Cell Production Rate Research Articles

The Production and Release of Living Root Cap Border Cells is a Function of Root Apical Meristem Type in Dicotyledonous Angiosperm Plants

The root apical meristems (RAM) of flowering plant roots are organized into recognizable pattern types. At present, there are no known ecological or physiological benefits to having one RAM organization type over another. Although there are phylogenetic distribution patterns in plant groups, the possible evolutionary advantages of different RAM organization patterns are not understood. Root caps of many flowering plant roots are known to release living border cells into the rhizosphere, where the cells are believed to have the capacity to alter conditions in the soil and to interact with soil micro-organisms. Consequently, high rates of border cell production may have the potential to benefit plant growth and development greatly, and to provide a selective advantage in certain soil environments. This study reports the use of several approaches to elucidate the anatomical and developmental relationships between RAM organization and border cell production. RAM types from many species were compared with numbers of border cells released in those species. In addition, other species were grown, fixed and sectioned to verify their organization type and capacity to produce border cells. Root tips were examined microscopically to characterize their pattern and some were stained to determine the viability of root cap cells. The first report of a correlation between RAM organization type and the production and release of border cells is provided: species exhibiting open RAM organization produce significantly more border cells than species exhibiting closed apical organization. Roots with closed apical organization release peripheral root cap cells in sheets or large groups of dead cells, whereas root caps with open organization release individual living border cells. This study, the first to document a relationship between RAM organization, root cap behaviour and a possible ecological benefit to the plant, may yield a framework to examine the evolutionary causes for the diversification of RAM organization types across taxa.

Epidermal Growth Factor Receptor Signaling Regulates Bax and Bcl-w Expression and Apoptotic Responses During Intestinal Adaptation in Mice

Normal intestinal adaptation to massive small-bowel resection requires intact epidermal growth factor receptor signaling and consists of increased enterocyte proliferation and apoptosis. Although emphasis has been placed on understanding the regulation of proliferation, few studies have evaluated the mechanism and contribution of apoptosis to the adaptation response. We sought to test the hypothesis that epidermal growth factor receptor signaling regulates specific Bcl-2 family members (Bax and Bcl-w) to direct apoptosis and adaptation after massive small-bowel resection. Laser capture microdissection microscopy permitted measurement of Bax and Bcl-w messenger RNA expression in crypt and villus enterocytes in control conditions and under epidermal growth factor receptor-inhibited (waved-2 mice) or stimulated (epidermal growth factor transgenic mice) conditions after a 50% small-bowel resection or sham operation. Resection-induced adaptation was then studied in Bax-null and Bcl-w-null mice under control circumstances and after epidermal growth factor receptor stimulation. When compared with Bcl-w, the most significant expression changes were observed with Bax and took place within crypt enterocytes. Epidermal growth factor receptor stimulation resulted in a decreased ratio of Bax to Bcl-w expression and decreased rates of apoptosis. Bax-null mice had no apoptosis response to small-bowel resection and displayed an amplified adaptation response to the administration of epidermal growth factor. Bcl-w-null mice had poor survival and impaired adaptation to small-bowel resection, an effect that was rescued by crossbreeding these mice with epidermal growth factor transgenic mice. The crypt expression of Bax and Bcl-w is influenced by epidermal growth factor receptor signaling and is key for the regulation of apoptosis. Epidermal growth factor receptor stimulation, coupled with apoptosis inhibition, may provide a novel strategy to amplify adaptation responses in patients after massive intestinal loss.

