Cell cycle oscillation of phosphatase inhibitor-2 in rat fibroblasts coincident with p34cdc2 restriction

Attention has focused on the regulation of the eucaryotic cell division cycle since the protein kinase p34cdc2 was identified as a key enzyme in mitotic induction. The level of this kinase remains constant throughout the cell cycle but its activity alters, particularly before M phase. Although the factors regulating cdc2 activity are still unknown, there is increasing evidence that it is influenced by p34cdc2 dephosphorylation. Protein phosphatase inhibitor-2 (I2) is a specific inhibitor of phosphatase type-1, which with type-2A is one of the two principal Ser(P) and Thr(P) phosphatases. Here we show that the level of I2, assayed by immunofluorescence staining, activity measurements, western immunoblotting and metabolic labelling, oscillates during the cell cycle in rat fibroblasts, peaking at S phase and mitosis. Moreover, when we inhibited I2 in vivo by microinjection of anti-I2 antibodies in S-phase cells, the pseudo-mitotic cellular response to injected p34cdc2 was restored, indicating that I2 might have a role in the modulation of p34cdc2 activity.     

Eukaryotic type II chaperonin CCT interacts with actin through specific subunits

Chaperonins assist the folding of other proteins. Type II chaperonins, such as chaperonin containing TCP-1(CCT), are found in archaea and in the eukaryotic cytosol. They are hexadecameric or nonadecameric oligomers composed of one to eight different polypeptides. Whereas type I chaperonins like GroEL are promiscuous, assisting in the folding of many other proteins, only a small number of proteins, mainly actin and tubulin, have been described as natural substrates of CCT. This specificity may be related to the divergence of the eight CCT subunits. Here we have obtained a three-dimensional reconstruction of the complex between CCT and alpha-actin by cryo-electron microscopy and image processing. This shows that alpha-actin interacts with the apical domains of either of two CCT subunits. Immunolabelling of CCT-substrate complexes with antibodies against two specific CCT subunits showed that actin binds to CCT using two specific and distinct interactions: the small domain of actin binds to CCTdelta and the large domain to CCTbeta or CCTepsilon (both in position 1,4 with respect to delta). These results indicate that the binding of actin to CCT is both subunit-specific and geometry-dependent. Thus, the substrate recognition mechanism of eukaryotic CCT may differ from that of prokaryotic GroEL.     

Probing the chemistry of thioredoxin catalysis with force

Thioredoxins are enzymes that catalyse disulphide bond reduction in all living organisms. Although catalysis is thought to proceed through a substitution nucleophilic bimolecular (S(N)2) reaction, the role of the enzyme in modulating this chemical reaction is unknown. Here, using single-molecule force-clamp spectroscopy, we investigate the catalytic mechanism of Escherichia coli thioredoxin (Trx). We applied mechanical force in the range of 25-600 pN to a disulphide bond substrate and monitored the reduction of these bonds by individual enzymes. We detected two alternative forms of the catalytic reaction, the first requiring a reorientation of the substrate disulphide bond, causing a shortening of the substrate polypeptide by 0.79 +/- 0.09 A (+/- s.e.m.), and the second elongating the substrate disulphide bond by 0.17 +/- 0.02 A (+/- s.e.m.). These results support the view that the Trx active site regulates the geometry of the participating sulphur atoms with sub-?ngstr?m precision to achieve efficient catalysis. Our results indicate that substrate conformational changes may be important in the regulation of Trx activity under conditions of oxidative stress and mechanical injury, such as those experienced in cardiovascular disease. Furthermore, single-molecule atomic force microscopy techniques, as shown here, can probe dynamic rearrangements within an enzyme's active site during catalysis that cannot be resolved with any other current structural biological technique.     

