Neurotrophic Factors Mediated Activation of Astrocytes Ameliorate Memory Loss by Amyloid Clearance after Transplantation of Lineage Negative Stem Cells.

Alzheimer's disease (AD) is one of the untreatable neurodegenerative disorders with associated societal burden. Current therapies only provide symptomatic relief without altering the rate of disease progression as reported by Lanctot et al. (Therapeutic Advances in Neurological Disorders 2 (3):163-180, 2009). The increased number of failed clinical trials in last two decades indicates the imperative need to explore alternative therapies for AD as reported by Tuszynski et al. (Nature Medicine 11 (5):551-555, 2005) and Liyanage et al. (Alzheimer's & Dementia 4:628-635, 2005). In this study, we aimed to decipher the role of neurotrophic factors in the reversal of memory loss by transplantation of lineage negative (Lin-ve) stem cells in a male mouse model of cognitive impairment induced by intrahippocampal injection of amyloid ß-42 (Aß-42). The efficacy of human umbilical cord blood (hUCB) derived Lin-ve stem cells were analyzed by neurobehavioral parameters, i.e., Morris water maze and passive avoidance after bilateral intra-hippocampal transplantation using stereotaxic surgery. Real-time PCR and immunohistochemistry was carried out in brain tissues in order to analyze the expression of neurotrophic factors, apoptotic, astrocytic, and other neuronal cell markers. The transplantation of Lin-ve stem cells led to reversal of memory loss associated with reduction of Aß-42 deposition from the brains. The molecular analysis revealed increase in neurotrophic factors, i.e., glial derived neurotrophic factor (GDNF), ciliary derived neurotrophic factor (CNTF), and Brain-derived neurotrophic factor (BDNF) after transplantation. The administration of ANA-12, a TrkB inhibitor, reversed the behavioral and molecular effects of stem cell transplantation suggesting involvement of BDNF-TrkB pathway in the rescue of memory loss. We believe that the amyloid clearance results from activation of astrocytes and anti-apoptotic pathways added by neurotrophic factors.

Germ cell-specific localization of immunoreactive riboflavin carrier protein in the male golden hamster: appearance during spermatogenesis and role in sperm function.

Riboflavin carrier protein (RCP) is a phosphoglycoprotein (37 kDa) that is well studied in chicken. An immunologically cross-reacting protein was identified in mammals and active immunization of male rats and bonnet monkeys with chicken RCP lead to an approximately 80% reduction in fertility. However, the physiological mechanism responsible for inhibition of male fertility has not been investigated. Moreover, information on the cell type-specific localization and the origin of immunoreactive RCP during spermatogenesis is extremely limited. Hence, studies were carried out to determine the pattern of expression of immunoreactive RCP during spermatogenesis and its role in sperm function in the golden hamster. Immunoreactive RCP was germ cell-specific, found to be associated with the acrosome-organizing region of early spermatids and showed interesting patterns of immunolocalization during late stages of spermiogenesis. Mature spermatozoa exhibited acrosome-specific localization, mainly in the peri-acrosomal membrane. The immunoreactive protein was undetectable in (non)gonadal somatic cells tested. The protein had a molecular mass of 45-55 kDa and was biosynthesized by round spermatids. The acrosome-specific localization of immunoreactive RCP was unchanged during capacitation, but it was substantially lost during acrosome reaction. Functional studies indicated that treatment of spermatozoa with anti-RCP antibodies did not have any effect on either capacitation or acrosome reaction, but markedly reduced the rate of sperm penetration into zona-free hamster oocytes. These results show the existence of male germ cell-specific immunoreactive RCP, having a potential role in sperm-egg interaction in hamsters. Also the pattern of immunoreactive-RCP localization makes it an ideal marker to monitor development of acrosome in mammalian spermatozoa.

Developmental and hormonal regulation of type II DNA topoisomerase in rat testis.

Type II DNA topoisomerase (topo II) is required for diverse biological functions including DNA replication, maintenance of genome stability, chromosome segregation and chromosome condensation. While the identity of topo II in rodent testis has been established, the regulation of topo II expression during the development of the postnatal testis and gametogenesis is unclear. Here, we report that rat testis topo II is developmentally and hormonally regulated. Topo IIalpha mRNA levels peaked prior to the onset of puberty, declined sharply thereafter and stabilized in adult testis. In contrast, the topo II enzyme content was lower in prepubertal testis but increased after the onset of puberty. Topo II was expressed in a cell-specific manner within germ cells, being detected only in pachytene spermatocytes. While testosterone markedly increased topo IIalpha mRNA levels in prepubertal testis, continued treatment failed to enhance topo IIalpha mRNA above postpubertal control levels. The extent of topo II activity remained steady regardless of the testosterone-induced increase in topo IIalpha mRNA levels. Inhibition of testosterone function in postpubertal animals by ethanedimethane sulphonate (EDS) and flutamide resulted in a significant decrease in topo IIalpha gene expression and topo II activity. The administration of exogenous testosterone (T) to EDS- and flutamide-treated rats restored topo IIalpha mRNA levels and topo II activity similar to the levels seen in the testis of age-matched control animals. Histochemical analyses of testes indicated that the effect of T on spermatogenesis was separable from its effect on topo IIalpha expression. Our results reveal that testosterone acts as a positive regulator of topo IIalpha gene expression and is required for the maintenance of topo IIalpha expression during the development of the postnatal testis and spermatogenesis.

