Subsurface iron and arsenic removal: low-cost technology for community-based water supply in Bangladesh.

The principle of subsurface or in situ iron and arsenic removal is that aerated water is periodically injected into an anoxic aquifer through a tube well, displacing groundwater containing Fe(II). An oxidation zone is created around the tube well where Fe(II) is oxidised. The freshly formed iron hydroxide surfaces provide new sorption sites for soluble Fe(II) and arsenic. The system's efficiency is determined based on the ratio between abstracted volume with reduced iron/arsenic concentrations (V) and the injected volume (V(i)). In the field study presented in this paper, the small-scale application of this technology was investigated in rural Bangladesh. It was found that at small injection volumes (<1 m³) iron removal was successful and became more effective with every successive cycle. For arsenic, however, the system did not prove to be very effective yet. Arsenic retardation was only limited and breakthrough of 10 µg/L (WHO guideline) was observed before V/V(i)=1, which corresponds to arrival of groundwater at the well. Possible explanations for insufficient arsenic adsorption are the short contact times within the oxidation zone, and the presence of competing anions, like phosphate.

Targeting RNA with peptidomimetic oligomers in human cells.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Here we report that two TAR RNA-binding peptidomimetics, oligourea and oligocarbamate, inhibit transcriptional activation by Tat protein in human cells with an IC50 of approximately 0.5 and 1 microM, respectively. Peptidomimetics that can target specific RNA structures provide novel molecules that can be used to control cellular processes involving protein-RNA interactions in vivo.

HIV-1 TAR RNA enhances the interaction between Tat and cyclin T1.

Human immunodeficiency virus, type 1 (HIV-1), Tat activates elongation of RNA polymerase II transcription at the HIV-1 promoter through interaction with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex, P-TEFb. Binding of Tat to CycT1 induces cooperative binding of the P-TEFb complex onto nascent HIV-1 TAR RNA. Here the specific interaction between Tat protein, human cyclin T1, and HIV-1 TAR RNA was analyzed by fluorescence resonance energy transfer, using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein synthesized through solid-phase chemistry. We find that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA and that TAR RNA further enhances the interaction between Tat and CycT1. We conclude that TAR RNA nucleates the formation of the Tat.P-TEFb complex through an induced fit mechanism.

Design, synthesis, and biological activity of a cyclic peptide: an inhibitor of HIV-1 tat-TAR interactions in human cells.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. A number of cyclic peptides are known to possess antibiotic activity and increased biological stability. Here we report the design, synthesis, and biological activity of a cyclic peptide (2), which inhibits transcriptional activation by Tat protein in human cells with an IC50 of approximately 40 nM. Cyclic peptides that can target specific RNA structures provide a new class of small molecules that can be used to control cellular processes involving RNA-protein interactions in vivo.

Inhibition of gene expression in human cells through small molecule-RNA interactions.

Small molecules that bind their biological receptors with high affinity and selectivity can be isolated from randomized pools of combinatorial libraries. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of HIV-1 gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5' end of all nascent HIV-1 transcripts. Here we demonstrate the isolation of small TAR RNA-binding molecules from an encoded combinatorial library. We have made an encoded combinatorial tripeptide library of 24,389 possible members from D-and L-alpha amino acids on TentaGel resin. Using on-bead screening we have identified a small family of mostly heterochiral tripeptides capable of structure-specific binding to the bulge loop of TAR RNA. In vitro binding studies reveal stereospecific discrimination when the best tripeptide ligand is compared with diastereomeric peptide sequences. In addition, the most strongly binding tripeptide was shown to suppress transcriptional activation by Tat protein in human cells with an IC(50) of approximately 50 nM. Our results indicate that tripeptide RNA ligands are cell permeable, nontoxic to cells, and capable of inhibiting expression of specific genes by interfering with RNA-protein interactions.

Proximity of a Tat peptide to the HIV-1 TAR RNA loop region determined by site-specific photo-cross-linking.

Transcriptional regulation in human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-ends of all HIV-1 mRNAs. We have used a site-specific cross-linking method based on 4-thio-uracil (4-thioU) photochemistry to determine the interactions of a Tat peptide, Tat(38-72), with the loop region of TAR RNA under physiological conditions. A TAR RNA construct with a single 4-thioU residue at positions U31 in the loop sequence was synthesized by chemical methods. Upon UV irradiation, 4-thioU at U31 formed a covalent cross-link with the Tat peptide. We did not observe any RNA-RNA cross-link formation. Competition experiments revealed that a specific RNA-protein complex formation was necessary for the RNA-protein cross-linking reaction. Our results demonstrate that, during RNA-protein recognition, the Tat peptide is located in close proximity to O4 of U31 in the TAR RNA loop sequence.

