CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4+ T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4+ T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989) Science 243, 1325–1330; Grant, K. L. & Klinman, J. P. (1989) Biochemistry 28, 6597–6605; and Bahnson, B. J. & Klinman, J. P. (1995) Methods Enzymol. 249, 373–397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991) J. Chem. Phys. 94, 3619–3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.

Infusion of α-galactosidase A reduces tissue globotriaosylceramide storage in patients with Fabry disease

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme α-galactosidase A (α-gal A). This enzymatic defect results in the accumulation of the glycosphingolipid globotriaosylceramide (Gb3; also referred to as ceramidetrihexoside) throughout the body. To investigate the effects of purified α-gal A, 10 patients with Fabry disease received a single i.v. infusion of one of five escalating dose levels of the enzyme. The objectives of this study were: (i) to evaluate the safety of administered α-gal A, (ii) to assess the pharmacokinetics of i.v.-administered α-gal A in plasma and liver, and (iii) to determine the effect of this replacement enzyme on hepatic, urine sediment and plasma concentrations of Gb3. α-Gal A infusions were well tolerated in all patients. Immunohistochemical staining of liver tissue approximately 2 days after enzyme infusion identified α-gal A in several cell types, including sinusoidal endothelial cells, Kupffer cells, and hepatocytes, suggesting diffuse uptake via the mannose 6-phosphate receptor. The tissue half-life in the liver was greater than 24 hr. After the single dose of α-gal A, nine of the 10 patients had significantly reduced Gb3 levels both in the liver and shed renal tubular epithelial cells in the urine sediment. These data demonstrate that single infusions of α-gal A prepared from transfected human fibroblasts are both safe and biochemically active in patients with Fabry disease. The degree of substrate reduction seen in the study is potentially clinically significant in view of the fact that Gb3 burden in Fabry patients increases gradually over decades. Taken together, these results suggest that enzyme replacement is likely to be an effective therapy for patients with this metabolic disorder.

Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins

Aβ1–42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer’s pathogenesis. Neurotoxicity of amyloid β protein (Aβ) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Aβ oligomers (referred to as ADDLs, for Aβ-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer’s disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer’s disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Aβ-derived diffusible ligands acting upon particular neural signal transduction pathways.

Chains of magnetite crystals in the meteorite ALH84001: Evidence of biological origin

The presence of magnetite crystal chains, considered missing evidence for the biological origin of magnetite in ALH84001 [Thomas-Keprta, K. L., Bazylinski, D. A., Kirschvink, J. L., Clemett, S. J., McKay, D. S., Wentworth, S. J., Vali, H., Gibson, E. K., Jr., & Romanek, C. S. (2000) Geochim. Cosmochim. Acta 64, 4049–4081], is demonstrated by high-power stereo backscattered scanning electron microscopy. Five characteristics of such chains (uniform crystal size and shape within chains, gaps between crystals, orientation of elongated crystals along the chain axis, flexibility of chains, and a halo that is a possible remnant of a membrane around chains), observed or inferred to be present in magnetotactic bacteria but incompatible with a nonbiological origin, are shown to be present. Although it is unlikely that magnetotactic bacteria were ever alive in ALH84001, decomposed remains of such organisms could have been deposited in cracks in the rock while it was still on the surface on Mars.

Logic gates using high Rydberg states

Connected logic gates can be operated on the levels of one molecule by making use of the special properties of high Rydberg states. Explicit experimental results for the NO molecule are provided as an example. A number of other options, including that of several gates concatenated so as to operate as a full adder, are discussed. Specific properties of high Rydberg states that are used are: their autoionization is delayed so that they can be distinguished from direct multiphoton ionization, during their long life such states also can decay by energy transfer to the molecular core in a way that can be controlled by the judicious application of very weak external electrical fields, and the Rydberg states can be detected by the application of an ionizing electrical field. The combination of two (or three) color photons with and without external weak fields allows the construction of quite elaborate logic circuit diagrams and shows that taking advantage of the different intramolecular dynamics of levels that differ by their excitation enables the compounding of logic operations on one molecular frame.

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Major histocompatibility class I presentation of soluble antigen facilitated by Mycobacterium tuberculosis infection.

Cell-mediated immune responses are essential for protection against many intracellular pathogens. For Mycobacterium tuberculosis (MTB), protection requires the activity of T cells that recognize antigens presented in the context of both major histocompatibility complex (MHC) class II and I molecules. Since MHC class I presentation generally requires antigen to be localized to the cytoplasmic compartment of antigen-presenting cells, it remains unclear how pathogens that reside primarily within endocytic vesicles of infected macrophages, such as MTB, can elicit specific MHC class I-restricted T cells. A mechanism is described for virulent MTB that allows soluble antigens ordinarily unable to enter the cytoplasm, such as ovalbumin, to be presented through the MHC class I pathway to T cells. The mechanism is selective for MHC class I presentation, since MTB infection inhibited MHC class II presentation of ovalbumin. The MHC class I presentation requires the tubercle bacilli to be viable, and it is dependent upon the transporter associated with antigen processing (TAP), which translocates antigenic peptides from the cytoplasm into the endoplasmic reticulum. The process is mimicked by Listeria monocytogenes and soluble listeriolysin, a pore-forming hemolysin derived from it, suggesting that virulent MTB may have evolved a comparable mechanism that allows molecules in a vacuolar compartment to enter the cytoplasmic presentation pathway for the generation of protective MHC class I-restricted T cells.

Synergistic up-regulation of the myeloid-specific promoter for the macrophage colony-stimulating factor receptor by AML1 and the t(8;21) fusion protein may contribute to leukemogenesis.

