Overproduction of isoprenoids by Saccharomyces cerevisiae in a synthetic grape juice medium in the absence of plant genes.

The objective of this work is to demonstrate if the hexaprenyl pyrophosphate synthetase Coq1p might be involved in monoterpenes synthesis in Saccharomyces cerevisiae, although its currently known function in yeast is to catalyze the first step in ubiquinone biosynthesis. However, in a BY4743 laboratory strain, the presence of an empty plasmid in a chemically defined grape juice medium results in a statistically significant increase of linalool, (E)-nerolidol and (E,E)-farnesol. When COQ1 is overexpressed from a plasmid, the levels of the volatile isoprenoids are further increased. Furthermore, overexpression of COQ1 in the same genetic context but with a mutated farnesyl pyrophosphate synthetase (erg20 mutation K197E), results in statistically significant higher levels of linalool (above 750 µg/L), geraniol, α-terpineol, and the sesquiterpenes, farnesol and nerolidol (total concentration of volatile isoprenoids surpasses 1300 µg/L). We show that the levels of monoterpenes and sesquiterpenes that S. cerevisiae can produce, in the absence of plant genes, depend on the composition of the medium and the genetic context. To the best of our knowledge, this is the highest level of linalool produced by S. cerevisiae up to now. Further research will be needed for understanding how COQ1 and the medium composition might interact to increase flavor complexity of fermented beverages.

T-level downstaging and complete pathologic response after preoperative chemoradiation for advanced rectal cancer result in decreased recurrence and improved disease-free survival.

PURPOSE: Preoperative chemoradiation therapy is used widely in the treatment of rectal cancer. The predictive value of response to neoadjuvant remains uncertain. We retrospectively evaluated the impact of response to preoperative and, specifically, of T-level downstaging, nodal downstaging, and complete pathologic response after chemoradiation therapy on oncologic outcome of patients with locally advanced rectal cancer. METHODS: There were 88 patients with ultrasound Stage T3/T4 midrectal (n = 37) and low rectal (n = 51) cancers (63 males; mean age 62.6 years). All patients were treated by preoperative 5-fluorouracil-based chemotherapy and pelvic radiation followed by surgical resection in six weeks or longer (56 sphincter-preserving resections). RESULTS: T-level downstaging after neoadjuvant treatment was demonstrated in 36 (41 percent) of 88 patients, and complete pathologic response was observed in 16 (18 percent) of the 88. Of the 42 patients with ultrasound-positive nodes, 27 had no evidence of nodal involvement on pathologic evaluation (64 percent). The overall response rate (T-level downstaging or nodal downstaging) was 51 percent. At a median follow-up of 33 months, 86.4 percent of patients were alive. The overall recurrence rate was 10.2 percent (three patients had local and six had metastatic recurrences). Patients with T-level downstaging and complete pathologic response were characterized by significantly better disease-free survival (P = 0.03, P = 0.04) and better overall survival (P = 0.07, P = 0.08), according to Wilcoxon's test comparing Kaplan-Meier survival curves. None of the patients with complete pathologic response developed recurrence or died during the follow-up period. CONCLUSION: T-level downstaging and complete pathologic response after preoperative chemoradiation therapy followed by definitive surgical resection for advanced rectal cancer resulted in decreased recurrence and improved disease-free survival. Advanced rectal cancers that undergo T-level downstaging and complete pathologic response after chemoradiation therapy may represent subgroups that are characterized by better biologic behavior.

Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis.

Both prokaryotes and eukaryotes respond to a decrease in temperature with the expression of a specific subset of proteins. Although a large body of information concerning cold shock-induced genes has been gathered, studies on temperature regulation have not clearly identified the key regulatory factor(s) responsible for thermosensing and signal transduction at low temperatures. Here we identified a two-component signal transduction system composed of a sensor kinase, DesK, and a response regulator, DesR, responsible for cold induction of the des gene coding for the Delta5-lipid desaturase from Bacillus subtilis. We found that DesR binds to a DNA sequence extending from position -28 to -77 relative to the start site of the temperature-regulated des gene. We show further that unsaturated fatty acids (UFAs), the products of the Delta5-desaturase, act as negative signalling molecules of des transcription. Thus, a regulatory loop composed of the DesK-DesR two-component signal transduction system and UFAs provides a novel mechanism for the control of gene expression at low temperatures.

