Biomedical subjects

M Lu

Publications and source records attributed to M Lu.

At least 109 records · Page 6Linked to original sources

PubMed2001

Treatment of traumatic brain injury in female rats with intravenous administration of bone marrow stromal cells.

OBJECTIVE: To study the effect of bone marrow stromal cells administered intravenously to female rats subjected to traumatic brain injury. METHODS: We injected marrow stromal cells harvested from male rat bone marrow (n = 24) into the tail vein of the female rat (n = 8) 24 hours after traumatic brain injury; the rats were killed at Day 7 or 14 after treatment. The neurological function of the rats was evaluated using the rotarod test and the neurological severity score. The distribution of the male donor cells in brain, heart, lung, kidney, liver, muscle, spleen, and bone marrow of the female recipient rats was measured by identifying Y chromosome-positive cells using fluorescent in situ hybridization. RESULTS: We found that marrow stromal cells injected intravenously significantly reduced motor and neurological deficits compared with control groups by Day 15 after traumatic brain injury (P < 0.05, analysis of covariance for repeated measures). The transplanted cells preferentially engrafted into the parenchyma of the injured brain and expressed the neuronal marker NeuN and the astrocytic marker glial fibrillary acidic protein. Marrow stromal cells were also found in other organs in female rats subjected to traumatic brain injury without any obvious adverse effects. CONCLUSION: These data suggest that the intravenous administration of marrow stromal cells may be a promising therapeutic strategy that warrants further investigation for patients with traumatic brain injury.

Animals↗

PubMed2000

PPARalpha agonists reduce 11beta-hydroxysteroid dehydrogenase type 1 in the liver.

11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) is an enzyme that converts cortisone to the active glucocorticoid, cortisol. Cortisol-cortisone interconversion plays a key role in the regulation of glucose metabolism, since mice deficient in 11betaHSD1 are resistant to diet-induced hyperglycemia. Peroxisome proliferator activator receptors (PPAR) are key regulators of glucose and lipid homeostasis. We observed a striking downregulation of murine hepatic 11betaHSD1 expression and activity after chronic treatment of wild-type mice with PPARalpha agonists, while 11betaHSD1 in the livers of PPARalpha knockout mice, or in mice treated for only 7 h with PPARalpha agonists, was unaltered. Our results are the first to show PPARalpha agonists can affect glucocorticoid metabolism in the liver by altering 11betaHSD1 expression after chronic treatment. Regulation of active glucocorticoid levels in the liver by PPARalpha agonists may in turn affect glucose metabolism, consistent with reports of their antidiabetic effects.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed2000

Transactivation of the p21 promoter by BRCA1 splice variants in mammary epithelial cells: evidence for both common and distinct activities of wildtype and mutant forms.

We have evaluated the transcriptional activation of a human p21 promoter reporter construct by transfection of BRCA1 expression constructs into tumorigenic and nontumorigenic human breast cell lines. Two cell lines with wildtype p53 (MCF-7 and MCF10A) demonstrated transcriptional activation of the p21 promoter by full-length BRCA1 (BRCA1L) as well as by two splice variants that lack most of exon 11 (BRCA1S and BRCA1S-9,10). In contrast, two cell lines with mutant p53 (MDA-231 and HCC1937) were inactive. Co-transfection of BRCA1L with BRCA1S or BRCA1S-9,10 exhibited synergistic p21 promoter activation, due to augmented expression of the cytomegalovirus promoter-based BRCA1 expression constructs. We examined the transcriptional activity of two known sequence alterations in BRCA1, one that results in a carboxy-terminal truncation of BRCA1 and is clearly pathogenic, and the other a missense mutation that is suspected of predisposing to cancer. Although both mutations have been shown to be defective in some assays of transactivation, we observed both mutations to be fully active in activation of the p21 promoter when incorporated in the full-length BRCA1L. In contrast, these mutations rendered BRCA1S inactive. These observations indicate that such transcriptional assays cannot serve as the basis for a functional appraisal of BRCA1 sequence alterations encountered in the course of genetic testing.

Alternative Splicing↗

PubMed2000

Early stroke treatment associated with better outcome: the NINDS rt-PA stroke study.

