Tissue-specific Signals Research Articles

Introduction on Angiogenesis in Breast Cancer

Angiogenesis is a complex process involving the growth and organization of blood vessels, and has been studied for over 30 years. Many excellent reviews about the distinct events that lead to the production of a vascular and microvascular tree exist already. Of course, much of the work has been focused on the angiogenesis present in pathologic states, notably oncogenesis and inflammatory diseases. Starting with the original work of Paget and then continued by many other researchers spearheaded by the original and groundbreaking work of Judah Folkman, Josh Fidler, and Mina Bissell, studies of abnormal angiogenesis have focused on understanding the complex interactions between the supporting stroma and the “diseased” index cell. In particular, a growing concern about tissue-specific signals has raised awareness that although governed by common principles, the details of abnormal angiogenesis require models that realistically reflect the intricacies of tissue specificity. We believe that this may derive from the fact that the growth of blood vessels in embryonic life develops following dynamic and plastic programs that depend on tissue differentiation. Because you are reading this journal, we surmise you have embraced the overarching hypothesis that tissue specificity must be considered when studying development and pathogenesis in the mammary gland. In this volume, we undertake to provide the readers of Mammary Gland Biology and Neoplasia with a series of articles that deal with novel hypotheses of the angiogenesis process in the breast. We strive to make this volume an excellent resource for researchers entering the field and to provide a starting point to ponder this fascinating process, which holds such great potential as a therapeutic target. Some of this potential is already being realized, as you will see explained in the following chapters. The chapters by Mercurio et al. and Merajver and Usmani identify novel aspects of signaling in angiogenesis in the mammary gland. The Mercurio group has been at the forefront of our understanding of integrin regulation. By now turning their attention to this regulation in mammary carcinogenesis, they clarify the potential for vascular endothelial growth factor to play a major role in breast cancer survival and success at metastatic sites. They further conceive a hypothesis for VEGF to have a role beyond its known effects on endothelial cell survival and angiogenesis per se. This chapter is especially useful for those concerned with the early steps in breast cancer metastasis and derangement of cell adhesion. Complementing this chapter, Merajver and Usmani review the impact of Rho proteins, which to a large extent are signaling upstream of integrins in response to G proteincoupled receptor stimulation. Although the Rho proteins have been traditionally associated with budding and membrane focal adhesions, it is now additionally known that they are required for metastasis in breast cancer; as such, they constitute especially attractive potential targets for therapeutic interventions. The Merajver lab has been studying the most lethal form of breast cancer—inflammatory breast cancer—for over 10 years; this chapter reflects some of the key insights they have gained through that work, and includes cutting edge research into angiogenesis-related targets that is reaching the clinic. J Mammary Gland Biol Neoplasia (2005) 10: 281–282 DOI 10.1007/s10911-006-9000-x

Combined imaging–histological study of cortical laminar specificity of fMRI signals

Since the commencement of functional magnetic resonance imaging (fMRI), great effort has been put into increasing its spatial resolution and signal specificity from vessel-weighted to more tissue-specific signals. The working assumption is that the “tissue” signals closely mirror changes at the neuronal level. While great progress has been made, the basic and most fundamental questions remain unanswered: where in the gray matter do these “tissue fMRI” changes occur? Recently, the temporal correspondence of hemodynamic-based fMRI signals and neurophysiological activity was explored. The data suggest, although not conclusively, that the local field potential (LFP) response gives a better estimate of changes that accompany increased neuronal activity. LFP are thought to be generated by synaptic activity reflecting input signals into layer IV within a cortical region. If so, the spatial distribution of the fMRI signal should be specific to the corresponding cortical lamina. Here, in a combined imaging and histological study, the spatial characteristics of fMRI signals across the lamina were explored. In a high-resolution fMRI study (0.15 × 0.15 × 2 mm 3), the spatial specificity of fMRI signals was correlated with the underlying cortical laminar cytoarchitectonic obtained within the same animal and tissue region. We demonstrate that when surface vessels are excluded high-resolution fMRI signals peak at cortical layer IV.

