Developmental ecdysteroid titers and DNA puffs in larvae of two sciarid species, Rhynchosciara americana and Rhynchosciara milleri (Diptera: Sciaridae).

Ecdysteroid titers, developmental landmarks and the presence of prominent amplifying regions (DNA puffs) have been compared during late larval to pupal development in four groups of Rhynchosciara americana larvae and in R. americana and Rhynchosciara milleri. Three prominent DNA puffs (B2, C3 and C8) expand and regress sequentially on the rising phase of the 20-hydroxyecdysone (20E) titer in R. americana as a firm, cellular cocoon is being constructed. A sharp rise in 20E coincides with the regression of these puffs. The shape of the 20E curve is similar in R. milleri, a species that does not construct a massive cocoon, but the behavior of certain DNA puffs and their temporal relationship to the curve differs. Regions corresponding to B2 and C3 can be identified in R. milleri by banding pattern similarity with R. americana chromosomes and, in the case of B2, by hybridization to an R. americana probe. A B2 puff appears in R. milleri as the 20E titer rises but remains small in all gland regions. A puff similar to the R. americana C3 puff occurs in posterior gland cells of R. milleri (C3(Rm)) after the B2 puff, but this site did not hybridize to R. americana C3 probes. C3(Rm) incorporated (3)H-thymidine above background, but showed less post-puff DNA accumulation than C3 of R. americana. R. americana C8 probes hybridized to a more distal region of the R. milleri C chromosome that did not appear to amplify or form a large puff. These differences can be related to developmental differences, in particular differences in cocoon construction between the two species.

Gastroesophageal Reflux and Microaspiration in Lung Transplant Recipients: The Utility of a Single Esophageal Manometry and pH Probe Monitoring Study.

BACKGROUND: Gastroesophageal reflux (GER) in recipients of lung transplant (LTX) is associated with chronic allograft rejection, presumably via microaspiration that damages airway epithelium. Most LTX programs perform a single post-LTX esophageal study to evaluate for GER; the efficacy of this test is unclear. METHODS: Patients with 1 year of post-LTX follow-up, including routine bronchoscopies with bronchoalveolar lavage fluid (BALF) samples as well as high-resolution esophageal manometry and pH probe monitoring (HREMpH), were evaluated. BALF samples were analyzed with competitive enzyme-linked immunosorbent assay to detect bile salts, which are indicative of aspiration. These results were compared to results of HREMpH studies post LTX. RESULTS: Ninety BALF samples were analyzed for bile salts and acted as disease positive for this evaluation. Of the 13 HREMpH cases, 8 were positive for GER, but only 3 were positive for bile salts via assay. Of the 5 HREMpH-negative cases, 2 experienced aspiration. A solitary HREMpH study had 60.0% sensitivity and 37.5% specificity with positive and negative likelihood ratios: 0.96 and 1.07, respectively. CONCLUSION: Microaspiration appears to be an intermittent phenomenon, and HREMpH screening poorly correlates with BALF evidence of aspiration; which may not be adequate. As aspiration detection is crucial in this population, further analysis is warranted.

Therapeutic efficacy of undenatured type-II collagen (UC-II) in comparison to glucosamine and chondroitin in arthritic horses.

