Efficacy and Nontarget Effects of Net Exclusion Enclosures on Apple Pest Management.

Management of direct apple pests, such as codling moth, continues to be problematic despite the widespread implementation of behavioral and chemical controls. Apple growers have increased their use of netting to protect fruit from environmental injury, with some structures enclosing the entire orchard. These enclosures represent a new pest management tactic through physical exclusion. We conducted a two-year trial to examine the effects of full exclusion netting on pests and natural enemies of apples. Insect densities and damage in trees under fully enclosed (net) cages were compared with conventionally (insecticide only) treated and untreated plots. Caged plots had 18.1- and 11.4-fold less codling moth damage than the check, and 4.9- and 4.2-fold less damage than the insecticide-only plots in 2016 and 2017, respectively. However, densities of woolly apple aphid and its parasitoid Aphelinus mali (Haldeman) (Hymenoptera: Aphelinidae) were significantly greater in the caged plots. Densities of earwigs, a typically flightless generalist predator, were not different among treatments, while adults of more mobile flying generalist predators, lacewings and syrphids, were significantly lower in cages compared with uncaged plots. These results demonstrate that although biological control may be partially disrupted, net enclosures have significant potential as a holistic apple management technique.

Frailty predicts morbidity, complications, and mortality in patients undergoing complex abdominal wall reconstruction.

PURPOSE: Frailty is becoming an increasingly established risk factor for adverse postoperative outcomes. Given the innately high morbidity involved in complex abdominal wall reconstruction (CAWR) and the propensity for co-morbidities among this patient population, we sought to determine the predictive utility of a frailty index in patients undergoing CAWR. METHODS: A retrospective analysis was conducted using the American College of Surgeons National Surgical Quality Improvement Project (ACS NSQIP) database. A total of 70,339 patients undergoing CAWR were identified using CPT codes for ventral hernia repair ± components separation, ± placement of mesh. A Modified Frailty Index (mFI) was calculated for each patient. Outcomes included overall morbidity, Clavien-Dindo Grade IV (CDIV) complications, and mortality. RESULTS: Overall, 9931 patients had at least one complication associated with their procedure and an average calculated mFI of 0.12 (± 0.11) which was significantly greater than the average mFI noted in patients with no complications (0.077 ± 0.85, p < 0.001). Similarly, average mFI score (0.16 ± 0.12) in patients with CDIV complications (n = 2541) was once again significantly greater than those without CDIV complications (0.080 ± 0.09; p < 0.001). Multivariable analyses also showed that all individual factors of the mFI were predictive of all-cause and CDIV complications (p < 0.001). Higher pre-operative mFI conferred a 7.77× likelihood of all-cause complications, 35.71× likelihood of CDIV complications, 3.85× likelihood of surgical site complications, and a 62.05× likelihood of death (p < 0.001 for all comparisons). CONCLUSION: We have shown that frailty as measured by mFI is an accurate predictor of morbidity and mortality in patients undergoing CAWR.

Using comparative genomics to develop a molecular diagnostic for the identification of an emerging pest Drosophila suzukii.

Drosophila suzukii (Spotted Wing Drosophila) has recently become a serious invasive pest of fruit crops in the USA, Canada, and Europe, leading to substantial economic losses. D. suzukii is a direct pest, ovipositing directly into ripe or ripening fruits; in contrast, other Drosophilids utilize decaying or blemished fruits and are nuisance pests at worst. Immature stages of D. suzukii are difficult to differentiate from other Drosophilids, posing problems for research and for meeting quarantine restrictions designed to prevent the spread of this pest in fruit exports. Here we used a combined phylogenetic and bioinformatic approach to discover genetic markers suitable for a species diagnostic protocol of this agricultural pest. We describe a molecular diagnostic for rapid identification of single D. suzukii larva using multiplex polymerase chain reaction. Our molecular diagnostic was validated using nine different species of Drosophila for specificity and 19 populations of D. suzukii from different geographical regions to ensure utility within species.

