Albumin induces CD44 expression in glomerular parietal epithelial cells by activating extracellular signal-regulated kinase 1/2 pathway

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a prosclerotic and migratory PEC phenotype in glomerulosclerosis. However, the regulatory mechanisms underlying CD44 expression by activated PECs remain largely unknown. This study was performed to examine the mediators responsible for CD44 induction in glomerular PECs in association with diabetes. CD44 expression and localization were evaluated in the glomeruli of Zucker diabetic rat kidneys and primary cultured PECs upon albumin stimulation. Real-time polymerase chain reaction confirmed an albuminuria-associated upregulation of the CD44 gene in the glomeruli of diabetic rats. Immunostaining analysis of diabetic kidneys further revealed an increase in CD44 in hypertrophic PECs, which often contain albumin-positive vesicles. Losartan treatment significantly attenuated albuminuria and lowered CD44 protein levels in the diabetic kidneys. In primary cultured rat PECs, rat serum albumin (0.25–1 mg/ml) caused a dose-dependent upregulation of CD44, claudin-1, and megalin protein expression, which was accompanied by an activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. Albumin-induced CD44 and claudin-1 expression were greatly suppressed in the presence of the ERK1/2 inhibitor, U0126. In addition, knockdown of megalin by small interfering RNA interference in PECs resulted in a significant reduction of albumin-induced CD44 and claudin-1 proteins. Taken together, our results demonstrate that albumin induces CD44 expression by PECs via the activation of the ERK signaling pathway, which is partially mediated by endocytic receptor megalin.     

Mutations in the S4 region isolate the final voltage-dependent cooperative step in potassium channel activation

Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% ( approximately 1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.     

Modulation of CD4 cell cytokine production by colon cancer-associated mucin

Mucins have been implicated in tumor-associated immunosuppression. The possibility that colon cancer mucin (CCM) may modulate T-helper 1 (TH1) activity was evaluated by investigating its effect on the production of interleukin-2 (IL-2) by CD4⁺ cells, a process that requires antigen-specific and costimulatory signals. Methods: CCM was purified from human colorectal cancer cells by gel-exclusion fast-pressure liquid chromatography. Cytokine production of purified CD4⁺ cells was evaluated at the protein and gene level in the presence of a phorbol ester or an anti-CD3 monoclonal antibody (mAb) plus mAb against the CD28 costimulatory receptor to mimic two-signal activation. Results: Soluble CCM, which contains mucins MUC2 as well as MUC1, inhibited IL-2 mRNA expression and secretion of CD4⁺ stimulated with a phorbol ester or an anti-CD3 mAb plus anti-CD28 mAb. Pretreatment of CD4⁺ cells with anti-CD28 mAb abrogated the suppressive effects of CCM on IL-2 production, and flow cytometry showed decreased binding of anti-CD28 mAb to its receptor in the presence of mucin. In addition, Ca²⁺ mobilization after T cell receptor cross-linking with anti-CD3 mAb was maintained in the presence of CCM. Although interferon γ production was also diminished, CCM did not induce a general inhibition of cytokine production, nor did it decrease cell viability. Macrophage inflammatory protein 1α production was up-regulated; the production of IL-10 and transforming growth factor β was unchanged. Conclusions: The results indicate that CCM can alter TH1 activity and suggest that the modulation of costimulatory interactions is involved. They provide another mechanism of immunosuppression mediated by these highly expressed tumor products. Received: 23 March 1999 / Accepted: 3 August 1999     

Influence of the mother's reproductive state on the hormonal status of daughters in marmosets (Callithrix kuhlii)

Behavioral and endocrine suppression of reproduction in subordinate females produces the high reproductive skew that characterizes callitrichid primate mating systems. Snowdon et al. [American Journal of Primatology 31:11-21, 1993] reported that the eldest daughters in tamarin families exhibit further endocrinological suppression immediately following the birth of siblings, and suggested that dominant females exert greater control over subordinate endocrinology during this energetically challenging phase of reproduction. We monitored the endocrine status of five Wied's black tufted-ear marmoset daughters before and after their mother delivered infants by measuring concentrations of urinary estradiol (E(2)), pregnanediol glucuronide (PdG), testosterone (T), and cortisol (CORT). Samples were collected from marmoset daughters 4 weeks prior to and 9 weeks following three consecutive sibling-litter births when the daughters were prepubertal (M=6.1 months of age), peripubertal (M=11.9 months), and postpubertal (M=17.6 months). The birth of infants was associated with reduced ovarian steroid excretion only in the prepubertal daughters. In contrast, ovarian steroid levels tended to increase in the postpubertal daughters. Urinary E(2) and T levels in the postpubertal daughters were 73.8% and 37.6% higher, respectively, in the 3 weeks following the birth of infants, relative to prepartum levels. In addition, peak urinary PdG concentrations in peri- and postpubertal daughters were equivalent to luteal phase concentrations in nonpregnant, breeding adult females, and all of the peri- and postpubertal daughters showed clear ovulatory cycles. Cortisol excretion did not change in response to the reproductive status of the mother, nor did the concentrations change across age. Our data suggest that marmoset daughters of potential breeding age are not hormonally suppressed during the mother's peripartum period or her return to fertility. These findings provide an additional example of species diversity in the social regulation of reproduction in callitrichid primates.     

Homozygous loss of menin is well tolerated in liver,a tissue not affected in MEN1

In recent studies from Sweden and the United States, a high vitamin A intake has been associated with low bone mineral density (BMD) and increased fracture risk. In Sweden and the United States, food items such as milk and breakfast cereals are fortified with vitamin A, whereas in Denmark there is no mandatory fortification with vitamin A. In the present study, we investigated relations between vitamin A intake and BMD and fracture risk in a Danish population consuming mostly unfortified food items. Within a population-based cohort study in 2,016 perimenopausal women, associations between BMD and vitamin A intake were assessed at baseline and after 5-year follow-up. Moreover, associations between baseline vitamin A intake and 5-year changes in BMD were studied. Finally, fracture risk was assessed in relation to vitamin A intake. In our cohort, dietary retinol intake (0.53 mg/day) was lower than the intake reported in recent studies form Sweden (0.78 mg/day) and the United States (1.66 mg/day). Cross-sectional and longitudinal analyses showed no associations between intake of vitamin A and BMD of the femoral neck or lumbar spine. Neither did BMD differ between those 5% who had the highest, and those 5% who had the lowest, vitamin A intake. During the 5-year study period, 163 subjects sustained a fracture (cases). Compared to 978 controls, logistic regression analyses revealed no difference in vitamin A intake. Thus, in a Danish population, average vitamin A intake is lower than in Sweden and the United States and not associated with detrimental effects on bone.     

Sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine

Background  

Sucrose density gradient centrifugation and cross-flow filtration methods have been developed and standardised for the safe and reproducible production of inactivated arbovirus antigens which are appropriate for use in diagnostic serological applications.