A bound iron porphyrin is redox active in hybrid bacterial reaction centers modified to possess a four-helix bundle domain.

In this paper we report the design of hybrid reaction centers with a novel redox-active cofactor. Reaction centers perform the primary photochemistry of photosynthesis, namely the light-induced transfer of an electron from the bacteriochlorophyll dimer to a series of electron acceptors. Hybrid complexes were created by the fusion of an artificial four-helix bundle to the M-subunit of the reaction center. Despite the large modification, optical spectra show that the purified hybrid reaction centers assemble as active complexes that retain the characteristic cofactor absorption peaks and are capable of light-induced charge separation. The four-helix bundle could bind iron-protoporphyrin in either a reduced and oxidized state. After binding iron-protoporphyrin to the hybrid reaction centers, light excitation results in a new derivative signal with a maximum at 402 nm and minimum at 429 nm. This signal increases in amplitude with longer light durations and persists in the dark. No signal is observed when iron-protoporphyrin is added to reaction centers without the four-helix bundle domain or when a redox-inactive zinc-protoporphyrin is bound. The results are consistent with the signal arising from a new redox reaction, electron transfer from the iron-protoporphyrin to the oxidized bacteriochlorophyll dimer. These outcomes demonstrate the feasibility of binding porphyrins to the hybrid reaction centers to gain new light-driven functions.

Identification of New Inhibitors with Potential Antitumor Activity from Polypeptide Structures via Hierarchical Virtual Screening.

Leukemias are neoplasms that affect hematopoietic cells, which are developed by genetic alterations (mutations) that lead to the loss of proliferation control mechanisms (maturation and/or cell death). The α4ß1 integrin receptor is a therapeutic target for inflammation, autoimmune diseases and lymphoid tumors. This study was carried out to search through the antagonists-based virtual screening for α4ß1 receptor. Initially, seventeen (17) structures were selected (based on the inhibitory activity values, IC50) and the structure with the best value was chosen as the pivot. The pharmacophoric pattern was determined from the online PharmaGist server and resulted in a model of score value equal to 97.940 with 15 pharmacophoric characteristics that were statistically evaluated via Pearson correlations, principal component analysis (PCA) and hierarchical clustering analysis (HCA). A refined model generated four pharmacophoric hypotheses totaling 1.478 structures set of Zinc_database. After, the pharmacokinetic, toxicological and biological activity predictions were realized comparing with pivot structure that resulted in five (ZINC72088291, ZINC68842860, ZINC14365931, ZINC09588345 and ZINC91247798) structures with optimal in silico predictions. Therefore, future studies are needed to confirm antitumor potential activity of molecules selected this work with in vitro and in vivo assays.

Perioperative brain shift and deep brain stimulating electrode deformation analysis: implications for rigid and non-rigid devices.

UNLABELLED: Deep brain stimulation (DBS) efficacy is related to optimal electrode placement. Several authors have quantified brain shift related to surgical targeting; yet, few reports document and discuss the effects of brain shift after insertion. OBJECTIVE: To quantify brain shift and electrode displacement after device insertion. Twelve patients were retrospectively reviewed, and one post-operative MRI and one time-delayed CT were obtained for each patient and their implanted electrodes modeled in 3D. Two competing methods were employed to measure the electrode tip location and deviation from the prototypical linear implant after the resolution of acute surgical changes, such as brain shift and pneumocephalus. In the interim between surgery and a pneumocephalus free postoperative scan, electrode deviation was documented in all patients and all electrodes. Significant shift of the electrode tip was identified in rostral, anterior, and medial directions (p < 0.05). Shift was greatest in the rostral direction, measuring an average of 1.41 mm. Brain shift and subsequent electrode displacement occurs in patients after DBS surgery with the reversal of intraoperative brain shift. Rostral displacement is on the order of the height of one DBS contact. Further investigation into the time course of intraoperative brain shift and its potential effects on procedures performed with rigid and non-rigid devices in supine and semi-sitting surgical positions is needed.

Biomechanical comparison of techniques to reduce the bulk of lacerated flexor tendon ends within digital sheaths of the porcine forelimb.

