根瘤菌生态学研究中两个数学模型的探讨

以自然重壤土和无菌砂为培养基质,栽种大豆,采用MPN法测数,证明了大豆根瘤菌数量的增长符合逻辑斯谛模型并编写了相应的BASIC程序。通过6组试验数据的计算,发现根瘤菌接种量和其占瘤率之间存在一定的数学关系,并编写了相应的BASIC程序。     

Reversal of muscle hypertrophy in the rat urinary bladder after removal of urethral obstruction

We studied the ultrastructure of the bladder musculature after first inducing hypertrophy by means of urethral obstruction and subsequently removing the obstruction. With hypertrophy the bladder musculature increases ten-fold or more in volume; after de-obstruction approximately 4/5 of the hypertrophic muscle weight and volume is lost within six weeks. In spite of this very large decrease in muscle mass there is no degeneration of muscle cells or nerve endings or of other cell types in the de-obstructed bladder either at 5 days or at 6 weeks. The individual muscle cells are smaller in size than in the hypertrophic bladder but still larger than control muscle cells. The decrease in muscle cell size is more substantial than the decrease in muscle cell surface. There are no lysosomes or other signs of intracellular degradation in any cells of the muscle layer. The musculature contains a very large amount of intercellular material, mainly collagen. This study documents the great plasticity of the musculature in the reduction of muscle mass after de-obstruction. However, some of the fine structural features are almost as different from the controls as in the hypertrophic muscle.     

Diverse expression profiles of glutathione-S-transferase subunits in mammalian urinary bladders

The hGSTM1 null genotype has been associated with increased susceptibility to urinary bladder cancer. However, the extent to which the GSTM1 subunit actually contributes to GST activities in mammalian urinary bladders is not clear. For adult mice, urinary bladders exhibited GST activity which was among the highest observed in the tissues tested. The mouse bladder GST activity with the 1-chloro 2,4-dinitrobenzene substrate was also more than 10-fold greater than that of rat and human bladders. A large increase in mouse bladder GST activity occurs during early development with the sharpest increase between 7 and 17 days of age. Subunit compositions of GSTs in adult mouse, human, and rat bladders are also markedly different. The mGSTM1 subunit is by far the predominant GST in mouse bladder, with increases in mGSTM1 between 7 and 17 days accounting for the sharp rise in GST activity during maturation. By contrast, Pi class GSTs predominate in both human and rat bladders. Investigators seeking to establish direct connections between susceptibility to bladder cancer and the hGSTM1 gene deletion should take into account the fact that the hGSTM1 subunit, even when present, represents a very minor fraction of the GST protein in human bladder.     

Known bacterial virulence factors do not explain the variation in urinary cytokine levels in patients with urosepsis

Abstract We measured urinary endotoxin, IL-6 and IL-8 levels in 23 patients with gram-negative urosepsis. The endotoxin and cytokine levels showed a 100–1000 fold range. No correlation was found between levels of urinary endotoxin, and IL-6 or IL-8 levels. In all cases bacterial numbers were ≥ 10⁵ CFU ml⁻¹ urine. The endotoxin content of the isolated microorganisms neither correlated with the urinary cytokine levels, nor with IL-6 and IL-8 levels obtained in vitro when 10³ log-phase CFU of each of the bacteria were incubated with heparinized whole blood of three healthy donors. Neither the haemolysin phenotype of the bacteria, nor the presence of the P-pili gene was correlated with the cytokine response in vivo or in vitro. Other factors than known bacterial virulence factors apparently contribute to the wide variation in urinary cytokine levels in urinary tract infection.     

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.     

