Clinical detection of Hepatitis C viral infection by yeast-secreted HCV-core:Gold-binding-peptide.

Access to affordable and field deployable diagnostics are key barriers to the control and eradication of many endemic and emerging infectious diseases. While cost, accuracy, and usability have all improved in recent years, there remains a pressing need for even less expensive and more scalable technologies. To that end, we explored new methods to inexpensively produce and couple protein-based biosensing molecules (affinity reagents) with scalable electrochemical sensors. Previous whole-cell constructs resulted in confounding measurements in clinical testing due to significant cross-reactivity when probing for host-immune (antibody) response to infection. To address this, we developed two complimentary strategies based on either the release of surface displayed or secretion of fusion proteins. These dual affinity biosensing elements couple antibody recognition (using antigen) and sensor surface adhesion (using gold-binding peptide-GBP) to allow single-step reagent production, purification, and biosensor assembly. As a proof-of-concept, we developed Hepatitis C virus (HCV)-core antigen-GBP fusion proteins. These constructs were first tested and optimized for consistent surface adhesion then the assembled immunosensors were tested for cross-reactivity and evaluated for performance in vitro. We observed loss of function of the released reagents while secreted constructs performed well in in vitro testing with 2 orders of dynamic range, and a limit of detection of 32 nM. Finally, we validated the secreted platform with clinical isolates (n = 3) with statistically significant differentiation of positive vs. non-infected serum (p < 0.0001) demonstrating the ability to clearly distinguish HCV positive and negative clinical samples.

New amber mutation in a beta-thalassemic gene with nonmeasurable levels of mutant messenger RNA in vivo.

We have identified a beta-thalassemia gene that carries a novel nonsense mutation in a Chinese patient. This mutation, a G to T substitution at the first position of codon 43, changes the glutamic acid coding triplet (GAG) to a terminator codon (TAG). Based on oligonucleotide hybridization studies of 78 Chinese and Southeast Asian beta-thalassemia chromosomes, we estimate that this mutation accounts for a small minority of the beta-thalassemia mutations in that population. Study of the expression of this cloned gene in a transient expression system demonstrated a 65% decrease in levels of normally spliced mutant beta-globin mRNA. However, the study of reticulocyte RNA isolated from an individual heterozygous for this mutation demonstrated a total absence of this mutant mRNA in vivo. The basis for this big discrepancy between the level of accumulated mRNA in vivo and in vitro is probably the result of differences in the stabilities of the mutant mRNA in erythroid cells.

A novel POU domain protein which binds to the T-cell receptor beta enhancer.

POU domain proteins have been implicated in the regulation of a number of lineage-specific genes. Among the first POU domain proteins described were the immunoglobulin octamer-binding proteins Oct-1 and Oct-2. It was therefore of special interest when we identified a novel lymphoid POU domain protein in Southwestern (DNA-protein) screens of T-cell lambda gt11 libraries. This novel POU protein, TCF beta 1, binds in a sequence-specific manner to a critical motif in the T-cell receptor (TCR) beta enhancer. Sequence analysis revealed that TCF beta 1 represents a new class of POU domain proteins which are distantly related to other POU proteins. TCF beta 1 is encoded by multiple exons whose organization is distinct from that of other POU domain proteins. The expression of TCF beta 1 in a tissue-restricted manner and its ability to bind to multiple motifs in the TCR beta enhancer support a role in regulating TCR beta gene expression. The expression of TCF beta 1 in both B and T cells and the ability of recombinant TCF beta 1 to bind octamer and octamer-related motifs suggest that TCF beta 1 has additional roles in lymphoid cell function. The ability of TCF beta 1 to transactivate in a sequence-specific manner is consistent with a role for regulating lymphoid gene expression.

A silencer element from the alpha-globin gene inhibits expression of beta-like genes.

We have studied the cis and trans interactions of the alpha- and beta-globin genes in a transient expression system. We found that the alpha-globin gene inhibited beta-globin expression in cis but not in trans. The silencer element responsible for this inhibition was localized to a 259-base-pair fragment at the 5' end of the alpha-globin gene.

Effect of p21waf1/cip1 transgene on radiation induced apoptosis in T cells.

The cyclin kinase inhibitor p21WAF1/Cip1 is upregulated by the tumor suppressor p53. While p21 is central for the G-1 arrest mediated by p53, it is still unclear if p21 also functions as a downstream effector of p53 dependent apoptosis. Apoptosis induced by DNA damage but not dexamethasone is p53 dependent in thymocytes. To investigate the physiological role of p21 in apoptosis, we have generated transgenic mice in which the p21 transgene is targeted for restricted expression in the T cell lineage. Thymocytes from p21 transgenic mice were hypersensitive to cell death induced by DNA damaging agents such as ionizing radiation and UV, but not be dexamethasone. Irradiated p21 transgenic thymocytes had approximately twofold more apoptotic cells as compared to irradiated age matched littermate control mice. Radiation induced death is comparable in thymocytes from p21 + Bcl2 + double transgenic mice and age matched littermate controls, indicating that the Bcl2 transgene rescues the radiation hypersensitivity imposed by p21. However, thymocytes from p53-/- mice even when they expressed the p21 transgene, were resistant to death induced by radiation. Together these results show that thymocytes from p21 transgenic mice are hypersensitive to radiation induced programmed cell death and suggest that the radiation hypersensitivity of p21 transgenic thymocytes involves p53 dependent pathway and signals in addition to p21.

