ATP regulation of recombinant type 3 inositol 1,4,5-trisphosphate receptor gating

A family of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channels plays a central role in Ca2+ signaling in most cells, but functional correlates of isoform diversity are unclear. Patch-clamp electrophysiology of endogenous type 1 (X-InsP3R-1) and recombinant rat type 3 InsP3R (r-InsP3R-3) channels in the outer membrane of isolated Xenopus oocyte nuclei indicated that enhanced affinity and reduced cooperativity of Ca2+ activation sites of the InsP3-liganded type 3 channel distinguished the two isoforms. Because Ca2+ activation of type 1 channel was the target of regulation by cytoplasmic ATP free acid concentration ([ATP](i)), here we studied the effects of [ATP]i on the dependence of r-InsP(3)R-3 gating on cytoplasmic free Ca2+ concentration ([Ca2+]i. As [ATP]i was increased from 0 to 0.5 mM, maximum r-InsP3R-3 channel open probability (Po) remained unchanged, whereas the half-maximal activating [Ca2+]i and activation Hill coefficient both decreased continuously, from 800 to 77 nM and from 1.6 to 1, respectively, and the half-maximal inhibitory [Ca2+]i was reduced from 115 to 39 microM. These effects were largely due to effects of ATP on the mean closed channel duration. Whereas the r-InsP3R-3 had a substantially higher Po than X-InsP3R-1 in activating [Ca2+]i (< 1 microM) and 0.5 mM ATP, the Ca2+ dependencies of channel gating of the two isoforms became remarkably similar in the absence of ATP. Our results suggest that ATP binding is responsible for conferring distinct gating properties on the two InsP3R channel isoforms. Possible molecular models to account for the distinct regulation by ATP of the Ca2+ activation properties of the two channel isoforms and the physiological implications of these results are discussed. Complex regulation by ATP of the types 1 and 3 InsP3R channel activities may enable cells to generate sophisticated patterns of Ca2+ signals with cytoplasmic ATP as one of the second messengers.     

Knockout mice: a paradigm shift in modern immunology

In the past decade, advances in genetic engineering and mouse knockout technology have transformed our understanding of the immune system. In particular, new perspectives on T-cell development, co-stimulation and activation have emerged from the study of single and multiple gene-knockout animals, as well as from conditional knockout and 'knock-in' mutants. Analysis of these animals has clarified important intracellular signalling pathways and has shed light on the regulatory mechanisms that govern normal immune responses and autoimmunity.     

Mortality and smoking in Hong Kong: case-control study of all adult deaths in 1998

ObjectiveTo assess the mortality currently associated with smoking in Hong Kong, and, since cigarette consumption reached its peak 20 years earlier in Hong Kong than in mainland China, to predict mortality in China 20 years hence.DesignCase-control study. Past smoking habits of all Chinese adults in Hong Kong who died in 1998 (cases) were sought from those registering the death.SettingAll the death registries in Hong Kong.Participants27 507 dead cases (81% of all registered deaths) and 13 054 live controls aged ⩾35 years.ResultsIn men aged 35-69 the adjusted risk ratios (and 95% confidence intervals) comparing smokers with non-smokers were 1.92 (1.70 to 2.16) for all deaths, 2.22 (1.94 to 2.55) for neoplastic deaths, 2.60 (2.10 to 3.21) for respiratory deaths (including tuberculosis, risk ratio 2.54), and 1.68 (1.43 to 1.97) for vascular deaths (each P<0.0001). In women aged 35-69 the corresponding risk ratios were 1.62 (1.40 to 1.88) for all deaths, 1.60 (1.33 to 1.93) for neoplastic deaths, 3.13 (2.21 to 4.44) for respiratory deaths, and 1.55 (1.20 to 1.99) for vascular deaths (each P<0.001). If these associations with smoking are largely or wholly causal then, among all registered deaths at ages 35-69 in 1998, tobacco caused about 33% (2534/7588) of all male deaths and 5% (169/3341) of all female deaths (hence 25% of all deaths at these ages). At older ages tobacco seemed to be the cause of 15% (3017/20 420) of all deaths.ConclusionsAmong middle aged men the proportion of deaths caused by smoking is more than twice as big in Hong Kong now (33%) as in mainland China 10 years earlier. This supports predictions of a large increase in tobacco attributable mortality in China as a whole.

What is already known on this topic

China, with 20% of the world''s population, smokes 30% of the world''s cigarettes. Men smoke most, and the proportion of male deaths at ages 35-69 attributable to tobacco has been predicted to rise over the next few decades from 13% (in 1988) to about 33%In Hong Kong cigarette consumption reached its peak 20 years earlier than in mainland China, so the epidemic of male deaths from tobacco should now be at a more advanced stage

What this study adds

In the general population of Hong Kong in 1998 tobacco caused about 33% of all male deaths at ages 35-69 plus 5% of all female deaths, and hence 25% of all deaths at these agesIn the male smokers tobacco caused about half of all deaths at ages 35-69The hazards now seen in Hong Kong foreshadow a substantial increase in tobacco deaths among middle aged men in mainland China over the next few decades if current smoking patterns persist     

