The origin of sickle cell alleles in Israel

Summary Molecular genetic studies were undertaken to determine the source of chromosomes carrying the sickle cell allele in Israeli patients. Analysis of restriction fragment length polymorphism (RFLP) patterns (haplotypes) along the -globin gene cluster was performed on 31 sickle chromosomes obtained from 10 unrelated families living in Israel. One is a Caucasian Jewish family, recently found to be carrying the sickle allele, and the other 9 are Arab families of various communities. The Jewish family, previously noted not to carry African red blood cell markers, was discovered to have the most common African haplotype of the -globin gene cluster, Benin. Similarly, 8 of the Arab families were also found to carry the Benin haplotype, whereas the ninth has the CAR (Central African Republic or Bantu) haplotype. The results suggest that sickle alleles in Israel originated in Africa, probably in two different regions, and migrated north into Arab and Jewish populations.     

Primaquine-induced superoxide production by beta-thalassemic red blood cells.

Primaquine, a prooxidant antimalarial drug, incubated with human red blood cells (RBC) induced marked superoxide generation in the cells as detected by exogenous cytochrome c reduction. In the presence of primaquine, beta-thalassemic RBC produced significantly more superoxide than normal RBC, thus reflecting the vulnerability of beta-thalassemic cells to oxidative stress.     

Demonstration of fluorescent X and Y bodies after antibody- and complement-mediated cytotoxicity

A technique for fluorescence staining of X and Y bodies (sex chromatin) after antibody- and complement-mediated cytotoxicity test has been developed. Cytotoxicity was quantitated by staining the dead cells with trypan blue (dye exclusion test). X bodies (Barr bodies) of human female fibroblast (stained with acridine orange) were observed in about 40 percent of the cells which survived cytotoxicity. Y bodies were studied on human male fibroblasts and in a hamster/human hybrid line which retained the human Y chromosome only. Fluorescent Y body was detectable in from 50 to 60 percent of the cells which survived the serological test. The double staining procedure did not significantly affect the proportion of killed (trypan blue-positive) cells. We suggest that this is a useful method for the detection of cytotoxic antibodies against the products of X and Y chromosomes, especially when mixed cell populations-such as tumor, sex chromosome mosaics, sperm, and artificially mixed human male and female cell lines-are tested.     

Functional atlas of the integrin adhesome

A detailed depiction of the 'integrin adhesome', consisting of a complex network of 156 components linked together and modified by 690 interactions is presented. Different views of the network reveal several functional 'subnets' that are involved in switching on or off many of the molecular interactions within the network, consequently affecting cell adhesion, migration and cytoskeletal organization. Examination of the adhesome network motifs reveals a relatively small number of key motifs, dominated by three-component complexes in which a scaffolding molecule recruits both a signalling molecule and its downstream target. We discuss the role of the different network modules in regulating the structural and signalling functions of cell-matrix adhesions.     

The interplay between pH sensitivity and label-free protein detection in immunologically modified nano-scaled field-effect transistor

We present experimental results in order to establish a correlation between pH sensitivity of immunologically modified nano-scaled field-effect transistor (NS-ImmunoFET) with their sensing capacity for label-free detection. The NS-ImmunoFETs are fabricated from silicon-on-insulator (SOI) wafers and are fully-depleted with thickness of ~20 nm. The data shows that higher sensitivity to pH entails enhanced sensitivity to analyte detection. This suggests that the mechanism of analyte detection as pure electrostatic perturbation induced by antibody-analyte interaction is over simplified. The fundamental assumption, in existing models for field-effect sensing mechanism assumes that the analyte molecules do not directly interact with the surface but rather stand 'deep' in the solution and away from the dielectric surface. Recent studies clearly provide contradicting evidence demonstrating that antibodies lie down flat on the surface. These observations led us to propose that the proteins that cover the gate area intimately interact with active sites on the surface thus forming a network of interacting sites. Since sensitivity to pH is directly correlated with the amount of amphoteric sites, we witness a direct correlation between sensitivity to pH and analyte detection. The highest and lowest threshold voltage shift for a label-free and specific detection of 6.5 nM IgG were 40 mV and 2.3 mV for NS-ImmunoFETs with pH sensitivity of 35 mV/decade and 15 mV/decade, respectively. Finally, physical modeling of the NS-ImmunoFET is presented and charge of a single IgG protein at pH 6 is calculated. The obtained value is consistent with charge of IgG protein cited in literature.     

