Gamma Actin Research Articles

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

BackgroundDiet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life.ResultsThe expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR.ConclusionsCtgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Expression of Subtype-Specific Group 1 Leiomyosarcoma Markers in a Wide Variety of Sarcomas by Gene Expression Analysis and Immunohistochemistry

Leiomyosarcomas (LMSs) constitute approximately one quarter of all sarcomas and are usually defined by morphologic criteria and/or immunoreactivity for actin or desmin. Among high-grade lesions, the distinction from undifferentiated pleomorphic sarcoma (UPS) can be problematic, and previous studies have shown that a significant number of LMS cases may be hiding under the diagnosis of UPS. We recently described 3 novel molecular LMS subtypes that are distributed similarly over LMSs of gyneocologic and non-gyneocologic origins. The group 1 subtype shows an improved disease-specific survival compared with the other 2 groups that is independent of histologic grade. Group 1 comprises approximately 25% of all LMSs, and is defined by a shared pattern of gene expression, a distinct pattern of genomic changes, and reactivity for at least 3 of 5 immunohistochemistry (IHC) markers (smooth muscle gamma actin, calsequestrin 2, human muscle cofilin2, myosin light chain kinase, and sarcolemmal membrane associated protein), as tested on 271 cases of LMS in tissue microarrays. These IHC markers have not been well characterized in non-LMS sarcomas. Here we provide a characterization of these 5 markers across normal tissues, an additional 59 cases of LMS, and a wide range of 565 non-LMS soft tissue tumors from 44 diagnostic categories, with a focus on UPS. When analyzed individually, the 5 markers were found to be expressed in many sarcomas other than LMSs. However, when analyzed by the same criteria used for the recognition of group 1 LMSs, in which a case is scored positive when at least 3 of 5 markers reacted, coordinate expression was seen in significant numbers of cases from only 3 diagnostic groups that included 22% of leiomyomas (n=22), 16% of gastrointestinal stromal tumors (n=43), and 18% of endometrial stromal sarcomas (n=11). In addition, 5% (n=57) of UPSs showed a staining pattern similar to that seen in group 1 LMSs. To further examine the possibility that group 1 LMS constitutes a small part of cases diagnosed as UPS, we examined the expression of the top 500 genes from the group 1 LMS expression signature in 29 UPSs by complementary DNA microarray. Unsupervised hierarchical clustering of 29 UPS expression showed that 2 (7%) had coordinated high levels of expression of genes from the group 1 LMS signature, a rate similar to that seen by IHC analysis. These findings show that group 1 LMS IHC markers smooth muscle gamma actin, calsequestrin 2, human muscle cofilin2, myosin light chain kinase, and sarcolemmal membrane associated protein when coordinately expressed have specificity for a subset of LMS when compared with other sarcomas, and may be useful for the recognition of group 1 LMS cases within cases diagnosed as UPS.

Cytoskeletal scaffolding proteins interact with Lynch‐Syndrome associated mismatch repair protein MLH1

The involvement of MLH1 in several mismatch repair-independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two-hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the "General Repository for Interaction Datasets" highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin beta 4 and Actin gamma, Cathepsin B and Annexin A2 as well as Spectrin alpha and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1-deficient and -proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1-Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.

Proteome Differences of Placenta Between Pre-Eclampsia and Normal Pregnancy

Placenta is a tissue unique to pregnancy and despite its major role in pregnancy, little is known about the proteome changes within placenta during pregnancy-related diseases such as pre-eclampsia (PE). Therefore, the aim of this study is the analysis of proteome differences between pre-eclamptic and normal full-term placentas. To achieve this goal, five normal and five severe pre-eclamptic placentas were included in this study. Total placental proteins were extracted and subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). After staining, the gels were scanned and the protein spots were analysed using Image Master 2D Platinum Software. Non-parametric Mann–Whitney test was used for analysis of the mean intensity differences of the spots between normal and pre-eclamptic placentas. Statistical analysis indicated that 17 spots were differently expressed in pre-eclamptic compared with normal placentas ( p < 0.05). Using Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI TOF/TOF) mass analysis, 11 out of 17 spots were identified. Among them, four proteins (chloride intracellular channel 3, apolipoprotein A-I, transthyretin (TTR) and protein disulphide isomerase) were up-regulated while seven (peroxiredoxin 2, peroxiredoxin 3, Hsc 70, Cu/Zn-superoxide dismutase (SOD-1), actin gamma 1 propeptide, chain A of enoyl-coenzyme A hydratase and HSP gp96) showed decreased expression in PE in comparison with normal placentas. In conclusion, down-regulation of proteins with anti-oxidant activities (peroxiredoxin 2 and peroxiredoxin 3) and altered expression of stress-response proteins (Hsc 70, Hsp gp96 and protein disulphide isomerase) might play an important role in the pathogenesis of PE.