Pronounced ecophysiological clonal differences of two common freshwater ciliates, Coleps spetai (Prostomatida) and Rimostrombidium lacustris (Oligotrichida), challenge the morphospecies concept

Cell volume, growth and production rates of two common planktonic freshwater ciliates, the prostomatid Coleps spetai and the oligotrich Rimostrombidium lacustris, were investigated in clonal laboratory cultures. The clones were isolated from oligo-mesotrophic alpine Lake Mondsee, Austria, during summer and kept in culture with the small cryptophyte Cryptomonas sp. as food. All parameters investigated revealed significant clonal differences among both species. The extent of the clonal differences was comparable to differences observed earlier between similar planktonic ciliate species. The ecological relevance of varying clonal growth rates was evaluated using a simple numerical model. The model outcome suggests that differences in growth rates by 10% may significantly alter the clonal composition in the course of a ciliate peak in temperate lakes. The experimental results and the model outcome are discussed in the context of the functional diversity of freshwater ciliates. It is concluded that the morphospecies concept, which is the most widely used concept by both ciliate taxonomists and ecologists, may severely underestimate the ecological plasticity and the functional diversity of aquatic ciliates.

Effect of defunction on cytokinetics and cancer at colonic suture lines

An intestinal suture line potentiates experimental carcinogenesis in its vicinity, probably due to adaptive hyperplasia. By contrast, a defunctioning colostomy causes distal hypoplasia, and fewer tumours develop. Male Sprague-Dawley rats (n = 160) were used to study adaptation (assessed by a stathmokinetic technique) and carcinogenesis (induced by azoxymethane) at an end-to-end anastomosis that was raised in either functioning or defunctioned left colon. Controls had no procedure and other rats had proximal colostomy alone. Defunction had a profound antitropic effect on the colon, reducing bowel length and weight and crypt cell production rate (CCPR) by 22-56 per cent. Anastomotic CCPR was increased by a factor of 2.6 over controls: mean(s.e.m.) values of 12.71(2.85) versus 4.87(0.41) cells/crypt/h (P less than 0.01), but defunction reduced this by 76 per cent to a mean(s.e.m.) value of 3.00(0.52) cells/crypt/h below that in the intact colon. In the left colon, 39-50 per cent of tumours were sited at the anastomosis. Compared with controls there were 77 per cent fewer tumours in defunctioned colon, but they still favoured the site of anastomosis. Neoplasms at the colostomy site accounted for 74-77 per cent of all right-sided tumours. Anastomosis and defunction have powerful but contrasting effects on colonic adaptation and carcinogenesis; when combined they tend to cancel each other out.

Effect of duodenogastric reflux on gastric mucosal proliferation after gastric surgery.

The effect of duodenogastric reflux on cell proliferation and mucosal mass in the stomach was studied. Male Wistar rats (n = 118) were submitted to Polya partial gastrectomy, partial gastrectomy with Roux-en-Y diversion of bile, total duodenogastric reflux or handling of the stomach alone (sham operation). Following oral administration of the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine for 6 months, animals were killed 6, 9 or 12 months postoperatively and their stomachs were examined for crypt cell production rate and mucosal DNA content. Compared with shams, crypt cell production rate was more than twice as high in the gastric remnant 12 months after Polya partial gastrectomy (P less than 0.001) and median DNA content was 31 per cent greater (P = 0.05). After total duodenogastric reflux, DNA content was 62 per cent greater than in shams (P = 0.02), while Roux-en-Y diversion reduced crypt cell production rate by 65 per cent (P less than 0.001). Only Polya gastrectomy increased the number of rats developing gastric carcinomas (9 versus 2 shams; P less than 0.05). Increased mucosal cell proliferation in rats with duodenogastric reflux may help to explain the development of gastric stump cancer.

Kinetic changes in experimental colonic urinary diversion.

An experimental model of colonic urinary diversion was performed on male Wistar rats to see if faeces, urine or a faeces and urine mixture produces tumours. Cell kinetic techniques, including the crypt cell production rate, and cellular DNA measurement were used to examine the effect of urine and of faeces on the colonic mucosa. Urinary diversion into the colon significantly increased colonic crypt activity. Tumours were found at the anastomosis when urine and faeces were mixed at 4 months, and at 6 months with urine alone.

Measurement of cell production rates in human gastro-intestinal cancer--a guide to treatment?