The present investigation evaluated arthritic pain in horses receiving daily placebo, undenatured type II collagen (UC-II) at 320, 480, or 640 mg (providing 80, 120, and 160 mg active UC-II, respectively), and glucosamine and chondroitin (5.4 and 1.8 g, respectively, bid for the first month, and thereafter once daily) for 150 days. Horses were evaluated for overall pain, pain upon limb manipulation, physical examination, and liver and kidney functions. Evaluation of overall pain was based upon a consistent observation of all subjects during a walk and a trot in the same pattern on the same surface. Pain upon limb manipulation was conducted after the walk and trot. It consisted of placing the affected joint in severe flexion for a period of 60 sec. The limb was then placed to the ground and the animal trotted off. The response to the flexion test was then noted with the first couple of strides the animal took. Flexion test was consistent with determining clinically the degree of osteoarthritis in a joint. Horses receiving placebo showed no change in arthritic condition, while those receiving 320 or 480 or 640 mg UC-II exhibited significant reduction in arthritic pain (P < 0.05). UC-II at 480 or 640 mg dose provided equal effects, and therefore, 480 mg dose was considered optimal. With this dose, reduction in overall pain was from 5.7 +/- 0.42 (100%) to 0.7 +/- 0.42 (12%); and in pain upon limb manipulation from 2.35 +/- 0.37 (100%) to 0.52 +/- 0.18 (22%). Although glucosamine and chondroitin treated group showed significant (P < 0.05) reduction in pain compared with pretreated values, the efficacy was less compared with that observed with UC-II. In fact, UC-II at 480 or 640 mg dose was found to be more effective than glucosamine and chondroitin in arthritic horses. Clinical condition (body weight, body temperature, respiration rate, and pulse rate), and liver (bilirubin, GGT, and ALP) and kidney (BUN and creatinine) functions remained unchanged, suggesting that these supplements were well tolerated.

Intravenous immunoglobulin in drug and device refractory patients with the symptoms of gastroparesis-an open-label study.

BACKGROUND: Gastroparesis is a complex clinical entity; many aspects of which remain unknown. Although most patients have idiopathic, diabetic, or postsurgical gastroparesis, many are thought to have measurable neuromuscular abnormalities. Immunotherapy has recently been utilized to treat suspected autoimmune gastrointestinal dysmotility. METHODS: Fourteen patients with symptoms of gastroparesis (Gp) who were refractory to drug/device were selected from 443 Gp patients from 2013 to 2015 who were treated at the University of Louisville motility center. All patients underwent a structural and psychiatric evaluation along with detailed psychological and behavioral examination to rule out eating disorders. We performed detailed neuromuscular evaluation and all 14 patients received at least 12 weeks of intravenous immunoglobulin (400 mg/kg infusion weekly). Response was defined subjectively (symptomatic improvement) using standardized IDIOM score system. KEY RESULTS: All 14 patients had serological evidence and/or tissue evidence of immunological abnormality. Post-IVIG therapy, there was a significant improvement in symptoms scores for nausea, vomiting, early satiety, and abdominal pain. CONCLUSIONS AND INFERENCES: Although limited by the absence of placebo group, the data illustrate the role of autoimmunity and neuromuscular evaluation in patients with gastroparesis and support the utility of a diagnostic trial of immunotherapy in an effort to improve therapeutic outcomes for such patients.

Vitamin E: non-antioxidant roles.

Vitamin E was originally considered a dietary factor of animal nutrition especially important for normal reproduction. The significance of vitamin E has been subsequently proven as a radical chain breaking antioxidant that can protect the integrity of tissues and play an important role in life processes. More recently alpha-tocopherol has been found to possess functions that are independent of its antioxidant/radical scavenging ability. Absorption in the body is alpha-tocopherol selective and other tocopherols are not absorbed or are absorbed to a lesser extent. Furthermore, pro-oxidant effects have been attributed to tocopherols as well as an anti-nitrating action. Non-antioxidant and non-pro-oxidant molecular mechanisms of tocopherols have been also described that are produced by alpha-tocopherol and not by beta-tocopherol. alpha-Tocopherol specific inhibitory effects have been seen on protein kinase C, on the growth of certain cells and on the transcription of some genes (CD36, and collagenase). Activation events have been seen on the protein phosphatase PP2A and on the expression of other genes (alpha-tropomyosin and Connective Tissue Growth Factor). Non-antioxidant molecular mechanisms have been also described for gamma-tocopherol, delta-tocopherol and tocotrienols.

X-ray structure of a lectin-bound DNA duplex containing an unnatural phenanthrenyl pair.

DNA duplexes containing unnatural base-pair surrogates are attractive biomolecular nanomaterials with potentially beneficial photophysical or electronic properties. Herein we report the first X-ray structure of a duplex containing a phen-pair in the center of the double helix in a zipper like stacking arrangement.

