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Objective: To evaluate the rate of vaginal hysterectomy and outcomes after initiation of a prospective decision-tree algorithm to determine the optimal surgical route of hysterectomy., Methods: A prospective algorithm to determine optimal route of hysterectomy was developed, which uses the following factors: history of laparotomy, uterine size, and vaginal access. The algorithm was implemented at our institution from November 24, 2015, to December 31, 2017, for patients requiring hysterectomy for benign indications. Expected route of hysterectomy was assigned by the algorithm and was compared with the actual route performed to identify compliance compared with deviation. Surgical outcomes were analyzed., Results: Of 365 patients who met inclusion criteria, 202 (55.3%) were expected to have a total vaginal hysterectomy, 57 (15.6%) were expected to have an examination under anesthesia followed by total vaginal hysterectomy, 52 (14.2%) were expected to have an examination under anesthesia followed by robotic-assisted total laparoscopic hysterectomy, and 54 (14.8%) were expected to have an abdominal or robotic-laparoscopic route of hysterectomy. Forty-six procedures (12.6%) deviated from the algorithm to a more invasive route (44 robotic, two abdominal). Seven patients had total vaginal hysterectomy when robotic-assisted total laparoscopic hysterectomy or abdominal hysterectomy was expected by the algorithm. Overall, 71% of patients were expected to have a vaginal route of hysterectomy per the algorithm, of whom 81.5% had a total vaginal hysterectomy performed; more than 99% of the total vaginal hysterectomies attempted were successfully completed., Conclusion: Vaginal surgery is feasible, carries a low complication rate with excellent outcomes, and should have a place in gynecologic surgery. National use of this prospective algorithm may increase the rate of total vaginal hysterectomy and decrease health care costs.

Objective: The aim of this study was to compare outcomes of vaginal hysterectomy between patients with and without the following perceived contraindications to vaginal surgery: uterine weight greater than 280 g, prior cesarean delivery, no vaginal parity, and obesity., Methods: Retrospective cohort of benign vaginal hysterectomies between 2009 and 2013 was obtained. Outcomes included uterine debulking, transfusion, intraoperative complications, length of stay, and Accordion grade 2+ postoperative complications. For each outcome, the association between the presence of each contraindication and the outcome was evaluated using univariate and multivariate logistic regression models., Results: Among 692 vaginal hysterectomies, 11% (76/691) had a uterine weight greater than 280 g, 11.3% (78/690) had no vaginal parity, 14.9% (103/690) had a history of cesarean delivery, and 37.7% (248/657) had a body mass index of 30 kg/m or greater; 110 (15.9%) had 2 or more contraindications. Uterine debulking occurred in 146 women (21.1%), and both uterine weight greater 280 g (adjusted odds ratio, 39.2; 95% confidence interval, 18.4-83.5) and prior cesarean delivery (adjusted odds ratio, 2.1; 95% confidence interval, 1.2-3.7) were significantly associated with an increased likelihood of uterine debulking after adjusting for age, hematologic disease, and preoperative diagnosis. None of the contraindications were significantly associated with need for a blood transfusion, presence of an intraoperative complication, length of stay greater than 2 days, or presence of an Accordion grade 2+ postoperative complication, which occurred in 2.7%, 2.5%, 14.0%, and 6.9% of all women, respectively., Conclusions: Vaginal hysterectomy can be safely performed with favorable outcomes, even in women with a uterus greater than 280 g, prior cesarean delivery, no vaginal parity, and obesity. Our findings challenge several perceived contraindications to vaginal hysterectomy.

