Comparative selective retention of particle size classes in the gastrointestinal tract of ponies and goats.

There is a discrepancy in the literature on potential digesta separation mechanisms in horses, with both a selective retention of fine and of large particles postulated in different publications. To assess the net effect of such mechanisms, we fed ponies on a hay-only diet a pulse dose of whole (unchopped) marked hay together with a solute marker, collected faeces on a regular basis, measured marker concentrations in whole faeces and in their large (2.0-16 mm), medium (0.5-1.0 mm) and small (0.063-0.25 mm) particle fraction, and calculated the corresponding mean retention times (MRTs). For comparison, the same experiment was performed in goats. In goats, as expected, MRTsolute (35 hr) was significantly shorter than MRTparticle (51 hr); only a very small fraction of particle marker was excreted as large particles (2%); and the MRT of these large particles was significantly shorter than that of small particles (with a relevant difference of 8.6 hr), indicating that those few large particles that escape the rumen do so mostly soon after ingestion. In ponies, MRTsolute (24 hr) did not differ from MRTparticle (24 hr); a higher fraction of particle marker was excreted as large particles (5%); and the MRT of these large particles was longer than that of small particles (but with a non-relevant difference of less than 1 hr). These results indicate that no relevant net separation of digesta phases occurs in horses and that selective particle retention mechanisms in the large intestine are unlikely to represent important characteristics of the horse's digestive physiology.

Effects of morcellation on long-term outcomes in patients with uterine leiomyosarcoma.

OBJECTIVES: Clinical long-term outcomes of women with uterine leiomyosarcoma (ULMS) with different types of hysterectomy (open abdominal, vaginal, laparoscopic and switch from laparoscopic to open abdominal) were compared according to morcellation and other factors. MATERIALS: The clinical cancer registry Regensburg (Germany) registered 64 patients between 2004 and 2013 with ULMS. A retrospective cohort analysis was performed using the Kaplan-Meier method to estimate 5-year overall survival (OAS), recurrence-free survival (RFS) and recurrence rates. To compare surgery with or without morcellation log rank test was used. To adjust for age, FIGO stage, grading and other factors multivariable Cox regression models were applied to estimate hazard ratios (HR). RESULTS: In the cohort of 64 patients 15 underwent morcellation, preferably during laparoscopic surgery. Although numbers were small we performed analysis for OAS and RFS. Median OAS for morcellation was 10.6 vs. 6.4 years for non morcellation. 5y-OAS was 76.0 % for morcellation compared to 54.8 % in patients without morcellation (p = 0.115). Cox regression models rendered an unadjusted (univariable) HR 0.428 for morcellation vs. non-morcellation (p = 0.125) and an adjusted (multivariable) HR 0.644 (p = 0.406). 5y-RFR was 64.0 % compared to 42.8 % in patients without morcellation (p = 0.104; unadjusted HR 0.484, p = 0.111; adjusted HR 0.607, p = 0.306). CONCLUSION: In general, the prognosis of patients with ULMS is poor. In our cohort, women who underwent hysterectomy with morcellation had a better cumulative OAS and RFS than women without morcellation. Although we adjusted for differences between women with and without morcellation regarding age, grading and stage, there were no statistically significant differences between the groups.

Short communication: milk output in llamas (Lama glama) in relation to energy intake and water turnover measured by an isotope dilution technique.

Despite the fact that llamas have become increasingly popular as companion and farm animals in both Europe and North America, scientific knowledge on their nutrient requirements is scarce. Compared with other livestock species, relatively little is known especially about the nutrient and energy requirements for lactating llamas. Therefore, we aimed to measure milk output in llama dams using an isotope dilution technique and relate it to energy intakes at different stages of lactation. We also validated the dilution technique by measuring total water turnover (TWT) directly and comparing it with values estimated by the isotope dilution technique. Our study involved 5 lactating llama dams and their suckling young. Milk output and TWT were measured at 4 stages of lactation (wk 3, 10, 18, and 26 postpartum). The method involved the application of the stable hydrogen isotope deuterium ((2)H) to the lactating dam. Drinking water intake and TWT decreased significantly with lactation stage, whether estimated by the isotope dilution technique or calculated from drinking water and water ingested from feeds. In contrast, lactation stage had no effect on dry matter intake, metabolizable energy (ME) intake, or the milk water fraction (i.e., the ratio between milk water excreted and TWT). The ratios between TWT measured and TWT estimated (by isotope dilution) did not differ with lactation stage and were close to 100% in all measurement weeks, indicating that the D(2)O dilution technique estimated TWT with high accuracy and only small variations. Calculating the required ME intakes for lactation from milk output data and gross energy content of milk revealed that, with increasing lactation stage, ME requirements per day for lactation decreased but remained constant per kilogram of milk output. Total measured ME intakes at different stages of lactation were similar to calculated ME intakes from published recommendation models for llamas.

