A piece of my mind. The veteran.
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Humans↗
Biomedical subjects
C Nguyen
Publications and source records attributed to C Nguyen.
Specific and irreversible cyclopeptide inhibitors of dipeptidyl peptidase IV activity of the T-cell activation antigen CD26.
The dipeptidyl peptidase IV (DPP IV) activity of CD26 is characterized by its post-proline-cleaving capacity that plays an important but not yet understood role in biological processes. Here we describe a new family of specific and irreversible inhibitors of this enzyme. Taking into account the substrate specificity of DPP IV for P2-P1><-P1' cleavage, we have designed and synthesized cyclopeptides c[(alphaH2N+)-Lys-Pro-Aba-(6-CH2-S+R2)-Glyn] 2TFA- (Aba = 3-aminobenzoic acid, R = alkyl) possessing a proline at the P1 position and a lysine in the P2 position, which allows the closing of the cycle on its side chain. These molecules show a free N-terminus, necessary for binding to the CD26 catalytic site, and a latent quinoniminium methide electrophile, responsible for inactivation. Treatment of c[alphaZ-Lys-Pro-Aba-(6-CH2-OC6H5)-Glyn], obtained by peptide synthesis in solution, with R2S/TFA simutaneously cleaved the Z protecting group and the phenyl ether function and led to a series of cyclopeptide sulfonium salts. These cyclopeptides inhibited rapidly and irreversibly the DPP IV activity of CD26, with IC50 values in the nanomolar range. Further studies were carried out to investigate the effect of the modification of the ring size (n = 2 or 4) and the nature of the sulfur substituents (R = Me, Bu, Oct). Cycle enlargement improved the inhibitory activity of the methylsulfonio cyclopeptide, whereas the increase of the alkyl chain length on the sulfur atom had no apparent effect. Other aminopeptidases were not inhibited, and a much weaker activity was observed on a novel isoform of DPP IV referred to as DPP IV-beta. Thus, this new family of irreversible inhibitors of DPP IV is highly specific to the peptidase activity of CD26.
Animals↗
Generation of intestinal mucosal lymphocytes in SCID mice reconstituted with mature, thymus-derived T cells.
Transfer of peripheral lymph node lymphocytes to SCID mice leads to the long term establishment of mucosal T lymphocytes within the epithelium and lamina propria of the small and large intestines. Analysis of engrafted intraepithelial lymphocytes (IEL) showed that they had acquired a surface phenotype that in several respects is typical of IEL. In addition, the functional profile of engrafted IEL derived from lymph node T cells was similar to that of normal IEL; as the donor-derived T cells exhibited a strong cytolytic activity, a poor proliferative response to mitogenic stimuli, and a tendency to home and expand specifically in the intestine upon transfer to secondary SCID recipients. Optimal engraftment of intestinal T cells required bacterial flora, as the number of lymphocytes was greatly reduced in SCID recipients with a reduced flora. These results demonstrate that mature, thymus-derived T cells can migrate to the intestine and become functionally specialized to the intestinal milieu. The acquisition of phenotypic markers characteristic of the intestinal microenvironment by engrafted cells suggests that T cell migration of lymphocytes to the SCID intestine is not aberrant, but it may reflect processes that are ongoing in immunocompetent mice. Furthermore, these data suggest that the homing and/or expansion of typical, thymus-derived T cells in the intestine may be driven by luminal Ags such as those derived from bacterial flora.
Animals↗
PMA alters folate receptor distribution in the plasma membrane and increases the rate of 5-methyltetrahydrofolate delivery in mature MA104 cells.
MA104 cells (a monkey kidney cell line) can internalize 5-methyltetrahydrofolate via a receptor mediated process termed potocytosis. Uptake is initiated by binding to an external folate receptor which cycles to an internal, but membrane bound compartment. These two pools can be measured by determining the amount of [3H]ligand removed by an acid-saline wash, i.e. acid labile and acid resistant pools. When assayed in confluent nonmitotic cells, 2/3 of the folate receptor pool is located in an internal (acid resistant) compartment, but phorbol 12-myristate 13-acetate (PMA) causes a shift such that 65-75% of the receptor pool resides on the surface of the plasma membrane. This new steady state is likely the result of an increased rate of receptor movement. In addition, PMA increases the rate of 5-methyl[3H]tetrahydrofolate delivery to the cytoplasm 1.8 fold. Using known inhibitors of potocytosis, we were able to show that the increased rate of delivery is receptor mediated. Comparison of the time courses of the PMA effects on folate receptor redistribution assessed by membrane binding of [3H]folic acid and 5-methyl[3H]tetrahydrofolate delivery to the cytoplasm suggests that PMA may be activating more than one protein kinase C independent signal transduction pathway. PMA is the first reported positive modulator of receptor mediated folate uptake.