Objective: To report rates and identify risk factors for urinary tract infection (UTI) after hysterectomy for benign conditions or combined with pelvic reconstructive surgery., Methods: This is a cohort study that included women who underwent hysterectomy either for benign gynecologic conditions or hysterectomy combined with pelvic reconstructive surgery from January 1, 2012, through June 30, 2014, at a single institution. The primary outcome was UTI within 8 weeks of surgery. Logistic regression modeling was used to develop a model for predicting UTI after surgery., Results: Of 1,156 women included in the study, 136 (11.8%, 95% CI 10.0-13.8) developed UTI within 8 weeks. Women who underwent hysterectomy for a benign gynecologic condition that was not combined with pelvic reconstructive surgery had an overall UTI rate of 7.3% (95% CI 5.6-9.3) vs 21.7% (95% CI 17.6-26.4) after hysterectomy combined with pelvic reconstructive surgery. After adjusting for hormone therapy use, the following were independent variables associated with postoperative UTI: premenopausal status with an adjusted odds ratio (OR) of 1.80 (95% CI 1.11-2.99), anterior vaginal wall prolapse with an adjusted OR of 4.39 (95% CI 2.77-6.97), and postvoid residual greater than 150 mL with an adjusted OR of 2.38 (95% CI 1.12-4.36). Using this model, postoperative UTI rates ranged from 4.3% to 59.4% with high postvoid residual and presence of anterior prolapse having the strongest association., Conclusion: There are wide variations in the rate of UTI after hysterectomy for begin disease including pelvic reconstructive surgery. These variations can be explained with a model based on available preoperative data.

Scope: The study aims at identifying 1) the most sensitive compartment among plasma phospholipids, erythrocytes, and LDL for studying alpha-linolenic acid (ALA) conversion, and 2) whether ALA incorporation and conversion is saturable after administration of 13 C-labeled ALA-rich linseed oil (LO). The effect of a daily intake of 7 g nonlabeled LO (>43% w/w ALA) for 1 month after bolus administration of 7 g 13 C-labeled LO on day 1, and for 2 months after bolus administration of 7 g 13 C-labeled LO on day 1 and day 29 on 13 C-ALA incorporation and conversion into its higher homologs is investigated in healthy volunteers., Methods and Results: Incorporation and conversion of LO-derived 13 C-labeled ALA is quantified by applying compartmental modeling. After bolus administration, a fractional conversion of approximately 30% from 13 C-ALA to 13 C-DHA is calculated as reflected by the LDL compartment. Treatment with LO for 8 weeks induces a mean reduction of 13 C-ALA conversion to 13 C-DHA by 48% as reflected by the LDL compartment, and a mean reduction of the 13 C-ALA incorporation into LDL by 46%., Conclusion: A 2-month dietary intake of a high dose of LO is sufficient to reach saturation of ALA incorporation into LDL particles, which are responsible for ALA distribution in the body., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Study Objective: To compare outcomes of vaginal hysterectomy (VH) and robotic-assisted hysterectomy (RH) among women with conditions perceived as contraindications to VH (uterine size ≥ 12 weeks' gestation, no vaginal parity, prior cesarean delivery, and obesity)., Design: Retrospective chart review (Canadian Task Force classification II-2)., Setting: Tertiary US medical center., Patients: Women with VH or RH. Women with conditions perceived as contraindications affecting surgical choice were excluded., Interventions: VH or RH for benign uterine disease at our institution during 2009 through 2013., Measurements and Main Results: Among women with the perceived contraindications, a logistic regression model was fit to compare each binary outcome between VH and RH. Models were weighted using inverse probability of treatment weights derived from propensity scores to adjust for covariate imbalance between procedures. The cohort had 692 VHs and 472 RHs. Among 160 women with uterine size ≥ 12 weeks' gestation, RH patients were less likely to have uterine debulking (adjusted odds ratio [aOR], .37; 95% confidence interval [CI], .15-.95]) than VH patients and more likely to have accordion grade ≥ 2 postoperative complications (aOR, 7.20; 95% CI, 1.46-35.42) and readmission (aOR, 15.55; 95% CI. .85-285.20). Among 272 women with prior cesarean section, RH patients were more likely to have grade ≥ 2 postoperative complications (aOR, 2.85; 95% CI, 1.29-6.30). No outcomes were significantly different between surgical routes among women with no vaginal parity or obesity. Mean operative time was significantly longer for RH., Conclusion: VH is a surgical option for patients with the conditions perceived as contraindications for vaginal surgery evaluated herein., (Copyright © 2017 American Association of Gynecologic Laparoscopists. Published by Elsevier Inc. All rights reserved.)

