Lys Substitution Research Articles

Functional Importance of Three Basic Residues Clustered at the Cytosolic Interface of Transmembrane Helix 15 in the Multidrug and Organic Anion Transporter MRP1 (ABCC1)

The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg1138, Lys1141, and Arg1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg1138 and Arg1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[32P]ADP. Mutation of Lys1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter.

Measles and rubella in Europe — reaching for elimination

Adenovirus type 7 (Ad7) is frequently responsible for severe respiratory infections, especially in young infants. Since the Ad7 epidemics have been associated with severe childhood pneumonia and significant mortality in Korea, 1995–1999, continuous surveillance was necessary for Ad7 related diseases.To characterize epidemiologic features of Ad7 in 1995–2004, genetic diversity of Ad7 were studied by determining genome types (GTs) and the fiber diversity.A total of 139 Ad7 strains were obtained from Korean children with pneumonia. Serotype specificity was confirmed by microneutralization assay. GTs were determined by restriction analysis with 12 enzymes. The variable region of the fiber was sequenced.Two GTs, Ad7d (N = 98, 71%) and Ad7l (N = 41, 29%) have been identified. In 1995–1996 and 2001–2002, Ad7d was dominant accounting for 98–100% of all Ad7; in 1999–2000, Ad7l was the prevalent GT accounting for 100% of all Ad7; in 1997–1998 and 2003–2004, both GTs circulated concurrently. The change in the relative predominance of GT occurred in 2 or 3 years. The Lys substitution for Arg at codon 280 of the fiber was identified in 31 Ad7d strains (32%) while no variations were observed among Ad7l. It was noteworthy that two fiber variants of Ad7d were not concurrently prevalent on any time after 1996. The shift in predominant fiber variants of Ad7d was also observed in 2–3 years.Our data demonstrated that the two GTs, Ad7d and Ad7l circulated in an alternating manner between outbreaks of Ad7 associated childhood pneumonia over 10 consecutive years in Korea. Fiber diversity at position 280 within Ad7d appeared to contribute to the annual distribution of Ad7d. This observation necessitates further studies to demonstrate an association between fiber variation and host cell specificity or neutralization antibody recognition.

Effect of alternating passage on adaptation of sindbis virus to vertebrate and invertebrate cells.

Mosquito-borne alphaviruses, which replicate alternately and obligately in mosquitoes and vertebrates, appear to experience lower rates of evolution than do many RNA viruses that replicate solely in vertebrates. This genetic stability is hypothesized to result from the alternating host cycle, which constrains evolution by imposing compromise fitness solutions in each host. To test this hypothesis, Sindbis virus was passaged serially, either in one cell type to eliminate host alteration or alternately between vertebrate (BHK) and mosquito (C6/36) cells. Following 20 to 50 serial passages, mutations were identified and changes in fitness were assessed using competition assays against genetically marked, surrogate parent viruses. Specialized viruses passaged in a single cell exhibited more mutations and amino acid changes per passage than those passaged alternately. Single host-adapted viruses exhibited fitness gains in the cells in which they specialized but fitness losses in the bypassed cell type. Most but not all viruses passaged alternately experienced lesser fitness gains than specialized viruses, with fewer mutations per passage. Clonal populations derived from alternately passaged viruses also exhibited adaptation to both cell lines, indicating that polymorphic populations are not required for simultaneous fitness gains in vertebrate and mosquito cells. Nearly all passaged viruses acquired Arg or Lys substitutions in the E2 envelope glycoprotein, but enhanced binding was only detected for BHK cells. These results support the hypothesis that arbovirus evolution may be constrained by alternating host transmission cycles, but they indicate a surprising ability for simultaneous adaptation to highly divergent cell types by combinations of mutations in single genomes.

Appropriate Function of 11β-Hydroxysteroid Dehydrogenase Type 1 in the Endoplasmic Reticulum Lumen Is Dependent on Its N-terminal Region Sharing Similar Topological Determinants with 50-kDa Esterase

By interconverting glucocorticoids, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11beta-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11beta-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11beta-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys(5) by Ser in 11beta-HSD1, but not of the analogous Lys(4) by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu(25)/Glu(26) in 11beta-HSD1 and Asp(25) in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys(35)/Lys(36) and Glu(25)/Glu(26) on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11beta-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11beta-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11beta-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11beta-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.

