Transport of thiamine in human intestine: mechanism and regulation in intestinal epithelial cell model Caco-2

The present studyexamined the intestinal uptake of thiamine (vitaminB₁) using the human-derivedintestinal epithelial cells Caco-2 as an in vitro model system.Thiamine uptake was found to be 1)temperature and energy dependent and occurred with minimal metabolicalteration; 2) pH sensitive;3)Na⁺ independent;4) saturable as a function ofconcentration with an apparent Michaelis-Menten constant of 3.18 ± 0.56 µM and maximal velocity of 13.37 ± 0.94 pmol · mgprotein¹ · 3 min¹;5) inhibited by the thiaminestructural analogs amprolium and oxythiamine, but not by unrelatedorganic cations tetraethylammonium, N-methylnicotinamide, and choline; and6) inhibited in a competitive mannerby amiloride with an inhibition constant of 0.2 mM. The role ofspecific protein kinase-mediated pathways in the regulation of thiamineuptake by Caco-2 cells was also examined using specific modulators ofthese pathways. The results showed possible involvement of aCa²⁺/calmodulin (CaM)-mediatedpathway in the regulation of thiamine uptake. No role for proteinkinase C- and protein tyrosine kinase-mediated pathways in theregulation of thiamine uptake was evident. These results demonstratethe involvement of a carrier-mediated system for thiamine uptake byCaco-2 intestinal epithelial cells. This system isNa⁺ independent and is differentfrom the transport systems of organic cations. Furthermore, aCaM-mediated pathway appears to play a role in regulating thiamineuptake in these cells.

     

Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells

After the liver, the pancreas contains the second highest level of folate among human tissues, and folate deficiency adversely affects its physiological function. Despite that, nothing is currently known about the cellular mechanisms involved in folate uptake by cells of this important exocrine organ or about folate uptake regulation. We have begun to address these issues, and in this report we present the results of our findings on the mechanism of folate uptake by the human-derived pancreatic MIA PaCa-2 cells. Our results show folic acid uptake to be 1) temperature and energy dependent; 2) pH dependent, with a markedly higher uptake at acidic pH compared with neutral or alkaline pH; 3) Na⁺ independent; 4) saturable as a function of substrate concentration (apparent K_m = 0.762 ± 0.10 µM); 5) inhibited (with similar affinity) by reduced, substituted, and oxidized folate derivatives; and 6) sensitive to the inhibitory effect of anion transport inhibitors. RT-PCR and Western blot analysis showed expression of the human reduced folate carrier (hRFC) at the RNA and protein levels, respectively. The functional contribution of hRFC in carrier-mediated folate uptake was confirmed by gene silencing using gene-specific small interfering RNA. Evidence also was found suggesting that the folate uptake process by MIA PaCa-2 cells is regulated by cAMP- and protein tyrosine kinase (PTK)-mediated pathways. These studies demonstrate for the first time the involvement of a specialized, acidic pH-dependent, carrier-mediated mechanism for folate uptake by human pancreatic MIA PaCa-2 cells. The results also show the involvement of hRFC in the uptake process and suggest the possible involvement of intracellular cAMP- and PTK-mediated pathways in the regulation of folate uptake. human reduced folate carrier; small interfering RNA; transport regulation     

Fermentation Kinetics of Media Optimization for the Production of Alpha Amylase by a New Isolate of Aspergillus Oryzae

The present study is concerned with the isolation and screening of different strains of Aspergillus oryzae for the production of alpha amylase. Ninety strains were isolated from soil and tested for the production of alpha amylase in shake flasks. Of all the strains tested, Aspergillus oryzae GCB-32 and Aspergillus oryzae GCB-35 gave maximum production of alpha amylase. Different culture media were screened for the production of alpha amylase by these two strains. M1 medium containing starch, yeast extract, NH₄Cl, MgSO₄·7H₂O and CaCl₂ gave the maximum production of alpha amylase by both the strains Aspergillus oryzae GCB-32 and Aspergillus oryzae GCB-35.Kinetic analysis revealed that the values of product yield coefficient(Y_p/x) and specific product yield coefficient(q_p) were found highly significant(p≤0.05) when medium M1 was used for the enzyme production.     

Exposure to Leishmania major modulates the proportion of CD4+ T cells without affecting cellular immune responses

The immune responses of individuals exposed to Leishmania major were evaluated and compared with those of non-exposed volunteers. Forty-one patients with active lesion(s), 43 healed individuals, 15 vaccinees 1 month or 1 year post vaccination, and 15 non-exposed volunteers were studied. Leishmanin skin test (LST) response, proliferative response of lymphocyte (PRL) to L. major antigen, IFN-gamma and IL-4 production, and percentage of L. major-specific CD4+, CD8+ and CD16+/CD56+ cells in peripheral blood mononuclear cells were assessed. Data showed positive LST (>5 mm) in 92% of patients, 98% of healed, and 80% or 43% of vaccinees 1 month and 1 year post vaccination, respectively. Positive PRL (SI>2.5) was displayed in 90%, 84%, 46% and 7% of patients, healed, vaccinated (post 1 year) and non-exposed donors, respectively. The mean +/-S.E. of IFN-gamma was 924 +/- 149, 1,278 +/- 185, 470 +/- 282 or 258 +/- 82 pg/ml in patients, healed cases and vaccinees after 1 month or 1 year, respectively. Positive IFN-gamma responders (>300 pg/ml) were shown in 72% of patients, 81% of healed cases, 31% or 39% of vaccinees and 0% of non-exposed donors. A reduced percentage of CD4+ T-cells and an increased percentage of NK cells were found in exposed individuals compared to non-exposed donors. The data indicated that exposure to L. major modulates the proportion of CD4+ T cells and increases NK cells percentage. However, the cellular immune responses including induction of LST, and IFN-gamma production are increased in exposed individuals.     

