Local energy and pre-envelope

We observe that the local energy is the pre-envelope for analytic function. The maxima and phase of this function can be used to compute and classify visual features such as motion and stereo disparity, texture, etc. We examine the construction of new filters for computing Local Energy, and compare these filters with the Gabor filters and the three-point-filter of Venkatesh and Owens.

Reinforcing and expanding the predictions of the disturbance vicariance hypothesis in Amazonian harlequin frogs: A molecular phylogenetic and climate envelope modelling approach

The disturbance vicariance hypothesis (DV) has been proposed to explain speciation in Amazonia, especially its edge regions, e. g. in eastern Guiana Shield harlequin frogs (Atelopus) which are suggested to have derived from a cool-adapted Andean ancestor. In concordance with DV predictions we studied that (i) these amphibians display a natural distribution gap in central Amazonia; (ii) east of this gap they constitute a monophyletic lineage which is nested within a pre-Andean/western clade; (iii) climate envelopes of Atelopus west and east of the distribution gap show some macroclimatic divergence due to a regional climate envelope shift; (iv) geographic distributions of climate envelopes of western and eastern Atelopus range into central Amazonia but with limited spatial overlap. We tested if presence and apparent absence data points of Atelopus were homogenously distributed with Ripley's K function. A molecular phylogeny (mitochondrial 16S rRNA gene) was reconstructed using Maximum Likelihood and Bayesian Inference to study if Guianan Atelopus constitute a clade nested within a larger genus phylogeny. We focused on climate envelope divergence and geographic distribution by computing climatic envelope models with MaxEnt based on macroscale bioclimatic parameters and testing them by using Schoener's index and modified Hellinger distance. We corroborated existing DV predictions and, for the first time, formulated new DV predictions aiming on species' climate envelope change. Our results suggest that cool-adapted Andean Atelopus ancestors had dispersed into the Amazon basin and further onto the eastern Guiana Shield where, under warm conditions, they were forced to change climate envelopes. © 2010 The Author(s).

EIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNAPhe tertiary structure, analogously to the EF-P monomer. © 2012 Springer-Verlag.

Aeroelastic stability analysis considering a continuous flight envelope

This paper presents a new methodology to analyze aeroelastic stability in a continuous range of flight envelope with varying parameter of velocity and altitude. The focus of the paper is to demonstrate that linear matrix inequalities can be used to evaluate the aeroelastic stability in a region of flight envelope instead of a single point, like classical methods. The proposed methodology can also be used to study if a system remains stable during an arbitrary motion from one point to another in the flight envelope, i.e., when the problem becomes time-variant. The main idea is to represent the system as a polytopic differential inclusion system using rational function approximation to write the model in time domain. The theory is outlined and simulations are carried out on the benchmark AGARD 445.6 wing to demonstrate the method. The classical pk-method is used for comparing results and validating the approach. It is shown that this method is efficient to identify stability regions in the flight envelope. (C) 2014 Elsevier Ltd. All rights reserved.

Probabilistic Envelope Curves for Extreme Rainfall Events - Curve Inviluppo Probabilistiche per Precipitazioni Estreme

A regional envelope curve (REC) of flood flows summarises the current bound on our experience of extreme floods in a region. RECs are available for most regions of the world. Recent scientific papers introduced a probabilistic interpretation of these curves and formulated an empirical estimator of the recurrence interval T associated with a REC, which, in principle, enables us to use RECs for design purposes in ungauged basins. The main aim of this work is twofold. First, it extends the REC concept to extreme rainstorm events by introducing the Depth-Duration Envelope Curves (DDEC), which are defined as the regional upper bound on all the record rainfall depths at present for various rainfall duration. Second, it adapts the probabilistic interpretation proposed for RECs to DDECs and it assesses the suitability of these curves for estimating the T-year rainfall event associated with a given duration and large T values. Probabilistic DDECs are complementary to regional frequency analysis of rainstorms and their utilization in combination with a suitable rainfall-runoff model can provide useful indications on the magnitude of extreme floods for gauged and ungauged basins. The study focuses on two different national datasets, the peak over threshold (POT) series of rainfall depths with duration 30 min., 1, 3, 9 and 24 hrs. obtained for 700 Austrian raingauges and the Annual Maximum Series (AMS) of rainfall depths with duration spanning from 5 min. to 24 hrs. collected at 220 raingauges located in northern-central Italy. The estimation of the recurrence interval of DDEC requires the quantification of the equivalent number of independent data which, in turn, is a function of the cross-correlation among sequences. While the quantification and modelling of intersite dependence is a straightforward task for AMS series, it may be cumbersome for POT series. This paper proposes a possible approach to address this problem.