Prolonged treatment of breast cancer cells with antiestrogens increases the activating protein-1-mediated response: involvement of the estrogen receptor.

At micromolar (pharmacological) concentrations, the action of tamoxifen on the proliferation of estrogen-dependent cells can be mediated not only by the estrogen receptor (ER), but also by other target molecules, such as protein kinase-C (PKC), which are easily inhibited by antiestrogens in cell-free experiments. By developing MTLN and MDT cell lines, in which any modulation of PKC activity is reflected by a variation of the expression of an activating protein-1 (AP-1)-controlled firefly luciferase gene, we investigated whether such antiestrogen inhibitory effects on PKC occurred in intact breast cancer cells. Firstly, in short term (4-h) treatment of both cell lines, antiestrogens only inhibited the 12-O-tetradecanoyl-phorbol-13-acetate-induced luciferase activity at very high concentrations (30 microM). A cytolytic effect was also observed. Secondly, in prolonged (4-day) treatments of MTLN (ER-positive) cells, low antiestrogen concentrations (nanomolar) decreased the basal AP-1 response by about 2 and increased the 12-O-tetradecanoyl-phorbol-13-acetate-stimulated AP-1 response by about 3-4. This stimulation was mediated by ER, because 1) dose-response curves established with tamoxifen and hydroxytamoxifen were in agreement with their affinity for ER; 2) when present with antiestrogens, estradiol abolished this phenomenon; and 3) this effect was not observed in MDT (ER-negative) cells. Such a latent activation of AP-1 pathway could appear in the course of breast cancer antiestrogen treatment, in conditions where natural PKC activators are abnormally produced with unexpected consequences on the results of a long term antiestrogen treatment.

Receptor-induced switch in site-site cooperativity during iron release by transferrin.

Iron removal by PPi from the N- and C-terminal binding sites of both free and receptor-complexed transferrin, when the partner site remains occupied with kinetically inert Co(III), has been studied at pH 7.4 and 5.6, at 25 degrees C. At extracellular pH, 7.4, the C-terminal site of free mixed-metal proteins is slightly more labile than its N-terminal counterpart in releasing iron to 0.05 M PPi. The rate and extent of iron removal are retarded from both sites when transferrins are receptor-bound. At endosomal pH, 5.6, the two sites exhibit greater kinetic heterogeneity in iron release to 0.005 M PPi. The N-terminal site is 6 times more facile in relinquishing iron than the C-terminal site when mixed-metal transferrins are free. However, the two sites are affected oppositely upon binding to the receptor. Iron release from the C-terminal site of receptor-complexed CoN-transferrin-FeC is 4 times faster than that from receptor-free protein. In contrast, iron removal from the N-terminal site of receptor-complexed FeN-transferrin-CoC is slowed by a factor of 2 compared to that from free protein. These results help explain our previous observation of a receptor-induced switch in site lability during iron removal from diferric transferrin at pH 5.6 (Bali & Aisen, 1991). Site-site cooperative interactions between the two sites of doubly-occupied transferrin during iron release are altered upon binding to receptor at pH 5.6. Iron in the otherwise weaker binding site of the N-terminal lobe is stabilized, while iron in the relatively stable binding site of the C-terminal lobe is labilized.

Receptor-modulated iron release from transferrin: differential effects on N- and C-terminal sites.

