Determining divergence times with a protein clock: Update and reevaluation

A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.

Functional analyses of troponin T mutations that cause hypertrophic cardiomyopathy: Insights into disease pathogenesis and troponin function

Mutations in a number of cardiac sarcomeric protein genes cause hypertrophic cardiomyopathy (HCM). Previous findings indicate that HCM-causing mutations associated with a truncated cardiac troponin T (TnT) and missense mutations in the β-myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. In contrast, Lin et al. [Lin, D., Bobkova, A., Homsher, E. & Tobacman, L. S. (1996) J. Clin. Invest. 97, 2842–2848] characterized a TnT point mutation (Ile79Asn) and concluded that it might lead to hypercontractility and, thus, potentially a different mechanism for HCM pathogenesis. In this study, three HCM-causing cardiac TnT mutations (Ile79Asn, Arg92Gln, and ΔGlu160) were studied in a myotube expression system. Functional studies of wild-type and mutant transfected myotubes revealed that all three mutants decreased the calcium sensitivity of force production and that the two missense mutations (Ile79Asn and Arg92Gln) increased the unloaded shortening velocity nearly 2-fold. The data demonstrate that TnT can alter the rate of myosin cross-bridge detachment, and thus the troponin complex plays a greater role in modulating muscle contractile performance than was recognized previously. Furthermore, these data suggest that these TnT mutations may cause disease via an increased energetic load on the heart. This would represent a second paradigm for HCM pathogenesis.

Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury

Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO−) and nitric oxide (NO⋅), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 μmol/kg S-nitrosoglutathione (GSNO) (an NO⋅ donor), or 3 μmol/kg Angeli’s salt (AS) (a source of NO−) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but, rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron oxidant that converts NO− to NO⋅, completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO. These results demonstrate that, although NO⋅ is protective, NO− increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute to postischemic myocardial injury.

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50°C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Disruption of the telomerase catalytic subunit gene from Arabidopsis inactivates telomerase and leads to a slow loss of telomeric DNA

Telomerase is an essential enzyme that maintains telomeres on eukaryotic chromosomes. In mammals, telomerase is required for the lifelong proliferative capacity of normal regenerative and reproductive tissues and for sustained growth in a dedifferentiated state. Although the importance of telomeres was first elucidated in plants 60 years ago, little is known about the role of telomeres and telomerase in plant growth and development. Here we report the cloning and characterization of the Arabidopsis telomerase reverse transcriptase (TERT) gene, AtTERT. AtTERT is predicted to encode a highly basic protein of 131 kDa that harbors the reverse transcriptase and telomerase-specific motifs common to all known TERT proteins. AtTERT mRNA is 10–20 times more abundant in callus, which has high levels of telomerase activity, versus leaves, which contain no detectable telomerase. Plants homozygous for a transfer DNA insertion into the AtTERT gene lack telomerase activity, confirming the identity and function of this gene. Because telomeres in wild-type Arabidopsis are short, the discovery that telomerase-null plants are viable for at least two generations was unexpected. In the absence of telomerase, telomeres decline by approximately 500 bp per generation, a rate 10 times slower than seen in telomerase-deficient mice. This gradual loss of telomeric DNA may reflect a reduced rate of nucleotide depletion per round of DNA replication, or the requirement for fewer cell divisions per organismal generation. Nevertheless, progressive telomere shortening in the mutants, however slow, ultimately should be lethal.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

Epidermal Growth Factor Signaling and Mitogenesis in Plcg1 Null Mouse Embryonic Fibroblasts

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-γ1, a ubiquitous tyrosine kinase substrate. Plcg1−/− embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1+/+ embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1−/− cells respond equivalently to PLcg1+/+ cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-γ1 for many responses to growth factors.

Cyclin D3-associated Kinase Activity Is Regulated by p27kip1 in BALB/c 3T3 Cells

We report that cyclin D3/cdk4 kinase activity is regulated by p27kip1 in BALB/c 3T3 cells. The association of p27kip1 was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27kip1/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27kip1-specific antibodies. The association of p27kip1 with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27kip1 protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27kip1 kinase activity was present throughout the cell cycle. Since we found that the p27kip1 activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27kip1 and the active cyclin D3 lacking p27kip1 by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27kip1/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27kip1 has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference.