Animals↗
The genomic organization of NKG2C, E, F, and D receptor genes in the human natural killer gene complex.
Interactions of natural killer cell receptors with their cognate ligands play a major role in regulating NK cell function. The NKG2 gene family encodes several highly similar proteins, which are known to form heterodimers with the CD94 receptor. These dimers play a role in the inhibition as well as the activation of NK cells. We have analyzed the gene structures of the NKG2C, D, E, and F genes, and determined their genomic organization. Restriction mapping and sequencing revealed the four genes to be closely linked to one another, and of the same transcriptional orientation. An exon duplication within the NKG2C and E genes was identified, although the duplicated version of this exon has not yet been found in mRNA sequences. The NKG2C, E, and F genes, despite being highly similar, are variable at their 3' ends. We show that NKG2C consists of six exons, whereas NKG2E has seven, and the splice acceptor site for the seventh exon occurs in an Alu repeat. NKG2F consists of only four exons and part of exon IV is in some cases spliced to the 5' end of the NKG2D transcript. NKG2D has only a low similarity to the other NKG2 genes.
Base Sequence↗
The utility of aminoglycosides in an era of emerging drug resistance.
As the problem of global antibiotic resistance continues to worsen, aminoglycosides have assumed increasing importance in clinical practice. Their broad antimicrobial spectrum, rapid bactericidal action, and ability to act synergistically with other drugs have made them especially useful in the treatment of serious nosocomial infections. However, as with other drugs, their overuse and misuse leads to the development of resistance in important microbial pathogens. The appropriate use of the aminoglycosides is essential to assure their continued efficacy. Therefore, physicians must familiarize themselves with both the clinical indications and the limitations of these drugs if they are to remain efficacious in the years to come.
Aminoglycosides↗
Differential use of transcription activation function 2 domain of the vitamin D receptor by 1,25-dihydroxyvitamin D3 and its A ring-modified analogs.
Analogs of 1,25-dihydroxyvitamin D3 (1,25D3) can be used to elucidate details of vitamin D receptor (VDR) activation. The A ring-modified analog, (TN-2) has 15-fold less affinity for VDR, but its transcriptional activity is diminished 1000-fold. Likewise, the ability of TN-2 to induce a protease-resistant conformation in VDR is 1/1000 that of 1,25D3. The stability of the VDR-TN-2 complexes is also significantly lower than VDR-1,25D3 complexes. Mapping the VDR-binding site of TN-2 showed that it had a significantly greater requirement for transcription activation function 2 (AF-2) residues than 1,25D3 did. These results suggest that the increased requirement for AF-2 residues that was induced by the A ring modifications is associated with diminished receptor activation. To determine whether restoring the potency of TN-2 by additional structural modifications would change the requirements for AF-2 residues, we synthesized hybrid analogs with 1beta-hydroxymethyl-3-epi groups and with dimethyl groups at positions 26 and 27 of the side chain, without or with a double bond between CD ring positions 16 and 17. We found that the side chain modification enhanced transcriptional activity 150-fold, increased the ability of the receptor to form a protease-resistant conformation 100-fold, and stabilized the VDR-analog complexes. The addition of the 16-ene group further reduced the analog's dissociation rate and increased its potency in the protease assays. These functional changes in the hybrid analogs were associated with a significant reduction in interaction with AF-2 residues. We conclude that there is an inverse relationship between analogs' potencies and their interaction with AF-2 residues of VDR.
Animals↗
The amino terminus of insulin-responsive aminopeptidase causes Glut4 translocation in 3T3-L1 adipocytes.