Evidence for an Unfolding/Threading Mechanism for Protein Disaggregation by Saccharomyces cerevisiae Hsp104

Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation.

Alteration of the basic amino acid domain of wild-type matrix protein reduced influenza virus replication at non-permissive temperature

The matrix protein (M1) of influenza virus plays an essential role in viral assembly and a variety of functions, including association with influenza virus ribonucleoprotein (RNP). Our previous studies show that the association of M1 with viral RNA and NP protein not only promotes formation of helical RNP, but also is required for export of RNP from the nucleus during viral replication. The RNA-binding domains of M1 have been mapped to two independent regions, a zinc-finger motif at amino acid positions 148 to 162, and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105, which is also involved in RNP-binding activity. To further understand the role of the RNP-binding domain of M1 on viral assembly and replication, mutations in the coding sequences of RKLKR and zinc-finger motif of M1 were constructed using a PCR technique and introduced into wild-type influenza virus by reverse genetics. Altering the zinc-finger motif of M1 only slightly affected viral growth. Substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR resulted in a lethal mutation. These results indicate that RKLKR domain of M1 protein plays an important role in viral replication.

Safety and immunogenicity in animals of subunit influenza vaccine given intranasally with mutants of Escherichia coli heat-labile enterotoxin (LT)

Mucosal delivery of inactivated vaccine against respiratory diseases offers several advantages over the traditional intramuscular injection. This requires the use of a potent adjuvant and/or of delivery systems to obtain strong local and systemic immune responses. Site-directed mutagenesis has allowed the generation of mutants such as LTK63 (Ser to Lys substitution at position 63 in the A subunit) and LTR72 (Ala to Arg at position 72) with abolished or strongly reduced toxicity while still retaining strong mucosal adjuvanticity. Both of them have been used to formulate a subunit influenza vaccine, administered intranasally together with nanoparticles and were shown to be able to potentiate its immunogenicity. These vaccine formulations were totally safe in different animal models.

Different expression pattern of hepcidin genes in the liver and pancreas of C57BL/6N and DBA/2N mice

Background/Aims Male C57BL/6 and DBA/2 mice differ in their liver iron content. The aim of this study was to examine possible differences in the expression of hepcidin genes ( Hamp and Hamp2) between the two strains. Methods Hepatic mRNAs were quantified by real-time PCR. Results Ferroportin1, transferrin receptor 2 and HAMP mRNA levels displayed no significant strain differences. However, HAMP2 mRNA levels were higher in DBA/2N mice. In both strains, HAMP2 mRNA content was sex-dependent, with higher values in female animals. Both hepatic HAMP and HAMP2 mRNA levels were elevated by iron overload, but treatment with lipopolysaccharide increased only HAMP mRNA. Lipopolysaccharide also elevated the amount of HAMP mRNA in the pancreas, while pancreatic HAMP2 mRNA levels were decreased. Sequence analysis of hepcidin amplicons from DBA/2N mice predicted an Asn→Lys substitution at position 73 of the HAMP peptide and a Ser→Phe substitution at position 76 of the HAMP2 peptide. Conclusions Hepatic Hamp2 expression displays considerable strain- and sex-dependent variation. Lipopolysaccharide increases expression of Hamp both in the liver and pancreas, but Hamp2 does not respond to lipopolysaccharide treatment. The significance of the amino acid substitutions in hepcidin peptides in DBA/2N mice is at present unknown.

Mutagenesis of the AT 1 receptor reveals different binding modes of angiotensin II and [Sar 1]-angiotensin II

Homology modeling of the structure of the AT 1 receptor, based on the high resolution rhodopsin crystal structure, indicated that it is unlikely that the binding of AngII to AT 1 involves simultaneously all the receptor's residues reported in the literature to participate in this process. Site-directed mutagenesis using Ala substitution of charged residues Lys 20, Arg 23, Glu 91 and Arg 93 was performed to evaluate the participation of their side-chains in ligand binding and in triggering the cell's response. A comparative analysis by competition binding and functional assays using angiotensin II and the analog [Sar 1]-angiotensin II suggests an important role for Arg 23 of AT 1 receptor in binding of the natural agonist. It is discussed whether some receptor's residues participate directly in the binding with AngII or whether they are part of a regulatory site.