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

The rab11 GTPase has been localized to both the Golgi and recycling endosomes; however, its Golgi-associated function has remained obscure. In this study, rab11 function in exocytic transport was analyzed by using two independent means to perturb its activity. First, expression of the dominant interfering rab11S25N mutant protein led to a significant inhibition of the cell surface transport of vesicular stomatitis virus (VSV) G protein and caused VSV G protein to accumulate in the Golgi. On the other hand, the expression of wild-type rab11 or the activating rab11Q70L mutant had no adverse effect on VSV G transport. Next, the membrane association of rab11, which is crucial for its function, was perturbed by modest increases in GDP dissociation inhibitor (GDI) levels. This led to selective inhibition of the trans-Golgi network to cell surface delivery, whereas endoplasmic reticulum–to–Golgi and intra-Golgi transport were largely unaffected. The transport inhibition was reversed specifically by coexpression of wild-type rab11 with GDI. Under the same conditions two other exocytic rab proteins, rab2 and rab8, remained membrane bound, and the transport steps regulated by these rab proteins were unaffected. Neither mutant rab11S25N nor GDI overexpression had any impact on the cell surface delivery of influenza hemagglutinin. These data show that functional rab11 is critical for the export of a basolateral marker but not an apical marker from the trans-Golgi network and pinpoint rab11 as a sensitive target for inhibition by excess GDI.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis

We have developed a coupled helicase–polymerase DNA unwinding assay and have used it to monitor the rate of double-stranded DNA unwinding catalyzed by the phage T4 DNA replication helicase (gp41). This procedure can be used to follow helicase activity in subpopulations in systems in which the unwinding-synthesis reaction is not synchronized on all the substrate-template molecules. We show that T4 replication helicase (gp41) and polymerase (gp43) can be assembled onto a loading site located near the end of a long double-stranded DNA template in the presence of a macromolecular crowding agent, and that this coupled “two-protein” system can carry out ATP-dependent strand displacement DNA synthesis at physiological rates (400 to 500 bp per sec) and with high processivity in the absence of other T4 DNA replication proteins. These results suggest that a direct helicase–polymerase interaction may be central to fast and processive double-stranded DNA replication, and lead us to reconsider the roles of the other replication proteins in processivity control.

The photoisomerization of retinal in bacteriorhodopsin: Experimental evidence for a three-state model

The primary events in the all-trans to 13-cis photoisomerization of retinal in bacteriorhodopsin have been investigated with femtosecond time-resolved absorbance spectroscopy. Spectra measured over a broad range extending from 7000 to 22,400 cm−1 reveal features whose dynamics are inconsistent with a model proposed earlier to account for the highly efficient photoisomerization process. Emerging from this work is a new three-state model. Photoexcitation of retinal with visible light accesses a shallow well on the excited state potential energy surface. This well is bounded by a small barrier, arising from an avoided crossing that separates the Franck–Condon region from the nearby reactive region of the photoisomerization coordinate. At ambient temperatures, the reactive region is accessed with a time constant of ≈500 fs, whereupon the retinal rapidly twists and encounters a second avoided crossing region. The protein mediates the passage into the second avoided crossing region and thereby exerts control over the quantum yield for forming 13-cis retinal. The driving force for photoisomerization resides in the retinal, not in the surrounding protein. This view contrasts with an earlier model where photoexcitation was thought to access directly a reactive region of the excited-state potential and thereby drive the retinal to a twisted conformation within 100–200 fs.

Retinoid-related orphan receptor γ (RORγ) is essential for lymphoid organogenesis and controls apoptosis during thymopoiesis

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/− mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+ and CD4+ lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/− mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+ subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/− mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/− thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+ thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/− mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.

Footprints on the viral DNA ends in Moloney murine leukemia virus preintegration complexes reflect a specific association with integrase

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3′ end processing.

Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia

A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuron-expressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.