Modified Hanley procedure for management of complex horseshoe fistulae.

PURPOSE: Horseshoe fistulae are challenging due to configuration and sphincter involvement. In view of the new treatment options for fistulae (e.g. collagen plug), aim of our study was to review the outcome of patients primarily treated with a traditional approach. METHODS: We retrospectively reviewed patients who presented between 2003 and 2008, with a posterior horseshoe abscess/fistula and were treated with a modified Hanley procedure and seton management. Excluded were Crohn's disease, fistulae from malignancy/surgical complications, and other treatment methods. Data collection included demographics, duration of the disease, and of the treatment, outcome, and incontinence. RESULTS: Twenty-three patients (M/F 20/3) were analyzed. Mean age was 50.3 + or - 10.2 years, median symptom duration 24 months; three patients (19%) had previously received colostomies without resolution. All patients received a posterior midline cutting seton. The average total number of setons was 3 + or - 1.3 (range 2-7), with removal after 1.6 + or - 1.2 months upon resolution of induration and suppuration. The cutting seton was tightened in monthly intervals on average 4.9 + or - 2.2 times. Follow-up was 15.4 + or - 9.7 months: 91.3% patients had complete healing time within 8.1 + or - 4.5 months; 19/23 patients recovered fast, and were able to work no later than 4 weeks postoperatively (2 patients retired, 2 on disability for other reasons). No incontinence was found. CONCLUSIONS: Fecal diversion alone did not resolve horseshoe fistulae. A modified Hanley procedure with drainage of the deep postanal space and cutting and draining setons proved to be safe, successful, and did not result in complaints of fecal incontinence. Completion of the treatment took months, but patients remained functional even with setons in place.

Stimulation of translation and cytoplasmic polyadenylation by the Xenopus c-mycI 3'-untranslated region.

Expression of messenger RNA (mRNA) in both embryonic and adult cells may be profoundly influenced by untranslated sequences in the 3'-end. Elements in the 3'-untranslated regions (UTRs) of messengers are known to influence messenger stability, polyadenylation, and translation. We have examined the effects of the 3'-UTR of Xenopus laevis c-mycI (either alone or in combination with the 5'-first exon) on the expression of a chloramphenicol acetyltransferase (CAT) reporter in Xenopus embryos. The Xenopus c-mycI 3'-UTR enhanced messenger translation independent of the 5'-UTR. RNase H analysis indicated that the Xenopus c-mycI 3'-UTR can promote the cytoplasmic polyadenylation of CAT mRNA in embryos. The result suggests that the post-fertilization enhancement of translation caused by the c-mycI 3'-UTR may be a consequence of cytoplasmic polyadenylation. A uridine (U)-rich sequence in the Xenopus c-mycI 3'-UTR that may be responsible for polyadenylation is similar to an element that destabilizes mammalian c-myc transcripts. We discuss the possibility that U-rich sequences may play a dual role by destabilizing growth-related transcripts in adult cells and stimulating their polyadenylation during development, and we propose that a switch in the role of such sequences in adult cells could lead to stabilization of these messengers, increased translation, and abnormal growth control.

Effects of cyclin A2 noncoding regions on reporter gene translation during early development of Xenopus laevis.

The repression of translation of Xenopus cyclin A2 transcripts during early development was examined by analyzing the effects of cyclin A2 noncoding regions using a CAT reporter system. On their own, the 5' and 3' UTRs (untranslated regions) were unable to inhibit reporter translation until approximately the time of the midblastula transition. Transcripts containing the 3' UTR were polyadenylated after fertilization and the midblastula transition. When both noncoding regions flanked a CAT reporter gene, translation was repressed at all stages of development examined in spite of their polyadenylation after fertilization. From these data, we conclude that the 5' and 3' UTRs interact synergically to prevent translation during early development and that the poly(A) tail is insufficient to promote their translation.

5'-3' interactions in regulation of translation in Xenopus early embryos.

