Crystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residues

BACKGROUND AND PURPOSE: Early and accurate diagnosis of brain edema in stroke patients is essential for the selection of appropriate treatment. We examined the correlations between the changes in the apparent diffusion coefficient (ADC), regional water content, and tissue ultrastructure during early focal cerebral ischemia. METHODS: The left middle cerebral arteries of cats were occluded with an intramagnet occlusion/recirculation device. T2-weighted, diffusion-weighted, and perfusion imaging were performed repeatedly during the initial 3 hours after occlusion. The ADCs obtained from ADC maps were compared with the corresponding tissue water content values determined by gravimetry and electron microscopic water localization. RESULTS: ADC reduction was detected in areas of low perfusion 15 minutes after occlusion and thereafter. The water content increase correlated linearly with the ADC decreases in both the gray and white matter. However, both the water content corresponding to an ADC value and the rate of ADC change of the gray and white matter differed significantly (P<.05) as follows: y = -10105x + 8533 (r=.86) and y = -6174x + 4611 (r=.67), respectively, where x is the water content (grams water per gram tissue) and y is the ADC (x 10(-6) mm2/s). Hydropic astrocytic swelling was seen in both structures, and in the white matter, oligodendroglial and myelinated axonal swelling and periaxonal space enlargement were observed. CONCLUSIONS: When early ischemic edema in experimental focal cerebral ischemia is evaluated with ADC mapping, the different slopes and intercepts of the water content and ADC correlation lines for the gray and white matter, which probably reflect different ultrastructural localization of water, should be taken into account.     

Effect of intracolonic benzalkonium chloride on trinitrobenzene sulphonic acid-induced colitis in the rat

BACKGROUND: We investigated the effects of benzalkonium chloride (BAC) on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats. METHODS: TNBS was administered intrarectally before and/or after BAC treatment. In the first study, the effects of treatment with BAC 6, 12 or 24 h after TNBS were examined. In the second study, animals were treated with BAC before, after or before and after TNBS, and were examined 7 days later. The severity of colitis was assessed by macroscopic and histological scoring of the colonic damage and by determination of colonic myeloperoxidase (MPO) activity. Macrophages and CD4+ and CD8+ T cells were examined by immunohistochemistry. RESULTS: When BAC was instilled into the colon 6, 12 or 24 h after TNBS, weight loss and macroscopic and histological features of the colon were similar to that of controls (TNBS alone). In contrast, MPO activity was significantly reduced in all three groups post-treated with BAC. In the groups examined 7 days after TNBS treatment, rats post-treated with BAC exhibited increased weight gain and significantly reduced macroscopic damage and MPO activity compared to the TNBS control group. Rats pre-treated with BAC exhibited less macroscopic damage of the colon than rats receiving only TNBS, but histological damage, MPO and weight gain were unchanged from TNBS controls. Immunohistochemistry revealed that BAC pre-treatment increased the numbers of macrophages and T cells in the colon. After TNBS treatment, macrophage accumulation was evident in the colon, but T cells were scarce. However, these cells were preserved or enhanced in the colonic mucosa in TNBS-treated rats that had been pre-treated with BAC. CONCLUSIONS: Treatment with BAC, particularly after induction of colitis, produces a significant reduction in the severity of tissue injury and inflammation through mechanisms that are not fully understood.     

Nonradioactive Northern blotting with biotinylated and digoxigenin-labeled RNA probes

The application of nonradioactive RNA probes for Northern blotting offers the advantage of a rapid turn-around time for results without the loss of sensitivity for target mRNA detection. However, a problem that has impeded the widespread use of nonradioactive RNA probes for use in Northern blotting is the difficulty in stripping these probes from nylon membranes after hybridization. In this report we describe two protocols for stripping digoxigenin (Dig)-labeled RNA probes from nylon membranes. One protocol utilizes a phosphate-buffered formamide stripping solution to remove nonchemically modified (regular) RNA probes while the other method utilizes strippable probes that were produced with a chemically modified nucleotide (CTP) and removed by a specific stripping solution. This latter method was developed by Ambion Inc. and is called Strip-EZ. We also describe a protocol for the detection of two separate rat mRNAs using both biotin and digoxigenin-labeled RNA probes that does not require stripping the membrane after hybridization. Finally, we describe the use of another new labeling technology, called Chem-Link, that quickly and conveniently labels RNA for use in Northern blotting.     

