Deletion Of Glutamic Acid Research Articles

Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime–avibactam due to a deletion in the blaKPC3 gene

ObjectivesCarbapenemase-producing strains of Klebsiella pneumoniae (KPC) are a great health concern, and therapy with ceftazidime–avibactam represents a choice for the treatment of infections involving these strains. We report a strain resistant to ceftazidime–avibactam due to a deletion of six nucleotides in the blaKPC gene sequence. MethodsTwo strains, namely AMP920 and AMP2009, were isolated from the same patient a month apart. Antimicrobial susceptibility testing was performed both by broth microdilution and by Etest. Immunoenzymatic assay to detect carbapenemase was performed for both strains. The blaKPC gene of both strains was amplified by PCR and sequenced. Enzyme activity towards carbapenems was tested by the CarbaNP test and hydrolysis spectrophotometer assay. ResultsThe two isolates differed in antimicrobial susceptibility. AMP920 showed meropenem and imipenem resistance (MIC 32 and 32 mg/mL). A month later the carbapenem MIC decreased to 8 and 1 mg/mL respectively, while the ceftazidime–avibactam MIC increased from 1 to 16 mg/mL. Both isolates showed a positive immunoenzymatic test for the KPC enzyme, but only AMP920 showed a positive CarbaNP test hydrolysing imipenem. The BlaKPC gene was amplified in both strains. After sequencing, the two amplicons showed a KPC3 variant. The gene of the second isolate showed a deletion of six nucleotides at 498–503, resulting in a mutant variant with the deletion of glutamic acid and leucine residues at positions 167 and 168. ConclusionsWe detected a new deletion in the blaKPC gene of a clinical strain of K. pneumoniae which resulted in resistance to ceftazidime–avibactam. The amino acids deleted are in the Ω loop (amino acids 165–179) of the KPC enzyme, enhancing ceftazidime affinity and preventing avibactam binding.

Effect of plants and their root exudate on bacterial activities during rhizobacterium–plant remediation of phenol from water

We investigated remediation of phenol from water using microbe–plant partnerships. Co-introduction of maize seedlings, Pseudomonas fluorescens rifampicin-resistant P13 and P. stutzeri P7 carrying self-transmissible TOL-like plasmids reduced phenol content in water at lower phenol concentrations (25, 50, and 75 mg/L), similar to individual introduction of the bacteria. Co-introduction of plants and bacteria significantly reduced phenol content in water at higher phenol concentrations (100, 125, and 150 mg/L) compared to using individual introduction of the bacteria. Moreover, TOL-like plasmids were transferred from P7 to P13. Addition of plants promoted the growth of both strains, leading to increased plasmid transfer. At higher phenol concentrations, addition of plants resulted in increases of catechol 2, 3-dioxygenase (C23O) activity and reduction in level of reactive oxygen species (ROS) of bacteria in the degradation experiments. Increased plasmid transfer and C23O activity and reduction in ROS level might be the major reasons why plants promote bacterial degradation of phenol at higher phenol concentrations. Furthermore, root exudate of maize seedlings and artificial root exudate (ARE) constructed using major components of the root exudate had the same effects on bacterial activities. Unlike the ARE, deletion of glucose, arabinose, or fructose or all the monosaccharides from ARE resulted in no increase in numbers of both strains and in plasmid transfer. At the higher phenol concentrations, deletion of glutamic acid, aspartic acid, alanine, or glycine or all the amino acids did not stimulate bacterial C23O activity. Deletion of fumaric, oxaloacetic or citric acids still reduced bacterial ROS level as ARE did, but, deletion of all the organic acids or DIMBOA, a hydroxamic acid, did not reduce bacterial ROS level as ARE did. The data showed that each monosaccharide might be important for sufficient numbers of plant-associated bacteria and increased plasmid transfer while each amino acid might be important for maintaining bacterial C23O activity and that DIMBOA might be responsible for the decrease in ROS levels. These results are the basis for efficient remediation of phenol from water by microbe–plant partnerships and further studies on the mechanism of rhizobacterium-plant interaction.