Cell production rates were measured in 19 cases of rectal carcinoma and in 11 cases of gastric carcinoma using a stathmokinetic technique. These measurements were compared with results from normal rectal and gastric mucosa. It was found that most of the cases of rectal cancer were proliferating more slowly than morphologically normal rectal mucosa. This was true for the majority of gastric cancers, but a small proportion appeared to be proliferating more rapidly. These results are discussed with their relevance to appropriate therapy.

Adaptation to jejunoileal bypass promotes experimental colorectal carcinogenesis.

The co-carcinogenic potential of 85 per cent jejunoileal bypass (JIB) was tested in male Sprague-Dawley rats (n = 81) given 6 preliminary injections of the selective intestinal carcinogen azoxymethane (total dose 90 mg/kg). Controls had sham JIB. Colorectal adaptation was studied 30 weeks postoperatively in rats given injections of vehicle alone. JIB caused 17-33 per cent increments in colonic length, weight and crypt depth; crypt cell production rate was more than doubled (P less than 0.01). Despite lowering body weight by 27 per cent, JIB increased the median number of colorectal tumours per rat from 3 to 8 (P less than 0.01). The findings suggest that evidence of hyperplasia and dysplasia should be sought in patients receiving subtotal jejunoileal bypass for obesity.

Experimental colonic carcinogenesis after gastric surgery

Peptic ulcer surgery may predispose to the subsequent development of colorectal cancer. This experimental study has investigated the effects of gastric operations on colonic cell proliferation, bile acid excretion and carcinogenesis. Male Sprague-Dawley rats (n = 105) underwent sham operation, Pólya partial gastrectomy or vagotomy and pyloroplasty. The carcinogen azoxymethane was administered weekly for 6 weeks thereafter (total dose 60 mg kg-1). When the animals were killed 24 weeks after operation, colons were examined for mucosal mass, crypt cell production rate (CCPR) and tumour yield; faeces were assayed for contents of neutral steroids and bile acids (both total and individual). Morphometric indices and mucosal DNA content were similar in all three groups. Pólya gastrectomy reduced: (1) CCPR throughout the colon (by 42-65 per cent, P less than 0.002); (2) the number of rats with colorectal tumours (26 per cent versus 63 per cent, P less than 0.05); (3) faecal levels of neutral steroids and bile acids, notably hyodeoxycholic acid (P less than 0.01). Although vagotomy and pyloroplasty increased caecal CCPR, there were no consistent differences in faecal steroids and no alteration in tumour yield after the operation. These results fail to support clinical studies suggesting that gastric surgery predisposes to colonic carcinogenesis. Indeed, Pólya partial gastrectomy exerts a protective effect, probably by inhibiting colonic cell proliferation.

The effect of altered luminal nutrition on cellular proliferation and plasma concentrations of enteroglucagon and gastrin after small bowel resection in the rat.

Luminal nutrition is known to have a trophic effect on small bowel mucosa after intestinal resection. Humoral agents, however, may also contribute to this process. Two of the proposed humoral agents, enteroglucagon and gastrin, were therefore investigated after intestinal resection and transection in the rat, and changes in their concentration in the plasma were related to cellular proliferation. Forty-eight male Wistar rats had either 75 per cent proximal small bowel resection or jejunal transection. The animals were further divided into three groups, each with a different nutritional intake. The first group were allowed food ad libitum. The second group were kept under hypothermic conditions which resulted in hyperphagia, while the last group were nourished intravenously. A further 8 animals had a laparotomy only (sham operation). All animals were killed 12 days after operation, plasma enteroglucagon and gastrin were measured, while determination of the crypt cell production rate (CCPR) was used to denote cellular proliferation. In each group resected rats had significantly higher crypt cell production rates and greater enteroglucagon levels compared with transected animals. However, only in the normally fed group was plasma gastrin increased in resected animals, there being no significant difference in the plasma concentration of this peptide in transected compared with resected rats, in both the intravenously fed and hyperphagic groups. In the models studied enteroglucagon appears to be a more likely candidate for a humoral trophic agent than gastrin in intestinal adaptation.

