Tissue DNA Content Research Articles

DNA Ploidy Analysis with Image Cytometry on Barrett's Esophagus-related Dysplasia and Esophageal Adenocarcinoma

*W1585 DNA Ploidy Analysis with Image Cytometry on Barrett's Esophagus-related Dysplasia and Esophageal Adenocarcinoma Chenggong Yu, Qin Huang, Michael Klein, Raj K. Goyal Background and Aims: Barrett’s esophagus is of great clinical importance to correctly grade precursor lesions histopathologically and to identify the patients with a high risk of developing invasive malignancy for early management. Regrettably, the pathological identification of early malignancy is subjective and has high inter-observer variations, and none of the molecular markers have been proven to be sensitive and specific for this purpose also. In our previous study, we found that DNA ploidy analysis was much better than histopathology for identifying premalignant lesions in the patients with Barrett’s esophagus. In the current study, we wish to expand the on-going image cytometry study using the Automated Cellular Image System (ACIS) to measure DNA content in tissue. Methods: The cases with Barrett’s esophagus or distal esophageal adenocarcinomawere retrieved from the computer files. The lesions were identified, classified, and marked on slides by two experienced pathologists. The corresponding tissue blocks were retrieved and cut at 7 mM. The sections were then Feulgen stained. DNA content was measured and analyzed with ACIS. The control nuclei of cells with DNA index (DI) between 0.90-1.10 consisted of those of benign stromal cells including endothelial cells, fibroblasts, macrophages, and large lymphocytes. DNA content histograms were classified into near diploidy/aneuploidy (DI: 1.111.30), low aneuploidy (DI: 1.31-1.90), tetraploidy (DI: 1.91-2.10), and high aneuploidy (DI: >2.11). Results: Of the 50 cases studied, 10 were gastric cardiac mucosa without dysplasia (CM), 10 with Barrett’s esophagus without dysplasia (BE), 5 with indefinite dysplasia (ID), 9 with low-grade dysplasia (LD), 8 with high-grade dysplasia (HD), and 8 with esophageal adenocarcinoma (EA). DNA ploidy status was found in various patterns and the cases with non-diploidy including aneuploidywere identified in 0/10CM, 8/10 BE, 4/5 ID, 6/9 LD, 8/8HD, and 8/8 EA cases. ThemeanDI for each disease was 1.05 for CM, 1.20 for BE, 1.38 for ID, 1.37 for LD, 1.83 for HD, and 1.97 for EA. The results indicate that nondiploidy is common in Barrett’s esophagus-related lesions and DNA image cytometry is able to identify malignant cells in the early premalignant diseases. Conclusion: DNA content image cytometry appears to be a sensitive method for identifying premalignant lesions in Barrett’s esophagus-related diseases. Unrecognized Barrett's Esophagus in Patients with Erosive Esophagitis Unveiled by Methylene Blue Chromoendoscopy Stephen N. Wong, Jose D. Sollano Jr., Melchor M. Chan, Rolando Lopez, Johnny T. Go, Victoriano Y. Lim, Carmelita D. Dalupang, Ramon E. Carpio BACKGROUND: Recent guidelines recommend surveillance for patients with Barrett’s esophagus (BE), especially if dysplasia is present. However, in the background of erosive esophagitis (EE), endoscopic recognition of BE may be difficult. Up to 50% of patients with EE have an underlying BE discovered after the EE has healed.Methylene blue (MB) is avidly taken up by goblet cells found in columnar intestinal metaplasia (CIM), a hallmark of BE, and will stain these areas blue while leaving areas without CIM unstained. Thus, utilizing MB as a staining agent in chromoendoscopy may offer an opportunity to increase the endoscopic yield for BE in patients with EE. METHODS: All patients with EE but with no endoscopic evidence of BE were recruited into the study. Once EE was identified, 15cc of 0.5% methylene blue solution (MB) was sprayed around the distal esophagus and allowed to stay for 1-2 minutes before flushing with 100-200cc tap water. Three biopsies were then taken from both the stained and the unstained esophagealmucosa. If the distal esophagealmucosa did not stain, randombiopsies from the unstained mucosa were taken. BE was confirmed if CIM was demonstrated on histopathology. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were computed. RESULTS: A total of 51 patients (male: female; 1.4:1) were recruited into the study. Smoking and alcohol intake were more common in males (p=0.005 and p=0.023, respectively). Over half (52.1%) of the patients had symptoms>6 months prior to endoscopy. There was no difference in demographic and clinical data among patients with and without BE (p>0.05). Distribution of esophagitis severity according to the Los Angeles classification was as follows: A (n=36, 75%); B (n=11, 22.9%); C (n=1, 2.1%). MB uptake was noted in 91.7% (n=44), 38 (86.4%) of whom were confirmed to have CIM on histopathology. In 3 patients who exhibited positive dye uptake, biopsies on both the stained and unstainedmucosa revealed CIM. The sensitivity, specificity, PPV andNPVofMBchromoendoscopy for the diagnosis of BE were 92.7%, 88.2%, 86,4% and 93.8%, respectively. There was no correlation between esophagitis severity and the presence of BE (p=0.294). CONCLUSIONS: Endoscopically unrecognized BE is highly prevalent in patients with EE. MB chromoendoscopy demonstrates high sensitivity and specificity in the diagnosis ofBEand is a valuable tool in unmasking this disease in patientswithEE.