A comparative study of backbone versus side chain peptide cyclization: application for HIV-1 integrase inhibitors

Peptide cyclization is an important tool for overcoming the limitations of linear peptides as drugs. Backbone cyclization (BC) has advantages over side chain (SC) cyclization because it combines N-alkylation for extra peptide stability. However, the appropriate building blocks for BC are not yet commercially available. This problem can be overcome by preparing SC cyclic peptide analogs of the most active BC peptide using commercially available building blocks. We have recently developed BC peptides that inhibit the HIV-1 integrase enzyme (IN) activity and HIV-1 replication in infected cells. Here we used this system as a model for systematically comparing the BC and SC cyclization modes using biophysical, biochemical and structural methods. The most potent SC cyclic peptide was active almost as the BC peptide and inhibited IN activity in vitro and blocked IN activity in cells even after 6 days. We conclude that both cyclization types have their respective advantages: The BC peptide is more active and stable, probably due to the N-alkylation, while SC cyclic peptides are easier to synthesize. Due to the high costs and efforts involved in preparing BC peptides, SC may be a more approachable method in many cases. We suggest that both methods are interchangeable.     

Consequences of N-acylation on structure and membrane binding properties of dermaseptin derivative K4-S4-(1-13)

Acyl conjugation to antimicrobial peptides is known to enhance antimicrobial properties. Here, we investigated the consequences of aminolauryl (NC(12)) conjugation to the dermaseptin derivative K(4)-S4-(1-13) (P) on binding properties to bilayer models mimicking bacterial plasma membrane, which is often cited as the ultimate site of action. Isothermal titration calorimetry revealed that acylation was responsible for enhancing the binding affinity of NC(12)-P compared with P (K = 13 x 10(5) and 1.5 x 10(5) m(-1), respectively). Surface plasmon resonance measurements confirmed the isothermal titration calorimetry results (K(app) = 12.6 x 10(5) and 1.53 x 10(5) m(-1), respectively) and further indicated that enhanced adhesion affinity (K(adhesion) = 3 x 10(5) and 1 x 10(5) m(-1), respectively) was coupled to enhanced tendency to insert within the bilayer (K(insertion) = 4.5 and 1.5, respectively). To gain insight into the molecular basis for these observations, we investigated the three-dimensional structures in the presence of dodecylphosphocholine using NMR. The ensemble of NMR-calculated structures (backbone root mean square deviation <0.6 A) showed that the acyl moiety was responsible for a significant molecular reorganization, possibly affecting the electrostatic potential distribution in NC(12)-P relative to that of P. The combined data present compelling evidence in support of the hypothesis that N-acylation affects antimicrobial properties by modifying the secondary structure of the peptide in a manner that facilitates contact with the membrane and consequently increases its disruption.     

Growth hormone receptor sequence changes do not play a role in determining height in children with idiopathic short stature

BACKGROUND/AIMS: In children with short stature, in whom growth hormone deficiency has been excluded, the presence of a normal or elevated growth hormone concentration concomitant with low insulin-like growth factor I suggests growth hormone insensitivity (GHI). Previous reports suggest that heterozygous mutations in the growth hormone receptor gene (GHR) may account for about 5% of children with idiopathic short stature (ISS). In the present study we have attempted to determine whether mutations in the GHR explain the short stature and growth retardation in a cohort of children with ISS and characteristics suggesting GHI. METHODS: For the present study 33 children with clinical and biochemical characteristics of GHI were selected from a cohort of 150 children of short stature. Molecular analysis of the GHR was performed using a single-strand conformation polymorphism technique and sequencing. Ten different sequence changes in 19 (58%) out of 33 children were identified, 9 of them novel and 1 that had been described previously. RESULTS: Two changes were found in exons 2 and 6. The known polymorphism of exon 6 (G168) was significantly more common in the control subjects than in our study group (63.5 vs. 30%; p < 0.0001). In the intronic sequences 8 previously undescribed DNA changes were found. The screening of the affected children's family members revealed that both normal and short stature members carried the same variants. The study group did not significantly differ from the controls in retention (GHRfl) or exclusion (GHRd3) of exon 3. CONCLUSION: Our study suggests that sequence changes of the GHR are common in children with ISS. The presence of these sequence changes in the control subjects as well as in normal stature family members indicates that these changes represent a simple polymorphism of the GHR. Such DNA changes are more prevalent than previously recognized, and they do not seem to play a contributory role in the etiology of short stature.     