VCAM-1 Expression in B-Lymphopoiesis and Leukemia.

Vascular Cell Adhesion Molecule-1 (VCAM; CD106), a member of the Ig Superfamily of molecules, binds to the β-1 integrin, Very Late Antigen-4 (VLA-4; CD49d); this interaction plays an integral role in leukocyte trafficking as well as lymphocyte-stromal cell interactions. VCAM can be shed from the surface of cells, and, in humans, serum levels of soluble VCAM (sVCAM) parallel activity and remission states in acute lymphocytic leukemia (ALL) and inflammation. Although widely investigated as a stromal-cell associated molecule, our lab and others have recently identified VCAM expression on normal bone-marrow derived B-lymphoid cells.Using FACS technology, we found that surface expression of VCAM is closely modulated at specific stages of B cell development, with relatively high levels on the pro-B cell population, down-modulation in pre-B cells at the onset of immunoglobulin (Ig) gene rearrangement, and subsequent re-expression at variable levels in immature and mature peripheral B cell subsets. We have verified VCAM transcripts by cDNA PCR in highly purified populations of murine precursor B cells. Normal human bone marrow precursor B-lymphoid populations (hematogones) also demonstrate VCAM surface protein expression. Finally, in an animal model of BCR/ABL+ ALL, we found that VCAM expression is dramatically increased on lymphoblasts when compared to normal reference populations in bone marrow and spleen. VCAM expression in human lymphoid malignancies is currently under investigation.Antibody-mediated VCAM cross-linking on primary B-cell precursors ex-vivo generates intracellular reactive oxygen species, demonstrating that signaling through this molecule has functional consequences. Intriguingly, in-vivo, VCAM expression is limited to B-lymphoid cells harvested from tissues such as bone marrow, spleen and lymph node; since, in the same animal, peripheral blood lymphocytes and peritoneal cells do not express readily detectable levels of the surface antigen. VCAM-expressing B-lymphoid cells cultured ex-vivo gradually lose surface expression over 24 hours. The tissue-associated modulation of VCAM expression is preserved in the murine Ph+ lymphoblasts; leukemia cells isolated from the peripheral blood express very low levels of surface VCAM compared to those harvested from bone marrow or spleen. Our data suggests that VCAM expression is dependent on tissue-specific microenvironmental signals in-vivo.B-lymphoid expression of both VCAM and its ligand VLA-4 is a surprising finding that has broad implications regarding leukemic cell interaction with endothelial cells, the bone marrow retention and trafficking of precursor- and leukemic-B cell populations, and the interpretation of an extensive experimental database predicated on the stromal-cell specificity of VCAM expression and function.

High-resolution functional magnetic resonance imaging of the animal brain

To fully understand brain function, one must look beyond the level of a single neuron. By elucidating the spatial properties of the columnar and laminar functional architectures, information regarding the neural processing in the brain can be gained. To map these fine functional structures noninvasively and repeatedly, functional magnetic resonance imaging (fMRI) can be employed. In this article the basic principles of fMRI are introduced, including specific hardware requirements and the equipment necessary for animal magnetic resonance research. Since fMRI measures a change in secondary hemodynamic responses induced by neural activity, it is critical to understand the principles and potential pitfalls of fMRI techniques. Thus, the underlying physics of conventional blood oxygenation, cerebral blood flow, and cerebral blood volume-based fMRI techniques are extensively discussed. Tissue-specific signal change is close to the site of neural activity, while signals from large vessels can be distant from the actual active site. Thus, methods to minimize large vessel contributions and to maximize tissue signals are described. The fundamental limitation of fMRI spatial resolution is the intrinsic hemodynamic response. Based on our high-resolution fMRI studies, the hemodynamic response is regulated at submillimeter functional domains and thus spatial resolution can be achieved to an order of 100 μm. Since hemodynamic responses are sluggish, it is difficult to obtain very high temporal resolution. By using an approach with multiple experiments with different stimulus conditions, temporal resolution can be improved on the order of 100 ms. With current fMRI technologies, submillimeter columnar- and laminar-specific specific functional images can be obtained from animal brains.

Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy.

The recirculation of T cells between the blood and secondary lymphoid organs requires that T cells are motile and sensitive to tissue-specific signals. T cell motility has been studied in vitro, but the migratory behavior of individual T cells in vivo has remained enigmatic. Here, using intravital two-photon laser microscopy, we imaged the locomotion and trafficking of naive CD4(+) T cells in the inguinal lymph nodes of anesthetized mice. Intravital recordings deep within the lymph node showed T cells flowing rapidly in the microvasculature and captured individual homing events. Within the diffuse cortex, T cells displayed robust motility with an average velocity of approximately 11 microm x min(-1). T cells cycled between states of low and high motility roughly every 2 min, achieving peak velocities >25 microm x min(-1). An analysis of T cell migration in 3D space revealed a default trafficking program analogous to a random walk. Our results show that naive T cells do not migrate collectively, as they might under the direction of pervasive chemokine gradients. Instead, they appear to migrate as autonomous agents, each cell taking an independent trafficking path. Our results call into question the role of chemokine gradients for basal T cell trafficking within T cell areas and suggest that antigen detection may result from a stochastic process through which a random walk facilitates contact with antigen-presenting dendritic cells.

Adaptor Protein Lad Relays PDGF Signal to Grb2 in Lung Cells: A Tissue-Specific PDGF Signal Transduction

Lad was previously identified as an adaptor protein binding to the SH2 domain of Lck (1). Specific detection of Lad mRNA in lung cells, as well as, in T cells led us to investigate the signaling pathways regulating Lad in lung cells. We found that (i) upon PDGF stimulation, Lad expression is induced in lung cells, especially in the bronchial epithelial cells; (ii) Lad is tyrosine phosphorylated upon PDGF stimulation and is associated with PDGF receptor; (iii) upon PDGF stimulation, Grb2 is recruited to Lad in human embryonic lung cells; (iv) overexpression of Lad elevated AP-1 promoter activity by two- to threefold, whereas dominant negative Lad abrogated PDGF-dependent activation of AP-1 promoter. These results provide a novel mechanism of PDGF-dependent signaling, in which Lad acts as an adaptor in a tissue-specific manner, linking PDGF signal to Grb2 and subsequent activation of AP-1.

Tissue-specific signal(s) activate the promoter of a metallocarboxypeptidase inhibitor gene family in potato tuber and berry.

The molecular basis of the differential expression of the GM7-type metallocarboxypeptidase inhibitor (MCPI) genes in tuberizing (StMCPI) and non-tuberizing Solanum species (SbMCPI) was investigated. It was shown that the StMCPI is encoded by a gene family in Solanum tuberosum (potato), but SbMCPI might be a single-copy gene in the non-tuberizing species Solanum brevidens. The StMCPI promoter shows evolutionary relatedness to the S. brevidens-derived SbMCPI and to the fruit-specific tomato promoter 2A11. Both StMCPI and SbMCPI promoter regions were able to confer tuber- and berry-specific expression for the beta-glucuronidase reporter gene in potato suggesting that the difference in MCPI gene expression is in trans regulatory factors between the tuberizing and the non-tuberizing Solanum species. The MCPI promoters did not respond to metabolic, environmental or hormonal signals in leaves. Thus, the MCPI genes are regulated in a different way than the other known tuber-specific genes and potentially are suitable for biotechnological application in potato to provide specific transgene expression in tuber and berry.

Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers

Sex determination is regulated by diverse pathways. Although upstream signals vary, a cysteine-rich DNA-binding domain (the DM motif) is conserved within downstream transcription factors of Drosophila melanogaster (Doublesex) and Caenorhabditis elegans (MAB-3). Vertebrate DM genes have likewise been identified and, remarkably, are associated with human sex reversal (46, XY gonadal dysgenesis). Here we demonstrate that the structure of the Doublesex domain contains a novel zinc module and disordered tail. The module consists of intertwined CCHC and HCCC Zn(2+)-binding sites; the tail functions as a nascent recognition alpha-helix. Mutations in either Zn(2+)-binding site or tail can lead to an intersex phenotype. The motif binds in the DNA minor groove without sharp DNA bending. These molecular features, unusual among zinc fingers and zinc modules, underlie the organization of a Drosophila enhancer that integrates sex- and tissue-specific signals. The structure provides a foundation for analysis of DM mutations affecting sexual dimorphism and courtship behavior.

Molecular genetic mechanisms of life span manipulation in Caenorhabditis elegans.

Aging and a limited life span are fundamental biological realities. Recent studies have demonstrated that longevity can be manipulated and have revealed molecular mechanisms underlying longevity control in the soil nematode Caenorhabditis elegans. Signals from both neurons and the gonad appear to negatively regulate longevity. One tissue-specific signal involves an insulin-like phosphatidylinositol 3-OH kinase pathway, dependent upon the DAF-16 forkhead transcription factor. These signals regulate mechanisms determining longevity that include the OLD-1 (formerly referred to as TKR-1) receptor tyrosine kinase. Interestingly, increased resistance to environmental stress shows a strong correlation with life extension.

Magnetic bead purification as a rapid and efficient method for enhanced antibody specificity for plant sample immunoblotting and immunolocalization

Molecular analysis of plant tissues with antibodies has traditionally been hindered by the presence of high non-specific binding by plant cell walls and other components along with significant contaminants within sera that retard identification of specific plant tissue targets. Methods which rely on immobile solid supports conjugated with high-affinity molecular entities, have been used to purify sera. Despite their wide use, traditional antibody purification methods can result in low yields or activity and can produce significant levels of secondary contaminants, resulting in high non-specific background and dilution of tissue-specific signals. Mobile support matrixes like magnetic beads conjugated with high-affinity antisera have recently become an efficient alternative method for isolating and identifying diverse molecular targets. In this study, rabbit anti-calreticulin (CRT) immunoglobulin G (IgG) was isolated from whole anti-CRT sera with magnetic beads and tested by Western blot and immunocytochemistry for CRT localization in Pistia stratiotes plant tissues. IgG protein quantitation and purity was compared between purified and non-purified pre-immune and anti-CRT sera using spectrophotometric, reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and fluorescence staining followed by quantitative densitometry analysis. Anti-CRT IgG binding specificity after purification was determined by Western blot of total soluble protein extract. Purified and non-purified pre-immune and anti-CRT samples were subsequently utilized for CRT immunogold localization in Pistia tissue sections and visualized with confocal microscopy. The results demonstrate that magnetic bead purified anti-CRT IgG from whole serum shows enhanced specificity and reduced background. The ease of use and speed of this IgG purification technique should find widespread use in the plant biology field.

Macrophage responses to hypoxia: relevance to disease mechanisms.

Macrophages are ubiquitous in the stromal compartment of tissues under normal physiological conditions and the number of these cells increases markedly with the onset and progression of many pathological states. The mechanisms underlying this response are well described in such conditions as wound healing and malignant tumors, where tissue-specific signals enhance the extravasation of blood monocytes and their subsequent differentiation into macrophages. Recent evidence suggests that macrophages may also be stimulated by microenvironmental factors present in diseased tissues to perform distinct, tissue-specific activities. One such factor, hypoxia (low oxygen tension), results from insufficient vascular perfusion of a given tissue. Various studies have shown that experimental hypoxia alters the morphology, expression of cell surface markers, viability, phagocytosis, metabolic activity, and release of cytokines by macrophages. Here we review the evidence for these macrophage responses to hypoxia, the involvement of co-stimuli, and their implications for the role of macrophages in various disease processes. Because the intracellular mechanisms mediating the effects of hypoxia on gene expression in other cell types have been characterized recently, we discuss their possible involvement in the effects of hypoxia on gene expression in macrophages.