Urinary bladder of rat: fine structure of normal and hypertrophic musculature

Summary The fine structure of the muscle of the urinary bladder in female rats is similar to that of other visceral muscles, although it is arranged in bundles of variable length, cross-section and orientation, forming a meshwork. When distended, the musculature is 100–120 m thick, with some variation and occasional discontinuity. Extended areas of cell-to-cell apposition with uniform intercellular space occur between muscle cells, whereas attachment plaques for mechanical coupling are less common than in other visceral muscles. There are no gap junctions between muscle cells. Many bundles of microfilaments and small elastic fibres run between the muscle cells. After chronic partial obstruction of the urethra, the bladder enlarges and is about 15 times heavier, but has the same shape as in controls; the growth is mainly accounted for by muscle hypertrophy. The outer surface of the hypertrophic bladder is increased 6-fold over the controls; the muscle is increased 3-fold in thickness, and is more compact. Mitoses are not found, but there is a massive increase in muscle cell size. There is a modest decrease in percentage volume of mitochondria, an increase in sarcoplasmic reticulum, and no appreciable change in the pattern of myofilaments. Gap junctions between hypertrophic muscle cells are virtually absent. Intramuscular nerve fibres and vesicle-containing varicosities appear as common in the hypertrophic muscle as in controls. There is no infiltration of the muscle by connective tissue and no significant occurrence of muscle cell death.     

A continuous optimization approach for inferring parameters in mathematical models of regulatory networks

Background

The advances of systems biology have raised a large number of sophisticated mathematical models for describing the dynamic property of complex biological systems. One of the major steps in developing mathematical models is to estimate unknown parameters of the model based on experimentally measured quantities. However, experimental conditions limit the amount of data that is available for mathematical modelling. The number of unknown parameters in mathematical models may be larger than the number of observation data. The imbalance between the number of experimental data and number of unknown parameters makes reverse-engineering problems particularly challenging.

Results

To address the issue of inadequate experimental data, we propose a continuous optimization approach for making reliable inference of model parameters. This approach first uses a spline interpolation to generate continuous functions of system dynamics as well as the first and second order derivatives of continuous functions. The expanded dataset is the basis to infer unknown model parameters using various continuous optimization criteria, including the error of simulation only, error of both simulation and the first derivative, or error of simulation as well as the first and second derivatives. We use three case studies to demonstrate the accuracy and reliability of the proposed new approach. Compared with the corresponding discrete criteria using experimental data at the measurement time points only, numerical results of the ERK kinase activation module show that the continuous absolute-error criteria using both function and high order derivatives generate estimates with better accuracy. This result is also supported by the second and third case studies for the G1/S transition network and the MAP kinase pathway, respectively. This suggests that the continuous absolute-error criteria lead to more accurate estimates than the corresponding discrete criteria. We also study the robustness property of these three models to examine the reliability of estimates. Simulation results show that the models with estimated parameters using continuous fitness functions have better robustness properties than those using the corresponding discrete fitness functions.

Conclusions

The inference studies and robustness analysis suggest that the proposed continuous optimization criteria are effective and robust for estimating unknown parameters in mathematical models.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-256) contains supplementary material, which is available to authorized users.     

Expression of prostaglandin E2 receptor subtypes in the canine lower urinary tract varies according to the gonadal status and gender

Locally-synthesised prostaglandin E₂ (PGE₂) is pivotal for the function of the lower urinary tract (LUT). This study aimed at investigating the expression and distribution pattern of the four PGE₂ receptor (EP) subtypes in the LUT of intact and gonadectomised male and female dogs. Expression for EP1, EP2, EP3, and EP4 and their mRNA (EP2, EP3, and EP4) was investigated. Twenty clinically healthy dogs were allotted into 4 groups based on their gonadal status and gender including 5 intact males, 5 anoestrous females, 4 castrated males, and 6 spayed females. In situ hybridization and immunohistochemistry showed variation in the expression of mRNA and protein for the EP subtypes among tissue layers (epithelium, sub-epithelial stroma, and muscle), regions (body and neck of the bladder as well as proximal and distal urethra) and between gonadal statuses and genders. The expression for the four EPs was intense in the luminal epithelium, intermediate to low in the muscle and the sub-epithelial stroma regardless of gonadal status or gender. Higher expression of all EPs and their mRNAs was observed in the proximal urethra compared to other regions in intact dogs. However, in gonadectomised dogs, the expression did not differ among different regions and was generally lower than in intact dogs particularly in the proximal urethra. Differences in the expression between genders were found and depended on EP subtypes. In conclusion, the results have shown that four subtypes of EP receptors and their mRNAs are present in the canine LUT and their expression was affected by the gonadal status and the gender. The results lead to suggest that an impaired LUT function post-neutering may partly be associated with differences in PGE₂ receptor expression between intact and gonadectomised dogs.     