We have investigated the effects of 3' noncoding elements in enhancing translation of messengers having translation-inhibiting 5' untranslated regions (UTRs). The translation of transcripts bearing the 5' UTRs of either human c-myc or a synthetic hairpin structure upstream of a chloramphenicol acetyltransferase (CAT) reporter sequence is greatly attenuated in early embryos of Xenopus laevis. Translation of transcripts bearing the human c-myc-5' UTR was markedly stimulated by the presence of 3' poly(A). Transcripts bearing the 5' hairpin element were insensitive to the presence of poly(A), but they were extremely sensitive to the composition of the 3' UTR. A GC-rich distal sequence repressed translation, whereas a proximal GGAAU sequence promoted translation of these transcripts. Our results support the concept that long-range interactions between the 5' and 3' ends of transcripts are important in regulating translation in Xenopus embryos.

Effects of c-myc first exons and 5' synthetic hairpins on RNA translation in oocytes and early embryos of Xenopus laevis.

Mammalian c-myc transcripts have long G/C-rich 5' untranslated regions (UTRs) that may fold into secondary structural elements that may impede translation. We have examined the effects of different c-myc first exons, which produce most of the 5' UTR of c-myc transcripts, on translation in Xenopus oocytes and embryos, by placing these structures upstream of a chloramphenicol acetyltransferase (CAT) reporter. Our results demonstrate that the human c-myc first exon inhibits reporter translation in both oocytes and embryos. Unlike their mammalian counterparts, Xenopus c-mycI first exons initiated at either promoter 1 or promoter 2 do not impede translation. We conclude that translation inhibition reported in a previous investigation (Lazarus, 1992. Oncogene, 7:1037) utilizing Xenopus c-mycI 5' non-coding elements was due to the inclusion of nonrelevant non-transcribed sequences. Previous investigators have reported that inhibition of translation in Xenopus oocytes by 5' secondary structure is alleviated after fertilization (Lazarus et al., 1988. Oncogene 3:517; Fu et al., 1991, Science 251:807). We repeated the experiments of Fu et al., examining the effects on translation by a highly stable synthetic hairpin. The hairpin severely [correction of severly] restricted translation in both oocytes and embryos, indicating that highly stable 5' secondary structure is equally inhibitory in oocytes and embryos.

Reliability of torque range of motion: a preliminary study.

The clinical reliability of torque range of motion (TROM) has not been tested. This preliminary review was performed to determine the intra- and interrater reliability of TROM. The proximal interphalangeal joints of the index and middle fingers of 14 normal subjects, 56 digits, were tested by applying extension forces of 200 g, 400 g, 600 g, and 800 g. Data were collected using both the hand-held Haldex orthotic gauge with the dial goniometer (manual) and the cantilever-beam force transducer and electrogoniometer (digital) as testing instruments. The testing order of digits, sides, and devices was randomized and tested separately by three different test teams. Results of the analysis of variance showed a significant difference of the means between raters at all force levels (p < 0.0001) and between instruments at 600-g and 800-g forces (p < 0.01). The Pearson product-moment correlation coefficient test, used to test the consistency, resulted in moderate consistency of measures of intrarater reliability between trials, and between instruments used (digital and manual). Intraclass correlation coefficients (ICCs) were calculated with results that were nearly identical to those of the Pearson. Intrarater reliability was in more consistent agreement at the 600-g and 800-g levels. There was significantly high agreement between the tests of tester B, compared with moderate correlations of the tests of testers A and C.

The induction of pyruvate kinase synthesis by heat shock in Xenopus laevis embryos.

