Transplantation of oligodendrocyte precursor cells (OPCs) is a promising potential therapeutic strategy for diseases affecting myelin. However, the derivation of engraftable OPCs from human pluripotent stem cells has proven difficult and primary OPCs are not readily available. Here we report the generation of induced OPCs (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to reprogram mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures resembling primary OPCs. More importantly, iOPCs gave rise to mature oligodendrocytes that could ensheath multiple host axons when co-cultured with primary dorsal root ganglion cells and formed myelin after transplantation into shiverer mice. We propose direct lineage reprogramming as a viable alternative approach for the generation of OPCs for use in disease modeling and regenerative medicine.

We report that the rate of transcription of cellular beta-tubulin genes increases during the early phase of adenovirus infection of HeLa cells, with kinetics very similar to those routinely found for viral genes. This activation depends upon adenovirus early region E1a, which encodes products that activate early virus transcription. To compare the responses of viral and cellular genes to E1a, we infected HeLa cells with dl312, a transcriptionally inactive deletion mutant that lacks a functional E1a gene. We then superinfected the cells with a helper virus, dl327, which encodes active E1a products, and measured changes in the rates of transcription of various cell and viral genes. Early region E3 of dl312 was activated 0 to 6 h postinfection and then repressed at 8 h postinfection, thus reproducing the two-step kinetics characteristic of a wild-type infection. Synthesis of beta-tubulin nuclear RNA was also transiently induced two- to six-fold, rising and falling in a manner similar to E3 transcription. An increase in helper virus multiplicity gave an increase in beta-tubulin stimulation, but dl312 alone, even at a high multiplicity of infection, gave no induction, confirming the requirement for E1a. beta-Actin nuclear RNA was actively synthesized before infection, but it was not further stimulated by the virus. Cellular beta-globin gene transcription was not stimulated by the virus, although transcription of a transfected beta-globin plasmid was induced by the virus or from a cotransfected E1a expression plasmid. We conclude that adenovirus 5 can stimulate beta-tubulin gene transcription. We discuss the significance for the viral life cycle of viral stimulation of cell genes and consider possible mechanisms in the light of the results obtained with beta-actin and beta-tubulin.

