Transient Appearance Research Articles

Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration

Changes in the expression patterns of genes involved in bile acid (BA) synthesis were investigated during rat liver regeneration that follows two-thirds partial hepatectomy. BAs in bile were measured by GC-MS and the absolute and relative abundance of specific mRNAs in the liver by RT-real-time quantitative PCR. Cyclin E mRNA, used as an indicator of liver cell proliferation, peaked at day 1. The levels of mRNA of α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) were first reduced (day 1) and then (days 2–3) increased, when those of farnesoid X receptor (FXR) were also transiently enhanced. The early (day 1) up-regulation of Cyp7a1, and Cyp8b1, together with the down-regulation of Cyp27, was consistent with an increased proportion of cholic acid versus chenodeoxycholic acid and a progressive recovery in total BAs secretion. The transient appearance of flat BAs ( allo-BAs plus Δ 4-unsaturated-BAs) during rat liver regeneration was probably due to the changes in the expression ratio of steroid 5α- versus 5β-reductase. Both were first (day 1) down-regulated and then up-regulated (5α-reductase more than 5β-reductase). In conclusion, changes in the expression patterns of nuclear receptors and enzymes involved in BA synthesis are consistent with the transient modifications that occur in BA pool during rat liver regeneration.

Fibroblasts Can Be Genetically Modified to Produce Excitable Cells Capable of Electrical Coupling

Cardiac conduction occurs in an electrical syncytium of excitable cells connected by gap junctions. Disruption of these electrophysiological properties causes conduction slowing or block. Depending on the location of affected cells within the heart, this has the potential to result in clinical syndromes such as atrioventricular block. With a view to developing gene therapy strategies for repairing cardiac conduction defects, we sought to establish whether the phenotype of fibroblasts can be modified by gene transfer to produce cells capable of electrical excitation and coupling. High-titer lentiviral vectors encoding MyoD, a myogenic transcription factor, and connexin43, a gap junction protein, were produced by established methods. Human dermal fibroblasts (HDFs) were efficiently (>80%) transduced at a multiplicity of infection of 50. HDFs transduced with the MyoD-encoding vector underwent myogenic conversion, as evidenced by myotube formation and detection of muscle-specific proteins. Importantly, calcium transients indicative of membrane excitability were observed in MyoD-induced myotubes after loading with a calcium-sensitive dye and electrical stimulation. Transients from adjacent myotubes displayed different excitation thresholds, indicating an absence of coupling between cells, consistent with skeletal muscle biology. In contrast, simultaneous transduction of HDFs with MyoD and connexin43-encoding vectors resulted in the appearance of transients in adjacent myotubes with identical thresholds, indicative of electrical coupling. Notably, dye transfer studies confirmed gap junctional intercellular communication. Fibroblasts can be genetically modified to produce excitable cells capable of electrical coupling. These observations strengthen the prospect of developing gene-based strategies for repairing cardiac conduction defects.

Rapid Assembly of Amyloid-β Peptide at a Liquid/Liquid Interface Produces Unstable β-Sheet Fibers

Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.

