Loop electrosurgical excision procedure with an intrauterine device in place.

Patients using an intrauterine device (IUD) who require a loop electrosurgical excision procedure (LEEP) for cervical dysplasia have traditionally had the IUD removed prior to the procedure. The only other options have been methods that lead to suboptimal sampling or risk cutting the strings. Our study suggests a procedure for performing the LEEP without removing the IUD, and review of the literature suggests that this method has not been reported before. The LEEP is performed using a conization electrode or a cone biopsy excisor. After noting that the IUD strings are of adequate length, a 0-polyglactin free tie is secured around the visible portion of the IUD strings without applying tension on the strings. A large, sterile absorbent-tipped applicator with a hollow handle becomes an 8 cm hollow plastic tube by removing the cotton tip with sterile scissors. The long end of the suture is threaded through the sterile tube. Without pulling on the IUD, the tube is then passed over the strings into the cervical canal approximately 2.5 cm to protect the strings from the excisor well into the cervical canal. Then, the LEEP is performed. After the specimen is removed, hemostasis can be obtained using a ball cautery electrode, keeping the protecting tube with the enclosed IUD strings out of the way. The tube is then carefully removed. The suture is now cut close to the polyglactin knot around the IUD strings, making certain not to shorten the IUD strings and making certain the visible length of the strings is the same as before the procedure. Ferric subsulfate is applied to the operative area to provide continued hemostasis. Follow-up for the LEEP is unchanged. This procedure may be performed on either levonorgestrel-releasing or copper IUDs.

Depot medroxyprogesterone acetate: a risk factor for cervical stenosis after loop electrosurgical excisional procedure management of cervical intraepithelial neoplasia?

OBJECTIVES: In our anecdotal experience and sporadically in the literature, gynecologists have observed a connection between patient's use of depot medroxyprogesterone acetate (DMPA) and increased occurrence of cervical stenosis during follow-up after loop electrosurgical excisional procedure (LEEP). We decided to formally examine this association in our clinic population. MATERIALS AND METHODS: We performed a chart review, enrolling 257 patients and tabulating data on demography, use of hormonal contraceptives, characteristics of the LEEP, and presence or absence of cervical stenosis at 1- and 6-month follow-up evaluations. Univariate tests of association between the independent variables and the dependent variable of cervical stenosis were examined via the chi and Student t tests for discrete and continuous variables, respectively. To characterize the relative importance of independent variables significantly associated with cervical stenosis, logistic regression was performed. RESULTS: Of the 257 charts reviewed, 127 patients (49.4%) completed 1 and 6 months after LEEP follow-up appointments, providing adequate data for analysis. In this population, we observed 25 cases of cervical stenosis, or an overall rate of 19.7%. Of patients using DMPA at the time of LEEP or during the follow-up period, 9 (41.0%) of 22 developed stenosis, whereas of those who did not use DMPA, 16 (15.2%) of 105 developed stenosis, indicating a significant difference (odds ratio = 3.85, 95% CI = 1.41-10.50). CONCLUSIONS: In our clinic population, use of DMPA was associated with higher rates of development of cervical stenosis, calling for larger studies of the association of DMPA in this LEEP complication.

Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity.

Recent studies suggest that HIV-1 budding occurs selectively from detergent-insoluble membrane domains, referred to as lipid rafts. Palmitoylation is thought to be one of the factors responsible for targeting membrane proteins to lipid rafts. The cytoplasmic domain of the HIV-1 envelope glycoprotein (gp160) contains two palmitoylated cysteine residues. In this work, we studied the solubility of gp160 after detergent extraction. We show that wild-type gp160 is mostly insoluble after ice-cold Triton X-100 extraction, but that it becomes almost completely soluble at 37 degrees C. In contrast, we find that a mutant gp160, in which the two palmitoylated cysteine residues are replaced by serine, is Triton X-100 soluble even under ice-cold extraction. These findings are consistent with the properties of proteins that localize to lipid rafts and strongly suggest that gp160 is associated with lipid rafts. Further, removal of both palmitoylation sites results in the formation of virus with low levels of gp160 incorporation as well as a decrease in viral infectivity by 60-fold. Our results strongly support the suggestion that HIV-1 buds from lipid rafts and point to a role for rafts as a viral assembly hub.

Expression of Brca1 and splice variant Brca1delta11 RNA levels in mouse mammary gland during normal development and tumorigenesis.