The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress.

BackgroundDystonia1 (DYT1) dystonia is caused by a glutamic acid deletion (ΔE) mutation in the gene encoding Torsin A in humans (HTorA). To investigate the unknown molecular and cellular mechanisms underlying DYT1 dystonia, we performed an unbiased proteomic analysis.ResultsWe found that the amount of proteins and transcripts of an Endoplasmic reticulum (ER) resident chaperone Heat shock protein cognate 3 (HSC3) and a mitochondria chaperone Heat Shock Protein 22 (HSP22) were significantly increased in the HTorAΔE– expressing brains compared to the normal HTorA (HTorAWT) expressing brains. The physiological consequences included an increased susceptibility to oxidative and ER stress compared to normal HTorAWT flies. The alteration of transcripts of Inositol-requiring enzyme-1 (IRE1)-dependent spliced X box binding protein 1(Xbp1), several ER chaperones, a nucleotide exchange factor, Autophagy related protein 8b (ATG8b) and components of the ER associated degradation (ERAD) pathway and increased expression of the Xbp1-enhanced Green Fluorescence Protein (eGFP) in HTorAΔE brains strongly indicated the activation of the unfolded protein response (UPR). In addition, perturbed expression of the UPR sensors and inducers in the HTorAΔEDrosophila brains resulted in a significantly reduced life span of the flies. Furthermore, the types and quantities of proteins present in the anti-HSC3 positive microsomes in the HTorAΔE brains were different from those of the HTorAWT brains.ConclusionTaken together, these data show that HTorAΔE in Drosophila brains may activate the UPR and increase the expression of HSP22 to compensate for the toxic effects caused by HTorAΔE in the brains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1518-0) contains supplementary material, which is available to authorized users.

CYP21A2 p.E238 Deletion as Result of Multiple Microconversion Events

More than 90% of congenital adrenal hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macroconversion or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbor rare mutations that do not originate from the pseudogene. A complete list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.imm.Ki.se/CYPalleles/cyp21.htm). In this report, we describe clinical and genetic findings regarding an Italian woman suffering from a classic salt-wasting form of CAH due to a severe 21-hydroxylase deficiency. A complex genetic family study was performed including a prenatal diagnosis. The patient was found to be heterozygous for p.I172N (exon 4), p.E238del (exon 6), p.M239K (exon 6), and p.F306insT (exon 7) mutations and homozygous for p.I236N (exon 6) and p.V237E (exon 6) mutations. The deletion of glutamic acid 238 is a new mutation not reported before in the literature. CYP21A2 genotyping has become a valuable complement to biochemical CAH investigation. We highlight the contribution of molecular genetic advancements to the clinical management of patients with 21-hydroxylase deficiency.

DYT1 Knock-In Mice Are Not Sensitized against Mitochondrial Complex-II Inhibition

DYT1 is caused by a partly penetrant dominant mutation in TOR1A that leads to a glutamic acid deletion (ΔE) in torsinA. Identifying environmental factors that modulate disease pathogenesis and penetrance could help design therapeutic strategies for dystonia. Several cell-based studies suggest that expression of torsinA(ΔE) increases the susceptibility of neuronal cells to challenges to their oxidative/energy metabolism. Based on those reports, we hypothesized that mice expressing torsinA(ΔE) would be more susceptible than control littermates to the effects of oxidative stress and ATP deficits caused by disruption of the mitochondrial respiratory chain in neurons. To test this hypothesis, we administered 20 or 50 mg/kg/day of the irreversible complex-II inhibitor 3-nitropropionic acid (3-NP) intraperitoneally for 15 consecutive days to young heterozygote DYT1 knock-in (KI) mice and wild type littermates. Repeated phenotypic assessments were performed at baseline, during and after the injections. Animals were then sacrificed and their brains processed for protein analysis. The administration of 20 mg/kg 3-NP led to increased levels of torsinA in the striatum, the main target of 3-NP, but did not cause motor dysfunction in DYT1 KI or control mice. The administration of 50 mg/kg/day of 3-NP caused the death of ∼40% of wild type animals. Interestingly, DYT1 KI animals showed significantly reduced mortality. Surviving animals exhibited abnormal motor behavior during and right after the injection period, but recovered by 4 weeks postinjection independent of genotype. In contrast to the findings reported in cultured cells, these studies suggest the DYT1 mutation does not sensitize central neurons against the toxic effects of oxidative stress and energy deficits.