3D Cryo‐Imaging: A Very High‐Resolution View of the Whole Mouse

We developed the Case Cryo-imaging system that provides information rich, very high-resolution, color brightfield, and molecular fluorescence images of a whole mouse using a section-and-image block-face imaging technology. The system consists of a mouse-sized, motorized cryo-microtome with special features for imaging, a modified, brightfield/fluorescence microscope, and a robotic xyz imaging system positioner, all of which is fully automated by a control system. Using the robotic system, we acquired microscopic tiled images at a pixel size of 15.6 microm over the block face of a whole mouse sectioned at 40 microm, with a total data volume of 55 GB. Viewing 2D images at multiple resolutions, we identified small structures such as cardiac vessels, muscle layers, villi of the small intestine, the optic nerve, and layers of the eye. Cryo-imaging was also suitable for imaging embryo mutants in 3D. A mouse, in which enhanced green fluorescent protein was expressed under gamma actin promoter in smooth muscle cells, gave clear 3D views of smooth muscle in the urogenital and gastrointestinal tracts. With cryo-imaging, we could obtain 3D vasculature down to 10 microm, over very large regions of mouse brain. Software is fully automated with fully programmable imaging/sectioning protocols, email notifications, and automatic volume visualization. With a unique combination of field-of-view, depth of field, contrast, and resolution, the Case Cryo-imaging system fills the gap between whole animal in vivo imaging and histology.

Smooth Muscle-Specific Gene Delivery in the Vasculature Based on Restriction of DNA Nuclear Import

The two currently employed approaches restricting gene delivery and/or expression to desired cell types in vivo rely on cell surface targeting or cell-specific promoters. We have developed a third approach based on cell-specific nuclear transport of the delivered plasmid DNA. We have previously shown that plasmid nuclear import in non-dividing cells is sequence-specific and have identified a set of cell-specific DNA nuclear targeting sequences that can be used to limit DNA nuclear import to desired cell types. Specifically we have identified elements of the smooth muscle gamma actin (SMGA) promoter that direct plasmid nuclear import selectively in smooth muscle cells (SMCs) in vitro (Vacik et al, 1999, Gene Therapy 6:1006-1014). In the present study, we demonstrate that the SMC-specific DNA nuclear targeting sequence from the SMGA promoter drives nuclear accumulation of plasmids and subsequent gene expression exclusively in the smooth muscle cell layer of the vessel wall in the intact vasculature of rats using electroporation mediated delivery. These results demonstrate that certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types providing a new, novel means of cell targeting for gene therapy.

Proteome analysis of human substantia nigra in Parkinson's disease

BackgroundParkinson's disease (PD) is the most common neurodegenerative disorder involving the motor system. Although not being the only region involved in PD, affection of the substantia nigra and its projections is responsible for some of the most debilitating features of the disease. To further advance a comprehensive understanding of nigral pathology, we conducted a tissue based comparative proteome study of healthy and diseased human substantia nigra.ResultsThe gross number of differentially regulated proteins in PD was 221. In total, we identified 37 proteins, of which 16 were differentially expressed. Identified differential proteins comprised elements of iron metabolism (H-ferritin) and glutathione-related redox metabolism (GST M3, GST P1, GST O1), including novel redox proteins (SH3BGRL). Additionally, many glial or related proteins were found to be differentially regulated in PD (GFAP, GMFB, galectin-1, sorcin), as well as proteins belonging to metabolic pathways sparsely described in PD, such as adenosyl homocysteinase (methylation), aldehyde dehydrogenase 1 and cellular retinol-binding protein 1 (aldehyde metabolism). Further differentially regulated proteins included annexin V, beta-tubulin cofactor A, coactosin-like protein and V-type ATPase subunit 1. Proteins that were similarly expressed in healthy or diseased substantia nigra comprised housekeeping proteins such as COX5A, Rho GDI alpha, actin gamma 1, creatin-kinase B, lactate dehydrogenase B, disulfide isomerase ER-60, Rab GDI beta, methyl glyoxalase 1 (AGE metabolism) and glutamine synthetase. Interestingly, also DJ-1 and UCH-L1 were expressed similarly. Furthermore, proteins believed to serve as internal standards were found to be expressed in a constant manner, such as 14-3-3 epsilon and hCRMP-2, thus lending further validity to our results.ConclusionUsing an approach encompassing high sensitivity and high resolution, we show that alterations of SN in PD include many more proteins than previously thought. The results point towards a heterogeneous aetiopathogenesis of the disease, including alterations of GSH-related proteins as well as alterations of proteins involved in retinoid metabolism, and they indicate that proteins involved in familial PD may not be differentially regulated in idiopathic Parkinson's disease.