Expression of Brca1 in mouse mammary cancer has yet to be analysed. We use a progressive model of neoplasia based on several mouse epithelial cell lines that represent distinct steps toward the fully tumorigenic state. Using RNase protection analysis because acceptable anti-Brca1 antibodies are not available we investigated the expression of Brca1 and a splice variant, Brca1Delta11, in several mammary hyperplasias and tumors that arose from them, and in normal mammary gland through pregnancy and involution. Expression of Brca1 was highest in rapidly proliferating cells. Expression of the full-length Brca1 was detectable in the virgin gland, was slightly elevated in the midpregnant gland, and decreased to levels similar to the age-matched virgin gland in the completely involuted gland. Expression of both forms of Brca1 was detectable in 9/9 paired hyperplasias and tumors, with levels of total Brca1, but not the splice variant Brca1Delta11, in tumors higher than those in the hyperplasias. While in disagreement with the observation that Brca1 levels decrease in human breast cancer progression, these patterns support the notion that Brca1 expression is associated with proliferating cells, and suggests that the link with differentiation seen in normal cells can be removed when cells become tumorigenic.

The A326S mutant of Gialpha1 as an approximation of the receptor-bound state.

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their alpha subunits. In search of an approximation of the receptor-alpha subunit complex, we have considered the properties of A326S Gialpha1, a mutation discovered originally in Gsalpha (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the alphai1beta1gamma2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Gialpha1 for GTPgammaS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the alpha subunit. The mutation also uncouples two activities of betagamma: stabilization of the GDP-bound alpha subunit (which is retained) and retardation of GDP dissociation from the heterotrimer (which is lost). For wild-type and mutant Gialpha1, beta gamma prevents irreversible inactivation of the alpha subunit at 30 degreesC. However, the mutation accelerates irreversible inactivation of alpha at 37 degreesC despite the presence of beta gamma. Structurally, the mutation weakens affinity for GTPgammaS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S Gialpha1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S Gialpha1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a Galpha subunit in the GDP-bound state.

Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon.

The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable. This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.

Enhanced tumor suppressor gene therapy via replication-deficient adenovirus vectors expressing an N-terminal truncated retinoblastoma protein.

The preclinical studies presented here demonstrate that treatment of human non-small cell lung carcinoma and bladder carcinoma cells by a recombinant adenovirus vector, AdCMVpRB94, expressing the N-terminal truncated retinoblastoma (RB) protein (pRB94) completely suppressed the tumorigenicity of the treated tumor cells in nude mice. Furthermore, gene therapy of established human RB- and RB+ bladder xenograft cancers in nude mice by AdCMVpRB94 resulted in regression of the treated tumors. Of note, although both the full-length and the truncated forms of the RB protein, when overexpressed in tumor cells via replication-deficient adenovirus vectors, were capable of suppression of tumor growth, the pRB94 was evidently more potent than the full-length RB protein.

Epidemiology of minority health.

Collecting epidemiologic data by ethnicity and race is a highly useful undertaking; but "bench mark" comparisons relative to majority Americans should not take priority over defining the determinants of health status within a minority group. Thus, it is necessary to identify factors contributing to the measured health status and to modify the environment, lifestyles, and behaviors to diminish the likelihood of undesirable health outcomes. This article presents an overview of the health status of African Americans, Asians and Pacific Islanders, and Hispanics. The goals are to provide a framework for the rational interpretation of both health status data and its determinants both within and between minority groups. This approach recognizes the heterogeneity of health status that exists within a minority group and encourages investigators to place more emphasis on the within-group health status differentials as they search for modifiable factors that underlie the risk for undesirable health outcomes.

Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis.

Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein alpha subunit, Gi alpha 1. The switch II and switch III segments become disordered, and linker II connecting the Ras and alpha helical domains moves, thus altering the structures of potential effector and beta gamma binding regions. Contacts between the alpha-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and beta gamma binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring alpha subunits in the lattice, suggesting that multimers of alpha subunits or heterotrimers may play a role in signal transduction.

Crystallization and preliminary crystallographic studies of Gi alpha 1 and mutants of Gi alpha 1 in the GTP and GDP-bound states.