Chromosomal variation in plant evolution

Chromosomal variation in plant evolution

Study on ceramide expression and DNA content in patients with healthy mucosa, leukoplakia, and carcinoma of the larynx.

To investigate the expression of ceramide produced by sphingomyelin and DNA content in patients with healthy laryngeal mucosa, leukoplakia, and laryngeal carcinoma. A retrospective review of the clinical and surgical records of 178 patients with leukoplakia of the larynx; 23 of them developed laryngeal carcinoma. University medical center. One hundred seventy-eight consecutive patients with leukoplakia of the larynx were identified from the archived pathology files of the Eye Ear Nose and Throat Hospital of Fudan University from January 1, 1990, to December 30, 2001. Among them, 23 developed laryngeal carcinoma. Flow cytometry and immunohistochemistry were performed to test DNA content and ceramide expression in healthy tissue, tissue with leukoplakia, and tissue with laryngeal carcinoma from the same patient. Among 23 patients with leukoplakia, 20 had aneuploidy and 3 had diploidy. The healthy tissues were all diploids, and the tissues with laryngeal carcinoma were all aneuploids. The expression of ceramide decreased gradually from healthy tissue to tissue with leukoplakia to tissue with laryngeal carcinoma (0, no staining; 1+, weak staining; 2+, mild staining; 3+, moderate staining; 4+, strong staining; and 5+, the highest staining intensity). The expression of ceramide in DNA diploid cells is stronger than in aneuploid cells. Ceramide, the second messenger in apoptosis, may associate with the progress of leukoplakia to carcinoma of the larynx. The reduction of ceramide may contribute to laryngeal carcinogenesis.

Developing tetraploid perennial ryegrass (Lolium perenne L.) populations

In a breeding programme aimed at developing tetraploids from Australian adapted germplasm, a root‐tip squash technique was initially used for ploidy determination. The highest recovery of tetraploids (39.7%) was obtained by treatment of 1‐week‐old seedlings with 0.1% colchicine concentration. Significant cultivar × colchicine concentration interaction was observed in seedling survival, highlighting the difference in response of the cultivars to colchicine treatment. Subsequently, flow cytometric estimation of the nuclear DNA content in young leaf tissues was used to screen the C1 and C2 generations. The speed and the efficiency of this method make it possible to examine a large number of plants. In the C1 population the percentage of tetraploids, diploids, and aneuploids was 25, 72.7, and 2.3% respectively, while in the C2 generation the percentage of tetraploids, diploids, and aneuploids was 43, 2, and 55% respectively.