Osteosarcoma after Preoperative Chemotherapy: Tissue Characterization with Specimen MR and the Role of Enhanced MR Imaging

Purpose : To evaluate the role of enhanced MR imaging in monitoring tumor response to preoperativechemotherapy for osteosarcomas. Materials and Methods : Fo r t y - s even patients (30 males and 17 females, witha mean age 17 years ; range 8 -44 years) with osteosarcomas were included in this study. We obtained spin echoT1-, T2-, and enhanced T1-weighted images before and after pre-operative chemotherapy and in all patientscorrelated changes in MR parameters with histopathologic response. We also obtained 19 specimen MR images,correlating these with histopathologic results in order to estimate tissue specific signals. Patients with morethan 10% viable tumor in the resected specimen were considered poor respon-ders(n=26), while those with 10% orless viable tumor were considered good respon-ders(n=21). Results : Four distinct patterns of signal intensitycorresponded, respectively to dead bone and dense fibrosis (low on T1- and T2-weighted images), viable tumor cells(in-termediate on T1- and high on T2-weighted images), necrosis (low on T1- and high on T 2 - weighted images),and hemorrhage (high on T1- and T2-weighted images), but a wide range of overlap was noted. In all four groups,viable tumor cells remained. Increased tumor vo l u m e, stable or increased edema and enhancement were goodpre-dictors of poor response (predictive values of 83%, 77%, and 89%, respectively). Decreased enhancement was theonly reliable predictor of good response (predictive value, 73%). Changes in tumor margin, homogeneity, signalintensity, and joint effu-sion did not correlate with histopathologic response. Conclusion : Signal intensities donot reflect histologic nature. Enhanced MR imaging is a useful predictor of tumor response to preoperativechemotherapy.

Unusual degradation of alpha-beta complexes in Xenopus oocytes by beta-subunits of Xenopus gastric H-K-ATPase.

The catalytic alpha-subunit of oligomeric P-type ATPases such as Na-K-ATPase and H-K-ATPase requires association with a beta-subunit after synthesis in the endoplasmic reticulum (ER) to become stably expressed and functionally active. In this study, we have expressed the beta-subunit of Xenopus gastric H-K-ATPase (betaHK) in Xenopus oocytes together with alpha-subunits of H-K-ATPase (alphaHK) or Na-K-ATPase (alphaNK) and have followed the biosynthesis, assembly, and cell surface expression of functional pumps. Immunoprecipitations of Xenopus betaHK from metabolically labeled oocytes show that it is well expressed and, when synthesized without alpha-subunits, can leave the ER and become fully glycosylated. Xenopus betaHK can associate with both coexpressed alphaHK and alphaNK, but the alpha-beta complexes formed are degraded rapidly in or close to the ER and do not produce functional pumps at the cell surface as assessed by 86Rb uptake. A possible explanation of these results is that Xenopus betaHK may contain a tissue-specific signal that is important in the formation or correct targeting of functional alpha-beta complexes in the stomach but that cannot be recognized in Xenopus oocytes and in consequence leads to cellular degradation of the alpha-beta complexes in this experimental system.

Structural characterization and tissue-specific expression of the mouse glucose-6-phosphate dehydrogenase gene.

Glucose-6-phosphate dehydrogenase (G6PD) activity differs among tissues and, in liver, with the dietary state of the mouse. Tissue-specific differences in G6PD activity in adipose tissue, liver, kidney, and heart were associated with similar differences in the amount of G6PD mRNA. Regulation of mRNA amount by dietary fat was only observed in liver. In mice fed a low-fat diet, the relative amounts of G6PD mRNA were 3:1:1:0.38, respectively, in the four tissues. Further, the amount of precursor mRNA for G6PD in liver, kidney, and heart reflected the amount of mature mRNA in these tissues, suggesting differing transcriptional activity. Our S1 nuclease and primer-extension analyses indicated that the same transcriptional start site is used in liver, kidney, and adipose tissue, resulting in a common 5' end of the mRNA in these tissues. Thus, differential regulation is not attributable to alternate promoter usage. A DNase hypersensitivity analysis of the 5' end of the G6PD gene identified three hypersensitive sites (HS): HS 1 and HS 2 were present in all tissues, whereas HS 3 was liver specific. Thus, regulation of G6PD expression involves both dietary and tissue-specific signals that appear to act via different mechanisms.