Objectives: Rectovaginal fistulae (RVF) often represent surgical challenges, and treatment must be individualized. We describe outcomes after primary surgical repair stratified by fistula etiology and surgical approach., Methods: This retrospective cohort study included women who underwent surgical management of RVF at a tertiary care center between July 1, 2001 and December 31, 2013. Cases were stratified according to the following etiology: cancer (RVF-C), inflammatory bowel disease or infectious (RVF-I), and other (RVF-O). Patients with prior surgical treatment of RVF were excluded. Surgical approaches included local (seton, plug), transvaginal or endorectal, abdominal, diversion alone, or definitive (completion proctocolectomy with permanent colostomy or pelvic exenteration). Recurrence-free survival was estimated using the Kaplan-Meier method, and comparisons between subgroups were evaluated based on fitting Cox proportional hazards models. Censoring occurred at last relevant clinical follow-up. Factors contributing to recurrence-free survival were evaluated including age, body mass index, smoking status, fistula etiology, ileostomy, and surgical approach., Results: During the study period, 107 women underwent surgical repair of RVF. The most common fistula etiology was RVF-I (54.2%), followed by RVF-O (23.4%), and RVF-C (22.4%). Ninety-four women underwent fistula repair by the local (29.9%), transvaginal/endorectal (25.2%), abdominal approach (19.6%), or diversion alone (13.1%), whereas 13 underwent definitive surgery (12.2%). Recurrence-free survival was significantly different depending on surgical approach (P < 0.001), but not etiology (P = 0.71). Recurrence-free survival (95% confidence interval) at 1 year after surgery was 35.2% (21.8%-56.9%) for the local approach, 55.6% (37.0%-83.3%) for the transvaginal or endorectal approach, 95% (85.9%-100%) for the abdominal approach, and 33.3% (15%-74.2%) for those with diversion only., Conclusions: Recurrence rates after RVF repair are high and did not differ by fistula etiology. Abdominal repair of RVF had significantly fewer recurrences.

Objectives: This study evaluated our experience after implementing a pelvic floor rehabilitation program including behavioral modification, biofeedback, and vaginal electrogalvanic stimulation (EGS)., Methods: This prospective cohort study evaluated outcomes of patients with pelvic floor dysfunction (urinary or defecatory dysfunction, pelvic pain/dyspareunia) who underwent pelvic floor rehabilitation. Patients received 4 to 7 sessions (1 every 2 weeks) including biofeedback and concluded with 30 minutes of vaginal EGS. Surveys assessed subjective changes in symptoms; success was evaluated using a 10-point visual analog scale (VAS) at the final session (10 = most successful). Paired comparisons of responses at baseline and final treatment were evaluated., Results: Ninety-four patients were followed up through therapy completion. Treatment indications included urinary (89.4%), defecatory (33.0%), and pelvic pain or dyspareunia (30.9%); 44.7% of patients had a combination of indications. Among women with urinary symptoms, the percentage reporting leakage decreased from 92.9% to 79.3% (P = 0.001), leakage at least daily decreased from 69.0% to 39.5% (P < 0.001), daily urgency with leakage decreased from 42.7% to 19.5% (P = 0.001), daily urgency without leakage decreased from 41.5% to 18.3% (P < 0.001), and median VAS rating (0 = not at all, 10 = a great deal) of daily life interference decreased from 5 to 1.5 (P < 0.001). The median success ratings were 8, 8, and 7 for treatment of urinary symptoms, pelvic pain/dyspareunia, and bowel symptoms, respectively., Conclusions: An aggressive pelvic rehabilitation program including biofeedback with vaginal EGS had a high rate of self-reported subjective success and satisfaction and should be considered a nonsurgical treatment option in patients with pelvic floor dysfunction.

