Reepithelialization of experimental scalds effected by topically applied superoxide dismutase: controlled animal studies

Highly reactive metabolites, such as oxygen free radicals, initiate a cascade of inflammatory processes in thermally damaged skin, leading to enhanced tissue loss and delayed wound healing. The extent of tissue necrosis in the zone of stasis is of prognostic significance in the wound healing process. In this study, the effect of oxygen free radical removal by recombinant human-Cu/Zn-superoxide dismutase, given in three different formulations during the inflammatory postburn phase and wound repair, was examined. Recombinant human superoxide dismutase was either injected directly into the lesions, spread as enzyme-containing gel onto the burned tissue, or encapsulated into liposomes consisting of 1,2 dipalmitoy-sn-glycero-3-phosphocholine, cholesterol and stearylamine, suspended into a hydrophilic gel and administered to burned animals immediately after trauma. Controls were treated with plain gel or kept untreated. Edema formation, size of lesions, deepening of necrosis, and reepithelialization were examined. Results indicate that superoxide dismutase treatment resulted in reduced and faster recruitment of edema formation, smaller wound sizes, and minor tissue necrosis compared to the controls, thus resulting in significantly faster reepithelialization after 3 weeks. These animal studies on the efficacy of liposomal oxygen free radical scavenger showed accelerated wound healing in all parameters tested.     

Renal findings on radiological followup of patients with Beckwith-Wiedemann syndrome

PURPOSE: The Beckwith-Wiedemann syndrome is most commonly characterized by macroglossia and abdominal wall defect(s), and it carries a predisposition to embryonal tumors, including Wilms tumor. We report our experience with the character and incidence of renal disease in patients with the Beckwith-Wiedemann syndrome, and discuss the role of radiological followup. MATERIALS AND METHODS: We reviewed the medical records of all patients diagnosed with the Beckwith-Wiedemann syndrome who were treated at our institution between March 1979 and February 1998. Radiological followup consisted of renal ultrasound at approximately 3 to 6-month intervals with the addition of computerized tomography or magnetic resonance imaging (MRI) in patients with an indeterminate lesion(s) or nephrogenic rest(s). RESULTS: A total of 29 patients were identified. Of these cases renal ultrasound showed normal kidneys bilaterally in 19 (70%), simple cysts in 5 (19%), indeterminate lesion(s) in 2 (7%) and nephrocalcinosis in 1 (4%). Nephrogenic rests were followed with MRI in 1 patient, and 1 in whom a 2 cm. mass was revealed by followup MRI underwent partial nephrectomy and chemotherapy for stage I Wilms tumor. CONCLUSIONS: The 3.7% incidence of Wilms tumor in our patients with the Beckwith-Wiedemann syndrome is similar to that in previously published reports. Aggressive follow-up by a sensitive radiological technique is warranted in cases of the Beckwith-Wiedemann syndrome, and associated hemihypertrophy and/or nephromegaly with or without evidence of a Wilms tumor precursor. The detection of suspected malignant disease at an early stage may permit curative nephron sparing surgery.     

The overactive bladder in childhood: long-term results with conservative management

PURPOSE: Idiopathic detrusor overactivity has not been thoroughly investigated and its natural history remains largely anecdotal. Bladder overactivity resulting from a neurogenic, anatomical or medical condition has been well described. Therefore, we assessed the long-term results of conservative treatment of children with idiopathic symptomatic refractory detrusor instability. MATERIALS AND METHODS: We reviewed the records of 58 patients who had an isolated finding of uninhibited contractions on urodynamics performed for refractory enuresis and daytime wetting between 1988 and 1994. Study exclusion criteria were chronic urinary tract infection, neurological lesion, anatomical abnormality of the lower urinary tract and less than 12 months of followup. RESULTS: Of the 30 children who met our study inclusion criteria 26 (87%) had complete (21) or significant (5) symptom resolution. Average time to resolution was 2.7 years (range 0.2 to 6.6). Patients with a 50% to 90% bladder capacity expected for age were more likely to benefit from therapy than those with a bladder capacity outside of this range. Age and gender were not significant predictors of resolution although girls were more likely to have resolution than boys. CONCLUSIONS: Idiopathic detrusor instability is amenable to conservative management in the majority of patients during a prolonged period. We advocate thorough urological and urodynamic evaluation to identify idiopathic detrusor instability as an etiology of enuresis and daytime wetting in complicated cases.     