Quantitative monitoring of colonization of olive genotypes by Verticillium dahliae pathotypes with real-time polymerase chain reaction

For Verticillium wilt diseases, quantitative measurement of the pathogen in infected tissues is necessary for characterizing the disease reaction of host genotypes to pathogen strains. The amount of pathogen DNA in the plant should be representative of the number of potential propagules. In this work, we quantified the DNA of highly virulent, defoliating (D) and mildly virulent, nondefoliating (ND) Verticillium dahliae in Verticillium wilt resistant (Acebuche-L), and -susceptible (Arbequina and Picual) olive genotypes by real-time polymerase chain reaction (PCR) assays. We show that the amount of pathogen DNA quantified in each olive genotype correlated with susceptibility to Verticillium wilt, but was influenced less by virulence of the infecting V. dahliae pathotype, suggesting that the extent of pathogen colonization does not clearly determine the virulence phenotype. Maximal pathogen DNA occurred in root and stem tissues before disease had fully developed in the susceptible olive genotypes, with pathogen DNA content in stem tissue being lower than that in root tissues. A boost in V. dahliae propagules in root tissues, as indicated by the amount of D and ND DNA, took place 1 week after inoculation, followed by a decrease over time. That decrease was sharp in Arbequina and Acebuche-L plants compared with a progressive reduction in Picual. The amount of V. dahliae DNA in roots of Picual plants remained at a high level for a long period of time following inoculation, but it eventually decreased too. Similar changes in pathogen DNA amounts over time occurred also in stem. Verticillium wilt hardly developed in Acebuche-L plants, but the amount of D and ND V. dahliae DNA in roots was comparable to that found in Arbequina plants. However, pathogen DNA could not be quantified in stems of Acebuche-L plants though it was detectable in them by nested-PCR. The results in our study show that real-time PCR is an excellent tool for quantitative diagnosis of olive infection by D and ND V. dahliae; as well as for monitoring pathogen colonization and assessing resistance and tolerance to Verticillium wilt in olive genotypes. This would help in olive breeding programs for resistance to the disease.

Serum lipids and tissue DNA content in Egyptian female breast cancer patients.

Several clinical studies suggest the prognostic significance of serum lipid levels and tissue DNA content in breast cancer. In the course of investigating the biological features of this disease among Egyptian female patients, we examined the serum lipid levels and tissue DNA content of premenopausal and postmenopausal breast cancer patients. Levels of total lipid, total cholesterol, and triglycerides were measured in the sera of women with breast cancer and compared with those of the control women. The DNA content in breast cancer tissue was also measured in these patients. Total lipid levels showed a significant increase in both premenopausal (follicular and luteal) and postmenopausal patients. Total cholesterol levels significantly increased in premenopausal (follicular and luteal) patients with no significant change in postmenopausal women. Triglyceride levels showed a significant increase in postmenopausal women, whereas no significant differences were observed in premenopausal patients. Tumors of premenopausal patients, in both follicular and luteal phases, showed a higher DNA content as compared with those of postmenopausal patients. Breast cancer tissues of grade III showed significantly higher DNA content than those of grade I and grade II. This study suggests an association between high levels of serum, total lipid and total cholesterol, and increased breast cancer risk in premenopausal women. Such an association is also suggested for the high total serum lipid and triglyceride levels in postmenopausal women. The DNA content in breast cancer tissue might be useful in determining a suitable therapy for individual cases, based on the malignancy grade.

Failure to detect Chlamydia pneumoniae DNA in cerebral aneursymal sac tissue with two different polymerase chain reaction methods

Objective:Chlamydia pneumoniae (C pneumoniae) is a common cause of a usually mild, community acquired pneumonia. This organism, however, can spread from the respiratory tract into other parts of the body...

Effect of oral oleoyl-estrone on adipose tissue composition in male rats.