A gene expression signature for high-risk multiple myeloma.

There is a strong need to better predict the survival of patients with newly diagnosed multiple myeloma (MM). As gene expression profiles (GEPs) reflect the biology of MM in individual patients, we built a prognostic signature based on GEPs. GEPs obtained from newly diagnosed MM patients included in the HOVON65/GMMG-HD4 trial (n=290) were used as training data. Using this set, a prognostic signature of 92 genes (EMC-92-gene signature) was generated by supervised principal component analysis combined with simulated annealing. Performance of the EMC-92-gene signature was confirmed in independent validation sets of newly diagnosed (total therapy (TT)2, n=351; TT3, n=142; MRC-IX, n=247) and relapsed patients (APEX, n=264). In all the sets, patients defined as high-risk by the EMC-92-gene signature show a clearly reduced overall survival (OS) with a hazard ratio (HR) of 3.40 (95% confidence interval (CI): 2.19-5.29) for the TT2 study, 5.23 (95% CI: 2.46-11.13) for the TT3 study, 2.38 (95% CI: 1.65-3.43) for the MRC-IX study and 3.01 (95% CI: 2.06-4.39) for the APEX study (P<0.0001 in all studies). In multivariate analyses this signature was proven to be independent of the currently used prognostic factors. The EMC-92-gene signature is better or comparable to previously published signatures. This signature contributes to risk assessment in clinical trials and could provide a tool for treatment choices in high-risk MM patients.

A non-BRCA1/2 hereditary breast cancer sub-group defined by aCGH profiling of genetically related patients.

Germline mutations in BRCA1 and BRCA2 explain approximately 25% of all familial breast cancers. Despite intense efforts to find additional high-risk breast cancer genes (BRCAx) using linkage analysis, none have been reported thus far. Here we explore the hypothesis that BRCAx breast tumors from genetically related patients share a somatic genetic etiology that might be revealed by array comparative genomic hybridization (aCGH) profiling. As BRCA1 and BRCA2 tumors can be identified on the basis of specific genomic profiles, the same may be true for a subset of BRCAx families. Analyses used aCGH to compare 58 non-BRCA1/2 familial breast tumors (designated BRCAx) to sporadic (non-familiar) controls, BRCA1 and BRCA2 tumors. The selection criteria for BRCAx families included at least three cases of breast cancer diagnosed before the age of 60 in the family, and the absence of ovarian or male breast cancer. Hierarchical cluster analysis was performed to determine sub-groups within the BRCAx tumor class and family heterogeneity. Analysis of aCGH profiles of BRCAx tumors indicated that they constitute a heterogeneous class, but are distinct from both sporadic and BRCA1/2 tumors. The BRCAx class could be divided into sub-groups. One subgroup was characterized by a gain of chromosome 22. Tumors from family members were classified within the same sub-group in agreement with the hypothesis that tumors from the same family would harbor a similar genetic background. This approach provides a method to target a sub-group of BRCAx families for further linkage analysis studies.

An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients.