Heat-shocked Xenopus embryos have an unusually complex heat shock response. The dominant heat shock protein (Hsp) has a relative molecular mass (M(r)) of 62,000 D (Hsp62). Affinity-purified IgGs against the glycolytic enzyme pyruvate kinase (PK; EC 2.7.1.40) specifically immunoprecipitated Hsp62 from extracts of embryos that had been heat-shocked at 37 degrees C for 30 min. Thus, Hsp62 and pyruvate kinase are immunologically cross-reacting. Electrophoretic separation of PK isoforms suggests that heat-shocked Xenopus embryos increase synthesis of an isoform of PK. Thermal denaturation studies suggest that this isoform has enhanced thermal stability. The identification of PK as an Hsp is discussed within the context of a physiological requirement for elevated levels of anaerobic glycolysis in heat-stressed cells as a vital component of the acquisition of thermotolerance.

A possible role for c-Myc oncoproteins in post-transcriptional regulation of ribosomal RNA.

Overexpression of members of the myc family of oncogenes contributes to the development of many vertebrate malignancies. Although several functions for myc gene products have been proposed, the targets for Myc activity during oncogenesis or normal development remain to be identified. Oocytes of Xenopus laevis, which are non-dividing cells that accumulate abundant c-Myc protein, provide an unusual opportunity to examine c-Myc function. We have investigated whether the accumulation of massive amounts of ribosomal RNA (rRNA) by Xenopus laevis oocytes may be related to their elevated expression of myc proto-oncogenes. Our data show that anti-Myc antibodies and some (but not all) c-Myc peptides from conserved regions of the c-Myc protein enhance the turnover of rRNA synthesized in homogenates of oocyte nuclei. These results suggest that one or more members of the Myc protein family may be involved in post-transcriptional regulation of rRNA metabolism. The regulation by c-Myc of rRNA could account, in part, for the generalized stimulation of cells during tumorigenesis.

Multiple antigens recognized by anti-c-myc antibodies in human cells and Xenopus oocytes.

We have investigated the localization, solubility, serum regulation, and phosphorylation of MYC antigens from Colo 320 cells, a human transformed cell line with an amplified c-myc gene, and from Xenopus oocytes, which express high levels of c-myc mRNA. Although MYC proteins are often reported to range from 60 to 68 kilodaltons, our panel of anti-MYC monoclonal antibodies recognized a number of higher and lower molecular mass antigens, in addition to proteins within this range. Based upon various criteria, including cross-recognition by several anti-MYC antibodies, we suggest that some of these antigens are bona fide MYC family proteins. Our results, as well as those of others reported previously, suggest that several MYC antigens may be simultaneously present in cells. The apparent diversity among members of the MYC family of antigens raises the possibility of multiple cellular functions and regulatory roles for these proteins.

Preparative labeling of proteins with [35S]methionine.

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.

Translational potentiation of messenger RNA with secondary structure in Xenopus.

Differential translation of messenger RNA (mRNA) with stable secondary structure in the 5' untranslated leader may contribute to the dramatic changes in protein synthetic patterns that occur during oogenesis and early development. Plasmids that contained the bacterial gene chloramphenicol acetyltransferase and which encoded mRNA with (hpCAT) or without (CAT) a stable hairpin secondary structure in the 5' noncoding region were transcribed in vitro, and the resulting mRNAs were injected into Xenopus oocytes, eggs, and early embryos. During early oogenesis, hpCAT mRNA was translated at less than 3 percent of the efficiency of CAT mRNA. The relative translational potential of hpCAT reached 100 percent in the newly fertilized egg and returned to approximately 3 percent after the midblastula transition.

Factors influencing the heat shock response of Xenopus laevis embryos.