INVITED COMMENTARY

Removal of lipid particles has become of interest after the alarming results of Moody and colleagues [1Moody D.M. Brown W.R. Challa V.R. Stump D.A. Reboussin D.M. Legault C. Brain microemboli associated with cardiopul-monary bypass a histologic and magnetic resonance imaging study.Ann Thorac Surg. 1995; 59: 1304-1307Abstract Full Text PDF PubMed Scopus (169) Google Scholar] on lipid embolization in brain arterioles after cardiac surgery. It resulted in various attempts to reduce lipids in retransfusion blood, which would be beneficial after cardiac surgery, but also after orthopedic surgery. Despite the known side effects of retransfusion from the wound area, autologous blood transfusion is increasingly used to reduce the use of allogenic blood. A major problem in justifying the efforts, time, and costs to clear retransfusion blood from lipid particles is to obtain evidence of clinically relevant brain damage and evidence for the dominant role of lipid particles. First of all, brain damage has been reported with very different significance. A number of studies could not show any deterioration in cognitive functions, whereas biochemical markers of brain damage, such as S100β and enolase, showed in some studies small and transient appearance. Clearly, the specificity of such tests is not sufficient or sensitive enough to demonstrate brain damage. Perhaps new markers, such as carnosinase, may appear relevant for monitoring brain damage. The second problem is that lipid particles are only one of the suspects of brain damage. Gaseous emboli, macromolecules, inflammatory agents, and leukocyte-platelet aggregates may be involved in occlusion of the small vasculature as well. In their discussion the authors indicated that, eg, macromolecules may also be removed by the current technique of particulate separation, but other potential sources of embolization possibly remain in retransfusion blood. The combination with other techniques to remove platelets, leukocytes, and products that affect hemostasis or inflammation may be a future option. Third, the reuse of shed blood is a matter of dispute. It depends on the operation time, preference of surgeons, and need for immediate volume during the procedure. To obtain the technique widely used, it must be simple, fast, and cost effective. Nevertheless, the PARSUS technique presented here offers new possibilities to improve the quality of retransfusion with high efficacy for removing potential dangerous lipid particles. Hopefully these qualifications will be supported by adequate monitoring instruments. It would be exciting to see the efficacy of a larger scale PARSUS device in clinical practice.

Modular Assembly Revealed by Tryptophan and Other Optical Probes in Staphylococcal Nuclease Folding

AbstractHow do different molecular events amalgamate to form kinetic funnels for protein folding? To probe local events and dissect folding of sub‐domains new tryptophan probes have been implanted in two locations of staphylococcal nuclease (SNase): the β‐barrel (W140F+L25W) and the C‐helix (W140F+L125W). 8‐Anilino‐l‐naphthalensulfonate (ANS) was also used to trace a transient appearance of hydrophobic clusters. By use of these fluorescence probes in addition to the CD222nm signal of protein, several molecular events for the folding of SNase and its mutants have been resolved and activation barriers for these reactions determined. Folding from a GdmHCl unfolded state begins a burst with the formation of 20% of the secondary structure (< 20 ms in CD mode). The next event (55 ms) is the clustering of hydrophobic side chains in the C‐ and the oligonucleotide binding (OB)‐domains. These hydrophobic clusters are capable of binding ANS and they continue to grow in 500–700 ms. ANS binding sites subsequently sequester and become inaccessible to ANS in a 1.7‐s reaction. Merging of the OB‐ and the C‐domains that follows is a complex tri‐phasic process (134 ms, 457 ms, and 31.3 s). Global folding measured by the CD222nm signal unveils yet another slow reaction (1,600 s) which was shown to originate from the trans/cis isomerization of Pro117. A modular assembly model depicting protein folding‐by‐parts or origami of folding is proposed and discussed in light of these observations.

Bench to bedside: HMGB1-a novel proinflammatory cytokine and potential therapeutic target for septic patients in the emergency department.

Overwhelming gram-negative bacterial infection and life-threatening systemic inflammation are widespread problems in critically ill emergency department patients. Currently, the treatment of these patients is largely supportive, focusing on antibiotics, fluids, hemodynamic and ventilatory support, and intensive monitoring. The only Food and Drug Administration-approved pharmaceutical agent for the treatment of sepsis is activated protein C, with its use largely relegated to the intensive care unit. The subject thus remains an active area of exploration for emergency medicine research. During sepsis and inflammation, innate immune cells release excessive amounts of proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1beta. If delivered early enough, anti-TNF antibodies can be an effective therapy in experimental models of septic shock. Anti-TNF antibodies have been developed for clinical use in rheumatoid arthritis and Crohn's disease. However, anti-TNF treatment for sepsis has been difficult to achieve in the clinical setting, perhaps because TNF's early release and transient appearance in the serum create a narrow therapeutic window. An alternative strategy would be to identify "late" mediators that may be clinically more accessible. High mobility group box 1 (HMGB1), a protein previously known only as a nuclear transcription factor, is now implicated as a late mediator of sepsis. Targeting late mediators of lethal systemic inflammation represents a novel approach that may widen the therapeutic window and lead to new strategies for inhibiting the deleterious effects of the inflammatory cascade. Here the authors review the studies that led to the discovery of HMGB1 as a late mediator of systemic inflammation and discuss the possibility of HMGB1 as a therapeutic target for septic patients in the emergency department.

