The cysteine-rich domains of the insulin and insulin-like growth factor I receptors are primary determinants of hormone binding specificity. Evidence from receptor chimeras

To delineate the structural determinants of the insulin receptor (IR) and insulin-like growth factor I receptor (IGFIR) which affect hormone binding specificity we have constructed seven chimeric receptor cDNAs and stably expressed them in Chinese hamster ovary cells. Clonal cell lines expressing high levels of each receptor chimera were analyzed for insulin and insulin-like growth factor I (IGFI) binding activity. Measurements of hormone binding and immunoprecipitation of metabolically labeled receptors showed that all chimeras were properly processed and expressed at the cell surface. The binding data indicate that 56 amino acids of the IR and 52 amino acids of the IGFIR located in corresponding regions of the cysteine-rich domains are the primary determinants of hormone binding specificity. These regions are located between amino acids Asn-230 and Ile-285 on the IR and between His-223 and Met-274 on the IGFIR. In addition, the alpha IR-3 antibody, which competes for IGFI binding, was found to interact with the same 52 amino acids of the IGFIR which determines hormone specificity. Other antibodies which interfere with insulin binding (5D9, MC51, and MA20) interact with epitopes in the COOH-terminal 288 amino acids of the alpha-subunit. We conclude that 56 and 52 amino acids of the cysteine-rich domains of the IR and IGFIR contain the major determinants of hormone binding specificity although other more COOH-terminal regions of both receptors contribute to hormone binding.     

Structural requirements for the binding of AMP to the allosteric site of NAD-specific isocitrate dehydrogenase from bakers' yeast

The specificity of yeast NAD-specific isocitrate dehydrogenase for the structures of the allosteric effector 5'-AMP was examined with analogues modified in the purine ring, pentosyl group, and 5'-phosphate group. An unsubstituted 6-amino group was essential for activation as was the phosphoryl group at the 5'-position. Activity was retained when an oxygen function of the 5'-phosphoryl was replaced by sulfur (Murry & Atkinson, 1968) or by nitrogen (phosphoramidates). 2-NH2-AMP, 2-azido-AMP, and 8-NH2-AMP were active; 8-azido-AMP and 8-Br-AMP were inactive. The configuration or nature of substituents about carbons 2' and 3' of the pentosyl portion of AMP was not critical for allosteric activation since AMP analogues containing, e.g., 2',3'-dideoxyribose or the bulky 2',3'-O-(2,4,6-trinitrocyclo-hexadienylidene) substituent (TNP-AMP) were active. TNP-AMP was bound to the enzyme with fluorescence enhancement and had an S0.5 for activation similar to the S0.5 for AMP. Positive effector activity was decreased when the pentosyl moiety of 5'-AMP was replaced by the six-membered nitrogen-containing morpholine group, indicating that the pentosyl group may be critical as a spacer for the proper geometry of binding to enzyme at the 6-amino and 5'-phosphoryl groups of 5'-AMP. A comparison of molecular models of 5'-AMP with 8,5'-cycloAMP suggests that the species of 5'-AMP required for binding to the enzyme contains the purine and ribose moieties in an anti conformation and positioning of the 5'-phosphate trans with respect to carbon 4'.(ABSTRACT TRUNCATED AT 250 WORDS)     

The photoreaction center of Rhodospirillum rubrum mutant strain F24.1

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665–670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7–8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7–8% of typical photoreaction center.     

The cellulolytic enzymes of Botryodiplodia theobromae Pat. Separation and characterization of cellulases and β-glucosidases

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.     

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver,fetal liver,cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123

The choleterol to phospholipid ratio in mitochodria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochonda is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane.In mitochondria from cholesterol-enriched liver and hepatomas, there occur a decrease in extent of hypoosmotic and phosphate-induced sweeling and decrease of conformational linked energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholopid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformation changes. No significant modifications of these parameters were found in fetal liver mitochondria.Cholesterol content does not influence K⁺ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K⁺ uptake, total mitochodrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42–48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochobdrial water content. Water content. Water is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is signifacantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspension during K⁺ uptake. The higher the ratio, the lower the extent and and initial rate of absorbance decrease.     