Growth-inhibiting N-substituted endogenous peptides.

Considering the large number of factors that modulate cell growth and differentiation, it might seem naive to assume that cell renewal is basically regulated according to a negative feedback principle, involving one tissue-specific chemical signal system in each organ/tissue. This concept is, nevertheless, about 60 years old. In 1937, Simms and Stillman found that some heat-labile factor, extracted from aorta, inhibited growth of tissue from chicken aorta. Twenty years later, Weiss and Kavanau (1957) formulated a broader hypothesis based on their own data, suggesting that differentiated cells in a tissue or organ generate an inhibitory signal that regulates the rate of cell division in the “proliferative pool” of that same tissue or organ, possibly also stimulating differentiation. Their work was continued by Iversen (1961) and Iversen and Evensen (1962), who analyzed epidermal cell kinetics during skin carcinogenesis, and Bullough (1962), who worked with epidermal regeneration after wounding. Bullough coined the name chalone, a name that was rapidly used for any growth-inhibitory activity that could be found in tissue/organ extracts, most of them more or less crude.KeywordsInhibitory PeptideEhrlich Ascites Tumor CellEhrlich Ascites Carcinoma CellEpidermal ChaloneEpidermal Cell ProliferationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A proposed role for the lutropin receptor in contact-inducible gonococcal invasion of Hec1B cells.

We previously reported the existence of a contact-inducible, enhanced invasion phenotype in the obligate human pathogen Neisseria gonorrhoeae. Our present studies showed that the ability of glutaraldehyde-fixed eucaryotic cells to convert gonococci (GC) to this invasive phenotype (Inv+) is limited to cells derived from reproductive tissues. We present evidence that GC recognize the lutropin receptor (LHr), which recognizes both luteinizing hormone and human chorionic gonadotropin (hCG), as the tissue-specific environmental signal that induces the conversion of GC to the Inv+ phenotype. By competitive binding studies, we showed that Inv+ GC bind to Hec1B cells, a human endometrial cell line, by a unique adhesin not present on noninduced GC and that this Inv+ GC-specific binding is completely blocked by the addition of hCG. We demonstrated that limiting the access of GC to LHr decreases the ability of the host cell to both convert GC to the Inv+ phenotype and serve as a target for Inv+ GC invasion. We propose a model of GC invasion of Hec1B cells in which the LHr plays a dual role both as an induction signal and as part of the internalization mechanism. This utilization of LHr could account for both the preponderance of complicated GC disease in women and the observed correlation of the disease with the onset of menses.

The HumanGATA-6Gene: Structure, Chromosomal Location, and Regulation of Expression by Tissue-Specific and Mitogen-Responsive Signals

GATA factors constitute a family of transcriptional regulatory proteins expressed with distinct developmental and tissue-specific profiles and thought to regulate cell-restricted programs of gene expression. Here we describe the molecular cloning, chromosomal location, and transcription of the humanGATA-6gene. TheGATA-6cDNA encodes a predicted 449-amino-acid protein, which is highly conserved among vertebrates, and includes the two adjacent zinc-finger/basic domains characteristic of the GATA factor family.GATA-6maps to human chromosome 18q11.1–q11.2 by fluorescencein situhybridization. The gene is transcribed in a pattern overlapping that ofGATA-4.Transcripts for both of these genes are prominent in heart, pancreas, and ovary, but onlyGATA-6mRNA is found in lung and liver.GATA-6transcripts are also detected in cultures of human and rat vascular smooth muscle cells (VSMCs). In VSMCs,GATA-6transcripts are down- regulated when quiescent cultures are stimulated to proliferate in response to mitogen activation. These data demonstrate thatGATA-6is subject to both tissue-specific and mitogen-responsive regulatory signals.GATA-6is a prime candidate for a gene that might regulate the differentiative state of VSMCs.