A novel regulatory element of the human alpha-globin gene responsible for its constitutive expression.

The alpha-globin gene is expressed at a constitutively high level upon gene transfer into both erythroid and nonerythroid cells. The beta-globin gene, on the other hand, is dependent on the presence of a linked viral enhancer for its efficient expression upon transfer into heterologous cells. In this report, we describe a novel regulatory element within the structural alpha-globin gene which can activate its own promoter to result in a high level of expression in both erythroid and non-erythroid cells. This regulatory element does not appear to have the properties of a classical enhancer. While this element exerts a positive effect on its own promoter, we have demonstrated in a previous study that the same element exerts a negative effect on heterologous genes such as the beta- and gamma-globin genes. In this study, we localize this element to a 259 nucleotide fragment immediately downstream from the translation initiation codon which is partially overlapped by a DNase I hypersensitive domain only in erythroid cells. We propose that this element may activate the alpha-globin gene promoter in all cell types in vivo as it does in vitro. The specificity of erythroid expression of the alpha-globin gene in vivo is probably determined by a "permissive" chromatin configuration in erythroid cells and a "nonpermissive" configuration in non-erythroid cells.

Experimental and mathematical models of Escherichia coli plasmid transfer in vitro and in vivo.

Little is known about the factors that govern plasmid transfers in natural ecosystems such as the gut. The consistent finding by earlier workers that plasmid transfer in the normal gut can be detected only at very low rates, if at all, has given rise to numerous speculations concerning the presence in vivo of various inhibitors of plasmid transfer. Plasmids R1, R1drd-19, and pBR322 were studied in Escherichia coli K-12 and wild-type E. coli hosts in two experimental systems: (i) gnotobiotic mice carrying a synthetic indigenous microflora (F-strains) which resemble in their function the normal indigenous microflora of the mouse large intestine, and (ii) anaerobic continuous-flow cultures of indigenous large intestinal microflora of the mouse, which can simulate bacterial interactions observed in the mouse gut. Mathematical models were developed to estimate plasmid transfer rates as a measure of the "fertility," i.e., of the intrinsic ability to transfer the plasmid under the environmental conditions of the gut. The models also evaluate the effects of plasmid segregation, reduction of the growth rates of plasmid-bearing bacterial hosts, repression of transfer functions, competition for nutrients, and bacterial attachment to the wall of the gut or culture vessel. Some confidence in the validity of these mathematical models was gained because they were able to reproduce a number of known phenomena such as the repression of fertility of the R1 plasmid, as well as known differences in the transmission and mobilization of the plasmids studied. Interpretation of the data obtained permitted a number of conclusions, some of which were rather unexpected. (i) Fertility of plasmid-bearing E. coli in the normal intestine was not impaired. The observed low rates of plasmid transfer in the normal gut can be explained on quantitative grounds alone and do not require hypothetical inhibitory mechanisms. (ii) Conditions for long-term spread and maintenance throughout human or animal populations of a diversity of conjugative and nonconjugative plasmids may be optimal among E. coli strains of low fertility, as are found among wild-type strains. (iii) E. coli strains carrying plasmid pBR322 plus R1drd-19 were impaired in their ability to transfer R1drd-19, but strains carrying pBR322 were significantly better recipients of R1drd-19 than a plasmid-free recipient E. coli. (iv) Long-term coexistence of plasmid-bearing and plasmid-free E. coli, in spite of undiminished fertility, appeared to be due to a detrimental effect of the plasmid on the growth rate of its host bacterium, rather than due to high rates of plasmid segregation. (v) Mathematical analysis of experimental data published by earlier investigators is consistent with the conclusion that plasmid transfer occurs consistently in the human gut, but that the resulting transconjugant E. coli populations are too small to be detected regularly with the culture methods used by earlier investigators. It is concluded that the long-term interactions observed were often the consequences of minor differences in parameters such as growth rates, fertility, rates of segregation, etc., which were too small to be detected except by precise mathematical analysis of long-term experiments, but which were nevertheless decisive determinants of the ultimate fates of the plasmids and their hosts.

Mechanisms that control bacterial populations in continuous-flow culture models of mouse large intestinal flora.