Heat shock-induced dendritic cell maturation is coupled by transient aggregation of ubiquitinated proteins independently of heat shock factor 1 or inducible heat shock protein 70

The transition of dendritic cells (DCs) from immature to mature states is critical for the optimal priming of the adaptive immune response. This highly regulated process is accompanied by structural and functional alterations of DCs, including rapid and dramatic redistributions of MHC class I and class II molecules to cell surfaces, nuclear translocation of NF-κB,and transient appearance of dendritic cell aggresome-like induced structures (DALIS) in the cytosol. We have previously found that DCs can be matured by a non-inflammatory stress stimulation, i.e., heat shock. In this study, we examined if thermomanipulation could induce DALIS formation. We found that heat shock of DCs, but not of other cell types, led to the appearance of aggresome-like structures that were structurally indistinguishable from DALIS induced by lipopolysaccharide (LPS). Furthermore, the induction of DALIS in DCs by heat, but not by LPS, correlated with the increased ability of DCs to cross-present exogenous antigens to MHC I. Since the canonical biochemical response to heat shock is the induction of heat shock proteins (HSP) by heat shock factors (Hsf), we studied the contribution of both HSP and Hsf in heat shock-mediated DALIS formation using gene knockout mice. We demonstrated that neither inducible HSP70 nor the major mammalian heat shock transcription factor Hsf1 was involved in the formation of DALIS. Our results highlighted the important roles of heat shock in modulating the function of DCs, and they further suggested that heat-mediated immune regulation can be uncoupled from heat shock response.

Prophylactic effect of IL-10 gene transfer on induced autoimmune dacryoadenitis.

To evaluate the effect of viral IL-10 on the lacrimal gland immunopathologic response in the ocular surface disease, induced autoimmune dacryoadenitis. Disease was induced in rabbits by injecting inferior lacrimal glands with peripheral blood lymphocytes activated by 5 days of coculture with autologous acinar cells in a mixed-cell reaction. In the treated group, an adenoviral vector carrying the vIL-10 gene was concurrently injected with activated lymphocytes. Tears were collected periodically for quantitation of IL-10 by ELISA. Two weeks after disease induction, tear production, tear film breakup time, and rose bengal staining score were determined. Sectioned glands were immunostained for expression of CD4, CD8, rabbit thymic lymphocyte antigen (RTLA), CD18 and major histocompatibility complex class II. The titer of vIL-10 in tears was at its maximum on day 3, started to decline by day 7, and was undetectable by day 14. In the diseased group, the tear production rate and tear film breakup time were significantly decreased, and rose bengal staining was significantly increased. Diseased glands had immune cell infiltrates containing CD4+, RTLA+, and CD18+ cells, and major histocompatibility complex class II expression was increased. These changes were significantly ameliorated by expression of vIL-10. In vivo transduction of the lacrimal gland with AdvIL-10 resulted in the transient appearance of vIL-10 in tears. The presence of vIL-10 partially suppressed the appearance of Sjögren-syndrome-like features of reduced tear production, accelerated tear breakup, ocular surface disease, and immunopathologic response. Anti-inflammatory cytokine gene expression may offer a therapeutic modality for the treatment of autoimmune dacryoadenitis, once suitable vectors become available.

Sorting nexin 17 accelerates internalization yet retards degradation of P-selectin.

The transient appearance of P-selectin on the surface of endothelial cells helps recruit leukocytes into sites of inflammation. The tight control of cell surface P-selectin on these cells depends on regulated exocytosis of Weibel-Palade bodies where the protein is stored and on its rapid endocytosis. After endocytosis, P-selectin is either sorted via endosomes and the Golgi apparatus for storage in Weibel-Palade bodies or targeted to lysosomes for degradation. A potential player in this complex endocytic itinerary is SNX17, a member of the sorting nexin family, which has been shown in a yeast two-hybrid assay to bind P-selectin. Here, we show that overexpression of SNX17 in mammalian cells can influence two key steps in the endocytic trafficking of P-selectin. First, it promotes the endocytosis of P-selectin from the plasma membrane. Second, it inhibits the movement of P-selectin into lysosomes, thereby reducing its degradation.