PURPOSE: Zone II flexor tendon repairs may create a bulging effect with increased bulk and resistance to tendon gliding. A biomechanical time 0 study was performed to assess 2 methods of tendon antibulking for work of flexion and strength characteristics. METHODS: We placed 24 fresh-frozen porcine forelimb tendons in a custom jig. Deep flexor tendon was sectioned just distal to the intact A1 and A2 pulleys. Specimens were divided into 3 groups before repair: group 1, nonmodified tendon; group 2, 30 degrees bilateral notch excised from both tendon ends; and group 3, triangular longitudinal central wedge excised from both tendon ends. All repairs used a 4-strand modified Kessler core suture and running circumferential epitendinous suture. Work of flexion, 2-mm gap formation, and ultimate load to failure were tested. RESULTS: Both antibulking techniques (groups 2 and 3) had significantly less work of flexion than group 1 (36.3 and 34.9 J vs 142.9 J, p < .001). There was no significant change in work of flexion between groups 2 and 3 (p > .05). There was no significant difference in terms of 2-mm gap formation among the 3 groups (p > .05). Groups 1 and 3 exhibited a significantly higher load to failure compared with group 2 (p < .05). CONCLUSIONS: The antibulking repair techniques used in this study decrease the work of flexion with no significant change in force to 2-mm gap formation. Group 2, however, did have significantly lower load to failure. These techniques might be beneficial in zone II flexor tendon injury, in which the tight annular pulley system restricts tendon gliding. However, this is a time 0 study and the potential adverse effects of increase tendon manipulation and trauma were not analyzed, which might increase adhesions and scar during the healing phase of tendon repair.

The receptor tyrosine kinase MerTK regulates dendritic cell production of BAFF.

The MerTK receptor tyrosine kinase is an important negative regulator of dendritic cell function and is required to prevent B cell autoimmunity in vivo. It is not currently known however, if any causal relationship exists between these two aspects of MerTK function. We sought to determine if dendritic cells (DC) from mice lacking MerTK (mertk(- / - ) mice) have characteristics that may aid in the development of B cell autoimmunity. Specifically, we found that mertk(- / - ) mice contain an elevated number of splenic DC, and this population contains an elevated proportion of cells secreting the critical B cell pro-survival factor, B cell activating factor (BAFF). Elevated numbers of BAFF-secreting cells were also detected among mertk(- / - ) bone marrow-derived dendritic cell (BMDC) populations. This was observed in both resting BMDC, and BMDC stimulated with lipopolysaccharide (LPS) or treated with exogenous apoptotic cells. We also found that DC in general have a pro-survival effect on resting B cells in co-culture. However, despite containing more BAFF-secreting cells, mertk(- / - ) BMDC were not superior to C57BL/6 or baff-deficient BMDC at promoting B cell survival. Furthermore, using decoy receptors, we show that DC may promote B cell survival and autoimmunity through a BAFF-and a proliferation-inducing ligand-independent mechanism.

Iron as a bound secondary electron donor in modified bacterial reaction centers.

The binding and oxidation of ferrous iron were studied in wild-type reaction centers and in mutants that have been modified to be both highly oxidizing and able to bind manganese [Thielges et al. (2005) Biochemistry 44, 7389-7394]. After illumination of wild-type reaction centers, steady-state optical spectroscopy showed that the oxidized bacteriochlorophyll dimer, P+, could oxidize iron but only as a second-order reaction at iron concentrations above 100 microM. In the modified reaction centers, P+ was reduced by iron in the presence of sodium bicarbonate with dissociation constants of approximately 1 microM for two mutants with different metal-binding sites. Transient optical spectroscopy showed that P+ was rapidly reduced with first-order rates of 170 and 275 s-1 for the two mutants. The dependence of the amplitude of this rate on the iron concentration yielded a dissociation constant of approximately 1 microM for both mutants, in agreement with the steady-state determination. The oxidation of bound iron by P+ was confirmed by the observation of a light-induced EPR signal centered at g values of 2.2 and 4.3 and attributed to high-spin Fe3+. Bicarbonate was required at pH 7 for low dissociation constants for both iron and manganese binding. The similarity between iron and manganese binding in these mutants provides insight into general properties of metal-binding sites in proteins.

Current and future strategies for the treatment of malignant brain tumors.

