Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.

Obesity and its associated comorbidities (e.g., diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually1 and are among the most prevalent and challenging conditions confronting the medical profession2,3. Neurotensin (NT), a 13-amino acid peptide predominantly localized in specialized enteroendocrine (EE) cells of the small bowel4 and released by fat ingestion5, facilitates fatty acid (FA) translocation in rat intestine6, and stimulates growth of various cancers7; the effects of NT are mediated through three known NT receptors (NTR1, 2 and 3)8. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality9; however, a role for NT as a causative factor in these diseases is unknown. Here, we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates FA absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3/sortilin. Consistent with the findings in mice, expression of NT in Drosophila midgut EE cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment.

Five mouse a-tubulin isotypes are described, each distinguished by the presence of unique amino acid substitutions within the coding region. Most, though not all of these isotype-specific amino acids, are clustered at the carboxy terminus. One of the a-tubulin isotypes described is expressed exclusively in testis and is encoded by two closely related genes (Ma3 and Ma7) which have homologous 3' untranslated regions but which differ at multiple third codon positions and in their 5' untranslated regions. We show that a subfamily of a-tubulin genes encoding the same testis-specific isotype also exists in humans. Thus, we conclude that (i) the duplication event leading to a pair of genes encoding a testis-specific ca-tubulin isotype predated the mammalian radiation, and (ii) both members of the duplicated sequence have been maintained since species divergence. A second ot-tubulin gene, Mca6, is expressed ubiquitously at a low level, whereas a third gene, MaL4, is unique in that it does not encode a carboxy-terminal tyrosine residue. This gene yields two transcripts: a 1.8-kilobase (kb) mRNA that is abundant in muscle and a 2.4-kb mRNA that is abundant in testis. Whereas the 1.8-kb mRNA encodes a distinct a-tubulin isotype, the 2.4-kb mRNA is defective in that the methionine residue required for translational initiation is missing. Patterns of developmental expression of the various at-tubulin isotypes are presented. Our data support the view that individual tubulin isotypes are capable of conferring functional specificity on different kinds of microtubules.Microtubules are assembled from heterodimers of a-and ,B-tubulin together with microtubule-associated proteins. They function in a wide variety of ways in eucaryotic cells; for example, they have specific functions in the mitotic and meiotic spindle, in the centriole, in the manchette and flagellar axoneme of spermatozoa, in axonal transport in neurons, and in the marginal bands of platelets. Clearly, associated proteins such as kinesin (28) play a crucial role in conferring these and other specific functions on microtubules. Additionally, a-and f-tubulin proteins themselves show significant heterogeneity, and the tubulin isotypes described to date vary in their patterns of expression in an evolutionarily conserved manner (16). This heterogeneity in a-and ,-tubulins offers the potential of contributing to diversity of microtubule function in eucaryotic cells, either through differential polymerization of the various tubulin subunits, or by virtue of unique interaction(s) with associated proteins.In mammals, a-and ,B-tubulins are encoded by large multigene families (5). A significant fraction of the genes in these families are pseudogenes (5, 13), a fact that makes it difficult to distinguish functional from nonfunctional genomic tubulin sequences. To circumvent this problem and to study the important question of the tubulin repertoire of mammals, we decided to study expressed mouse tubulin genes at the mRNA level by exhaustive screening of cDNA libraries. We re...

To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement.