Transcriptional control of the low-temperature-inducible des gene, encoding the delta5 desaturase of Bacillus subtilis.

The Bacillus subtilis des gene encodes the cold-inducible Delta5 lipid desaturase involved in the formation of unsaturated fatty acids from saturated phospholipid precursors. Here, we describe the expression pattern of the des gene in response to a temperature downshift from 37 to 20 degrees C. We found that the synthesis of des mRNA is undetectable at 37 degrees C but dramatically induced upon the temperature downshift. Decay characteristics of the des transcript as well as the in vivo decay of B. subtilis bulk mRNA were investigated. The results showed that the stability of the des transcript as well as of bulk mRNA lasted substantially longer at 20 degrees C than at 37 degrees C. Functional expression of des at 37 degrees C was achieved by exchanging its promoter with the non-cold shock spac promoter. These data provide the first direct evidence that temperature-mediated control of transcription is the major mechanism regulating the mRNA levels of the B. subtilis desaturase. The present results also demonstrate that the only component of the desaturation system regulated by temperature is the desaturase enzyme.

Reconstruction of rectovaginal fistula with sphincter disruption by combining rectal mucosal advancement flap and anal sphincteroplasty.

PURPOSE: This study evaluated the effectiveness of combining advancement flap with sphincteroplasty in patients symptomatic with rectovaginal fistula and anal sphincter disruption. METHODS: Twenty patients with rectovaginal fistulas and anal sphincter disruptions after vaginal deliveries underwent combined rectal mucosal advancement flap and anal sphincteroplasty between July 1986 and July 1993. The mean age of the patients was 30 (range, 18-40) years and the mean duration of symptoms was 54.8 weeks (range, 7 weeks to 6 years). In addition to mucosal advancement flap repair, 13 patients underwent two-layer repair of anal sphincters (with reapproximation of the puborectalis in 8 of the patients); 6 patients underwent one-layer overlap repair of anal sphincters (with reapproximation of the puborectalis in 2 of the patients); and 1 patient underwent reapproximation of internal anal sphincter alone because squeeze pressures were adequate, as determined by anal manometry. RESULTS: Postoperatively, vaginal discharge of stool and flatus was eliminated entirely in all 20 patients. Perfect anal continence of stool and flatus was restored in 14 patients (70 percent). Incontinence was improved but not eliminated in six patients (4 incontinent to liquid stool and 2 to flatus), and two patients required perineal pads. Subjectively, 19 patients (95 percent) reported the result as excellent or good. There were no complications. CONCLUSION: The combination of mucosal advancement flap and anal sphincteroplasty is a safe and highly effective procedure for correcting rectovaginal fistula with sphincter disruption after obstetrical injuries.

Colonoscopy-assisted colostomy--an alternative to laparotomy: report of two cases.

PURPOSE: The objective of this study was to evaluate the feasibility of performing fecal diversion with the help of a colonoscope without a concomitant laparotomy. METHODS: Colostomies were performed on two patients who needed fecal diversion and who would benefit from avoiding the morbidity of laparotomy. A colonoscope was used in each case to guide the surgeon in selecting the appropriate bowel segment. RESULTS: No complications related to the colostomy were noted in either patient. CONCLUSIONS: The technique of colonoscopy-assisted colostomy that we have described offers an acceptable method of creating a stoma without the need for laparotomy.

A Bacillus subtilis gene induced by cold shock encodes a membrane phospholipid desaturase.

Bacillus subtilis grown at 37 degrees C synthesizes saturated fatty acids with only traces of unsaturated fatty acids (UFAs). However, when cultures growing at 37 degrees C are transferred to 20 degrees C, UFA synthesis is induced. We report the identification and characterization of the gene encoding the fatty acid desaturase of B. subtilis. This gene, called des, was isolated by complementation of Escherichia coli strains with mutations in either of two different genes of UFA synthesis. The des gene encodes a polypeptide of 352 amino acid residues containing the three conserved histidine cluster motifs and two putative membrane-spanning domains characteristic of the membrane-bound desaturases of plants and cyanobacteria. Expression of the des gene in E. coli resulted in desaturation of palmitic acid moieties of the membrane phospholipids to give the novel mono-UFA cis-5-hexadecenoic acid, indicating that the B. subtilis des gene product is a delta5 acyl-lipid desaturase. The des gene was disrupted, and the resulting null mutant strains were unable to synthesize UFAs upon a shift to low growth temperatures. The des null mutant strain grew as well as its congenic parent at 20 or 37 degrees C but showed severely reduced survival during stationary phase. Analysis of operon fusions in which the des promoter directed the synthesis of a lacZ reporter gene showed that des expression is repressed at 37 degrees C, but a shift of cultures from 37 to 20 degrees C resulted in a 10- to 15-fold increase in transcription. This is the first report of a membrane phospholipid desaturase in a nonphotosynthetic organism and the first direct evidence for cold induction of a desaturase.