BACKGROUND: The National Institute of Neurological Disorders and Stroke (NINDS) rt-PA Stroke Study showed a similar percentage of intracranial hemorrhage and good outcome in patients 3 months after stroke treatment given 0 to 90 minutes and 91 to 180 minutes after stroke onset. At 24 hours after stroke onset more patients treated 0 to 90 compared to 91 to 180 minutes after stroke onset had improved by four or more points on the NIH Stroke Scale (NIHSS). The authors performed further analyses to characterize the relationship of onset-to-treatment time (OTT) to outcome at 3 months, early improvement at 24 hours, and intracranial hemorrhage within 36 hours. METHODS: Univariate analyses identified potentially confounding variables associated with OTT that could mask an OTT-treatment interaction. Tests for OTT-treatment interactions adjusting for potential masking confounders were performed. An OTT-treatment interaction was considered significant if p < or = 0.10, implying that treatment effectiveness was related to OTT. RESULTS: For 24-hour improvement, there were no masking confounders identified and there was an OTT-treatment interaction (p = 0.08). For 3-month favorable outcome, the NIHSS met criteria for a masking confounder. After adjusting for NIHSS as a covariate, an OTT-treatment interaction was detected (p = 0.09): the adjusted OR (95% CI) for a favorable 3-month outcome associated with recombinant tissue-type plasminogen activator (rt-PA) was 2.11 (1.33 to 3.35) in the 0 to 90 minute stratum and 1.69 (1.09 to 2.62) in the 91 to 180 minute stratum. In the group treated with rt-PA, after adjusting for baseline NIHSS, an effect of OTT on the occurrence of intracranial hemorrhage was not detected. CONCLUSIONS: If the NINDS rt-PA Stroke Trial treatment protocol is followed, this analysis suggests that patients treated 0 to 90 minutes from stroke onset with rt-PA have an increased odds of improvement at 24 hours and favorable 3-month outcome compared to patients treated later than 90 minutes. No effect of OTT on intracranial hemorrhage was detected within the group treated with rt-PA, possibly due to low power.

Double-Blind Method↗

PubMed2000

Naturally occurring woodchuck hepatitis virus (WHV) deletion mutants in chronically WHV-infected woodchucks.

Deletion mutants of hepatitis B virus (HBV) are often found in chronically HBV-infected patients. It has not been possible to study the significance of such deletion mutants on liver diseases in a suitable animal model. In this study, we characterized naturally occurring deletion mutants of woodchuck hepatitis virus (WHV) in 11 chronically WHV-infected woodchucks. Deletions within the WHV preS region (nt 2992-338) had a length of 72 or 84 bp and were located in the amino terminal part of preS1. Internal deletions within the core gene (CID) had variable lengths (103 to 312 bp) and were identified within the center of this gene (nt 2021-2587). Four of seven CIDs were in-frame deletions, whereas the remaining three CIDs were out-of-frame deletions and led to the interruption of the reading frame. Sequence analysis of cloned PCR products of CIDs showed that heterogeneous WHV deletion mutants coexisted in single woodchucks. In addition, WHV genomes with double deletions in the preS1 and the core region could be found. We were unable to detect the expression of truncated core proteins in transfection experiments. The CID mutations led to a marked increase of the expression of the luciferase gene which was fused to the start codon of WHV polymerase, probably due to the shortening of the untranslated region or the removal of AUGs preceding the polymerase start codon. The characterization of naturally occurring WHV deletion mutants will allow us to study their biological and pathogenic properties in the woodchuck model in the future.

Animals↗

PubMed2000

The amino-terminal domain of the B subunit of vacuolar H+-ATPase contains a filamentous actin binding site.

Vacuolar H(+)-ATPase (V-ATPase) binds actin filaments with high affinity (K(d) = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.

Actin Cytoskeleton↗

PubMed2000

Predicting prognosis after stroke: a placebo group analysis from the National Institute of Neurological Disorders and Stroke rt-PA Stroke Trial.

BACKGROUND: Physicians are often asked to predict outcome after acute stroke. Very little information is available that can reliably predict the likelihood of severe disability or death. OBJECTIVE: To develop a practical method for predicting a poor outcome after acute ischemic stroke. METHODS: Data from the placebo arms of Parts 1 and 2 of the National Institute of Neurological Disorders and Stroke rt-PA [recombinant tissue plasminogen activator] Stroke Trial were used to identify variables that could predict a poor outcome, defined as moderately severe disability, severe disability, or death (Modified Rankin Scale score >3) 3 months after stroke. RESULTS: Baseline variables that predicted poor outcome were the NIH Stroke Scale (NIHSS) >17 plus atrial fibrillation, yielding a positive predictive value (PPV) of 96% (95% CI, 88 to 100%). The best predictor at 24 hours was NIHSS >22, yielding a PPV of 98% (95% CI, 93 to 100%). The best predictor at 7 to 10 days was NIHSS >16, yielding a PPV of 92% (95% CI, 85 to 99%). CONCLUSIONS: Patients with a severe neurologic deficit after acute ischemic stroke, as measured by the NIHSS, have a poor prognosis. During the first week after acute ischemic stroke, it is possible to identify a subset of patients who are highly likely to have a poor outcome. These findings require confirmation in a separate study.