Effects of a higher incubation temperature between embryonic day 9 and 12 on growth and meat quality characteristics of turkeys.

1. The study investigated the influence of manipulating incubation temperature for a short period on the post-hatch development up to week 16 in male and female BUT Big 6 turkeys. 2. Eggs were incubated at a control temperature of 37·5°C and 55% RH until d 18 when transferred to a hatcher at 37·5°C and 85% RH. For a 4 d period between embryonic day 9 (ED 9) and 12, eggs were incubated at 38·5°C and 55% RH (HT). 3. Birds were slaughtered at 16 weeks of age to analyse meat quality parameters of the Musculus pectoralis superficialis (MPS). 4. Across both incubation treatments, the turkey males had significantly higher live and breast weights, but lower breast yields than the females. The sex of the animals only influenced the yellowness of the MPS with lower values in the males. 5. Temperature manipulation resulted in significantly decreased live weights of HT birds compared with the control animals across all ages in both sexes. No impact of incubation treatment on meat quality characteristics was found. 6. The results indicate a negative effect of higher incubation temperature on the post-hatch growth, possibly by influencing the mechanisms that regulate the hypertrophic growth of the muscle fibres.

Predictive preoperative clinical score for patients with liver-only oligometastatic colorectal cancer.

BACKGROUND: Resection of liver metastases from colorectal cancer (CRC) in the oligometastatic stage improves survival and is a potentially curative treatment. Thus, predictive scores that reliably identify those patients who especially benefit from surgery are essential. PATIENTS AND METHODS: In this multicenter analysis, 512 patients had undergone surgery for liver metastases from CRC. We investigated distinct cancer-specific risk factors that are routinely available in clinical practice and developed a predictive preoperative score using a training cohort (TC), which was thereafter tested in a validation cohort (VC). RESULTS: Inflammatory response to the tumor, a right-sided primary tumor, multiple liver metastases, and node-positive primary tumor were significant adverse variables for overall survival (OS). Patients were stratified in five groups according to the cumulative score given by the presence of these risk factors. Median OS for patients without risk factors was 133.8 months [95% confidence interval (CI) 81.2-not reached (nr)] in the TC and was not reached in the VC. OS decreased significantly for each subsequent group with increasing number of risk factors. Median OS was significantly shorter (P < 0.0001) for patients presenting all four risk factors: 14.3 months (95% CI 10.5 months-nr) in the TC and 16.6 months (95% CI 14.6 months-nr) in the VC. CONCLUSIONS: Including easily obtainable variables, this preoperative score identifies oligometastatic CRC patients with prolonged survival rates that may be cured, and harbors potential to be implemented in daily clinical practice.

Intermediate high index layer for laser mode tuning in organic semiconductor lasers.

We modified the optical properties of organic semiconductor distributed feedback lasers by introducing a high refractive index layer consisting of tantalum pentoxide between the substrate and the active material layer. A thin film of tris-(8-hydroxyquinoline) aluminium doped with the laser dye 4-dicyanomethylene-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran was used as the active layer. By varying the intermediate layer thickness we could change the effective refractive index of the guided laser mode and thus the laser wavelength. With this technique we were able to tune the laser emission range between 613 nm and 667 nm. For high index layer thicknesses higher than 40 nm the laser operated on the TE(1)-mode rather than the fundamental TE(0)-mode.

Adaptation capacity of Boer goats to saline drinking water.