A mathematical model of hematopoiesis: II. Cyclical neutropenia

Cyclical neutropenia is a dynamical disease of the hematopoietic system marked by an oscillation in circulating leukocyte (e.g. neutrophil) numbers to near zero levels and then back to normal. This oscillation is also mirrored in the platelets and reticulocytes which oscillate with the same period. Cyclical neutropenia has an animal counterpart in the grey collie. Using the mathematical model of the hematopoietic system of Colijn and Mackey [A mathematical model of hematopoiesis: I. Periodic chronic myelogenous leukemia. Companion paper to the present paper.] we have determined what parameters are necessary to mimic laboratory and clinical data on untreated grey collies and humans, and also what changes in these parameters are necessary to fit data during treatment with granulocyte colony stimulating factor (G-CSF). Compared to the normal steady-state values, we found that the major parameter changes that mimic untreated cyclical neutropenia correspond to a decreased amplification (increased apoptosis) within the proliferating neutrophil precursor compartment, and a decrease in the maximal rate of re-entry into the proliferative phase of the stem cell compartment. For the data obtained during G-CSF treatment, good fits were obtained only when parameters were altered that would imply that G-CSF led to higher amplification (lower rate of apoptosis) in the proliferating neutrophil precursors, and a elevated rate of differentiation into the neutrophil line.     

Differences in the proportion of collagen and muscle in the canine lower urinary tract with regard to gonadal status and gender

Gonadectomy not only affects hormonal homeostasis but also alters the turnover of different components of the extracellular matrix in urogenital tissues. Collagen is an important component of the bladder and urethral walls and thus crucial for the mechanical properties of normal lower urinary tract (LUT) functions. This study aimed at investigating the possibility of differences in the proportion of collagen and muscle tissues in the LUT of intact and gonadectomised male and female dogs. Twenty clinically healthy dogs were used including 10 sexually intact dogs (5 males, 5 anoestrus females) and 10 gonadectomised dogs (4 males and 6 females). Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra were collected. The tissue sections were stained with Masson's Trichrome. Quantitative evaluation of the blue-stained area for collagen and red-counterstained area for muscle was performed using colour image analysis. The relative proportion of collagen and muscle significantly differed with the gonadal status, the gender and the region. Overall, gonadectomised dogs had a higher (P < 0.001) proportion of collagen and consequently a lower (P < 0.001) proportion of muscle than intact dogs. Regardless of gonadal statuses, females had a higher (P < 0.05) proportion of collagen and a lower (P < 0.05) proportion of muscle tissues than males. Gender differences were found in all four regions of the LUT in intact dogs but only in proximal urethra in gonadectomised dogs where spayed females had a higher (P < 0.05) proportion of collagen and less muscle (P < 0.05). Regional differences were observed in females; a higher proportion of collagen and therefore less muscle were found in the urethra compared with the bladder. Proportional differences in collagen and muscle between intact and gonadectomised animals suggest a relation of different hormonal statuses to structural changes in the canine LUT. Excessive collagen deposits and less muscular volume may impair structural and functional integrity of the LUT which may associate with the development of post-neutering urinary incontinence in the dog.     

Intramural neurons in the urinary bladder of the guinea-pig

Summary The urinary bladder of adult female guinea-pigs was stained histochemically to detect the presence of intramural ganglion neurons. Counts on wholemount preparations of entire bladders revealed the presence of 2000–2500 neurons per bladder, either as individual nerve cells or, more often, as ganglia containing up to 40 neurons. Both ganglia and single neurons lie along nerve trunks and are interconnected to form a plexus. Ganglia occur in every part of the bladder; they are more numerous on the dorsal than on the ventral wall, and they are especially abundant in an area within a radius of 800 m from the point of entry into the bladder wall of ureters and urinary arteries. The ganglia are located inside the muscle coat and close to muscle bundles; they usually lie nearer the mucosa than the serosa. Ultrastructurally, each ganglion is surrounded by a capsule; in addition to neurons and glial cells, the ganglia contain capillaries, collagen fibrils and fibroblasts; ganglion neurons are individually wrapped by glial cells and are separated from one another by connective tissue.     