Aged↗

PubMed2000

Optically defined multifunctional patterning of photosensitive thin-film silica mesophases.

Photosensitive films incorporating molecular photoacid generators compartmentalized within a silica-surfactant mesophase were prepared by an evaporation-induced self-assembly process. Ultraviolet exposure promoted localized acid-catalyzed siloxane condensation, which can be used for selective etching of unexposed regions; for "gray-scale" patterning of refractive index, pore size, surface area, and wetting behavior; and for optically defining a mesophase transformation (from hexagonal to tetragonal) within the film. The ability to optically define and continuously control both structure and function on the macro- and mesoscales is of interest for sensor arrays, nanoreactors, photonic and fluidic devices, and low-dielectric-constant films.

Journal Article↗

PubMed2000

Spinal cord injury in rat: treatment with bone marrow stromal cell transplantation.

We tested the hypothesis that transplantation of bone marrow stromal cells (MSCs) into the spinal cord after a contusion injury promotes functional outcome. Rats (n = 31) were subjected to a weight driven implant injury. MSCs or phosphate buffered saline was injected into the spinal cord 1 week after injury. Sections of tissue were analyzed by double-labeled immunohistochemistry for MSC identification. Functional outcome measurements using the Basso-Beattie-Bresnehan score were performed weekly to 5 weeks post-injury. The data indicate significant improvement in functional outcome in animals treated with MSC transplantation compared to control animals. Scattered cells derived from MSCs expressed neural protein markers. These data suggest that transplantation of MSCs may have a therapeutic role after spinal cord injury.

Animals↗

PubMed2000

CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage.

The CaMKIIalpha mRNA extends into distal hippocampal dendrites, and the 3' untranslated region (3'UTR) is sufficient to mediate this localization. We labeled the 3'UTR of the CaMKIIalpha mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage tagging system. The CaMKIIalpha 3'UTR formed discrete granules throughout the dendrites of transfected cells. The identity of the fluorescent granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites. These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization. So that motile granules could be tracked, shorter periods of observation were required. The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde. Colocalization of CaMKIIalpha 3'UTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses. Neuronal depolarization increased the number of granules in the anterograde motile pool. Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli. Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

3' Untranslated Regions↗

PubMed2000

Core structure of the outer membrane lipoprotein from Escherichia coli at 1.9 A resolution.

The outer membrane lipoprotein of the Escherichia coli cell envelope has characteristic lipid modifications at an amino-terminal cysteine and can exist in a form bound covalently to the peptidoglycan through a carboxyl-terminal lysine. The 56-residue polypeptide moiety of the lipoprotein, designated Lpp-56, folds into a stable, trimeric helical structure in aqueous solution. The 1.9 A resolution crystal structure of Lpp-56 comprises a parallel three-stranded coiled coil including a novel alanine-zipper unit and two helix-capping motifs. The amino-terminal motif forms a hydrogen-bonding network anchoring an umbrella-shaped fold. The carboxyl-terminal motif uses puckering of the tyrosine side-chains as a unique docking arrangement in helix termination. The structure provides an explanation for assembly and insertion of the lipoprotein molecules into the outer membrane of gram-negative bacteria and suggests a molecular target for antibacterial drug discovery.

Alanine↗

PubMed2000

Detection of germ-cell tumor cells in the peripheral blood by nested reverse transcription-polymerase chain reaction for alpha-fetoprotein-messenger RNA and beta human chorionic gonadotropin-messenger RNA.

By establishing sensitive nested reverse transcription-PCRs for the detection of mRNA of alpha-fetoprotein (AFP) and beta human chorionic gonadotropin (betahCG), we investigated the presence of circulating tumor cells in the peripheral blood of 119 patients with germ-cell tumor. A total of 336 blood samples obtained before and during therapy were examined with regard to clinical applicability. The overall ratio of positive PCR results was 26.5% and was independent of the serum concentration of AFP and hCG/betahCG. No correlation of the positivity for AFP-mRNA to serum AFP level was found. In contrast, positive results in betahCG-PCR were twice as frequent in patients with elevated serum hCG/betahCG levels as in those with normal serum hCG/betahCG levels (P = 0.012). To develop a valid correlation to tumor stage, tumor histology, and serum level of tumor markers, a subgroup of 36 patients was evaluated before definite therapy. The subgroup revealed an overall ratio of 33.3% positive PCR results. The serum level of both of the markers did not correlate with the detection of corresponding mRNA in peripheral blood samples. However, positive betahCG-PCR results were found exclusively in patients with elevated serum hCG/betahCG (6 of 18 versus 0 of 18; P = 0.019). Patients with stage IIC/III germ-cell tumor demonstrated nearly twice the frequency of positive PCR results as patients with stage I tumor [7 (41.2%) of 17 versus 4 (23.5%) of 17] in this subgroup. With regard to histology, positive PCR results were found mostly in embryonal carcinoma.