Due to global climatic changes, water and soil salinization is an increasing worldwide phenomenon, thus creating new threats for farm animal production. The present study was designed to investigate the adaptation capacity of goats towards sodium chloride (NaCl) in drinking water. Twelve non-pregnant Boer goats with an average body weight of 50.5 ± 9.0 kg were kept in individual pens. The study was conducted in four phases applying a two-choice preference test. In the control phase (phase 1) only fresh water was supplied in two containers. In phase 2, water with different salt concentrations (0.25%, 0.5%, 0.75%, 1.0%, 1.25% and 1.5%) was offered in one container and tap water in the other (sensitivity test). During the third phase (adaptation), goats were stepwise habituated to saline water by offering only saline water with different increasing concentrations (between 0% and 1.5% NaCl) in both containers. Subsequently, in phase 4 (sensitivity re-test) the same treatment as in phase 2 was repeated. Goats had ad libitum access to hay, water and a mineral licking block. Individual water and feed intake were recorded daily, while body weight and body condition score were measured every 2nd week. Body weight was not affected by saline water intake, whereas dry matter intake and body condition scores decreased significantly during the experiment. Water intake was significantly (P<0.001) higher in phase 2 (sensitivity test) and phase 3 (adaptation), compared to phase 1 (control) and phase 4 (sensitivity re-test). Total sodium intake followed the same pattern. In phase 2, when goats had the choice between fresh and saline water for the first time they preferred higher salt concentrations and consumed significantly (P<0.001) higher amounts of saline water (75.4 ± 53.2 g/kg BW0.82 per day) than in the re-test (40.4 ± 34.0 g/kg BW0.82 per day) after the habituation period. Thus, salt discrimination rejection thresholds were lowered to 1.25% in phase 4 compared to 1.5% in phase 2. The results suggest that a stepwise adaptation to saline drinking water in goats is an effective method to habituate the animals to saline water intake when concentrations were below 1.5%. Goats reacted more sensitively to the salinity of drinking water after prolonged exposure to saline water indicating flexible regulation mechanisms depending on the total sodium balance of the animal.

Reactions to saline drinking water in Boer goats in a free-choice system.

Salinization of groundwater and soil is a prevalent global issue with serious consequences on animal health and production. The present study was conducted to investigate the capacity of Boer goats to adjust their salt intake from saline drinking water in a free-choice system. In total, 12 non-pregnant Boer goats aged between 1 and 8 years with an average BW of 46.4±8.3 kg were kept in individual pens for 4 weeks. In the control phase (1 week), only fresh water was supplied in five identical buckets for each pen. During the subsequent treatment phase (3 weeks), fresh water and four different concentrations (0.75, 1.0, 1.25 and 1.5% NaCl) of saline water were offered simultaneously in a free-choice system. The positions of the concentrations were changed daily at random. Cut hay and water were provided ad libitum, and a mineral supplement was allocated. Feed and water intake, mineral supplement intake, ambient temperature and relative humidity were recorded daily, whereas BW and body condition score were measured weekly. Dry matter intake, total water intake and total sodium intake were significantly (P<0.001) higher during the treatment phase. Body weight and body condition were not affected by saline water intake. Across the treatment phase, saline water consumption was significantly (P<0.001) lower in young (19.6±27.1 g/kg BW0.82 per day) than in adult goats (27.9±31.5 g/kg BW0.82 per day), indicating that young goats were more sensitive towards the saline water. All goats had a significant preference for fresh water (0% salt) over saline water. At the first offering of the simultaneous choice situation (week 2), animals did not differentiate between the salt concentration of 0.75% and 1.0%. However, with successive treatment (weeks 3 and 4), animals distinguished between saline water concentrations and preferred the 0.75% salt concentration. Salt concentrations of 1% to 1.5% were avoided. The total sodium intake of the goats ranged between 0.37 and 0.55 g /kg BW0.75 per day during the treatment phase, being 8- to 11-fold higher than the daily requirements of sodium for body maintenance. The results suggest that goats are able to differentiate between saline water concentrations and to adjust their sodium intake by quick adjustments in self-selection in a free-choice system. Compared with two-choice preference tests, the present free-choice situation allows evaluating changes in saline water acceptance with prolonged exposure.