To determine whether the oral administration of oleoyl-estrone has similar mass-decreasing effects on the main different sites of white adipose tissue (WAT). Adult male Zucker lean rats were given a daily oral gavage of oleoyl-estrone (OE, 10 micromol/kg) in 0.2 ml of sunflower oil for 10 days, and were compared with controls receiving only the oil. The mass of the main WAT sites: subcutaneous, epididymal, mesenteric, retroperitoneal, gluteal, perirenal and interscapular, as well as perirenal and interscapular brown adipose tissue (BAT), were dissected and studied. The tissue weight, DNA, protein, lipid and total cholesterol content, together with the levels of leptin and acyl-estrone in the larger WAT and BAT masses, were measured. The weights of WAT depots were correlated with body weight but those of BAT were not. Cell size was maximal for epididymal and mesenteric and minimal for subcutaneous and retroperitoneal WAT and BAT. Differences were detected in DNA, and in protein and lipid content between distinct WAT sites. OE treatment tended to decrease cell number and cell size in WAT; only small differences in composition were found between WAT locations inside the visceral cavity and those outside. Decreases in lipid content were maximal in mesenteric fat. Leptin and acyl-estrone content were fairly uniform at the different WAT sites, except for high concentrations in gluteal WAT. OE induced a greater decrease in leptin and acyl-estrone than in DNA and lipids; changes in these hormones were fairly parallel in all sites. In general, the differences in composition between visceral and peripheral subcutaneous WAT and their responses to OE were less marked than the individual differences observed between specific sites, regardless of location. WAT sites are fairly diverse in composition, but their response to OE treatment was uniform. OE decreased the weight of WAT through reduction of both cell numbers and size; but did not change the mass or composition of BAT significantly. The effects of OE are more marked in the hormonal signals (leptin and acyl-estrone) from the tissue than in its composition and mass.

In Vivo Investigation of Progressive Alterations in Rat Mammary Gland Tumors by Near-Infrared Spectroscopy

We have investigated mammary gland tissues of female rats treated with 7,12-dimethylbenz[a]anthracene in sesame oil by a near infrared (NIR) spectroscopy finding that the DNA and water contents in the cancerous tissues were larger than those in the normal tissues but that the lipid content in the former was less than that in the latter. With protein contents, however, little difference was observed between the two. Thus, we used a lipid band around 1725 nm (the first overtone of n-alkane) and a protein band around 2054 nm (a combination band of amide A and amide II of polypeptides) for a quantitative evaluation of malignant changes in the mammary gland tissues. The lipid/protein band intensity ratios were calculated from the spectra of the mammary glands in the control animals and those of the noncancerous and cancerous sites in the treated animals. The lipid/protein ratios in the control animals, in the noncancerous sites, and in the cancerous sites were 1.452 ± 0.221 (n = 5), 0.728 ± 0.069 (n = 5), and 0.362 ± 0.060 (n = 5), respectively. These values were significantly different from each other (P < 0.001). The lipid changes observed by near-infrared (NIR) spectroscopy were confirmed by the results obtained from chemical methods for the evaluation of lipid levels in the same samples. Thus, our NIR spectroscopic method would be able not only to discriminate between cancerous and normal tissues but also to distinguish animals with cancers from normal animals. In addition, as the cancer grew, the lipid band intensity decreased, this band was shifted to higher wavelengths, and collagen peaks appeared in the tissues. These findings were supported by histological examinations of the cancerous and normal tissues. The present study indicates that NIR spectroscopy has high specificity and sensitivity in discriminating cancerous tissues from normal mammary glands in animals and it may offer potential for noninvasive, in vivo diagnosis of female breast cancer in the near future.

Influence of Terminalia chebula on dermal wound healing in rats.

The effects of topical administration of an alcohol extract of the leaves of an evergreen plant, Terminalia chebula, on the healing of rat dermal wounds, in vivo, was assessed. T. chebula treated wounds healed much faster as indicated by improved rates of contraction and a decreased period of epithelialization. Biochemical studies revealed a significant increase in total protein, DNA and collagen contents in the granulation tissues of treated wounds. The levels of hexosamine and uronic acid in these tissues, also increased upto day 8 post-wounding. Reduced lipid peroxide levels in treated wounds, as well as ESR measurement of antioxidant activity by DPPH radical quenching, suggested that T. chebula possessed antioxidant activities. The tensile strength of tissues from extract-treated incision wounds increased by about 40%. In addition, T. chebula possessed antimicrobial activity and was active largely against Staphylococcus aureus and Klebsiella. These results strongly document the beneficial effects of T. chebula in the acceleration of the healing process.