Controlling human immunodeficiency virus type 1 gene expression by unnatural peptides.

Small unnatural peptides that target specific RNA structures have the potential to control biological processes. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of human immunodeficiency virus type 1 (HIV-1) gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all nascent HIV-1 transcripts. We report here a synthetic peptide derived from Tat sequence (37-72), containing all D-amino acids, that binds in the major groove of TAR RNA and interferes with transcriptional activation by Tat protein in vitro and in HeLa cells. Our results indicate that unnatural peptides can inhibit the transcription of specific genes regulated by RNA-protein interactions.

Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis ofHIV-1 Tat-TAR complex.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans -activation responsive region (TAR) RNA, a 59 base stem-loop structure located at the 5'-end of all HIV transcripts. We have used an intramolecular RNA self-cleaving strategy to determine the folding of TAR RNA and its interactions with a Tat peptide. We incor-porated an EDTA analog at position 24 in the HIV-1 Tat binding site of the TAR RNA. After isolation and purification of the EDTA-TAR conjugate, RNA self-cleavage was initiated by the addition of an iron salt, ascorbate and hydrogen peroxide. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Sites of RNA cleavage were mapped by sequencing reactions. A Tat fragment, Tat(38-72), specifically inhibited RNA self-cleavage. To determine the structural changes caused by the Tat peptide, we performed Fe(II)-EDTA footprinting experiments on Tat-TAR complex. Our high-resolution footprinting results suggest that the inhibition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage and (ii) RNA goes through a structural change where EDTA-U24 is rigidly positioned out of the helix and cannot get access to other nucleotides in the loop of TAR RNA, which are not protected by the Tat peptide. Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures and RNA-protein interactions.

Probing the proximity of the core domain of an HIV-1 Tat fragment in a Tat-TAR complex by affinity cleaving.

Transactivation of human immunodeficiency virus (HIV) gene expression depends upon the interaction of the viral regulatory protein Tat with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all mRNAs. We have used a site-directed RNA-cleaving strategy to determine the neighborhood of the core domain of a Tat fragment in the Tat-TAR complex. We synthesized a 35-amino acid fragment containing arginine-rich RNA-binding domain of Tat(38-72) and attached an EDTA analog to its amino terminus. A derivative of (p-aminobenzyl)-EDTA tetra-tert-butyl ester was synthesized and attached to the amino terminus of the Tat peptide by standard peptide coupling methods. Cleavage from the resin and deprotection of the peptide were carried out in trifluoroacetic acid which also generated unprotected metal binding EDTA moieties. We used this EDTA-Tat conjugate to form a specific complex with TAR RNA. This sequence-specific RNA-binding peptide was converted into a sequence-specific RNA-cleaving peptide by the addition of Fe(II) salt, ascorbate, and H2O2. Hydroxyl radicals generated from the tethered Fe(II) cleaved the TAR RNA backbone in two localized regions. Site-specific cleavage of TAR RNA was observed at the bulge residues (U23, C24, and U25), in the loop region (G34 and A35), and at the strand opposite the bulge (U40 and C41). These results demonstrate that, in the three-dimensional structure of the Tat-TAR complex, the Phe38 of Tat(38-72) is located in the proximity of the bulge region and two nucleotides from the loop sequence.

Isoleucine 368 is involved in low-affinity binding of N6-modified cAMP analogues to site B of the regulatory subunit of cAMP-dependent protein kinase I.

The regulatory (R) subunit of cAMP-dependent protein kinase has a well-defined domain structure including the two in-tandem cAMP-binding sites that constitute the C-terminus of the protein. The N-terminal binding site (A) has a considerably higher affinity for analogues of cAMP that are substituted with bulky and hydrophobic substituents at the 6-amino group of the adenine ring compared to the affinity observed at the second site (B). On the basis of the crystal structure of the catabolite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A could be involved in a high-affinity hydrophobic interaction, whereas a corresponding isoleucine (I368) in domain B could lead to steric hindrance in the binding of bulky N6-substituted analogues. Site-directed mutagenesis was used to construct mutations in Y244 and I368. Binding displacement experiments showed that replacing the tyrosine in site A with isoleucine (Y244I) did not affect the interaction of either N6-substituted or otherwise modified analogues with this site. However, replacing I368 with tyrosine (I368Y) led to a 3-4-fold increase in affinity for those N6-modified analogues that had a hydrophobic group attached directly or close to the 6-amino molecule. We conclude that I368 is involved in the molecular interaction between binding domain B and the 6-amino group of the adenine moiety of cAMP and that this residue is partly responsible for the reduced affinity of N6-substituted cAMP analogues for this site.