ANALYSIS OF MID-GESTATION BABOON FRONTAL CORTEX TRANSCRIPTOMES IN FETUSES OF AD LIBITIUM FED (CON) COMPARED TO 70% OF AD LIBITUM FED (NR) MOTHERS

Previous work has shown that fetuses of dams fed 70% of the diet consumed by the ad libitum fed controls show an imbalance of apoptotic cell loss in target regions of the frontal cortex for migrating cells compared with controls. In addition, fetuses of nutrient restricted (NR) dams showed a decrease in neurite density and myelination. Taken together these data indicate multiple impairments of cortical development during the first half of gestation. The purpose of this study was to define molecular mechanisms by which NR impacts frontal cortex development in the mid-gestation baboon fetus. Fetal baboon brains were collected at necropsy, the frontal cortex regions dissected and snap frozen from 0.5 gestation (G) CON and NR (n=6 for each group) baboons. Extracted frontal cortex RNA was used to interrogate each transcriptome using human Affymetrix GeneChips. Data were analyzed to identify differential gene expression and differential gene expression in the context of biological pathways. T-tests were used to assess differential gene expression and z-scores (z) to assess differences in biological pathways. Z≥2 was considered significant. Comparison of CON versus NR 0.5G frontal cortex RNA shows 318 significantly differently expressed genes with 157 up- and 161 down-regulated in the NR group. Furthermore, pathway analysis shows significant down-regulation in pathways considered vital to brain development such as: anti-apoptosis (z = 2.41); axonogenesis (z = 3.7); neuron development and differentiation (z = 2.51); extracellular matrix organization and biogenesis (z = 5.07); cell motility (z = 3.85); and regulation of neurogenesis (z = 7.55). Analysis of transcriptome data shows differences between CON and MNR frontal cortex in individual genes and pathways known to be important for developmental processes such as: 1) axonogenesis and neurogenesis, e.g., neuronal cell adhesion molecule (NCAM) and 2) genes known to be important for cell motility, e.g. beta actin and gamma actin. This study is a first step towards understanding the molecular mechanisms that underlie our previously observed alterations in apoptosis, cell proliferation and neurite density that accompany NR. (platform)

Differential arginylation of actin isoforms: the mystery of the actin N-terminus

Actin is one of the most abundant, essential and well studied intracellular proteins, yet its regulation in vivo is still not completely understood. One of the mysteries around actin concerns the existence of multiple actin isoforms that are extremely similar to each other except for their N-termini but have been shown in multiple studies to preferentially incorporate into different actin networks and are suggested to have different roles in vivo. The mechanisms of this actin isoform segregation are unknown. My colleagues and I recently showed that beta but not gamma actin in cultured fibroblasts undergoes N-terminal arginylation, which regulates actin polymerization and lamella formation in motile cells. Here, I propose that arginylation could be a general mechanism that regulates actin isoform segregation in vivo and participates in the formation of loose beta-actin network at the leading edge of the cell.

Correction: Human MicroRNA Targets

In PLoS Biology, volume 2, issue 11: DOI: 10.1371/journal.pbio.0020363 We incorrectly named the products of two genes, gamma actin and betaAPP, as FMRP ligands because of a misreading of Table 1 from [1]. BetaAPP is incorrectly referred to as an FMRP ligand in Tables 2 and S9, gamma actin in Table S9 only. This does not change the use of FMRP binding as a way to support the target predictions, as the statistics remain virtually the same; the enrichment of FMRP targets within the predicted microRNA target set changes from 5.31 to 5.26 as a result of our misclassification. In addition, it does not change the fact that betaAPP is a predicted microRNA target. Our Web site at http://www.microrna.org/ contains the updated information with changes flagged. We thank Dr. Denman for pointing out this error to us.

