Ecological opportunity and the origin of adaptive radiations.

Ecological opportunity--through entry into a new environment, the origin of a key innovation or extinction of antagonists--is widely thought to link ecological population dynamics to evolutionary diversification. The population-level processes arising from ecological opportunity are well documented under the concept of ecological release. However, there is little consensus as to how these processes promote phenotypic diversification, rapid speciation and adaptive radiation. We propose that ecological opportunity could promote adaptive radiation by generating specific changes to the selective regimes acting on natural populations, both by relaxing effective stabilizing selection and by creating conditions that ultimately generate diversifying selection. We assess theoretical and empirical evidence for these effects of ecological opportunity and review emerging phylogenetic approaches that attempt to detect the signature of ecological opportunity across geological time. Finally, we evaluate the evidence for the evolutionary effects of ecological opportunity in the diversification of Caribbean Anolis lizards. Some of the processes that could link ecological opportunity to adaptive radiation are well documented, but others remain unsupported. We suggest that more study is required to characterize the form of natural selection acting on natural populations and to better describe the relationship between ecological opportunity and speciation rates.

Encoding and retrieval are differentially processed by the anterior cingulate and prelimbic cortices: a study based on trace eyeblink conditioning in the rabbit.

A growing body of literature suggests that structures along the midline of the prefrontal cortex (mPFC), including Brodmann's area 32 (prelimbic cortex) and area 24 (anterior cingulate cortex) in the rabbit play a role in retrieval of learned information. The present studies compared the effects of post-training lesions produced either immediately or 1-week following learning, to either prelimbic (area 32) or anterior cingulate (area 24) cortex on trace eyeblink (EB) conditioning. Further, because recent evidence suggests that the mPFC may play an even greater role in learning and memory when emotional arousal is low, these studies compared the effects of lesions in groups conditioned with either a relatively low-arousal corneal airpuff, or a more aversive periorbital eyeshock unconditioned stimulus (US). A total of six groups were tested, which received selective ibotenic acid or "sham" control lesions to either area 32 or 24, immediately or 1-week following asymptotic learning, and conditioned with an eyeshock US or an airpuff US. Results showed that the greatest lesion deficits were found when conditioning with the less aversive airpuff US. Further, lesions produced to area 32 one-week, but not immediately following learning, caused significant deficits in performance, while lesions produced to area 24 immediately, but not 1-week following learning, caused significant deficits in performance. These findings add to the body of evidence which shows that area 32 of the mPFC regulates retrieval, but not acquisition or storage of information, while area 24 mediates a less specific reacquisition process, but not permanent storage or retrieval of information during relearning of memories abolished by mPFC damage. These findings were, however, specific to those experiments in which the relatively non-aversive airpuff was the US.

[Myocardial infarction as cause of an accident. The role of multislice CT in polytrauma management, differential diagnosis and insurance aspects]. / Der akute Myokardinfarkt als Unfallursache. Stellenwert der Ganzkörper-CT im Schockraum, Differenzialdiagnosen und versicherungsrechtliche Aspekte.

We present for the first time the use of contrast-enhanced multislice computed tomography in trauma care to detect acute myocardial infarction and verify it as the cause of a traffic accident. In addition to the case report, cardiac contusion, coronary dissection, and facets of insurance law are discussed. The determination of acute myocardial infarction, cardiac contusion, and coronary dissection can be challenging, but answers can be found in the medical history and accident details. The trauma surgeon in the emergency department must always be interested in clarifying the cause of trauma and keeping a secondary diagnosis in mind to strive for the goal of optimal and complete polytrauma care.

Prefrontal control of trace eyeblink conditioning in rabbits: role in retrieval of the CR?

Rabbits (Oryctolagus cuniculus) were trained on a trace eyeblink (EB) conditioning task to a criterion of 10 consecutive EB conditioned responses (CRs). One week later, ibotenic acid or sham lesions were made in the mPFC centered on the prelimbic region (Brodmann's area 32) or the cingulate cortex (Brodmann's area 24). Following a 1-week postoperative recovery period, all animals were retrained for 4 consecutive days using the same parameters as during acquisition, given 1 week off, and retrained for another 4 days. Mean EB conditioning deficits in the group with area 32 lesions occurred on the first and second days of each retraining period. However, by the third and fourth days of retraining, these lesioned animals were performing at a level comparable to that of the sham group. Lesions of area 24 did not produce deficits at either retesting period. These findings were interpreted to indicate that area 32, but not area 24, is involved in retrieval processes, rather than consolidation or storage, in that the animals were impaired at both retesting times, but were able to relearn the task.