Iron release to PPi from N- and C-terminal monoferric transferrins and their complexes with transferrin receptor has been studied at pH 7.4 and 5.6 in 0.05 M HEPES or MES/0.1 M NaCl/0.01 M CHAPS at 25 degrees C. The two sites exhibit kinetic heterogeneity in releasing iron. The N-terminal form is slightly less labile than its C-terminal counterpart at pH 7.4, but much more facile in releasing iron at pH 5.6. At pH 7.4, iron removal by 0.05 M pyrophosphate from each form of monoferric transferrin complexed to the receptor is considerably slower than from the corresponding free monoferric transferrin. However, at pH 5.6, complexation of transferrin to its receptor affects the two forms differently. The rate of iron release to 0.005 M pyrophosphate by the N-terminal species is substantially the same whether transferrin is free or bound to the receptor. In contrast, the C-terminal form releases iron much faster when complexed to the receptor than when free. Urea/PAGE analysis of iron removal from free and receptor-complexed diferric transferrin at pH 5.6 reveals that its C-terminal site is also more labile in the complex, but its N-terminal site is more labile in free diferric transferrin. Thus, the newly discovered role of transferrin receptor in modulating iron release from transferrin predominantly involves the C-terminal site. This observation helps explain the prevalence of circulating N-terminal monoferric transferrin in the human circulation.

Prevalence of high risk factors in mothers in urban slums of Delhi.

Point prevalence of 'High Risk' factors was assessed in 450 mothers of reproductive age group residing in two urban slum communities. Major risk factors prevalent were non-pregnant weight less than 43.5 kg (24%), elderly grand multipara (17%), and history of previous still births and/or intrauterine death (3%).

A new role for the transferrin receptor in the release of iron from transferrin.

Iron removal by pyrophosphate from human serum diferric transferrin and the complex of transferrin with its receptor was studied in 0.05 M HEPES or MES buffers containing 0.1 M NaCl and 0.01 M CHAPS at 25 degrees C at pH 7.4, 6.4, and 5.6. At each pH, the concentration of pyrophosphate was adjusted to achieve rates of release amenable to study over a reasonable time course. Released iron was separated from protein-bound iron by poly(ethylene glycol) precipitation of aliquots drawn from the reaction mixture at various times during the course of a kinetic run. The amount of 59Fe label associated with the protein and pyrophosphate was determined from the radioactivity of precipitate and supernatant, respectively, in each aliquot. Iron removal of 0.05 M pyrophosphate at pH 7.4 from diferric transferrin bound to the receptor is considerably slower than that from free diferric transferrin, with observed pseudo-first-order rate constants of 0.020 and 0.191 min-1, respectively. For iron removal by 0.01 M pyrophosphate at pH 6.4, corresponding rate constants are 0.031 and 0.644 min-1. However, at pH 5.6, iron removal by 0.001 M pyrophosphate is faster from diferric transferrin bound to its receptor than from free transferrin (observed rate constants of 0.819 and 0.160 min-1, respectively). Thus, the transferrin receptor not only facilitates the removal of iron from diferric transferrin at the low pH that prevails in endocytic vesicles but may also reduce its accessibility to iron acceptors at extracellular pH, thereby minimizing the likelihood of nonspecific release of iron from transferrin at the cell surface.

Site-specific rate constants for iron removal from diferric transferrin by nitrilotris(methylenephosphonic acid) and pyrophosphate.

The kinetics of iron removal from human serum diferric transferrin by nitrilotris(methylenephosphonic acid) (NTP) and pyrophosphate (PPi) have been studied in 0.1 M, pH 7.4, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonate buffer at 25 degrees C using urea/polyacrylamide gel electrophoresis. The four microscopic rate constants required for a complete description of iron removal from the two transferrin metal-binding sites have been measured at 100 mM concentrations of NTP and PPi. There is very good agreement between the rate constants determined by electrophoresis in this study and the corresponding rate constants determined spectrophotometrically for monoferric transferrins that have been labeled at the empty binding site with substitutionally inert Co(III). The results validate the use of cobalt-labeled transferrins as models for kinetic studies on iron removal from diferric transferrin.

Care of the newborn by traditional birth attendants.

The study was carried out in a slum cum resettlement colony (Area-I) and four villages (Area-II) of Delhi. Management of the newborn by the 25 functioning Traditional Birth Attendants (TBA's) who conducted 83.64% deliveries in Area-I and 16.22% in Area-II was studied. Majority of TBA's did not have the concept of washing hands before conducting per vaginum (P/V) examinations or deliveries. Most of the TBA's, i.e., 21 out of 25 used a razor blade to cut the umbilical cord of which 9 used a fresh blade. No TBA left the cord untied. Vigorous patting in upright and also after holding the baby upside down was the commonest (68%) method of neonatal resuscitation. All TBA's massaged and bathed the baby everyday. Majority of the TBA's (18 out of 25) referred the baby to a health agency for immunization though they did not know the exact schedule.

Nutritional status of pre-school children of urban slum communities in Delhi.

Nutritional status of 486 preschool children residing in urban slums was assessed by making domiciliary visits. The overall prevalence of protein energy malnutrition (PEM) was found to be 81.8%, while 31.8, 44.1, 5.7 and 0.2% of children had Grades I, II, III and IV PEM, respectively. Age, sex and education had a significant association with PEM.