Endorectal mucosal advancement flap: the preferred method for complex cryptoglandular fistula-in-ano.

Cryptoglandular fistula-in-ano is a common affliction that usually responds well to conventional surgical procedures such as fistulectomy, fistulotomy, and seton placement. These procedures, however, can be associated with varying degrees of fecal incontinence. Endorectal mucosal advancement flap has been advocated as an alternative procedure that avoids this problem. This study was undertaken to determine the risks and benefits associated with endorectal mucosal advancement flap in the treatment of complex fistula-in-ano. One hundred sixty-four patients underwent 167 endorectal mucosal advancement flap procedures for complex cryptoglandular fistula-in-ano between January 1982 and December 1990. There were 126 men and 38 women whose mean age was 42.1 years (range 20 to 79 years). The majority of the patients (70%) had complex fistulas (transsphincteric, suprasphincteric, or extrasphincteric). Fifteen patients (9%) had an intersphincteric fistula. All patients were available for short-term follow-up (6 weeks). Postoperative morbidity was minimal and included urinary retention in 13 patients (7.8%) and bleeding in one patient. Healing time averaged 6 weeks. Long-term follow-up, ranging from 19 to 135 months, was carried out in 61 patients. There were two recurrences (3.28%). Nine patients (15%) complained of varying degrees of fecal incontinence. Six patients complained of incontinence to flatus and three patients complained of incontinence to liquid stool. No patient was incontinent of solid stool. Sixty patients (98%) rated their functional result as excellent or good. Endorectal mucosal advancement flap is a safe and effective technique for the treatment of complex cryptoglandular fistula-in-ano. It can be performed with minimal morbidity, no mortality, an acceptable recurrence rate, and little alteration in anorectal continence.

Colonoscopic bowel preparations--which one? A blinded, prospective, randomized trial.

UNLABELLED: For the past decade peroral, orthograde, polyethylene glycol-electrolyte lavage solutions (PEG-ELS) have been the preferred bowel-cleansing regimens before diagnostic and therapeutic procedures on the colon and rectum. The large volume and unpalatibility of these solutions may lead to troubling side effects and poor patient compliance. PURPOSE: This study was undertaken to determine which of various colon-cleansing methods before colonoscopy would provide greater patient acceptance while maintaining similar or improved effectiveness and safety. METHODS: Three hundred twenty-nine patients undergoing elective ambulatory colonoscopy were prospectively randomized to one of three bowel preparation regimens. Group 1 received 41 of PEG-ELS (n = 124). Group 2, in addition to PEG-ELS, received oral metoclopramide (n = 99). Group 3 received oral sodium phosphate (n = 106). All groups were evenly matched according to age and sex. RESULTS: Ninety-one percent of all patients completed the preparation received. Sixteen percent of patients suffered significant sleep loss with a bowel preparation. When comparing the three groups, there was no difference in the assessment of nausea, vomiting, abdominal cramps, anal irritation, or quality of the preparation. Compared with other preparations, oral sodium phosphate was better tolerated. More patients completed the preparation (P < or = 0.001). Fewer patients complained of abdominal fullness (P < or = 0.001). More patients were willing to repeat their preparation (P < or = 0.02). Also, sodium phosphate was found to be four times less expensive than either of the PEG-ELS preparations. CONCLUSION: All regimens were found to be equally effective. Abdominal symptoms and bowel preparation were not influenced by the addition of metoclopramide. The oral sodium phosphate preparation was less expensive, better tolerated, and more likely to be completed than either of the other preparations.

Delayed repair of obstetric injuries of the anorectum and vagina. A stratified surgical approach.