The stretch receptor organs of Alexandrowicz in lobster and crayfish possess sensory neurons which have their cell bodies in the periphery. The cell bodies send dendrites into a fine nearby muscle strand and at the opposite pole they give rise to an axon running to the central nervous system. Mechanisms of excitation between dendrites, cell soma, and axon have been studied in completely isolated receptor structures with the cell components under visual observation. Two sensory neuron types were investigated, those which adapt rapidly to stretch, the fast cells, and those which adapt slowly, the slow cells. 1. Potentials recorded from the cell body of the neurons with intracellular leads gave resting potentials of 70 to 80 mv. and action potentials which in fresh preparations exceeded the resting potentials by about 10 to 20 mv. In some experiments chymotrypsin or trypsin was used to make cell impalement easier. They did not appreciably alter resting or action potentials. 2. It has been shown that normally excitation starts in the distal portion of dendrites which are depolarized by stretch deformation. The changed potential within the dendritic terminals can persist for the duration of stretch and is called the generator potential. Secondarily, by electrotonic spread, the generator potential reduces the resting potential of the nearby cell soma. This excitation spread between dendrites and soma is seen best during subthreshold excitation by relatively small stretches of normal cells. It is also seen during the whole range of receptor stretch in neurons in which nerve conduction has been blocked by an anesthetic. The electrotonic changes in the cells are graded, reflecting the magnitude and rate of rise of stretch, and presumably the changing levels of the generator potential. Thus in the present neurons the resting potential and the excitability level of the cell soma can be set and controlled over a wide range by local events within the dendrites. 3. Whenever stretch reduces the resting membrane potential, measured in the relaxed state in the cell body, by 8 to 12 mv. in slow cells and by 17 to 22 mv. in fast cells, conducted impulses are initiated. It is thought that in slow cells conducted impulses are initiated in the dendrites while in fast cells they arise in the cell body or near to it. In fresh preparations the speed of stretch does not appreciably influence the membrane threshold for discharges, while during developing fatigue the firing level is higher when extension is gradual. 4. Some of the specific neuron characteristics are: Fast receptor cells have a relatively high threshold to stretch. During prolonged stretch the depolarization of the cell soma is not well maintained, presumably due to a decline in the generator potential, resulting in cessation of discharges in less than a minute. This appears to be the basis of the relatively rapid adaptation. A residual subthreshold depolarization can persist for many minutes of stretch. Slow cells which resemble the sensory fibers of vertebrate spindles are excited by weak stretch. Their discharge rate remains remarkably constant for long periods. It is concluded that, once threshold excitation is reached, the generator potential within slow cell dendrites is well maintained for the duration of stretch. Possible reasons for differences in discharge properties between fast and slow cells are discussed. 5. If stretch of receptor cells is gradually continued above threshold, the discharge frequency first increases over a considerable range without an appreciable change in the firing level for discharges. Beyond that range the membrane threshold for conducted responses of the cell soma rises, the impulses become smaller, and partial conduction in the soma-axon boundary region occurs. At a critical depolarization level which may be maintained for many minutes, all conduction ceases. These overstretch phenomena are reversible and resemble cathodal block. 6. The following general scheme of excitation is proposed: stretch deformation of dendritic terminals ->> generator potential ->> electrotonic spread toward the cell soma (prepotential) ->> dendrite-soma impulse ->> axon impulse. 7. Following release of stretch a transient hyperpolarization of slow receptor cells was seen. This off effect is influenced by the speed of relaxation. 8. Membrane potential changes recorded in the cell bodies serve as very sensitive detectors of activity within the receptor muscle bundles, indicating the extent and time course of contractile events.

The CAG repeats in the human Huntington's disease (HD) gene exhibit striking length-dependent intergenerational instability, typically small size increases or decreases of one to a few CAGs, but little variation in somatic tissues. In a subset of male transmissions, larger size increases occur to produce extreme HD alleles that display somatic instability and cause juvenile onset of the disorder. Initial efforts to reproduce these features in a mouse model transgenic for HD exon 1 with 48 CAG repeats revealed only mild intergenerational instability ( approximately 2% of meioses). A similar pattern was obtained when this repeat was inserted into exon 1 of the mouse Hdh gene. However, lengthening the repeats in Hdh to 90 and 109 units produced a graded increase in the mutation frequency to >70%, with instability being more evident in female transmissions. No large jumps in CAG length were detected in either male or female transmissions. Instead, size changes were modest increases and decreases, with expansions typically emanating from males and contractions from females. Limited CAG variation in the somatic tissues gave way to marked mosaicism in liver and striatum for the longest repeats in older mice. These results indicate that gametogenesis is the primary source of inherited instability in the Hdh knock-in mouse, as it is in man, but that the underlying repeat length-dependent mechanism, which may or may not be related in the two species, operates at higher CAG numbers. Moreover, the large CAG repeat increases seen in a subset of male HD transmissions are not reproduced in the mouse, suggesting that these arise by a different fundamental mechanism than the small size fluctuations that are frequent during gametogenesis in both species.