Inhibition of pressure induced bladder smooth muscle cell hyperplasia using CRM197

PURPOSE: In vivo the effects of sustained hydrostatic pressure on the bladder wall and its components are evident under physiological and pathological conditions. We previously reported that exposure of bladder smooth muscle cells to 20 and 40 cm. H2O hydrostatic pressure for as little as 1 hour resulted in the up-regulation of heparin binding epidermal growth factor messenger RNA in a time dependent fashion as well as in activation of the heparin binding epidermal growth factor growth factor gene. In our current study we investigated the use of CRM197 as an agent for blocking undesirable cellular level events, such as smooth muscle cell hyperplasia, eliminating the irreversible alterations in bladder and kidney function that result from chronic and/or severe bladder outlet obstruction. MATERIALS AND METHODS: Control and experimental neonatal ovine smooth muscle cells were exposed to 0.3 pressure and 8.5 cm. H2O, respectively, for 7 days. We evaluated the mitogenic activity of the supernatant medium from bladder smooth muscle cells exposed to 8.5 cm. H2O for 5 days (conditioned medium) before and after the addition of 0.1 mg./ml. CRM197. Bladder smooth muscle cell apoptosis was also assessed after CRM197 exposure. Statistical analysis was performed using the Student t test with p <0.05 considered significant. RESULTS: Exposing bladder smooth muscle cells to sustained 8.5 cm. H2O hydrostatic pressure for 7 days resulted in increased cell proliferation. Conditioned medium contained mitogenic activity, which was ablated after CRM197 was added. No direct toxic effect of CRM197 on bladder smooth muscle cell growth was appreciated (no apoptosis). CONCLUSIONS: We demonstrated a proliferative response of neonatal bladder smooth muscle cells after exposure to sustained hydrostatic pressure. This response was partially due to the release of heparin binding epidermal growth factor and was blocked by adding CRM197. These data support the potential use of CRM197 in drug targeted therapy for diseases involving bladder outlet obstruction.     

Trends in management and outcomes of pulmonary embolism with a multidisciplinary response team

Journal of Thrombosis and Thrombolysis - Multidisciplinary pulmonary embolism (PE) response teams have garnered widespread adoption given the complexities of managing acute PE and provide a...     

A cross-sectional study of newborns over a 20-year period in Szeged,Hungary

Objective: Records of metric data of birth, serve not only the medical needs of the newborn baby, but are also indicators to assess the status of public health.

Methods: This is a retrospective study of 4946 newborns (singleton: 2508 boys and 2365 girls) born in 1989 and in 2009 at the Department of Obstetrics and Gynaecology of the University of Szeged. We aimed as to compare and map the metrical changes over 20 years, and to describe the averages of four body parameters of the normal birth weight (2500–4000?g) subgroup (3993 singleton babies) in both years. Statistical analysis was performed with SPSS 17.0.

Results: In 1989, the mean birth weight was 3223.770?±?559.595?g, birth length 49.551?±?2.729?cm, chest circumference 32.181?±?2.231?cm, and head circumference 34.122?±?1.688?cm. In 2009, the birth weight was 3309.673?±?582.630?g, birth length 49.515?±?2.658?cm, chest circumference 32.736?±?2.392?cm and head circumference 33.854?±?1.768?cm. The mean birth weight, chest circumference and the maximum value of birth weight have thus increased. The mean maternal age shifted to 30.21?±?4.863 years, which is an increase of 3.57 years in 20 years.

Conclusion: The body parameters of newborns changed significantly between 1989 and 2009. As underlying causes changes in eating habits and lifestyle of the mother are to be mentioned.     

Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli

Crystallography has advanced our understanding of G protein–coupled receptors, but low expression levels and instability in solution have limited structural insights to very few selected members of this large protein family. Using neurotensin receptor 1 (NTR1) as a proof of principle, we show that two directed evolution technologies that we recently developed have the potential to overcome these problems. We purified three neurotensin-bound NTR1 variants from Escherichia coli and determined their X-ray structures at up to 2.75 Å resolution using vapor diffusion crystallization experiments. A crystallized construct was pharmacologically characterized and exhibited ligand-dependent signaling, internalization, and wild-type–like agonist and antagonist affinities. Our structures are fully consistent with all biochemically defined ligand-contacting residues, and they represent an inactive NTR1 state at the cytosolic side. They exhibit significant differences to a previously determined NTR1 structure (Protein Data Bank ID code 4GRV) in the ligand-binding pocket and by the presence of the amphipathic helix 8. A comparison of helix 8 stability determinants between NTR1 and other crystallized G protein–coupled receptors suggests that the occupancy of the canonical position of the amphipathic helix is reduced to various extents in many receptors, and we have elucidated the sequence determinants for a stable helix 8. Our analysis also provides a structural rationale for the long-known effects of C-terminal palmitoylation reactions on G protein–coupled receptor signaling, receptor maturation, and desensitization.Neurotensin is a 13-amino-acid peptide, which plays important roles in the pathogenesis of Parkinson’s disease, schizophrenia, antinociception, and hypothermia and in lung cancer progression (1–4). It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A G protein–coupled receptors (GPCRs) (5, 6), whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol (5, 7). Activated NTR1 is rapidly phosphorylated and internalizes by a β-arrestin– and clathrin-mediated process (8), which is crucial for desensitizing the receptor (9). Several lines of evidence suggest that internalization is also linked to G protein–independent NTR1 signaling (10, 11). To improve our mechanistic understanding of NTR1 and to gain additional insight into GPCR features such as helix 8 (H8), we were interested in obtaining a structure of this receptor in a physiologically relevant state.To date, by far the most successful strategy for GPCR structure determination requires the replacement of the intracellular loop 3 by a fusion protein, as the intracellular domain is otherwise too small to provide crystal contacts. The fusion protein approach has provided a wealth of valuable structural data on GPCRs, but as it renders the crystallized constructs signaling-inactive, the most important functionality—the activation of G proteins—cannot be confirmed for these structures. This leads inevitably to a degree of uncertainty regarding the physiological relevance of intracellular structural aspects, and it also impedes the elucidation of signaling mechanisms, as functional assays and structure determination cannot be performed with the same GPCR constructs.Crystallization in the absence of fusion proteins was so far mainly possible for rhodopsin (12), the A_2A adenosine receptor (A2AR) (13), and the β₁-adrenergic receptor (14). Together, they share a high stability, which is either given naturally (rhodopsin) or it is due to stabilizing mutations. High stability appeared to be crucial for crystallographic success, as it allowed the application of harsh short-chain detergents. These tend to form small micelles, which may explain why crystal contact formation can occur under these conditions despite the small extra- and intracellular domains of class A GPCRs.Besides the stability requirement and/or the necessity of fusion proteins, structural studies of GPCRs have also been complicated by the need of eukaryotic expression systems [e.g., Spodoptera frugiperda (Sf9) insect cells], as prokaryotes exhibit generally low functional expression levels of wild-type GPCRs. However, prokaryotes such as Escherichia coli offer several advantages compared with insect cells, including quick genetic modification strategies, growth to high cell densities, fast doubling times, inexpensive media, absence of glycosylation, and robust handling. Furthermore, E. coli is well suited for producing fully isotope-labeled proteins—a crucial requirement for many NMR studies, which are limited to date.To exploit these advantages, we recently developed a directed evolution method for high functional GPCR expression levels in E. coli (15). In contrast to screening a few hundred mutants one by one, this strategy allows the simultaneous, competitive testing of >10⁸ different protein variants for highest prokaryotic expression and functionality. Briefly, diverse libraries of NTR1 variants were either obtained synthetically (16, 17) or by error-prone PCR on the wild-type sequence (15). The libraries were ligated to a plasmid encoding an inducible promoter, which was subsequently used to transform E. coli. Selection pressure for high functional expression levels was applied by incubating the induced cells with fluorescently labeled neurotensin, which allowed enrichment of the best expressing cells by fluorescence-activated cell sorting (FACS). The outlined procedure was performed in cycles, leading to a gradual adaptation of the NTR1 population toward high functional expression levels, and additionally, it gave rise to an increase in thermostability for certain variants.In a second technology, termed CHESS (cellular high-throughput encapsulation, solubilization and screening), we adapted this concept to directly evolve NTR1 variants for high thermostability in short-chain detergent micelles—a property that is not only beneficial for structural studies but also for in vitro drug screening (18). The crucial development of CHESS was to surround, simultaneously, every E. coli cell by a semipermeable polysaccharide capsule. This allows us to solubilize the receptor mutants with harsh short-chain detergents, each mutant inside its own encapsulated cell, all at once and in the same test tube. Both the solubilized receptors and their encoding plasmids are maintained within the same capsules. Long-term incubation under these conditions followed by labeling of the encapsulated solubilized receptors with fluorescent neurotensin and rounds of FACS enrichment ensured a strong selection pressure and a gradual adaption of the NTR1 population toward high stability in harsh short-chain detergents (18).In this work, we present the crystal structures of three evolved NTR1 variants, which were either obtained by evolving high functional expression levels in E. coli or by directed evolution for stability in detergent micelles. In contrast to the majority of crystallized GPCRs, our NTR1 variants are devoid of bulky modifications at the cytoplasmic face and can thus remain signaling-active, which allows us to gain unique insights into the structure–function relationship of NTR1.