Effect of cholesterol content on some physical and functional properties of mitochondria isolated from adult rat liver, fetal liver, cholesterol-enriched liver and hepatomas AH-130, 3924A and 5123.

The cholesterol to phospholipid ratio in mitochondria from hepatomas AH-130, 3924A and 5123 is higher than in the particles isolated from adult or fetal rat livers. Nearly all the cholesterol of hepatoma mitochondria is located in membranes. As in liver mitochondria, in the particles isolated from hepatoma AH-130 there is more cholesterol in the outer than in the inner membrane. In mitochondria from cholesterol-enriched liver and hepatomas, there occurs a decrease in extent of hypoosmotic and phosphate-induced swelling and a decrease of conformational changes linked to energy states. The phenomenon is more marked in particles which exhibit higher cholesterol to phospholipid ratios. A statistically significant negative correlation exists between the cholesterol to phospholipid ratio and extent of volume or conformational changes. No significant modifications of these parameters were found in fetal liver mitochondria. Cholesterol content does not influence K+ uptake by cholesterol-enriched or hepatoma mitochondria. Nor does cholesterol content affect the respiratory increment related to this uptake. As a consequence of K+ uptake, total mitochondrial water exchangeable with tritiated water rises 20% while sucrose-impermeable water rises 42-48% in both adult rat liver and hepatoma AH-130 mitochondria. Absorbance changes linked to ion uptake do not correspond merely to variations in mitochondrial water content. Water content is apparently not influenced by the cholesterol to phospholipid ratio. However, the ratio is significantly correlated to both extent and initial rate of absorbance decrease of mitochondrial suspensions during K+ uptake. The higher the ratio, the lower the extent and initial rate of absorbance decrease.     

School racial segregation and long-term cardiovascular health among Black adults in the US: A quasi-experimental study

BackgroundCardiovascular disease (CVD) disproportionately affects Black adults in the United States. This is increasingly acknowledged to be due to inequitable distribution of health-promoting resources. One potential contributor is inequities in educational opportunities, although it is unclear what aspects of education are most salient. School racial segregation may affect cardiovascular health by increasing stress, constraining socioeconomic opportunities, and altering health behaviors. We investigated the association between school segregation and Black adults’ CVD risk.Methods and findingsWe leveraged a natural experiment created by quasi-random (i.e., arbitrary) timing of local court decisions since 1991 that released school districts from court-ordered desegregation. We used the Panel Study of Income Dynamics (PSID) (1991 to 2017), linked with district-level school segregation measures and desegregation court order status. The sample included 1,053 Black participants who ever resided in school districts that were under a court desegregation order in 1991. The exposure was mean school segregation during observed schooling years. Outcomes included several adult CVD risk factors and outcomes. We fitted standard ordinary least squares (OLS) multivariable linear regression models, then conducted instrumental variables (IV) analysis, using the proportion of schooling years spent in districts that had been released from court-ordered desegregation as an instrument. We adjusted for individual- and district-level preexposure confounders, birth year, and state fixed effects. In standard linear models, school segregation was associated with a lower probability of good self-rated health (−0.05 percentage points per SD of the segregation index; 95% CI: −0.08, −0.03; p < 0.001) and a higher probability of binge drinking (0.04 percentage points; 95% CI: 0.002, 0.07; p = 0.04) and heart disease (0.01 percentage points; 95% CI: 0.002, 0.15; p = 0.007). IV analyses also found that school segregation was associated with a lower probability of good self-rated health (−0.09 percentage points; 95% CI: −0.17, −0.02, p = 0.02) and a higher probability of binge drinking (0.17 percentage points; 95% CI: 0.04, 0.30, p = 0.008). For IV estimates, only binge drinking was robust to adjustments for multiple hypothesis testing. Limitations included self-reported outcomes and potential residual confounding and exposure misclassification.ConclusionsSchool segregation exposure in childhood may have longstanding impacts on Black adults’ cardiovascular health. Future research should replicate these analyses in larger samples and explore potential mechanisms. Given the recent rise in school segregation, this study has implications for policies and programs to address racial inequities in CVD.

Min Hee Kim and colleagues investigate the association between exposure to school racial segregation in childhood and long-term cardiovascular health among Black adults in the United States.