1H and 15N NMR resonance assignments and preliminary structural characterization of Escherichia coli apocytochrome b562

The 1H and 15N resonances of uniformly enriched apocytochrome b562 (106 residues) have been assigned. The assignment work began with the identification of the majority of HN-H alpha-H beta subspin systems in two-dimensional DQF-COSY and TOCSY spectra of unlabeled protein in D2O and in 95% H2O/5% D2O buffer. Intraresidue and interresidue NOE connectivities were then searched for in two-dimensional homonuclear NOESY spectra recorded on unlabeled protein and in the three-dimensional NOESY-HMQC spectrum recorded on uniformly 15N-enriched protein. Those data, combined with the main-chain-directed assignment strategy (MCD), led to the assignment of the main-chain and many side-chain resonances of 103 of the 106 residues. Qualitatively, the helical conformation is found to be the dominant secondary structure in apocytochrome b562 as it is in holocytochrome b562. The helical segments in apocytochrome b562 overlap extensively with the helical regions defined in the crystal structure of ferricytochrome b562. In addition, a number of tertiary NOEs have been identified which indicate that the global fold of the apoprotein at least partially resembles the four-helix bundle of the holoprotein. The results presented here, together with the evidence obtained with other methods [Feng and Sligar (1991) Biochemistry (submitted)], support the notion that the interior of the protein is fluid and may correspond to a molten globule state.     

Evaluation of the amplitude of changes in the distance between membranes in contact exposed to an external voltage

A quantitative evaluation of the amplitude of changes in the distance between two membranes due to changes in transmembrane voltage has been theoretically performed. The results are in good agreement with the data of bilayer lipid membrane experiments.     

Biochemical profiles of membranes from x-ray and neutron diffraction.

X-ray and neutron diffraction methods provide some information about the distribution of mass in biological membranes and lipid-water systems. Scattering density profiles obtained from these systems, however, usually are not directly interpretable in terms of the relative amounts of chemical constituents (e.g., lipid, protein, and water) as a function of position in the membrane. We demonstrate here that the combined use of x-ray and neutron-scattering profiles, together with information on the total amounts of each of the major membrane components, are sufficient to calculate unambiguously the volume fractions of these components at well-defined regions of the lamellar unit. Three cases are considered: a calculated model membrane pair, dipalmitoylphosphatidylcholine-water multilayers, and rabbit sciatic nerve myelin. For the model system, we discuss the limitations imposed by finite resolution in the diffraction patterns. For the lipid-water multilayers, we calculate water volume fractions in the hydrocarbon tail, lipid headgroup, and interlamellar regions; estimates of these values by various methods are in good agreement with our results. For the nerve myelin, we predict new results for the distribution of protein through the membrane.     

Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.     

Relationship between beta-lactamase activity and resistance to beta-lactam antibiotics in Mycobacterium smegmatis

Penicillin-susceptible mutants and beta-lactamase-negative mutants were isolated from Mycobacterium smegmatis after nitrosoguanidine mutagenesis. Both the mutants were found to be susceptible to low levels of penicillin and cephalosporins by twofold dilution testing. Clavulanic acid reduced the minimal inhibitory concentrations of beta-lactamase-labile beta-lactams for the penicillin-susceptible mutants and the parent strain, but had no effect on the susceptibility of the beta-lactamase-negative mutants. Comparison of the beta-lactamase activities found in these mutants and the parent strain indicated that there was a rough correlation between the beta-lactamase level in these organisms and their susceptibility to beta-lactams.     

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.     

The gamma-subunit of the principal G-protein from squid (Loligo forbesi) photoreceptors contains a novel N-terminal sequence.

The squid (Loligo forbesi) visual system presents as accessible a system for study of G-protein mediated signal transduction as the vertebrate rod outer segment with the added advantage that the major G-protein is a member of the Gq-class. Here the cDNA clone encoding the gamma-subunit of this G-protein is reported, thereby completing the molecular cloning of the heterotrimeric G-protein. The deduced protein structure of G-gamma has relatively little sequence identity with known mammalian counterparts particularly in comparison with the relatively high degree found for both the alpha- and beta-subunits of this protein. In particular, the N-terminus of the squid visual G-gamma contains a repetitive, highly charged region, rich in lysine and glutamate, that has no parallel in other G-proteins. The amino acid sequence of a number of peptides derived by chemical cleavage of G-gamma accounted for much of the protein sequence predicted from the cDNA, including the unusual N-terminal region.     

Gamma-hydroxybutyric acid induces oxidative stress in cerebral cortex of young rats

GHB is a naturally occurring compound in the central nervous system (CNS) whose tissue concentration are highly increased during drug abuse and in the inherited deficiency of succinic semialdehyde dehydrogenase (SSADH) activity. SSADH deficiency is a neurometabolic-inherited disorder of the degradation pathway of gamma-aminobutyric acid (GABA). It is biochemically characterized by increased concentrations of gamma-hydroxybutyric acid (GHB) in tissues, cerebrospinal fluid (CSF), blood and urine of affected patients. Clinical manifestations are variable, ranging from mild retardation of mental, motor, and language development to more severe neurological symptoms, such as hypotonia, ataxia and seizures, whose underlying mechanisms are practically unknown. In the present study, the in vitro and in vivo effects of GHB was investigated on some parameters of oxidative stress, such as chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), as well as the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in homogenates from cerebral cortex of 15-day-old Wistar rats. In vitro, GHB significantly increased chemiluminescence and TBA-RS levels, while TRAP and TAR measurements were markedly diminished. In contrast, the activities of the antioxidant enzymes SOD, CAT and GPX were not altered by GHB in vitro. Acute administration of GHB provoked a significant enhance of TBA-RS levels and a decrease of TRAP and TAR measurements. These results indicate that GHB induces oxidative stress by stimulating lipid peroxidation and decreasing the non-enzymatic antioxidant defenses in cerebral cortex of young rats. If these effects also occur in humans, it is possible that they might contribute to the brain damage found in SSADH-deficient patients and possibly in individuals who consume GHB or its prodrug gamma-butyrolactone.     

In the trenches: lessons for scientists from California's Proposition 71 campaign

I describe a number of valuable lessons I learned from participating in California's Proposition 71 effort about the role that scientists and rigorous scientific advice can play in a public political process. I describe how scientists can provide valuable information and advice and how they can also gain a great deal from the experience that is valuable to a practicing research scientist. Finally, I argue that in the future, building similar broad coalitions to support biomedical and other areas of scientific research will be essential to protect publicly funded science. Thus, a key lesson from the Proposition 71 experience is that engagement of scientists with diverse nonscientific groups can make a big difference and that scientists must actively engage with the public in the future if we are to contribute robustly to the medical and economic health of our communities.