A plant basal in vitro system supporting accurate transcription of both RNA polymerase II- and III-dependent genes: supplement of green leaf component(s) drives accurate transcription of a light-responsive rbcS gene.

An in vitro transcription initiation system has been developed from nuclei of rapidly growing, non-green tobacco (Nicotiana tabacum) cultured (BY-2) cells. Conditions for nuclear extraction and in vitro transcription reaction have been optimized with a tobacco beta-1,3-glucanase gene, a constitutively expressed gene in BY-2 cells. The in vitro system supports accurate transcription of RNA polymerase II-dependent promoters from not only plant genes (tobacco beta-1,3-glucanase gene, cauliflower mosaic virus 35S promoter) but also animal genes (adenovirus 2 major late promoter, simian virus 40 early major promoter). In addition, this system drives accurate transcription of an RNA polymerase III-dependent Arabidopsis thaliana U6 snRNA gene. As BY-2 cells do not differentiate in response to light or any other stimuli, they would provide a basal transcription system which lacks tissue-specific and light-responsive nuclear signals as well as chloroplast-derived signals. Consequently, the BY-2 cell-free system is unable to transcribe the tomato gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS3C) whose expression is tissue-specific and light-inducible. However, the transcription of rbcS3C was obtained by supplementing the BY-2 system with a nuclear extract of light-grown tomato seedlings. The promoter regions necessary for rbcS transcription was mapped in vitro using a series of 5' deletion mutants. The 351 bp upstream sequence is essential and the further upstream region from -351 to -441 enhances its transcription. The in vitro basal system will be useful to identify specific signals from both the nucleus and chloroplast in green leaves and other organs/tissues.

Binding of Vav to Grb2 through dimerization of Src homology 3 domains.

The protooncogenic protein Vav has the structure of an intracellular signal transducer. It is exclusively expressed in cells of hematopoietic lineage and plays a crucial role in hematopoietic cell differentiation. Here we report that both in cell extracts and within intact mammalian cells Vav binds to Grb2 (Sem-5/ASH/Drk), an adaptor molecule which plays a key role in Ras activation. The interaction became evident from a yeast two-hybrid screen and its specificity was demonstrated by in vitro binding assays. It is mediated by an unusual protein-protein binding reaction: dimerization of specific intact Src homology 3 domains of each of the partners. Signaling during hematopoietic lineage differentiation may therefore involve the tissue-specific signal transducer Vav linking into the ubiquitous pathway involving Grb2 and ultimately Ras.

Alternative splicing: a mechanism for phenotypic rescue of a common inherited defect.

Approximately 2% of Caucasians and African-Americans are homozygous for a nonsense mutation in exon 2 of the AMPD1 (AMP deaminase) gene. These individuals have a high grade deficiency of AMPD activity in their skeletal muscle. More than 100 patients with AMPD1 deficiency have been reported to have symptoms of a metabolic myopathy, but it is apparent many individuals with this inherited defect are asymptomatic given the prevalence of this mutant. Results of the present study provide a potential molecular explanation for "correction" of this genetic defect. Alternative splicing eliminates exon 2 in 0.6-2% of AMPD1 mRNA transcripts in adult skeletal muscle. Expression studies document that AMPD1 mRNA, which has exon 2 deleted, encodes a functional AMPD peptide. A much higher percentage of alternatively spliced transcripts are found during differentiation of human myocytes in vitro. Transfection studies with human minigene constructs demonstrate that alternative splicing of the primary transcript of human AMPD1 is controlled by tissue-specific and stage-specific signals. Alternative splicing of exon 2 in individuals who have inherited this defect provides a mechanism for phenotypic rescue and variations in splicing patterns may contribute to the variability in clinical symptoms.