Samuel M. Newman

The influence of culture time and passage number on the morphological and physiological development of Caco-2 cells.

Abstract The Caco-2 cell line is used by many investigators as a model of the intestinal epithelium to study nutrient uptake and transport. Our goal was to create an awareness of inherent variabilities in the Caco-2 cell line which may influence their suitability as a model or their application to specific problems. To study the influence of passage on the model, cultures were monitored from passage 20 to 109. Transepithelial electrical resistance (TEER) and sucrase activity (measured in 21-day-old cultures) increased through about passage 36. TEER values declined after about passage 60; sucrase remained elevated but variable. Cells at passage 22, 33, and 72 were grown simultaneously for 24 days. Older-passaged cells reached plateau phase sooner. Before Day 15, passage 72 cells had higher TEER and lower permeability to 14C-mannitol than passages 22 and 33; however, after Day 15 all passages showed similar permeability. On Day 21, passage 72 cells had significantly lower alkaline phosphatase activity than did the other passages. Electron microscopy did not reveal any major morphological differences between the passages; however, it did show that some areas of cells grown on membranes were not monolayers but were several cells thick with varied morphology. Investigation of the formation of these multilayered areas showed them to be an inherent part of cell growth under the conditions used. These results emphasize the inherent variability in Caco-2 cell models and emphasize the need to monitor closely the culture characteristics during growth and differentiation under specific experimental conditions.

Dietary zinc reduces osteoclast resorption activities and increases markers of osteoblast differentiation, matrix maturation, and mineralization in the long bones of growing rats.

The nutritional influence of zinc on markers of bone extracellular matrix resorption and mineralization was investigated in growing rats. Thirty male weanling rats were randomly assigned to consume AIN-93G based diets containing 2.5, 5, 7.5, 15 or 30 microg Zn/g diet for 24 days. Femur zinc increased substantially as zinc increased from 5 to 15 microg/g diet and modestly between 15 and 30 microg/g (P<.05). By morphological assessment, trabecular bone increased steadily as dietary zinc increased to 30 microg/g. Increasing dietary zinc tended to decrease Zip2 expression nonsignificantly and elevated the relative expression of metallothionen-I at 15 but not 30 microg Zn/g diet. Femur osteoclastic resorption potential, indicated by matrix metalloproteinases (MMP-2 and MMP-9) and carbonic anhydrase-2 activities decreased with increasing dietary zinc. In contrast to indicators of extracellular matrix resorption, femur tartrate-resistant acid and alkaline phosphatase activities increased fourfold as dietary zinc increased from 2.5 to 30 microg Zn/g. Likewise, 15 or 30 microg Zn/g diet resulted in maximum relative expression of osteocalcin, without influencing expression of core-binding factor alpha-1, collagen Type 1 alpha-1, or nuclear factor of activated T cells c1. In conclusion, increased trabecular bone with additional zinc suggests that previous requirement estimates of 15 microg Zn/g diet may not meet nutritional needs for optimal bone development. Overall, the up-regulation of extracellular matrix modeling indexes and concomitant decrease in resorption activities as dietary zinc increased from 2.5 to 30 microg/g provide evidence of one or more physiological roles for zinc in modulating the balance between bone formation and resorption.

Inadequate dietary copper increases tumorigenesis in the Min mouse

Multiple intestinal neoplasia (Min) mice are a good model for the investigation of the effects of dietary alterations on genetic susceptibility for intestinal cancer. In the current study, nursing dams and their pups were placed on an AIN-93G diet containing either 1 or 6 ppm copper. The pups were maintained on the same concentration of dietary copper after weanling until they were 13-weeks-old. Animals fed copper deficient diets had a significantly (P<0.0003) higher small intestine tumor incidence and a significantly (P<0.04) higher small intestine tumor burden than animals fed adequate dietary copper. Therefore, inadequate dietary copper can increase the spontaneous tumorigenesis that occurs in the Min mouse.