Antimicrobial peptides derived from heme-containing proteins: Hemocidins

Deprived of heme and partially unfolded hemoglobin, myoglobin and cytochrome c display microbicidal activity against a broad spectrum of microorganisms with half maximal lethal dose estimated at micromolar concentrations. The intact proteins were ineffective. Antibacterial activity of these apohemoproteins was also sustained after digestion to approximately 50 amino acids long peptides but further fragmentation abolished microbicidal properties. The most active fragment of apomyoglobin (corresponding to 56–131 region) showed a pronounced effect on the E. coli membrane permeabilization and its action was sensitive to salt as well as to divalent cations concentrations. The membrane-directed effect was specific toward bacteria but no lipopolysaccharide binding properties were observed. No hemolytic properties, even at high peptide concentrations were found; however, a slight but dose-independent cytotoxic effect was observed on fibroblasts and hepatoma cells. The presented data suggest a `carpet-like' mechanism of the membrane-directed activity and may result from exceptional abilities of hemoprotein-derived peptides to form alpha-helical structures. We postulate that the antimicrobial peptides obtained from the heme-containing proteins should be named hemocidins, in contrast to, e.g., hemorphins displaying opioid-like activity.     

Highly Amyloidogenic Two-chain Peptide Fragments Are Released upon Partial Digestion of Insulin with Pepsin

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A–Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to “explosive” fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein.     

Staphylococcal SplB Serine Protease Utilizes a Novel Molecular Mechanism of Activation

Staphylococcal SplB protease belongs to the chymotrypsin family. Chymotrypsin zymogen is activated by proteolytic processing at the N terminus, resulting in significant structural rearrangement at the active site. Here, we demonstrate that the molecular mechanism of SplB protease activation differs significantly and we characterize the novel mechanism in detail. Using peptide and protein substrates we show that the native signal peptide, or any N-terminal extension, has an inhibitory effect on SplB. Only precise N-terminal processing releases the full proteolytic activity of the wild type analogously to chymotrypsin. However, comparison of the crystal structures of mature SplB and a zymogen mimic show no rearrangement at the active site whatsoever. Instead, only the formation of a unique hydrogen bond network, distant form the active site, by the new N-terminal glutamic acid of mature SplB is observed. The importance of this network and influence of particular hydrogen bond interactions at the N terminus on the catalytic process is demonstrated by evaluating the kinetics of a series of mutants. The results allow us to propose a consistent model where changes in the overall protein dynamics rather than structural rearrangement of the active site are involved in the activation process.     

The Bipolar II Depression Questionnaire: A Self-Report Tool for Detecting Bipolar II Depression

Bipolar II (BP-II) depression is often misdiagnosed as unipolar (UP) depression, resulting in suboptimal treatment. Tools for differentiating between these two types of depression are lacking. This study aimed to develop a simple, self-report screening instrument to help distinguish BP-II depression from UP depressive disorder. A prototype BP-II depression questionnaire (BPIIDQ-P) was constructed following a literature review, panel discussions and a field trial. Consecutively assessed patients with a diagnosis of depressive disorder or BP with depressive episodes completed the BPIIDQ-P at a psychiatric outpatient clinic in Hong Kong between October and December 2013. Data were analyzed using discriminant analysis and logistic regression. Of the 298 subjects recruited, 65 (21.8%) were males and 233 (78.2%) females. There were 112 (37.6%) subjects with BP depression [BP-I = 42 (14.1%), BP-II = 70 (23.5%)] and 182 (62.4%) with UP depression. Based on family history, age at onset, postpartum depression, episodic course, attacks of anxiety, hypersomnia, social phobia and agoraphobia, the 8-item BPIIDQ-8 was constructed. The BPIIDQ-8 differentiated subjects with BP-II from those with UP depression with a sensitivity/specificity of 0.75/0.63 for the whole sample and 0.77/0.72 for a female subgroup with a history of childbirth. The BPIIDQ-8 can differentiate BP-II from UP depression at the secondary care level with satisfactory to good reliability and validity. It has good potential as a screening tool for BP-II depression in primary care settings. Recall bias, the relatively small sample size, and the high proportion of females in the BP-II sample limit the generalization of the results.     

Caldesmon-binding sites on tropomyosin

The interaction of chicken gizzard caldesmon with fragments of tropomyosin, generated by chemical, enzymatic, and mutational means, was studied to determine the caldesmon-binding site(s) on tropomyosin. Binding was examined by fluorescence spectroscopy and affinity chromatography. Removal of residues 1-141 and 228-284, respectively, from the NH2 and COOH ends of tropomyosin did not affect its binding to caldesmon significantly, indicating that the major, caldesmon-binding region lies between residues 142-227. The Escherichia coli produced chicken gizzard beta-tropomyosin mutant, CSM-beta (1/8/12-227), bound caldesmon about 2-fold stronger than a similar mutant of residues 8-200. This further focused the primary caldesmon-binding site to residues 201-227. Cleavage of tropomyosin at CYS-190 weakened markedly the binding of the two resulting fragments, residues 1-189 and 190-284, to caldesmon suggesting the requirement for the integrity of the caldesmon-binding region between residues 142227 of tropomyosin for strong interaction with caldesmon. Based on data from this study and others, we have proposed models for the interaction of tropomyosin with caldesmon in vitro, as well as the possible arrangement of the smooth muscle thin filament proteins in vivo.