Extraction with 0 04% (w/v) Triton X-100 removes the flagellar membrane from sea urchin sperm while leaving the motile apparatus apparently intact When reactivated in a suitable medium containing exogenous adenosine triphosphate (ATP), nearly 100% of the sperm are motile and they swim in a manner resembling that of live sperm. Under standard conditions, with 1 mM ATP at 25 degrees C, the reactivated sperm had an average frequency of 32 beats/sec and progressed forward a distance of 2.4 microm/beat; comparable figures for live sperm in seawater were 46 beats/sec and 3 9 microm/beat. The adenosine triphosphatase (ATPase) activity of the reactivated sperm was measured with a pH-stat in the presence of oligomycin to inhibit residual mitochondrial ATPase. The motile sperm had an ATPase activity of 0.16 micromole P(i)/(min x mg protein), while sperm that had been rendered non-motile by homogenizing had an activity of 0 045 micromole P(i)/(min x mg protein). The difference between the ATPase activities of the motile and nonmotile sperm was tentatively interpreted as the amount of activity coupled to movement, and under optimal conditions it amounted to about 72% of the total ATPase activity Under some conditions the movement-coupled ATPase activity was proportional to the beat frequency, but it was possibly also affected by other wave parameters. The coupled ATPase activity decreased to almost zero when movement was prevented by raising the viscosity, or by changing the pH or salt concentration. The motility of reactivated sperm was wholly dependent on the presence of ATP; other nucleotides gave very low phosphatase activity and no movement. The requirement for a divalent cation was best satisfied with Mg(++), although some motility was also obtained with Mn(++) and Ca(++). The coupled ATPase activity had a Michaelis constant (K(m)) of 0.15 mM. The beat frequency of the reactivated sperm varied with the ATP concentration, with an effective "K(m)" of 0.2 mM.

We describe the cloning and function of the human XRCC1 gene, which is the first mammalian gene isolated that affects cellular sensitivity to ionizing radiation. The CHO mutant EM9 has 10-fold-higher sensitivity to ethyl methanesulfonate, 1.8-fold-higher sensitivity to ionizing radiation, a reduced capacity to rejoin single-strand DNA breaks, and a 10-fold-elevated level of sister chromatid exchange compared with the CHO parental cells. The complementing human gene was cloned from a cosmid library of a tertiary transformant. Two cosmid clones produced transformants that showed approximately 100% correction of the repair defect in EM9 cells, as determined by the kinetics of strand break repair, cell survival, and the level of sister chromatid exchange. A nearly full-length clone obtained from the pcD2 human cDNA expression library gave approximately 80% correction of EM9, as determined by the level of sister chromatid exchange. Based on an analysis of the nucleotide sequence of the cDNA insert compared with that of the 5' end of the gene from a cosmid clone, the cDNA clone appeared to be missing approximately 100 bp of transcribed sequence, including 26 nucleotides of coding sequence. The cDNA probe detected a single transcript of approximately 2.2 kb in HeLa polyadenylated RNA by Northern (RNA) blot hybridization. From the open reading frame and the positions of likely start sites for transcription and translation, the size of the putative XRCC1 protein is 633 amino acids (69.5 kDa). The size of the XRCC1 gene is 33 kb, as determined by localizing the endpoints on a restriction endonuclease site map of one cosmid clone. The deduced amino acid sequence did not show significant homology with any protein in the protein sequence data bases examined.

Gain-of-function NOTCH1 mutations are found in 50%-70% of human T cell acute lymphoblastic leukemia/lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show "addiction" to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.