Objective: To evaluate practice change after initiation of a robotic surgery program using a clinical algorithm to determine the optimal surgical approach to benign hysterectomy., Methods: A retrospective postrobot cohort of benign hysterectomies (2009-2013) was identified and the expected surgical route was determined from an algorithm using vaginal access and uterine size as decision tree branches. We excluded the laparoscopic hysterectomy route. A prerobot cohort (2004-2005) was used to evaluate a practice change after the addition of robotic technology (2007). Costs were estimated., Results: Cohorts were similar in regard to uterine size, vaginal parity, and prior laparotomy history. In the prerobot cohort (n=473), 320 hysterectomies (67.7%) were performed vaginally and 153 (32.3%) through laparotomy with 15.1% (46/305) performed abdominally when the algorithm specified vaginal hysterectomy. In the postrobot cohort (n=1,198), 672 hysterectomies (56.1%) were vaginal; 390 (32.6%) robot-assisted; and 136 (11.4%) abdominal. Of 743 procedures, 38 (5.1%) involved laparotomy and 154 (20.7%) involved robotic technique when a vaginal approach was expected. Robotic hysterectomies had longer operations (141 compared with 59 minutes, P<.001) and higher rates of surgical site infection (4.7% compared with 0.2%, P<.001) and urinary tract infection (8.1% compared with 4.1%, P=.05) but no difference in major complications (P=.27) or readmissions (P=.27) compared with vaginal hysterectomy. Algorithm conformance would have saved an estimated $800,000 in hospital costs over 5 years., Conclusion: When a decision tree algorithm indicated vaginal hysterectomy as the route of choice, vaginal hysterectomy was associated with shorter operative times, lower infection rate, and lower cost. Vaginal hysterectomy should be the route of choice when feasible.

Management of the constricted or obliterated vagina demands an understanding and recognition of the potential etiologies leading to this presentation. A thorough and comprehensive medical and surgical review is required to arrive at an accurate diagnosis, which then will guide medical or surgical intervention. It is paramount to recognize when underlying medical conditions are contributing to these conditions and to begin medical therapy; failure to do so will often yield suboptimal results. When these conditions arise after surgical interventions, compensatory surgical techniques that correct upper and lower vaginal strictures or obliteration include incision through the stricture, vaginal advancement, Z-plasty, skin grafts, perineal flaps, and abdominal flaps and grafts. Postoperative surveillance and dilation are critical to optimize long-term success.

Introduction and Hypothesis: The objective was to demonstrate a surgical technique for the management of a small vesicovaginal fistula (VVF) involving a combination of cystoscopic holmium laser ablation and vaginal repair., Methods: A 55-year-old morbidly obese female presented with complaints of menometrorrhagia and complex adnexal mass. She underwent an attempted robotic hysterectomy, which was converted to open hysterectomy, omentectomy, and lymphadenectomy owing to an intraoperative diagnosis of endometrioid carcinoma of the endometrium and dense pelvic adhesions. Postoperatively, the patient developed intermittent urinary leakage associated with position change. On evaluation, a speculum examination did not reveal any fistulous tract or leakage of fluid in the vagina. A tampon test was positive, but no evidence of a fistula was noted on a CT urogram. Cystourethroscopy was performed and identified a small VVF. The patient subsequently underwent repair of her VVF using a combination of cystoscopic holmium laser ablation and transvaginal excision of the suspected fistula opening., Results: About 2 weeks after the surgery, a tampon test was negative and cystourethroscopy revealed healing bladder mucosa. The patient remains fistula-free at 12 months post-operatively., Conclusion: Holmium laser ablation combined with partial vaginal excision may be considered as a management option for a small VVF.

Introduction and Hypothesis: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome includes vaginal agenesis with varied uterine development. The objective of this video is to illustrate our surgical technique to create a cervical and vaginal canal to relieve menstrual obstruction for a teenager with a functional uterus and vaginal agenesis., Methods: Using vaginal dissection and a mini laparotomy, a sound placed through the fundus of the uterus created an endocervical and vaginal channel to relieve her menstrual obstruction. A Foley catheter stented the cervical canal and a red rubber chest tube catheter stented the vagina until epithelization was achieved., Results: No complications were encountered. The patient was examined with intermittent hysteroscopy with gentle dilation of the cervix. She had the red rubber catheter removed at 3 months, and she started using a small dilator. Her menses were suppressed with a gonadotropin releasing-hormone agonist allowing for complete healing. She is now 17. Her vaginal canal is well-epithelialized. Hysteroscopy confirmed a patent endocervical canal and uterine cavity., Conclusion: MRKH is rare. A small percentage of affected women has a functional endometrium requiring intervention for menstrual obstruction. Full vaginal reconstruction may be considered, but creation of a small canal to provide menstrual relief can be a temporary solution in those not desiring sexual function.