The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins.

Work in Saccharomyces cerevisiae has shown that Atp12p binds to unassembled alpha subunits of F(1) and in so doing prevents the alpha subunit from associating with itself in non-productive complexes during assembly of the F(1) moiety of the mitochondrial ATP synthase. We have developed a method to prepare recombinant Atp12p after expression of its human cDNA in bacterial cells. The molecular chaperone activity of HuAtp12p was studied using citrate synthase as a model substrate. Wild type HuAtp12p suppresses the aggregation of thermally inactivated citrate synthase. In contrast, the mutant protein HuAtp12p(E240K), which harbors a lysine at the position of the highly conserved Glu-240, fails to prevent citrate synthase aggregation at 43 degrees C. No significant differences were observed between the wild type and the mutant proteins as judged by sedimentation analysis, cysteine titration, tryptophan emission spectra, or limited proteolysis, which suggests that the E240K mutation alters the activity of HuAtp12p with minimal effects on the physical integrity of the protein. An additional important finding of this work is that the equilibrium chemical denaturation curve of HuAtp12p shows two components, the first of which is associated with protein aggregation. This result is consistent with a model for Atp12p structure in which there is a hydrophobic chaperone domain that is buried within the protein interior.

The 3′ region of the DRD2 gene is involved in genetic susceptibility to schizophrenia

The gene coding for the D2 dopamine receptor ( DRD2) is considered as one of the most relevant candidate genes in schizophrenia. Previous genetic studies focusing on this gene yielded conflicting results, for example because of differences in methodology (linkage versus association studies) and variability in the loci analyzed (the DRD2 gene having many polymorphic sites). We used a progressive strategy with two different approaches (case-control and transmission disequilibrium test) and investigated six genetic polymorphisms spanning the DRD2 gene in 103 patients with DSM-IV criteria of schizophrenia, their 206 parents and 83 matched healthy control subjects. We found a significant excess of the A2 allele in subject with schizophrenia compared to unaffected controls. An excess of transmission of the A2 allele (and haplotypes containing this marker) from the parents to the affected children was also observed. Interestingly, the TaqI A1/A2 polymorphism, located 9.5 kb downstream from the DRD2 gene, maps in a novel gene, untitled “X-kinase”, and leads to a 713Glu→Lys substitution in exon 8. As the analysis of the other markers within the DRD2 gene does not improve the strength of the association, our data are in favor of a specific role of the 3′ chromosomic region of the DRD2 gene in the vulnerability to schizophrenia.

Cotton rat Sihi-M3 is a minimally oligomorphic Mhc I-b molecule that binds the chemotactic peptide fMLF under stringent conditions. Evidence that positive selection drives inter-species diversity of residues interacting with the termini of short peptides.

The leading model for class I-b evolution suggests non-polymorphic I-b genes evolve by gene duplication from polymorphic I-a genes. We recently found N-formyl peptide-specific orthologs of the class I-b gene H2-M3 in the rodent subfamily Sigmodontinae. To test if sigmodont M3 is a I-b gene, we sequenced M3 from wild cotton rats ( Sigmodon hispidus) diverse at the class II locus, Sihi-DQA. These haplotypes carry a single allele of M3 that closely resembles H2-M3. However, peptide-binding assays showed that cotton rat M3 bound the chemotactic N-formylpeptide fMLF better than did rat or mouse M3. The Ala116-->Lys substitution in cotton rat M3 might enhance binding of fMLF and is one of eight residues of M3 that interact with ligand residues P3 and P4 and that are positively selected, with a d(N) /d(S) ratio of 1.8. Thus, M3 is a class I-b gene in both sigmodontine and murine murids, but positive selection operates on a small subset of residues in the traditionally defined antigen recognition site.

Tetratricopeptide Repeat Motif-mediated Hsc70-mSTI1 Interaction: MOLECULAR CHARACTERIZATION OF THE CRITICAL CONTACTS FOR SUCCESSFUL BINDING AND SPECIFICITY

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.