2,7-Dichlorodihydrofluorescein (DCDHF), commonly known as dichlorofluorescin, and dihydrorhodamine 123 (DHR) are often used to detect the production of reactive nitrogen and oxygen species in cells via oxidation to their respective fluorescent products. To determine which biological oxidants might be involved, DCDHF and DHR were exposed to a number of oxidants in vitro to determine which are capable of oxidizing these compounds. Formation of dichlorofluorescein (DCF) and rhodamine is typically monitored by measuring their intrinsic fluorescence, however, absorbance can also be utilized (epsilon500 nm = 59,500 and 78,800 M(-1) cm(-1) for DCF and rhodamine, respectively). Peroxynitrite (ONOO-) readily oxidized both compounds with an efficiency equal to 38% of added ONOO- for DCDHF and 44% for DHR. Addition of nitric oxide (NO) to a superoxide-generating system resulted in DCDHF and DHR oxidation which was inhibitable by superoxide dismutase (SOD). SIN-1-mediated oxidation of DCDHF and DHR was also SOD-inhibitable, suggesting that peroxynitrite is the primary oxidant formed from SIN-1 decomposition. Aerobic addition of NO resulted in DCDHF oxidation in a manner consistent with nitrogen dioxide (.NO2) formation. NO did not oxidize DHR and actually inhibited UV-light-induced DHR oxidation. Simultaneous addition of NO and ONOO- resulted in an apparent inhibition of indicator oxidation; however, subsequent addition of ONOO- alone 20 s later produced a higher than average amount of oxidized indicator. Addition of indicator after NO + ONOO- followed by subsequent ONOO- addition gave similar results, suggesting the formation of a relatively stable, oxidant-activated NO/ONOO- adduct. At pH 7.4, hypochlorous acid was 66% efficient at oxidizing DHR but only 9% with DCDHF. Neither H2O2 (1 mM) nor superoxide flux alone produced significant indicator oxidation. Oxidation of DCDHF by horseradish peroxidase (HRP) plus H2O2 was considerably less efficient than oxidation of DHR. At 20-fold higher concentrations, HRP alone oxidized DHR but the rate was much lower than when H2O2 was present. Catalase largely inhibited HRP-mediated oxidation of DHR but not DCDHF, suggesting a direct effect of the peroxidase on DCDHF. These results reveal that peroxynitrite, hypochlorous acid, and H2O2 plus peroxidase all oxidize DCDHF and DHR to varying degrees but that neither superoxide, H2O2 alone, nor physiological levels of nitric oxide are capable of indicator oxidation. Thus, DCDHF or DHR oxidation in any given cell type may involve more than one oxidant. In cell systems where nitric oxide production occurs, oxidation of either DCDHF or DHR is likely to include a peroxynitrite component. Identification of relevant oxidants will best be achieved with a combined experimental approach which exploits the differential reactivities of DCDHF and DHR and the judicious use of inhibitors and oxidant scavengers.

In infected cells, hepatitis C virus (HCV) core protein is targeted to lipid droplets, which serve as intracellular storage organelles. Using a tissue culture system to generate infectious HCV, we have shown that the coating of lipid droplets by the core protein occurs in a time-dependent manner and coincides with higher rates of virus production. At earlier times, the protein was located at punctate sites in close proximity to the edge of lipid droplets. Investigations by using Z-stack analysis have shown that many lipid droplets contained a single punctate site that could represent positions where core transfers from the endoplasmic reticulum membrane to droplets. The effects of lipid droplet association on virus production were studied by introducing mutations into the domain D2, the C-terminal region of the core protein necessary for droplet attachment. Alteration of a phenylalanine residue that was crucial for lipid droplet association generated an unstable form of the protein that could only be detected in the presence of a proteasome inhibitor. Moreover, converting two proline residues in D2 to alanines blocked coating of lipid droplets by core, although the protein was directed to punctate sites that were indistinguishable from those observed at early times for wild-type core protein. Neither of these virus mutants gave rise to virus progeny. By contrast, mutation at a cysteine residue positioned 2 aa upstream of the phenylalanine residue did not affect lipid droplet localization and produced wild-type levels of infectious progeny. Taken together, our findings indicate that lipid droplet association by core is connected to virus production.

In cultures of embryonic and adult mouse striatum, we previously demonstrated that EGF induces the proliferation of putative stem cells, which give rise to spheres of undifferentiated cells that can generate neurons and astrocytes. We report here that the spheres of undifferentiated cells contain mRNA and protein for the FGF receptor (FGFR1). Indirect immunocytochemistry demonstrated that many of the cells within the EGF-generated spheres were immunoreactive for FGFR1. Exogenous application of bFGF to the EGF-generated cells induced the proliferation of two progenitor cell types. The first, a bipotent progenitor cell, gave rise to cells with the antigenic and morphological properties of neurons and astrocytes; the other gave rise to cells with neuronal characteristics only. bFGF-generated cells with neuronal morphology exhibited electrophysiological properties indicative of immature central neurons. These results support the hypothesis that sequential actions of growth factors play a role in regulating the generation of neurons and astrocytes in the developing CNS.