Molecular characterization of the DNA puff C-8 gene of Rhynchosciara americana.

We have mapped the only transcription unit known to be present in the C-8 DNA puff of Rhynchosciara americana and describe the isolation and sequence of a cDNA clone, pRa C-8-22, which contains a nearly complete copy of the mRNA transcribed from this DNA puff and part of the sequence of genomic clone BSC8-0.9, which contains the promotor region and the remainder of the transcription unit. The characteristics of the protein predicted from the ORF present in the cDNA indicate that it is unique and secreted.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana.

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

Tocopherol-binding proteins: their function and physiological significance.

The present review is a continuation of earlier essays on the uptake mechanisms and the biological function of vitamin E. There are eight naturally occurring homologues of vitamin E, which differ in their structure and in biological activity in vivo and in vitro. Various studies have suggested that after normal gastrointestinal absorption of dietary vitamin E specific mechanisms favor the preferential accumulation of one of its homologues, alpha-tocopherol, in the human body. This process is thought to be mediated in part by the alpha-tocopherol transfer protein (alpha-TTP) in the liver cytoplasm. The mechanism and pathway by which alpha-TTP specifically incorporates alpha-tocopherol into plasma lipoproteins is not yet fully understood. Because alpha-tocopherol is widely distributed in tissues in various concentrations but alpha-TTP resides only in liver, its role as intracellular carrier of alpha-tocopherol seems unlikely. However, recent data indicate that a system of alpha-tocopherol-binding proteins is involved in these processes that favor the localization of alpha-tocopherol at the sites where it is required. The current status of the evidence for the regulation of alpha-tocopherol levels and their impact on cellular signaling is discussed.

Quinazoline antifolates inhibiting thymidylate synthase: variation of the N10 substituent.

The synthesis of 12 new 5,8-dideazafolates with isopropyl, cyclopropylmethyl, 2-fluoroethyl, carbamoylmethyl, phenacyl, 3-fluorobenzyl, 5-uracilylmethyl, carboxymethyl, 2-carboxyethyl, 3-cyanopropyl, 3-hydroxypropyl, and cyanomethyl substituents at N10 is described. In general, the synthetic route involved monoalkylation of diethyl N-(4-amino-benzoyl)-L-glutamate, coupling of the resulting secondary amine with 2-amino-6-(bromomethyl)-4-hydroxyquinazoline hydrobromide in N,N-dimethylacetamide with calcium carbonate as the base, and deprotection using mild alkali. The cyanomethyl derivatives was found to be unexpectedly base labile and was therefore prepared by mild acid deprotection of a di-tert-butyl ester. The compounds were tested as inhibitors of purified L1210 thymidylate synthase (TS). Four members of the series were more potent that the N10-hydrogen compound, but none was superior to the previously described N10-propargyl-5,8-dideazafolic acid. Selected compounds were examined as inhibitors of purified L1210 dihydrofolate reductase (DHFR). As desired, N10 substitution in general reduced DHFR inhibitory activity; these results are discussed. As a measure of cytotoxicity, the compounds were examined for their inhibition of the growth of L1210 cells in culture. None of the new substituents conferred enhanced potency relative to N10-propargyl-5,8-dideazafolic acid (ID50 = 5 microM), which, as the best TS inhibitor and a relatively poor DHFR inhibitor, continues to lead this series.

Structure-function relationship in the tocopherol transfer protein.

The role of specific amino acid residues in mediating the biochemical functions of tocopherol transfer protein (TTP) was investigated using site-directed mutagenesis and functional assays. These findings further current understanding of TTP mechanism of action and its role in human health.

Active neurocysticercosis, parenchymal and extraparenchymal: a study of 38 patients.