Flow cytometric evidence for endopolyploidization in cabbage ( Brassica oleracea L.) flowers

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from flowers of cabbage (Brassica oleracea L.) revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in mature flowers. The distribution of cells with the differerent ploidy levels is tissue-specific and is characteristic of the stage of development. Nuclei of young flower buds exclusively gave 2C and 4C peaks, indicating that the tissues maintained diploid level. Endoreduplication was consistently detected during flower development. Endopolyploidy is probably common in differentiation of cabbage plants. Implications of this original feature are discussed.

Lactation failure in crossbred Sahiwal Friesian cattle.

Milk producers in Malaysia make extensive use of crossbred Sahiwal Friesian dairy cattle. These animals have, however, been found susceptible to lactation failure. A survey of cows in an experimental herd of F1 Sahiwal Friesian animals indicated that, in 30% of animals, milk yield decreased to negligible levels within the first 8 weeks post partum. Lactation failure was associated with a progressive increase in the amount of residual milk left in the udder after normal milking. By week 3 of lactation, residual milk volume was significantly greater than that in animals that, based on previous lactation history were not susceptible to lactation failure, and accounted for up to 30% of milk available at the morning milking. The cellular consequences of residual milk accumulation were evident in the activities of acetyl-CoA carboxylase, fatty acid synthetase and galactosyltransferase, key enzyme markers of cellular differentiation, which decreased in glands undergoing lactation failure and were lower than values measured in tissue of control cows. Mammary cell number, estimated by tissue DNA content, was also reduced in animals undergoing lactation failure. These indices of mammary development indicate that lactation failure is the result of premature involution in susceptible animals. Premature involution is a predictable consequence of progressive milk stasis in failing lactation, and attributable to an increase in autocrine feedback by inhibitory milk constituents. The progressive increase in residual milk is, on the other hand, unlikely to be attributable to impaired mammary development. Measurements of milk storage during milk accumulation showed no differences between control and lactation failure cows in the distribution of milk between alveolar and cisternal storage compartments. We conclude that lactation failure in Sahiwal Friesian cows is due to a failure of milk removal, and probably the result of an impaired milk ejection reflex rather than to the glands' milk storage characteristics.

DNA content for Asian pines parallels New World relatives

This hypothesis is based on an observed correlation between DNA content and thermal regime for fish, zooplankton, salamanders, and some flowering plants. The Asian pine results provided no support for the latitudinal hypothesis; Asian tropical pine species did not have smaller genomes than their temperate or boreal relatives. DNA content of haploid megagametophyte tissue varied from 21.85 pg/C for hard pine Pinus densiflora Sieb. &amp; Zucc. to 29.59 pg/C for soft pine Pinus bungeana Zucc. Pinus merkusii Jung. &amp; De Vriese (29.63 pg/C) was the exceptional hard pine, with a genome size larger than many soft pines. The mean DNA content of Asian soft pines exceeded Asian hard pines (Δ 3.22 pg/C), a parallel to the previously reported trends for New World pines. No continental effect was detected. Based on 46 pines species sampled in centers of species diversity in Asian and the New World, soft pines had mean DNA content which exceeded hard pines by 4.97 pg/C.Key words: gymnosperms, conifers, laser flow cytometry, megagametophytes, C values, phylogeny.

Patterns of Endopolyploidy During Seedling Development in Cabbage (Brassica oleracea L.)

Flow cytometric analysis of nuclear DNA contents of somatic tissues of cabbage (Brassica oleracea L.) has revealed extensive endopolyploidization, resulting in tissues that comprise mixtures of cells with different DNA contents, ranging from 2C to 16C. Patterns of endopolyploidy are specific to each developmental stage. Multiple polyploidy was not present in the embryos of dry seeds. Rapid endoreduplication occurred in the radicle and the hypocotyl of the embryos during seed germination. Further endoreduplication cycles were detected in all tissues except those of the shoot tips. In five cabbage cultivars tested, seedlings contained cells of four ploidy levels, corresponding to 2C, 4C, 8C and 16C. Multiploidy may be an integral part of differentiation programs in cabbage plants. The biological significance of endoreduplication in cabbage plants is discussed.