Basophils and mast cells, which are selectively endowed with the high-affinity IgE receptor and mediate a range of adaptive and innate immune responses, have an unknown developmental relationship. Here, by evaluating the expression of the beta7 integrin, a molecule that is required for selective homing of mast cell progenitors (MCPs) to the periphery, we identified bipotent progenitors that are capable of differentiating into either cell type in the mouse spleen. These basophil/mast cell progenitors (BMCPs) gave rise to basophils and mast cells at the single-cell level and reconstituted both mucosal and connective tissue mast cells. We also identified the basophil progenitor (BaP) and the MCP in the bone marrow and the gastrointestinal mucosa, respectively. We further show that the granulocyte-related transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) plays a primary role in the fate decision of BMCPs, being expressed in BaPs but not in MCPs. Thus, circulating basophils and tissue mast cells share a common developmental stage at which their fate decision might be controlled principally by C/EBPalpha.

We have recently established a novel expression cloning system using retroviral vectors. The system is based on a high-efficiency packaging cell line, BOSC23, and a simplified retroviral vector, pBabeX, carrying no selection marker. cDNA libraries, constructed in the pBabeX vector, are transiently transfected into BOSC23 cells. The supernatant contains more than 3X10(6)/mL, which would cover large complexities of cDNA libraries. The retrovirus stock gave 100% infection efficiency in NIH3T3 cells and 5-40% infection efficiency in various hematopoietic cell lines. In contrast to the conventional expression cloning system, in which it is necessary to transfect cDNA libraries transiently into particular cell types such as COS cells, retrovirus-mediated expression cloning allows us to transduce cDNAs into a wide variety of cell types. This method therefore makes it possible to select cells expressing a cDNA of interest by various functional assays. When combined with polymerase chain reaction (PCR)-driven random mutagenesis, this system is also useful in searching for mutations of various molecules that will result in alterations of their functions.

Patients who show the "strategy application disorder" can show deficits restricted to situations requiring multitasking, but the precise neuroanatomical and cognitive correlates of this problem have been rarely investigated. In this study, 60 people with circumscribed cerebral lesions and 60 age- and IQ-matched controls were given a multitasking procedure which allowed consideration of the relative contributions of task learning and remembering, planning, plan-following and remembering one's actions to multitasking performance. Lesions to the left posterior cingulate and forceps major regions gave deficits on all measures except planning. Remembering task contingencies after a delay was also affected by lesions in the region of the left anterior cingulate, and rule-breaking and failures of task switching were additionally found in people with lesions affecting the medial and more polar aspects of Brodmann's areas 8, 9 and especially 10. Planning deficits were associated with lesions to the right dorsolateral prefrontal cortex (RDLPFC). A theory of the relationships between the cognitive constructs underpinning multitasking was tested using structural equation modelling. The results suggest that there are three primary constructs that support multitasking: retrospective memory, prospective memory, and planning, with the second two drawing upon the products of the first. It is tentatively suggested that the left anterior and posterior cingulates together play some part in the retrospective memory demands, while the prospective memory and planning components make demands on processes supported by the left areas 8, 9 and 10 and the RDLPFC respectively.