Milk proteins and milk protein aggregates are among the most important nanovehicles in food technology. Milk proteins have various functional properties that facilitate their ability to carry hydrophobic nutraceutical substances. The main functional transport properties that were examined in the reviewed studies are binding of molecules or ions, surface activity, aggregation, gelation, and interaction with other polymers. Hydrophobic binding has been investigated using caseins and isolated β-casein as well as whey proteins. Surface activity of caseins has been used to create emulsion-based carrier systems. Furthermore, caseins are able to self-assemble into micelles, which can incorporate molecules. Gelation and interaction with other polymers can be used to encapsulate molecules into protein networks. The release of transported substances mainly depends on pH and swelling behavior of the proteins. The targeted use of nanocarrier systems requires specific knowledge about the binding mechanisms between the proteins and the carried substances in a certain food matrix., (© 2015 Institute of Food Technologists®)

The sn position of fatty acids in seed oil lipids affects physiological function in pharmaceutical and dietary applications. In this study the composition of acyl-chain substituents in the sn positions of glycerol backbones in triacylglycerols (TAG) have been compared. TAG from native and transgenic medium-chain fatty acid-enriched rape seed oil were analyzed by reversed-phase high performance liquid chromatography coupled with online atmospheric-pressure chemical ionization ion-trap mass spectrometry. The transformation of summer rape with thioesterase and 3-ketoacyl-[ACP]-synthase genes of Cuphea lanceolata led to increased expression of 1.5% (w/w) caprylic acid (8:0), 6.7% (w/w) capric acid (10:0), 0.9% (w/w) lauric acid (12:0), and 0.2% (w/w) myristic acid (14:0). In contrast, linoleic (18:2n6) and alpha-linolenic acid (18:3n3) levels decreased compared with the original seed oil. The TAG sn position distribution of fatty acids was also modified. The original oil included eleven unique TAG species whereas the transgenic oil contained sixty. Twenty species were common to both oils. The transgenic oil included trioctadecenoyl-glycerol (18:1/18:1/18:1) and trioctadecatrienoyl-glycerol (18:3/18:3/18:3) whereas the native oil included only the latter. The transgenic TAG were dominated by combinations of caprylic, capric, lauric, myrisitic, palmitic (16:0), stearic (18:0), oleic (18:1n9), linoleic, arachidic (20:0), behenic (22:0), and lignoceric acids (24:0), which accounted for 52% of the total fat. In the original TAG palmitic, stearic, oleic, and linoleic acids accounted for 50% of the total fat. Medium-chain triacylglycerols with capric and lauric acids combined with stearic, oleic, linoleic, alpha-linolenic, arachidic, and gondoic acids (20:1n9) accounted for 25% of the transgenic oil. The medium-chain fatty acids were mainly integrated into the sn-1/3 position combined with the essential linoleic and alpha-linolenic acids at the sn-2 position. Eight species contained caprylic, capric, and lauric acids in the sn-2 position. The appearance of new TAG in the transgenic oil illustrates the extensive effect of genetic modification on fat metabolism by transformed plants and offers interesting possibilities for improved enteral applications.

We report the induction of laughter and smiling by cortical electrical stimulation of the frontal lobe of two patients: an 18-month-old boy with a left frontal cortical lesion extending to the vertex and the central gyrus, and a 35-year-old woman with a lesion in the right supplementary sensorimotor area (SSMA). The subjects underwent presurgical epilepsy evaluation with subdural grid electrodes to determine surgical candidacy. Stimulation of the prefrontal area reproducibly induced laughter. The adult patient reported absence of emotional content. Slowing of speech occurred under stimulation of electrodes in the upper and posterior vicinity. In this patient laughter was elicited in the anterior part of the SSMA. In the child, this response was induced by stimulation of the lateral prefrontal cortex near the midline. We conclude that the anterior portion of the SSMA/lateral premotor cortex is involved in generating the motor pattern of laughter.