Ibotenic acid lesions to ventrolateral thalamic nuclei disrupts trace and delay eyeblink conditioning in rabbits.

Intact cerebellar structures (i.e., deep nuclei and perhaps cortex) are essential for acquisition of both simple delay and trace eyeblink (EB) conditioning. However, successful trace conditioning also requires intact cortico-limbic structures (i.e., hippocampus, medial thalamus, and medial prefrontal cortex, mPFC). A direct connection between the cerebellum and ventrolateral thalamic nuclei (VLTN) has been demonstrated in several species. Since VLTN projects to both premotor and prefrontal cortex, it may be an essential link in a cerebellar-thalamic-prefrontal circuit that provides the CNS substrate for acquisition of the trace EB CR. The current studies thus assessed the role of the VLTN on trace EB conditioning in New Zealand albino rabbits. We first verified afferent connections to the mPFC (Brodmann's area 32) from the VLTN, by injecting the retrograde tracer Flourogold(c) into area 32. Strong labeling in VLTN from terminal projections to mPFC were found. We next assessed the role of VLTN in trace eyeblink conditioning in animals that received either sham or ibotenic acid VLTN lesions. EB conditioning began with 10 consecutive daily sessions of trace conditioning, followed immediately by 4 days of extinction, and then 4 days of delay conditioning. VLTN lesions significantly impaired acquisition of both trace and delay conditioning, and impaired extinction. These findings, thus confirm the importance of the VLTN in a postulated cerebellar-thalamic-prefrontal circuit that underlies successful trace, as well as delay EB conditioning.

Novel diode laser-compatible fluorophores and their application to single molecule detection, protein labeling and fluorescence resonance energy transfer immunoassay.

We describe a series of new long-wave absorbing and fluorescing cyanine dyes and labels (based on a general logic for the design of such dyes), their spectra, covalent and noncovalent linkage to proteins, their use in single molecule detection (SMD) and as donors and acceptors, respectively, in fluorescence resonance energy transfer studies. The new labels represent water-soluble and reactive fluorophores whose quantum yields increase substantially if noncovalently or covalently bound to proteins. Due to their strong absorptions between 550 and 700 nm they are excitable by light-emitting diodes or diode lasers. Their high absorbances (epsilon around 100,000) and adequate fluorescence quantum yields (phi up to 0.68 if bound to proteins) along with their availability as reactive NHS esters make them viable labels for proteins and oligomers, e.g. in context with SMD or fluorescence energy transfer immunoassay which is demonstrated for the system HSA/anti-HSA.

Vaccinia virus late transcription is activated in vitro by cellular heterogeneous nuclear ribonucleoproteins.

Vaccinia virus gene expression is temporally regulated, and three gene classes have been identified: early, intermediate, and late. Several virus-encoded proteins and an activity designated VLTF-X are required for maximum transcription in vitro of a template containing a late promoter. VLTF-X is present in both cytoplasmic and nuclear extracts prepared from uninfected mammalian cells and co-purifies with a late promoter DNA-binding activity. Here, extensive purification of VLTF-X has revealed that heterogeneous nuclear ribonucleoproteins A2/B1 and RBM3 co-purified with in vitro late transcription stimulation. Overexpression and purification of these proteins from Escherichia coli demonstrated that they both complemented for VLTF-X activity in in vitro transcription reactions. These studies identify two host cell factors potentially contributing to poxvirus replication in vivo.

Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes.

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of approximately 25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

Red laser-induced fluorescence energy transfer in an immunosystem.

We describe two near-infrared fluorescent squaraine dyes (Sq635 and Sq660), their spectra, their covalent linkage to proteins, and their use as donor and acceptor, respectively, in a fluorescence resonance energy transfer (FRET) immunoassay based on the use of red lasers. The dyes show quantum yields of around 10% in the free form and up to 68% when bound to proteins. If converted into their N-hydroxysuccinimide esters, they can be linked to free amino groups of proteins. To improve water solubility, two sulfo groups were introduced. The emission spectrum of Sq635 overlaps the absorption spectrum of Sq660, a fact that makes them a useful pair of dyes for use in FRET immunoassay which is demonstrated for human serum albumin/anti-human serum albumin.

Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels.

We describe the synthesis and spectral characterization of two reactive long-wavelength fluorescence labels (Sq635-m and Sq635-b), having either one or two N-hydroxysuccinimidyl esters. Both are squaraine derivatives and consist of a cyanine-type chromophore and a central squarate bridge. To improve water solubility, we introduced two sulfonic acid groups into the heterocyclic ring systems, and for covalent attachment to proteins, a reactive N-hydroxy-succinimide ester (NHS ester) was synthesized. The squaraine markers exhibit low quantum yields in water (phi = 0.15) and high quantum yields (phi = 0.6-0.7) when bound to proteins. The absorption maxima at 635 nm in water and at approximately 645 nm when bound to proteins allow excitation with commercially available diode lasers. The detection limit of a representative squaraine dye in blood was estimated to be half that of a commonly used fluorophore.

Managing organizational challenge and change: closing an inpatient unit.

The effects of a turbulent health care delivery market have impacted the day-to-day reality of acute care hospitals. One effect is that the supply of acute care hospital beds currently exceeds the demand, a trend that is expected to continue to the year 2000 and beyond. Nursing administrators at St. Marys Hospital Medical Center made the decision to close an inpatient unit in order to better match acute care bed supply to existing demand. Decision support for closure, organizational change, and lessons learned from the closure process are discussed.

Dynamin-mediated internalization of caveolae.

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.

Ethanol-induced alterations of the microtubule cytoskeleton in hepatocytes.

Ethanol has been predicted to alter vesicle-based protein traffic in hepatocytes, in part, via a disruption of the microtubule (MT) cytoskeleton. However, information on the effects of chronic ethanol exposure on MT function in vivo is sparse. Therefore the goal of this study was to test for ethanol-induced changes in rat liver tubulin expression, assembly, and cellular organization, using molecular, biochemical and morphological methods. The results of this study showed that tubulin mRNA and protein levels were not altered by ethanol. Tubulin, isolated from control and ethanol-fed rats, showed similar polymerization characteristics as assessed by calculation of the critical concentration for assembly and morphological structure. In contrast, the total amount of assembly-competent tubulin was reduced in livers from ethanol-fed rats compared with control rats when assessed by quantitative immunoblot analysis using a tubulin antibody. In addition, we observed that MT regrowth and organization in cultured hepatocytes treated with cold and nocodazole was markedly impaired by chronic ethanol exposure. In summary, these results indicate that tubulin levels in liver are not reduced by ethanol exposure. While there is a substantial amount of tubulin protein capable of assembling into functional MTs in ethanol-damaged livers, a marked portion of this tubulin is polymerization incompetent. This may explain why these hepatocytes exhibit a reduced number of MTs with an altered organization.

A vaccinia virus late transcription factor copurifies with a factor that binds to a viral late promoter and is complemented by extracts from uninfected HeLa cells.

We have previously described a vaccinia virus late transcription factor, VLTF-X, which we found to be present in cells at early and late times in infection. In this study, transcription complementation assays were used to demonstrate that VLTF-X activity is also present in virion extracts and in the cytoplasm of uninfected HeLa cells. Mobility shift assays performed on various VLTF-X preparations revealed that a late promoter DNA-binding activity cochromatographed and cosedimented with VLTF-X activity. Competition experiments demonstrated that this binding was specific for the late promoter region of the probe and that late transcription was dramatically reduced by an oligonucleotide that blocked factor-DNA complex formation but was only minimally affected by an oligonucleotide that did not inhibit complex formation. These results suggest that a cellular factor may participate in vaccinia virus late transcription. These findings also confirm the requirement for VLTF-X and distinguish it from any of the previously described vaccinia virus late transcription factors, which have all been mapped to the viral genome. Finally, these studies also suggest that the biochemical role for VLTF-X may be in late promoter recognition.

Establishment and molecular characterization of five cell lines derived from renal and extrarenal malignant rhabdoid tumors.