In this retrospective study we have analysed a series of 38 patients seen from 1983 to 1992 (mean follow-up, 4.5 years) with active neurocysticercosis (NCC), 23 (60.5%) with parenchymal and 15 (39.5%) with extraparenchymal NCC. Classification into these two forms of NCC was based on computed tomography and magnetic resonance imaging criteria. The enzyme-linked immunosorbent assay performed in cerebrospinal fluid, for anti-Taenia solium antibodies, was positive in 18 of 23 (78%) cases. Epilepsy and/or intracranial hypertension were the most common clinical presentation (92%). Twenty-three (60.5%) of 38 patients were treated with praziquantel and/or albendazole. In parenchymal NCC, the efficacy of medical therapy was complete in 13 of 16 (81%) and partial in 3 of 16 (19%) patients. In contrast, in all cases of extraparenchymal NCC treated with cysticidal drugs the results were disappointing. A ventriculoperitoneal shunt was performed in 9 of 13 patients with extraparenchymal NCC and hydrocephalus. Severe complications, including two deaths, associated with the natural evolution of the disease or with surgery, occurred only in extraparenchymal NCC. Therefore, we confirm the existence of the two forms of active NCC, parenchymal and extraparenchymal, which are strikingly different in clinical presentation, medical therapy response, complications, morbidity and mortality.

High-flow, small-hole arteriovenous fistulas: treatment with electrodetachable coils.

We present one case of carotid-cavernous fistula caused by percutaneous treatment of trigeminal neuralgia and one case of vertebrovertebral fistula caused by percutaneous internal jugular vein cannulation. Each fistula had a small arteriovenous communication that prevented the use of detachable balloons. Endovascular transarterial treatment of these two iatrogenic fistulas with electrically detachable platinum coils was performed. Both fistulas were occluded with preservation of the parent artery, and the patients have fully recovered.

Vitamin E mediated response of smooth muscle cell to oxidant stress.

Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed in the presence of alpha-tocopherol, the data supports the conclusion of a transcriptional control exerted by alpha-tocopherol on alpha-tropomyosin. Generally, the data strongly suggests the existence of a ligand/receptor type of mechanism at the basis of alpha-tocopherol action. It is concluded that an oxidative stress-induced diminution of alpha-tocopherol in smooth muscle cell activates a reaction cascade leading to changes in gene expression and increase in cell proliferation by a non-antioxidant mechanism.

DNA sequence amplification in sciarid flies: results and perspectives.

The discovery of DNA sequence amplification in sciarid flies and investigations into its control and biological significance are reviewed. Results thus far show that amplification of specific salivary gland polytene chromosome bands is a general phenomenon in sciarids. It is brought about as part of a final endoreplication cycle by the rising titer of ecdysterone that occurs as the larvae approach the prepupal period. Amplification and transcription of these bands is a late, multistep effect of this hormone. The DNA puffs which form in amplified regions produce mRNAs which are translated into polypeptides that appear to be involved in cocoon formation. Application of molecular cloning techniques to the study of DNA amplification has allowed precise quantitation of amplification for several DNA puffs and is yielding maps of their transcription units. These techniques will ultimately help to define the origins of DNA puff replication and contribute to an understanding of the mechanism and control of the amplification phenomenon in Sciaridae. Projections for future experimental approaches are presented.

In vivo effects of ecdysterone on puff formation, and RNA and protein synthesis in the salivary glands of Rhynchosciara americana.

1. Fourth-instar larvae of Rhynchosciara americana were injected with the insect molting hormone, ecdysterone, giving final hemolymph concentrations from 4.46 to 223 microM. 2. Induction of the DNA puff, B2b, in the proximal (S1) region of the salivary glands of Rhynchosciara americana by 22.6 microM ecdysterone, was accompanied by the production of an mRNA and a polypeptide with the same characteristics as B2b products produced during normal development. This mRNA and polypeptide were restricted to the proximal region of the gland, as is the B2b puff. 3. Synthesis of other poly(A)+RNAs was also stimulated in S1 by ecdysterone, and other puffs that appear during normal development were induced. However, rRNA production in S1 goes through a pattern of inhibition, followed by recovery when B2b is puffed, and subsequent inhibition. 4. Low molecular weight RNA, with a peak in the region of 4S, is stimulated after ecdysterone administration.

