The ATP/2e ratio during photosynthesis in intact spinach chloroplasts.

Addition of ribose-5-phosphate to intact spinach chloroplasts in the absence of added P_i resulted in a conversion of part of the Benson-Calvin cycle into a linear sequence so that triose phosphate accumulated during CO₂ fixation stoichiometrically with the O₂ evolved (triose phosphate / O₂ ratio was 2.0). The fortunate consequence of this effect is that the $\text{ATP}\text{2e}$ ratio may be calculated from the 3-phosphoglycerate and triose phosphate accumulated and the O₂ evolved. In this way the $\text{ATP}\text{2e}$ ratio was shown to be 2.0, with cyclic or pseudocyclic phosphorylation contributing less than 9% to the total phosphorylation.     

Photocross-linking analysis of the contact surface of tRNA Met in complexes with Escherichia coli methionine:tRNA ligase.

Photoinduced covalent cross-linking has been used to identify a common surface of four methionine-accepting tRNAs which interact specifically with the Escherichia coli methionine:tRNA ligase (EC 6.1.1.10). tRNA--ligase mixtures were irradiated, and the covalently linked complexes were isolated and digested with T1 RNase (Schimmel & Budzik, 1977). The fragments lost from the elution profile of the T1 RNase digest were considered to have been cross-linked to the protein and therefore in intimate contact with the enzyme. Only specific cognate tRNA--ligase pairs produce covalently linked complexes. The four substrate tRNAs used in this study have substantially different sequences, but all showed a common cross-linking pattern, supporting the view that the sites cross-linked to the enzyme reflect the functionally common contact surface rather than particularly photoreactivity regions of tRNA. The cross-linked contact surface is comprised of three regions: (1) the narrow groove of the anticodon stem and its extension into the anticodon loop; (2) the 3' terminal residues; and (3) the 3' side of the "T arm". Unlike previous studies with other tRNAs, the D arm is not involved and significant radiation damage is suffered by the tRNA which must be taken into account in the analysis. The results are consistent with and complement chemical modification studies [Schulman, L. H., & Pelka, H. (1977) Biochemistry 16, 4256].     

Sea-blue histiocytosis and beta-thalassemia in the same family.

Among 5 families with SBH of Neapolitan origin, herediatry glyco-lipidosis was accompanied in one by beta-thalassemia. All 10 members of this family, namely parents and 8 siblings, were investigated. The mother and two children were found to be carriers of both SBH and beta-thalassemia, while three other siblings were carriers of SBH alone. None of the six patients conformed the classic clinical picture often observed in genotypical SBH. The present state of genotypic transmission of the stigma is discussed on the basis of the author's experience as well as the data in the literature. As for the combination SBH-thalassemia in the same individual it may be concluded that the two genes are most likely independent and certainly not linked.     

A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1^R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1a^R117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a^+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a^+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a^+/ki Tert^-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a^+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a^+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.     

Substance P and acetylcholine are co-localized in the pathway mediating mucociliary activity in Rana pipiens

Mucociliary activity is an important clearance mechanism in the respiratory system of air breathing vertebrates. Substance P (SP) and acetylcholine play a key role in the stimulation of the mucociliary transport in the frog palate. In this study, retrograde neuronal tracing was combined with immunocytochemistry for SP and choline acetyl transferase (ChAT) in the trigeminal ganglion and for neurokinin-1 receptor (NK1R) in the palate of Rana pipiens. The cells of origin of the palatine nerve were identified in the trigeminal ganglion using the retrograde tracer Fluorogold (FG). Optimal labeling of FG cells in the trigeminal ganglion was obtained at 96 h of exposure. Immunoflorescent shows that SP and acetylcholine are co-localized in 92% of the cells labeled with FG in the trigeminal ganglion. NK1 receptors were found in the membrane of epithelial and goblet cells of the palate. Ultrastructural study of the palate showed axonal-like endings with vesicles in connection with epithelial and goblet cells. These results further support the concerted action of both neurotransmitters in the regulation of mucociliary activity in the frog palate.     

Local multiple sequence alignment using dead-end elimination

MOTIVATION: Local multiple sequence alignment is a basic tool for extracting functionally important regions shared by a family of protein sequences. We present an effectively polynomial-time algorithm for rigorously solving the local multiple alignment problem. RESULTS: The algorithm is based on the dead-end elimination procedure that makes it possible to avoid an exhaustive search. In the framework of the sum-of-pairs scoring system, certain rejection criteria are derived in order to eliminate those sequence segments and segment pairs that can be mathematically shown to be inconsistent (dead-ending) with the globally optimal alignment. Iterative application of the elimination criteria results in a rapid reduction of combinatorial possibilities without considering them explicitly. In the vast majority of cases, the procedure converges to a unique globally optimal solution. In contrast to the exhaustive search, whose computational complexity is combinatorial, the algorithm is computationally feasible because the number of operations required to eliminate the dead-ending segments and segment pairs grows quadratically and cubically, respectively, with the total number of sequence elements. The method is illustrated on a set of protein families for which the globally optimal alignments are well recognized. AVAILABILITY: The source code of the program implementing the algorithm is available upon request from the authors. CONTACT: alex_lukashin@biogen.com.     

Capsaicin Modulates Proliferation,Migration, and Activation of Hepatic Stellate Cells

Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro.     

Characterization of Ca2+-activated, phospholipid-dependent protein kinase C/protein kinase M in guinea pig peritoneal exudate macrophages

Protein kinase C (C-kinase) is shown to be present in the cytosolic and particulate fractions of mineral oil induced peritoneal macrophages of guinea pigs. By omission or use of a high concentration of leupeptin, three forms of the enzyme were obtained: stimulant/Ca2+/phospholipid-dependent C-kinase, eluted from DEAE 52 cellulose at 0.08-0.16 M NaCl; stimulant/Ca2+/phospholipid-independent protein kinase M (M-kinase), and Ca2+-inhibited & stimulant/phospholipid-dependent form of protein kinase, both eluted from DEAE 52 cellulose at 0.18-0.22 M NaCl. Phorbol ester or 1,2-diacylglycerol were used as stimulants. It is suggested that Ca2+-inhibited & stimulant/phospholipid-dependent protein kinase represents the in vivo form of the M-kinase in intact cells.