Ordered and dynamic assembly of single spliceosomes

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.     

Gate-variable optical transitions in graphene

Two-dimensional graphene monolayers and bilayers exhibit fascinating electrical transport behaviors. Using infrared spectroscopy, we find that they also have strong interband transitions and that their optical transitions can be substantially modified through electrical gating, much like electrical transport in field-effect transistors. This gate dependence of interband transitions adds a valuable dimension for optically probing graphene band structure. For a graphene monolayer, it yields directly the linear band dispersion of Dirac fermions, whereas in a bilayer, it reveals a dominating van Hove singularity arising from interlayer coupling. The strong and layer-dependent optical transitions of graphene and the tunability by simple electrical gating hold promise for new applications in infrared optics and optoelectronics.     

Localized control of ligand binding in hemoglobin: effect of tertiary structure on picosecond geminate recombination

The picosecond geminate rebinding of molecular oxygen was monitored in a variety of different human, reptilian, and fish hemoglobins. The fast (100 to 200 picoseconds) component of the rebinding is highly sensitive to protein structure. Both proximal and distal perturbations of the heme affect this rebinding process. The rebinding yield for the fast process correlates with the frequency of the stretching motion of the iron-proximal histidine mode (VFe-His) observed in the transient Raman spectra of photodissociated ligated hemoglobins. The high-affinity R-state species exhibit the highest values for VFe-His and the highest yields for fast rebinding, whereas low affinity R-state species and T-state species exhibit lower values of VFe-His and correspondingly reduced yields for this geminate process. These findings link protein control of ligand binding with events at the heme.     

Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.

An expression cloning strategy was devised to isolate the keratinocyte growth factor (KGF) receptor complementary DNA. NIH/3T3 fibroblasts, which secrete this epithelial cell-specific mitogen, were transfected with a keratinocyte expression complementary DNA library. Among several transformed foci identified, one demonstrated the acquisition of specific high-affinity KGF binding sites. The pattern of binding competition by related fibroblast growth factors (FGFs) indicated that this receptor had high affinity for acidic FGF as well as KGF. The rescued 4.2-kilobase complementary DNA was shown to encode a predicted membrane-spanning tyrosine kinase related to but distinct from the basic FGF receptor. This expression cloning approach may be generally applicable to the isolation of genes that constitute limiting steps in mitogenic signaling pathways.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.     

Hepatozoon spp.: report of some cases in dogs in Brasília, Brazil

Canine hepatozoonosis is a disease caused by the tick-borne protozoan Hepatozoon spp. It has been reported in the United States, southern Europe, the Middle East, Africa and the Far East. In Brazil, canine hepatozoonosis is an emerging protozoal tick-borne disease, and is characterized by distinct clinical signs. The objective of this study was to analyze the laboratory findings of some hepatozoonosis cases in dogs in Brasília, Brazil, and their clinical signs. The animals of this experiment showed low parasitemia, similar to H. americanum, but the clinical signs presented were similar to H. canis. According to our observations and in agreement with O'Dwyer et al. [Vet. Parasitol. 94 (2001) 143], the Brazilian Hepatozoon appears more to resemble the species found in the eastern Hemisphere than with H. americanum of North America, or could be caused by a new species. Our data revealed that hepatozoonosis could be considered endemic in Brasília.     

Trehalose regulation of glucose-6-phosphate hydrolysis in blowfly extracts

The presence of trehalose enhances the rate of glucose-6-phosphate hydrolysis in extracts of fat body and other tissues of adult Phormia regina Meigen. The activation appears to be specific, and increasing the concentration of trehalose changes both V(max) and K(m) for glucose-6-phosphate. There is, however, no easily recognizable phosphotransferase in the extract.