An extracytoplasmic function sigma factor operon regulates Myxococcus xanthus developmental gene expression

Myxococcus xanthus is a Gram-negative soil bacterium that undergoes multicellular development when high-density cells are starved on a solid surface. Expression of the 4445 gene, predicted to encode a periplasmic protein, commences 1.5 h after the initiation of development and requires starvation and high density conditions. Addition of crude or boiled supernatant from starving high-density cells restored 4445 expression to starving low-density cells. Addition of L-threonine or L-isoleucine to starving low-density cells also restored 4445 expression, indicating that the high-density signaling activity present in the supernatant might be composed of extracellular amino acids or small peptides. To investigate the circuitry integrating these starvation and high-density signals, the cis- and trans-acting elements controlling 4445 expression were identified. The 4445 transcription start site was determined by primer extension analysis to be 58 by upstream of the predicted translation start site. The promoter region contained a consensus sequence characteristic of e&barbelow;xtrac&barbelow;ytoplasmic f&barbelow;unction (ECF) sigma factor-dependent promoters, suggesting that 4445 expression might be regulated by an ECF sigma factor-dependent pathway, which are known to respond to envelope stresses. The small size of the minimum regulatory region, identified by 5′-end deletion analysis as being only 66 by upstream of the transcription start site, suggests that RNA polymerase could be the sole direct regulator of 4445 expression. To identify trans-acting negative regulators of 4445 expression, a strain containing a 4445-lacZ was mutagenized using the Himar1-tet transposon. The four transposon insertions characterized mapped to an operon encoding a putative ECF sigma factor, ecfA; an anti-sigma factor, reaA; and a negative regulator, reaB. The reaA and the reaB mutants expressed 4445 during growth and development at levels almost 100-fold higher than wild type, indicating that these genes encode negative regulators. The ecfA mutant expressed 4445-lacZ at basal levels, indicating that ecfA is a positive regulator. High Mg2+ concentrations over-stimulated this ecfA pathway possibly due to the depletion of exopolysaccharides and assembled type IV pili. These data indicate that the ecfA operon encodes a new regulatory stress pathway that integrates and transduces starvation and cell density cues during early development and is also responsive to cell-surface alterations.^

Ripple cancellation technique applied to a synchronous buck converter to achieve very high bandwidth and very high efficiency envelope amplifier

This work presents a single stage converter for a high bandwidth and a high efficiency envelope amplifier. The current ripple cancellation technique is applied to a synchronous buck converter to cancel the output current ripple and to decrease the switching frequency without a reduction in the large signal bandwidth. The converter is modeled and the new design with ripple cancellation circuit is detailed. The advantages of the proposed design are presented and validated experimentally. The transfer function of the output filter of the buck converter with ripple cancellation circuit has been modeled and compared to measurements, showing a good correspondence. Experimental validation is provided at 4MHz of switching frequency for DC and variable output voltage for a sinusoidal and a 64QAM signal. Additional experimental validation of the efficiency improvement is provided, compared to the equivalent design (same bandwidth and output voltage ripple) of the conventional buck converter.