Static retention of the lumenal monotopic membrane protein torsinA in the endoplasmic reticulum

TorsinA is a membrane-associated enzyme in the endoplasmic reticulum (ER) lumen that is mutated in DYT1 dystonia. How it remains in the ER has been unclear. We report that a hydrophobic N-terminal domain (NTD) directs static retention of torsinA within the ER by excluding it from ER exit sites, as has been previously reported for short transmembrane domains (TMDs). We show that despite the NTD's physicochemical similarity to TMDs, it does not traverse the membrane, defining torsinA as a lumenal monotopic membrane protein and requiring a new paradigm to explain retention. ER retention and membrane association are perturbed by a subset of nonconservative mutations to the NTD, suggesting that a helical structure with defined orientation in the membrane is required. TorsinA preferentially enriches in ER sheets, as might be expected for a lumenal monotopic membrane protein. We propose that the principle of membrane-based protein sorting extends to monotopic membrane proteins, and identify other proteins including the monotopic lumenal enzyme cyclooxygenase 1 (prostaglandin H synthase 1) that share this mechanism of retention with torsinA.

The Osaka FAD Mutation E22Δ Leads to the Formation of a Previously Unknown Type of Amyloid β Fibrils and Modulates Aβ Neurotoxicity

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cerebral deposition of amyloid fibrils formed by the amyloid β (Aβ) peptide. Aβ has a length of 39–43 amino acid residues; the predominant Aβ isoforms are Aβ1–40 and Aβ1–42. While the majority of AD cases occur spontaneously, a subset of early-onset familial AD cases is caused by mutations in the genes encoding the Aβ precursor protein or presenilin 1/presenilin 2. Recently, a deletion of glutamic acid at position 22 within the Aβ sequence (E22Δ) was identified in Japanese patients with familial dementia, but the aggregation properties of the deletion variant of Aβ are not well understood. We investigated the aggregation characteristics and neurotoxicity of recombinantly expressed Aβ isoforms 1–40 and 1–42 with and without the E22Δ mutation. We show that the E22Δ mutation strongly accelerates the fibril formation of Aβ1–42 E22Δ compared to Aβ1–42 wild type (wt). In addition, we demonstrate that fibrils of Aβ1–40 E22Δ form a unique quaternary structure characterized by a strong tendency to form fibrillar bundles and a strongly increased thioflavin T binding capacity. Aβ1–40 E22Δ was neurotoxic in rat primary neuron cultures as compared to nontoxic Aβ1–40 wt. Aβ1–42 E22Δ was less toxic than Aβ1–42 wt, but it significantly decreased neurite outgrowth per cell in neuronal primary cultures. Because Aβ1–40 is the major Aβ form in vivo, the gain of toxic function caused by the E22 deletion may explain the development of familial AD in mutation carriers.

Exploring the Influence of TorsinA Expression on Protein Quality Control

DYT1 dystonia is caused by a glutamic acid deletion (ΔE) in the endoplasmic reticulum (ER) protein torsinA. Previous studies suggest that torsinA modulates the aggregation of cytosolic misfolded proteins and ER stress responses, although the mechanisms underlying those effects remain unclear. In order to investigate the bases of these observations, we analyzed the interaction between torsinA expression, protein aggregation and ER stress in PC6.3 cells. Unexpectedly, we found that expression of torsinA(wt) or (ΔE) does not influence the inclusion formation by an expanded polyglutamine reporter protein in this cellular model. Furthermore, torsinA does not prevent the activation of ER stress induced by thapsigargin or the reducing agent DTT. Interestingly, DTT induces post-translational changes in torsinA, more prominently for torsinA(wt) than (ΔE). This work highlights the importance of model system selection for the study of torsinA function. Furthermore, it provides additional evidence suggesting that torsinA is sensitive to changes in the cellular redox potential.