Functional polymorphisms in the IL6 gene promoter and the risk of urinary bladder cancer in India

BackgroundInterleukin-6 is a multifunctional cytokine, which plays a key role in tumor proliferation and differentiation. Variations in its gene (IL6) sequence may affect the risk of developing various cancers, including urinary bladder cancer. The present study was done to find the association of functional polymorphisms in the IL6 promoter with urinary bladder cancer.Materials and methodsSingle nucleotide polymorphisms were genotyped in histologically confirmed 232 cases of urinary bladder cancer and 250 healthy controls. The controls subjects were matched to the cases by age, sex, and ethnicity. Genotyping of the polymorphisms (−174G>C; −572G>C, −596A>G) was undertaken by direct DNA sequencing. The level of association between the genotypes and urinary bladder cancer risk was estimated by odds ratios and 95% confidence intervals generated by applying the chi-square test. Linkage disequilibrium (LD) between SNPs and haplotype analysis were performed using Haploview software.ResultSignificantly higher number of smokers (p = 0.047), tobacco chewers (p = <0.001) and those with non-vegetarian food habits (p = 0.016) were seen in the case group. The distribution of genotypes at −174G>C locus differed significantly between cases and controls and the variant genotypes GC + CC were significantly rarer in the cases (p = 0.00073; OR = 0.52 95% CI 0.35–0.75). Variant genotypes (GC + CC) were more common in grade I than grade III tumors (p = 0.032), further suggesting a protective effect. No LD was found between the SNPs; however, the frequency of haplotype AGC was significantly lesser in the cases than controls (p = 0.0103), suggesting a protective effect. Genotype distribution at the other two loci (−572G>C and −596A>G) did not show association with bladder cancer.ConclusionsIL6 (−174G>C) substitution confers significant protection against the risk of urinary bladder cancer in the study population, while other substitutions in this gene (−572G>C and −596A>G) do not affect the risk. In general, there is a lack of studies on the cytokine gene polymorphisms in urinary bladder cancer.     

Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

A mathematical model of canine granulocytopoiesis

Summary The granulocyte cell renewal system of the dog is represented by a mathematical model consisting of the following compartments: The pool of pluripotential stem cells, the committed stem cell pool, divided into a blood and a bone marrow compartment, the proliferation pool, the maturation pool, the reserve pool and the blood pool of functional granulocytes. This chain of compartments is described by a system of non-linear differential equations. Cell losses anyplace in the system provoke increased production in all pools containing cells capable to divide. A reduced number of granulocytes in the blood pool stimulates production of a granulocyte releasing factor which mobilizes a rising number of cells to transit from the marrow reserve into the blood pool.The model was simulated on a digital computer. It was found to be capable to reproduce the steady state conditions and it also fits the data of two distinct experimental perturbations of the system both equally well. These perturbations are a loss of proliferating cells as it occurs after the administration of cytostatic drugs and losses of functional cells as they are induced by leukapheresis experiments of differing leukapheresis rates.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 112)     

Measurement of DNA damage in rat urinary bladder transitional cells: Improved selective harvest of transitional cells and detailed Comet assay protocols

Urinary bladder transitional epithelium is the main site of bladder cancer, and the use of transitional cells to study carcinogenesis/genotoxicity is recommended over the use of whole bladders. Because the transitional epithelium is only a small fraction of the whole bladder, the alkaline single cell gel electrophoresis assay (Comet assay), which requires only a small number of cells per sample, is especially suitable for measuring DNA damage in transitional cells. However, existed procedures of cell collection did not yield transitional cells with a high purity, and pooling of samples was needed for Comet assay. The goal of this study was to develop an optimized protocol to evaluate DNA damage in the urinary bladder transitional epithelium. This was achieved by an enzymatic stripping method (trypsin–EDTA incubation plus gentle scraping) to selectively harvest transitional cells from rat bladders, and the use of the alkaline Comet assay to detect DNA strand breaks, alkaline labile sites, and DNA–protein crosslinks. Step by step procedures are reported here. Cells collected from a single rat bladder were sufficient for multiple Comet assays. With this new protocol, increases in DNA damage were detected in transitional cells after in vitro exposure to the positive control agents, hydrogen peroxide or formaldehyde. Repair of the induced DNA damage occurred within 4 h. This indicated the capacity for DNA repair was maintained in the harvested cells. The new protocol provides a simple and inexpensive method to detect various types of DNA damage and to measure DNA damage repair in urinary bladder transitional cells.     