Deuterium for estimating total body water and turnover rates in turkeys exposed to different incubation treatments.

Total water intake (TWI) in poultry can be influenced by various factors. Recommendations for water requirements are usually reported on a flock basis without considering individual variation. In the present study, a total of 18 turkeys were used to measure water intake over a 1-wk period starting at 15 wk of age by applying the deuterium dilution technique. Poults originated from eggs exposed to different incubation treatments, with eggs incubated at normal temperature (37.5 degrees C) and eggs subjected to 38.5 degrees C at embryonic d 9 to 12. Experimental birds were kept in flocks of 22 to 30 birds separated by sex and treatment. Feed and water were provided ad libitum. Incubation treatment had no significant effect on any of the parameters investigated (BW, daily gain, water turnover rate, total body water, TWI), whereas sex exerted a significant effect on nearly all traits. Total body water ranged between 60 and 65% of BW, with significantly (P < 0.05) greater values for toms (63.2%) than for hens (60.9%). Males had approximately 30% greater water influxes than females (1,054 +/- 198 vs. 742 +/- 153 mL/d, mean +/- SD). However, the significant influence of sex was eliminated (P = 0.464) when TWI was expressed as grams per kilogram of BW (76 +/- 18 vs. 70 +/- 12 mL/kg of BW; males vs. females). Water consumed averaged 837 mL in male and 569 mL per day in female birds. The present results suggest that the isotope dilution method offers a viable method to measure individual water intake, which can be used for establishing reference values for water consumption in group-housed turkeys.

Long-term adaptation capacity of ponies: effect of season and feed restriction on blood and physiological parameters.

Domesticated horses are increasingly kept under semi-natural housing conditions, whereas their adaptation capacity is not fully investigated. In all, 10 Shetland pony mares were held under semi-extensive conditions for 1 year. In winter animals were allocated into two feeding groups (60% and 100% of maintenance energy requirement, respectively). Triiodothyronine, thyroxine, non-esterified fatty acids (NEFA), total bilirubin, total protein, triglyceride, glucose, insulin and hair length were measured at monthly intervals, whereas BW, body condition score, cresty neck score and resting heart rate were recorded every 2 weeks. From summer to winter all Ponies showed a reduction in resting heart rate (P0.05). Refeeding of restrictively fed ponies resulted in a rapid increase in resting heart rate and BW and a return of blood parameters to reference values. Adequately supplied animals adapted without difficulty to varying environmental conditions, whereas feed restriction in ponies during winter resulted in reduced resting heart rates suggesting a reduced basal metabolic rate. The energy restriction was compensated by mobilizing body fat reserves which led to changes in blood parameters. Refeeding in feed restricted animals revealed a remarkably quick recovery of physiological and blood parameters to reference values. We therefore suggest that year round-outdoor housing can be a suitable housing system for robust horse breeds provided that an adequate food supply is available.

[Treatment strategy for early stage rectal cancer (T1 carcinoma)]. / Therapeutische Strategie bei Frühkarzinomen im Rektum (T1­Karzinom).

BACKROUND: A common consensus for the definition for early rectal cancer does not exist. This item is used in cases of histological findings including pTis, pT1 or pT2 tumors. The term early rectal cancer is not mentioned in the German S3 guidelines on colorectal cancer. The pTis tumors are located at the mucosa level of the intestinal wall and they have nearly no tendency to develop metastases but pT2 tumors have a high risk of local metastases; therefore, the term early rectal cancer is not adequate for pT2 tumors. OBJECTIVE: This focus of this article is exclusively on pT1 rectal cancer. Following the histological definition, pT1 tumors of the rectum are located at the level of the mucosa and submucosa of the intestinal wall. CONCLUSION: With respect to the nature of the tumor (e.g. size, grading, invasion of lymphatic and/or blood vessels, Kikuchi classification) local methods (endoscopic procedure, surgical techniques) or radical resections are recommended. Tumor budding is of increasing interest and importance. Depending on the severity of the tumor budding classification (bd1-bd3) there is an association with a more frequent occurrence of lymph node metastases and should therefore be taken into consideration in treatment decisions in the future.

