Experimental Biology and Medicine

Because resolving human complex diseases is difficult, appropriate biomedical models must be developed and validated. In the past, researchers have studied diseases either by characterizing a human clinical disease and choosing the most appropriate animal model, or by characterizing a naturally occurring or induced mutant animal and identifying which human disease it best resembled. Although there has been a great deal of progress through the use of these methods, such models have intrinsic faults that limit their relevance to clinical medicine. The recent advent of techniques in molecular biology, genomics, transgenesis, and cloning furnishes investigators with the ability to study vertebrates (e.g., pigs, cows, chickens, dogs) with greater precision and utilize them as model organisms. Comparative and functional genomics and proteomics provide effective approaches for Identifying the genetic and environmental factors responsible for complex diseases and in the development of prevention and treatment strategies and therapeutics. By identifying and studying homologous genes across species, researchers are able to accurately translate and apply experimental data from animal experiments to humans. This review supports the hypothesis that associated enabling technologies can be used to create, de novo, appropriate animal models that recapitulate the human clinical manifestation. Comparative and functional genomic and proteomic techniques can then be used to identify gene and protein functions and the Interactions responsible for disease phenotypes, which aids in the development of prevention and treatment strategies.

Intestinal crypts are composed of a well-defined hierarchy of epithelial cells, and proliferating epithelial cells reside close to the bottom of the crypts—even in the large intestine. We investigated whether CD8⁺ and CD4⁺ intraepithelial lymphocytes (IELs) and CD161⁺ natural killer (NK) cells localized in proliferating or differentiated epithelial region of cecum and colon. Both proliferating epithelial layer cells and the immune cells along the longitudinal crypt axis of the large intestine were measured histochemically. Dietary intervention revealed that the physiological localization of the immune cells in the longitudinal crypt axis depended on the immune cell type. CD8⁺ IELs were preferentially located among differentiated epithelial cells. In contrast, CD161⁺ NK cells were located adjacent to the epithelial cells at the bottom of crypt. Cecal crypts contained significantly larger numbers of CDS⁺ IELs than did colonic crypts. However, there was only a minor population of CD4⁺ IEL in the cecal and colonic epithelia. Some dietary fibers increased the densities of CD8⁺ IELs and CD161⁺ NK cells in the cecum, with the magnitude of response varying among the types of fiber. There was a significant relationship between SCFA and the localization of Immune cells, especially CD8⁺ IEL and CD161⁺ NK celts, which are considered to be involved in the maintenance of epithelial homeostasis.

Cytochrome c oxidase (CCO) is the Cu-dependent, terminal respiratory complex of the mitochondrial electron transport chain. Inhibition of CCO can promote oxidative stress by increasing mitochondrial production of reactive oxygen species (ROS). Because mitochondria have an important role in apoptosis as both a target and source for ROS, enhanced ROS production resulting from inhibition of CCO by Cu deficiency may trigger apoptosis. The present study focuses on the mitochondrial effects of N,N'-bis(2-aminoethyl)-1, 3-propanediamine (TET), which inhibits CCO by causing cellular Cu deficiency, and the antioxidants ascorbate and α-tocopherol in a human promyelocyte leukemia cell line (HL-60). The following effects were observed: (i) TET reduced both cell growth and viability only in the presence of ascorbate or α-tocopherol; (ii) TET reduced CCO activity and increased mitochondrial ROS production as indicated by increased expression of Mn superoxide dismutase, but the induction of Mn superoxide dismutase was not affected by ascorbate or α-tocopherol; (iii) TET acted independently of ascorbate or α-tocopherol in disrupting mitochondrial membrane potential; (iv) TET did not increase caspase-8 activity in the absence of ascorbate or α-tocopherol; and (v) TET did not increase transfer of cytochrome c from mitochondria to the cytosol unless α-tocopherol was present. These findings indicate that reduction in CCO activity by TET-Induced Cu deficiency increased oxidative stress in HL-60 cells sufficiently to disrupt the electrochemical gradient of the inner mitochondrial membrane but did not trigger cell death. Also, ascorbate and α-tocopherol did not alleviate oxidative stress but may have become pro-oxidants, adding to the oxidant burden sufficiently to trigger cell death in TET-treated cells.