A comparison of cuticle deposition during the pre- and posteclosion stages of the adult weevil, Anthonomus grandis Boheman (Coleoptera : Curculionidae)

Abstract An ultrastructural analysis of cuticle deposition before and after adult eclosion of the cotton boll weevil, Anthonomus grandis Boheman (Coleoptera : Curculionidae), is made to monitor the intra- and extracellular events that accompany the shift in cuticle architecture. A typical 5-layered epicuticle and a multi-lamellate procuticle are deposited during the pharate adult stage. Secretion of the epicuticle and the procuticle begins about 2 and 4 days after pupation, respectively. Following eclosion, a lattice-like endocuticle is secreted in the form of layers of parallel rod- or beam-shaped macrofibers. Deposition of endocuticle over the first week after emergence, is at a rate of 3 or 4 layers per day. The imaginal endocuticle accounts for the major portion of the cuticle mass as there is about a 14-fold increase in sclerite thickness and an overall 4-fold increase in non KOH-extractable extskeletal mass during the first week after emergence. Extensive cytoskeletal and surface remodeling plus a change in secretory product packaging occurs at the apical region of the epidermal cells upon shifting to deposition of the endocuticle. Intralayer orientation of the macrofibers is under cellular control and is accomplished by the formation of templates consisting of membrane placque-bearing, canal-like depressions on the apical surface extending across cell borders. Comparisons of cuticle sections to simulated plots drawn via computer graphics, show that each successive layer of macrofibers is rotated with respect to the overlying layer by an angle of about 72°. Except for vertical columns of cuticular fibers that support pore canals, microfibril orientation within the procuticle/exocuticle generally follows the Bouligand model for a typical lamellate arthropod cuticle. Direct cellular control over the interlaminar orientation of the microfibrils forming the procuticle could not be discerned in this study.

Antennal sexual dimorphism in Diabrotica virgifera virgifera (Le Conte) (Coleoptera : Chrysomelidae): Male specific structures, ultrastructure of a unique sensillum, and sites of esterase activity☆

Morphological and histochemical studies were performed on the flagellar antennal segments of adult Diabrotica virgifera virgifera LeConte (Coleoptera : Chrysomclidae). The work was part of a program to investigate the biological significance of a soluble esterase (Est-a) found only in the antennae. Est-a was known to be produced initially it the distal flagellar segments and age-related increases in concentration have been recorded in male beetles in comparison to females. The ventral surface of the 5 distal segments of male antennae possess a field of raised, smooth cuticle having pit-like depressions. The pits contain unusual cup-shaped sensilla abundantly supplied with pore tubules, typical of those associated with olfactory processes in other species. The epicuticle of the cur) sensilla is thin but lacks the pores commonly found in olfactory sensilla. The duct of at least one dermal gland opens into each pit. When sectioned antennae were stained fcr general esterase activity, it was the cells of the dermal glands that stained positive. The pit fields are unique to males. However, cup sensilla and dermal glands with esterase activity are present on female antennae and at sites outside the pit fields on male antennae. Circumstantial evidence suggests that the observed sensillum type functions in olfaction. The sensilla and associated glands in the pit fields of males may be involved in detection of the female-produced sex pheromone, 8-methyl-2-decyl propanoate.

Uric acid storage in the epidermal cells of Manduca sexta: Localization and movement during the larval-pupal transformation

Abstract In the integument of Manduca sexta (L.) larvae, uric acid is shown to be preferentially stored within the white pigmented epidermal cells. Uric acid concentrations in the epidermal tissues are higher in the dorso-lateral white stripes (>500 nmol/mg) than in adjacent blue-green pigmented regions (30 nmol/mg). The distribution and temporal movements of uric acid were determined by feeding larvae artificial diet containing [ 14 C]hypoxanthine for the first 48 h of the fifth instar. Day-3 larvae (86 h after larval ecdysis) contained label mainly in the white-pigmented epidermal tissues with only small quantities in the gut and no radioactivity in the fat body or haemolymph. For day-4 larvae, about 80% of the label in the epidermis was either translocated to storage within fat body or excreted before the onset of wandering. HPLC analysis and reactivity with uricase confirmed that the labelled material in the tissues was urica acid. Ultrastructurally, the white-pigmented epidermal cells contain an abundance of lightly-stained urate granules, that coalesce into large accumulations toward the basal side of cells during the initial stages of metamorphosis. By day 4 these cells are essentially devoid of urate granules. The possibility of hormonal control of uric acid movement is discussed.