Expression in Escherichia coli of a rat cDNA encoding an apurinic/apyrimidinic endonuclease.

A rat cDNA (rAPEN) with 85% DNA identity to the major human apurinic/apyrimidinic (AP) endonuclease gene was used to construct a fusion between it and glutathione-S-transferase (GST). The GST-rAPEN fusion was subsequently overexpressed in Escherichia coli, purified on glutathione-agarose affinity columns, and the purified protein tested for AP endonuclease activity. DNA nicks were found to be specifically introduced into AP DNA in a reaction that was dependent upon the time of incubation and the amount of GST-rAPEN added. The DNA scissions produced by GST-rAPEN were determined to be adjacent and 5' to an AP site. The purified fusion protein was also able to efficiently remove 3'-(4 hydroxy-5-phospho-2-pentenal) residues, and to a lesser extent 3'-phosphoglycolate residues. The GST-rAPEN activity failed to exhibit any 3'-5' exonuclease activity, a characteristic shared by the major AP endonuclease in bovine and human.

Cancer in Libya--a retrospective study (1981-1985).

Between 1981 and 1985, 1124 patients with histopathologically confirmed malignant disease were registered at the sole oncology clinic of Libya, 664 (59%) were males and 460 (41%) were females. Overall, malignant lymphoma (ML) was the most common cancer (180/1124, 16%), with non-Hodgkin's lymphoma (NHL) being more common (57.2%) than Hodgkin's disease (HD) (42.8%). Considered separately, lung cancer was the most common tumour (22.4%) in males with a male to female ratio among the highest in the world (18.6:1), 85% of our male patients were smokers and more than 50% had been smoking heavily for 20 years or more. Breast cancer was the most frequent tumour (29.8%) of females and the majority of our patients were of a younger age group (72.3% below 50 years). Almost all our patients were multiparous and had breast-fed their babies. Cancer of the cervix uteri was less frequent (4.5%) than ovarian cancer (7.8%). The incidence of colorectal cancer was higher (4.6%) than other African countries. Contrarily the primary tumours of liver (1.9%) and bladder (0.5%) were less frequent. Among the children, aged less than 10 years, the common solid tumours of childhood occurred in the following frequency, ML 31.2%, nervous system 19.2%, Wilm's tumour 16.8% and bone tumours 9.6%.

Purification to homogeneity and characterization of a redoxyendonuclease from calf thymus.

A redoxyendonuclease from calf thymus was purified to apparent homogeneity. The redoxyendonuclease recognized and induced cleavage of DNA damaged by ultraviolet light. The enzyme preparation produced a single band of a relative molecular mass of approximately 34 kDa upon SDS/PAGE. The apurinic/apyrimidinic endonuclease and the DNA glycosylase activities remained associated in the apparently homogeneous preparation of the enzyme. The redoxyendonuclease activity displayed a broad pH optimum between pH 5.0-8.5 and exhibited no requirement for divalent cations. By application of FPLC columns Mono-S, Mono-Q and Mono-P, the isoelectric point (pI) of the enzyme was found to be approximately 8.0. Using the DNA sequencing procedure of Maxam and Gilbert [Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560] the purified enzyme was found to incise ultraviolet-light-irradiated DNA at pyrimidine sites as observed previously with a more crude form of the enzyme. While the most frequently cleavaged sites for the crude preparation were at cytosine residues, the apparently homogeneous enzyme preparation frequently induced cleavage sites at both cytosine and guanine residues. Predominant incision induced by the apparently homogeneous preparation was observed at guanine residues when a particular DNA sequence was used as substrate. Furthermore, the 16 N-terminal amino acid residues of the purified enzyme were identified. The sequence did not show any significant similarity to other known proteins.

Plasmids and factors associated with virulence in environmental isolates of Vibrio cholerae non-O1 in Bangladesh.

Plasmid profiles and factors associated with toxigenicity in 51 strains of Vibrio cholerae non-O1 isolated from water samples collected in Bangladesh were analysed. Eleven (21.5%) strains were found to harbour at least one plasmid of 1.7-115 Mda; seven of these strains shared a 115-Mda plasmid. Six of 13 strains tested gave positive cytotoxic and enterotoxic responses. However, two non-cytotoxic strains were enterotoxigenic. Only three of the six cytotoxic and enterotoxic strains caused haemagglutination of human erythrocytes which indicated that toxin production and haemagglutinating activity were unrelated in these V. cholerae non-O1 strains. Conjugal transfer assays demonstrated that the 115-Mda plasmid harboured by some of the toxigenic V. cholerae non-O1 strains carried genes coding for antibiotic resistance and cytotoxin production but not for enterotoxin production. However, this plasmid was also carried by non-toxigenic strains. Some other strains carrying no plasmids or only small-mol.-wt plasmids, were found to be toxigenic. Therefore, toxin production is not plasmid-mediated in all V. cholerae non-O1 strains. Regardless of their pathogenic potential, V. cholerae non-O1 strains possessed the capacity to grow in conditions of iron limitation and, under these conditions, synthesis of at least two new outer-membrane proteins was induced.