The sn-position of FA in membrane lipids has an influence on the physiological function of cells, is predictive for diseases, and therefore is useful for diagnostics. The current study compares the compositions of acyl chain substituents in the sn-1 and sn-2 positions of the glycerol backbones of phospholipids derived from human erythrocytes by using RP-HPLC coupled with on-line electrospray ionization ion trap MS. Preferential loss of the acyl group in the sn-1 position was used to determine the degree of regiospecific preference exhibited by the phospholipid molecules. The identities of the molecular species and the positions of the acyl substituents were identified using product-ion spectra of major precursor ions selected from the mass spectra averaged across peaks in the total ion chromatogram. Saturated FA were found to be located mainly in the sn-1 position of the glycerol backbones of erythrocyte phospholipids, whereas PUFA were found primarily in the sn-2 position. All measured phospholipids revealed palmitic acid (16:0) at the sn-1 position. Linoleic acid (18:2n-6) and arachidonic acid (20:4n-6) were found to be attached exclusively to the sn-2 position of the backbone, whereas eicosadienoic (20:2n-6) and eicosatrienoic acid (20:3n-9) occurred in both positions of the backbone of PC. Oleic (18:1n-9), linoleic (18:2n-6), and octadecatrienoic (18:3) acids of PE and PS were linked to both positions. Lignoceric acid (24:1 n-9) was found to be strictly localized at the sn-2 position, whereas nervonic (24:1n-9) acid of PS was associated with both positions of the backbone. A detailed analysis of the blood cell membrane lipids by MS might be helpful to characterize postprandial kinetics of pharmacological or dietary lipid applications, as well as environmental influences on cell membranes.

We report the occurrence of subacute sclerosing panencephalitis (SSPE) in two brothers two years after measles infection. The diagnosis was confirmed by compatible data from medical history, occurrence of autochthonic measles virus (MV) IgG production in the central nervous system (CNS), and pathognomonic EEG changes. Pathogenetically, SSPE is caused by a genome mutation of intracellularly persisting MV, causing viral nucleocapsides to accumulate in the brain cells. A specific predisposing immune defect is not known. The occurrence of two cases in one family is suggestive of a genetic predisposing factor.

Epigastric sensations are the most frequent type of aura in medial temporal lobe epilepsy. Until now, the site of the symptomatogenic zone of the epigastric aura remains controversial. The temporal lobe has been discussed, as well as the insular cortex. The case presented here supports the assumption of an insular origin of the epigastric aura. A 44-year-old male suffered from typical psychomotor seizures preceded by an epigastric aura. Imaging revealed a cavernoma located in the right insular region, the ipsilateral temporal lobe being otherwise unremarkable. The lesion was surgically removed, leaving the temporal lobe untouched. Apart from one clinical episode that was unlike his habitual seizures, the patient remained free of seizures (including auras). We conclude that the insular cortex was most likely the symptomatogenic zone for this patient's epigastric auras. Although it can not be excluded that this patient's aura resulted from ictal activation of other brain structures, the successful lesionectomy is in favor of our hypothesis.

In this study 27 patients with right hemisphere lesions, 25 patients with left hemisphere lesions and 26 normal control subjects were investigated for unimodal and simultaneous multimodal recognition of emotional attitude. All subjects were shown 330 videotaped items of 4 seconds duration, each of which was to be judged in terms of facial expression, emotional prosody and the emotional meaning of the underlying spoken sentence. In a preceding experiment comparable unimodal emotional stimuli were applied. The results suggest (a) right hemisphere superiority for recognition of emotions conveyed by facial and prosodic information, (b) a right hemisphere dominance for the recognition of fear and (c) no significant enhancement of right hemisphere superiority under multimodal presentation of emotional stimuli.

The use of a disposable affinity column and low-melting-temperature agarose for the quantitative preparation of DNA restriction fragments is presented. After electrophoretic separation of DNA, the band(s) are excised and the DNA/agarose melted in a low-salt buffer. After cooling, the DNA is bound to an Elutip-d affinity column. Fragments are eluted at high salt and concentrated by ethanol precipitation. Recoveries greater than 80% are achieved with purity suitable for most applications in molecular biology.