The role of pancreatico-biliary secretions in intestinal adaptation after resection, and its relationship to plasma enteroglucagon.

Two groups, each containing 16 male Wistar rats, had either 75 per cent small bowel resection or jejunal transection; 8 animals from each group; had previously been subjected to pancreatico-biliary diversion. All animals were killed 12 days after the operation, plasma enteroglucagon levels were measured and crypt cell production rate (CCPR) at different sites of the remaining small intestine was measured using a metaphase arrest technique with vincristine. In each of the resected groups there was a significant increase in the CCPR and enteroglucagon levels compared with the transected groups. Furthermore it was found that the CCPR and enteroglucagon levels were higher in the resected group without the pancreatico-biliary diversion compared with the resected group with the diversion. This study, although it confirms the importance of pancreatico-biliary secretions in intestinal adaptation, could also indicate that a humoral factor may be important in the control of intestinal cell proliferation. Our findings do not exclude the possibility that enteroglucagon could be a candidate for such a role.

Relative effects of ileal resection and bypass on intestinal adaptation and carcinogenesis.

Since ileal resection and ileal bypass are commonly performed in man and might stimulate colonic hyperplasia, their co-carcinogenic potential was explored in male Sprague-Dawley rats (n = 135). One week after 33 per cent distal small-bowel resection, 33 per cent distal small-bowel bypass or distal ileal transection (control), animals started a 6-week course of azoxymethane injections (total dose 90 mg/kg ip). Findings in rats killed at 20 and 25 weeks were similar: bypass produced a higher yield of colorectal tumours (4.0 +/- 0.6 per rat: mean +/- s.e.) than controls (2.4 +/- 0.4; P less than 0.05), but resection caused maximal enhancement (5.2 +/- 0.5: P less than 0.01). In rats killed at 30 weeks, however, tumour yields were almost identical. Overall, resection increased colonic tumour yield by 55 per cent (P less than 0.02) and bypass by 32 per cent. Stathmokinetic measurements of crypt cell production rate (CCPR) at 20 weeks showed similar increases after resection and bypass both in residual functioning small bowel (109-200 per cent: P less than 0.01) and in colorectum (63-100 per cent: P less than 0.05). At 30 weeks these adaptive effects persisted, despite an overall increase in CCPR with age. Loss of functioning ileum enhances experimental colorectal carcinogenesis principally by reducing the latent period for tumour development. Resection has a greater effect than bypass probably by producing earlier hyperplasia, though later adaptive effects are similar.

Hypoplasia of defunctioned rectum.

The adaptive response of the large bowel to surgical defunction in man is essentially unknown, although in the rat there is progressive hypoplasia and a reduced propensity to experimental carcinogenesis. Mucosal biopsies were taken from the upper rectum completely defunctioned by a proximal stoma from 2 months to 5 years earlier in 11 patients and from 14 controls without abdominal operations or disease. Samples were established in organ culture and, after 16 h, crypt cell production rate (CCPR) was determined by a stathmokinetic technique. Crypt morphometry was also undertaken. CCPR in defunctioned large bowel was less than half that of controls: (mean (s.d.)-1.96 (0.68) versus 4.65 (0.54) cells crypt-1 h-1, P less than 0.0001). Likewise, crypt length was 24 per cent lower (0.34 (0.05) versus 0.44 (0.04) mm, P less than 0.0001) and crypt width was 38 per cent lower (0.04 (0.01) versus 0.07 (0.01) mm, P less than 0.0001). Rectal defunction causes profound and persistent hypoplasia in man.

Mucosal morphology, cell proliferation and faecal bacteriology in acute pouchitis.