Adolescent↗

PubMed2000

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant.

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.

Arginine↗

PubMed2000

Mutational analysis of basic residues in the rat vesicular acetylcholine transporter. Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation.

The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.

Acetylcholine↗

PubMed2000

Structure of Escherichia coli ribosomal protein L25 complexed with a 5S rRNA fragment at 1.8-A resolution.

The crystal structure of Escherichia coli ribosomal protein L25 bound to an 18-base pair portion of 5S ribosomal RNA, which contains "loop E," has been determined at 1.8-A resolution. The protein primarily recognizes a unique RNA shape, although five side chains make direct or water-mediated interactions with bases. Three beta-strands lie in the widened minor groove of loop E formed by noncanonical base pairs and cross-strand purine stacks, and an alpha-helix interacts in an adjacent widened major groove. The structure of loop E is largely the same as that of uncomplexed RNA (rms deviation of 0.4 A for 11 base pairs), and 3 Mg(2+) ions that stabilize the noncanonical base pairs lie in the same or similar locations in both structures. Perhaps surprisingly, those residues interacting with the RNA backbone are the most conserved among known L25 sequences, whereas those interacting with the bases are not.

Conserved Sequence↗

PubMed2000

Helical interactions in the HIV-1 gp41 core reveal structural basis for the inhibitory activity of gp41 peptides.

The HIV-1 gp41 envelope protein mediates membrane fusion that leads to virus entry into the cell. The core structure of fusion-active gp41 is a six-helix bundle in which an N-terminal three-stranded coiled coil is surrounded by a sheath of antiparallel C-terminal helices. A conserved glutamine (Gln 652) buried in this helical interface replaced by leucine increases HIV-1 infectivity. To define the basis for this enhanced membrane fusion activity, we investigate the role of the Gln 652 to Leu substitution on the conformation, stability, and biological activity of the N34(L6)C28 model of the gp41 ectodomain core. The 2.0 A resolution crystal structure of the mutant molecule shows that the Leu 652 side chains make prominent contacts with hydrophobic grooves on the surface of the central coiled coil. The Gln 652 to Leu mutation leads to a marginal stabilization of the six-helix bundle by -0.8 kcal/mol, evaluated from thermal unfolding experiments. Strikingly, the mutant N34(L6)C28 peptide is a potent inhibitor of HIV-1 infection, with 10-fold greater activity than the wild-type molecule. This inhibitory potency can be traced to the corresponding C-terminal mutant peptide that likely has greater potential to interact with the coiled-coil trimer. These results provide strong evidence that conserved interhelical packing interactions in the gp41 core are important determinants of HIV-1 entry and its inhibition. These interactions also offer a test-bed for the development of more potent analogues of gp41 peptide inhibitors.

Amino Acid Sequence↗

PubMed2000

Buried polar interactions and conformational stability in the simian immunodeficiency virus (SIV) gp41 core.

For human (HIV) and simian (SIV) immunodeficiency viruses, the gp41 envelope protein undergoes a receptor-activated conformational change from a labile native structure to an energetically more stable fusogenic conformation, which then mediates viral-cell membrane fusion. The core structure of fusion-active gp41 is a six-helix bundle in which three antiparallel carboxyl-terminal helices are packed against an amino-terminal trimeric coiled coil. Here we show that a recombinant model of the SIV gp41 core, designated N36(L6)C34, forms an alpha-helical trimer that exhibits a cooperative two-state folding-unfolding transition. We investigate the importance of buried polar interactions in determining the overall fold of the gp41 core. We have replaced each of four polar amino acids at the heptad a and d positions of the coiled coil in N36(L6)C34 with a representative hydrophobic amino acid, isoleucine. The Q565I, T582I, and T586I variants form six-helix bundle structures that are significantly more stable than that of the wild-type peptide, whereas the Q575I variant misfolds into an insoluble aggregate under physiological conditions. Thus, the buried polar residues within the amino-terminal heptad repeat are important determinants of the structural specificity and stability of the gp41 core. We suggest that these conserved buried polar interactions play a role in governing the conformational state of the gp41 molecule.

Amino Acid Sequence↗

PubMed2000

Interactions between HIV-1 gp41 core and detergents and their implications for membrane fusion.

The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergent n-octyl-beta-D-glucopyranoside (betaOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly alpha-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion.

Amino Acid Sequence↗