1年间 ,我们为 15例起搏器心内膜炎患者 ,施行了体外循环直视下心内起搏电极撤除术。本文对这些病人的临床资料进行回顾性研究。结果　本组病例中 ,有 2例院内死亡 ,术后随访期内 (中间值 =31 3个月 ;区间 ,1 8～ 5 8 7个月 )无远期死亡 ,总死亡率为 13 3%。所有出院病人均无感染复发。大部分病人心功能明显改善 (P <0 0 1)。只有 6例患者术后因持续存在的心律失常需要重新安装起搏器。本组病例中常见的外科手术指征为 :1)附着于起搏电极的大型赘生物 ,2 )顽固感染 ,3)三尖瓣架构损坏 ,4)肺栓塞 ,5 )存在需要外科处理的基础的或并存的心脏病变。结论　根据我们的结果及相关文献报道 ,我们认为对患起搏器心内膜炎的病人 ,应尽早采用外科手术撤除植入的心内起搏器硬件。手术治疗的远期效果是值得信赖的。     

Diagnosis and treatment of pancreatic pseudocysts in chronic pancreatitis

Pancreatic pseudocysts are a well-known complication of acute or chronic pancreatitis, with a higher incidence in the latter. Diagnosis is accomplished most often by computed tomographic scanning, by endoscopic retrograde cholangiopancreatography, or by ultrasound, and a rapid progress in the improvement of diagnostic tools enables detection with high sensitivity and specificity. Different strategies contribute to the treatment of pancreatic pseudocysts: endoscopic transpapillary or transmural drainage, percutaneous catheter drainage, or open surgery. The feasibility of endoscopic drainage is highly dependent on the anatomy and topography of the pseudocyst, but provides high success and low complication rates. Percutaneous drainage is used for infected pseudocysts. However, its usefulness in chronic pancreatitis-associated pseudocysts is questionable. Internal drainage and pseudocyst resection are frequently used as surgical approaches with a good overall outcome, but a somewhat higher morbidity and mortality compared with endoscopic intervention. We therefore conclude that pseudocyst treatment in chronic pancreatitis can be effectively achieved by both endoscopic and surgical means. This review entails publications referring to the classification of pancreatic pseudocysts, epidemiology, diagnostic tools, and therapeutic options for pancreatic pseudocysts. Only full articles were considered for the review. Based on a search in PubMed, the MeSH terms "pancreatic pseudocysts and classification," "diagnosis," and "endoscopic, percutaneous, and surgical treatment" were used either alone or in combination.