Glioblastoma (GB) is the most common subtype of primary brain tumor in adults. These tumors are highly invasive, very aggressive, and often infiltrate critical neurological areas within the brain. The mean survival time after diagnosis of GB has remained unchanged during the last few decades, in spite of advances in surgical techniques, radiotherapy, and also chemotherapy; patients' survival ranges from 9 to 12 months after initial diagnosis. In the same time frame, with our increasing understanding and knowledge of the physiopathology of several cancers, meaningful advances have been made in the treatment and control of several cancers, such as breast, prostate, and hematopoietic malignancies. Although a number of the genetic lesions present in GB have been elucidated and our understanding of the progressions of this cancer has increased dramatically over the last few years, it has not yet been possible to harness this information towards developing effective cures. In this review, we will focus on the classical ways in which GB is currently being treated, and will introduce a novel therapeutic modality, i.e., gene therapy, which we believe will be used in combination with classical treatment strategies to prolong the life-span of patients and to ultimately be able to control and/or cure these brain tumors. We will discuss the use of several vector systems that are needed to introduce the therapeutic genes within either the tumor mass, if these are not resectable, or the tumor bed, after successful tumor resection. We also discuss different therapeutic modalities that could be exploited using gene therapy, i.e., conditional cytotoxic approach, direct cytotoxicity, immunotherapy, inhibition of angiogenesis, and the use of pro-apoptotic genes. The advantages and disadvantages of each of the current vector systems available to transfer genes into the CNS are also discussed. With the advances in molecular techniques, both towards the elucidation of the physiopathology of GB and the development of novel, more efficient and less toxic vectors to deliver putative therapeutic genes into the CNS, it should be possible to develop new rationale and effective therapeutic approaches to treat this devastating cancer.

Regulated, adenovirus-mediated delivery of tyrosine hydroxylase suppresses growth of estrogen-induced pituitary prolactinomas.

Prolactin-secreting adenomas are one of the most common types of intracranial neoplasm found in humans. The modalities of clinical treatment currently in use include D(2)-dopamine receptor agonists, surgery, and radiotherapy, and the success rates for treatment are good. However, there are prolactinomas that are difficult to treat. As an alternative, we have developed a gene therapy strategy in which the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is overexpressed in the anterior pituitary (AP) gland. Because dopamine is known to have an inhibitory effect on lactotroph growth and prolactin secretion, we developed a system that would enable its local synthesis from freely available precursor amino acids. A dual adenovirus tetracycline-regulatable expression system was generated to control the production of TH. In the absence but not presence of the tetracycline analog doxycycline, TH expression was observed in AP tumor cell lines AtT20, GH3, and MMQ. In both primary AP cell cultures and the AP gland, in situ expression of TH was seen in lactotrophs, somatotrophs, corticotrophs, thyrotrophs, and gonadotrophs in the absence but not presence of doxycycline. The ability of this system to inhibit hyperprolactinemia and pituitary lactotroph hyperplasia was then assessed in a model of estrogen- or estrogen/sulpiride-induced pituitary tumors. In the absence but not presence of doxycycline, a 49% reduction in pituitary growth and 58% reduction in the increase of circulating prolactin levels were observed in estrogen, but not estrogen/sulpiride, treated rats. These results indicate that in situ dopamine enhancement gene therapy can be a useful tool for the treatment of prolactinoma. Dopamine synthesis can be tightly regulated and the therapeutic benefit of the system is only inhibited when local dopamine signaling is impaired.

Switching on and off transgene expression within lactotrophic cells in the anterior pituitary gland in vivo.

To further develop our understanding of anterior pituitary (AP) function and to aid the development of gene therapy strategies for the treatment of pituitary diseases, adenovirus (Ad)-mediated gene transfer to the AP gland will be a useful tool. Although successful widespread gene transfer within the AP has been achieved using first generation Ads the ability to control transgene expression would be very beneficial when studying AP regulatory functions and delivering a potentially therapeutic gene into the AP gland. A dual adenoviral vector system encoding for cell type-specific and regulatable transcription units was developed to achieve transcriptionally targeted transgenesis within specific cell populations in the adult AP gland. To achieve regulatable transgene expression within predetermined AP cells, the tetracycline-responsive transcriptional elements have been engineered to be under the control of human, lactotroph-specific PRL (hPRL) promoter elements within a dual adenoviral vector system. The inducibility, cell type specificity, and levels of transgene expression were characterized in vitro and in vivo and compared with the strong ubiquitous beta-actin/human cytomegalovirus (CAG) promoter. Inducible expression of the marker gene beta-galactosidase under the control of the hPRL promoter was restricted to lactotrophic tumor cell lines and lactotrophic cells within primary AP cultures. Lactotroph cell type specificity and inducible transgene expression were also observed within the AP gland in vivo, and this could be switched on or off. Administration of doxycycline abrogated transgene expression both in vitro and in vivo. Our results also provide evidence that an excess of trans-activator is needed to achieve maximal transgene expression. Our data indicate that combined transcriptional and inducible transgenesis can be achieved using adenoviral vectors that allow spatial and temporal restriction of transgene expression within the adult AP gland in vivo.

Long-term transgene expression within the anterior pituitary gland in situ: impact on circulating hormone levels, cellular and antibody-mediated immune responses.