Hemodynamic characteristics, myocardial kinetics and microvascular rheology of FS-069, a second-generation echocardiographic contrast agent capable of producing myocardial opacification from a venous injection

OBJECTIVES: We sought to 1) study the effects of FS-069 on cardiac and systemic hemodynamic function, myocardial blood flow, left ventricular wall thickening and pulmonary gas exchange when injected intravenously; and 2) compare the myocardial kinetics and microvascular rheology of FS-069 and Albunex when injected directly into a coronary artery. BACKGROUND: FS-069 is a second-generation echocardiographic contrast agent composed of perfluoropropane-filled albumin microspheres; it is capable of consistent and reproducible myocardial opacification from a venous injection. METHODS: Nine dogs were used to study the effects of FS-069 on hemodynamic function, pulmonary gas exchange, left ventricular wall thickening and myocardial blood flow and to characterize its myocardial kinetics when injected intravenously. These dogs were also used to compare the myocardial kinetics of FS-069 with those of Albunex during intracoronary injections. Nine Sprague-Dawley rats were used to compare the microvascular rheology of these two contrast agents, and in vitro modeling was performed to assess whether the microvascular findings of FS-069 can explain its echocardiographic behavior during direct coronary injections. RESULTS: There were no effects of 30 rapid venous injections of FS-069 (every 20 s) on cardiac output; mean aortic, pulmonary or left atrial pressures; and peak positive and negative first derivative of left ventricular pressure (dP/dt). Similarly, there were no effects of this agent on radiolabeled microsphere-measured regional myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange. When injected intravenously, the myocardial transit of this agent resembled a gamma-variate form. When diluted FS-069 was injected directly into the coronary artery; however, its transit resembled the integral of gamma-variate function, with persistent myocardial opacification lasting several minutes, which was different from that of Albunex. Intravital microscopy revealed that, unlike Albunex, when no bubbles are entrapped within the microcirculation after an arterial injection, a very small fraction of the diluted, larger FS-069 microbubbles are entrapped. In vitro modeling confirmed that this small fraction of microbubbles can result in persistent myocardial opacification. CONCLUSIONS: FS-069 produces no changes in hemodynamic function, myocardial blood flow, left ventricular wall thickening or pulmonary gas exchange when injected intravenously in large amounts. When diluted FS-069 is injected into the coronary artery, a very small fraction of the larger bubbles are entrapped within the microcirculation, resulting in a persistent contrast effect. Thus, although FS-069 is a safe intravenous echocardiographic contrast agent, it cannot provide information on myocardial blood flow when injected directly into a coronary artery.     

Comparison of ras-p21 bound to GDP and GTP: differences in protein and ligand dynamics

This paper documents the first essential dynamics analysis of ras protein ligands and of the protein itself, showing important features of their dynamic properties. Essential dynamics analysis of 300 ps of full solvent molecular dynamics simulations revealed differences in structure and dynamics between GDP- and GTP-bound forms of H-ras-p21. Regions in the protein which exhibited a structural shift correspond to the switch regions described previously. Differences in dynamics between H-ras-p21 GDP and H-ras-p21 GTP may be related to interactions of ras with GAP and its receptor and effector. Molecular dynamics of free GDP (in the absence of protein) were performed in water for 2 ns and analysed using essential dynamics. The conformations of GDP and GTP when bound to the protein were compared with free GDP, revealing that the ligands bind to the protein in an energetically unfavourable conformation. GDP and GTP molecules from various other protein crystal structures were also analysed. These ligands adopt similar conformations to those seen in H-ras-p21.      

Production of two-material micro-assemblies by two-component powder injection molding and sinter-joining

In the field of micro-technology the production of metallic and ceramic micro-components by powder injection molding (PIM) has become a more and more established fabrication method. But in order to fulfill the demand for more complex-shaped high-precision micro-components further development work has to be performed. This is especially true if more efficient production routes for multi-component-micro-assemblies consisting of different materials or sub-components are envisaged. To meet these challenges, investigations are performed to realize and to establish two primary shape micro-processes. These are two-component micro-injection molding (2C-MicroPIM) and sinter-joining. The realization of these technologies will lead to a markedly reduction of the efforts for handling, adjustment, and assembling of metallic and ceramic micro-assemblies. Furthermore, an increased integration level and functionality can be yielded. For an effective transfer of scientific results to industrial applications the whole process chain must be considered, from development and construction of the tooling as well as of the components to the quality assurance and determination of the properties of the assemblies after sintering. These primary shape processes shall enable the mutual processing of different materials within the fabrication process, so avoiding separate mounting or assembling steps. Additionally fixed and loose junctions between at least two components shall be realized. The progress in research and development will be demonstrated especially by the implementation of shaft-to-collar connections between micro-gearwheels and corresponding shafts. Regarding two-component micro-injection molding, the tool construction for shaft-to-collar connections will be presented as well as first experimental results on the properties of selected ceramic powders and feedstocks for the special requirements of the 2C-MicroPIM process. With the assembly step being performed outside the injection molding tool before sinter-joining different parts and geometries can be combined quite easily. The presented article gives an overview on the concept and on preliminary testing results for the fabrication of a shaft-to-collar-connection. Additionally, a solution for an automated assembly of a shaft and a toothed wheel outside the injection molding tool is presented.     