Preceding studies have revealed that gum arabic (GA), a natural proteoglycan (≥250, 000 Da), has proabsorptive properties—as shown by increased sodium and water absorption—in normal rats, and especially in two animal models of diarrhea. Because nitric oxide (NO) metabolism is linked to gastrointestinal Physiology, the goals of this study were to determine whether GA modulated NO and to determine intestinal function in vivo when NO production was enhanced by l-arginine (Arg), added at either 1 or 20 mM. Mechanistically, the goal was also to determine whether GA was a NO scavenger and a small intestinal NO synthase (NOS) inhibitor. Using a glucose-electrolyte solution in rat jejunal perfusions we found that GA at ± 10 μM (2.5 g/l) decreased nitrite and nitrate formation, tending to normalize water, sodium, and glucose absorption when modified by Arg addition. In vitro tests, with oxyhemoglobin as a marker, showed that GA at ≥5 μM scavenged NO. For GA effects on NOS, small intestinal homogenate supematants (10, 000 g) from frozen tissues of either adult or 2-day-old rats were incubated for 1 hour at 37°C in the presence of 2 mM Arg and increasing GA concentrations (0-100 μM). GA produced a concentration-dependent inhibition of NOS, reaching approximately 31% inhibition with 5 μM GA and up to 51% with 50 μM GA. GA at 100 μM produced no further inhibition. The data indicate that GA, in addition to its ability to remove NO diffused into the intestinal lumen, may also partially inhibit intestinal NOS end thus modulate intestinal absorption through these mechanisms. Use of GA as a food additive may help in restoring or improving small Intestinal function in conditions where functional damage has occurred.

Particular intestinal bacteria metabolize the soy isoflavone daidzein to equol and O-desmethylangolensin (O-DMA), metabolites that can be identified in urine. Individuals that harbor bacteria capable of producing equol or O-DMA are known as equol producers (approximately 30%-50% of the population) and O-DMA producers (approximately 80%-90% of the population), respectively. The equol-producer phenotype has been associated with sex hormone-related outcomes in several studies. However, the bacteria responsible for these phenotypes have not yet been identified and factors that influence the manifestation of these phenotypes are not well understood. To evaluate familial clustering of and nongenetic factors associated with these phenotypes, 410 individuals from 112 families participated in phenotyping (3-day soy challenge and Day 4 spot urine collection). In segregation analyses of the equol-producer phenotype, the Mendelian dominant model provided the most parsimonious fit to the data, suggesting that the pattern of inheritance of the equol-producer phenotype is consistent with an autosomal dominant trait. This phenotype was positively associated with education (p trend = 0.01), but not with sex, smoking, or several dietary factors. Results of the segregation analyses of the O-DMA-producer phenotype were inconclusive; no other models provided a more parsimonious fit to the data than the general model. This phenotype was inversely associated with age in a nonlinear model (p = 0.01), positively associated with age- and sex-adjusted height (odds ratio [OR] 10-cm increase = 0.38, 95% confidence interval [CI] = 0.15, 0.95) and body mass index (kg/m²) (OR = 0.91, 95% CI = 0.85, 0.96), but not with sex, education, smoking, or several dietary factors. These results suggest the equol-producer phenotype may be under some degree of genetic control and that there are likely other environmental factors not evaluated in the present analysis that contribute to both of these phenotypes. These results provide a foundation for further work to refine our understanding of heritable and environmental determinants of daidzein-metabolizing phenotypes.

We have reported that dietary inorganic phosphate (Pi) deprivation induces a Pi-seeking behavior in juvenile male rats. The purpose of the present study was to determine whether the Pi appetite is present in adult animals, and if so, whether it is altered during times of increased demand for Pi, such as pregnancy and lactation. Both male and female animals fed a low-phosphate diet (LPD) ingested significantly greater amounts of PiH₂0 daily than their normal phosphate diet (NPD) controls, and per 100 g of body weight (BW), the female animals fed LPD tended to ingest greater amounts of PiH₂0 than male rats fed LPD. Pregnant and lactating rats fed LPD ingested significantly more PiH₂0 than those fed NPD, however, neither group displayed a Pi appetite different than virgin females. However, lactation further reduced Pi levels in plasma and cerebral spinal fluid compared with control values. Despite the additional Pi from the PiH₂0 in the mothers fed LPD, pup birth weight was significantly lower than in NPD litters, and this was exacerbated 9 days after birth. This attenuated BW gain was associated with lower plasma Pi levels in the pups. In conclusion, a mild but consistent Pi-seeking behavior is induced in adult male and female rats after only 2 days of dietary Pi restriction. On a relative basis, the amount of PiH₂0 ingested is greater in female than in male animals, but does not increase further during pregnancy and lactation.

