4E-binding protein phosphorylation and eukaryotic initiation factor-4E release are required for airway smooth muscle hypertrophy

The molecular mechanisms of airway smooth muscle hypertrophy, a feature of severe asthma, are poorly understood. We previously established a conditionally immortalized human bronchial smooth muscle cell line with a temperature-sensitive SV40 large T antigen. Temperature shift and loss of large T cause G1-phase cell cycle arrest that is accompanied by increased airway smooth muscle cell size. In the present study, we hypothesized that phosphorylation of eukaryotic initiation factor-4E (eIF4E)-binding protein (4E-BP), which subsequently releases eIF4E and initiates cap-dependent mRNA translation, was required for airway smooth muscle hypertrophy. Treatment of cells with chemical inhibitors of PI 3-kinase and mammalian target of rapamycin blocked protein synthesis and cell growth while decreasing the phosphorylation of 4E-BP and increasing the binding of 4E-BP to eIF4E, consistent with the notion that 4E-BP1 phosphorylation and eIF4E function are required for hypertrophy. To test this directly, we infected cells with a retrovirus encoding a phosphorylation site mutant of 4E-BP1 (AA-4E-BP-1) that dominantly inhibits eIF4E. Upon temperature shift, cells infected with AA-4E-BP-1, but not empty vector, failed to undergo hypertrophic growth. We conclude that phosphorylation of 4E-BP, eIF4E release, and cap-dependent protein synthesis are required for hypertrophy of human airway smooth muscle cells.     

Utilization of the 2017 diagnostic criteria for hEDS by the Toronto GoodHope Ehlers–Danlos syndrome clinic: A retrospective review

The new 2017 diagnostic criteria for hypermobile Ehlers–Danlos Syndrome (hEDS) provide a framework for diagnosing hEDS but are more stringent than the previous Villefranche criteria. Our clinical experience at the GoodHope EDS clinic was that the 2017 criteria left many highly symptomatic patients without a diagnosis of hEDS. We conducted a retrospective cohort study to confirm our clinic experience and assess the accuracy of the 2017 diagnostic criteria for hEDS in patients who had a previous hEDS diagnosis based on the Villefranche criteria. Our study found that 15% (n = 20 of 131) of patients with a prior diagnosis of hEDS met the 2017 diagnostic criteria, and many of the traits used to distinguish hEDS were not significantly more frequent in patients who met 2017 criteria versus those who did not. In both groups objective systemic manifestations were found less frequently than subjective systemic manifestations. Beighton score (BS) as assessed by primary care practitioner was found to be higher than assessment by EDS practitioner in 81% (n = 74 of 91) of cases. Generalized joint hypermobility was confirmed in only 46% (n = 51 of 111) of patients who had a previous diagnosis of hEDS. Higher BS did not correlate with increased number of systemic manifestations in our cohort. Common comorbidities of hEDS were found with similar frequency in those who met 2017 criteria and those who did not. Based on our cohort, the 2017 hEDS diagnostic criteria require refinement to improve its diagnostic accuracy.     

BAG1 over-expression in brain protects against stroke

The co-chaperone BAG1 binds and regulates 70 kDa heat shock proteins (Hsp70/Hsc70) and exhibits cytoprotective activity in cell culture models. Recently, we observed that BAG1 expression is induced during neuronal differentiation in the developing brain. However, the in vivo effects of BAG1 during development and after maturation of the central nervous system have never been examined. We generated transgenic mice over-expressing BAG1 in neurons. While brain development was essentially normal, cultured cortical neurons from transgenic animals exhibited resistance to glutamate-induced, apoptotic neuronal death. Moreover, in an in vivo stroke model involving transient middle cerebral artery occlusion, BAG1 transgenic mice demonstrated decreased mortality and substantially reduced infarct volumes compared to wild-type littermates. Interestingly, brain tissue from BAG1 transgenic mice contained higher levels of neuroprotective Hsp70/Hsc70 protein but not mRNA, suggesting a potential mechanism whereby BAG1 exerts its anti-apoptotic effects. In summary, BAG1 displays potent neuroprotective activity in vivo against stroke, and therefore represents an interesting target for developing new therapeutic strategies including gene therapy and small-molecule drugs for reducing brain injury during cerebral ischemia and neurodegenerative diseases.     

Seasonal malaria transmission and variation of anopheline density in two distinct endemic areas in Brazilian Amazonia

Studies on seasonal anopheline fauna variation were performed in two distinct settlements in the State of Rond?nia, Brazil: one at the Madeira River banks (Portuchuelo) with stable native Amazonian population; the other at an inland lumber-extracting farm (Urupá) in dry land, in which adults are mostly migrants. During a 6-yr period (1994-2000), 8,638 adult anophelines were collected: 2,684 in Urupá and 5,954 in Portuchuelo. Anopheles darlingi represented >95% of total mosquitoes caught. Dissection of 4,424 A. darlingi females yielded a very low sporozoite infection index below 0.1%. Oocysts were found in both localities in approximately 0.1% of dissected mosquitoes. Determination of the hour biting rates disclosed seasonal variations in both localities. However, in Portuchuelo, mosquito density peaked at the acme of the rainy season, whereas at Urupá it peaked in the dry season. The increase in mosquito density and incidence of malaria cases were coincident. The high mosquito densities observed in the riverine settlement of Portochuelo sector B, which permits evaluation in > 10,000 mosquitoes' bites/person/year, could explain, in spite of the low mosquito's infection index, the previously described development of natural immunity in the local population that is not observed in the dry land agroindustrial settlement of Urupá.     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