The search for migraine genes: an overview of current knowledge

Migraine is a complex familial condition that imparts a significant burden on society. There is evidence for a role of genetic factors in migraine, and elucidating the genetic basis of this disabling condition remains the focus of much research. In this review we discuss results of genetic studies to date, from the discovery of the role of neural ion channel gene mutations in familial hemiplegic migraine (FHM) to linkage analyses and candidate gene studies in the more common forms of migraine. The success of FHM regarding discovery of genetic defects associated with the disorder remains elusive in common migraine, and causative genes have not yet been identified. Thus we suggest additional approaches for analysing the genetic basis of this disorder. The continuing search for migraine genes may aid in a greater understanding of the mechanisms that underlie the disorder and potentially lead to significant diagnostic and therapeutic applications. Received 16 December 2005; received after revision 9 October 2006; accepted 13 November 2006     

Multiple ADH genes are associated with upper aerodigestive cancers

Alcohol is an important risk factor for upper aerodigestive cancers and is principally metabolized by alcohol dehydrogenase (ADH) enzymes. We have investigated six ADH genetic variants in over 3,800 aerodigestive cancer cases and 5,200 controls from three individual studies. Gene variants rs1229984 (ADH1B) and rs1573496 (ADH7) were significantly protective against aerodigestive cancer in each individual study and overall (P = 10(-10) and 10(-9), respectively). These effects became more apparent with increasing alcohol consumption (P for trend = 0.0002 and 0.065, respectively). Both gene effects were independent of each other, implying that multiple ADH genes may be involved in upper aerodigestive cancer etiology.     

Spatial patterns of the semi-aquatic rodent Nectomys squamipes in Atlantic forest streams

Nectomys squamipes is a semi-aquatic rodent with a wide geographic range in Brazilian forests and savannas. This species is a host for several parasites associated with aquatic environments, especially Schistosoma mansoni, and thus its spatial pattern may influence some diseases’ patterns. Here we present important aspects of water rat spatial behaviour, including technical and ecological aspects. Water rats were studied by live trapping in rivers and in gallery forests between January 2005 and August 2008, with a total sampling effort of 9511 trap-nights. We also studied some animals through radio tracking and the spool-and-line technique. Water rat movement was distributed evenly between the terrestrial and aquatic environments. Nectomys squamipes occurrence in small tributaries was higher than in larger and lotic rivers. Both home range, the conventional two-dimensional approach, and home length, a one-dimensional approach, proved to be useful to study N. squamipes, depending on the shape of the water body used by the individual. Although Nectomys squamipes’ home length was positively correlated with body weight, sex was also important to explain home length variation; at similar body weights, males have larger home lengths than females. Males explored larger areas during the rainy season, likely in search of females, as this is the water rat reproductive season in Atlantic forest. In contrast, females explored larger areas during the dry season, likely looking for additional food supplies as a result of deficient water rat food supplies during the Atlantic forest dry season. Females overlapped home lengths with more than one male, and males with more than one female, suggesting a promiscuous mating system for this species.     

IgE-mediated degranulation of mast cells does not require opening of ion channels

Rat peritoneal mast cells respond to antigenic stimulation by releasing histamine through exocytosis. The dynamics of exocytosis can be investigated by dialysing single cells with patch pipettes using the whole-cell recording configuration of the patch-clamp technique. However, dialysed cells fail to respond to external stimuli such as compound 48/80 or antigens, suggesting that essential cytoplasmic components have been washed out. We have developed a new patch-clamp configuration in which the patch under the pipette tip is not disrupted but instead permeabilized, preventing the diffusion of large molecules out of the cell. In this configuration the cell responds to external stimulation, and the capacitance as well as the conductance of the cell membrane can be recorded during degranulation. On antigenic stimulation, the cell capacitance (proportional to plasma membrane area), after an initial delay, increases by a factor of about 3. This increase in capacitance is often preceded by a transient increase in conductance. Agents that block Ca-activated channels inhibit this conductance change without affecting the amplitude and time course of degranulation. We therefore conclude that, in contrast to excitable secretory cells such as chromaffin cells, mast cells do not use ion channels in stimulus-secretion coupling.     

Reverse engineering of the giant muscle protein titin

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.