Wide bandwidth envelope trackers with reduced switching frequency for RF power amplifier

El requerimiento de proveer alta frecuencia de datos en los modernos sistema de comunicación inalámbricos resulta en complejas señales moduladas de radio-frequencia (RF) con un gran ancho de banda y alto ratio pico-promedio (PAPR). Para garantizar la linealidad del comportamiento, los amplificadores lineales de potencia comunes funcionan típicamente entre 4 y 10 dB de back-o_ desde la máxima potencia de salida, ocasionando una baja eficiencia del sistema. La eliminación y restauración de la evolvente (EER) y el seguimiento de la evolvente (ET) son dos prometedoras técnicas para resolver el problema de la eficiencia. Tanto en EER como en ET, es complicado diseñar un amplificador de potencia que sea eficiente para señales de RF de alto ancho de banda y alto PAPR. Una propuesta común para los amplificadores de potencia es incluir un convertidor de potencia de muy alta eficiencia operando a frecuencias más altas que el ancho de banda de la señal RF. En este caso, la potencia perdida del convertidor ocasionado por la alta frecuencia desaconseja su práctica cuando el ancho de banda es muy alto. La solución a este problema es el enfoque de esta disertación que presenta dos arquitecturas de amplificador evolvente: convertidor híbrido-serie con una técnica de evolvente lenta y un convertidor multinivel basado en un convertidor reductor multifase con control de tiempo mínimo. En la primera arquitectura, una topología híbrida está compuesta de una convertidor reductor conmutado y un regulador lineal en serie que trabajan juntos para ajustar la tensión de salida para seguir a la evolvente con precisión. Un algoritmo de generación de una evolvente lenta crea una forma de onda con una pendiente limitada que es menor que la pendiente máxima de la evolvente original. La salida del convertidor reductor sigue esa forma de onda en vez de la evolvente original usando una menor frecuencia de conmutación, porque la forma de onda no sólo tiene una pendiente reducida sino también un menor ancho de banda. De esta forma, el regulador lineal se usa para filtrar la forma de onda tiene una pérdida de potencia adicional. Dependiendo de cuánto se puede reducir la pendiente de la evolvente para producir la forma de onda, existe un trade-off entre la pérdida de potencia del convertidor reductor relacionada con la frecuencia de conmutación y el regulador lineal. El punto óptimo referido a la menor pérdida de potencia total del amplificador de evolvente es capaz de identificarse con la ayuda de modelo preciso de pérdidas que es una combinación de modelos comportamentales y analíticos de pérdidas. Además, se analiza el efecto en la respuesta del filtro de salida del convertidor reductor. Un filtro de dampeo paralelo extra es necesario para eliminar la oscilación resonante del filtro de salida porque el convertidor reductor opera en lazo abierto. La segunda arquitectura es un amplificador de evolvente de seguimiento de tensión multinivel. Al contrario que los convertidores que usan multi-fuentes, un convertidor reductor multifase se emplea para generar la tensión multinivel. En régimen permanente, el convertidor reductor opera en puntos del ciclo de trabajo con cancelación completa del rizado. El número de niveles de tensión es igual al número de fases de acuerdo a las características del entrelazamiento del convertidor reductor. En la transición, un control de tiempo mínimo (MTC) para convertidores multifase es novedosamente propuesto y desarrollado para cambiar la tensión de salida del convertidor reductor entre diferentes niveles. A diferencia de controles convencionales de tiempo mínimo para convertidores multifase con inductancia equivalente, el propuesto MTC considera el rizado de corriente por cada fase basado en un desfase fijo que resulta en diferentes esquemas de control entre las fases. La ventaja de este control es que todas las corrientes vuelven a su fase en régimen permanente después de la transición para que la siguiente transición pueda empezar muy pronto, lo que es muy favorable para la aplicación de seguimiento