Hybridization of poly(A)+RNA from salivary glands of Rhynchosciara americana to restriction DNA fragments and polytene chromosomes.

The poly(A)+RNAs produced during DNA puff formation in the salivary gland of R. americana were used to detect the DNA sequences involved in their transcription, using the Southern hybridization and in situ hybridization techniques. DNA prepared from salivary gland after DNA puff regression and carcass were cleaved with EcoRI and hybridized to poly(A)+RNA. After hybridization two major bands corresponding to sizes of 3.0 and 6.0 kb were detected. The hybridization level in the salivary gland DNA was approximately 5-fold that observed with carcass DNA. After in situ hybridization, approximately 10 chromosome regions were labelled. The most highly labelled chromosome regions were C3d and C8e. These regions have been described as DNA puffs that undergo amplification at a specific stage of larval development.

Signalling functions of alpha-tocopherol in smooth muscle cells.

alpha-Tocopherol but not beta-tocopherol, activates protein phosphatase 2A, decreases protein kinase C activity and attenuates smooth muscle cell proliferation at physiological concentrations. beta-Tocopherol prevents the effects of alpha-tocopherol. Inhibition of protein kinase C alpha, but not of the other isoforms, by the inhibitor Gö6976 prevents the effect of alpha-tocopherol. Protein kinase C alpha, immunoprecipitated from alpha-tocopherol treated cells, is less phosphorylated and inactive. It is proposed that the specific activation of protein phosphatase 2A by alpha-tocopherol results in dephosphorylation and inactivation of protein kinase C alpha. Finally, this cascade of events leads to smooth muscle cell proliferation inhibition.

[Endovascular treatment of dissecting aneurysms of the posterior cerebral artery]. / Tratamento endovascular de aneurismas dissecantes da artéria cerebral posterior.

In the literature, there are few reports of posterior cerebral artery aneurysms resulting from isolated dissections. In these cases the treatment is still a matter of discussion and includes anticoagulation, surgical intervention, endovascular techniques and conservative management. We present five cases of Posterior Cerebral Artery (PCA) aneurysms, with angiographic criteria of dissection (double lumen, proximal narrowing), all being situated at the P2 segment, three being close to the P1/P2 junction. In one case, a large aneurysm ruptured during the attempted catheterization with subsequent death of the patient. In three patients occlusion of the parent vessel was performed with Guglielmi detachable coils located proximal to the aneurysm, with no new neurological deficits. In the other case there was spontaneous thrombosis of the aneurysm. Occlusion of the parent vessel by endovascular techniques in dissecting PCA aneurysms has a low probability of neurological deficits and seems to be an appropriate approach for these aneurysms.

[Dural fistulas of the anterior cranial fossa]. / Fístulas durais do andar anterior.

Dural arterio-venous shunts (DAVS) of the anterior cranial fossa are quite rare. There are only a few cases reported in the literature. The authors present 5 cases of dural arterio-venous shunts (DAVS) of the anterior cranial fossa, allowing more data for later reviews of this rare and aggressive localisation of DAVS. The clinical set, imaging findings (with emphasis in diagnostic angiography), treatment and outcome in these 5 patients are described. Haemorrhage was the presenting form in 4 patients; the other case presented was investigated for headache. There were bilateral symmetric feeders in two patients, unilateral feeders in another two and unilateral predominant feeders in one; contribution of the external carotid artery, when present, was always minimal. Venous drainage included one or combinations of these: superior sagittal sinus, sylvian veins and cavernous sinus. Three patients had venous aneurysms in anterior cranial fossa; of these, two presented with haemorrhage, but the third one of them has been asymptomatic for 7 years. Three patients were treated by surgical exclusion of the shunt and became clinical and angiographicaly normal. According to the literature, our cases confirm the highly aggressive behaviour of these DAVS. Surgical treatment is an efficacious method of therapy and should be done as soon as possible. Embolization can be attempted but is technically difficult and eventually too expensive.