Flow cytometric evidence for endopolyploidy in seedlings of some Brassica species

Flow cytometric analysis of the nuclear DNA contents of somatic tissues from seedlings of Brassica rapa L. and B. oleracea L. revealed extensive endoreduplication, resulting in tissues that contain cells with multiple ploidy levels (also called ’endopolyploidy’). Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C and 64C were observed in Brassica rapa, while B. oleracea exhibited a mixture of cells with five ploidy levels, 2C, 4C, 8C, 16C and 32C. The distribution of cells with the different ploidy levels was tissue-specific and characteristic of the stage of development. Multiploidy was not found in the embryos of dry seeds. Rapid endoreduplication occurred during seedling development. It is most probable that multiploidy is, if not a general feature, at least very common in Brassica species. The physiological and genetic implications of this original feature are discussed.

Local injection of IFN-gamma reduces the number of myofibroblasts and the collagen content in palatal wounds.

Wound contraction and scar formation after cleft palate surgery impair maxillary growth and dentoalveolar development. Since myofibroblast numbers and scar formation are reduced by interferon-gamma (IFN-gamma) in the healing of dermal wounds, the hypothesis was tested that local administration of IFN-gamma reduces the numbers of myofibroblasts and the elevated amount of collagen during palatal mucoperiosteal wound healing. Standardized mucoperiosteal excision wounds were made in the palatal mucoperiosteum of young rats. Either IFN-gamma or vehicle alone (sham group) was repeatedly injected into the wound site between 4 and 29 days post-wounding. The results were compared with unmanipulated control wounds. Samples of wound tissue were prepared for biochemical and microscopic analysis. The hydroxyproline, sulfated glycosaminoglycan and DNA contents of the wound tissues were analyzed biochemically. The degree of re-epithelialization, tissue thickness, the numbers of myofibroblasts, and the amounts of elastin and collagen types I and III were evaluated on histological sections. Injection of vehicle alone affected almost all healing parameters, compared with the controls, and delayed the wound-healing process. IFN-gamma stimulated re-epithelialization and decreased the numbers of myofibroblasts when compared with vehicle-treated wounds. It also decreased the hydroxyproline and glycosaminoglycan contents of 60-day-old wound tissue, but the histological characteristics of scar tissue persisted. Therefore, IFN-gamma is able to reduce the numbers of myofibroblasts and the collagen content of scar tissue after palatal wound healing. It may be a promising pharmaceutical agent for the reduction of wound contraction and scarring after cleft palate surgery.

Unreduced diploid nuclei in Cupressus dupreziana A. Camus pollen

Size and DNA content of pollen of Cupressus dupreziana A. Camus, a highly endangered Mediterranean conifer, were assessed by cytomorphological observations and flow cytometric analyses and then compared to C. sempervirens L. pollen. Mature C. dupreziana pollen was composed of two uninucleated types of pollen grains differing in size. Around 35% of the grains exhibited a size similar to C. sempervirens pollen, while 65% exhibited a larger diameter. However only one peak of fluorescence was detected by flow cytometry. DNA content of C. dupreziana pollen was twice the DNA content of C. sempervirens pollen. Comparison of DNA contents of mature and germinating pollen revealed that mature pollen of both species were arrested in the G2 phase. Comparison with the DNA content of somatic tissue (2C) provided evidence for the production of unreduced pollen in C. dupreziana. This unexpected feature in gymnosperms is discussed in terms of reproductive strategy of this species.

Castration-induced apoptotic cell death in the Brown Norway rat prostate decreases as a function of age.