A study was performed to investigate whether acute reservoir ileitis (pouchitis) is associated with specific changes in mucosal morphology, crypt cell kinetics and faecal bacteriology in the ileal pouch. Forty-six patients were studied (ileal reservoir, 36; end ileostomy, ten) using clinical grading, sigmoidoscopy and biopsy; 24 patients with a reservoir were restudied after therapy for 1 month with metronidazole 400 mg three times daily. An index of villus atrophy and crypt cell production rate (CCPR) were determined in all biopsy material. Faecal bacteriology was assessed in 12 patients with a pouch before and after metronidazole therapy. The mucosa of patients with pouchitis was associated with a lower villus atrophy index (P = 0.052), a higher CCPR (P = 0.03) and a higher grade of acute inflammation than that in those without pouchitis. There was no difference in faecal bacterial counts between patients with and without pouchitis. A low atrophy index correlated with a high CCPR (P < 0.001), worse functional score (P < 0.001) and more severe pouch mucosal acute inflammation (P < 0.001), but not with faecal bacteriology. Following metronidazole therapy there was resolution of acute pouch inflammation, increased villus atrophy index (P = 0.049), decreased CCPR (P = 0.049) but no differences in faecal bacterial counts apart from Bacteroides species. These data show that metronidazole therapy does not specifically alter the growth of common faecal bacteria in patients with pouchitis, apart from Bacteroides species. However, metronidazole causes resolution of the typical changes in pouch mucosal morphology and crypt cell kinetics associated with pouchitis.

Individuals with a strong family history of colorectal cancer demonstrate abnormal rectal mucosal proliferation.

Thirty-seven individuals with an increased lifetime risk of colorectal cancer because of family history and a control group (n = 21) with no such family history underwent colonoscopy. No neoplastic lesion was found in any individual. Rectal biopsies of macroscopically normal mucosa were taken 8 cm from the anal margin and mucosal proliferation assessed by the crypt cell production rate (CCPR). There was no difference in CCPR between the control group (median 10 (95 per cent confidence interval 7-11) cells per crypt per h) and those at intermediate risk (lifetime risk between 1:17 and 1:10, n = 14). However, there was a significant difference in CCPR between the control group and those at higher risk (lifetime risk > 1:10, n = 23) (median CCPR 13 (95 per cent confidence interval 10-17) cells per crypt per h, P = 0.004). A high risk of colorectal cancer derived from family history correlates with an increased colorectal mucosal proliferation rate.

Inhibition of intestinal carcinogenesis by dietary supplementation with calcium.

Dietary supplementation with calcium reduces colonic crypt cell production rates in both normal and hyperplastic mucosa. Calcium can bind intraluminally with bile salts and fatty acids thus reducing their mitogenic effect. The protective role of oral calcium on intestinal carcinogenesis (induced by azoxymethane) was tested in 60 male Sprague-Dawley rats submitted to either 80 per cent mid jejuno-ileal resection (n = 30) or jejunal transection (n = 30). Half the rats in each group received calcium lactate 24 g/l added to their drinking water. Rats were killed 25-27 weeks postoperatively. Enterectomy increased colonic tumour yield by 60-106 per cent (P = 0.002-0.005) and duodenal tumour yield by 70-86 per cent. Calcium abolished this effect at both sites, halving intestinal tumour yields in rats with both transection and resection (P less than 0.05). Doubling the dietary intake of calcium inhibits experimental carcinogenesis.

Regulation of erythropoietin production

The glycoprotein hormone erythropoietin (EPO) is an essential growth and survival factor for erythroid progenitor cells, and the rate of red blood cell production is normally determined by the serum EPO concentration. EPO production is inversely related to oxygen availability, so that an effective feedback loop is established, which controls erythropoiesis. Since recombinant EPO became available as an effective therapeutic agent, significant progress has also been made in understanding the basis of this feedback control. The main determinant of EPO synthesis is the transcriptional activity of its gene in liver and kidneys, which is related to local oxygen tensions. This control is achieved by hypoxia-inducible transcription factors (HIF), consisting of a constitutive beta-subunit and one of two alternative oxygen-regulated HIFalpha subunits (HIF-1alpha and HIF-2alpha). In the presence of oxygen (normoxia) the HIFalpha subunits are hydroxylated, which targets them for proteasomal degradation. Under hypoxia, because of the lack of molecular oxygen, HIF cannot be hydroxylated and is thereby stabilized. Although HIF-1alpha was the first transcription factor identified through its ability to bind to an enhancer sequence of the EPO gene, more recent evidence suggests that HIF-2alpha is responsible for the regulation of EPO. Although EPO is a prime example for an oxygen- regulated gene, the role of the HIF system goes far beyond the regulation of EPO, because it operates widely in almost all cells and controls a broad transcriptional response to hypoxia, including genes involved in cell metabolism, angiogenesis and vascular tone. Further evidence suggests that apart from its effect as an erythropoietic hormone EPO acts as a paracrine, tissue-protective protein in the brain and possibly also in other organs.