BACKGROUND: Breast cancer cells deficient for BRCA1 are hypersensitive to agents inducing DNA double-strand breaks (DSB), such as bifunctional alkylators and platinum agents. Earlier, we had developed a comparative genomic hybridisation (CGH) classifier based on BRCA1-mutated breast cancers. We hypothesised that this BRCA1-like(CGH) classifier could also detect loss of function of BRCA1 due to other causes besides mutations and, consequently, might predict sensitivity to DSB-inducing agents. PATIENTS AND METHODS: We evaluated this classifier in stage III breast cancer patients, who had been randomly assigned between adjuvant high-dose platinum-based (HD-PB) chemotherapy, a DSB-inducing regimen, and conventional anthracycline-based chemotherapy. Additionally, we assessed BRCA1 loss through mutation or promoter methylation and immunohistochemical basal-like status in the triple-negative subgroup (TN subgroup). RESULTS: We observed greater benefit from HD-PB chemotherapy versus conventional chemotherapy among patients with BRCA1-like(CGH) tumours [41/230 = 18%, multivariate hazard ratio (HR) = 0.12, 95% confidence interval (CI) 0.04-0.43] compared with patients with non-BRCA1-like(CGH) tumours (189/230 = 82%, HR = 0.78, 95% CI 0.50-1.20), with a significant difference (test for interaction P = 0.006). Similar results were obtained for overall survival (P interaction = 0.04) and when analyses were restricted to the TN subgroup. Sixty-three percent (20/32) of assessable BRCA1-like(CGH) tumours harboured either a BRCA1 mutation (n = 8) or BRCA1 methylation (n = 12). CONCLUSION: BRCA1 loss as assessed by CGH analysis can identify patients with substantially improved outcome after adjuvant DSB-inducing chemotherapy when compared with standard anthracycline-based chemotherapy in our series.

A multiplex PCR predictor for aCGH success of FFPE samples.

Formalin-fixed, paraffin-embedded (FFPE) tissue archives are the largest and longest time-spanning collections of patient material in pathology archives. Methods to disclose information with molecular techniques, such as array comparative genomic hybridisation (aCGH) have rapidly developed but are still not optimal. Array comparative genomic hybridisation is one efficient method for finding tumour suppressors and oncogenes in solid tumours, and also for classification of tumours. The fastest way of analysing large numbers of tumours is through the use of archival tissue samples with first, the huge advantage of larger median follow-up time of patients studied and second, the advantage of being able to locate and analyse multiple tumours, even across generations, from related individuals (families). Unfortunately, DNA from archival tissues is not always suitable for molecular analysis due to insufficient quality. Until now, this quality remained undefined. We report the optimisation of a genomic-DNA isolation procedure from FFPE pathology archives in combination with a subsequent multiplex PCR-based quality-control that simply identified all samples refractory to further DNA-based analyses.

Resistance and cross-resistance in populations of the leafrollers, Choristoneura rosaceana and Pandemis pyrusana , in Washington apples.

Insecticide bioassays of the leafrollers, Choristoneura rosaceana (Harris), and Pandemis pyrusana Kearfott (Lepidoptera: Tortricidae), were used to investigate resistance and cross-resistance between azinphosmethyl and other insecticides. Comparisons of field-collected populations with susceptible laboratory colonies of both leafroller species were made in 1996-97, prior to registration and field introduction of several of insecticides, and were re-tested in 2000-2001 following several years of use in the field. Insecticides tested included azinphosmethyl, chlorpyrifos, methyl parathion, tebufenozide, methoxyfenozide, spinosad, indoxacarb, acetamiprid, Bacillus thuringiensis , and azadirachtin. Azinphosmethyl-susceptible laboratory colonies were used for comparison to field populations. Resistance to azinphosmethyl was found in all populations of C. rosaceana (5.2-26.8 fold) and P. pyrusana (8.4-24.9 fold) collected from commercial orchards. Cross-resistance between azinphosmethyl and the insect growth regulators tebufenozide and methoxyfenozide was found in all but one population of the two leafroller species. No cross-resistance was found to chlorpyrifos. Some of the populations tested were cross-resistant to spinosad and indoxacarb, but the responses to these materials were more variable.

Role of neonicotinyl insecticides in Washington apple integrated pest management. Part I. Control of lepidopteran pests.