CSN complex controls the stability of selected synaptic proteins via a torsinA-dependent process

DYT1 dystonia is caused by an autosomal dominant mutation that leads to a glutamic acid deletion in torsinA (TA), a member of the AAA+ ATPase superfamily. In this study, we identified a novel-binding partner of TA, the subunit 4 (CSN4) of CSN signalosome. TA binds CSN4 and the synaptic regulator snapin in neuroblastoma cells and in brain synaptosomes. CSN4 and TA are required for the stability of both snapin and the synaptotagmin-specific endocytic adaptor stonin 2, as downregulation of CSN4 or TA reduces the levels of both proteins. Snapin is phosphorylated by the CSN-associated kinase protein kinase D (PKD) and its expression is decreased upon PKD inhibition. In contrast, the stability of stonin 2 is regulated by neddylation, another CSN-associated activity. Overexpression of the pathological TA mutant (ΔE-TA) reduces stonin 2 expression, causing the accumulation of the calcium sensor synaptotagmin 1 on the cell surface. Retrieval of surface-stranded synaptotagmin 1 is restored by overexpression of stonin 2 in ΔE-TA-expressing cells, suggesting that the DYT1 mutation compromises the role of TA in protein stabilisation and synaptic vesicle recycling.

Transcriptional and proteomic profiling in a cellular model of DYT1 dystonia

DYT1, the most common inherited dystonia, is caused by a common dominant mutation in the TOR1A gene that leads to a glutamic acid deletion in the protein torsinA. Wild-type torsinA locates preferentially in the endoplasmic reticulum while the disease-linked mutant accumulates in the nuclear envelope. As a result, it has been proposed that DYT1 pathogenesis could result either from transcriptional dysregulation caused by abnormal interactions of mutant torsinA with nuclear envelope proteins, or from a loss of torsinA function in the endoplasmic reticulum that would impair specific neurobiological pathways. Aiming to determine whether one or both of these potential mechanisms are implicated in DYT1 pathogenesis, we completed unbiased transcriptional and proteomic profiling in well-characterized neural cell lines that inducibly express wild-type or mutant torsinA. These experiments demonstrated that the accumulation of mutant torsinA in the nuclear envelope is not sufficient to cause transcriptional dysregulation. However, we detected expression changes at the protein level that, together with other reports, suggest a potential implication of torsinA on energy metabolism and regulation of the redox state. Furthermore, several proteins identified in this study have been previously linked to other forms of dystonia. In conclusion, our results argue against the hypothesis of transcriptional dysregulation in DYT1 dystonia, suggesting potential alternative pathogenic pathways.

Familial breast cancer screening reveals an alteration in the RAP80 UIM domain that impairs DNA damage response function

Germline mutations in two major susceptibility genes, BRCA1 and BRCA2, account for nearly 20% of familial breast cancers. A majority of the remaining genetic factors involved in heritable breast cancer susceptibility are, however, unknown. Recently, a new BRCA1-interacting protein, receptor associated protein 80 (RAP80), was identified. RAP80 plays an important role in BRCA1-mediated DNA damage responses (DDRs) by recruiting BRCA1 to DNA double-strand breaks (DSBs). A comprehensive screening of DNA from affected index cases of 112 BRCA1/BRCA2 mutation-negative Finnish breast cancer families revealed altogether 10 alterations in RAP80, one of which, c.241-243delGAA, resulted in a single glutamic acid deletion at residue 81 in a highly conserved region of ubiquitin interaction motif 1. The resultant delE81 protein product displayed significantly reduced ubiquitin binding and DSB localization. Expression of the RAP80 delE81 allele impaired both BRCA1 and ABRA1 DSB recruitment, thus compromising BRCA1-mediated DDR signaling. Compared with wild-type RAP80, expression of the delE81 allele was associated with a significant increase in cytogenetically detectable chromosomal aberrations, particularly chromatid breaks. Although evidently quite rare, these results suggest that critical constitutional mutations in RAP80 abrogate DDR function and may be involved in genetic predisposition to cancer.