Genetic and physiological architecture of early vigor in Aegilops tauschii, the D-genome donor of hexaploid wheat. A quantitative trait loci analysis.

Plant growth can be studied at different organizational levels, varying from cell, leaf, and shoot to the whole plant. The early growth of seedlings is important for the plant's establishment and its eventual success. Wheat (Triticum aestivum, genome AABBDD) seedlings exhibit a low early growth rate or early vigor. The germplasm of wheat is limited. Wild relatives constitute a source of genetic variation. We explored the physiological and genetic relationships among a range of early vigor traits in Aegilops tauschii, the D-genome donor. A genetic map was constructed with amplified fragment-length polymorphism and simple sequence repeat markers, and quantitative trait loci (QTL) analysis was performed on the F(4) population of recombinant inbred lines derived from a cross between contrasting accessions. The genetic map consisted of 10 linkage groups, which were assigned to the seven chromosomes and covered 68% of the D genome. QTL analysis revealed 87 mapped QTLs (log of the odds >2.65) in clusters, 3.1 QTLs per trait, explaining 32% of the phenotypic variance. Chromosomes 1D, 4D, and 7D harbored QTLs for relative growth rate, biomass allocation, specific leaf area, leaf area ratio, and unit leaf rate. Chromosome 2D covered QTLs for rate and duration of leaf elongation, cell production rate, and cell length. Chromosome 5D harbored QTLs for the total leaf mass and area and growth rate of the number of leaves and tillers. The results show that several physiological correlations between growth traits have a genetic basis. Genetic links between traits are not absolute, opening perspectives for identification of favorable alleles in A. tauschii to improve early vigor in wheat.

Polyploidy and Cellular Mechanisms Changing Leaf Size: Comparison of Diploid and Autotetraploid Populations in Two Species of Lolium

Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.

Regulation of Fruit Growth and Fruit Size in Apple

Fruit development in apple cultivars varying in their ultimate fruit size was analyzed using cytology, flow cytometry (FCM), and semi-quantitative RT-PCR. Fruit size variation across cultivars was largely explained by variation in cell number. The cell division phase lasted for less than 30 days in all varieties, less than previously believed. A distinct overlap between the cell division and cell expansion phases was present. Analysis of the relative cell production rate (rCPR) showed a major peak about 10 days after full bloom (DAFB) after which it declined. Comparison of the rCPR across varieties suggested distinct patterns of cell production with `Gala' having a low but sustained rCPR, `Pixy Crunch' a short but high rCPR, and `Golden Delicious' having a high and sustained rCPR. FCM analysis also showed similar patterns with a peak in the proportion of dividing cells about 10 DAFB followed by a decline. To further understand regulation of cell number, four cell cycle related genes were cloned from `Gala'. Cyclin Dependent Kinase B (CDK B) and Cyclin B were found to be highly cell division phase specific in their expression. Analysis of gene expression by semi-quantitative RT-PCR indicated peak expression of these two genes at 5-10 DAFB, consistent with the peaks in rCPR and proportion of dividing cells. Comparison of gene expression across the varieties showed higher peak expression of the above genes in the larger-fruited `Golden Delicious' than in the smaller-fruited `Gala.' This study provides novel insight into the regulation of fruit development in apple and also suggests a role for the cell cycle genes in fruit size regulation.