de tensión multinivel. Además, el control es independiente de la carga y no es afectado por corrientes de fase desbalanceadas. Al igual que en la primera arquitectura, hay una etapa lineal con la misma función, conectada en serie con el convertidor reductor multifase. Dado que tanto el régimen permanente como el estado de transición del convertidor no están fuertemente relacionados con la frecuencia de conmutación, la frecuencia de conmutación puede ser reducida para el alto ancho de banda de la evolvente, la cual es la principal consideración de esta arquitectura. La optimización de la segunda arquitectura para más alto anchos de banda de la evolvente es presentada incluyendo el diseño del filtro de salida, la frecuencia de conmutación y el número de fases. El área de diseño del filtro está restringido por la transición rápida y el mínimo pulso del hardware. La rápida transición necesita un filtro pequeño pero la limitación del pulso mínimo del hardware lleva el diseño en el sentido contrario. La frecuencia de conmutación del convertidor afecta principalmente a la limitación del mínimo pulso y a las pérdidas de potencia. Con una menor frecuencia de conmutación, el ancho de pulso en la transición es más pequeño. El número de fases relativo a la aplicación específica puede ser optimizado en términos de la eficiencia global. Otro aspecto de la optimización es mejorar la estrategia de control. La transición permite seguir algunas partes de la evolvente que son más rápidas de lo que el hardware puede soportar al precio de complejidad. El nuevo método de sincronización de la transición incrementa la frecuencia de la transición, permitiendo que la tensión multinivel esté más cerca de la evolvente. Ambas estrategias permiten que el convertidor pueda seguir una evolvente con un ancho de banda más alto que la limitación de la etapa de potencia. El modelo de pérdidas del amplificador de evolvente se ha detallado y validado mediante medidas. El mecanismo de pérdidas de potencia del convertidor reductor tiene que incluir las transiciones en tiempo real, lo cual es diferente del clásico modelos de pérdidas de un convertidor reductor síncrono. Este modelo estima la eficiencia del sistema y juega un papel muy importante en el proceso de optimización. Finalmente, la segunda arquitectura del amplificador de evolvente se integra con el amplificador de clase F. La medida del sistema EER prueba el ahorro de energía con el amplificador de evolvente propuesto sin perjudicar la linealidad del sistema. ABSTRACT The requirement of delivering high data rates in modern wireless communication systems results in complex modulated RF signals with wide bandwidth and high peak-to-average ratio (PAPR). In order to guarantee the linearity performance, the conventional linear power amplifiers typically work at 4 to 10 dB back-off from the maximum output power, leading to low system efficiency. The envelope elimination and restoration (EER) and envelope tracking (ET) are two promising techniques to overcome the efficiency problem. In both EER and ET, it is challenging to design efficient envelope amplifier for wide bandwidth and high PAPR RF signals. An usual approach for envelope amplifier includes a high-efficiency switching power converter operating at a frequency higher than the RF signal's bandwidth. In this case, the power loss of converter caused by high switching operation becomes unbearable for system efficiency when signal bandwidth is very wide. The solution of this problem is the focus of this dissertation that presents two architectures of envelope amplifier: a hybrid series converter with slow-envelope technique and a multilevel converter based on a multiphase buck converter with the minimum time control. In the first architecture, a hybrid topology is composed of a switched buck converter and a linear regulator in series that work together to adjust the output voltage to track the envelope with accuracy. A slow envelope generation algorithm yields a waveform with limited slew rate that is lower than the maximum slew rate of the original envelope. The buck converter's output follows this waveform