Malignant rhabdoid tumor (MRT) is a rare, enigmatic childhood cancer characterized by extreme aggressiveness and resistance to chemotherapy. To understand better the origin of the tumor and the mechanisms by which it develops and resists treatment, five cell lines were established from patients presenting with MRT (two renal and three extrarenal tumors). All of the cell lines display the light microscopic and ultrastructural features, as well as the variable immunohistochemical profile, characteristic of MRT. All are capable of forming tumors in nude mice. Three of the cell lines have detectable abnormalities of chromosome 22: one a t(22, 22) unbalanced translocation and two others a loss of heterozygosity of polymerase chain reaction-based microsatellite markers. Northern blot analysis showed that overexpression of the c-myc message was a consistent characteristic of the five MRTs evaluated. Although mutations of the p53 gene were not detectable by sequence analysis, all of the cell lines showed nuclear accumulation of the p53 protein by an immunocytochemical analysis in a minority of the cells. This result suggests that dysfunction in a p53-dependent apoptotic pathway might play a role in the multiple drug resistance phenotype of these tumors.

Vesicle movement in rat hepatocytes is reduced by ethanol exposure: alterations in microtubule-based motor enzymes.

BACKGROUND & AIMS: Ethanol is known to alter vesicle-mediated protein trafficking in hepatocytes by undefined mechanisms. In this study, the effects of long- and short-term ethanol exposure on vesicle movements were measured in isolated hepatocytes, and alterations in the function of the microtubule-associated motor enzymes dynamin, kinesin, and dynein, which are believed to support the transport and/or budding of vesicles along microtubules, were tested. METHODS: Vesicular movements in isolated hepatocytes exposed to short- and long-term ethanol treatment were measured. Motor adenosine triphosphatase activities and their association with specific membrane organelles were assessed in response to long-term administration of ethanol in vivo or acetaldehyde in vitro. RESULTS: Hepatocytes exposed to short- or long-term ethanol treatment showed a significant reduction in the number of motile vesicles. No alterations in the levels of motor messenger RNA, protein, or enzymatic activity were observed. Interestingly, ethanol had no effect on the association of dynein and kinesin with membranes, whereas there was a significant increase in the amount of dynamin associated specifically with Golgi membranes. CONCLUSIONS: Long- and short-term ethanol exposure markedly reduces hepatocellular vesicle transport by a mechanism apparently independent of any alteration in the enzymatic activity of molecular motors, possibly involving a change in the function of dynamin.

[Possibilities for interpreting digital migration analysis of cement-free PM total hip endoprostheses]. / Interpretationsmöglichkeiten der digitalen Wanderungsanalyse zementfreier PM-Hüfttotalendoprothesen.

PURPOSE: Aim of this study was the validation of a digital measurement device for hip implants. METHODS: From 43 patients with a minimum of four subsequent roentgenograms 246 roentgenograms of PM-shafts (an average 5.72/shaft) and 142 roentgenograms of PM-cups (an average 6.45/cup) were digitized on a DiagnostiX 2048 basis station (Pace Systems, Germany). Modified Sutherland/Nunn methods were chosen for measuring purposes. Regression analysis was done and the results were correlated to implant loosening. RESULTS: Concerning the total vertical migration of the cup there is a significant (p < 0.04) difference between the loosened components (6.02 +/- 2.59 mm) and the fixed cups (2.26 +/- 2.89 mm). The average annual vertical migration rate of the stem shows a highly significant difference (p < 0.007) when comparing loosened stems (0.82 +/- 0.43 mm, total migration 4.78 +/- 2.89 mm) and fixed ones (0.20 +/- 0.54 mm, total migration 1.72 +/- 3.02 mm) CONCLUSIONS: Using the regression analysis and a sufficient number of pictures the system allows us to give migration values that make loosening likely. However for the single implant an individual prognosis of loosening is not possible.

The hospital financing system in Germany.

In common with other western coutries, German health expenditure had been increasing at a rapid rate in recent years, especially in the hospital sector. This paper describes the reaction of the German legislator and summarises what has happened over the last few years following the introduction of the extensive Legal Reform Act. The paper puts the main emphasis on a new differentiated benefit system for hospitals, which is a requirement from 1996 onwards, after a transitional period. It shows the single components and the modalities of the new system and the possibilities of combining the new types of payment.