Altered nucleotide content and changes in mitochondrial energy states associated with copper deficiency in rat platelets

Dietary copper deficiency severely reduces platelet cytochrome c oxidase activity, but the effect of this perturbation on platelet energy metabolism is unknown. In this study, the effect of copper deficiency on platelet adenine nucleotide and GTP content was determined. Structural changes in platelet mitochondria during copper deficiency also were examined. Copper deficiency caused a 24% reduction in adenosine triphosphate (ATP) content, a 113% increase in adenosine monophosphate (AMP) content, and a 36% reduction in GTP content. Because the secretory pool of ATP was not affected by copper deficiency, the changes observed in platelet nucleotide content most likely reflect an effect on the metabolic nucleotide pool. Furthermore, the number of platelet mitochondria exhibiting swollen cristae and high matrical density, characteristics suggesting that mitochondria are engaged in oxidative phosphorylation, was increased by copper deficiency. These findings indicate that copper deficiency can alter energy metabolism in platelets in a manner that is consistent with partial blockage of mitochondrial electron transport and reduced ATP production.

Copper deficiency: A potential model for determining the role of mitochondria in cardiac aging.

Heart mitochondria experience age-related declines in cytochrome c oxidase (CCO) activity and increases in the generation of reactive oxygen species (ROS) that may contribute to loss of cardiac function and the development of disease that occur with advancing age. In a manner similar to aging, copper deficiency also suppresses heart CCO activity and has cardiovascular consequences related to increased peroxidation. Food restriction is often used as a tool to study oxidative mechanisms of aging and the present study examines the potential of copper deficiency to model the role of mitochondria in cardiac aging by determining if the effect of food restriction on CCO activity and oxidative stress in heart mitochondria parallels its effect on cardiac mitochondria during aging. Overall, copper deficiency severely inhibited CCO activity and increased both Mn superoxide dismutase (MnSOD) and glutathione peroxidase (GPX) in isolated heart mitochondria. However, a 20% reduction in food intake by copper-deficient rats increased CCO activity by 65% and decreased MnSOD activity by 25% but had no effect in rats fed adequate copper. Copper deficiency also reduced the carbonyl content of 80–100 kDa mitochondrial proteins, but the reduction in carbonyl content was unaffected by food restriction. Food restriction did, however, completely prevent the enlargement of cardiac mitochondria in copper-deficient rats. Together, these findings indicate that copper deficiency induces mitochondrial antioxidant enzyme activity and hypertrophy in cardiac tissue in response to reduced CCO activity and that food restriction may counteract these changes by reducing oxidative stress. Because the action of food restriction on CCO activity and mitochondrially generated oxidative stress are similar in copper deficiency and aging, copper deficiency may serve as a short-term model for studying the potential roles of mitochondria in cardiac aging.

Ultrastructural changes in the intestine of rats fed high-zinc diets

The feeding of high-zinc diets to humans is often used as therapy for patients with Wilson’s disease, an autosomal recessive disorder of copper accumulation. There seem to be no outward adverse effects of this treatment; however, preliminary studies in our laboratory have shown apparent weaknesses in the intestinal wall of rats fed high-zinc diets. As a consequence, this study was carried out to determine if feeding high-zinc diets to rats would affect the ultrastructural morphology of the small intestine. The effects of treatment on copper status of the rats also were determined. Weanling male rats were fed diets containing either 35 or 350 mg of zinc/kg. After 7 weeks, blood and various tissues were collected to measure copper status indicators, and portions of the upper duodenum were excised and prepared for light and electron microscopy. Results showed that rats fed high-zinc diets had significantly lower copper status as indicated by low serum copper, serum ceruloplasmin activity, and liver copper, than rats fed normal-zinc diets. Liver superoxide dismutase or cytochrome c oxidase activities were not affected by high zinc. Observations of sections of the duodenum by electron microscopy showed that non-assembled collagen molecules of the lamina propria were more often disorganized and formed tangled masses in rats fed the high-zinc diet than in those fed normal-zinc diets. This suggests that low copper status caused by high-zinc feeding might be affecting the activity of lysyl oxidase, a copper-dependent enzyme, and thus crosslinking of the collagen molecules. However, these observations did not always correlate with low copper status. Other possible explanations include a direct competition between zinc and copper for sites on lysyl oxidase, zinc blocking of aldehyde residues on the collagen molecule, or some unrecognized process involving other enzymes or other aspects of collagen assembly. Whether such processes or affinities actually exist is still under investigation. J. Trace Elem. Exp. Med. 10: 37–46, 1997. Published 1997 Wiley-Liss, Inc.1