Growth and differentiation of the prostate gland depends upon androgens, yet overgrowth of the human prostate occurs later in life when serum levels of testosterone are declining. We have reported a similar phenomenon in the Brown Norway rat, but the age-dependent overgrowth of the prostate is confined to the dorsal and lateral lobes and, hence, is lobe specific. Because tissue growth depends upon the balance between proliferation and death of cells, the present study was designed to investigate whether cell death differed in the various prostatic lobes of Brown Norway rats as a function of age. Apoptosis of cells in the ventral, dorsal, lateral, and anterior lobes of the prostate was examined in young (4-month-old) and old (24-month-old) Brown Norway rats after castration. Whereas castration caused tissue weights of all four prostatic lobes to decrease over the course of 10 days, this occurred more rapidly and to a greater magnitude in the ventral than in the dorsal, lateral, and anterior lobes. Tissue DNA content, a measure of cell number, decreased only in the ventral lobe after castration. DNA fragmentation, indicative of apoptotic cell death, was detected by in situ labeling using the terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling method and as intranucleosomal cleavage of genomic DNA analyzed by agarose gel electrophoresis. Both methods demonstrated the correlation between loss of DNA content and apoptotic cell death in the ventral lobe, whereas only the highly sensitive terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) method revealed relatively few dying cells in the dorsal, lateral, and anterior lobes after castration. Moreover, when examined as a function of age, less cell death occurred in all four lobes of old rats compared with young rats. In both young and old rat prostates, cell death was observed in epithelial and stromal cells within the ventral lobe where apoptotic cells were detected throughout the branched ductal network and were not restricted to a particular region. Taken together, these studies demonstrate the marked differences in cell death and survival between the different rat prostatic lobes in response to castration and further suggest that the androgen-sensitive apoptotic response is age dependent. Hence, the lower rates of cell death observed for the dorsal and lateral lobes, accompanied by the further decline that occurs with increasing age, are important components of the age-dependent and lobe-specific overgrowth observed for these lobes. Moreover, the age-dependent decline in apoptotic cell death observed in the prostates of old rats suggests that prostatic cells develop androgen independence as a function of age, and survival of these cells does not require androgen.

Molecular and Cell Biology of Skin Wound Healing in a Pig Model

To define the pattern of change at the molecular and cellular levels during the healing of exci-sional skin wounds in the skeletally immature pig, mRNA levels for relevant molecules were assessed by semiquantitative RT-PCR using porcine specific primer sets and RNA isolated from normal skin and samples at various time post-wounding. Analysis of cellular change was assessed by DNA quantification and histology of tissue sections. The results demonstrated that the changes in the pattern of RNA and DNA content of the scar tissue were consistent with the observed increasing cellularity. The mRNA levels for collagen I, III, HSP47, IL-1, TGF-P, MMP-1, -2 and -9, TIMP-1, -2, and-4, PAI-1, versican were significantly elevated during healing; levels for biglycan and fibromodulin were not significantly altered; and the mRNA levels for TIMP-3 were depressed. These findings suggest that skin wound healing is a series of complex matrix-cell interactions that involve cellular migration and inflammation, followed by proliferation of fibroblasts with new collagen synthesis, and lastly tissue remodeling of the scar.

Study of Dermal Wound Healing Activity of Trigonella foenum graceum Seeds in Rats.

The influence of seed suspension of Trigonella foenum graceum (commonly known as fenugreek) was investigated for wound healing properties in excision, incision, and dead space wound models in rats. The seed suspension of T. foenum graceum 1ml/kg was given either topically or orally once a day. The granulation tissues formed were removed after 4, 8, 12, and 16 days of post excision wounding; and biochemical parameters such as DNA, total protein, total collagen, and hexosamine were estimated. The suspension increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increases in DNA, protein, and collagen contents of the granulation tissues. Quicker and better maturation and cross-linking of collagen was ob-served in the suspension-treated rats, as indicated by the high stability of acid soluble collagen and increase in aldehyde content and tensile strength. The orally treated wound was found to epithelize faster, and the rate of wound contraction was higher, as compared with the control wounds. The results show that T. foenum graceum produced different actions on the various phases of wound repair.