The Canadian American Ticlopidine Study (CATS) is a randomised, double-blind, placebo-controlled trial to assess the effect of ticlopidine (250 mg twice daily) in reducing the rate of subsequent occurrence of stroke, myocardial infarction, or vascular death in patients who have had a recent thromboembolic stroke. Twenty-five centres entered 1072 patients into the study between 1 week and 4 months after their qualifying stroke. The patients were treated and followed for up to 3 years (mean 24 months). In the efficacy analysis, the event rate per year for stroke, myocardial infarction or vascular death, considered together, was 15.3% in the placebo group and 10.8% in the ticlopidine group, representing a relative risk reduction with ticlopidine of 30.2% (95% confidence interval 7.5-48.3%; p = 0.006). Ticlopidine was beneficial for both men and women (relative risk reductions 28.1%, p = 0.037, and 34.2%, p = 0.045, respectively). Analysis by intention-to-treat gave a smaller estimate of risk reduction (23.3%, p = 0.020) for stroke, myocardial infarction, or vascular death. Adverse experiences associated with ticlopidine included neutropenia (severe in about 1% of cases) and skin rash and diarrhoea (severe in 2% of cases each); all were reversible. This study provides evidence of a beneficial effect of ticlopidine in both men and women with a recent thromboembolic stroke.

Antigen- and mitogen-stimulated cytokine production by peripheral blood mononuclear cells (PBMC) of 50 pregnant women and 31 age- and sex-matched non-pregnant controls were analysed to determine whether changes in cytokine production occur during normal and pathologic human gestation. The pregnant women, consecutively enrolled during a 3-month period, were undergoing a normal, non-pathologic pregnancy at the time of entry into the study, and underwent ultrasound examination to ascertain the exact week of pregnancy and the vitality of the fetus. Forty of the 50 pregnancies (80%) terminated physiologically with the birth of normal babies. Spontaneous abortions were observed in 5/50 (10%) women, and five women gave birth to newborns small for gestational age (SGA). A decrease in the production of IL-2 and interferon-gamma (IFN-gamma) accompanied by an increase in production of IL-4 and IL-10, was observed in normal pregnancy, with the lowest quantities of IL-2 and IFN-gamma and the highest quantities of IL-4 and IL-10 present in the third trimester of pregnancy. Statistically significant increased production of both IL-2 and IFN-gamma and reduced production of IL-10 characterized pathologic pregnancies and distinguished them from normal pregnancies. These preliminary data suggest that a type 2 cytokine profile may be associated with normal human pregnancy, whereas the lack of a dominant type 2 cytokine profile may be indicative of a pathologic pregnancy.

BACKGROUND

Insufficient data are available about the safety of oral anticoagulant therapy. The specialized organization of thrombosis services in the Netherlands can provide important information on the bleeding risk and various risk factors for bleeding in patients receiving oral anticoagulant therapy.

METHODS

In a follow-up study over a 12-month period beginning in January 1988 on all patients treated by the Leiden Thrombosis Service, the frequency of bleeding complications was assessed. A Poisson regression model was used to assess the relative contribution to the bleeding risk of age, sex, target zone (intensity of anticoagulant effect aimed at), achieved intensity of anticoagulant therapy (International Normalized Ratio), and the type of coumarin derivative used.

RESULTS

Six thousand eight hundred fourteen patients experienced 1003 bleeding complications (16.5 per 100 treatment-years), 162 of which were major bleeds (2.7 per 100 treatment-years). Bleeding increased significantly with age (32% increase for all bleeding, 46% for major bleeding for every 10-year increase in age in comparison with age < 40 years). Women had more minor bleeding complications than men, whereas both sexes experienced major bleeding in an equal frequency. There was no influence of target zone, while every one-point increase in International Normalized Ratio gave 42% more major bleeding (54% more regarding all bleeding). Use of acenocoumarol resulted in fewer bleeds (26% less regarding all bleeding and 46% less regarding major bleeding) than use of phenprocoumon.

CONCLUSIONS

The risk of anticoagulant therapy in a routine, real-life situation is similar as in the setting of several well-organized clinical trials. The risk of bleeding complications rises significantly with age and with the achieved intensity of anticoagulation, and is dependent on the type of coumarin derivative that is used.