Biosurfactant production by Mucor circinelloides: Environmental applications and surface‐active properties

Biosurfactants are structurally a diverse group of surface‐active molecules widely used for various purposes in industry. In this study, among 120 fungal isolates, M‐06 was selected as a superior biosurfactant producer, based on different standard methods, and was identified as Mucor circinelloides on the basis of its nucleotide sequence of the internal transcribed spacer (ITS) gene. M. circinelloides reduced the surface tension to 26 mN/m and its EI₂₄ index was determined to be 66.6%. The produced biosurfactant exhibited a high degree of stability at a high temperature (121°C), salinity (40 g/L), and acidic pH (2–8). The fermentation broth's ability to recover oil from contaminated sand was 2 and 1.8 times higher than those of water and Tween 80, respectively. The ability of biosurfactant to emulsify crude oil in the sea and fresh water was 64.9 and 48% respectively. This strain could remove 87.6% of crude oil in the Minimal Salt Medium (MSM) crude oil as the sole carbon source. The results from a primary chemical characterization of crude biosurfactant suggest that it is of a glycolipid nature. The strain and its biosurfactant could be used as a potent candidate in bioremediation of oil‐contaminated water, soil, and for oil recovery processes.     

Insights into in vitro spermatogenesis in mammals: Past,present, future

Considering the self‐renewal and differentiation ability of pluripotent stem cells, some studies have pointed out the possibility of stem cell‐derived sperm production. Most studies that test this hypothesis have been conducted on rodents, with some promising results; however, studies on humans are progressing slowly, and have encountered technical and ethical hurdles. Established methods to differentiate stem cells—including embryoid bodies, co‐culturing, and various feeder cells—may provide a niche that is similar to in vivo conditions and resolve epigenetic abnormalities, but a gonadal‐like three‐dimensional structure is still required to produce germ cells with the correct imprinting. In the last few years, sperm‐like cells with fertilizing capacity were produced from mouse embryonic stem cells, and the resulting embryos from these cells yielded live offspring. Future research should move towards the use of adult stem cells, however, owing to the unavailability of embryonic cells in adults. More intensive research and techniques are required since in vitro spermatogenesis provides hope to individuals without mature sperm who cannot be treated, and may be a useful system to study the precise mechanism of spermatogenesis. In this review, we describe recent studies of in vitro spermatogenesis mechanisms and related techniques in mammals. We also discuss the possible cell surface markers and culture conditions that might improve in vitro spermatogenesis.     

Impaired spermatogenesis associated with changes in spatial arrangement of Sertoli and spermatogonial cells following induced diabetes

The current study was conducted to assess the relationship between testicular cells in spermatogenesis, through which the production of healthy and mature sperm is essential. However, it seems necessary to obtain more information about the three-dimensional pattern of the testis cells arrangement, which is directly related to the function of the testis after induction of diabetes. Twelve adult mice (28-30 g) were assigned into two experimental groups: (1) control and (2) diabetic (40 mg/kg STZ). The epididymal sperm collected from the tail of the epididymis and testes samples were taken for stereology, immunocytochemistry and RNA extraction. Our data showed that diabetes could notably decrease the number of testicular cells, together with a reduction of total sperm count. In addition, the results from the second-order stereology indicated the significant changes in the spatial arrangement of Sertoli cells and spermatogonial cells in the diabetic groups, in comparison with the control (P < .05). Moreover, the immunohistochemistry results showed a significant reduction in Sex-determining Region Y (SRY) box 9 gene (SOX9), vimentin, occludin, and connexin-43 positive cells in the diabetic groups compared with the control (P < .05). Furthermore, our data showed that the expression of steroidogenic acute regulatory protein steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine receptor peripheral benzodiazepine receptor (PBR) was significantly reduced in the diabetic groups, in comparison with the control (P < .05). These findings suggest that structural and functional changes of testis cells after induction of diabetes cause the alterations in the spatial arrangement of Sertoli and spermatogonial cells, ultimately influencing the normal spermatogenesis in mice.     

Localization of neural cell adhesion molecule (N-CAM) immunoreactivity in adult rat tissues

Neural cell adhesion molecule (N-CAM) mediates homophilic adhesion between cells and heterophilic adhesion between cells and extracellular matrix in a Ca²⁺-independent manner. N-CAM is widely expressed during development and plays a crucial role in cell division, migration, and differentiation, but its expression is restricted in adults. The distribution of N-CAM immunoreactivity in adult rat tissues was investigated in the present study. N-CAM immunoreactivity was present in the nervous system in the molecular layer of the cerebellum, ependymal cells surrounding the central canal, axons of the white matter, and in Lamina X of the gray matter of the spinal cord. N-CAM immunoreactivity also was found in autonomic nerves. In the digestive system, N-CAM immunoreactivity was found in the stratified squamous epithelium and nerve plexus of the esophagus, glandular cells of the stomach and pylorus, lamina propria, and epithelium of the villi of the duodenum, jejunum, and ileum. N-CAM immunoreactivity was demonstrated in the secretory cells of the adenohypophysis, islets of Langerhans, and acinar cells of the exocrine pancreas. Alveolar cells of the lung were also N-CAM immunoreactive. In the urinary system, N-CAM immunoreactivity was seen in the proximal convoluted tubules of the kidney. In the male reproductive system, N-CAM immunoreactivity was demonstrated in the nerve plexus around the urethral epithelium and in the nerve fibers around the smooth muscle cells of the corpus cavernosum penis. In the visual system, N-CAM immunoreactivity was seen in the epithelial cells of the corpus ciliaris. Cornea and lens epithelium also showed positive immunoreactivity. Our results suggest that cells in many tissues and organs of the adult rat synthesize N-CAM.