We hypothesized that the phosphodiesterase 5 inhibitor, sildenafil, and the guanosine cyclase stimulator, atrial natriuretic peptide (ANP), would act synergistically to increase cGMP levels and blunt hypoxic pulmonary hypertension in rats, because these compounds act via different mechanisms to increase the intracellular second messenger. Acute hypoxia: Adult Sprague-Dawley rats were gavaged with sildenafil (1 mg/kg) or vehicle and exposed to acute hypoxia with and without ANP (10-⁸ -10-⁵ M). Sildenafil decreased systemic blood pressure (103 ± 10 vs. 87 ± 6 mm Hg, P < 0.001) and blunted the hypoxia-induced increase in right ventricular systolic pressure (RVSP; percent increase 73.7% ± 9.4% in sildenafil-treated rats vs. 117.2% ± 21.1% in vehicle-treated rats, P = 0.03). Also, ANP and sildenafil had synergistic effects on blunting the hypoxia-induced increase in RVSP (P < 0.001) and on rising plasma cGMP levels (P < 0.05). Chronic hypoxia: Other rats were exposed to prolonged hypoxia (3 weeks, 0.5 atm) after subcutaneous implantation of a sustained-release pellet containing lower (2.5 mg), or higher (25 mg) doses of sildenafil, or placebo. Higher-dose, but not lower-dose sildenafil blunted the chronic hypoxia-induced increase in RVSP (P = 0.006). RVSP and plasma sildenafil levels were inversely correlated in hypoxic rats (r² = 0.68, P = 0.044). Lung cGMP levels were increased by both chronic hypoxia and sildenafil, with the greatest increase achieved by the combination. Plasma and right ventricular (RV) cGMP levels were increased by hypoxia, but sildenafil had no effect. RV hypertrophy and pulmonary artery muscularization were also unaffected by sildenafil. In conclusion, sildenafil and ANP have synergistic effects on the blunting of hypoxia-induced pulmonary vasoconstriction. During chronic hypoxia, sildenafil normalizes RVSP, but in the doses used, sildenafil has no effect on RV hypertrophy or pulmonary vascular remodeling.

In the present investigation, 17ß-estradiol (E₂) and tamoxifen, an antiestrogen, were evaluated for their effects on the release of ascorbic acid (AA) and luteinizing hormone-releasing hormone (LHRH). Medial basal hypothalami (MBH) from adult male rats were incubated with graded concentrations of E₂ (10-⁹ to 10-⁶ M) or a combination of E₂ (10-⁷ M) and tamoxifen (10-⁷ and 10-⁶ M) in 0.5 ml of Krebs Ringer bicarbonate buffer for 1 hr. AA and LHRH in the incubation medium were measured by high-performance liquid chromatography and radioimmunoassay, respectively. E₂ significantly elevated both AA and LHRH release and the minimal effective dose was 10-⁷ M. A combination of E₂ (10-⁷ M) and tamoxifen (10-⁶ M) totally blocked E₂-induced AA and LHRH release. The stimulatory effect of E₂ was also suppressed in the presence of N^G-monomethyl-l-arginine, a competitive inhibitor of nitric oxide synthase (NOS), illustrating that the release is mediated by nitric oxide (NO). To further characterize the role of NO, the tissues were incubated with E₂ or a combination of E₂ + (6 anllino-5, 8-quinolinedione) LY 83583 (10-⁶ and 10-⁵ M), an inhibitor of NOS. LY 83583 was effective in suppressing E₂-induced AA and LHRH release, demonstrating that the effect was mediated by cyclic GMP. Incubation of the tissues with E₂ or a combination of E₂ + 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxalin-1-one (O.D.Q.) (10-⁵ and 10-⁴ M), a specific inhibitor of soluble guanylyl cyclase failed to alter AA release but significantly suppressed LHRH release. The role of a prostaglandin synthesis blocker in E₂-lnduced AA and LHRH release was tested by incubating the tissues with E₂ or a combination of E₂ + Indomethacin (1.8 × 10-⁷ or 1.8 × 10-⁶ M). Indomethacin produced a significant decrease in E₂-induced AA and LHRH release, suggesting that the release process required prostaglandins as an intracellular mediator. In conclusion, E₂ stimulated both AA and LHRH release and the effect was mediated by NO and prostaglandins.