DeltaFY mutation in human torsin A [corrected] induces locomotor disability and abberant synaptic structures in Drosophila.

We investigate the molecular and cellular etiologies that underlie the deletion of the six amino acid residues (DeltaF323-Y328; DeltaFY) in human torsin A (HtorA). The most common and severe mutation involved with early-onset torsion dystonia is a glutamic acid deletion (DeltaE 302/303; DeltaE) in HtorA which induces protein aggregates in neurons and cells. Even though DeltaFY HtorA forms no protein clusters, flies expressing DeltaFY HtorA in neurons or muscles manifested a similar but delayed onset of adult locomotor disability compared with flies expressing DeltaE in HtorA. In addition, flies expressing DeltaFY HtorA had fewer aberrant ultrastructures at synapses compared with flies expressing DeltaE HtorA. Taken together, the DeltaFY mutation in HtorA may be responsible for behavioral and anatomical aberrations in gDrosophila.

Consequences of the DYT1 mutation on torsinA oligomerization and degradation

DYT1 is the most common inherited dystonia, a neurological syndrome that causes disabling involuntary muscle contractions. This autosomal dominant disease is caused by a glutamic acid deletion near the carboxy-terminus in the protein torsinA. Cell- and animal-based studies have shown how the DYT1 mutation causes mutant torsinA to redistribute from the endoplasmic reticulum to the nuclear envelope, acting through a dominant negative effect over the wild type protein. As a result, the wild type:mutant torsinA expression ratio would be important for disease pathogenesis, and events that influence it, such as a differential degradation process for each protein, might modulate DYT1 pathobiology. The DYT1 mutation also triggers the formation of abnormal intermolecular disulfide bonds in torsinA, although the significance of this finding is unclear. How the protein quality control machinery handles torsinA, and whether this process is affected by its abnormal oligomerization remain unknown. Here, we first explored how the disease-linked mutation influences the catabolic process of human torsinA, demonstrating that the differences in subcellular localization between both forms of torsinA lead to divergences in their degradation pathways and, whereas torsinA is normally recycled through autophagy, the proteasome is also required for the efficient clearance of the mutated form. Subsequently, we determined that the abnormal disulfide bond-dependent oligomerization of mutant torsinA is not a result of its redistribution to the nuclear envelope, but a direct consequence of the mutation. Finally, we established that the presence of disulfide links in mutant torsinA oligomers interfere with their degradation by the proteasome, thus relying on autophagy as the main pathway for clearance. In conclusion, the abnormal subcellular localization and oligomerization of DYT1-linked torsinA influences its catabolic process, opening the door to the modulation of the wild type:mutant torsinA ratio through pharmacological manipulation of protein degradation pathways.

The mutant leucine-zipper domain impairs both dimerization and suppressive function of Foxp3 in T cells.

Regulatory T cells that express the Foxp3 transcription factor play important roles in preventing autoimmune diseases. Although several studies have demonstrated that the lack of the forkhead DNA-binding domain of Foxp3 caused severe autoimmune disease in scurfy mutant mice, the other functional domains of Foxp3 are less well characterized. Here, we show that the deletion of glutamic acid (DeltaE250) in the leucine-zipper domain of Foxp3 causes a loss of hyporesponsiveness when compared with wild-type Foxp3 upon antigenic stimulation. CD4 T cells that ectopically express the glutamic acid mutant show significant losses of suppressor activity both in vitro and in vivo. We also demonstrate that regulation of both Th1- and Th2-type cytokine secretion in CD4 T cells that express wild-type Foxp3 is significantly altered by the deletion of glutamic acid. Defects are also observed in the expression of adhesion molecules, such as l-selectin (CD62L) and CD103, suggesting an important role of glutamic acid in the migratory behavior of regulatory T cells. Finally, this mutation reduces transcriptional repressor activity and impairs the homodimerization of Foxp3. Taken together, our results provide insight into the mechanism that controls autoimmune diseases via the deletion of this single glutamic acid residue in the leucine-zipper domain of Foxp3.