Rafting across polarized cells

For raft proteins, direct delivery is not the travel route of choice, according to Roman Polishchuk, Jennifer Lippincott-Schwartz (NIH, Bethesda, MD), and colleagues. They find that, contrary to earlier evidence, glycosyl phosphatidylinositol (GPI)–anchored raft proteins make a detour to the basolateral membrane of polarized MDCK cells before crossing the cell to their final destination in the apical membrane. Figure Apical and basolateral proteins (red and green) leave the Golgi together. Direct delivery had been plausible, as sorting of GPI-anchored proteins into rafts, and thus away from basolateral proteins, occurs in the trans-Golgi network (TGN) before either departs for plasma membranes. Few or no apical proteins were ever seen in the basolateral membrane. And finally, transport across the cell seemed unlikely: the main method of departure, clathrin-mediated endocytosis, did not transport rafts. Lippincott-Schwartz recently found that rafts are continually endocytosed by a nonclathrin pathway, overcoming the clathrin objection. And at the basolateral surface, she says, “all of these biochemical experiments would have potentially missed a transient appearance.” The NIH team found that GPI-YFP and basolateral proteins were segregated as they left the TGN, but nevertheless shared the same tube-shaped carriers. (Others have claimed one cargo type per carrier, but did not prebleach to reduce background.) Those carriers must be paying a visit to the basolateral membrane, as both proteins got stuck intracellularly when fusion to the basolateral membrane was prevented with tannic acid, a cell-impermeable fixative. When tannic acid was applied to the apical side, only the apical cargo was intracellular, presumably after making its transient basolateral stop. Evidence for this trip across the cell came from a tracer and antibody, which were applied to the basolateral side but travelled with GPI-YFP apically. A nonraft apical protein went straight to the apical side of the cell. Why should the cell bother with the more circuitous route taken by GPI-YFP? Lippincott-Schwartz suggests that raft-associated proteins may help carry other cargos across the cell so they can do their job in the gut or elsewhere. Bacteria, some of which are known to attach to rafts, may even grab on and get a free ride. ▪ Reference: Polishchuk, R., et al. 2004. Nat. Cell Biol. 6:297–307. [PubMed]

The effect of oral 5-HTP administration on 5-HTP and 5-HT immunoreactivity in monoaminergic brain regions of rats

5-Hydroxytryptophan (5-HTP), which is the rate-limiting precursor in serotonin (5-hydroxytryptamine (5-HT)) biosynthesis, is used as an oral supplement to enhance serotonin levels in humans. To evaluate its effects on serotonin levels and localization, 5-hydroxytryptophan was administered to Sprague–Dawley rats either orally or via intraperitoneal injection. 5-Hydroxytryptophan-immunoreactivity was co-localized with serotonin-immunoreactivity in the serotonergic dorsal raphe nucleus of control animals and this was not changed in animals given 5-hydroxytryptophan. Oral 5-HTP administration increased the intensity of both 5-HTP and serotonin immunoreactivity in raphe neurons. However, 5-HTP treatment also caused ectopic 5-hydroxytryptophan-immunoreactivity and serotonin-immunoreactivity in normally dopaminergic neurons of the substantia nigra par compacta. Serotonin-immunoreactivity was confined to neurons that also displayed amino acid decarboxylase immunoreactivity, but in a small percentage of substantia nigra neurons, serotonin immunoreactivity was not co-localized with tyrosine hydroxylase-immunoreactivity. The intensity of the immunoreactivity to serotonin and 5-hydroxytryptophan in the substantia nigra was maximal within 2 h of 5-hydroxytryptophan administration and returned to control levels by 24 h. This time course mirrored changes in HPLC measurements of 5-hydroxytryptophan, serotonin, and the metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the urine. 5-Hydroxytryptophan administration did not cause ectopic appearance of either serotonin or 5-hydroxytryptophan in the noradrenergic locus coeruleus. These results suggest that a single oral dose of 5-HTP increases the 5-HTP and serotonin content of serotonergic neurons and causes the transient ectopic appearance of serotonin in some normally non-serotonergic neurons.