Three neonicotinyl insecticides, acetamiprid, thiacloprid and clothianidin, were evaluated for their impact on four species of lepidopteran pests of apple in Washington, the codling moth, Cydia pomonella (L.), the Pandemis leafroller, Pandemis pyrusana Kearfott, and the obliquebanded leafroller, Choristoneura rosaceana (Harris), and Lacanobia subjuncta (Grote & Robinson). None of the neonicotinyl insecticides demonstrated sufficient activity against P. pyrusana, C. rosaceana, or L. subjuncta to warrant field trials. Conversely, all had some activity against one or more stages of C. pomonella. Acetamiprid was highly toxic to larvae in laboratory bioassays, and had relatively long activity of field-aged residues (21 days). It also showed some toxicity to C. pomonella eggs (via topical exposure) and adults. Acetamiprid provided the highest level of fruit protection from C. pomonella attack in field trials conducted over five years in experimental orchards with extremely high codling moth pressure. Thiacloprid performed similarly in bioassays, but fruit protection in field trials was slightly lower than acetamiprid. Clothianidin showed moderate to high toxicity in bioassays, depending on the C. pomonella stage tested, but poor fruit protection from attack in field trials. None of the neonicotinyl insecticides were as toxic to larvae or effective in protecting fruit as the current standard organophosphate insecticide used for C. pomonella control, azinphosmethyl. However, both acetamiprid and thiacloprid should provide acceptable levels of C. pomonella control in commercial orchards where densities are much lower than in the experimental orchards used for our trials. The advantages and disadvantages of the neonicotinyl insecticides as replacements for the organophosphate insecticides and their role in a pest management system for Washington apple orchards are discussed.

Role of neonicotinyl insecticides in Washington apple integrated pest management. Part II. Nontarget effects on integrated mite control.

The effect of neonicotinyl insecticides on integrated mite control in Washington apple was examined from 0 In a series of 20 field trials (54 treatments) designed primarily to look at efficacy against the codling moth, Cydia pomonella, nearly half of the treatments using four or more applications of acetamiprid had peak mite densities exceeding the economic threshold of 5 mites per leaf. Overall, acetamiprid treatments had 4.6-fold higher mite densities than the standard organophosphate insecticide treatment. Of the treatments with high mite populations, Panonychus ulmi, the European red mite, and Tetranychus urticae, the twospotted spider mite, were the dominant species in roughly equal numbers of cases. Only 11.1% of the thiacloprid treatments exceeded 5 mites per leaf; these experimental treatments included eight applications, whereas the current label restricts the number of applications at the rate for C. pomonella to two applications. One out of six clothianidin treatments caused a significantly higher mite density than the standard treatment; however, this material appeared to suppress predatory mites. Neonicotinyl insecticides did not eliminate predatory mites, but they inhibited their ability to respond normally to increasing prey populations. In field trials designed specifically to examine mite population densities where neonicotinyl insecticides were used, significantly higher levels of tetranychid mites occurred in one or more acetamiprid treatments (one, two or four applications) in five out of six trials. In the sixth trial (in a commercial orchard), only two acetamiprid applications were made, and mite populations were low in all treatments. While elevated mite densities were more likely to occur with four applications, in one case it occurred following a single application. The predominant tetranychid mite species (either P. ulmi or T. urticae) varied from trial to trial; however, there was no apparent bias regarding stimulation of the two species. Horticultural mineral oil was used with acetamiprid in some trials in an attempt to mitigate mite outbreaks. However, the addition of oil did not counteract the tendency of acetamiprid to increase tetranychid mite populations, and in one trial, had a negative effect on predatory mite densities. Seasonal tetranychid mite density was positively related to the total grams AI (or number of applications) of acetamiprid, thus reducing the number of applications per season should lower the probability of mite outbreaks.

Effects of seasonal mineral oil applications on the pest and natural enemy complexes of apple.