The AAA+ protein torsinA interacts with a conserved domain present in LAP1 and a novel ER protein

A glutamic acid deletion (ΔE) in the AAA+ protein torsinA causes DYT1 dystonia. Although the majority of torsinA resides within the endoplasmic reticulum (ER), torsinA binds a substrate in the lumen of the nuclear envelope (NE), and the ΔE mutation enhances this interaction. Using a novel cell-based screen, we identify lamina-associated polypeptide 1 (LAP1) as a torsinA-interacting protein. LAP1 may be a torsinA substrate, as expression of the isolated lumenal domain of LAP1 inhibits the NE localization of “substrate trap” EQ-torsinA and EQ-torsinA coimmunoprecipitates with LAP1 to a greater extent than wild-type torsinA. Furthermore, we identify a novel transmembrane protein, lumenal domain like LAP1 (LULL1), which also appears to interact with torsinA. Interestingly, LULL1 resides in the main ER. Consequently, torsinA interacts directly or indirectly with a novel class of transmembrane proteins that are localized in different subdomains of the ER system, either or both of which may play a role in the pathogenesis of DYT1 dystonia.

Synthetic peptide derived from alpha-fetoprotein inhibits growth of human breast cancer: investigation of the pharmacophore and synthesis optimization.

Asynthetic peptide that inhibits the growth of estrogen receptor positive (ER+) human breast cancers, growing as xenografts in mice, has been reported. The cyclic 9-mer peptide, cyclo[EMTOVNOGQ], is derived from alpha-fetoprotein (AFP), a safe, naturally occurring human protein produced during pregnancy, which itself has anti-estrogenic and anti-breast cancer activity. To determine the pharmacophore of the peptide, a series of analogs was prepared using solid-phase peptide synthesis. Analogs were screened in a 1-day bioassay, which assessed their ability to inhibit the estrogen-stimulated growth of uterus in immature mice. Deletion of glutamic acid, Glu1, abolished activity of the peptide, but glutamine (Gln) or asparagine (Asn) could be substituted for Glu1 without loss of activity. Methionine (Met2) was replaced with lysine (Lys) or tyrosine (Tyr) with retention of activity. Substitution of Lys for Met2 in the cyclic molecule resulted in a compound with activity comparable with the Met2-containing cyclic molecule, but with a greater than twofold increase in purity and corresponding increase in yield. This Lys analog demonstrated anti-breast cancer activity equivalent to that of the original Met-containing peptide. Therefore, Met2 is not essential for biologic activity and substitution of Lys is synthetically advantageous. Threonine (Thr3) is a nonessential site, and can be substituted with serine (Ser), valine (Val), or alanine (Ala) without significant loss of activity. Hydroxyproline (Hyp), substituted in place of the naturally occurring prolines (Pro4, Pro7), allowed retention of activity and increased stability of the peptide during storage. Replacement of the first Pro (Pro4) with Ser maintains the activity of the peptide, but substitution of Ser for the second Pro (Pro7) abolishes the activity of the peptide. This suggests that the imino acid at residue 7 is important for conformation of the peptide, and the backbone atoms are part of the pharmacophore, but Pro4 is not essential. Valine (Val5) can be substituted only with branched-chain amino acids (isoleucine, leucine or Thr); replacement by d-valine or Ala resulted in loss of biologic activity. Thus, for this site, the bulky branched side chain is essential. Asparagine (Asn6) is essential for activity. Substitution with Gln or aspartic acid (Asp), resulted in reduction of biologic activity. Removal of glycine (Gly8) resulted in a loss of activity but nonconservative substitutions can be made at this site without a loss of activity indicating that it is not part of the pharmacophore. Cyclization of the peptide is facilitated by addition of Gln9, but this residue does not occur in AFP nor is it necessary for activity. Gln9 can be replaced with Asn, resulting in a molecule with similar activity. These data indicate that the pharmacophore of the peptide includes side chains of Val5 and Asn6 and backbone atoms contributed by Thr3, Val5, Asn6, Hyp7 and Gly8. Met2 and Gln9 can be modified or replaced. Glu1 can be replaced with charged amino acids, and is not likely to be part of the binding site of the peptide. The results of this study provide information that will be helpful in the rational modification of cyclo[EMTOVNOGQ] to yield peptide analogs and peptidomimetics with advantages in synthesis, pharmacologic properties, and biologic activity.