Adenoviral vectors have been identified as useful tools for gene transfer to the pituitary gland with the aim of providing therapeutic treatments for pituitary diseases. Although successful adenovirus-mediated gene transfer to the pituitary has been shown, the duration of transgene expression, local immune responses and consequences on circulating pituitary hormone levels have not been investigated. These are critical not only for the successful implementation of these gene transfer techniques both for physiological and/or therapeutic applications but also for assessing the safety of these approaches. We have therefore assessed duration and levels of transgene expression 3 days, 14 days, 1, 2, and 3 months after delivery of adenoviruses expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK), under the control of the major immediate early human cytomegalovirus (RAd-hCMV/TK) or human PRL (RAd-hPrl/TK) promoters, to the anterior pituitary (AP) gland in situ. The presence of vector genome and cellular immune infiltrates within the AP gland were also studied along with the levels of circulating anti-adenovirus neutralizing antibodies and AP hormones in sera. Ubiquitous or cell-type specific expression of HSV1-TK within the AP gland was seen from RAd-hCMV/TK and RAd-hPrl/TK respectively at all time points, although a reduction in expression was seen over time. PCR amplification of HSV1-TK specific sequences showed the persistence of adenoviral genomes for up to 3 months. Analysis of the AP showed the presence of a virus-induced inflammation that peaked around day 14 and was resolved between 2-3 months. ED1-positive macrophages, CD8-positive T-cells and CD161-positive NK cells were identified up to 1 month after virus administration. A virus-induced humoral immune response was also present as anti-adenovirus neutralizing antibodies were detected from 14 days after virus administration. Levels of circulating pituitary hormones were unaffected by virus administration with the exception of the stress hormone ACTH which was increased at 3 days but normalized by 14 days. In conclusion, our data indicates that adenovirus-mediated delivery to the AP gland in situ may be a useful tool for the treatment of pituitary diseases as no major cytotoxicity or disruption of AP hormonal functions are seen. Despite of this, further developments to this approach still need to be made to combat the reduced transgene expression seen over time and the induction of virus-induced immune responses.

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles.

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.

Helical CT of urinary calculi: effect of stone composition, stone size, and scan collimation.

OBJECTIVE: Helical CT has become the preferred methodology for identifying urinary calculi. However, the ability to predict stone composition, which influences patient treatment, depends on the accurate measurement of the radiographic attenuation of stones. We studied the effects of stone composition, stone size, and scan collimation width on the measurement of attenuation in vitro. MATERIALS AND METHODS: One hundred twenty-seven human urinary calculi of known composition and size were scanned at 120 kVp, 240 mA, and a 1:1 pitch at different collimations. A model, based on the physics of helical CT, was used to predict the effect of scan collimation width and stone size on measured attenuation. RESULTS: At a 1-mm collimation, stone groups could be differentiated by attenuation: the attenuation of uric acid was less than that of cystine or struvite, which overlapped; these were less than the attenuation of calcium oxalate monohydrate, which was in turn lower than that of brushite and hydroxyapatite, which overlapped and showed the highest values. At a wider collimation, attenuation was lower and the ability to differentiate stone composition was lost. Attenuation also decreased with smaller stones. At a 10-mm collimation, some uric acid stones (

Clinical utility of three-dimensional US.

Three-dimensional (3D) ultrasonography (US) is rapidly gaining popularity as it moves out of the research environment and into the clinical setting. This modality offers several distinct advantages over conventional US, including 3D image reconstruction with a single pass of the US beam, virtually unlimited viewing perspectives; accurate assessment of long-term effects of treatment; and more accurate, repeatable evaluation of anatomic structures and disease entities. In obstetric imaging, 3D US provides a novel perspective on the fetal anatomy, makes anomalies easier to recognize, facilitates maternal-fetal bonding, and helps families better understand fetal abnormalities. Three-dimensional pelvic US allows volume data sets to be acquired with both transvaginal and transabdominal probes. Viewing multiple 3D power Doppler US images in a fast cine loop has proved useful in angiographic applications. Three-dimensional prostate US can help make accurate volume assessments for dosimetry planning or for estimating prostate-specific antigen levels. In breast imaging, 3D US has the capacity to demonstrate lesion margins and topography, thereby helping differentiate benign from malignant masses. Three-dimensional US can also help determine the need for biopsy and help facilitate needle localization and guidance during biopsy. With recent advances in computer technology and display techniques, 3D US will likely play an increasingly important role in medicine.

Giant cell reparative granuloma of the petrous temporal bone: a case report and literature review.