A variety of modified electrofusion protocols designed to improve the efficiency of hybridoma production have recently appeared in the literature. We undertook to maximize the number of antibody secreting murine hybridomas by optimizing the temperature and fusion strength parameters of the conventional electrofusion technique. Anti-DNP secreting hybridomas were generated by fusing SP2/0 to immunized mouse splenic lymphocytes using an unmodified electrofusion protocol consisting of washing in a weakly conducting sorbitol fusion medium supplemented with bovine serum albumin, calcium and magnesium ions. This was followed by dielectrophoretic alignment and application of 3 short duration, high intensity field pulses in helical chambers. Optimal efficiencies of hybridomas were generated by the application of 2000 V/cm pulses at 25 degrees C (2.45 hybridomas x 10(-4) splenocytes) and as many as 63% of resulting hybridomas secreted anti-DNP monoclonal antibodies, the majority of which were IgG's. These data show that modification of the electrofusion protocol by pretreatment of fusion partners with proteolytic enzymes or the use of antigen bridging is not required for the successful and efficient production of specific monoclonal antibodies by electrofusion.

Electrofusion of mammalian cells in strongly hypo-osmolar media containing sorbitol, small amounts of divalent cations and albumin resulted in high yields of hybrids. The number of viable hybrids was higher than any value for chemically- or electrically-mediated fusion reported in the literature. Optimum clone numbers were obtained for fusion of osmotically-stable subclones of murine myeloma cells with DNP-Hy-stimulated lymphocytes provided that the osmolarity of the fusion medium was as low as 75 mosmol/l. Similar results were obtained for fusion of osmotically stable subclones of myeloma cells with the murine hybridoma cell line G8. Due to the dramatic increase in volume the field strength of the breakdown pulse (leading to fusion of the dielectrophoretically aligned cells) has to be reduced, as predicted by theory. The efficacy of hypo-osmolar electrofusion allowed the use of very few cells (about 10(5) lymphocytes or G8 cells per fusion chamber). This figure is considerably smaller than that reported in the literature for iso-osmolar electrofusion. It is significant that, in contrast to iso-osmolar conditions, the fusion yield in hypo-osmolar electrofusion was reproducible over long periods of time and less dependent of variations between cultures. At suspension densities of about 10(6) cells per fusion chamber (normally used in iso-osmolar electrofusion) hypo-osmolar electrofusion of homogeneous cell suspensions resulted in the formation of many giant cells when the appropriate field conditions were applied. Similar high or, at some field strengths, even higher numbers of clones at low cell suspension density were obtained when G8 and myeloma cells were first exposed during the washing procedure to strongly hypo-osmolar media, but then transferred to iso-osmolar solutions for electrofusion. Similar experiments with lymphocytes and myeloma cells failed because of destruction of many lymphocytes by the two osmotic shock steps in rapid succession. Volume distribution measurements of G8 and myeloma cells showed that after re-incubation of the osmotically pre-stressed cells the original volume distribution is largely, but not completely re-established. This and other results indicate that osmotic pressure gradients and associated tensions in the membrane do not play a primary role in the initiation of the electrofusion process. The experiments suggest that due to the osmotic (pre-) stress the membrane permeability is slightly and uniformly increased presumably due to the dissolution of membrane- and cell-skeleton proteins. Obviously, this facilitates electrofusion in hypo-osmolar or subsequently in iso-osmolar solutions.

The psbA gene from higher plants, which codes for the atrazine herbicide binding protein of photosystem II (QB protein), has been recently sequenced by various laboratories. From these data there are two potential translation sites, one yielding a protein of 38,500 kd and another a protein of 34,500 kd. In the present study, cloned psbA gene sequences from maize, tobacco, and pea have been expressed in a highly defined E. coli in vitro transcription/translation system. In order to determine the start site of translation, we also have employed a simplified E. coli system designed to synthesize the first di- or tripeptide of the gene product. From these results, it is clear that the first ATG of the longest open reading frame of the psbA gene, that begins fMet-Thr, is not recognized in vitro. Instead, the next downstream Met at position 37 is the initiation site, since the expected dipeptide fMet-Ile is synthesized from all psbA clones. These data are in accord with the in vivo results that the gene product is a precursor protein of 34,500 kd.