Cofilin Binding to Muscle and Non-muscle Actin Filaments: Isoform-dependent Cooperative Interactions

I have monitored equilibrium binding of human cofilin to rabbit skeletal muscle (alpha) and human non-muscle (85% beta, 15% gamma) actin filaments from the quenching of pyrene actin fluorescence. Filament binding is cooperative and stoichiometric (i.e. one cofilin molecule per actin subunit) for both actin isoforms. The Hill coefficient for binding to betagamma-actin filaments (n(H)=3.5) is greater than for muscle actin (n(H)=2.3). Analysis of equilibrium binding using a nearest-neighbor cooperativity model indicates that the intrinsic affinities for binding to an isolated site are comparable (10-14 microM) for both filament isoforms but the cooperative free energy is greater for binding betagamma-actin filaments. The predicted cofilin cluster sizes and filament binding densities are small at concentrations of cofilin where efficient filament severing is observed, indicating that a few bound cofilin molecules are sufficient to destabilize the filament lattice and promote fragmentation. The analysis used in this study provides a framework for evaluating proton and ion linkage and effects of regulatory proteins on cofilin binding and severing of actin filaments.

A mutation in the gamma actin 1 (ACTG1) gene causes autosomal dominant hearing loss (DFNA20/26)

Linkage analysis in a multigenerational family with autosomal dominant hearing loss yielded a chromosomal localisation of the underlying genetic defect in the DFNA20/26 locus at 17q25-qter. The 6-cM critical region...

27. Analysis of a SAGE (serial analysis of gene expression) catalogue of human granulosa cells

Mammalian ovarian follicles are composed of an oocyte surrounded by granulosa (or follicle) cells. Granulosa cells are essential for successful oocyte maturation and ovulation. A gene expression profile, or transcriptome, provides a catalogue of the genes expressed by a cell or tissue and the levels of individual transcript expression. In the case of the granulosa cell, its transcriptome should allow insight into granulosa cell function and potentially lead to markers of follicle quality for use in human assisted reproduction such as in vitro fertilization (IVF). We used SAGE (serial analysis of gene expression) as a technique to determine the transcriptome of granulosa cells. SAGE relies on the assumption that a 14base sequence whose position is defined in relation to the 3'-end of any transcript uniquely identifies the gene from which the transcript originated (1). Human granulosa cells were obtained from Otago Fertility Services, Dunedin, New Zealand from 4 adult females, at the time of IVF treatment. After purification, total RNA (5.96 μg) was extracted from these cells using the Qiagen RNeasy kit. The SAGE library was constructed with the Invitrogen I-SAGE kit. Briefly, SAGEtags were extracted from concatemerized ditag sequences using SAGE2000 software (Invitrogen, version B). In total 59 clones were sequenced, yielding 1339 SAGEtags. Selected human SAGE libraries (normal ovary, heart, liver, lung, pancreas and white blood cell) were downloaded from the NIH Sagemap website (ftp.ncbi.nih.gov/pub/sage/seq/) and their relative SAGEtag abundances were compared to that of the human granulosa SAGE library. It was found that human granulosa cells had a unique pattern of gene expression and a number of tags were found that had high abundance in the human granulosa SAGE library, but were absent from other libraries (for example, hydroxysteroid (11-beta) dehydrogenase I and scavenger receptor class B member 1). Those genes that were common to many cell and tissue libraries tended to be structural and house-keeping genes (for example gamma actin). PCR techniques were used to independently validate the SAGE catalogue. (1) Velculescu, V. E., Zhang, L., Vogelstein, B., Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270, 484– 487.

Neonatal pulmonary hypertension prevents reorganisation of the pulmonary arterial smooth muscle cytoskeleton after birth.

The pulmonary arterial smooth muscle cell (SMC) cytoskeleton was studied in tissue from 36 piglets aged from within 5 min of birth to 21 d of age, and in 8 adults. An additional 16 piglets were made pulmonary hypertensive by exposure to hypobaric hypoxia (50.8 kPa) for 3 d. In conduit intrapulmonary elastic arteries alpha, beta and gamma actin, the 204, 200 and 196 kDa myosin heavy chain (MHC) isoforms and vinculin were localised by immunohistochemistry. The total actin content, the proportion of monomeric to filamentous alpha and gamma actin and changes in the proportions of the MHC isoforms were determined biochemically. Dividing SMCs were localised and quantified using Ki-67. We found a transient reduction in immunohistochemical expression of gamma actin, 204 kDa MHC isoform and vinculin at 3 and 6 d in the inner media, associated with a transient increase in Ki-67 labelling. The actin content also decreased at 3 and 6 d (P < 0.05), but there was a postnatal, permanent increase in monomeric actin, first the alpha then the gamma isoform. The relative proportions of the MHC isoforms did not change between birth and adulthood in elastic pulmonary arteries but in muscular arteries the 200 kDa isoform increased between 14 d and adulthood. Pulmonary hypertension prevented both the immunohistochemical changes and the postnatal burst of SMC replication and prevented the transient postnatal reduction in actin content. These findings suggest that rapid remodelling of the actin cytoskeleton is an essential prerequisite of a normal postnatal fall in pulmonary vascular resistance.

