Lawrence J. Berliner

α‐Lactalbumin: structure and function

Small milk protein α‐lactalbumin (α‐LA), a component of lactose synthase, is a simple model Ca2+ binding protein, which does not belong to the EF‐hand proteins, and a classical example of molten globule state. It has a strong Ca2+ binding site, which binds Mg2+, Mn2+, Na+, and K+, and several distinct Zn2+ binding sites. The binding of cations to the Ca2+ site increases protein stability against action of heat and various denaturing agents, while the binding of Zn2+ to the Ca2+‐loaded protein decreases its stability. Functioning of α‐LA requires its interactions with membranes, proteins, peptides and low molecular weight substrates and products. It was shown that these interactions are modulated by the binding of metal cations. Recently it was found that some folding variants of α‐LA demonstrate bactericidal activity and some of them cause apoptosis of tumor cells.

A novel reversible thiol-specific spin label: Papain active site labeling and inhibition☆

Abstract A new, highly reactive, thiol-specific spin label, (1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)methanethiosulfonate was synthesized. Its unique specificity was demonstrated with the active thiol protease, papain, which was stoichiometrically inhibited within 5 min, resulting in a conformationally sensitive spectrum, which was identical over the pH range 4.5–7.5. The spin-label modification yielded a mixed disulfide between Cys 25 of papain and the 3-methylpyrroline nitroxide which was rapidly and completely reversed by exposing the labeled papain to mild concentrations of dithiothreitol. The concentration of released nitroxide corresponded exactly to the number of reactive thiol groups in the original enzyme. Full enzymatic activity was restored after the spin label was removed. This spin label is useful as a sensitive thiol titrating agent as well as a specific conformational probe of thiol site structure by virtue of its minimal rotational freedom and distance from the covalent disulfide linkage to the macromolecule under study.

α-Lactalbumin unfolding is not sufficient to cause apoptosis, but is required for the conversion to HAMLET (human α-lactalbumin made lethal to tumor cells)

HAMLET (human α‐lactalbumin made lethal to tumor cells) is a complex of human α‐lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis‐like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from α‐lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of α‐lactalbumin is sufficient to induce cell death. We used the bovine α‐lactalbumin Ca2+ site mutant D87A, which is unable to bind Ca2+, and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET‐like complex in the presence of oleic acid. BAMLET (bovine α‐lactalbumin made lethal to tumor cells) and D87A‐BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca2+site, as HAMLET maintained a high affinity for Ca2+ but D87A‐BAMLET was active with no Ca2+ bound. We conclude that partial unfolding of α‐lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca2+‐binding site is not required for conversion of α‐lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca2+site.

Ultra high field magnetic resonance imaging

Ultra High Field Magnetic Resonance Imaging: A Historical Perspective.- Design Considerations for Ultra High Field MRI Magnet Systems.- Hardware Considerations in Ultra High Field MRI.- Aspects of Clinical Imaging at 7 T.- The Challenges of Integrating A 9.4T MR Scanner for Human Brain Imaging.- Ultra High Field MRI: High-Frequency Coils.- A Perspective into Ultra High Field MRI RF Coils.- Radiofrequency Field Calculations for High Field MRI.- Magnetic Susceptibility Effects in High Field MRI.- High Magnetic Fields for Imaging Cerebral Morphology, Function, and Biochemistry.- High-Resolution and Microscopic Imaging at High Field.- In-Vivo NMR Spectroscopy of the Brain at High Fields.- Clinical Promise: Clinical Imaging at Ultra High Field.

Manganese Complexes of Curcumin Analogues: Evaluation of Hydroxyl Radical Scavenging Ability, Superoxide Dismutase Activity and Stability towards Hydrolysis

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO•) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 μM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2-• where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2-• to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37 mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO• scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+releasing, respectively.

THE SPIN-LABEL APPROACH TO LABELING MEMBRANE PROTEIN SULFHYDRYL GROUPS*

Labeling experiments with viable membrane preparations are optimally undertaken where conditions are extremely mild (minimal excess of modification reagent, very short reaction times) in order to reduce the possibilities of introducing artifacts (cell aging and death, denaturation, etc.). Furthermore, specificity of labeling is a critical requirement where the system is complex and contains many proteins. The spin label IV described above offers several advantages over previously employed reagents. By virtue of its reversibility and ease of quantitation, one may repetitively label and remove label from the same membrane preparation in order to check reproducibility while using the same sample as a control.