AML1 is involved in the (8;21) translocation, associated with acute myelogenous leukemia (AML)-type M2, which results in the production of the AML1-ETO fusion protein: the amino-terminal 177 amino acids of AML1 and the carboxyl-terminal 575 amino acids of ETO. The mechanism by which AML1-ETO accomplishes leukemic transformation is unknown; however, AML1-ETO interferes with AML1 transactivation of such AML1 targets as the T-cell receptor beta enhancer and the granulocyte-macrophage colony-stimulating factor promoter. Herein, we explored the effect of AML1-ETO on regulation of a myeloid-specific AML1 target, the macrophage colony-stimulating factor (M-CSF) receptor promoter. We found that AML1-ETO and AML1 work synergistically to transactivate the M-CSF receptor promoter, thus exhibiting a different activity than previously described. Truncation mutants within the ETO portion of AML1-ETO revealed the region of ETO necessary for the cooperativity between AML1 and AML1-ETO lies between amino acids 347 and 540. Endogenous M-CSF receptor expression was examined in Kasumi-1 cells, derived from a patient with AML-M2 t(8;21) and the promonocytic cell line U937. Kasumi-1 cells exhibited a significantly higher level of M-CSF receptor expression than U937 cells. Bone marrow from patients with AML-M2 t(8;21) also exhibited a higher level of expression of M-CSF receptor compared with normal controls. The upregulation of M-CSF receptor expression by AML1-ETO may contribute to the development of a leukemic state in these patients.

Acyl tunichlorins: a new class of nickel chlorins isolated from the Caribbean tunicate Trididemnum solidum.

A new class of nickel-containing chlorins (acyl tunichlorins) has been isolated from the Caribbean tunicate Trididemnum solidum. The structures of 28 of these nickel (II) hydroporphyrins were elucidated using mass spectrometry, one- and two-dimensional NMR spectroscopy, and chemical degradation/derivatization. Unique structural features of these compounds include the diversity of aliphatic side chains, which are derived from C14:0 to C22:6 fatty acids, and their location at an unprecedented position at C-2a on the hydroporphyrin nucleus. No chlorins with ester-linked acyl side chains at C-2a have been reported previously. Although the exact biological role that these compounds play in T. solidum remains unknown, acyl tunichlorins represent the only nickel-containing chlorins to be isolated from a living system and are the C-2a acyl derivatives of tunichlorin, a nickel chlorin reported by this laboratory in 1988.

Intracellular pH in adipocytes: effects of free fatty acid diffusion across the plasma membrane, lipolytic agonists, and insulin.

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.

The differential adhesion forces of anterior cruciate and medial collateral ligament fibroblasts: effects of tropomodulin, talin, vinculin, and alpha-actinin.

We have determined the effects of tropomodulin (Tmod), talin, vinculin, and alpha-actinin on ligament fibroblast adhesion. The anterior cruciate ligament (ACL), which lacks a functional healing response, and the medial collateral ligament (MCL), a functionally healing ligament, were selected for this study. The micropipette aspiration technique was used to determine the forces needed to separate ACL and MCL cells from a fibronectin-coated surface. Delivery of exogenous tropomodulin, an actin-filament capping protein, into MCL fibroblasts significantly increased adhesion, whereas its monoclonal antibody (mAb) significantly decreased cell adhesiveness. However, for ACL fibroblasts, Tmod significantly reduced adhesion, whereas its mAb had no effect. mAbs to talin, vinculin, and alpha-actinin significantly decreased the adhesion of both ACL and MCL cells with increasing concentrations of antibody, and also reduced stress fiber formation and cell spreading rate as revealed by immunofluorescence microscopy. Disruption of actin filament and microtubule assembly with cytochalasin D and colchicine, respectively, also significantly reduced adhesion in ACL and MCL cells. In conclusion, both ACL and MCL fibroblast adhesion depends on cytoskeletal assembly; however, this dependence differs between ACL and MCL fibroblasts in many ways, especially in the role of Tmod. These results add yet another possible factor in explaining the clinical differences in healing between the ACL and the MCL.

Increased tricarboxylic acid cycle flux in rat brain during forepaw stimulation detected with 1H[13C]NMR.

NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation.

16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the Green Non-Sulfur bacteria.

Microorganisms play an important role in the biogeochemistry of the ocean surface layer, but spatial and temporal structures in the distributions of specific bacterioplankton species are largely unexplored, with the exceptions of those organisms that can be detected by either autofluorescence or culture methods. The use of rRNA genes as genetic markers provides a tool by which patterns in the growth, distribution, and activity of abundant bacterioplankton species can be studied regardless of the ease with which they can be cultured. Here we report an unusual cluster of related 16S rRNA genes (SAR202, SAR263, SAR279, SAR287, SAR293, SAR307) cloned from seawater collected at 250 m in the Sargasso Sea in August 1991, when the water column was highly stratified and the deep chlorophyll maximum was located at a depth of 120 m. Phylogenetic analysis and an unusual 15-bp deletion confirmed that the genes were related to the Green Non-Sulfur phylum of the domain Bacteria. This is the first evidence that representatives of this phylum occur in the open ocean. Oligonucleotide probes were used to examine the distribution of the SAR202 gene cluster in vertical profiles (0-250 m) from the Atlantic and Pacific Oceans, and in discrete (monthly) time series (O and 200 m) (over 30 consecutive months in the Western Sargasso Sea. The data provide robust statistical support for the conclusion that the SAR202 gene cluster is proportionately most abundant at the lower boundary of the deep chlorophyll maximum (P = 2.33 x 10(-5)). These results suggest that previously unsuspected stratification of microbial populations may be a significant factor in the ecology of the ocean surface layer.