Expression of human coagulation factor VIII in adipocytes transduced with the simian immunodeficiency virus agmTYO1-based vector for hemophilia A gene therapy.

We demonstrate that transduction of adipocytes with a simian immunodeficiency virus agm TYO1 (SIVagm)-based lentiviral vector carrying the human coagulation factor VIII gene (SIVhFVIII) resulted in expression of the human FVIII transgene in vitro and in db/db mice in vivo. Cultured human adipocytes were transduced with the SIVagm vector carrying the GFP gene in a dose-dependent manner and transduction of adipocytes with SIVhFVIII resulted in efficient expression of human coagulation factor VIII (hFVIII; 320 +/- 39.8 ng/10(6) adipocytes/24 h) in vitro. Based upon successful transduction of adipocytes by SIV vectors carrying the lacZ gene in vivo in mice, the adipose tissue of db/db mice was transduced with SIVhFVIII. There was a transient appearance of human FVIII in mouse plasma (maximum 1.8 ng/ml) on day 11 after the injection. Transcripts of human FVIII transgene and human FVIII antigen also were detected in the adipose tissue by RT-PCR and immunofluorescence, respectively, on day 14. Emergence of anti-human FVIII antibodies 14 days after the injection of SIVhFVIII may explain the disappearance of human FVIII from the circulation. These results suggest that transduction of the adipocytes with vectors carrying the human FVIII gene may be potentially applicable for gene therapy of hemophilia A.

Photometric study of new southern SU UMa-type dwarf novae and candidates - III. NSV 10934, MM Sco, AB Nor and CAL 86

We photometrically observed four southern dwarf novae in outburst (NSV 10934, MM Sco, AB Nor and CAL 86). NSV 10934 was confirmed to be an SU UMa-type dwarf nova with a mean superhump period of 0.07478(1) d. This star also showed transient appearance of quasi-periodic oscillations (QPOs) during the final growing stage of the superhumps. Combined with the recent theoretical interpretation and with the rather unusual rapid terminal fading of normal outbursts, NSV 10934 may be a candidate intermediate polar showing SU UMa-type properties. The mean superhump periods of MM Sco and AB Nor were determined to be 0.06136(4) d and 0.08438(2) d, respectively. We suggest that AB Nor belongs to a rather rare class of long-period SU UMa-type dwarf novae with low mass-transfer rates. We also observed an outburst of the suspected SU UMa-type dwarf nova CAL 86. We identified this outburst as a normal outburst and determined the mean decline rate of 1.1 mag d 1 .

Paraproteinemia After Hematopoietic Stem Cell Transplantation

The frequency and clinical significance of paraproteinemia in patients receiving hematopoietic stem cell (HSC) transplants were assessed. Of 66 patients with hematologic malignancies, excluding multiple myeloma who received an allogeneic or autologous HSC transplant, paraproteins were detected in 12 patients using immunoelectrophoresis. None of the patients showed paraproteinemia before HSC transplantation. The class of paraproteins most commonly seen was IgG. In 9 of these 12 patients (75%), a paraprotein was detected continuously after HSC transplantation for an average duration of 464 days, while others demonstrated a transient appearance of the protein. Paraproteinemia after HSC transplantation was not related to the stem cell source, (allograft vs. autograft), age, gender, viral infection and graft-vs.-host disease (GVHD). None of the patients developed plasma cell dyscrasia after the appearance of the paraprotein, while 1 patient developed secondary acute lymphoblastic leukemia. These findings indicate that paraproteinemia after HSC transplantation may be caused by an aberrant immune reconstitution after both allogeneic and autologous HSC transplantation. A long-term follow-up of patients with paraproteinemia after HSC transplantation is needed to confirm this finding in a larger series of patients.

Transient DNA strand breaks during mouse and human spermiogenesis new insights in stage specificity and link to chromatin remodeling.