Measurement of milk intake in suckling llamas (Lama glama) using deuterium oxide dilution.

The objective of the study was to estimate daily milk intake in llama crias and relate nutrient intakes at peak lactation to growth data. Milk intake in 11 suckling llamas was estimated from water kinetics using deuterium oxide (D2O) at d 17, 66, and 128 postpartum. Daily milk intakes averaged 2.6, 2.3, and 2.0 kg at 17, 66, and 128 d postpartum, respectively. Milk intake decreased with age when expressed as daily amount, percentage of body weight (BW), or per kilogram of metabolic size, but the influence of age was eliminated when expressed per gram of daily gain. Because llamas only have one young per parturition, milk intake was equivalent to the daily milk output of the dam, which ranged from 27.6 to 96.9 g/kg of maternal BW(0.75). Compared with different ruminant species, milk production in llamas appears to lie between wild and domestic ruminants used for meat production. Nutrients (dry matter, fat, protein, and lactose) and energy intakes from the milk calculated by combining milk intake and milk composition data decreased with age when expressed as daily amount or per 100 g of BW, but when expressed per gram of daily gain, no clear trend was observed. Maintenance requirement for suckling llamas at peak lactation (17 d postpartum) was 312 kJ of ME/kg of BW(0.83). Combined with milk composition data, the present milk intake estimations at different stages of the lactation can be used to establish recommendations for nutrient and energy requirements of suckling llamas.

Measurements of the bodyweight and other physical characteristics of 11 llamas (Lama glama) from birth to weaning.

The bodyweight (bw), thoracic circumference, length and height of 11 llamas were recorded weekly from birth for 27 weeks. Their mean (sd) birth weight was 11.60 (2.35) kg and their weight increased in curvilinear fashion reaching 61.05 (13.75) kg after 27 weeks. Their average daily gains reached the highest value three weeks after birth and then decreased, reaching the lowest value of 262 g per day at 27 weeks. On a metabolic bw(0.83) basis, the growth of the llamas when their dams were at peak lactation was comparable with that of calves, fawns and lambs. All the measurements were significantly affected by the age, but not by the sex of the young or the parity of the dam. The regression models chosen to predict the bodyweight and weight gain of the llamas yielded R(2) ranging from 0.97 to 0.99. Correlations between bodyweight and other single body measurements were between r=0.68 and 0.77. Single or combined body measurements were good predictors of bodyweight, but additional body measurements added little to the already good fit provided by a single measurement (R(2) ranging from 0.93 to 0.98). The thoracic circumference (R(2)=0.98) and length (R(2)=0.96) were good predictors of the bodyweight of the llamas between birth and 27 weeks old.

Survival benefit of pelvic and paraaortic lymphadenectomy in high-grade endometrial carcinoma: a retrospective population-based cohort analysis.

OBJECTIVE: The standard therapy for high-grade endometrial cancer is surgery but the therapeutic effects of pelvic and paraaortic lymph node dissection (LND) are poorly investigated. In this study, we retrospectively evaluated overall survival, recurrence rates and recurrence-free survival among patients with high-grade type I and II endometrial carcinoma who underwent LND. METHODS: This study included 284 patients who are recorded in the German Tumor Centre Regensburg form 1998 to 2015 and were selected by cancer grading, the absence of secondary tumors, primary surgery including hysterectomy and available follow-up. 244 of the 284 patients in this cohort were unequivocally classified as R0 after resection. RESULTS: A significantly increased overall survival was observed for systematic LND of 25 or more paraaortic and pelvic lymph nodes versus patients who did not undergo such intervention (p < 0.001) or had elective LND of 1-24 lymph nodes both in univariable (p = 0.016) and multivariable (p = 0.014) analysis. A similar observation was made for recurrence-free survival of patients in the cohort who underwent complete tumor resection (R0). In addition, a reduced cumulative recurrence rate was observed for patients with systematic LND. CONCLUSIONS: Our study provides evidence that the systematic removal of 25 or more pelvic and paraaortic lymph nodes reduces the recurrence rate and that it is beneficial for the long-term overall and recurrence-free survival of patients with high-grade endometrial cancer.

Changes in Llama (Lama glama) milk composition during lactation.