Giant cell reparative granuloma (GCRG) is an unusual, benign bone lesion that most commonly affects the maxilla and mandible; skull involvement is rare. The etiology is uncertain but may be related to trauma. GCRG is difficult to distinguish from giant cell tumor of the bone and has a lower recurrence rate. Thirteen reports of temporal bone GCRG in 11 patients have been reported. One report of a petrous GCRG in a 3-year-old girl has been identified. A 38-year-old male presented with a 2-year history of fullness in his left ear, ipsilateral hearing loss, and intermittent cacosmia. Computed tomography and magnetic resonance imaging revealed a large left-sided anterior temporal extradural mass. The patient underwent a left frontotemporal craniotomy and resection of a left temporal fossa tumor that involved the petrous and squamous parts of the temporal bone. The patient's post-operative course was uneventful, except for increased hearing loss secondary to opening of the epitympanum. Follow-up at one month revealed no other problems. Histopathology of the specimen was consistent with a giant cell reparative granuloma.

Effect of macroscopic air bubbles on cell lysis by shock wave lithotripsy in vitro.

In studies of cells or stones in vitro, the material to be exposed to shock waves (SWs) is commonly contained in plastic vials. It is difficult to remove all air bubbles from such vials. Because SWs reflect at an air-fluid interface, and because existing gas bubbles can serve as nuclei for cavitation events, we sought to determine in our system whether the inclusion of small, visible bubbles in the specimen vial has an effect on SW-induced cell lysis. We found that even small bubbles led to increased lysis of red blood cells (1- to 3-mm diameter bubbles, 9.8+/-0.5% lysis, n = 7; no bubbles, 4.4+/-0.8%, n = 4), and that the degree of lysis increased with bubble size. Damage could not be reduced by centrifuging the cells to the opposite end of the vial, away from the bubble. B-scan ultrasound imaging of blood in polypropylene pipette bulbs showed that, with each SW, bubbles were recruited from the air interface, mixing throughout the fluid volume, and these appeared to serve as nuclei for increased echogenicity during impact by subsequent SWs; thus, bubble effects in vials could involve the proliferation of cavitation nuclei from existing bubbles. Whereas injury to red blood cells was greatly increased by the presence of bubbles in vials, lytic injury to cultured epithelial cells (LLC-PK1, which have a more complex cytoarchitecture than red blood cells) was not increased by the presence of small air bubbles. This suggests different susceptibility to SW damage for different types of cells. Thus, the presence of even a small air bubble can increase SW-induced cell damage, perhaps by increasing the number of cavitation nuclei throughout the vial, but this effect is variable with cell type.

Perineal prostate cancer seeding along needle tract following cryosurgery.

We report a case of tumor seeding along the path of a cryoprobe during cryotherapy for prostate cancer. Seeding was reported as a potential problem in the era of transperineal biopsies, but we suspect that it may again resurface in modern minimally invasive surgical treatments that develop a transperineal tract. Minimizing prostate trauma and bleeding during minimally invasive therapies such as cryosurgery and brachytherapy may decrease the risk of tumor seeding. We believe that the perineum of all patients who have undergone transperineal procedures should be thoroughly checked during follow-up.

Interstitial laser coagulation of the prostate: introduction of a volume-based treatment formula with 12-month follow-up.

The objective of the present study was to assess the efficacy of interstitial laser coagulation (ILC) of the prostate using a low-volume treatment formula as a minimally invasive therapy for symptomatic benign prostatic hyperplasia (BPH). A total of 25 men underwent ILC of the prostate for symptomatic BPH between February 1997 and June 1997. The Indigo 830e laser system from Indigo Medical Inc. and the factory preset therapy regimen was used for ILC of the prostate. The number of treatments or punctures was determined by the formula 0.5 x total prostate volume (ml)/8 ml, rounded to the closest even whole number. The treatment outcome was evaluated at 3-, 6-, and 12-month intervals using the American Urological Association's (AUA) BPH symptom score, maximal urinary flow, prostate size, and postvoid residual urine volume. The AUA symptom score decreased from 23.2 (range 17-28) prior to treatment to 9.4 (range 4-14) at 3 months, 6.6 (range 5-12) at 6 months, and 7.2 (range 4-11) at 12 months. The maximal flow rate improved from 8.4 (range 5-10) ml/s pretreatment to 14.1 (range 10-20) ml/s at 3 months, 14.8 (range 10-18) ml/s at 6 months, and 16.8 (range 12-25) ml/s at 12 months after treatment. There was no significant postprocedure complication. The 1-year clinical results suggest that the ILC procedure using the Indigo 830e device in conjunction with a low-volume treatment formula has outcomes similar to those obtained following ILC based on high-volume coagulation.