The origin of VIP-containing nerves in the urinary bladder of rat

The innervation of the urinary bladder is known to include a considerable number of nerves containing vasoactive intestinal polypeptide (VIP). The origin of such nerves in the bladder of rat was investigated in this study using the methods of immunocytochemistry and radioimmunoassay combined with surgical sectioning of the hypogastric and/or pelvic nerves to the bladder. Eight days after pelvic nerve sectioning proximal to the main pelvic ganglion, VIP-immunoreactive nerves and VIP content were markedly increased from the level in the sham-operated rat bladder. Sectioning of hypogastric or both nerve pathways led to a less significant increase. It was therefore postulated that the majority of VIP-immunoreactive nerves originate from ganglia located either close to the bladder or within the bladder wall. It is interesting that in these experiments the VIP content of the bladder nerves is inversely related to the changes in motility that would be expected to result from the nerve sections.     

A vascular network closely linked to the epithelium of the urinary bladder of the rat

Summary Scanning electron microscopy was used on the mucosa of the rat urinary bladder after digestion with strong alkali and microdissection. The underside of the epithelium (and the plane of the epithelium-tunica propria interface) is not smooth but is scored by grooves-10 m wide and 3–4 m deep—connected into a fine mesh. A net of blood capillaries located in the uppermost part of the tunica propria occupies these grooves. They measure 3–9 m in diameter, are separated from the epithelium by a gap of 0.3 m, often show fenestrations, and are accompanied by numerous and extensive pericytes and by some fibroblasts. We discuss these observations in the light of current knowledge of blood flow in the bladder, contraction and distension of the bladder wall and formation of mucosal folds, transport of solutes through the epithelium, and plasma extravasation from mucosal blood vessels in neurogenic inflammation.     

Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder

The expression of a number of enzymes involved in drug metabolism, membrane function etc. was compared in hyperplastic and neoplastic lesions of the rat bladder and in human bladder tumours. Transitional cell carcinomas (TCC) in both rat and Man were characterized by decreased alkaline phosphatase (ALP) and increased gammaglutamyl transpeptidase (GGT), β-glucuronidase (β-Gl), succinate dehydrogenase (SD) and glucose-6-phosphate dehydrogenase (G6PD) activities. In addition, binding for antibodies specific for different cytochrome P-450 species (UT₅₀, PB_3a, MC₁, MC₂) and microsomal epoxide hydrolase (mEHb) was elevated in both murine and human tumours. Comparison of the enzyme phenotype in hyperplastic lesions induced by freeze ulceration or uracil administration with that in preneoplastic papillary or nodular hyperplasia (PNH) and TCC suggested, however, that most of the alteration in enzyme content or activity was non-specific and related to requirements for epithelial cell proliferation. On the other hand, the decreased ALP, and increased GGT and β - Gl activity appeared more directly related to neoplastic transformation. The results suggested that qualitative differences exist between reactive hyperplasia and preneoplastic or neoplastic lesions in the urinary bladder. The finding of increased cytochrome P-450, in clear contrast to the reduction characteristic of preneoplastic hepatic lesions, may be important with regard to the observed difference in neoplastic transformation between the bladder and liver in response to drug metabolising enzyme inducers. Supported by Grants-in-aid for Cancer Research and a Comprehensive 10 Year Strategy for Cancer Control from the Ministry of Health and Welfare, for Cancer Research and Scientific Research from the Ministry of Education, Science and Culture, and by a grant from the Deutsche Forschungsgemeinschaft