Myxoid leiomyosarcoma of the ovary: Analysis of three cases

The first three cases of myxoid leiomyosarcoma occurring in the ovary are reported. Two cases in stage III were found in postmenopausal patients and a further case was found in stage I in a 32-year-old. All masses were large and gelatinous with cystic change, necrosis, and hemorrhage, but both uteri and ligaments and contralateral adnexa appeared normal. Microscopically, the tumors showed a predominantly reticular meshwork of elongated cells surrounded by abundant basophilic material. While electron microscopy proved inconclusive due to nondifferentiation, the use of monoclonal antibodies against smooth muscle actin demonstrated a smooth muscle type of differentiation. The differential diagnosis of this rare ovarian condition includes other myxoid ovarian lesions, such as ovarian edema, myxoma, endodermal sinus tumors, and the sarcomatous component of malignant mixed müllerian tumor and carcinosarcoma, as well as lymphovascular tumors. Since mitotic count due to decreased cellular density is unusually low in myxoid leiomyosarcoma, capsular rupture and clinical stage seem to be more reliable prognostic markers. The highly aggressive behavior of myxoid leiomyosarcoma parallels that of typical ovarian leiomyosarcoma. Two of the three patients in this series died of tumor at 13 and 24 months after diagnosis; the other patient is free of disease at 3 years after diagnosis.     

Squash Procedure for Protein Immunolocalization in Meiotic Cells

Several techniques have been developed for protein immunolocalization in meiotic cells. However, most of them include treatments that lead to cell disruption and are only suitable for prophase-I cells. We describe a novel squash procedure of cell preparation for protein immunolabelling of different meiotic stages. This procedure is an alternative to both cryosectioning and whole spreading procedures. We present results obtained in mouse spermatocytes with three different antibodies: the MPM-2 mAb against mitotic phosphoepitopes, an anticentromere serum and a polyclonal serum against the SCP3 protein of the axial elements and lateral elements of the synaptonemal complex. The procedure was tested for single and double immunolabelling. With this technique a large number of cells at different meiotic stages can be analysed. Cell stages are easily identified and cell and chromosome structures are preserved. Thus, it allows the study of chromosome behaviour and the relations hips between the different structural elements of the cell throughout meiotic divisions. Our procedure is also suitable for three-dimensional (3D) analyses and proved to be reliable in a wide range of systems including insects and mammals. In addition, the procedure may be interesting to obtain a rapid immunological diagnosis.     

Multiple myeloma presenting as a sellar plasmacytoma and mimicking a pituitary tumor: Report of a case and a review of the literature

A case of multiple myeloma presenting as a plasmacytoma and mimicking a pituitary tumor is reported. The importance of recognizing the existence of plasmacytoma in the differential diagnosis of sellar and parasellar tumors is stressed, as it leads to important differences in management. The literature is reviewed. Patients presenting with plasmacytoma with symptoms of a pituitary adenoma have an average age of 58 years; the male to female ratio is 3:1; diplopia, headache, and progressive visual loss are the most important symptoms; third, sixth, and fourth cranial nerve palsies are the most common signs; and pituitary function is usually normal. CT scan of the sellar region is the imaging modality of choice; pathologically, light and electron microscopy are invaluable in making the correct diagnosis. Making the correct diagnosis allows further appropriate investigation for systemic disease and consequent management.     

Experimental determination of the anisotropy function for the model 200 103Pd "light seed" and derivation of the anisotropy constant based upon the linear quadratic model

Since the publication of the AAPM Task Group 43 report in 1995, Model 200 103Pd seed, which has been widely used in prostate seed implants and other brachytherapy procedures, has undergone some changes in its internal geometry resulting from the manufacturer's transition from lower specific activity reactor-produced 103Pd ("heavy seeds") to higher specific activity accelerator-produced radioactive material ("light seeds"). Based on previously reported theoretical calculations and measurements, the dose rate constants and the radial dose functions of the two types of seeds are nearly the same and have already been reported. In this work, the anisotropy function of the "light seed" was experimentally measured and an averaging method for the determination of the anisotropy constant from distance-dependent values of anisotropy factors is presented based upon the continuous low dose rate irradiation linear quadratic model for cell killing. The anisotropy function of Model 200 103Pd "light seeds" was measured in a Solid Water phantom using 1 X 1 x 1 mm micro LiF TLD chips at radial distances of 1, 2, 3, 4, 5, and 6 cm and at angles from 0 to 90 degrees with respect to the longitudinal axis of the seeds. At a radial distance of 1 cm, the measured anisotropy function of the 103Pd "light seed" is considerably lower than that of the 103Pd "heavy seed" reported in the TG 43 report. Our measured values at all radial distances are in excellent agreement with the results of a Monte Carlo simulation reported by Weaver, except for points along and near the seed longitudinal axis. The anisotropy constant of the 103Pd "light seed" was calculated using the linear quadratic biological model for cell killing in 30 clinical implants. For the model 200 "light seed," it has a value of 0.865. However, our biological model calculations lead us to conclude that if the anisotropy factors of an interstitial brachytherapy seed vary significantly over radial distances anisotropy constant should not be used as an approximation for anisotropy characteristics of a brachytherapy seed.