The natural history of Precambrian life is still unknown because of the rarity of microbial fossils and biomarkers. However, the composition of modern-day genomes may bear imprints of ancient biogeochemical events. Here we use an explicit model of macroevolution including gene birth, transfer, duplication and loss events to map the evolutionary history of 3,983 gene families across the three domains of life onto a geological timeline. Surprisingly, we find that a brief period of genetic innovation during the Archaean eon, which coincides with a rapid diversification of bacterial lineages, gave rise to 27% of major modern gene families. A functional analysis of genes born during this Archaean expansion reveals that they are likely to be involved in electron-transport and respiratory pathways. Genes arising after this expansion show increasing use of molecular oxygen (P = 3.4 × 10(-8)) and redox-sensitive transition metals and compounds, which is consistent with an increasingly oxygenating biosphere.

Analysis of organellar genomes strongly supports the idea that chloroplasts and mitochondria originated in evolution as eubacteria-like endosymbionts, whose closest contemporaries are cyanobacteria and purple photosynthetic bacteria, respectively. However, there is still much debate about whether a single endosymbiotic event or multiple ones gave rise to each organelle in different eukaryotes, and considerable uncertainty about what has happened to the genomes of chloroplasts and mitochondria since their appearance in the eukaryotic cell.

The Na(+)/H(+) exchanger (NHE) is involved in intracellular pH homeostasis of many mammalian cell types. To date seven NHE isoforms (NHE1-NHE7) have been identified. NHE1 is the most predominant isoform expressed in heart where it contributes to cardiomyocyte pH homeostasis. Although the NHE activation is essential for the restoration of physiological pH, hyperactivation of NHE1 during ischemia-reperfusion episodes disrupts the intracellular ion balance, leading to cardiac dysfunction and damage. Beside its ability to inhibit a conductive Na(+) channel and the Na(+)/Ca(++) exchanger, amiloride was the first drug described as NHE inhibitor. Double substitution of the nitrogen of the 5-amino group of amiloride gave DMA, EIPA, MIBA and HMA. Later, several acylguanidines were prepared to selectively inhibit NHE1. The replacement of the pyrazine ring of amiloride by a pyridine ring or by a phenyl increased the potency and the NHE selectivity. The simultaneous replacement of the pyrazine ring by a phenyl, of the 6-chloro by a sulfomethyl led to drugs such as HOE-694, cariporide, eniporide and BIIB-513 which also selectively inhibited NHE1. In the last decade several bicyclic guanidines were prepared: zoniporide, MS-31038, SM-20220, SM-20550, SMP-300, KB-R9032, BMS-284640, T-162559, TY-12533, S-3226 or SL-591227. Extensive pre-clinical studies indicated that NHE inhibitors afford substantial protection in different animal models of myocardial ischemia (MI) and reperfusion, but the results of clinical trials involving eniporide and cariporide were mixed.

Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.

A collaborative comparison of macro- and microdilution antifungal susceptibility tests was performed in five laboratories. MICs of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined in all five centers against 95 coded isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. A standard protocol with the following National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Testing recommendations was used: an inoculum standardized by spectrophotometer, buffered (RPMI 1640) medium (pH 7.0), incubation at 35 degrees C, and an additive drug dilution procedure. Two inoculum sizes were tested (1 x 10(4) to 5 x 10(3) to 2.5 x 10(3) CFU/ml) and three scoring criteria were evaluated for MIC endpoint determinations, which were scored as 0 (optically clear), < or = 1 (slightly hazy turbidity), and < or = 2 (prominent decrease in turbidity compared with that of the growth control). Overall intra- and interlaboratory reproducibility was optimal with the low-density inoculum, the second-day readings, and MICs scored as either 1 or 2. The microdilution MICs demonstrated interlaboratory agreement with most of the four drugs higher than or similar to that of the macrodilution MICs. In general, there was good interlaboratory agreement with amphotericin B, fluconazole, and flucytosine; ketoconazole gave more variable results.