Several different crystal forms of Gi alpha 1 have been grown and analyzed. Crystals of native protein containing bound GTP gamma S belong to space group P3(1)2(1) or P3(2)2(1) with cell dimensions a,b = 80.6 A and c = 106.3 A and diffract to a resolution of 1.9 A using synchrotron radiation. Crystals of native protein containing bound GDP belong to space group I4 with cell dimensions a,b = 121.3 A, and c = 67.7 A and diffract to 3.0 A. Data sets from crystals grown using mutant proteins have also been obtained and characterized.

GRAMP 100: a membrane protein concentrated on secretory granule membranes and the apical cell surface in exocrine acinar cells.

Using a monoclonal antibody (SG10A6) raised against secretion granule membranes of the rat parotid gland, we have identified an antigen that is a common component of both exocrine pancreatic and parotid granule membranes. SG10A6 (an IgM) immunoprecipitates antigen that migrates as a single band (M(r) approximately 80 KD unreduced; M(r) approximately 100 KD reduced) and immunoblots at least two polypeptides that are similar to the reduced and nonreduced immunoprecipitated antigen. This granule-associated membrane polypeptide (GRAMP 100; named for the apparent M(r) in reduced form) is also a prominent component of plasma membrane fractions. Immunocytochemical localization at the electron microscopic level demonstrates the presence of GRAMP 100 on granule membranes, especially condensing vacuoles and exocytotic figures, and the apical plasma membrane. Lower levels of antigen are detected on basolateral plasma membrane and on peri-Golgi membranes that may be part of the endosomal system. Both the cell fractionation and immunocytochemical localization indicate that GRAMP 100 differs in distribution from GRAMP 92 and 30K SCAMPs, two other components of exocrine granule membranes identified with monoclonal antibodies. To date, no polypeptides have been identified with this approach that are exclusive components of exocrine granule membranes.

A secretion granule membrane protein (GRAMP 92) is found in non-granule membranes including those of the endocytic pathway.

GRAMP 92, a secretion granule-associated membrane protein, has been identified in exocrine and endocrine storage granule membranes using a monoclonal antibody against rat parotid secretion granule membranes. This integral membrane glycoprotein has a M(r) of 92,000 in pancreatic zymogen granule membranes, and is slightly smaller in endocrine granule membranes. In both cases, deglycosylation produces core proteins of M(r) 52,000, that have identical peptide fingerprints. Unlike the slightly smaller zymogen granule membrane glycoprotein GP-2, GRAMP 92 does not appear to be bound to the membrane by a glycophosphatidyl inositol anchor, is not found on the plasma membrane and is not released into the secretion. Within acinar cells, low levels of antigen are observed immunocytochemically over the membranes of most granules. Antigen is highly concentrated on small vesicles that are closely apposed to (and possibly interact with) granules. As well, antigen is localized to organelles in the Golgi and basolateral regions that are part of the endocytic pathway. In hepatocytes a glycoprotein similar if not identical to GRAMP 92 marks the endocytic pathway including lysosomes. These findings indicate that GRAMP 92 is a widely distributed endocytic component and suggest that cells specialized for regulated secretion may adapt such components for storage granule function. Granule-associated GRAMP 92-rich membranes may link the exocytotic and endocytic pathways.

Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes.

A novel compositional overlap between membranes of exocrine and endocrine granules, synaptic vesicles, and a liver Golgi fraction has been identified using a monoclonal antibody (SG7C12) raised against parotid secretion granule membranes. This antibody binds secretory carrier membrane proteins with apparent Mr 31,000, 33,000 and 35,000 (designated SCAMPs 31, 33, 35). The proteins are nonglycosylated integral membrane components, and the epitope recognized by SG7C12 is on the cytoplasmic side of the granule membrane. SCAMP 33 is found in all secretory carrier membranes studied so far while SCAMP 35 is found in exocrine and certain endocrine granules and liver Golgi membranes and SCAMP31 only in exocrine granules. They are not related to other similar-sized proteins that have been studied previously in relation to vesicular transport and secretion. Immunocytochemical staining shows that these SCAMPs are highly concentrated in the apical cytoplasm of exocrine cells. Antigens are present not only on exocrine granules and synaptic vesicles but also on other smooth membrane vesicles of exocrine and neural origin as revealed by immunolocalization in subcellular fractions and immunoadsorption to antibody-coated magnetic beads. The wide tissue distribution and localization to secretory carriers and related membranes suggest that SCAMPs 31-35 may be essential components in vesicle-mediated transport/secretion.