In the course of mammalian spermiogenesis, a unique chromatin remodeling process takes place within elongating and condensing spermatid nuclei. The histone-to-protamine exchange results in efficient packaging and increased stability of the paternal genome. Although not fully understood, this change in chromatin architecture must require a global but transient appearance of endogenous DNA strand breaks because most of the DNA supercoiling is eliminated in the mature sperm. To establish the extent of DNA strand breakage and the stage specificity at which these breaks are created and repaired, we performed a sensitive terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay to detect in situ DNA strand breaks on both mice and human testis cross sections. In the mouse, we established that DNA strand breaks are indeed detected in the whole population of elongating spermatids between stages IX and XI of the seminiferous epithelium cycle perfectly coincident with the chromatin remodeling as revealed by histone H4 hyperacetylation. Similarly, TUNEL analyses performed on human testis sections revealed an elevated and global increase in the levels of DNA strand breaks present in nuclei of round-shaped spermatids also coincident with chromatin remodeling. The demonstration of the global character of the transient DNA strand breaks in mammalian spermiogenesis suggests that deleterious consequences on genetic integrity of the male gamete may arise from any disturbance in the process. In addition, this investigation may shed some light on the origin of the low success rate that has been encountered so far with intracytoplasmic injection procedures making use of round spermatids in humans.

Molecular impact of the M184V mutation in human immunodeficiency virus type 1 reverse transcriptase.

The generation of drug-resistant variants of human immunodeficiency virus (HIV) type 1 (HIV-1) in vivo as well as in vitro is a consequence of the error proneness of the HIV-1 reverse transcriptase (RT) enzyme. During viral replication, RT copies the single-stranded RNA genome into double-stranded DNA. Due to the lack of 3′ exonuclease proofreading activity, the incorporation of missense nucleotides occurs at a relatively high frequency. The misincorporation rate of the RT enzyme ranges from 10−4 to 10−5, depending on the nature of the template and the source of the RT (4, 7, 31, 39, 54). During in vivo and in vitro selections, drug-resistant variants emerge; competition for replication then results in outgrowth of the fittest variant. The M184V mutation in HIV-1 RT is associated with high-level resistance to the antiviral drug 2′,3′-dideoxy-3′-thiacytidine (3TC) and has been extensively studied from the clinical, biological, and enzymatic perspectives. In fact, the first report of a change from a methionine to valine at residue 184 (M184V) in HIV was in regard to selection for resistance in tissue culture to didanosine (ddI) (23). Later, this mutation was shown to be responsible for both high-level resistance to 3TC (6, 17, 18, 22, 57, 63, 69) and low-level resistance to almost all the molecules that act as nucleoside analog RT inhibitors (NRTIs) (Table ​(Table1)1) (25, 53, 64). The appearance of the M184V substitution was found to be transiently preceded by another mutation, M184I, that also confers high-level resistance (about 1,000-fold) to 3TC (6) and occurs in patients treated with 3TC (58). Longitudinal sampling revealed a transient appearance of the M184I variant, which subsequently disappeared from the viral population due to the outgrowth of the M184V variant (3, 37). Eventual outgrowth of the M184V variant at the expense of M184I during therapy is consistent with superior RT polymerase function (3, 8, 11) and a higher viral replication rate of the M184V variant in primary cells (3). Inspection of the nucleotide sequences of both 3TC-resistant variants indicates that M184V (GTG) originates from wild-type (WT) Met (ATG) and not from the initial M184I variant (ATA) (34). Both variants are generated from the WT ATG sequence by transitional substitutions (G to A for 184I and A to G for 184V). The reason that M184I appears before M184V is that the G→A substitution is the type of mutation that most commonly occurs during HIV-1 replication (13, 24). TABLE 1. Resistance profile of the M184V mutation in RT

Bone marrow stimulation and left ventricular function in acute myocardial infarction.