Behavioral phenotypes of knockout mice are often interpreted as the effects of the absence of the gene product on adult behavior, yet behavioral differences among genotypes may be exaggerated or blurred by the postnatal environment. For example, mice develop in litters of varying sex ratios and genotypes, and it is possible that some of these behavioral differences may result from the composition of the litter. To determine whether these factors might play a role in the development of the behavioral characteristics that have become diagnostic of the knockout, offspring of parents heterozygous for a null mutation of estrogen receptor α (ERKO) were sexed and genotyped within 2 days of birth. Litters were then reconstituted, forming same-sex litters of equal numbers of ERKO and wild-type (WT) individuals that were tested in a standard resident-intruder paradigm. In this manner the effect of genotype would be evident without the potential confound of the presence of the opposite sex in the litter. Behavioral differences between the genotypes were more sharply defined than reported previously. ERKO females displayed only aggressive behavior whereas their WT littermates displayed only mounting behavior; both aggression and mounting behavior were greatly reduced in ERKO males. These data suggest that the postnatal environment such as litter composition may influence the development of sociosexual behaviors in ERKO mice.

This study examined whether charting daily weight patterns can predict weight regain in obese patients. The subjects were 98 moderately obese Japanese women aged 23 to 66 years who were obliged to precisely record their daily weights during the initial 4-month education period, but not thereafter. The patients were followed up at 8, 12, and 16 months. Abdominal fat areas and blood samples were assessed in the outpatient clinic at 0, 4, and 16 months. The standard deviations (SDs) of the differences in body weight between “after waking up” and “after breakfast” (SDa), “after dinner” (SDb), and “before going to bed” (SDc) were calculated, which were parameters reflecting the fluctuations in the daily weight patterns during the first 4 months. SDc, but not SDa or SDb, was correlated positively with weight regain at 8, 12, and 16 months (P = 0.049, P = 0.002, and P = 0.001, respectively). There were significant differences in temporal change in body weight and abdominal visceral fat between the small SDc group (SDc ≤25th percentile) and the large SDc group (SDc >75th percentile), but not for subcutaneous abdominal fat or the serum concentrations of glucose, insulin, or lipids. The results indicate that fluctuation of body weight immediately before going to bed is useful for predicting the rebound in body weight.

Earlier studies indicate that J6-1 human leukemic cells proliferate and propagate via the membrane-bound macrophage colony-stimulating factor (M-CSF)-mediated auto-juxtacrine mechanism. Matrix metalloproteinases (MMPs) can modulate the activity of cell membrane molecules and influence many cellular behaviors. Therefore, we hypothesized that MMP may also be involved in the membrane-bound M-CSF-mediated juxtacrine mechanism. First, we investigated whether blocking of membrane-bound M-CSF by neutralizing antibody to M-CSF or M-CSF receptor and adding of exogenous M-CSF are able to influence MMP-9 release. Next, we determined whether MMP-9 participated in J6-1 cells proliferation and influence the shedding of membrane-bound M-CSF and its receptor. Current studies show that blockade of the interaction between membrane-bound M-CSF and M-CSF receptor by antibody to M-CSF or M-CSF receptor promotes MMP-9 release. Moreover, we demonstrated that because of M-CSF mediated juxtacrine, lack of MMP-9 promotes J6-1 cell proliferation, in which a decrease in the shedding of cell-surface M-CSFR is involved. Hence, we suggest that membrane-bound M-CSF inhibit MMP-9 release and down-regulated MMP-9 contribute to juxtacrine stimulating in leukemic cell growth.

