The barriers and facilitators for recognising distress in people with severe dementia on general hospital wards.

Introduction: psychological symptoms and delirium are common, but underreported in people with dementia on hospital wards. Unrecognised and untreated symptoms can manifest as distress. Identifying distress accurately therefore could act as a trigger for better investigation and treatment of the underlying causes. The challenges faced by healthcare professionals to recognise and report distress are poorly understood. Methods: semi-structured interviews with a purposive sample of 25 healthcare professionals working with older people in general hospitals were conducted. Interviews were analysed generating themes that describe the facilitators and barriers of recognising and caring for distress in dementia. Results: regardless of training or experience all participants had a similar understanding of distress, and identified it as a term that is easily understood and communicated. All participants believed they recognised distress innately. However, the majority also believed it was facilitated by experience, being familiar with their patients and listening to the concerns of the person's usual carers. Barriers to distress recognition included busy ward environments, and that some people may lack the skill to identify distress in hypoactive patients. Conclusion: distress may be a simple and easily identified marker of unmet need in people with dementia in hospital. However, modifiable and unmodifiable barriers are suggested that reduce the chance of distress being identified or acted on. Improving our understanding of how distress is identified in this environment, and in turn developing systems that overcome these barriers, may improve the accuracy with which distress is identified on hospital wards.

Characterization of the Vibrio fischeri Fatty Acid Chemoreceptors, VfcB and VfcB2.

Bacteria use a wide variety of methyl-accepting chemotaxis proteins (MCPs) to mediate their attraction to or repulsion from different chemical signals in their environment. The bioluminescent marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and encodes a large repertoire of MCPs that are hypothesized to be used during different parts of its complex, multistage lifestyle. Here, we report the initial characterization of two such MCPs from V. fischeri that are responsible for mediating migration toward short- and medium-chain aliphatic (or fatty) acids. These receptors appear to be distributed among only members of the family Vibrionaceae and are likely descended from a receptor that has been lost by the majority of the members of this family. While chemotaxis greatly enhances the efficiency of host colonization by V. fischeri, fatty acids do not appear to be used as a chemical cue during this stage of the symbiosis. This study presents an example of straight-chain fatty acid chemoattraction and contributes to the growing body of characterized MCP-ligand interactions.

The incidence and mortality for meningococcal disease associated with area deprivation: an ecological study of hospital episode statistics.

AIMS: To determine whether incidence, mortality, and case fatality for meningococcal disease (MD) differs by area deprivation, and if this has changed over time. METHODS: The population of children aged less than 5 years with MD was analysed as quintiles of area deprivation scores over two time periods, 1995-99 and 1991-94. Annual age standardised rates were calculated and the association between incidence, mortality, and area deprivation quintiles assessed using Poisson regression and the risk ratios determined. Case fatality was calculated from the odds ratio of mortality by area deprivation score for the two time periods. RESULTS: There were 10,524 cases of MD and 441 deaths (4.2%). Incidence rates were higher for 1995-99 (45.4 per 100,000) compared to 1991-94 (27.4 per 100,000). Mortality rates remained stable over time, indicating a decline in risk of death of around 40%. The incidence rates for the most deprived quintile were around twice those for the most affluent quintile, but this gradient declined over time. A threefold gradient was seen for mortality rates across the top and bottom quintiles, which was constant over time. The odds of mortality did not show a linear pattern, with mortality being lowest in the first and highest in the second and fifth area deprivation quintiles. CONCLUSIONS: These data show that MD incidence and mortality are socially patterned. The determinants of case fatality are more complex and require further investigation.

Broad-complex, but not ecdysone receptor, is required for progression of the morphogenetic furrow in the Drosophila eye.

The progression of the morphogenetic furrow in the developing Drosophila eye is an early metamorphic, ecdysteroid-dependent event. Although Ecdysone receptor-encoded nuclear receptor isoforms are the only known ecdysteroid receptors, we show that the Ecdysone receptor gene is not required for furrow function. DHR78, which encodes another candidate ecdysteroid receptor, is also not required. In contrast, zinc finger-containing isoforms encoded by the early ecdysone response gene Broad-complex regulate furrow progression and photoreceptor specification. br-encoded Broad-complex subfunctions are required for furrow progression and proper R8 specification, and are antagonized by other subfunctions of Broad-complex. There is a switch from Broad complex Z2 to Z1 zinc-finger isoform expression at the furrow which requires Z2 expression and responds to Hedgehog signals. These results suggest that a novel hormone transduction hierarchy involving an uncharacterized receptor operates in the eye disc.

Identification of a new HIV-2 subtype based on phylogenetic analysis of full-length genomic sequence.

Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus from sooty mangabey (SIV(SM) form one of the six primate lentivirus lineages. The close phylogenetic relationship and geographic coincidence indicate that HIV-2 originated from cross-species transmission of SIV(SM) to humans. HIV-2 exhibits considerable genetic diversity, with subtypes A-F identified. Previously, we reported the partial gag and env sequences of an unusual HIV-2 isolate, Abt96. Abt96 was collected in Ivory Coast from an asymptomatic blood donor. Here we describe the near full-length genomic sequence of Abt96. The genome was assembled from overlapping PCR fragments amplified from viral RNA isolated from plasma. Phylogenetic analysis of sequences derived from segments of the Abt96 genome demonstrate that the Abt96 isolate branches independently of all other characterized HIV-2 isolates. On the basis of the phylogenetic data being presented, we propose that Abt96 is a new HIV-2 subtype and designate it subtype G.

Determination of Drosophila photoreceptors: timing is everything.

This review covers recent findings concerning the specification of the photoreceptor subtypes in the Drosophila eye. Particular attention is paid to aspects of retinal patterning and differentiation where relative timing of events seems to be tightly controlled and essential for proper assembly of the compound eye. For example, specification of the founding photoreceptors of each cluster requires sequential positive and negative signaling through the Notch pathway, and reiterated signaling through the epidermal growth factor receptor leads to the pairwise recruitment of the distinct types of photoreceptors in discrete zones across the eye. Results suggest that different signaling environments for these two receptors may exist across the disc, and that receiving cells may constantly shift their predisposition to respond to such signals by adopting given fates. In addition, considerable data exist that the rate of expansion of retinal patterning across the disc is restricted to allow the orderly patterning of retinal precursors, and that one mechanism for controlling this rate may be the co-ordinated expression anterior to the furrow of factors which both inhibit and promote the expansion of retinal patterning. Finally, this review considers the possibility that the morphogenetic furrow serves as a moving source of morphogens which supply spatial information to both anterior and posterior tissue, providing temporal cues that regulate the many events involved in orderly assembly of the precise array of retinal cell types in the compound eye.

Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected transmembrane HIV antigens provides a simple and reliable test that is capable of distinguishing HIV infections on the basis of viral type.

Ecdysone pathway is required for furrow progression in the developing Drosophila eye.

In Drosophila, secretion of the steroid hormone ecdysone from the prothoracic ring gland coordinates and triggers events such as molting and metamorphosis. In the developing Drosophila compound eye, pattern formation and cell-type specification initiate at a moving boundary known as the morphogenetic furrow. We have investigated the role of ecdysone in eye development and report here that the ecdysone signaling pathway is required for progression of the morphogenetic furrow in the eye imaginal disc of Drosophila. Genetic disruption both of the ecdysone signal in vivo with the ecdysoneless1 (ecd1) mutant and of ecdysone response with a Broad-Complex mutant result in disruption of morphogenetic furrow progression. In addition, we show that ecdysone-dependent gene expression, both of a reporter of transcriptional activity of the Ecdysone Receptor and of the Z1 isoform of the Broad Complex, are localized in and close to the furrow. These results suggest that, in the morphogenetic furrow, temporal hormonal signals are integrated into genetic pathways specifying spatial pattern.

Serologic and phylogenetic characterization of HIV-1 subtypes in Uganda.

OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.

PIP: Extensive sequence heterogeneity between HIV-1 isolates has led to the classification of HIV-1 into group M (major) subtypes A-J, and group O (outlier). Some isolates have also been found to be the result of recombination between different group M subtypes. Findings are reported from a study conducted to determine the various HIV genetic subtypes in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate the serologic detection of infection by commercial immunoassays. 64 HIV-seropositive plasma samples were collected from the Nakasero Blood Bank in Kampala and evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. 63 of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of group O infections. According to phylogenetic analysis, 25 samples were subtype A, 3 subtype C, 22 subtype D, and 9 heterogenous for subtypes A and D. Despite the sequence variation observed in this study population, commercial EIA based upon HIV-1 subtype B proteins detected all of the infections. A peptide-based assay failed to detect 3 infections by subtype D viruses.

Sequence of gp41env immunodominant region of HIV type 1 group O from west central Africa.