We have determined the absolute translation rates for four individual codons in Escherichia coli. We used our previously described system for direct measurements of in vivo translation rates using small, in-frame inserts in the lacZ gene. The inserts consisted of multiple synthetic 30 base-pair DNA oligomers with high densities of the four individual codons, GAA (Glu), GAG (Glu), CCG (Pro) and CGA (Arg). Our method is independent of expression level, of mRNA half-life and of transcription rate. Codon GAA was found to be translated with a rate of 21.6 codons/second whereas codon GAG was translated 3.4-fold slower (6.4 codons/s). These two codons are read by the same tRNA species. Codon CCG and CGA are both read by abundant tRNA species but nevertheless we found them to be translated slowly with rates of 5.8 and 4.2 codons/second, respectively. The context of these codons were varied, but we found no significant influence of context on their translation rates and we suggest a mechanism for why context may not affect translation rates. One insert with a low translation rate gave results that most readily can be explained by assuming queue formation of ribosomes on the insert. Such a queue was found to reduce the expression level by approximately 35%. Our experiments allowed us to estimate the average distance between ribosomes and thereby the translation initiation frequency on the wild-type lacZ mRNA. This was found to be one per three seconds.

Human colonic biota is a complex microbial ecosystem that serves as a host defense. Unlike most microbial ecosystems, its composition has been studied extensively by relatively efficient culture methods. We have compared an established culture-based method with direct amplification and partial sequencing of cloned 16S rRNA genes from a human fecal specimen. Nine cycles of PCR were also compared with 35 cycles. Colonies and cloned amplicons were classified by comparing their ribosomal DNA (rDNA; DNA coding for rRNA) sequences with rDNA sequences of known phylogeny. Quantitative culture recovered 58% of the microscopic count. The 48 colonies identified gave 21 rDNA sequences; it was estimated that 72% of the rDNA sequences from the total population of culturable cells would match these 21 sampled sequences (72% coverage). Fifty 9-cycle clones gave 27 sequences and 59% coverage of cloned rDNAs. Thirty-nine rDNAs cloned after 35 cycles of PCR gave 13 sequences for 74% coverage. Thus, the representation of the ecosystem after 35 cycles of PCR was distorted and lacked diversity. However, when the number of temperature cycles was minimized, biodiversity was preserved, and there was good agreement between culturing bacteria and sampling rDNA directly.

Platelet-derived growth factor has been purified from human outdated platelet-rich plasma with a 21% overall yield. This five-step procedure represents a 500,000-fold purification over serum. 6.4 ng/ml (2 X 10(-10) M) of purified platelet-derived growth factor stimulated DNA synthesis in quiescent, density arrested cultures of Swiss 3T3 cells to a level equivalent to that produced by 5% calf serum. The growth-promoting activity and specific binding to 3T3 cells has been shown to be associated with four entities of molecular weights: 31,000, 29,000, 28,500, and 27,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. In the presence of 2-mercaptoethanol, the four molecular species are converted to three inactive chains of lower molecular weights: 17,500, 16,000, and 14,400. Other methods of chemically cleaving the disulfide bonds were investigated: reductive cleavage by S-sulfonation, reductive cleavage with dithiothreitol, and performic acid oxidation. In all cases, mitogenic activity was reduced by 80-100%. Attempts to restore mitogenic activity after reduction were unsuccessful. Two-dimensional 125I-peptide mapping of the nonreduced and reduced multiple forms was also investigated. The four nonreduced moieties gave basically identical maps. The maps of the reduced 17,500- and 16,000-dalton chains were also essentially identical, but the map of the reduced 14,400-dalton chain was significantly different. We propose that platelet-derived growth factor consists of two polypeptide chains: a 14,400-dalton chain and either a 17,500- or a 16,000-dalton chain.