The insulin-responsive aminopeptidase (IRAP) is a constituent of the vesicles that contain the insulin-regulated glucose transporter (Glut4). Like Glut4, IRAP translocates to the cell surface in response to insulin. Microinjection into 3T3-L1 adipocytes of a glutathione S-transferase (GST) fusion protein containing the cytosolic portion of IRAP (GST-IRAP-(1-109)), resulted in translocation of Glut4 to the cell surface. Immunostaining of 3T3-L1 adipocytes for Glut4 showed that the percentage of cells with substantial cell surface Glut4 was 10% in unstimulated cells, 8% following injection of GST, and 27% following injection of GST-IRAP-(1-109). Increased cell surface Glut4 occurred within 5-10 min following injection and was maintained for at least 4 h. A fusion protein containing only 28 amino acids from IRAP (GST-IRAP-(55-82)) was as effective in increasing cell surface Glut4 as stimulation with 100 nM insulin (44% versus 43%, respectively). In contrast to insulin-stimulated Glut4 translocation, the redistribution of Glut4 following injection of GST-IRAP-(55-82) was not blocked by wortmannin or co-injection with a SH2 domain from the regulatory subunit of phosphatidylinositol 3-kinase. These data suggest that the amino terminus of IRAP interacts with a retention/sorting protein that also regulates the distribution of Glut4 in insulin-responsive cells.
3T3 Cells↗
Laparoscopic assisted fusion of the lumbosacral spine. A biomechanical and histologic analysis of the open versus laparoscopic technique in an animal model.
STUDY DESIGN: An animal model for laparoscopic lumbosacral fusion. OBJECTIVES: To compare the biomechanical and histologic results of open to laparoscopic lumbosacral discectomy and fusion in an animal model. BACKGROUND DATA: Early clinical reports of laparoscopic lumbosacral fusions are encouraging, but animal experiments have not been reported. METHODS: Ten pigs (50-80 kg) were divided into two groups. Group 1 underwent an open anterior lumbosacral discectomy and fusion at L7-S1 using autologous bone graft and a titanium MOSS (DePuy Motech) cage. Group 2 was identical to Group 1 except that a laparoscopic technique was used. The animals were killed at 3 months, and the lumbosacral spines were harvested for biomechanical and histologic testing. RESULTS: Estimated blood loss and average length of operation, respectively, for the two groups were: Group 1, 50 mL, 2 hours 50 minutes; and Group 2, 40 mL, 3 hours 40 minutes. There were no perioperative or postoperative complications in either group. Motion analysis results showed less motion in lateral bending, flexion, and extension than in the intact specimen in both groups. Tensile testing showed that the stiffness was significantly greater in the open group than in the laparoscopic group (P < 0.004). Histologic examination showed a less extensive discectomy and less bone growth in the implant in the laparoscopic group. Inadequate decortication of end-plates occurred in two animals who underwent laparoscopy. CONCLUSIONS: Although lumbosacral discectomy and implant insertion can be performed using the laparoscopic technique, the construct may not have the same biomechanical strength as that attained with the open procedure. Laparoscopic-assisted lumbosacral fusion surgery requires additional investigation before it is widely used in clinical situations.
Animals↗
The active sites of fructose 6-phosphate,2-kinase: fructose-2, 6-bisphosphatase from rat testis. Roles of Asp-128, Thr-52, Thr-130, Asn-73, and Tyr-197.
To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.
Amino Acids↗
Differential mRNA expression in untreated and TNF-alpha elicited murine dendritic cells precursors.
We have compared the pattern of gene expression in long term cultured precursor dendritic cells (DC), either untreated (immature) or cultured for two days in the presence of recombinant murine (rm)-TNF alpha (mature). The hybridization signature of complex cDNA probes prepared from total RNA extracted from immature and mature DC were analyzed using a mouse thymic cDNA library, gridded on high density filters. For each clone spotted on the filters, we have measured using an imaging plate device the hybridization signals of the complex probe obtained from immature or mature DC. Comparative analysis of these values allowed us to identify differentially expressed gene products. Our goal is to identify a new set of genes induced or repressed during DC maturation elicited by rmTNF alpha treatment.
Animals↗
Automated chemical synthesis: from resins to instruments.
Solid-phase organic synthesis is now a prevalent activity in drug discovery. In keeping with this keen interest is the need to develop reliable automated synthesis instrumentation as well as polymeric supports and linkers suitable for the full range of organic synthesis applications. In this paper, we review our activities in the development of new and enabling tools for automated chemical synthesis, including the following: (i) new solid supports such as ArgoGel (PS-PEG-based) and Argo-X203 (PS-based); and (ii) the Nautilus 2400 system, a fully closed and inert automated chemistry development workstation. Selected chemistry optimization and synthesis examples performed on the Nautilus and new solid supports will be described.
Automation↗
Modulation of mRNA levels in the presence of thymocytes and genome mapping for a set of genes expressed in mouse thymic epithelial cells.