Ultrastructural and histochemical characterization of special muscle cells in the monkey small intestine.

The ultrastructure, three-dimensional arrangement, and histochemical features of special muscle cells in the monkey small intestine were investigated. The cells formed a special layer separated from the main part by a connective tissue space along the submucosal surface of the circular muscle coat. Scanning electron microscopy using alkali maceration demonstrated this inner sublayer to be a continuous thin sheet consisting of irregularly-shaped muscle cells equipped with many cytoplasmic projections and caveolae. Other ultrastructural features included direct contact with interstitial cells, due to their close association with nerve fibers of the deep muscular plexus. Histochemical examination revealed significant alkaline phosphatase activity and immunoreactivity for vascular smooth muscle alpha actin in these muscle cells, whereas the ordinary circular muscle cells were immunopositive for enteric smooth muscle gamma actin. These findings suggest that the special muscle cells play an important role in regulating the radial stretch of the monkey small intestinal wall.

Protein-protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain.

Numerous functions have been proposed for the testis brain RNA-binding protein (TB-RBP) and its human homologue, Translin, ranging from mRNA transport and translational regulation to DNA rearrangement and repair. To gain insight into the likely functions of this 26 kDa protein, immunoprecipitation was used to identify proteins that interact with TB-RBP in mouse cytosolic extracts. Three proteins, the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin, and Trax, were specifically immunoprecipitated with an affinity-purified antibody to recombinant mouse TB-RBP. In vitro binding assays with recombinant proteins and EM immunocytochemistry confirm that TB-RBP interacts with the TER ATPase in vitro and in vivo. Confocal microscopy has demonstrated that TB-RBP colocalizes with actin in the cytoplasm of male germ cells. The immunoprecipitation of Trax with TB-RBP confirms a published report demonstrating protein interactions between the two proteins in a yeast two-hybrid assay. These data support the hypothesis that TB-RBP serves as a link in attaching specific mRNAs to cytoskeletal structures and suggests an involvement for the ubiquitously expressed TER ATPase in intracellular and/or intercellular mRNA transport.

Cell-specific nuclear import of plasmid DNA.

One factor limiting the success of non-viral gene therapy vectors is the relative inability to target genes specifically to a desired cell type. To address this limitation, we have begun to develop cell-specific vectors whose specificity is at the level of the nuclear import of the plasmid DNA. We have recently shown that nuclear import of plasmid DNA is a sequence-specific event, requiring the SV40 enhancer, a region known to bind to a number of general transcription factors (Dean DA, Exp Cell Res 1997; 230: 293). From these studies we developed a model whereby transcription factor(s) bind to the DNA in the cytoplasm to create a protein-DNA complex that can enter the nucleus using the protein import machinery. Our model predicts that by using DNA elements containing binding sites for transcription factors expressed in unique cell types, we should be able to create plasmids that target to the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscle cells, we have created a series of reporter plasmids that are expressed selectively in smooth muscle cells. Moreover, when injected into the cytoplasm, plasmids containing portions of the SMGA promoter localize to the nucleus of smooth muscle cells, but remain cytoplasmic in fibroblasts and CV1 cells. In contrast, a similar plasmid carrying the SV40 enhancer is transported into the nuclei of all cell types tested. Nuclear import of the SMGA promoter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected CV1 cells, supporting our model for the nuclear import of plasmids. Finally, these nuclear targeting sequences were also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specific manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors for any desired cell type.

Prognostic value of intratumoral interferon gamma messenger RNA expression in invasive cervical carcinomas.