Species specificity and lack of production of STb enterotoxin by Escherichia coli strains isolated from humans with diarrheal illness.

Escherichia coli strains produce at least two heat-stable enterotoxins, STa and STb. STa is well known to be important in the pathogenesis of human diarrheal disease; the role of STb has not been defined. Fifty-two E. coli strains recovered from human diarrheal illness in northeast Brazil or Bangladesh were examined in weaned porcine ligated intestinal segments for STb activity. A total of 113 E. coli strains from human diarrheal disease in northeast Brazil and 28 E. coli strains from Bangladesh were examined for DNA hybridization to a STb gene probe. None of these strains produced STb as detected by enterotoxic activity or by the gene probe. We also examined adult human ileal mucosa for responses to STb in the Ussing chamber in vitro. In contrast to piglet jejunum, which consistently responds electrogenically to crude STb, human ileal tissue showed no response to STb but responded electrogenically to theophylline (10 mM). These results suggest that STb-producing E. coli strains are not a major cause of diarrheal illness in humans.

Fourteen years of shigellosis in Dhaka: an epidemiological analysis.

We examined whether the proportion of Shigellae patients among diarrhoeal cases, the distribution, species, case-fatality rates and hospital visits changed over time in Dhaka. We isolated 19639 Shigella strains from 822812 diarrhoea cases treated at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), between 1969 and 1982. The number of cases increased from 209 (2.5%) in 1969 to 4833 (7.7%) in 1976. Extrapolating from a 4% vigorous systematic sample of ICDDR,B hospital visits shigellosis cases and their proportion among diarrhoea cases increased to more than 9500 (12.0%) in 1981. The prevalence of various shigellae species altered over time. For example: in 1969 Shigella flexneri predominated in 74% of all Shigella cases; in 1973 Shigella dysenteriae accounted for 56%, and in 1981 Shigella flexneri again predominated (75%). More than 20% of all Shigella isolations were from infants: 60% in males and 40% in females. Over 7% of severe cases of Shigella infection referred from the outpatient department and admitted for treatment died. Nearly 40% of all the Shigella deaths were in infants of less than a year old while 49% were in 1-4 year old children. Increasing prevalence of shigellosis appears to be an important cause of diarrhoea in Dhaka especially among children. Areas with poor sanitation and water supply had higher prevalence. However, hospitalized cases represented a fraction of the actual problem. Resistance to antibiotics appears to be increasing and the development of new drugs and preventive methods within economic reach of less developed countries are crucial for reduction of the disease and related deaths.

Nutritional status, body size and severity of diarrhoea associated with rotavirus or enterotoxigenic Escherichia coli.

Children with severe malnutrition have an increased risk of death from diarrhoea. To determine if the clinical manifestations of specific types of diarrhoea differed according to the nutritional status or size (weight and length) of the patient, we studied children with acute diarrhoea associated with rotavirus or enterotoxigenic Escherichia coli. In this study we found that a child's body size, which was determined by his age and nutritional status, was a significant predictor of his rate of stool output per kilogram of body weight. Thus, children who are small because of young age and/or malnutrition appear to lose a greater proportion of their total fluid volume during diarrhoea and might be expected to have a higher frequency of severe dehydration and death, if untreated.

PIP: This study assessed the effects of undernutrition (measured by weight-for-length) and low body weight on stool output rates in 82 children under 5 years of age with diarrhea associated with rotavirus or enterotoxigenic Escherichia coli. Although weight-for-length status did not affect stool output, children appeared to lose fluid as a greater rate if they had low body weight. Lower-weight children had rates of stool loss 14-61% greater than higher-weight children. Multiple regressionanalysis aimed at determining whether age, weight, or length was the best predictor of stool output found that length was the best predictor. However, weight and age are likely to be indirectly related to diarrheal severity, since malnutrition and young age are reasons for smaller body weight or length. It is hypothesized that the small intestine of an underweight child is greater in proportion to body weight than that of a normal weight child, resulting in larger stool losses per kg/body weight. The finding that children of small body size lose a greater proportion of their body fluid during diarrhea suggests that these children should be regarded as a high risk group during diarrhea. Special attention should be given to water and electrolyte replacement to prevent dehydration and death among these children.