A novel nonhydrolyzable ether derivative of RRR-α-tocopherol, RRR-α-tocopherol ether acetic acid analog [2, 5, 7, 8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (α-TEA)], and a hydrolyzable ester derivative RRR-α-tocopheryl succinate (vitamin E succinate; VES) inhibited BALB/c mouse 66cl-4-GFP mammary tumor cell growth in vitro and in vivo. Treatment of 66cl-4-GFP cells in culture with α-TEA or VES Induced dose-dependent DNA synthesis arrest and apoptosis and inhibited colony formation. Liposomal formulations of α-TEA delivered orally or by aerosol significantly reduced subcutaneous 66cl-4-GFP tumor burden and metastasis to lung and lymph nodes. Liposomal formulations of VES delivered by aerosol significantly reduced tumor burden and lung metastasis, but not lymph node metastasis. Unlike α-TEA, VES was ineffective in reducing tumor burden and metastasis to lungs and lymph nodes when administered orally. Analyses of tumor sections showed that α-TEA delivered by either method significantly reduced tumor cell proliferation as measured by KI67, and increased apoptosis as measured by terminal deoxynucleotldyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), whereas VES delivered by aerosol reduced tumor cell proliferation and increased apoptosis, but not significantly. In summary, the nonhydrolyzable ether vitamin E derivative α-TEA was effective in reducing tumor burden and metastasis when delivered either by aerosol or orally, whereas the hydrolyzable ester vitamin E derivative VES was effective only when delivered by aerosol.

Recently, the novel podocyte proteins podocin, nephrin, and α-actinin-4 have been identified in three congenital/family nephrotic syndromes, respectively. Further studies showed that these podocyte proteins were involved in some acquired nephrotic syndromes and various experimental models of proteinuria. However, the molecular interactions among these podocyte proteins remain unclear. In this study, to investigate the molecular interactions among podocin, nephrin, and α-actinin-4, we reconstructed the RNA interference (RNAI) expression vector, pSilencer 2.1-U6, specifically targeting podocin mRNA, and it was transfected into the mouse podocyte clone (MPC5). Immunofluorescence staining, double-immunolabeling, confocal microscopy, semiquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the distribution and expression of podocin, nephrin, α-actinin-4, and glyseraldehyde-3-phosphate dehydrogenase (GAPDH)/β-actin. The fluorescence intensity of podocin and nephrin decreased obviously, along with the evident distribution change from the cell membrane surface to the nucleus circumference in podocyte. In relation to GAPDH, the mRNA reductions of podocin and nephrin were observed by about 65% and 70%, respectively. The expression of podocin protein was too low to be detected in the interference group. In relation to β-actin, the protein level of nephrin decreased by about 78%. The distribution and the mRNA and protein level of α-actinin showed no appreciable change. Alpha-actinin localized mainly in the cytoplasm and also extended to the processes. Thus, the significant decreased expression of nephrin along with the redistribution were detected with the knockdown of podocin mRNA, whereas the expression and distribution of α-actinin-4 showed no change. These results suggest that podocin may interact directly with nephrin, but not with α-actinin.

The heart is one of a number of organs that may be affected in systemic lupus erythematosus (SLE), a prototypic autoimmune disease. Potential anatomical sites of involvement include the myocardium, pericardium, endocardium, valves, conduction system and blood vessels that subserve the heart. Typically, the severity of cardiovascular disease in lupus correlates with the degree of systemic inflammation, which is mirrored by the level of C-reactive protein (CRP) in the plasma. C-reactive protein, in turn is regulated by proinflammatory cytokines, such as interleukins (ILs) 1ß and 6. These cytokines have been found in functionally and/or structurally damaged areas of the heart and have been implicated in disease pathogenesis. It has been assumed that the source of these putatively pathogenetically relevant cytokines in the compromised heart is infiltrating mononuclear cells. This study tests the hypothesis that cardiomyocytes per se may contribute to proinflammatory cytokine production in the setting of systemic inflammation. Using as the experimental model MRL/MpJ-rnfrs6^lpr (MRL-lpr/ lpr) mice, which spontaneously manifest an autoimmune syndrome that has clinical features of SLE, we show that ventricular homogenates and ventricular cardiomyocytes constitutively overexpress genes encoding the proinflammatory cytokines IL-1ß, IL-6, IL-10, and gamma interferon. The results suggest the possibility that proinflammatory cytokines emanating from the heart may actually contribute to the high levels of CRP that appear to aid in predicting subsequent cardiac events. Viewed in this setting, CRP becomes a footprint of an ongoing pathogenic process mediated, in part, by the heart muscle itself.