Many skin disorders are associated with increased numbers of activated mast cells and are worsened by stress; however, the mechanism underlying these processes is not understood. Corticotropin-releasing hormone (CRH) is secreted under stress from the hypothalamus, but also in the skin, where it induces mast cell activation and vascular permeability. We investigated the effect of CRH in a number of animal models by using i.v. Evans blue extravasation as a marker of vascular permeability. Intradermal CRH is among the most potent peptides at 100 nM, its effect being nearly comparable to that of neurotensin (NT). Pretreatment of skin injection sites with the NT receptor antagonist SR48692 blocks CRH-induced vascular permeability, which is diminished in NT؊͞؊ mice, implying that NT is necessary for the effect of CRH. CRH and NT precursor mRNA are shown to be expressed in both dorsal root ganglia and skin, whereas the latter also expresses mRNA for prohormone convertase 5, an enzyme that cleaves pro-NT into its active form. We also show that the effect of both CRH and NT is absent in W͞W v mast cell-deficient mice; however, only a fraction of skin mast cells express CRH receptors, as shown by FACS analysis of CRH receptor (CRHR) and c-kit double-positive disaggregated mouse skin mast cells. These findings suggest that CRH induces skin vascular permeability through NT acting on mast cells and that both peptides should be considered in the pathogenesis of skin disorders exacerbated by stress.inflammation ͉ mast cells ͉ stress ͉ urticaria A cute emotional stress in humans precipitates or worsens skin conditions that involve mast cells (1), including atopic dermatitis (2), psoriasis (3), and urticaria (4). Acute restraint stress in rats has been shown to induce degranulation of skin mast cells (5), an effect mimicked by intradermal injection of corticotropin-releasing hormone (CRH) (6). CRH also increases vascular permeability when injected intradermally, an effect absent in W͞W v mast cell-deficient mice and blocked by the mast cell stabilizer disodium cromoglycate (cromolyn) (7). CRH also induces mast celldependent vasodilation (8) in the microvasculature of human skin (9). CRH mRNA and peptide are expressed in human skin; in contrast, mouse skin apparently does not express mRNA for CRH and contains only CRH peptide (10). CRH receptors (CRHR) are expressed in both human and rodent skin (10, 11).Skin mast cells may have important functions as ''sensors'' of environmental and emotional stress (12), possibly through direct activation by CRH and related peptides (13). As a result, mast cells could play an important role in the pathophysiology of inflammatory diseases worsened by stress (14). However, it is not yet known whether CRH acts alone to activate skin mast cells.Neuropeptides, especially neurotensin (NT), could be involved in the pathogenesis of inflammatory skin disorders, especially those exacerbated by stress (15). NT is one of the most potent inducers of vascular permeability when injected into rodent skin (7), ...

Sepsis is a complex, incompletely understood and often fatal disorder, typically accompanied by hypotension, that is considered to represent a dysregulated host response to infection. Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension. We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe cecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, show reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell-dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.

The sequence of a human P-tubulin cDNA clone (D,B-1) is described; our data revealed 95.6% homology compared with the sequence of a human f-tubulin processed pseudogene derived by reverse transcription of a processed mRNA (Wilde et al., Nature [London] 297:83-84, 1982). However, the amino acid sequence encoded by this cDNA showed less homology with pig and chicken 1-tubulin sequences than the latter did to each other, with major divergence within the 15 carboxy-terminal amino acids. On the other hand, an independently isolated, functionally expressed genomic human 1-tubulin sequence (5,B) possessed a very high degree of homology with chicken and pig 3-tubulins in this region. Thus, human cells appear to contain two distinct ,B-tubulin isotypes. Both the intact ,B-tubulin cDNA clone and a subclone containing only the 3' untranslated region detected two mRNA species in HeLa cells; these mRNAs were 1.8 and 2.6 kilobases long and were present in about equal amounts. Two independently subcloned probes constructed from the 3' untranslated region of the 53 genomic sequence also detected a 2.6-kilobase 1-tubulin mRNA. However, the 3'-untranslated-region probes from the cDNA clone and the genomic sequence did not cross-hybridize. Thus, at least two human ,B-tubulin genes, each specifying a distinct isotype, are expressed in HeLa cells, and the 2.6-kilobase mRNA band is a composite of at least two comigrating f-tubulin mRNAs.a-and P-tubulins are the major soluble proteins of microtubules. These long filamentous structures are found in all eucaryotic cells and are involved in a remarkable diversity of cellular functions (11). In humans, each of the genes encoding a-and ,-tubulins constitutes a large multigene family of about 15 to 20 members (8), the majority of which have been isolated in recombinant fragments (9, 41). Sequence analyses of several of these genes have shown that many of them are pseudogenes (that is, sequences which contain one or more genetic lesions that preclude the synthesis of a functional protein product). Among these pseudogenes is a novel class that is characterized by (i) the absence of intervening sequences, (ii) the presence of a short polyadenylic acid [poly(A)] tract 3' to the poly(A) signal, and (iii) the existence of short flanking direct repeat sequences (22,42,43). These pseudogenes most probably arose by reintegration into the host germ line of a cDNA transcript synthesized on a processed mRNA template.Because a significant number, perhaps the majority, of human a-and P-tubulin genes are pseudogenes, an important question concerns the number offunctionally expressed sequences.One approach to this problem is to investigate the complexity of tubulin mRNAs by constructing cDNA clones. In this paper we describe the sequence of a human P-tubulin cDNA clone that encompasses 98.4% of the coding region. Our data revealed very extensive homology with the sequence of a processed human P-tubulin pseudogene and enabled us to estimate the time of insertion of the mRNA-derived molecule into the germ li...

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