The peptide binding site of the substance P (NK-1) receptor localized by a photoreactive analogue of substance P: presence of a disulfide bond.

Substance P (SP) is a neuropeptide that mediates multiple physiological responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence. To identify the regions of the SP receptor, also termed the NK-1 receptor, involved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa). In the present study, we used radioiodinated Bpa8-SP to covalently label with high efficiency the rat SP receptor expressed in a transfected mammalian cell line. To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled receptor was subjected to partial enzymatic cleavage by trypsin. A major digestion product of 22 kDa was identified. Upon reduction with 2-mercaptoethanol the mass of this product decreased to 14 kDa. The 22-kDa tryptic fragment was purified in excellent yield by preparative SDS/PAGE under nonreducing conditions. Subcleavage with Staphylococcus aureus V8 protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa, respectively. Upon reductive cleavage, the V8 protease fragment decreased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3.2 kDa. Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, the V8 protease fragment is Thr-173 to Glu-183, while the endoproteinase ArgC fragment is Val-178 to Arg-190. These two fragments share the common sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa8-SP is thus restricted to a residue within this overlap sequence. The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participates in a disulfide bond that links the first and second extracellular loops of the receptor.

A model for the transcriptional regulation of the CYP2B1/B2 gene in rat liver.

The phenobarbitone-responsive minimal promoter has been shown to lie between nt -179 and nt + 1 in the 5' (upstream) region of the CYP2B1/B2 gene in rat liver, on the basis of the drug responsiveness of the sequence linked to human growth hormone gene as reporter and targeted to liver as an asialoglycoprotein-DNA complex in vivo. Competition analyses of the nuclear protein-DNA complexes formed in gel shift assays with the positive (nt -69 to -98) and negative (nt -126 to -160) cis elements (PE and NE, respectively) identified within this region earlier indicate that the same protein may be binding to both the elements. The protein species purified on PE and NE affinity columns appear to be identical based on SDS/PAGE analysis, where it migrates as a protein of 26-28 kDa. Traces of a high molecular weight protein (94-100 kDa) are also seen in the preparation obtained after one round of affinity chromatography. The purified protein stimulates transcription of a minigene construct containing the 179 nt on the 5' side of the CYP2B1/B2 gene linked to the I exon in a cell-free system from liver nuclei. The purified protein can give rise to all the three complexes (I, II, and III) with the PE, just as the crude nuclear extract, under appropriate conditions. Manipulations in vitro indicate that the NE has a significantly higher affinity for the dephosphorylated form than for the phosphorylated form of the protein. The PE binds both forms. Phenobarbitone treatment of the animal leads to a significant increase in the phosphorylation of the 26- to 28-kDa and 94-kDa proteins in nuclear labeling experiments followed by isolation on a PE affinity column. We propose that the protein binding predominantly to the NE in the dephosphorylated state characterizes the basal level of transcription of the CYP2B1/B2 gene. Phenobarbitone treatment leads to phosphorylation of the protein, shifting the equilibrium toward binding to the PE. This can promote interaction with an upstream enhancer through other proteins such as the 94-kDa protein and leads to a significant activation of transcription.

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.

Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms.

Of the approximately 380 families of angiosperms, representatives of only 10 are known to form symbiotic associations with nitrogen-fixing bacteria in root nodules. The morphologically based classification schemes proposed by taxonomists suggest that many of these 10 families of plants are only distantly related, engendering the hypothesis that the capacity to fix nitrogen evolved independently several, if not many, times. This has in turn influenced attitudes toward the likelihood of transferring genes responsible for symbiotic nitrogen fixation to crop species lacking this ability. Phylogenetic analysis of DNA sequences for the chloroplast gene rbcL indicates, however, that representatives of all 10 families with nitrogen-fixing symbioses occur together, with several families lacking this association, in a single clade. This study therefore indicates that only one lineage of closely related taxa achieved the underlying genetic architecture necessary for symbiotic nitrogen fixation in root nodules.

Architecture of a Bench-Type Carbonate Lake Margin and Its Relation to Fluvially Dominated Deltas, Las Minas Basin, Upper Miocene, Spain

The Upper Miocene stratigraphic succession of the Las Minas Basin, located at the external zone of the Betic Chain in SE Spain, preserves several examples of lake carbonate bench deposits. Excellent exposures of the carbonate benches allow detailed observation of the architecture of these sediments and provide new insights for the ‘‘steep-gradient bench margin–low energy’’ model proposed by Platt and Wright (1991). The lake carbonate benches developed in close association with fluvially dominated shallow deltas that exhibit typical Gilbert-type profiles. The delta sequences comprise bottomset prodelta marl facies, distal to proximal foreset facies, deposited mainly in a delta-front environment, and topset facies, the latter reflecting both subaqueous delta-front and subaerial delta-plain environments. The development of the carbonate benches was constrained by the convexupward morphology of the deltaic deposits, which led to the available accommodation space for the growth of the steep-gradient platforms. The benches display a progradational pattern characterized by sigmoid-oblique internal geometries and offlap upper boundary relationships, which suggests that the carbonate benches developed under slow though continuous lake-level rise. Both the dimensions of the benches and the dominant carbonate components (i.e., encrusted charophyte stems and calcified cyanobaterial remains), allow comparisons with the progradational marl benches recognized in modern temperate hardwater lakes. Accordingly, the case study presented here provides a good ancient sedimentary analog for low-energy lake carbonate benches. Moreover, the evolutionary trend inferred from the fossil example offers new insights into the depositional conditions of this type of sediment and allows recognition of the transitional pattern from bench to ramp carbonate lake margins.

Reconfigurable computing for Monte Carlo simulations: results and prospects of the Janus project

We describe Janus, a massively parallel FPGA-based computer optimized for the simulation of spin glasses, theoretical models for the behavior of glassy materials. FPGAs (as compared to GPUs or many-core processors) provide a complementary approach to massively parallel computing. In particular, our model problem is formulated in terms of binary variables, and floating-point operations can be (almost) completely avoided. The FPGA architecture allows us to run many independent threads with almost no latencies in memory access, thus updating up to 1024 spins per cycle. We describe Janus in detail and we summarize the physics results obtained in four years of operation of this machine; we discuss two types of physics applications: long simulations on very large systems (which try to mimic and provide understanding about the experimental non equilibrium dynamics), and low-temperature equilibrium simulations using an artificial parallel tempering dynamics. The time scale of our non-equilibrium simulations spans eleven orders of magnitude (from picoseconds to a tenth of a second). On the other hand, our equilibrium simulations are unprecedented both because of the low temperatures reached and for the large systems that we have brought to equilibrium. A finite-time scaling ansatz emerges from the detailed comparison of the two sets of simulations. Janus has made it possible to perform spin glass simulations that would take several decades on more conventional architectures. The paper ends with an assessment of the potential of possible future versions of the Janus architecture, based on state-of-the-art technology.