Biocontrol potential of the entomogenous fungi Beauveria bassiana and Metarhizium anisopliae for tsetse flies (Glossina spp.) at developmental sites

Belonging to the Annonaceae family, marolo (Annona coriaceae) is a native species of the Brazilian "cerrado" región (Minas Gerais, Goiás and Distrito Federal) and can be found in South American tropical zones. Its fruits are highly consumed by local people and commercialized in markets or street stalls. There is, however, a tendency for the extinction of marolo due to deforestation and the large scale plantation of monocultures instead of native plants. The literature still offers no data on the chemical composition of the proximate composition and vitamin C, A and tannin contents were carried out on the yellow marolo pulp as well as the determination of the physico-chemical characteristics of the seed oil. Five batches of fruit from the Alfenas region--south of Minas Gerais State--were analysed in this work and their average composition were: humidity 77%, total sugar 15%, reducing sugar 11%, crude protein 1%, lipids 3%, fiber 5% and fixed mineral residue 1%. The contents of vitamin C and A were 8.2 mg/100g and 117.5 RE/100g, respectively, and the tannin content was 245 mg/100g. The results showed high fiber and lipid contents of marolo pulp in comparison with many other tropical fruit pulps. The vitamin C contents were equivalent to those found in avocado, pineapple and watermelon, while the vitamin A contents were equivalent to papaya, peach, guava and several other tropical fruits. Marolo seed contains 45% of oil on a dry basis. Its composition and physico-chemical characteristics showed the possibility of producing a good quality oil, with great potential for the fine oil market. However the presence of alkaloids in the oil needs to be further studied. Their elimination could be done by refining or extraction in a continuous press. The results exalt the high quality of marolo pulp, showing that the preservation of native species should be stimulated.     

Tennessee's failed managed care program for mental health and substance abuse services

In July 1996, Tennessee initiated a managed mental health and substance abuse program called TennCare Partners. This publicly funded "carve-out" experiment started chaotically and soon deteriorated into a crisis. Many patients did not receive care or lost continuity of care, and the traditional "safety net" mental health system nearly disintegrated. This qualitative case study sought to ascertain the impact of the TennCare Partners program. It points out that the program's difficulties stemmed directly from a flawed design that spread funds previously earmarked for severely mentally ill patients across the entire Medicaid population. States contemplating similar reforms should strive to protect vulnerable patients by risk-adjusting capitation payments and by focusing resources on care for severely mentally ill persons. States should also minimize program complexity and ensure the accountability of managed care networks for their patients' behavioral health care needs.     

Prenatal sonographic findings associated with malignant astrocytoma following normal early third-trimester ultrasonography

We present an unusual case in which sonographic assessment at 33 weeks' gestation, 5 weeks following a normal fetal anatomical survey and biometry, demonstrated a large, irregular-shaped, echogenic, suprasellar midline intracranial mass occupying the anterior and middle fossas. Associated severe obstructive hydrocephalus with "dangling" choroid plexus bilaterally was noted with a markedly thin cortical mantle and increased cranial biometry. Elective cesarean delivery was performed due to the associated craniomegaly at 37 weeks' gestation. Although breathing spontaneously at delivery, the infant subsequently required mechanical ventilation and developed neonatal seizures. A ventriculoperitoneal shunt was placed on Day 3 of life. Transcranial needle biopsy demonstrated malignant astrocytoma (glioblastoma multiforme). This case suggests the rapid development of an intracranial malignant astrocytoma over a relatively short period of time. The significant ultrasonographic finding of an intracranial, destructive fetal deformation, following a normal examination 5 weeks previously, demonstrates the limitations of screening ultrasonography in predicting perinatal outcome.     

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit

Amino acid changes S180A (S-->A at site 180), H197Y, Y277F, T285A, and A308S are known to shift the maximum wavelength of absorption (lambda max) of red and green visual pigments toward blue, essentially in an additive fashion. To test the generality of this "five-sites" rule, we have determined the partial amino acid sequences of red and green pigments from five mammalian orders (Artiodactyla, Carnivora, Lagomorpha, Perissodactyla, and Rodentia). The result suggests that cat (Felis catus), dog (Canis familiaris), and goat (Capra hircus) pigments all with AHYTA at the five critical sites have lambda max values of approximately 530 nm, whereas rat (Rattus norvegicus) pigment with AYYTS has a lambda max value of approximately 510 nm, which is accurately predicted by the five-sites rule. However, the observed lambda max values of the orthologous pigments of European rabbit (Oryctolagus cuniculus), white-tailed deer (Odocoileus virginianus), gray squirrel (Sciurus carolinensis), and guinea pig (Cavia procellus) are consistently more than 10 nm higher than the predicted values, suggesting the existence of additional molecular mechanisms for red and green color vision. The inferred amino acid sequences of ancestral organisms suggest that the extant mammalian red and green pigments appear to have evolved from a single ancestral green-red hybrid pigment by directed amino acid substitutions.