MD Simulations on the Influence of Disease-Related Amino Acid Mutations in the Human Prion Protein

We have performed molecular dynamics simulations on the human prion protein to study the effect of point mutations in relation to the inherited form of Creutzfeldt-Jakob disease. Three model structures of the human prion protein are examined with a focus on their dynamics and conformational changes. Model 1 is an NMR structure of the globular domain (125-228) of a wild-type prion. The model consists of three α-helices and an anti-parallel β-sheet. Models 2 and 3 are mutant structures containing a Glu200→Asp and a Glu200→Lys substitution, respectively. These models are derived from NMR structures. The Glu200→Lys in model 3 is a disease-related amino acid exchange. The results of about 2-million time step calculations have shown that the globular domains of models 1 and 2 are stable, whereas, for model 3, we observe a partial unfolding and reorganization of the protein structure by insertion of the disease-related mutation Glu200→Lys.

Restriction of viral replication by mutation of the influenza virus matrix protein.

The matrix protein (M1) of influenza virus plays an essential role in viral assembly and has a variety of functions, including association with influenza virus ribonucleoprotein (RNP). Our previous studies show that the association of M1 with viral RNA and nucleoprotein not only promotes formation of helical RNP but also is required for export of RNP from the nucleus during viral replication. The RNA-binding domains of M1 have been mapped to two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105, which is also involved in RNP-binding activity. To further understand the role of the RNP-binding domain of M1 in viral assembly and replication, mutations in the coding sequences of RKLKR and the zinc finger motif of M1 were constructed using a PCR technique and introduced into wild-type influenza virus by reverse genetics. Altering the zinc finger motif of M1 only slightly affected viral growth. Substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR resulted in a lethal mutation. These results indicate that the RKLKR domain of M1 protein plays an important role in viral replication.

The carp muscle-specific sub-isoenzymes of creatine kinase form distinct dimers at different temperatures.

We have previously cloned three muscle-specific sub-isoforms of creatine kinase (CK, EC 2.7.3.2) from the common carp ( Cyprinus carpio ), designated M1-CK, M2-CK, and M3-CK. The enzyme has a key role in maintaining the energy homoeostasis of cells with fluctuating energy requirements. In the present paper, we report that all three M-CKs in the red and white muscle of different temperature-acclimatized carp were ubiquitously distributed in the cytosol and along membranes. In addition, the expression levels of these isoforms were not significantly altered in response to the temperature acclimatization. Interestingly, our studies showed that the formation of distinct homo- or heterodimers among these three M-CKs was found at various temperatures. At higher temperature, the M1M1-CK and M2M2-CK homodimers, and the M1M3-CK heterodimer are the predominant MM-CKs, whereas the M3M3-CK achieves its homodimeric state at lower temperature. We postulated testable homology models to investigate the chemical properties of these dimeric interfaces. M1M1-CK was used as a reference to compare the structural differences with the M3M3-CK dimer. The calculated solvent accessible surface area that was buried in the contact interfaces of the M1M1-CK and M3M3-CK dimers showed an overall decrease of 12% in the M3M3-CK interface. The modelling analysis also suggested a net decrease of twelve hydrophobic residues and a Phe(3)-->Lys substitution in the M3M3-CK interface. An increase in thermolability of the M3M3-CK homodimer might be due to the decrease in subunit ion pairs and buried surface area in this dimer. Based on our findings, we propose that the carp-muscle-specific CK isoenzymes could undergo shuffling to form distinct M-CK homo- or heterodimers in acclimatization to environmental temperatures.

A Leu–Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1–8)–magainin 2 (1–12) hybrid peptide (CA–MA). In particular, CA–MA analogue P5 (P5), designed by flexible region (GIG→P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.