We have further characterized the heat shock response of Xenopus laevis embryos. Xenopus embryos respond to heat shock by consistently synthesizing four major heat shock proteins (hsps) of 62, 70, 76, and 87 kilodaltons. In addition to these hsps, heat-shocked embryos also exhibit the synthesis of several minor hsps. The synthesis of these hsps is often variable. We have monitored the effects of different temperatures and lengths of heat shock on the pattern and intensity of hsp synthesis. In general, the four major hsps are induced more strongly at higher temperatures and during increasing intervals of heat shock. The temperature and duration of heat shock can affect the synthesis of the minor hsps, however. Some hsps are synthesized at lower temperatures only (i.e., below 37 degrees C), whereas others are synthesized only at higher temperatures (i.e., above 37 degrees C). We have extensively examined the characteristics of hsp 35 synthesis, one of the most variably synthesized hsps. This hsp is characteristically synthesized at temperatures above 35 degrees C and usually during the first 40 min of heat shock, after which it becomes undetectable. In some experiments, its synthesis is restimulated during later intervals of heat shock. Hsp 35 is also under developmental regulation. It is not synthesized by heat-shocked embryos until the late blastula to early gastrula stage. After this brief period of inducibility, its synthesis is dramatically reduced in mid- to late gastrulae, but reappears in heat-shocked neurulae. We have previously demonstrated that hsp 35 is related to the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The induction of hsp 35 synthesis is inversely correlated with the constitutive levels of GAPDH specific activity. In this paper we document further correlations between the synthesis of hsp 35 and GAPDH specific activity during early Xenopus development.

A role for glyceraldehyde-3-phosphate dehydrogenase in the development of thermotolerance in Xenopus laevis embryos.

During heat shock, Xenopus laevis embryos exhibit an increase in the rate of accumulation of lactate and a loss of ATP relative to non-heat-shocked control embryos. These results suggest that heat shock stimulates a shift in energy metabolism to anaerobic glycolysis while at the same time causing an increase in the demand for ATP. We have evidence indicating that the embryo may meet such demands placed on it by increasing the levels of some glycolytic enzymes. In this report, we show that heat shock stimulates increases in the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase [( EC 1.2.1.12] GAPDH). The specific activity of GAPDH shows a significant increase after heat shock, which correlates with the accumulation of GAPDH in heat-shocked embryos as detected by immunoblotting. Increases in GAPDH-specific activity are variable, however, and are inversely proportional to the levels of specific activity in control embryos; i.e., constitutive enzyme activity. We further analyzed the heat-enhanced accumulation of GAPDH by electrophoretically separating GAPDH isozymes on nondenaturing polyacrylamide gels. Control embryos exhibit a single isozyme of GAPDH, whereas heat-shocked embryos exhibit two isozymes of GAPDH. When these isozymes are labeled with [35S]methionine, separated by nondenaturing gel electrophoresis, and analyzed by fluorography, a heat-shock protein is found to comigrate with the isozyme unique to the heat-shocked sample. Enzyme activity assays at different temperatures suggest that this isozyme has optimum enzymatic activity only at heat-shock temperatures. We have correlated a 35-kD heat-shock protein (hsp35) with GAPDH using the following evidence: this hsp comigrates with GAPDH on one-dimensional SDS polyacrylamide gels; heat-enhanced increases in GAPDH specific activity correlate with hsp35 synthesis; and hsp35 and GAPDH have similar peptide maps. This relationship also provides a compelling explanation for the restriction of hsp35 synthesis to the vegetal hemisphere cells of heat-shocked early gastrulae reported previously (Nickells, R. W., and L. W. Browder. 1985. Dev. Biol. 112:391-395).

Changes in heat shock protein synthesis and hsp70 gene transcription during erythropoiesis of Xenopus laevis.