The production of the cytokine interferon gamma (IFN gamma) by activated peripheral blood mononuclear cells may be reduced in patients with invasive cervical carcinoma. This study was designed to assess the prognostic value of intratumoral IFN gamma messenger RNA (mRNA) levels in such patients. Biopsy specimens of primary cervical lesions were obtained from 27 patients with invasive squamous cell carcinoma before they received any therapy. Two prognostic groups were considered: 1) a group of 14 patients who had no apparent disease recurrence and who were alive 2 years after diagnosis (good-prognosis group) and 2) a group of 13 patients who had disease recurrence or died during the 2-year follow-up (poor-prognosis group). A competitive reverse transcription-polymerase chain reaction assay was used to measure levels of IFN gamma and beta actin mRNA. The expression of human leukocyte antigen (HLA) class II proteins (which is stimulated by IFN gamma) in tumor cells was studied by immunostaining. Tumor specimens from all 14 patients in the good-prognosis group contained more than 10(3) IFN gamma mRNA copies per 5 x 10(5) beta actin mRNA copies, whereas tumor specimens from only six of the 13 patients in the poor-prognosis group contained this level of IFN gamma mRNA (two-sided P = .006). No clear relationship was observed between levels of IFN gamma mRNA and T-cell or natural killer cell infiltration in tumors; however, a statistically significant association was observed between HLA class II expression on tumor cells and IFN gamma mRNA levels (two-sided P = .01). A subgroup of poor-prognosis cervical carcinoma patients who have low levels of intratumoral IFN gamma mRNA was identified.

ALTERATIONS IN INTESTINAL SMOOTH MUSCLE IN THE ABSENCE OF PACEMAKER ACTIVITY

Interstitial Cells of Cajal [ICC] generate pacemaker slow waves in the murine gastrointestinal [GI] tract which conduct through the gap junctions to the smooth muscle cells. ICC express the proto-oncogene c-kit. W/Wv mice have a mutation of c-kit and show a marked reduction in the number of ICC and an absence of pacemaker waves. To test the hypothesis that loss of ICC alters the smooth muscle phenotype, we compared the GI tract in W/Wv mice with that of age and sex matched controls. Smooth muscle alpha actin [SMAA] and smooth muscle gamma actin [SMGA] were quantified and compared in the fundus, body, pylorus, duodenum, jejunum, ileum, proximal and distal colon. In both the mutant and control animals at 8 weeks of age, the levels of these proteins varied in a segment specific pattern with pylorus, doudenum and jejunum showing a much lower expression of both actins relative to the other segments. However, W/Wv mice demonstrated decreased levels of SMGA and SMAA throughout the GI tract. The mutants also displayed an enlarged proximal gut but no changes were observed in smooth muscle histology. At 4 weeks of age, no differences were noted by these criteria. This study represents the first comprehensive analysis of smooth muscle isoactin expression at the protein level in the adult murine GI tract. The results show that smooth muscle isoactin content is segment-specific and that the level of these proteins is reduced in the absence of pacemaker activity. Although the basis for the late onset of these changes and the enlargement of the stomach and duodenum remains to be determined, this may reflect pseudo-obstruction of the gut and W/Wv mice could provide an animal model for this disorder.

Leptin secretion from adipose tissue in women. Relationship to plasma levels and gene expression.

The role of expression and secretion of the ob gene product, leptin, for the regulation of plasma leptin levels has been investigated in vitro using abdominal subcutaneous adipose tissue of 20 obese, otherwise healthy, and 11 nonobese women. Body mass index (BMI, mean+/-SEM; kg/m2) in the two groups was 41+/-2 and 23+/-1, respectively. Fat cell volume was 815+/-55 pl in the obese and 320+/-46 pl in the nonobese group. In the obese group, plasma leptin concentrations and adipose leptin mRNA (relative to gamma actin) were increased five and two times, respectively. Moreover, adipose tissue secretion rates per gram lipid weight or per fat cell number were also increased two and seven times, respectively, in the obese group. There were strong linear correlations (r = 0.6-0.8) between plasma leptin, leptin secretion, and leptin mRNA. All of these leptin measurements correlated strongly with BMI and fat cell volume (r = 0.7- 0.9). About 60% of the variation in plasma leptin could be attributed to variations in leptin secretion rate, BMI, or fat cell volume. We conclude that elevated circulating levels of leptin in obese women above all result from accelerated secretion rates of the peptide from adipose tissue because of increased ob gene expression. However, leptin mRNA, leptin secretion, and circulating leptin levels are all more closely related to the stored amount of lipids in the fat cells of adipose tissue than they are to an arbitrary division into obese versus nonobese.