instead of the original envelope using lower switching frequency, because the waveform has not only reduced slew rate but also reduced bandwidth. In this way, the linear regulator used to filter the waveform has additional power loss. Depending on how much reduction of the slew rate of envelope in order to obtain that waveform, there is a trade-off between the power loss of buck converter related to the switching frequency and the power loss of linear regulator. The optimal point referring to the lowest total power loss of this envelope amplifier is identified with the help of a precise power loss model that is a combination of behavioral and analytic loss model. In addition, the output filter's effect on the response is analyzed. An extra parallel damping filter is needed to eliminate the resonant oscillation of output filter L and C, because the buck converter operates in open loop. The second architecture is a multilevel voltage tracking envelope amplifier. Unlike the converters using multi-sources, a multiphase buck converter is employed to generate the multilevel voltage. In the steady state, the buck converter operates at complete ripple cancellation points of duty cycle. The number of the voltage levels is equal to the number of phases according the characteristics of interleaved buck converter. In the transition, a minimum time control (MTC) for multiphase converter is originally proposed and developed for changing the output voltage of buck converter between different levels. As opposed to conventional minimum time control for multiphase converter with equivalent inductance, the proposed MTC considers the current ripple of each phase based on the fixed phase shift resulting in different control schemes among the phases. The advantage of this control is that all the phase current return to the steady state after the transition so that the next transition can be triggered very soon, which is very favorable for the application of multilevel voltage tracking. Besides, the control is independent on the load condition and not affected by the unbalance of phase current. Like the first architecture, there is also a linear stage with the same function, connected in series with the multiphase buck converter. Since both steady state and transition state of the converter are not strongly related to the switching frequency, it can be reduced for wide bandwidth envelope which is the main consideration of this architecture. The optimization of the second architecture for wider bandwidth envelope is presented including the output filter design, switching frequency and the number of phases. The filter design area is restrained by fast transition and the minimum pulse of hardware. The fast transition needs small filter but the minimum pulse of hardware limitation pushes the filter in opposite way. The converter switching frequency mainly affects the minimum pulse limitation and the power loss. With lower switching frequency, the pulse width in the transition is smaller. The number of phases related to specific application can be optimized in terms of overall efficiency. Another aspect of optimization is improving control strategy. Transition shift allows tracking some parts of envelope that are faster than the hardware can support at the price of complexity. The new transition synchronization method increases the frequency of transition, allowing the multilevel voltage to be closer to the envelope. Both control strategies push the converter to track wider bandwidth envelope than the limitation of power stage. The power loss model of envelope amplifier is detailed and validated by measurements. The power loss mechanism of buck converter has to include the transitions in real time operation, which is different from classical power loss model of synchronous buck converter. This model estimates the system efficiency and play a very important role in optimization process. Finally, the second envelope amplifier architecture is integrated with a Class F amplifier. EER system measurement proves the power saving with the proposed envelope amplifier without disrupting the linearity performance.