This 3-yr study examined the use of two different apple, Malus domestica Borkhausen, pest management programs based on horticultural mineral oil. Whereas oil provided some additional control of codling moth, Cydia pomonella (L.), when targeting eggs of both generations (Oil/Direct Pest program, typically six applications per season), the additional benefit was difficult to detect when densities were high. With moderate densities, oil reduced the number of fruit infestations, but not stings (unsuccessful entries). There also were some measurable benefits to leafroller, Pandemis pyrusana Kearfott control. Oil was most useful, however, in suppression of secondary pests. White apple leafhopper, Typhlocyba pomaria McAtee, was the primary target of oil applications in the Oil/Indirect Pest program (typically three applications per season). However, leafhopper suppression in the Oil/Direct Pest program was generally greater because of the higher number of applications. Phytophagous tetranychid and eriophyid mites also were suppressed by more oil applications. Predatory mite populations were lower in both oil programs than in the check, but it is difficult to determine whether direct toxicity or reduction of prey was responsible for lower predator populations. There also was some evidence that oil suppressed woolly apple aphid, Eriosoma lanigerum Hausman. The six-spray oil program largely prevented a woolly apple aphid outbreak that occurred in July and August 1998 in the check, although the three-spray program seemed to provide some suppression despite the nonspecific spray timing.

Effect of pesticides on Colpoclypeus florus (Hymenoptera: Eulophidae) and Trichogramma platneri (Hymenoptera: Trichogrammatidae), parasitoids of leafrollers in Washington.

Pesticides were evaluated for their effect on two parasitoid species, Colpoclypeus flouts and Trichogramma platneri, that are potential biological control agents of leafrollers in apple orchards. Organophosphate and carbamate insecticides were highly toxic to both parasitoids in topical applications, but foliar residues of some products were nontoxic after 7 d. At reduced rates, topically applied pyrethroids were low in toxicity to C. florus were highly toxic to T. platneri, and foliar residues were nontoxic after about 7 d. Imidacloprid and abamectin were highly toxic when applied topically to both parasitoids but were not toxic as 1-d-old residues. Insect growth regulators did not cause mortality either as topical applications or residues; however, diflubenzuron caused severe sublethal effects, completely blocking the production of C. florus offspring. Biorational pesticides, such as soap, oil, and B. thuringiensis products, caused no toxicity to C. florus but had a direct impact on T. platneri as topical applications through physical immobilization. The potential to integrate different pesticides with biological control of leafrollers and the need for a step-wise approach to evaluate the impact of pesticides against natural enemies is discussed.

Influence of timing and prey availability on fruit damage to apple by Campylomma verbasci (Hemiptera: Miridae).

Campylomma verbasci Meyer is a zoophytophagous mirid that feeds on small arthropods as well as apple (Malus domestica Borkhausen) fruits, causing economic damage to some cultivars. The influence of timing and prey availability on the amount of fruit damage was studied to determine whether either factor could be used to refine a management program. C. verbasci nymphs were caged on branches of fruiting 'Golden Delicious' apple trees during the period from bloom through early fruit set. The greatest amount of fruit damage occurred during the bloom period; little or no damage occurred after fruit reached approximately equals 13 mm in diameter. The availability of prey did not reduce the incidence of fruit damage by C. verbasci, nor did it influence the survival of nymphs. Nymphal survival was higher, however, in cages where a blossom or fruitlet was present versus a vegetative spur. These data support the hypothesis that post petal fall insecticide applications (those made after the fruit is greater than approximately equals 10-13 mm in diameter) are not useful in preventing economic levels of fruit damage in Washington State, and that petal fall applications would only prevent a fraction of the total amount of damage by this pest. The data from this study do not support the hypothesis that manipulating arthropod prey species of C. verbasci will prevent fruit damage. There was evidence to support the hypothesis that nymphs can survive a relatively short period (7 d) without arthropod prey.

Sloppy paired acts as the downstream target of wingless in the Drosophila CNS and interaction between sloppy paired and gooseberry inhibits sloppy paired during neurogenesis.

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. In the ventral nerve cord of the Drosophila embryo, Wg is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. In this paper, we show that sloppy paired 1 and sloppy paired 2 function as the downstream targets of the Wg signaling to generate the NB4-2 cell. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. That slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, previous results show that Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, we also show that gsb interacts with slp and prevents Slp from specifying NB4-2 identity. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that cell.