PIP: HIV-1 group O is endemic in the west central region of Africa, where the frequency of infection is estimated to be 3-10% of all HIV-1-infected individuals. However, international travel and immigration have led to group O cases being identified in France, Germany, Belgium, Spain, and the US. With the exception of an infected French woman, all reported group O-infected individuals originate from or have a connection to west central Africa. Since most immunoassay reagents are based upon HIV-1 group M, many HIV immunoassays have lower sensitivity for the detection of group O infections. Serum samples were collected from patients at hospitals, tuberculosis (TB) clinics, and STD clinics in endemic regions of Cameroon and Equatorial Guinea in a study of the sequence divergence with group O isolate infections. Screening of the 1086 samples using a range of research and commercial immunoassays found 255 to be HIV-1 seropositive. On the basis of differential reactivity in the various immunoassays, 8 individuals were identified as potentially being infected with group O virus, of which 4 were drawn from TB patients. 7 of the group-O samples were then subjected to polymerase chain reaction (PCR) amplification to verify group O infection. The gp41(env) immunodominant region was successfully amplified and sequenced from 4 of the 7 samples, 2 of which were from the TB patients; 4 of 1086 samples were definitely infected with HIV-1 group O.^ieng

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Transgenic mice expressing either bovine growth hormone (bGH) or human GH releasing hormone (hGRH) have increased splenic progenitor cell colony formation and DNA synthesis in vitro and in vivo.

To investigate the potential effects of growth hormone (GH) on the hematopoietic system, mice transgenic for bovine GH (bGH) or human growth hormone releasing hormone (hGRH) genes, each of which can result in the constitutive overproduction of GH, were analyzed for splenic and bone marrow (BM) localized hematopoietic progenitor cells. These transgenic mice had splenic hyperplasia with increased absolute numbers of splenic erythroid and megakaryocytic progenitor cells as assessed by in vitro assay and megakaryocyte development as seen in spleens. As an in vivo indication of multilineage progenitor cell effects in hGRH mice, the number of day-10 CFU-S colonies derived from the donor spleen was significantly higher than in nontransgenic littermate controls. A high proportion (54-71%) of splenic erythroid, granulocyte-macrophage, and megakaryocyte progenitors were in cycle in transgenic mice in contrast to < or = 30% in nontransgenic control littermates. Compared to controls, splenocytes from hGRH mice had a significantly higher proliferative index when infused into irradiated nontransgenic controls. With the exception of the megakaryocyte colony assay and in vivo proliferative index, none of these findings were evident when identical assays were performed on the BM from the same mice. Consistent with the BM data, peripheral blood leukocyte, erythroid, and platelet numbers were comparable in transgenic and nontransgenic control littermates. We conclude that the constitutive expression of bGH or hGRH leads largely to splenic hematopoietic effects involving progenitor cell populations from at least two lineages.

A molecular sensor system based on genetically engineered alkaline phosphatase.

Binding and signaling proteins based on Escherichia coli alkaline phosphatase (AP; EC 3.1.3.1) were designed for the detection of antibodies. Hybrid proteins were constructed by using wild-type AP and point mutants of AP [Asp-101 --> Ser (D101S) and Asp-153 --> Gly (D153G)]. The binding function of the hybrid proteins is provided by a peptide epitope inserted between amino acids 407 and 408 in AP. Binding of anti-epitope antibodies to the hybrid proteins modulates the enzyme activity of the hybrids; upon antibody binding, enzyme activity can increase to as much as 300% of the level of activity in the absence of antibody or can decrease as much as 40%, depending on the presence or absence of the point mutations in AP. The fact that modulation is altered from inhibition to activation by single amino acid changes in the active site of AP suggests that the mechanism for modulation is due to structural alterations upon antibody binding. Modulation is a general phenomenon. The properties of the system are demonstrated by using two epitopes, one from the V3 loop of human immunodeficiency virus type 1 gp120 protein and one from hepatitis C virus core protein, and corresponding monoclonal antibodies. The trend of modulation is consistent for all hybrids; those in wild-type AP are inhibited by antibody, while those in the AP mutants are activated by antibody. This demonstrates that modulation of enzyme activity of the AP-epitope hybrid proteins is not specific to either a particular epitope sequence or a particular antibody-epitope combination.

Association of Langerhans cell histiocytosis with malignant neoplasms.

BACKGROUND: The association of Langerhans cell histiocytosis (LCH) with a malignant neoplasm is rare and generally has been the subject of isolated case reports. METHODS: A recent case of LCH seen at the University of Minnesota in combination with acute lymphoblastic leukemia led the authors to review their own charts from 1960 onward, in addition to the literature for other reported associations of LCH and malignant neoplasms. RESULTS: In addition to the presented case and 3 cases from the files of the authors, the literature contained 87 reported cases. Of the 91 patients, 39 had LCH with malignant lymphoma (ML); 25 of these cases were Hodgkin disease. In 11 of these 39 patients, the LCH was diagnosed from 12 months to 33 years after the ML was diagnosed. In 62% of the patients with LCH-ML (24 patients), the diagnosis was made concurrently and the Langerhans cells were found in the same lymph nodes. In the remaining four patients, the diagnosis of LCH preceded that of ML by 6-24 months. In 22 patients, including 2 patients in the files of the authors, LCH was reported in association with leukemia; 16 (73%) of these cases were associated with acute nonlymphoblastic leukemia. In two cases the leukemia preceded the LCH. In 6 patients both diagnoses were made concurrently, and in 14 patients (64%) the diagnosis of LCH preceded the diagnosis of leukemia by 8 months to 17 years. In the remaining 30 patients, LCH was associated with a variety of solid tumors, including a lung carcinoma in 12 patients. In all of these 12 cases the LCH was confined to the lung, and in 75% (9 of 12) of patients the diagnoses were made concurrently. In the 16 patients in whom the LCH preceded the solid tumor, the malignant diseases in 69% (11 of 16) developed within the radiation field used for the treatment of the LCH. CONCLUSIONS: The intimate and simultaneous association of LCH with ML and lung carcinomas suggests strongly that the process that leads to the association is a reactive one. However, in the patients with leukemia and the other solid tumors, the latency of the malignant neoplasm after the diagnosis of LCH is suggestive of a therapy-related process.