Milk samples were collected weekly from 10 llamas during the first 27 wk after parturition under controlled stable conditions. Mean values for the concentrations of the major milk components across the lactation period were 4.70% fat, 4.23% protein, 5.93% lactose, 15.61% dry matter, and 22.62 mg/dL of milk urea N. All constituents were affected by the stage of lactation. There was an increase in fat to protein ratio as protein concentration declined and fat concentration increased. Fat, protein, and lactose concentrations changed during the transition from colostrum to milk. In the first month postpartum, fat concentration remained constant, protein decreased, and lactose increased. Starting with wk 5 postpartum, fat and protein increased and lactose decreased until the end of lactation. Among the major constituents fat had the highest variation. The mean gross energy concentration of milk was 3.88 kJ/g and showed a similar course as protein. Fat contributed 48.0%, protein 26.3%, and lactose 25.7% to the gross energy in the milk. Milk urea N values were higher than those found in ruminants and increased with stage of lactation, whereas the pH decreased. The analyzed milk components were not affected by the lactation number of the animal, except milk urea N. Somatic cell counts indicated the absence of mastitis and revealed that the average somatic cell count of uninfected llamas is lower than in animals usually used for milk production. The 2 algebraic models fitted by a nonlinear regression procedure to the data resulted in suitable prediction curves for the constituents (R2 = 0.76 to 0.94). The courses of major milk constituents in llamas during lactation are similar to those in domesticated ruminants, although different in their values. The established curves facilitate the composition of milk replacers at different stages of lactation for nursing llamas whose dams died or are agalactic.

Elucidation of the mechanism enabling tumor selective prodrug monotherapy.

Elucidation of the mechanism enabling tumor selective PMT in vivo with appropriate glucuronyl-spacer-doxorubicin prodrugs, such as HMR 1826, is important for the design of clinical studies, as well as for the development of more selective drugs. Enzyme histochemistry, immunohistochemistry, and the terminal deoxytransferase technique were applied using human cryopreserved cancer tissues, normal human, monkey, and mouse tissues, and human tumor xenografts to examine mechanisms underlying the selectivity of successful PMT with HMR 1826. It could unambiguously be shown by enzyme histochemistry that necrotic areas in human cancers are the sites in which lysosomal beta-glucuronidase is liberated extracellularly in high local concentrations. The cells responsible for the liberation of the enzyme are mainly acute and chronic inflammatory cells, as shown by IHC. Furthermore, it could be demonstrated that beta-glucuronidase liberated in necrotic areas of tumors can activate HMR 1826, resulting in increased doxorubicin deposition in human tumor xenografts or in human lung cancers subjected to extracorporal perfusion, compared to chemotherapy with doxorubicin. Additionally, the doxorubicin load to normal tissues was significantly reduced compared to chemotherapy with doxorubicin. Surprisingly, the increased doxorubicin deposition in tumors also resulted in strong antitumor effects also in cancers resistant to maximum tolerated doses of systemic doxorubicin. Finally, toxicity studies in mice and monkeys revealed an excellent tolerability of HMR 1826, up to a dose of 3 g/m2 (monkeys). These data suggest that HMR 1826 is a promising candidate for clinical development.

Diffusion behavior of gap junction hemichannels in living cells.

Due to its non-invasive character, fluorescence correlation spectroscopy (FCS) is particularly suited for the investigation of diffusion behavior of proteins in living cells. In this study we have investigated the diffusion properties of CFP-labeled gap junction hemichannels in the plasma membrane of living HeLa cells. Gap junction hemichannels or connexons are the precursors for the cell-cell- or gap junction channels that form large plaques at the contact areas between two adjacent cells. It has been proposed that new channels are recruited into a gap junction structure from a pool of hemichannels that can freely diffuse over the entire plasma membrane. The statistical approach shows that the geometry of the membrane within the focus is the most important property for the form of the autocorrelation curve and in turn for the determination of the diffusion coefficient. On the other hand binding-unbinding events which lead to anomalous diffusion have only a minor effect to the position and shape of the correlation curve compared to the geometry of the membrane.

Prodrugs of anthracyclines for use in antibody-directed enzyme prodrug therapy.