Regulation of lactase and sucrase-isomaltase gene expression in the duodenum during childhood.

BACKGROUND: In children, lactase and sucrase-isomaltase are essential intestinal glycohydrolases, and insufficiency of either enzyme causes diarrhea and malnutrition. Little is known about the regulation of lactase and sucrase-isomaltase expression in the duodenum during childhood. In this study, the mechanisms of regulation of duodenal expression of both enzymes were examined in a study population with ages ranging from 1 to 18 years. METHODS: Duodenal biopsy specimens from 60 white children were used to analyze tissue morphology and to quantify lactase and sucrase-isomaltase mRNA and protein. RESULTS: Among healthy subjects, high interindividual variability was noted in both mRNA and protein levels for lactase and sucrase-isomaltase. Lactase mRNA level per subject did not correlate with sucrase-isomaltase mRNA level and thus appeared independent. Both lactase and sucrase-isomaltase protein levels correlated significantly with their respective mRNA levels. For each enzyme, a significant inverse correlation was observed between the degree of villus atrophy and mRNA levels. Aging from 1 to 18 years did not result in significant changes in mRNA or protein levels of either enzyme. Immunostaining patterns within the duodenal epithelium for lactase differed from sucrase-isomaltase in adjacent sections, illustrating independent regulation at the cellular level. CONCLUSIONS: In the duodenum of white children, lactase and sucrase-isomaltase seem primarily regulated at the transcriptional level. The expression of each enzyme in the intestinal epithelium is regulated by an independent mechanism. Lactase and sucrase-isomaltase exhibit stable mRNA and protein levels in healthy children as they grow to adulthood. Mucosal damage affected levels of both enzymes negatively.

Intestinal carbamoyl phosphate synthase I in human and rat. Expression during development shows species differences and mosaic expression in duodenum of both species.

The clinical importance of carbamoyl phosphate synthase I (CPSI) relates to its capacity to metabolize ammonia, because CPSI deficiencies cause lethal serum ammonia levels. Although some metabolic parameters concerning liver and intestinal CPSI have been reported, the extent to which enterocytes contribute to ammonia conversion remains unclear without a detailed description of its developmental and spatial expression patterns. Therefore, we determined the patterns of enterocytic CPSI mRNA and protein expression in human and rat intestine during embryonic and postnatal development, using in situ hybridization and immunohistochemistry. CPSI protein appeared during human embryogenesis in liver at 31-35 e. d. (embryonic days) before intestine (59 e.d.), whereas in rat CPSI detection in intestine (at 16 e.d.) preceded liver (20 e.d.). During all stages of development there was a good correlation between the expression of CPSI protein and mRNA in the intestinal epithelium. Strikingly, duodenal enterocytes in both species exhibited mosaic CPSI protein expression despite uniform CPSI mRNA expression in the epithelium and the presence of functional mitochondria in all epithelial cells. Unlike rat, CPSI in human embryos was expressed in liver before intestine. Although CPSI was primarily regulated at the transcriptional level, CPSI protein appeared mosaic in the duodenum of both species, possibly due to post-transcriptional regulation.

Pediatric duodenal biopsies: mucosal morphology and glycohydrolase expression do not change along the duodenum.