A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction.

The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37 degrees C for 1 hour, (125)I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas (125)I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the alpha-helix content of lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%) of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I Nichinan and normal r-proapoA-I showed similar particle size. To study cholesterol efflux, human skin fibroblasts were labeled with [(3)H]cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex. Fractional cholesterol efflux from [(3)H]cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation. Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I. Our data demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts.

Extensive analysis of 40 infertile patients with congenital absence of the vas deferens: in 50% of cases only one CFTR allele could be detected

Mutations in the cystic fibrosis (CF) conductance transmembrane regulator (CFTR) gene have been detected in patients with CF and in males with infertility attributable to congenital bilateral absence of the vas deferens (CBAVD). Thirty individuals with CBAVD and 10 with congenital unilateral absence of the vas deferens (CUAVD) were analyzed by single-strand conformation analysis and denaturing gradient gel electrophoresis for mutations in most of the CFTR gene. All 40 individuals were pancreatic sufficient, but twenty patients had recurrent or sporadic respiratory infections, asthma/asthmatic bronchitis, and/or rhino-sinusitis. Agenesia or displasia of one or both seminal vesicles was detected in 30 men and other urogenital malformations were present in six subjects. Among the 40 samples, we identified 13 different CFTR mutations, two of which were previously unknown. One new mutation in exon 4 was the deletion of glutamic acid at codon 115 (delta E115). A second new mutation was found in exon 17b, viz., an A --> C substitution at position 3311, changing lysine to threonine at codon 1060 (K1060T). CFTR mutations were detected in 22 out of 30 (73.3%) CBAVD patients and in one out of 10 (10%) CUAVD individuals, showing a significantly lower incidence of CFTR mutations in CBAVD/CUAVD patients (P << 0.0001), compared with that found in the CF patient population. Only three CBAVD patients were found with more than one CFTR mutation (delta F508/L206W, delta F508/R74W + D1270N, R117H/712-1G --> T), highlighting L206W, R74W/D1270N, and R117H as benign CF mutations. Sweat electrolyte values were increased in 76.6% of CBAVD patients, but three individuals without CFTR mutations had normal sweat electrolyte levels (10% of the total CBAVD patients), suggesting that factors other than CFTR mutations are involved in CBAVD. The failure to identify a second mutation in exons and their flanking regions of the CFTR gene suggests that these mutations could be located in introns or in the promoter region of CFTR. Such mutations could result in CFTR levels below the minimum 6%-10% necessary for normal protein function.

Opossum ( Didelphis virginiana) “little” and “big” gastrins

1. 1. “Little” gastrin from most mammalian species are 17 amino acid peptides and the precursor “big” gastrins are 34 amino acid peptides. 2. 2. “Little” gastrins of the New World hystricomorphs, guinea-pig and chinchilla, are 16 amino acid peptides due to deletion of a glutamic acid in the region 6–9 from their NH 2-terminus and the corresponding “big” gastrins are 33 amino acid peptides. 3. 3. Antral gastrins from the opossum, a New World marsupial, have a glutamic acid deletion in the same region as the hystricomorph gastrins. 4. 4. Opossum “big” gastrin is a 33 amino acid peptide with the following sequence: 〈ELGPQDLPYLTADLSKKQGPWLEEEEAYGWMDF#.