A Gnotobiotic Transgenic Mouse Model for Studying Interactions between Small Intestinal Enterocytes and Intraepithelial Lymphocytes

The mouse intestinal epithelium undergoes continuous renewal throughout life. Intraepithelial lymphocytes (IELs) represent a significant fraction of this epithelium and play an important role in intestinal mucosal barrier function. We have generated a germ-free transgenic mouse model to examine the effects of a genetically engineered proliferative abnormality in the principal epithelial cell lineage (enterocytes) on IEL census and on IEL-enterocytic cross-talk. SV40 large T antigen (TAg(Wt)) or a mutant derivative (TAg(K107/8)) that does not bind pRB was expressed in small intestinal villus enterocytes under the control of elements from the intestinal fatty acid binding protein gene (Fabpi). Quantitative immunohistochemical and flow cytometric analyses of conventionally raised and germ-free FVB/N Fabpi-TAg(Wt), Fabpi-TAg(K107/8), and nontransgenic mice disclosed that forced reentry of enterocytes into the cell cycle is accompanied by an influx of thymically educated alphabeta T cell receptor (TCR)(+) CD4(+) and alphabeta TCR(+) CD8alphabeta(+) IELs and a decrease in intestinally derived gammadelta TCR(+) CD8alphaalpha IELs. Real time quantitative reverse transcriptase-PCR studies of jejunal villus epithelium recovered from germ-free transgenic and normal mice by laser capture microdissection and gammadelta TCR(+) jejunal IELs purified by flow cytometry disclosed that the proliferative abnormality is accompanied by decreased expression of enterocytic interleukin-7 as well as IEL interleukin-7Ralpha and transforming growth factor beta3. The analysis also revealed that normal villus epithelium expresses Fms-like tyrosine kinase 3 (Flt3), a known regulator of hematopoietic stem cell proliferation and neuronal cell survival, and its ligand (Flt3L). Epithelial expression of this receptor and its ligand is reduced by the proliferative abnormality, whereas IEL expression of Flt3L remains constant. Together, these findings demonstrate that changes in the proliferative status of the intestinal epithelium affects maturation of gammadelta TCR(+) IELs and produces an influx of alphabeta TCR(+) IELs even in the absence of a microflora.

Muscarinic toxin-like proteins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene organization.

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37-Ala38 in BM8; Lys37-Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchR-binding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with alpha-neurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the protein-coding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.

In vivo evaluation of 188Re-labeled alpha-melanocyte stimulating hormone peptide analogs for melanoma therapy.

The purpose of our study was to optimize melanoma tumor uptake of 188Re-CCMSH and reduce its nonspecific kidney retention. Nephrotoxicity is often a serious problem associated with targeted radiotherapy, therefore, increasing the tumor/kidney uptake ratio of 188Re-CCMSH is crucial for optimizing its therapeutic efficacy. Structural modification of the peptide and amino acid co-infusion were investigated as strategies to improve the tumor/kidney uptake ratio of 188Re-CCMSH. The substitution of Lys11 with Arg11 was examined to determine if removal of lysine from the peptide would improve kidney clearance without sacrificing tumor uptake. The pharmacokinetics of 188Re-CCMSH and 188Re-(Arg(11))CCMSH were determined in B16/F1 murine melanoma-bearing C57 mice. Tumor uptake values of (188)Re-CCMSH and 188Re-(Arg(11))CCMSH were 15.03 +/- 5.20% ID/g and 20.44 +/- 1.91% ID/g at 1 hr postinjection and 1.94 +/- 0.47% ID/g and 3.50 +/- 2.32% ID/g at 24 hr postinjection. Renal retention of 188Re-(Arg(11))CCMSH was 11.79 +/- 1.29 ID/g and 3.67 +/- 0.51 ID/g at 1 hr and 4 hr postinjection, which was a greater than 50% reduction compared to 188Re-CCMSH. The Arg for Lys substitution in 188Re-(Arg(11))CCMSH resulted in improved tumor uptake and reduced kidney retention. Renal retention of both 188Re-CCMSH and 188Re-(Arg(11))CCMSH were significantly reduced by co-injection of 20 mg of L-lysine, L-arginine and a combination of L-lysine:L-arginine. Tumor/kidney uptake values for 188Re-CCMSH and 188Re-(Arg(11))CCMSH were maximally reduced by 52.9% and 46.3%, respectively. However, even with amino acid co-injection, the tumor/kidney ratio of 188Re-CCMSH was lower than that of 188Re-(Arg(11))CCMSH. Improved tumor uptake and reduced kidney retention of 188Re-(Arg(11))CCMSH will facilitate targeted irradiation of melanoma tumors while minimizing the dose to the kidneys.