BACKGROUND: Duodenal mucosal biopsies are routinely taken for diagnosis in children with complaints of the upper gastrointestinal tract. Surprisingly, little is known about the usefulness of proximal duodenal versus distal duodenal biopsies for routine diagnostic purposes. This study evaluated the comparability of proximal and distal duodenal biopsies with respect to mucosal morphology as well as glycohydrolase expression as an indicator of intestinal epithelial function. METHODS: Specimens obtained in duodenal endoscopic biopsies from 64 children, ranging in age from 3 months to 18 years with normal or affected mucosa, were studied. Biopsies were performed in anatomically defined regions in the bulbus duodeni (the very proximal part of the duodenum) and distally of the papilla of Vater (distal of the pancreatic duct). Biopsy specimens were paraformaldehyde-fixed for histologic examination and immunohistochemical evaluation or were homogenized to isolate RNA. Crypt/villus morphology was assessed as is routinely determined by pathologists. In addition, several aspects of lactase and sucrase-isomaltase expression as paradigms of intestinal brush border enzymes were assessed: localization at the cellular level, semiquantitative immunohistochemistry, and quantitative measurement of the messenger RNA levels of the respective brush border glycohydrolases. RESULTS: As anticipated, there was a wide interpatient variation in mucosal morphology and expression of lactase and sucrase-isomaltase. Nonetheless, the consistent finding was that in each patient, measurements of morphology and lactase and sucrase-isomaltase gene expression were very similar between samples obtained in the proximal and distal biopsies. CONCLUSIONS: Biopsies performed in either location in the duodenum are equally suitable for diagnostic workup of patients suspected of mucosal abnormalities affecting morphology or small intestinal brush border glycohydrolase activities.

Lactase and sucrase-isomaltase gene expression during Caco-2 cell differentiation.

The Caco-2 cell line is derived from a human colon adenocarcinoma and differentiates in vitro into small-intestinal enterocyte-like cells, expressing the hydrolases lactase and sucrase-isomaltase. We cultured Caco-2 cells on permeable supports from 0 to 37 days after plating to study endogenous lactase and sucrase-isomaltase gene expression in relation to cell differentiation. Profiles of lactase and sucrase-isomaltase mRNA, protein and enzyme activity were analysed on a per-cell basis, using immunocytochemistry, RNase protection assays, metabolic polypeptide labelling and enzyme activity assays. Tight-junction formation was complete 6 days after plating. Immunocytochemistry of Caco-2 cross-sections showed lactase and sucrase-isomaltase predominantly in the microvillar membrane of polarized cells. mRNA, protein and enzyme activity of lactase appeared consecutively, reaching maximum levels 8-11 days after plating. Whereas lactase mRNA and protein biosynthesis showed a sharp decline after peak levels, lactase activity remained high until 37 days after plating. In contrast, mRNA and protein biosynthesis and activity of sucrase-isomaltase peaked successively 11-21 days after plating, and exhibited comparable levels throughout the entire experiment. The following conclusions were reached. (1) In Caco-2 cells, biosynthesis of lactase and sucrase-isomaltase is regulated by the amount of their mRNAs, indicating transcriptional control. (2) Sucrase-isomaltase activity is most probably transcriptionally controlled at all time points. (3) In contrast, lactase activity is initially regulated by its level of biosynthesis. After its peak at 8 days, the slow decline in activity compared with its biosynthesis indicates high stability. (4) Different mRNA profiles for lactase and sucrase-isomaltase indicate different mechanisms of transcriptional regulation of these genes.

Intestinal brush border glycohydrolases: structure, function, and development.

The hydrolytic enzymes of the intestinal brush border membrane are essential for the degradation of nutrients to absorbable units. Particularly, the brush border glycohydrolases are responsible for the degradation of di- and oligosaccharides into monosaccharides, and are thus crucial for the energy-intake of humans and other mammals. This review will critically discuss all that is known in the literature about intestinal brush border glycohydrolases. First, we will assess the importance of these enzymes in degradation of dietary carbohydrates. Then, we will closely examine the relevant features of the intestinal epithelium which harbors these glycohydrolases. Each of the glycohydrolytic brush border enzymes will be reviewed with respect to structure, biosynthesis, substrate specificity, hydrolytic mechanism, gene regulation and developmental expression. Finally, intestinal disorders will be discussed that affect the expression of the brush border glycohydrolases. The clinical consequences of these enzyme deficiency disorders will be discussed. Concomitantly, these disorders may provide us with important details regarding the functions and gene expression of these enzymes under specific (pathogenic) circumstances.