Program review. The Interdisciplinary Biophysics Graduate Program at the University of Michigan

The Michigan Biophysics Graduate Program (MBGP) was established in 1949, making it one of the first such programs in the world. The intellectual base of the program was significantly broadened in the 1980 when faculty members from a number of other units on campus were invited to join. Currently over forty faculty members from a variety of disciplines participate as mentors for the Ph.D. students enrolled in the MBGP providing our students with rich opportunities for academic learning and research. The MBGP has two main objectives: 1) to provide graduate students with both the intellectual and technical training in modern biophysics, 2) to sensitize our students to the power and unique opportunities of interdisciplinary work and thinking so as to train them to conduct research that crosses the boundaries between the biological and physical sciences. The program offers students opportunities to conduct research in a variety of areas of contemporary biophysics including structural biology, single molecule spectroscopy, spectroscopy and its applications, computational biology, membrane biophysics, neurobiophysics and enzymology. The MBGP offers a balanced curriculum that aims to provide our students with a strong academic base and, at the same time, accommodate their different academic backgrounds. Judging its past performance through the success of its former students, the MBGP has been highly successful, and there is every reason to believe that strong training in the biophysical sciences, as provided by the MBGP, will become even more valuable in the future both in the academic and the industrial settings. in the academic and the industrial settings.     

Identification of Products of the Uridinediphospho-N-acetyl-d-Glucosamine Oxidoreductase System from Citrobacter freundii ATCC 10053

Uridinediphospho-N-acetyl glucosamine is converted to one or more uridine-diphospho-4-keto-6-deoxy-2-acetamidohexose derivatives by an enzyme from Citrobacter freundii ATCC 10053. Borohydride reduction of reaction products followed by acid hydrolysis yielded several amino sugars. Two of these were identified as fucosamine and quinovosamine by chromatography and ninhydrin degradation.     

Relation between fractures and mortality: results from the Canadian Multicentre Osteoporosis Study

Background

Fractures have largely been assessed by their impact on quality of life or health care costs. We conducted this study to evaluate the relation between fractures and mortality.

Methods

A total of 7753 randomly selected people (2187 men and 5566 women) aged 50 years and older from across Canada participated in a 5-year observational cohort study. Incident fractures were identified on the basis of validated self-report and were classified by type (vertebral, pelvic, forearm or wrist, rib, hip and “other”). We subdivided fracture groups by the year in which the fracture occurred during follow-up; those occurring in the fourth and fifth years were grouped together. We examined the relation between the time of the incident fracture and death.

Results

Compared with participants who had no fracture during follow-up, those who had a vertebral fracture in the second year were at increased risk of death (adjusted hazard ratio [HR] 2.7, 95% confidence interval [CI] 1.1–6.6); also at risk were those who had a hip fracture during the first year (adjusted HR 3.2, 95% CI 1.4–7.4). Among women, the risk of death was increased for those with a vertebral fracture during the first year (adjusted HR 3.7, 95% CI 1.1–12.8) or the second year of follow-up (adjusted HR 3.2, 95% CI 1.2–8.1). The risk of death was also increased among women with hip fracture during the first year of follow-up (adjusted HR 3.0, 95% CI 1.0–8.7).

Interpretation

Vertebral and hip fractures are associated with an increased risk of death. Interventions that reduce the incidence of these fractures need to be implemented to improve survival.Osteoporosis-related fractures are a major health concern, affecting a growing number of individuals worldwide. The burden of fracture has largely been assessed by the impact on health-related quality of life and health care costs.^1,2 Fractures can also be associated with death. However, trials that have examined the relation between fractures and mortality have had limitations that may influence their results and the generalizability of the studies, including small samples,^3,4 the examination of only 1 type of fracture,^4–10 the inclusion of only women,^8,11 the enrolment of participants from specific areas (i.e., hospitals or certain geographic regions),^{3,4,7,8,10,12} the nonrandom selection of participants^3–11 and the lack of statistical adjustment for confounding factors that may influence mortality.^3,5–7,12We evaluated the relation between incident fractures and mortality over a 5-year period in a cohort of men and women 50 years of age and older. In addition, we examined whether other characteristics of participants were risk factors for death.     

Amyloid-β Membrane Binding and Permeabilization are Distinct Processes Influenced Separately by Membrane Charge and Fluidity

The 40 and 42 residue amyloid-β (Aβ) peptides are major components of the proteinaceous plaques prevalent in the Alzheimer's disease-afflicted brain and have been shown to have an important role in instigating neuronal degeneration. Whereas it was previously thought that Aβ becomes cytotoxic upon forming large fibrillar aggregates, recent studies suggest that soluble intermediate-sized oligomeric species cause cell death through membrane permeabilization. The present study examines the interactions between Aβ40 and lipid membranes using liposomes as a model system to determine how changes in membrane composition influence the conversion of Aβ into these toxic species. Aβ40 membrane binding was monitored using fluorescence-based assays with a tryptophan-substituted peptide (Aβ40 [Y10W]). We extend previous observations that Aβ40 interacts preferentially with negatively charged membranes, and show that binding of nonfibrillar, low molecular mass oligomers of Aβ40 to anionic, but not neutral, membranes involves insertion of the peptide into the bilayer, as well as sequential conformational changes corresponding to the degree of oligomerization induced. Significantly, while anionic membranes in the gel, liquid crystalline, and liquid ordered phases induce these conformational changes equally, membrane permeabilization is reduced dramatically as the fluidity of the membrane is decreased. These findings demonstrate that binding alone is not sufficient for membrane permeabilization, and that the latter is also highly dependent on the fluidity and phase of the membrane. We conclude that binding and pore formation are two distinct steps. The differences in Aβ behavior induced by membrane composition may have significant implications on the development and progression of AD as neuronal membrane composition is altered with age.     

Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

Location of Trp265 in metarhodopsin II: implications for the activation mechanism of the visual receptor rhodopsin

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265(6.48) with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the beta-ionone ring allows Trp265(6.48) and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly 13C-labeled Trp265(6.48). The retinal C20-Trp265(6.48) contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 A toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265(6.48) side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265(6.48) and Gly121(3.36) is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120(3.35) on H3 and Met86(2.53) on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.     

Dual Neuroprotective and Neurotoxic Effects of Cannabinoid Drugs in Vitro

Either protective or toxic effects of cannabinoids on cell survival have been reported extensively in the literature; however, the factors that determine the direction of the effect are still obscured. In this study we have used the neuroblastoma cell line N18TG2 that expresses CB1 cannabinoid receptors to investigate several factors that may determine the consequences of exposure to cannabinoid agonists. Cells that were grown under optimal, stressful, or differentiating conditions were exposed to cannabinoid agonists and then assayed for cell viability by measuring MTT, LDH, and caspase-3 activity. Various cannabinoid agonists (CP 55,940, ∆9-THC, HU-210, and WIN 55,212-2) failed to affect cell viability when the cells were grown under optimal conditions. On the other hand, the same agonists significantly reduced cell viability when the cells were grown under stressful conditions (glucose- and serum-free medium), while enhancing the viability of cells grown in differentiation medium (0.5% serum and 1.5% DMSO). The toxic/protective profile was not dependent on the type or the concentration of the cannabinoid agonist that was applied. The cannabinoid agonist CP 55,940 similarly affected the non-neuronal HEK-293 cells that were grown under stressful conditions only when they expressed CB1 receptors. Our results shed light on the conflicting reports regarding the protective or toxic effects of cannabinoids in vitro and indicate that cannabinoids may activate different intracellular signaling mechanisms, depending on the state of the cell, thus leading to different physiological consequences.