Helices and surface coils for low-field in vivo ESR and EPR imaging applications

Abstract Single-turn, flat-loop surface coils are described for low-field electron paramagnetic resonance studies at L band with a home-built spectrometer on aqueous nitroxide samples. The loops were compared with helix resonators and found to be more sensitive, especially for applications where the microwave probe surrounds or is immersed into the aqueous sample. The overall signal to noise for a flat loop of dimensions 0.7 cm diameter, 4.1 cm length, and 0.8 mm gap at 1.86 GHz was equal to or better than that in an X -band cavity aqueous flat cell. The only disadvantage of this surface coil is the depth sensitivity, which falls off exponentially with the distance from the coil plane.

Consequences of Nitric Oxide Generation in Epileptic- Seizure Rodent Models as Studied by In Vivo EPR

The role of nitric oxide (NO) in epileptogenesis was studied in pentylenetetrazole (PTZ)‐treated animals using in vivo and ex vivo EPR spectroscopy. NO generation was measured directly in the brain of a PTZ‐induced mouse in vivo by an L‐band EPR spectrometer. An elevation in NO production in the brain was observed during convulsions, and more NO was generated in the tonic seizure vs. the clonic seizure. NO content in several brain tissues (including the cerebral cortex (CR), cerebellum (CL), olfactory bulb (OB), hippocampus (HI), and hypothalamus (HT)) of PTZ‐doped rats was analyzed quantitatively ex vivo by X‐band EPR. To test the involvement of NO in seizure development, pharmacological analyses were performed using the NO synthase (NOS) inhibitors NG‐nitro‐L‐arginine (L‐NNA), NG‐monomethyl‐L‐arginine (L‐NMMA), and 3‐bromo‐7‐nitroindazole (3Br‐7NI). All of these inhibitors suppressed the convulsions, holding them at the clonic level, and prevented development of a tonic convulsion in rats doped with up to 80 mg/kg PTZ. 3Br‐7NI completely inhibited NO production, but L‐NNA and L‐NMMA showed only 70% inhibition of NO production in PTZ‐doped rats. In order to examine the contributions of NO in convulsions, rats were treated with anticonvulsants (phenytoin and diazepam) before PTZ treatment. Both drugs completely suppressed tonic convulsion in PTZ‐doped rats at doses up to 80 mg/kg, but NO levels were similar to those detected in a clonic convulsion. These results support the notion that NO does not directly induce a clonic convulsion, but may be generated as a consequence of onset of seizure. Magn Reson Med 48:1051–1056, 2002.

Cooperative thermal transitions of bovine and human apo-α-lactalbumins: evidence for a new intermediate state

The thermal denaturation of bovine and human apo‐α‐lactalbumins at neutral pH has been studied by intrinsic protein fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC) methods. Apo‐α‐lactalbumin possesses a thermal transition with a midpoint about 25–30°C under these conditions (pH 8.1, 10 mM borate, 1 mM EGTA), which is reflected in changes in both fluorescence emission maximum and quantum yield. However, the CD showed a decrease in ellipticity at 270 nm with a midpoint at about 10–15°C, while DSC shows the transition within the region of 15–20°C. The non‐coincidence of transition monitored by different methods suggests the existence of an intermediate state in the course of the thermal denaturation process. This intermediate state is not the classical molten globule state which occurs at higher temperature (i.e. denatured state at these conditions) [D.A. Dolgikh, R.I. Gilmanshin, E.V. Brazhnikov, V.E. Bychkova, G.V. Semisotnov, S.Y. Venyaminov and O.B. Ptitsyn, FEBS Letters, 136 (1981) 311–315] and has physical properties intermediate between the native and molten globule states.

In vivo imaging of spin-trapped nitric oxide in rats with septic shock: MRI spin trapping

This paper reports the first in vivo NMR image of the distribution of NO using the “MRI spin‐trapping” technique. NO was complexed with the Fe(II)‐chelate spin trap, N‐methyl‐d‐glucamine dithiocarbamate (MGD), verified as (MGD)2‐Fe(II)‐NO by EPR, and the radical distribution was “visualized” by MR images. In rats, the (MGD)2‐Fe(II)‐NO complex was concentrated in the liver displaying significantly enhanced contrast in the vascular structure such as hepatic vein and inferior vena cava. Nitric oxide synthase was verified as the source of NO in rats with septic shock by pre‐administration of the competitive inhibitor N‐monomethyl‐l‐arginine, resulting in reduced enhancement. The NO complex was more stable in vivo and a more effective MRI contrast agent than other stable nitrogen containing radicals, such as nitroxides. The MRI spin‐trapping method should be a powerful tool for visualizing spatial distributions of free radicals in pathologic organs and tissues when combined with the appropriate radical complexing agent, such as (MGD)2‐Fe(II) used in these studies. Magn Reson Med 42:235–239, 1999.