Identification of an insulin-responsive, slow endocytic recycling mechanism in Chinese hamster ovary cells

In adipocytes, the insulin-regulated aminopeptidase (IRAP) is trafficked through the same insulin-regulated recycling pathway as the GLUT4 glucose transporter. We find that a chimera, containing the cytoplasmic domain of IRAP fused to transmembrane and extracellular domains of the transferrin receptor, is slowly recycled and rapidly internalized in Chinese hamster ovary cells. Morphological studies indicate that the chimera is slowly trafficked through the general endosomal recycling compartment rather than being sorted to a specialized recycling pathway. A chimera in which a di-leucine sequence within the cytoplasmic domain of IRAP has been mutated to alanines is rapidly internalized and rapidly recycled, indicating that this di-leucine is required for the slow recycling but not for the rapid internalization. Insulin stimulates a 2-3-fold increase in the recycling of the chimera and only a 1.2-fold increase in the recycling of the transferrin receptor. The effect of insulin on the recycling of the chimera is blocked by wortmannin, a phosphatidylinositol 3'-kinase inhibitor. GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) increases the recycling of the chimera by 50% but has no effect on the recycling of the transferrin receptor. In these studies, we have identified in Chinese hamster ovary cells a novel, slow endocytic recycling mechanism that is regulated by insulin.     

Estrogen replacement therapy and worsening of radiographic knee osteoarthritis: the Framingham Study

OBJECTIVE: To examine whether estrogen replacement therapy (ERT) prevents worsening of radiographic knee osteoarthritis (OA) in elderly women. METHODS: A total of 551 women ages 63-91 years (mean age 71) in the Framingham Study were followed up from biennial examination 18 (1983-1985) to examination 22 (1992-1993). Data on postmenopausal ERT were obtained every 2 years. Subjects were classified into 3 groups according to their estrogen use at biennial examination 18: never users (n = 349), past users (n = 162), and current users (n = 40). Women received anteroposterior weight-bearing knee radiographs at examinations 18 and 22. Using the Kellgren and Lawrence criteria, global radiographic knee OA was assessed, (grade range 0-4) and individual radiographic features, such as osteophytes and joint space narrowing, were scored from 0 to 3. Worsening was defined as either development of radiographic OA that was not present at baseline (incident OA) or progression of baseline radiographic OA by > or =1 Kellgren and Lawrence grade (progressive OA). Potential confounding factors included age, body mass index, weight change, smoking, knee injury, physical activity level, and bone mineral density at the femoral neck. RESULTS: During 8 years of followup, 17.4% of knee radiographic scores worsened by 1 grade and 5.8% by 2 or 3 grades among never users of ERT. Among current estrogen users, only 11.7% of knee radiographic scores worsened by 1 grade and none worsened by more than 1 grade. After adjusting for age and other potential confounding factors, the relative risk of incident radiographic knee OA in comparison with never users of estrogen was 0.8 (95% confidence interval [95% CI] 0.5-1.4) in past users and 0.4 (95% CI 0.1-3.0) in current users. Current use of estrogen also showed a trend toward decreased risk of progressive knee OA compared with never use (odds ratio [OR] 0.5, 95% CI 0.1-2.9). When both incident and progressive radiographic knee OA cases were combined, current ERT use had a 60% decreased risk compared with never use (OR 0.4, 95% CI 0.1-1.5). CONCLUSION: This is the first prospective cohort study to examine the effects of ERT on radiographic knee OA. The results indicate that current use of ERT had a moderate, but not statistically significant, protective effect against worsening of radiographic knee OA among elderly white women. These findings corroborate those of cross-sectional studies and point further to a potential benefit of female hormones in OA.     

Indications that the Cgamma2 homology region is not a regular domain

The Cgamma2 homology region of rabbit IgG does not behave like a domain. Thus, there is no trans-interaction between the two Cgamma2 regions; instead there is an unusual cis-interaction between Cgamma2 and Cgamma3 regions. The observations were made on the plasmin digestion products Facb (IgG minus the Cgamma3 region) and pFc' (Cgamma3 region), which did not dissociate under neutral conditions but dissociated in 3M guanidine solution (that is, cis-interaction between Cgamma2 and Cgamma3). The Facb fragment split into subunits with equal molecular weights under neutral conditions on partial reduction and alkylation (that is, lack of trans-interaction between the two Cgamma2 in the molecule).     

A statistical model of the dynamics of a mosquito vector (Culex tarsalis) population

A model of the dynamics of a mosquito Culex tarsalis is derived that includes the life states through which the mosquito proceeds. Transition probabilities from one state (egg, larva, pupa and adult) to another are derived and they depend on the duration of stay and mortality in each state. A formula is derived for the expected number of mosquitoes alive at any time during the spring or summer. This formula depends on the number of eggs oviposited and the transition probabilities. Data are used to estimate the parameters and to illustrate the usefulness of this model in examining the effect of changes in mosquito survival on the dynamics of the population.     

The membrane affinities of the aliphatic amino acid side chains in an alpha-helical context are independent of membrane immersion depth

Understanding, predicting, and designing the binding of peptides and proteins to bilayers require quantifying the intrinsic propensities of individual amino acid residues to bind membranes as a function of structural context and bilayer depth. A host-guest study was performed using the peptide host named helix5 in order to determine the membrane affinities of the aliphatic side chains both in an alpha-helical context and as a function of bilayer depth. Use of the alpha-helical host with a constrained geometry allowed the placement of guest sites at three different depths in bilayers and minimized secondary structural changes due to guest substitutions. Circular dichroism and electron paramagnetic resonance (EPR) were used to characterize the aqueous and bilayer-bound structures of the peptide variants. EPR was also used to measure the bilayer-water partition constants of the peptide variants, and the Delta DeltaGtr values (relative to Gly) of the aliphatic amino acid side chains were subsequently calculated. Surprisingly, the DeltaDeltaGtr values did not significantly vary as a function of the guest site depth in bilayers. In addition, the Delta DeltaGtr values determined in an alpha-helical context are reduced to approximately two-thirds of Delta DeltaGtr values determined in other studies for the bilayer-water and octanol-water partitioning of amino acid side chains in extended and unstructured hosts. Both the relative reduction in Delta DeltaGtr values in the context of an alpha-helical host and the invariance of Delta DeltaGtr values with respect to bilayer depth are consistent with the membrane affinities of the aliphatic residues being largely determined by the classical hydrophobic effect.