Identification of a Feline Leukemia Virus Variant That Can Use THTR1, FLVCR1, and FLVCR2 for Infection

The pathogenic subgroup C feline leukemia virus (FeLV-C) arises in infected cats as a result of mutations in the envelope (Env) of the subgroup A FeLV (FeLV-A). To better understand emergence of FeLV-C and potential FeLV intermediates that may arise, we characterized FeLV Env sequences from the primary FY981 FeLV isolate previously derived from an anemic cat. Here, we report the characterization of the novel FY981 FeLV Env that is highly related to FeLV-A Env but whose variable region A (VRA) receptor recognition sequence partially resembles the VRA sequence from the prototypical FeLV-C/Sarma Env. Pseudotype viruses bearing FY981 Env were capable of infecting feline, human, and guinea pig cells, suggestive of a subgroup C phenotype, but also infected porcine ST-IOWA cells that are normally resistant to FeLV-C and to FeLV-A. Analysis of the host receptor used by FY981 suggests that FY981 can use both the FeLV-C receptor FLVCR1 and the feline FeLV-A receptor THTR1 for infection. However, our results suggest that FY981 infection of ST-IOWA cells is not mediated by the porcine homologue of FLVCR1 and THTR1 but by an alternative receptor, which we have now identified as the FLVCR1-related protein FLVCR2. Together, our results suggest that FY981 FeLV uses FLVCR1, FLVCR2, and THTR1 as receptors. Our findings suggest the possibility that pathogenic FeLV-C arises in FeLV-infected cats through intermediates that are multitropic in their receptor use.Feline leukemia viruses (FeLVs) are pathogenic retroviruses of domestic cats that induce proliferative, degenerative, and immunosuppressive disorders (17, 18, 27). Infected cats often contain a mixture of FeLVs that have been categorized into subgroups A, B, and C based on their interference and in vitro host range properties (39). These subgroups have been shown to be tightly associated with distinct feline diseases (17, 27). A fourth subgroup (T) that is highly related to FeLV-A has also been reported (26) and has been shown to be associated with immune deficiency (30). FeLV-A is the primary strain that is transmitted between cats and is the progenitor virus from which other subgroups arise. FeLV-B arises by recombination between endogenous retroviral sequences present in the cat genome and the gene encoding the FeLV-A envelope (Env) protein (32, 40, 42) that is responsible for host cell surface receptor recognition. FeLV-C and FeLV-T are formed by mutations in the FeLV-A Env gene (10, 27, 38). Env recombination and mutations are a major determinant of the host receptors used by the different FeLV subgroups (3, 23, 34, 46, 47).The emergence of pathogenic FeLV-C in cats infected with FeLV-A provides a classic example of Env mutations that switch the host receptor used for infection, leading to a fatal pathogenic disease in the host. The emergence of FeLV-C is tightly associated with red blood cell aplasia, a fatal feline anemia characterized by a specific disruption in erythroid progenitor cell development (2, 12, 19, 28). Moreover, FeLV-C emergence coincides with a switch in the host receptor used for infection from the thiamine transporter THTR1 (FeLV-A receptor) (23) to the heme exporter FLVCR1 (FeLV-C receptor) (34, 35, 46). Previous studies have suggested that the feline anemia is caused by the FeLV-C Env protein binding to, and disrupting, the cellular function of FLVCR1 (1, 34, 46). Indeed, expression of FeLV-C Env in hematopoietic stem cells (36) or disruption of FLVCR1 (21, 35) specifically disrupts early erythropoiesis, which mimics the anemia observed in cats with FeLV-C. Interestingly, the emergence of FeLV-C from FeLV-A is analogous to the emergence of cytopathic X4 strains of human immunodeficiency virus type 1 (HIV-1) in individuals infected with the R5 strain of HIV-1. Analogous to the emergence of FeLV-C, X4 HIV-1 arises by Env mutations in the R5 HIV-1 strain, which leads to a switch in the coreceptor used for infection allowing an expansion in HIV-1 cell tropism and subsequently to accelerated AIDS (4, 8). However, whereas HIV-1 pathogenesis also involves emergence of variants or X4/R5 intermediates that can use multiple related coreceptors for infection (5, 8, 11), the mechanism of how FeLV-C emerges, in terms of the presence of FeLV-A/FeLV-C intermediates, has yet to be elucidated.To better understand the emergence of pathogenic FeLV-C in infected cats and potential FeLV variants/intermediates that may arise, we isolated and characterized FeLV Env sequences from a primary FeLV isolate derived from a cat with pure red cell aplasia. In this study, we report the isolation and characterization of the novel FY981 Env, a hybrid FeLV-A/FeLV-C Env that, when pseudotyped, can use FLVCR1, THTR1, and the FLVCR1-related FLVCR2 for infection. Our findings suggest that the FY981 Env could represent a potential FeLV intermediate that arises during the emergence of pathogenic FeLV-C.