A series of new prodrugs of daunorubicin and doxorubicin which are candidates for antibody-directed enzyme prodrug therapy (ADEPT) is reported. These compounds (25a,b,c and 32a,b,c) have been designed to generate cytotoxic drugs after activation with beta-glucuronidase. As expected, recovery of the active drug was observed after enzymatic cleavage by Escherichia coli beta-glucuronidase as well as by a fusion protein which has been obtained from human beta-glucuronidase and humanized CEA-specific binding region. The six prodrugs are highly stable and are more than 100-fold less cytotoxic than doxorubicin against murine L1210 cell lines. The ortho-substituted phenyl carbamates 25a,b,c are better substrates for beta-glucuronidase than the corresponding para-substituted analogues. After taking into account additional factors such as stability in plasma and kinetics of enzymatic cleavage, we selected the o-nitro prodrug 25c for clinical trials.

Expression and function of beta-glucuronidase in pancreatic cancer: potential role in drug targeting.

Improvement of non-surgical strategies is a pivotal task in the treatment of pancreatic cancer. Response to treatment with most anticancer agents has been very poor, probably due to insufficient drug concentration in tumor tissue. Increased response rates during chemotherapy might be achieved by dose escalation; however, this approach is often hampered by severe side effects. One strategy to overcome these adverse effects is application of nontoxic glucuronide prodrugs from which the active moiety is released by beta-glucuronidase within or near the tumor. The use of glucuronide prodrugs in pancreatic cancer requires increased expression of the enzyme in the diseased tissue, a problem that has not been addressed so far. We therefore investigated function and expression of beta-glucuronidase in tissue samples from human healthy pancreas (n=7) and pancreatic adenocarcinoma (n=8), respectively. Comparing the ability of tissue homogenates to cleave the standard substrate 4-methylumbelliferyl-beta-D-glucuronide, we found a significantly increased specific beta-glucuronidase activity (P<0.05) in pancreatic cancer (median: 133; 75% percentile: 286; 25% percentile: 111 nmol/mg per h) as compared to healthy pancreas (median: 74; 75% percentile: 113; 25% percentile: 71 nmol/mg per h). Enzyme kinetic experiments with the model prodrug N-[4-beta-glucuronyl-3-nitrobenzyloxycarbonyl] doxorubicin (HMR 1826) demonstrated bioactivation of HMR 1826 by pancreatic beta-glucuronidase. Enzymatic activity was found to be closely related to enzyme contents (r=0.87) as assessed by Western blot analysis. Our data indicate that increased beta-glucuronidase activity in pancreatic cancer seems to be due to an elevated steady-state level of the protein. This may be the basis for new therapeutic strategies in treatment of pancreatic carcinoma by using glucuronide prodrugs of anticancer agents.

Synthesis of antigenic determinants of the Brucella A antigen, utilizing methyl 4-azido-4,6-dideoxy-alpha-D-mannopyranoside efficiently derived from D-mannose.

A strategy for the synthesis of Brucella O-antigenic determinants containing 2-linked 4,6-dideoxy-4-formamido-alpha-D-mannopyranosyl residues is described. The approach adopted also permits the N-acyl moiety to be varied. A high-yield synthesis of methyl 4-azido-4,6-dideoxy-alpha-D-mannopyranoside from D-mannose on the 10-20-g scale provided the key intermediate. Regioselective acetylation of 7 gave the 2-acetate 8, which, on treatment with benzyl trichloroacetimidate, provided methyl 2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside. This compound served as a common precursor to the glycosyl donor 12 and acceptor 10 molecules. Silver trifluoromethanesulphonate-promoted glycosylation of 10 by 12 gave a disaccharide derivative, hydrogenolysis of which gave methyl 4-amino-2-O-(4-amino-4,6-dideoxy-alpha-D-mannopyranosyl)-4,6-dideoxy- alpha-D-mannopyranoside from which the N-formyl and N-acetyl derivatives were obtained. Deacetylation of 13 followed by glycosylation with 12 gave a trisaccharide derivative. The N-formylated disaccharide 17 inhibited the binding of Brucella O-polysaccharide to Brucella-specific monoclonal antibodies.