Highly Stoichiometric, Stable, and Specific Association of Integrin α3β1 with CD151 Provides a Major Link to Phosphatidylinositol 4-Kinase, and May Regulate Cell Migration

Here we describe an association between α3β1 integrin and transmembrane-4 superfamily (TM4SF) protein CD151. This association is maintained in relatively stringent detergents and thus is remarkably stable in comparison with previously reported integrin–TM4SF protein associations. Also, the association is highly specific (i.e., observed in vitro in absence of any other cell surface proteins), and highly stoichiometric (nearly 90% of α3β1 associated with CD151). In addition, α3β1 and CD151 appeared in parallel on many cell lines and showed nearly identical skin staining patterns. Compared with other integrins, α3β1 exhibited a considerably higher level of associated phosphatidylinositol-4-kinase (PtdIns 4-kinase) activity, most of which was removed upon immunodepletion of CD151. Specificity for CD151 and PtdIns 4-kinase association resided in the extracellular domain of α3β1, thus establishing a novel paradigm for the specific recruitment of an intracellular signaling molecule. Finally, antibodies to either CD151 or α3β1 caused a ∼88–92% reduction in neutrophil motility in response to f-Met-Leu-Phe on fibronectin, suggesting an functionally important role of these complexes in cell migration.

Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease

For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 × 106 infectious units of recombinant adeno-associated virus encoding the human β-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.

Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts

This paper describes three distinct estrogen receptor (ER) subtypes: ERα, ERβ, and a unique type, ERγ, cloned from a teleost fish, the Atlantic croaker Micropogonias undulatus; the first identification of a third type of classical ER in vertebrate species. Phylogenetic analysis shows that ERγ arose through gene duplication from ERβ early in the teleost lineage and indicates that ERγ is present in other teleosts, although it has not been recognized as such. The Atlantic croaker ERγ shows amino acid differences in regions important for ligand binding and receptor activation that are conserved in all other ERγs. The three ER subtypes are genetically distinct and have different distribution patterns in Atlantic croaker tissues. In addition, ERβ and ERγ fusion proteins can each bind estradiol-17β with high affinity. The presence of three functional ERs in one species expands the role of ER multiplicity in estrogen signaling systems and provides a unique opportunity to investigate the dynamics and mechanisms of ER evolution.

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Capture of a protein synthesis-dependent component of long-term depression

Hippocampal-based behavioral memories and hippocampal-based forms of synaptic plasticity, such as long-term potentiation, are divisible into short- and long-term phases, with the long-term phase requiring the synthesis of new proteins and mRNA for its persistence. By contrast, it is less clear whether long-term depression (LTD) can be divisible into phases. We here describe that in stable hippocampal organotypic cultures, LTD also is not a unitary event but a multiphase process. A prolonged stimulus of 900 stimuli spaced at 1 Hz for 15 min induces a late phase of LTD, which is protein- and mRNA synthesis-dependent. By contrast, a short train of the same 900 stimuli massed at 5 Hz for 3 min produces only a short-lasting LTD. This short-lasting LTD is capable of capturing late-phase LTD. The 5-Hz stimulus or the prolonged 1-Hz stimulus in the presence of protein synthesis inhibitors each can be transformed into an enduring late phase of depression when the prolonged stimulus is applied to another input in the same population of neurons.

NEEDLY, a Pinus radiata ortholog of FLORICAULA/LEAFY genes, expressed in both reproductive and vegetative meristems

The LEAFY/FLORICAULA genes from Arabidopsis and Antirrhinum are necessary for normal flower development and play a key role in diverse angiosperm species. A homologue of these flower meristem-identity genes, NEEDLY (NLY), has been identified in Pinus radiata. Although the NLY protein shares extensive sequence similarity with its angiosperm counterparts, it is lacking the proline-rich and acidic motifs thought to function as transcriptional activation domains. NLY already is expressed during vegetative development at least 5 years before the transition to the reproductive phase. Expression of NLY in transgenic Arabidopsis promotes floral fate, demonstrating that, despite its sequence divergence, NLY encodes a functional ortholog of the FLORICAULA/LEAFY genes of angiosperms. Expression of the LFY∷NLY transgene can largely complement the defects in flower development caused by a severe lfy allele.

Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods

A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.

A New Model for Nuclear Envelope BreakdownV⃞

Nuclear envelope breakdown was investigated during meiotic maturation of starfish oocytes. Fluorescent 70-kDa dextran entry, as monitored by confocal microscopy, consists of two phases, a slow uniform increase and then a massive wave. From quantitative analysis of the first phase of dextran entry, and from imaging of green fluorescent protein chimeras, we conclude that nuclear pore disassembly begins several minutes before nuclear envelope breakdown. The best fit for the second phase of entry is with a spreading disruption of the membrane permeability barrier determined by three-dimensional computer simulations of diffusion. We propose a new model for the mechanism of nuclear envelope breakdown in which disassembly of the nuclear pores leads to a fenestration of the nuclear envelope double membrane.

Anatomy of word and sentence meaning

Reading and listening involve complex psychological processes that recruit many brain areas. The anatomy of processing English words has been studied by a variety of imaging methods. Although there is widespread agreement on the general anatomical areas involved in comprehending words, there are still disputes about the computations that go on in these areas. Examination of the time relations (circuitry) among these anatomical areas can aid in understanding their computations. In this paper, we concentrate on tasks that involve obtaining the meaning of a word in isolation or in relation to a sentence. Our current data support a finding in the literature that frontal semantic areas are active well before posterior areas. We use the subject’s attention to amplify relevant brain areas involved either in semantic classification or in judging the relation of the word to a sentence to test the hypothesis that frontal areas are concerned with lexical semantics and posterior areas are more involved in comprehension of propositions that involve several words.

Biologic consequences of Stat1-independent IFN signaling

Although Stat1 is required for many IFN-dependent responses, recent work has shown that IFNγ functions independently of Stat1 to affect the growth of tumor cells or immortalized fibroblasts. We now demonstrate that both IFNγ and IFNα/β regulate proliferative responses in cells of the mononuclear phagocyte lineage derived from Stat1-null mice. Using both representational difference analysis and gene arrays, we show that IFNγ exerts its Stat1-independent actions on mononuclear phagocytes by regulating the expression of many genes. This result was confirmed by monitoring changes in expression and function of the corresponding gene products. Regulation of the expression of these genes requires the IFNγ receptor and Jak1. The physiologic relevance of IFN-dependent, Stat1-independent signaling was demonstrated by monitoring antiviral responses in Stat1-null mice. Thus, the IFN receptors engage alternative Stat1-independent signaling pathways that have important physiological consequences.