Tumor cells expressing a retroviral envelope escape immune rejection in vivo

A model system for the in vivo control of tumor cell proliferation by the immune system has been used to assay for the possible immunosuppressive activity of retroviral proteins. Expression vectors for the entire or the transmembrane subunit of the Moloney murine leukemia virus envelope protein were constructed, as well as control vectors for irrelevant transmembrane proteins—or no protein. They were introduced either into MCA205 murine tumor cells, which do not proliferate upon s.c. injection into an allogeneic host, or into CL8.1 murine tumor cells, which overexpress class I antigens and are rejected in a syngeneic host. In both cases, expression of the complete envelope protein or of the transmembrane subunit resulted in tumor growth in vivo, with no effect of control vectors. Tumor cell growth results from inhibition of the host immune response, as the envelope-dependent effect was no more observed for MCA205 cells in syngeneic mice or for CL8.1 cells in x-irradiated mice. This inhibition is local because it is not observed at the level of control tumor cells injected contralaterally. These results suggest a noncanonical function of retroviral envelopes in the “penetrance” of viral infections, as well as a possible involvement of the envelope proteins of endogenous retroviruses in tumoral processes.

The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2–1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

The long cytoplasmic tail of gp41 is required in a cell type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions

Lentiviruses, including HIV-1, have transmembrane envelope (Env) glycoproteins with cytoplasmic tails that are quite long compared with those of other retroviruses. However, mainly because of the lack of biochemical studies performed in cell types that are targets for HIV-1 infection, no clear consensus exists regarding the function of the long lentiviral Env cytoplasmic tail in virus replication. In this report, we characterize the biological and biochemical properties of an HIV-1 mutant lacking the gp41 cytoplasmic tail. We find that the gp41 cytoplasmic tail is necessary for the efficient establishment of a productive, spreading infection in the majority of T cell lines tested, peripheral blood mononuclear cells, and monocyte-derived macrophages. Biochemical studies using a high-level, transient HIV-1 expression system based on pseudotyping with the vesicular stomatitis virus glycoprotein demonstrate that in HeLa and MT-4 cells, mutant Env incorporation into virions is reduced only 3-fold relative to wild type. In contrast, gp120 levels in virions produced from a number of other T cell lines and primary macrophages are reduced more than 10-fold by the gp41 truncation. The Env incorporation defect imposed by the cytoplasmic tail truncation is not the result of increased shedding of gp120 from virions or reduced cell-surface Env expression. These results demonstrate that in the majority of T cell lines, and in primary cell types that serve as natural targets for HIV-1 infection in vivo, the gp41 cytoplasmic tail is essential for efficient Env incorporation into virions.

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98−/− cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98−/− cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation

Jaagsiekte sheep retrovirus (JSRV) can induce rapid, multifocal lung cancer, but JSRV is a simple retrovirus having no known oncogenes. Here we show that the envelope (env) gene of JSRV has the unusual property that it can induce transformation in rat fibroblasts, and thus is likely to be responsible for oncogenesis in animals. Retrovirus entry into cells is mediated by Env interaction with particular cell-surface receptors, and we have used phenotypic screening of radiation hybrid cell lines to identify the candidate lung cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2 was previously described as a lysosomal hyaluronidase, but we show that HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. Furthermore, we could not detect hyaluronidase activity associated with or secreted by cells expressing HYAL2, whereas we could easily detect such activity from cells expressing the related serum hyaluronidase HYAL1. Although the function of HYAL2 is currently unknown, other GPI-anchored proteins are involved in signal transduction, and some mediate mitogenic responses, suggesting a potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer induced by JSRV closely resembles human bronchiolo-alveolar carcinoma, a disease that is increasing in frequency and now accounts for ≈25% of all lung cancer. The finding that JSRV env is oncogenic and the identification of HYAL2 as the JSRV receptor provide tools for further investigation of the mechanism of JSRV oncogenesis and its relationship to human bronchiolo-alveolar carcinoma.

Protein kinase C chimeras: catalytic domains of alpha and beta II protein kinase C contain determinants for isotype-specific function.

Protein kinase C (PKC) is involved in the proliferation and differentiation of many cell types. In human erythroleukemia (K-562) cells, the PKC isoforms alpha and beta II play distinct functional roles. alpha PKC is involved in phorbol 12-myristate 13-acetate-induced cytostasis and megakaryocytic differentiation, whereas beta II PKC is required for proliferation. To identify regions within alpha and beta II PKC that allow participation in these divergent pathways, we constructed chimeras in which the regulatory and catalytic domains of alpha and beta II PKC were exchanged. These PKC chimeras can be stably expressed, exhibit enzymatic properties similar to native alpha and beta II PKC in vitro, and participate in alpha and beta II PKC isotype-specific pathways in K-562 cells. Expression of the beta/alpha PKC chimera induces cytostasis in the same manner as overexpression of wild-type alpha PKC. In contrast, the alpha/beta II PKC chimera, like wild-type beta II PKC, selectively translocates to the nucleus and leads to increased phosphorylation of the nuclear envelope polypeptide lamin B in response to bryostatin-1. Therefore, the catalytic domains of alpha and beta II PKC contain determinants important for alpha and beta II PKC isotype function. These results suggest that the catalytic domain represents a potential target for modulating PKC isotype activity in vivo.