PURPOSE: We categorized the various types of post-obstetric injuries of the anorectum and vagina encountered in a five-year period. The operative procedures used to repair these injuries and the functional outcome after surgery were assessed. METHODS: Between 1986 and 1991, 52 patients were surgically treated for obstetric injuries of the anorectum and vagina; 48 patients were available for follow-up study. Four clinical injury types were identified: Type I, incontinent and sphincter (11 patients); Type II, rectovaginal fistula (16 patients); Type III, rectovaginal fistula and incontinent and sphincter (11 patients); and Type IV, cloaca-like defect (10 patients). The mean age of the patients was 30 years, the mean duration of symptoms before surgery was 13 months, and the mean follow-up period was 16 months. The major component of surgical repair for each injury type was: Type I, overlap repair of external anal sphincter; Type II, rectal mucosal advancement flap; Type III, overlap repair of external anal sphincter and rectal mucosal advancement flap; and Type IV, overlap repair of external anal sphincter, anterior levatorplasty, and anal and vaginal mucosal reconstruction. Fecal diversion was not performed in any patient. Specific questions were asked at the most recent follow-up assessment to determine results. RESULTS: Continence status postoperatively was classified as perfect, impaired, or poor; poor was defined as no improvement or worse. Postoperative continence (perfect impaired, or poor) was, respectively: Type I (11 patients), 64 percent, 36 percent, and 0 percent; Type II (16 patients), 56 percent, 0 percent, and 44 percent; Type III (11 patients), 64 percent, 36 percent, and 0 percent; and Type IV (10 patients), 90 percent, 10 percent, and 0 percent. Vaginal discharge of stool was eliminated in all patients with a rectovaginal fistula. Subjectively, 92 percent of the patients had excellent or good results. Complications included wound hematoma (n = 2), fecal impaction (n = 2), urinary retention (n = 1), and urinary tract infection (n = 1). CONCLUSION: Patients with Type II injuries had the worst results (P < 0.001). These patients should be evaluated for anal incontinence before surgery to assess the need for a concomitant sphincteroplasty.

Abdominal colon and rectal operations in the elderly.

Sixty-seven abdominal operations for colon and rectal disorders were performed on 56 patients 80 years of age or older from January 1, 1984 to June 30, 1989. Nine patients required multiple operations. Sixty-two procedures (92 percent) were performed on patients in their ninth decade; two operations were performed on patients 95 years of age or older. Forty-five patients (80 percent) were operated upon for carcinoma. Operations included segmental colectomy (33 patients), low anterior resection (12 patients), total abdominal colectomy (3 patients) and abdominoperineal resection (2 patients). Forty patients were classified as ASA Class III; the majority were monitored in the surgical intensive care unit for a mean of 2.84 days. Thirty patients were monitored with arterial catheters and 21 with central invasive monitoring. Operative mortality was 7 percent (4 patients). Two patients died from diffuse carcinomatosis; one patient had a fatal myocardial infarction. The final death occurred from multisystem organ failure following anastomotic dehiscence. Twenty-seven operations were performed without postoperative complications; 18 operations were followed by a single minor complication. The average hospital stay was 18.96 days. All patients were admitted from home. Thirty-three returned home postoperatively; 16 were discharged to an extended care facility. In conclusion, elderly patients with colon and rectal disorders can be operated upon with acceptable morbidity and mortality. Age alone should not interdict surgical therapy.

Squamous-cell carcinoma of the anal canal: management with combined chemo-radiation therapy.

Twenty patients with squamous-cell carcinoma of the anal canal received combined chemo-radiation therapy as their primary treatment. There were 18 women and two men with a mean age of 63 years (range, 34-91 years). The mean follow-up was 34 months (range, 6-62 months). Anal margin cancers and adenocarcinomas were excluded. Fourteen of 20 patients treated had a complete response. There were six local failures: three with residual disease at the end of treatment and three with recurrent disease at a later date. Of the three with residual disease, one underwent abdominoperineal resection and two received salvage therapy (one with chemo-radiation and one with radiation alone). All three patients with recurrent disease were treated with abdominoperineal resection. All six were disease free at the end of the study. Of the 14 patients with complete local response, one presented with liver metastases 19 months later. Sixteen patients (80 percent) were alive at the end of the study, and 19 patients (95 percent) had no evidence of disease. These data add support for salvage therapy in the treatment of patients with residual disease following initial chemo-radiation therapy. Salvage options for patients with squamous-cell carcinoma of the anus who fail the Nigro protocol will be discussed.

Surgical treatment of low rectovaginal fistulas.