A previous study had established that anaerobic continuous-flow (CF) cultures of conventional mouse cecal flora were able to maintain the in vivo ecological balance among the indigenous bacterial species tested. This paper describes experiments designed to determine the mechanisms which control the population sizes of these species in such CF cultures. One strain each of Escherichia coli, Fusobacterium sp., and Eubacterium sp. were studied. Growth of these strains in filtrates of CF cultures was considerably more rapid than in the CF cultures themselves, indicating that the inhibitory activity had been lost in the process of filtration. Growth rates to match those in CF cultures could be obtained, however, by restoring the original levels of H(2)S in the culture filtrates. The inhibitory effect of H(2)S in filtrates and in dialysates of CF cultures could be abolished by adding glucose or pyruvate, but not formate or lactate. The fatty acids present in CF cultures matched those in the cecum of conventional mice in both quality and concentration. These acids could not account for the slow rates of growth of the tested strains in CF cultures, but they did cause a marked increase in the initial lag phase of E. coli growth. The results obtained are compatible with the hypothesis that the populations of most indigenous intestinal bacteria are controlled by one or a few nutritional substrates which a given strain can utilize most efficiently in the presence of H(2)S and at the prevailing conditions of pH and anaerobiosis. This hypothesis consequently implies that the populations of enterobacteria, such as the E. coli strain tested, and those of the predominant anaerobes are controlled by analogous mechanisms.

Survival and implantation of Escherichia coli in the intestinal tract.

Preliminary experiments established that a 0.5-ml inoculum that is introduced directly into the stomach of mice was cleared rapidly into the small intestine. Bicarbonate buffer, but not skim milk, protected such an inoculum from stomach acid until at least 90% of it had entered the small intestine. Passage and survival of various Escherichia coli strains through the mouse gut were tested by introducing a buffered bacterial inoculum directly into the stomach, together with the following two intestinal tracers: Cr(51)Cl(3) and spores of a thermophilic Bacillus sp. Quantitative recovery of excreted bacteria was accomplished by collecting the feces overnight in a refrigerated cage pan. The data show that wild-type E. coli strains and E. coli K-12 are excreted rapidly (98 to 100% within 18 h) in the feces without overall multiplication or death. E. coli varkappa1776 and DP50supF, i.e., strains certified for recombinant DNA experiments underwent rapid death in vivo, such that their excretion in the feces was reduced to approximately 1.1 and 4.7% of the inoculum, respectively. The acidity of the stomach had little bactericidal effect on the E. coli K-12 strain tested, but significantly reduced the survival of more acidsensitive bacteria (Vibrio cholerae) under these conditions. Long-term implantation of E. coli strains into continuous-flow cultures of mouse cecal flora or into conventional mice was difficult to accomplish. In contrast, when the E. coli strain was first inoculated into sterile continuous-flow cultures or into germfree mice, which were subsequently associated with conventional mouse cecal flora, the E. coli strains persisted in a large proportion of the animals at levels resembling E. coli populations in conventional mice. Metabolic adaptation contributed only partially to the success of an E. coli inoculum that was introduced first. A mathematical model is described which explains this phenomenon on the basis of competition for adhesion sites in which an advantage accrues to the bacterium which occupies those sites first. The mathematical model predicts that two or more bacterial strains that compete in the gut for the same limiting nutrient can coexist, if the metabolically less efficient strains have specific adhesion sites available. The specific rate constant of E. coli growth in monoassociated gnotobiotic mice was 2.0 h(-1), whereas the excretion rate in conventional animals was -0.23 h(-1). Consequently, limitation of growth must be regarded as the primary mechanism controlling bacterial populations in the large intestine. The beginnings of a general hypothesis of the ecology of the large intestine are proposed, in which the effects of the competitive metabolic interactions described earlier are modified by the effects of bacterial association with the intestinal wall.

Abnormal processing of beta-Malay globin RNA.

Hemoglobin Malay (alpha 2 beta 2 19 Asn----Ser) has been observed in a few Malaysian patients with thalassemia intermedia. The beta Malay substitution increases the homology of the cryptic splice site at codons 17/18/19 of the beta-globin gene to the donor consensus splice sequence, suggesting that the beta-thalassemia associated with this mutation may be due to the generation of a new splice site. To test this hypothesis, we constructed a hybrid gene where we replaced part of a normal beta-globin gene with a PCR amplified region of the beta Malay gene. The expression of this mutant gene was studied in a heterologous transient expression system. The data show that nearly 25% of globin mRNA produced by this gene is abnormally spliced at the new splice site, providing a molecular mechanism for the beta-thalassemia associated with the mutation.

Increased expression of the G gamma and A gamma globin genes associated with a mutation in the A gamma enhancer.

We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3' A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.

p70 lupus autoantigen binds the enhancer of the T-cell receptor beta-chain gene.