Modulation of gene expression in mouse thymic epithelium upon culture in the presence of thymocytes (coculture) was studied by comparison of hybridization signatures on a set of nearly 5000 mouse thymus cDNA clones. Forty-nine differentially expressed clones (usually down-regulated in coculture) were characterized by tag sequencing. Many of them corresponded to entities that had not been described previously in the mouse, and were further characterized by genome mapping. This set of genes appears to be involved in growth regulation and differentiation within the thymus.
Animals↗
Angiogenesis at the site of neuroma formation in transected peripheral nerve.
Investiture of new microvessels within an injured peripheral nerve trunk may determine the success that the local environment has in promoting axonal sprouting and regeneration. We therefore examined microvessel investment of 24 h-14 d proximal nerve stump preparations in rat sciatic nerves. The stumps, later destined to form neuromas, were created by sciatic nerve transection with resection of distal branches to prevent distal reinnervation. Microvessels were studied in the proximal stump in semithin whole mount sections of nerve and by analysis of India ink perfused microvessel profiles. Quantitative image analysis was made of the luminal profiles of vessels perfused with India ink from unfixed sections of the stumps, contralateral uninjured nerves and sham-exposed but uninjured nerves. Evidence of angiogenesis was observed in stumps 7 d after transection, indicated by a rise in the total numbers of perfused microvessels and in the numbers of 2-6 microns diameter perfused microvessels. There was a shift in the histogram of the percentage of perfused microvessels towards the 2-4 microns range and a reduction in the mean microvessel luminal area in the stumps. By 14 d, new microvessels were larger, indicated by an increase in total luminal area. New microvessels were prominent in the epineurial connective tissue or between layers of perineurial cells of former fascicles. Microvessels probably share a battery of trophic signals with other proliferating cellular elements in the milieu of the injured peripheral nerve trunk.
Analysis of Variance↗
Myeloperoxidase deficiency manifesting as pustular candidal dermatitis.
Myeloperoxidase deficiency is the most common neutrophilic lysosomal enzyme deficiency. Case studies indicate that individuals with myeloperoxidase deficiency are not susceptible to serious infection in the absence of coexisting conditions such as diabetes mellitus. We present a case of myeloperoxidase deficiency manifesting as disseminated pustular candidal dermatitis in a nondiabetic male. Ceftriaxone therapy was administered to the patient for 8 days after he received a closed head injury and before the development of fever and pustular dermatitis. Candida albicans was isolated from the skin lesion. His neutrophils demonstrated a qualitative lack of myeloperoxidase. Patients who develop rapidly disseminated fungal dermatitis while they are receiving antimicrobial therapy that is relatively limited in coverage should be evaluated for myeloperoxidase deficiency.
Adult↗
Development of genetic vaccines for pathogenic genes: construction of attenuated vif DNA immunization cassettes.
OBJECTIVE: To develop a putative immunization cassette using HIV-1 vif accessory gene derived from HIV-1 clinical specimens as a component of a DNA vaccine for HIV-1. METHODS: vif genes were cloned from HIV-1-infected patients and the sequence variation present within the patients was analyzed. Prototypic genetic variants were selected and the ability of these clones to induce humoral and cellular immune responses was studied in animals. The selected protective genetic variants were biologically characterized through transcomplementation assays using primary cells infected with a vif-defective HIV-1 proviral clone. RESULTS: Analysis of vif variants from different patients revealed that vif is highly conserved with the open reading frame remaining intact in vivo. It was shown that attenuated vif clones from HIV-1-infected subjects can effectively induce both humoral and cellular responses against Vif protein in mice. Evaluation of the cellular responses in vitro using human cellular targets infected with a clinical HIV-1 isolate showed that vif clones could induce cellular responses capable of destroying the virus. CONCLUSIONS: The vif variants developed in this study exhibited non-productive phenotypes, yet were capable of inducing specific immune responses against HIV-1. These constructs could be used as part of a DNA vaccine strategy for HIV-1. This vaccine adaptation strategy could be used for the development of immunogens for any pathogen resulting in cross-reactive immunity and attenuated gene pathogenesis.
AIDS Vaccines↗
[The risk of suicide and its prevention in the elderly].
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Aged↗
Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death.
In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNFalpha induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNFalpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNFalpha-mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNFalpha. These findings suggest that the elevated TNFalpha levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNFalpha-induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors.
Analysis of Variance↗