Mutations in an RNP1 consensus sequence of Rho protein reduce RNA binding affinity but facilitate helicase turnover.

Escherichia coli rho protein facilitates transcription termination by a mechanism that involves rho binding to the nascent RNA, activation of rho's RNA-dependent ATPase activity, and release of the mRNA from the DNA template. The initial step, formation of a rho-RNA complex, is mediated primarily by an RNA binding domain included within the amino-terminal 151 amino acids of rho protein. We have now identified one specific portion of this region that is involved in RNA binding, by photocross-linking and by site-directed mutagenesis. UV irradiation of rho-RNA complexes results in covalent attachment of the RNA to a single peptide in rho that apparently spans amino acids 45-100. Within this peptide is a ribonucleoprotein (RNP1) consensus sequence, Gly-Phe-Gly-Phe, that is present in many RNA-binding proteins. Mutagenesis of the phenylalanine residues in this consensus to leucine or alanine results in mutant proteins that are defective for RNA binding and have altered ATPase and RNA-DNA helicase activities. The weakened affinity but increased salt sensitivity of RNA binding by the mutant proteins suggests that they have lost more than just a set of nonionic interactions and are consistent with a change in the conformation of the RNA binding site. Whatever the changes, they appear localized primarily to the RNA binding domain because the mutants retain much of their RNA-dependent ATPase activity. We infer that the Phe residues themselves do not play a substantial role in the activation of ATP hydrolysis. Our results indicate that several different components of RNA interaction are required for rho activity and support a role for the RNP1 consensus region of rho in at least one specific aspect of RNA binding.

A short intervening structure can block rho factor helicase action at a distance.

We have characterized the helicase activity of transcription termination factor rho on a variety of substrates. Helicase activity requires specific recognition of a single-stranded region of RNA upstream (5') of the nucleic acid duplex on which rho acts. Spacer sequences of at least 450 nucleotides can be inserted between the rho-binding signals and the duplex region with little effect on activity. RNA-DNA helices of up to 120 base pairs, but not as long as 210 base pairs, can be disrupted efficiently by rho. The stoichiometry of release of substrates with long spacer sequences, as with the standard substrate, approaches a value of one RNA released per rho hexamer; thus cooperative binding by rho does not account for action at a distance. Instead, these results are consistent with a model in which a single rho hexamer binds initially to terminator sequences and then either loops out or tracks along the intervening RNA to reach the duplex region. Results with complex substrates are inconsistent with looping and support the tracking model: under conditions that allow disruption of RNA-DNA, but not RNA-RNA helices (0.4 mM Mg2+), the presence of a short RNA-RNA helix acts as a block to the disruption of an RNA-DNA helix downstream. These findings are discussed in relation to the mechanism of the helicase activity as well as its role in rho-dependent transcription termination.

Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis.

The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.

Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor.

We have utilized oligonucleotide site-directed mutagenesis to test our prediction that Escherichia coli rho factor has an ATP-binding domain separate from its RNA-binding domain and similar to that of adenylate kinase. Single amino acid substitutions were generated in regions thought to be within the active site and catalytically important for the ATPase activity, changing lysine 181 and/or lysine 184 to glutamine, and aspartate 265 to valine and asparagine. The altered proteins were purified and characterized in vitro for RNA- and ATP-binding ability, ATPase activity, helicase activity, and ability to catalyze transcription termination. Our results indicate that 1) these amino acid alterations in the proposed ATP-binding domain do not interfere with RNA binding; 2) substitution of lysine 184 by glutamine actually improves the ATPase and related activities while the same substitution at lysine 181 reduces but does not eliminate activity; 3) the double mutation changing both lysine 181 and lysine 184 to glutamine eliminates ATPase activity; and 4) the aspartate at 265 is also required for ATP hydrolysis but not for ATP binding. These results are consistent with our proposal that the general tertiary structure of rho's ATP-binding domain is similar to that of adenylate kinase.