The p70 (Ku) autoantigen has been described as a nonhistone nuclear protein recognized by antibodies from lupus patients. In our studies on the regulation of T-cell receptor (TCR) beta-chain gene expression we have identified the p70 lupus autoantigen as a DNA-binding protein that binds the enhancer of the TCR beta-chain gene. This enhancer is essential for expression of the TCR beta gene. The core TCR beta enhancer contains the E3 motif, which we show here is essential for enhancer activity. The protection of the E3 motif in T cells and the marked reduction in enhancer activity when the E3 motif is mutated underline its physiological importance in regulating beta enhancer activity. The p70 lupus autoantigen gene was identified by screening T-cell lambda gt11 libraries with an E3 probe. The gene encodes a protein which binds the E3 motif in a sequence-specific manner. The identification of a 70-kDa protein as a major E3-binding protein by UV crosslinking is consistent with the conclusion that the p70 lupus autoantigen binds the beta enhancer. Finally, we have shown that T-cell nuclear proteins which bind the E3 motif bear p70 (Ku) lupus autoantigenic determinants. Together these data suggest that the p70 autoantigen binds a critical motif in the beta enhancer and probably regulates TCR beta gene expression.

Regulated expression of p18, a major phosphoprotein of leukemic cells.

p18 is a phosphoprotein that is present in great abundance in acute leukemia blasts and in less abundance in proliferating lymphocytes. This protein undergoes major changes in its state of phosphorylation upon induction of differentiation of leukemic cells in culture. The same protein appears to be involved in a variety of other cellular processes that include regulation of hormone secretion, T cell activation, muscle differentiation, and brain development. In this report, we describe our studies of the regulation of expression of this gene in leukemic cells. We show that the expression of this gene is markedly reduced upon induction of differentiation of a variety of leukemic cells in culture. We use a cDNA clone that we constructed earlier which encodes this protein as a probe to isolate the human chromosomal p18 gene. We characterize the 5' end of this gene in detail and identify its promoter element. We also identify a regulatory element in the first intervening sequence (IVS-1) of this gene which loses its DNase I hypersensitivity upon induction of differentiation of leukemic cells in culture. Our DNase I footprinting experiments demonstrate nuclear protein binding to multiple sequence motifs within its promoter element and its IVS-1 regulatory element. Functional studies using a transient expression system show that deletion of these sequence motifs has profound effects on the expression of this gene. These studies begin to shed some light on the mechanism of regulation of a gene that may be involved in control of cell growth and differentiation and in a variety of other vital cellular processes.

The beta-globin gene on the Chinese delta beta-thalassemia chromosome carries a promoter mutation.

A new type of delta beta-thalassemia characterized by decreased expression of the beta-globin gene and increased expression of both G gamma and A gamma globin gene in the absence of a detectable deletion has recently been described in the Chinese population. In this study we characterize the mutant beta-globin gene from this delta beta-thalassemia chromosome. An A to G transversion is identified in the "ATA" sequence of the promoter region that leads to decreased expression of the beta-globin gene in vivo and in vitro. We also demonstrate the presence of this mutation in every individual with a high fetal hemoglobin phenotype in this family and its absence in every individual with a normal hemoglobin phenotype. This same promoter mutation has recently been detected in Chinese beta-thalassemia genes where it is present on chromosomes of the same haplotype as that of the delta beta-thalassemia chromosome we are studying. These data support the hypothesis that an as yet unidentified mutation occurred on the ancestral chromosome carrying the promoter mutation and subsequently gave rise to the delta beta-thalassemia phenotype.

A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C. The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C. The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation. The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.

The large subunit of replication factor C promotes cell survival after DNA damage in an LxCxE motif- and Rb-dependent manner.

Retinoblastoma (Rb) protein promotes cell survival after DNA damage. We show here that the LxCxE binding site in Rb mediates both cell survival and cell-cycle arrest after DNA damage. Replication factor C (RF-C) complex plays an important role in DNA replication. We describe a novel function of the large subunit of RF-C in promoting cell survival after DNA damage. RF-Cp145 contains an LxCxE motif, and mutation of this motif abolishes the protective effect of RF-Cp145. The inability of wild-type RF-Cp145 to promote cell survival in Rb-null cells is rescued by Rb but not by Rb mutants defective in binding LxCxE proteins. RF-C thus enhances cell survival after DNA damage in an Rb-dependent manner.

Microdevices in medicine.

The application of microelectromechanical systems (MEMS) to medicine is described. Three types of biomedical devices are considered, including diagnostic microsystems, surgical microsystems, and therapeutic microsystems. The opportunities of MEMS miniaturization in these emerging disciplines are considered, with emphasis placed on the importance of the technology in providing a better outcome for the patient and a lower overall health care cost. Several case examples in each of these areas are described. Key aspects of MEMS technology as it is applied to these three areas are described, along with some of the fabrication challenges.