Experimental studies have demonstrated that bone marrow stem cells can migrate into peripheral blood and regenerate damaged myocardium. Bone marrow stimulation might improve myocardial functional recovery after acute myocardial infarction (AMI). In all, 104 consecutive patients with anterior wall AMI treated by primary coronary angioplasty and stenting within 12 h after onset, and who underwent left ventriculography on admission and 6 months after discharge, were studied. Among these, 23 patients (Group 1) demonstrated transient appearance of immature blood cells including myelocytes, promyelocytes, and myeloblasts during hospital stay. Thirty-eight matched patients in whom no immature blood cells were detected were studied as a control group (Group 2). There was no significant difference in baseline characteristics between the two groups. There was no significant difference in left ventricular ejection fraction (EF) on admission between Group 1 (33 +/- 12%) and Group 2 (34 +/- 8%). In contrast, EF was significantly better in Group 1 (47 +/- 12%) than in Group 2 (40 +/- 10%, p = 0.016) 6 months after discharge. The study suggests significantly greater improvement in left ventricular function in patients with AMI with sign of bone marrow stimulation than in matched patients with no sign of bone marrow stimulation.

Uncovering multiple axonal targeting pathways in hippocampal neurons.

Neuronal polarity is, at least in part, mediated by the differential sorting of membrane proteins to distinct domains, such as axons and somata/dendrites. We investigated the pathways underlying the subcellular targeting of NgCAM, a cell adhesion molecule residing on the axonal plasma membrane. Following transport of NgCAM kinetically, surprisingly we observed a transient appearance of NgCAM on the somatodendritic plasma membrane. Down-regulation of endocytosis resulted in loss of axonal accumulation of NgCAM, indicating that the axonal localization of NgCAM was dependent on endocytosis. Our data suggest the existence of a dendrite-to-axon transcytotic pathway to achieve axonal accumulation. NgCAM mutants with a point mutation in a crucial cytoplasmic tail motif (YRSL) are unable to access the transcytotic route. Instead, they were found to travel to the axon on a direct route. Therefore, our results suggest that multiple distinct pathways operate in hippocampal neurons to achieve axonal accumulation of membrane proteins.

Hemin-dependent induction and internalization of CD38 in K562 cells.

The cell surface antigen, CD38, is a bifunctional ecto-enzyme, which is predominantly expressed on hematopoietic cells during differentiation. In the present study, it is shown that hemin treatment of K562 cells gives rise to induction of enzymatic activities inherent to CD38. GDP-ribosyl cyclase activity, an indicator of CD38, increased initially in response to hemin in a time-dependent manner, reached a maximum level on the 5th day and, thereafter, declined sharply to the initial level. The increase in NAD(+) glycohydrolase and ADP-ribose uptake activities followed a similar time course. However, the decline in the latter activities after the 5th day of induction appeared to be rather slow in contrast to GDP-ribosyl cyclase activity. The time course of these changes was well correlated with the FACScan findings obtained by use of anti-CD38 monoclonal antibody. SDS-PAGE and Western blot analyses by use of the monoclonal antibody OKT10 revealed a transient hemin-dependent appearance of a 43 kDa membrane protein with maximum signal intensity on the first 4 days of incubation. There was subsequently a gradual decrease on the 5th day, concomitant with a reciprocal increase in activity of the internalized protein fraction. The results together indicated that hemin-induced expression of CD38 was followed by its down-regulation.

Enrichment and properties of an anaerobic mixed culture that reductively deiodinates 5-amino-2,4,6-triiodoisophthalic acid, an X-ray contrast agent precursor.

5-Amino-2,4,6-triiodoisophthalic acid (ATIA), both a precursor and a degradative intermediate of triiodinated contrast media, was anaerobically converted by sludge from a wastewater treatment plant. ATIA conversion took place only when an electron donor such as ethanol was added. A stable mixed culture was established by transfer to a defined synthetic mineral medium with ATIA and ethanol. It could be maintained for 1 year when the sulfate concentration was kept below 30 microM. Transient appearance of 5-amino-2,4-diiodoisophthalic acid, iodide release (2.7 mol iodide/mol ATIA) and accumulation of 5-aminoisophthalic acid indicated that ATIA was reductively dehalogenated. The enriched mixed culture also dehalogenated ATIA derivatives but deiodination remained incomplete. ATIA was the sole terminal electron acceptor used by the mixed culture during deiodination. The ratio of electrons transferred to ATIA, 0.83, was consistent with a respiratory metabolism. Formate, acetate, lactate, butyrate and hydrogen were also used as electron donors. Deiodination was inhibited by a headspace of air or by addition of nitrate, sulfite or thiosulfate. The reaction was 2.6 times slower with sulfate than without.