Forty women with low rectovaginal fistulas were operated upon over a 9-year period. The etiology of the fistula in the majority was obstetric. Nine women had prior attempts to repair the fistula. All 40 women were managed with endorectal advancement flap with the addition of sphincteroplasty or perineal body reconstruction in 15 patients and rectocele repair in six patients. Postoperative complications included urinary difficulties (two patients) and wound complications (three patients). There were two recurrences. All women treated with sphincteroplasty or perineal body reconstruction were continent. Seven women complained of varying degrees of incontinence postoperatively; none had undergone sphincter or perineal body reconstruction. Endorectal advancement flap is a safe and effective operation for women with rectovaginal fistulas. Concomitant sphincteroplasty or perineal body reconstruction should be performed in women with historical, physical, or manometric evidence of incontinence.

Rectocele repair. Four years' experience.

A retrospective review of 64 rectocele repairs done over a four-year period was performed. The most common indication for repair was constipation. Thirty-five patients were repaired transanally, and 29 were repaired transvaginally. The overall morbidity was 34 percent, and the overall mortality was 0 percent. The most common complication was urinary retention in 12.5 percent. There was no difference in complications between techniques. Of 46 patients contacted for follow-up, 25 (54 percent) still complained of constipation, 17 (34 percent) had partial incontinence, 8 (17 percent) noted persistent rectal pain, 15 (32 percent) mentioned occasional rectal bleeding, and 10 (22 percent) complained of vaginal tightness or sexual dysfunction. Thirty-seven (80 percent) patients stated that they had improved after surgery. Except for persistent rectal pain, there was no difference in results between transanal and transvaginal repairs. Those undergoing transvaginal repair had a much greater problem with pain. Our relatively poor results may be due to an unselective approach to rectocele repair. The presence of both constipation and a rectocele does not imply an association, and a complete anorectal physiologic examination should precede repair. There is no functional difference between transvaginal and transanal rectocele repair.

Preoperative versus postoperative adjuvant radiotherapy for surgically curable carcinoma of the rectum and distal sigmoid colon.

From January 1979 to October 1986, 86 patients with surgically resectable adenocarcinoma of the rectum or rectosigmoid were treated with adjuvant radiotherapy consisting of preoperative 2,400 cGy (22 patients), preoperative 4,000 cGy (14 patients), "sandwich" technique (27 patients), and postoperative irradiation (23 patients). Average follow-up was 42.9 months. The local recurrence rate was 4.5%, 9.1%, 7.4%, and 34.8%, respectively. The distant metastasis rate was 18.2%, 18.2%, 7.4%, and 30.4%, respectively. Preoperative radiotherapy with adequate surgical resection appears more effective in reducing the incidence of local recurrence.

Vaginography: an easy and safe technique for diagnosis of colovaginal fistulas.

Colovaginal fistulas are often difficult to demonstrate. Vaginography is a simple, safe, and effective technique.

Complete obstruction of the sigmoid colon treated by primary resection and anastomosis--an improved technique (preliminary report). Report of three cases.

Three patients with complete colonic obstruction treated by primary resection and anastomosis with intraoperative colon tube decompression and bowel lumen sterilization without a protective colostomy are presented. An improved colonic decompressor was used. It is postulated that this procedure is an alternative safe technique in patients with colonic obstruction in whom an end-colostomy, mucous fistula, or Hartmann pouch would be necessary.

A new silicone-prosthesis in the modified Thiersch operation.

Experience with a new silicone prosthesis in the modified Thiersch operation for rectal procidentia in 16 extremely poor-risk patients is presented. The technique of implantation, structural details of the prosthesis, and the clinical results are described. The use of a new silicone prosthesis in the modified Thiersch procedure is a viable alternative in this group of patients. Surgical technique is a primary determining factor in preventing complications.

The efficacy of barium-enema examinations in patients with anorectal disease.

This retrospective study examines the value of an air contrast barium enema examination in detecting proximal neoplasia in the patient presenting with benign anorectal disease as determined by history, physical examination, rigid, and flexible sigmoidoscopy. In 428 of these patients, the roentgenographic studies showed proximal colonic cancer or polyps in less than 1 percent of patients reviewed. In addition, a review of 402 patients with known colon and rectal cancer were surveyed using the same criteria for diagnosis, and less than 1 percent were misinterpreted as having benign anorectal disease.