As erythroid cells of Xenopus laevis differentiate, their capacity for transcription and protein synthesis becomes progressively restricted. Mature erythrocytes are thought to be transcriptionally inert and have very low levels of protein synthesis (Maclean et al., 1973, Cell Differ. 2, 261-269). We have examined actual and potential synthetic activity during erythropoiesis by comparing the patterns of protein synthesis and measuring the transcriptional activity of selected genes under ambient and heat shock conditions. As erythropoiesis proceeds, there is a progressive decrease in the number of heat shock proteins (hsps) whose synthesis is induced at elevated temperatures. Despite this repression, mature erythrocytes are able to respond to heat shock with the induction of synthesis of one protein, hsp70. In addition, an hsp70-like protein is synthesized constitutively in orthochromatic erythroblasts, the final erythroid cell stage before maturation into erythrocytes. An in vitro nuclear run-off transcription assay was used to study transcription of the hsp70, beta-globin, and ribosomal genes during erythropoiesis. beta-Globin and ribosomal gene transcription were detected at all stages of erythropoiesis, including the erythrocyte stage. However, transcription of the beta-globin gene relative to ribosomal gene transcription declines substantially in erythrocytes as compared to earlier stages. Hsp70 gene transcription shows both stage-specific and heat shock-dependent transcription. Orthochromatic erythroblasts and erythrocytes have heat shock-dependent hsp70 gene transcription, whereas the gene is transcribed at the earlier erythroblast stages at ambient temperature as well as during heat shock. The constitutive hsp70 transcripts made during these earlier stages may be stored for utilization in constitutive synthesis of the hsp70-like protein in orthochromatic erythroblasts. These data indicate that although erythrocytes become synthetically repressed during differentiation, they are less inert than previously thought. The data also reveal that the synthesis of hsps and related proteins is subject to both transcriptional and post-transcriptional control during erythropoiesis.

The effects of heat shock on the morphology and protein synthesis of the epidermis of Xenopus laevis larvae.

By scanning electron microscopy, we have observed that a 20-min heat shock at 37 degrees C, although not lethal, causes extensive damage to the epidermis of 30-h and 2-d (post-fertilization) Xenopus laevis larvae. The primary effects of heat shock are the apical swelling of the epidermal cells, giving the epidermis a "cobblestone" appearance, and the selective shedding of the ciliated cells. The shed cells may be cell fragments, however, because some of them are anucleate. Shed cells also exhibit the enriched synthesis of a group of heat shock proteins of 62,000 D molecular weight, suggesting that these proteins are specific to the shed cells. Prolonged heat shock of these larvae (i.e., 30 min at 37 degrees C) results in the complete disintegration of the epidermis, followed by larval death. At later stages of development (3-d and 4-d post-fertilization), the epidermis becomes more resistant to heat-induced damage inflicted by a 20-min heat shock. This increase in resistance coincides with the development of large secretory cells and the loss of ciliated cells in the epidermis and thus parallels a change in the state of histological differentiation.

Decay of the oocyte-type heat shock response of Xenopus laevis.

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.

Heat-shock gene expression in animal embryonic systems.

We have examined the expression of heat shock or stress genes in fish, echinoderm, amphibian, and mammalian embryonic systems. In a Chinook salmon embryo cell line, elevation of the incubation temperature or exposure to metal ions (e.g., cadmium and zinc) induced a set of heat-shock proteins HSPs. Transcriptional inhibitor, in vitro translation, and Northern hybridization studies suggest that fish HSP synthesis is regulated at the transcriptional level. The synthesis of HSPs during early development of Arbacia punctulata, Xenopus laevis, mouse, and rabbit is a stage-dependent phenomenon. In each of the developmental systems, HSP synthesis could not be induced until after cleavage stages. The ability of the embryo to undergo a heat-shock response (i.e., HSP synthesis) was correlated with the ability to detect HSP mRNA accumulation by either in vitro translation or Northern hybridization assays. Thus, the stage-dependent synthesis of HSPs appears to be controlled at the transcriptional level. Finally, in all of the organisms studied, the capacity to synthesize HSPs and accumulate HSP mRNA also coincides with acquisition of thermotolerance.

Acquisition of the heat-shock response and thermotolerance during early development of Xenopus laevis.

The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.

Aldehyde oxidase cross-reacting material in the Aldoxn, cin, mal, and lxd mutants of Drosophila melanogaster.

Rocker immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldoxn, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldoxn larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldoxn flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extract. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.