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We previously used a mycobacteriophage L5-derived integrating vector to demonstrate that glnE and aroK are essential genes in Mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. We tested three systems to replace the integrated copy with alternative alleles. The most efficient method was to transform the strain with a second copy of the integrating vector. Excision of the resident vector and integration of the incoming vector occurred at an extremely high efficiency. This technique will allow us to study the role and functionality of essential genes in this important human pathogen.

The ability of Mycobacterium tuberculosis auxotrophs to survive long-term starvation was measured. Tryptophan and histidine auxotrophs did not survive single-amino-acid starvation, whereas a proline auxotroph did. All three auxotrophs survived complete starvation. THP-1 cells were also able to restrict the growth of the tryptophan and histidine auxotrophs.

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3-regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3-regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.

The acetamidase of Mycobacterium smegmatis is an inducible enzyme which enables the organism to utilise several amides as sole carbon sources. The acetamidase structural gene (amiE) is located downstream of four other genes, of which three form a probable operon with amiE; the fourth (amiC) is divergently transcribed. We constructed deletion mutants in two of these genes in order to determine their role in acetamidase expression. Both AmiC and AmiD were shown to be positive regulators of acetamidase expression required for induction. Combinations of regulatory gene deletions were made which revealed that AmiC interacts with the previously characterised negative regulator AmiA, whereas AmiD does not.

Two-component regulatory signal transduction systems are widely distributed among bacteria and enable the organisms to make coordinated changes in gene expression in response to a variety of environmental stimuli. The genome sequence of Mycobacterium tuberculosis contains 11 complete two-component systems, four isolated homologous regulators, and three isolated homologous sensors. We have constructed defined mutations in six of these genes and measured virulence in a SCID mouse model. Mice infected with four of the mutants (deletions of devR, tcrXY, trcS, and kdpDE) died more rapidly than those infected with wild-type bacteria. The other two mutants (narL and Rv3220c) showed no change compared to the wild-type H37Rv strain. The most hypervirulent mutant (devRdelta) also grew more rapidly in the acute stage of infection in immunocompetent mice and in gamma interferon-activated macrophages. These results define a novel class of genes in this pathogen whose presence slows down its multiplication in vivo or increases its susceptibility to host killing mechanisms. Thus, M. tuberculosis actively maintains a balance between its own survival and that of the host.

Motivation: Microarray experiments are inherently noisy. Replication is the key to estimating realistic fold-changes despite such noise. In the analysis of the various sources of noise the dependency structure of the replication needs to be taken into account., Results: We analyzed replicate data sets from a Mycobacterium tuberculosis trcS mutant in order to identify differentially expressed genes and suggest new methods for filtering and normalizing raw array data and for imputing missing values. Mixed ANOVA models are applied to quantify the various sources of error. Such analysis also allows us to determine the optimal number of samples and arrays. Significance values for differential expression are obtained by a hierarchical bootstrapping scheme on scaled residuals. Four highly upregulated genes, including bfrB, were analyzed further. We observed an artefact, where transcriptional readthrough from these genes led to apparent upregulation of adjacent genes., Availability: All methods and data discussed are available in the package YASMAhttp://www.cryst.bbk.ac.uk/wernisch/yasma.html for the statistical data analysis system R (http://www.R-project.org).

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.

To begin to understand the role of Mycobacterium smegmatis dnaA in DNA replication, the dnaA gene was characterized at the genetic level. Western analyses revealed that DnaA accounts for approximately 0.18% of the total cellular protein during both the active and stationary growth periods. Expression of antisense dnaA RNA reduced viability, indicating that dnaA is an essential gene in replication. To further understand the role(s) of dnaA in replication, a conditionally expressing strain was constructed in which expression of dnaA was controlled by acetamide. Growth in the presence of 0.2% acetamide elevated the intracellular levels of DnaA and increased cell length, but did not affect viability. Visualization of DNA by fluorescence microscopy revealed that DnaA-overproducing cells were multinucleoidal, indicating a loss of synchrony between the replication and cell-division cycles. Withdrawal of acetamide resulted in the depletion of the intracellular levels of DnaA, reduced viability and gradually blocked DNA synthesis. Acetamide-starved cells were very filamentous, several times the size of the parent cells and showed either abnormal or multi-nucleoid morphology, indicating a blockage in cell-division events. The addition of acetamide to the starved cells restored their viability and shortened the lengths of their filaments back to the size of the parent cells. Thus, both increasing and decreasing the levels of DnaA have an effect on the cells, indicating that the level of DnaA is critical to the maintenance of coordination between DNA replication and cell division. It is concluded that DNA replication and cell-division processes in M. smegmatis are linked, and it is proposed that DnaA has a role in both of these processes.

Attempts to construct Mycobacterium tuberculosis strains with a defect in the common aromatic amino acid biosynthesis pathway were made. In other bacteria the genes of this pathway (aro) can be disrupted in the presence of suitable media supplements. The genomic organization of the aro genes in M. tuberculosis reveals that there is one operon (aroCKBQ) and three isolated aro genes (aroE, aroG and aroA). The aroK gene was chosen as a target for disruption; this encodes shikimate kinase, which catalyses the fifth step in chorismate biosynthesis. Attempts to replace the wild-type aroK gene with a disrupted allele (aroKDelta::hyg) by a two-step homologous recombination procedure were unsuccessful in a wild-type strain. When a second functional copy of aroK was integrated into the chromosome, it was possible to isolate a strain carrying the disrupted gene. Excision of the L5-integrated copy of aroK by the L5 excisionase could be not be achieved in the strain carrying the disrupted copy, but was possible in a strain carrying a wild-type copy. These results demonstrate that the chorismate pathway is essential for the viability of M. tuberculosis.

The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements. To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains. Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M. tuberculosis. Furthermore, by using a surface plasmon resonance technique we showed that purified M. tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that M. tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M. tuberculosis oriC replication are different for chromosomes and plasmids.

Objective and Importance: The presence of a residual arteriovenous malformation (AVM) on postoperative angiograms is typically an indication for prompt return to the operating room to complete resection, because of the risk of early hemorrhage. This approach, however, may involve risks of neurological deficits when the residual AVM is in eloquent cortex. We present a case of complete thrombosis of a residual AVM after surgery. This residual AVM tissue was located in eloquent cortex. Complete spontaneous thrombosis of residual AVMs after surgery has only rarely been reported. This phenomenon raises questions regarding the most appropriate management for residual AVMs in eloquent cortex., Clinical Presentation: The patient was a 43-year-old, right-handed, male patient with an AVM centered in the left precentral gyrus. The patient presented with medically intractable seizures and progressive right hemiparesis. After AVM resection, angiography revealed a residual AVM with early venous drainage. Angiography performed 1 week later demonstrated a persisting AVM nidus without early venous drainage. Angiography performed 3 months later demonstrated complete thrombosis of the residual AVM., Intervention: The patient has been monitored for more than 1 year, without additional symptoms or therapy., Conclusion: We continue to advocate prompt return to the operating room when postoperative angiography reveals a residual AVM with persistent shunting. When the residual AVM is in eloquent cortex and is small, with a single draining vein, however, observation of the patient (with strict blood pressure control) and repeat angiography after 1 week represent an alternative strategy that is supported by this case report. As this case demonstrates, it is possible for small residual AVMs to thrombose. This may avert the need for reoperation for residual AVMs in eloquent cortex, with the potential for neurological deficits.

Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

Background: The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation., Results: We constructed a deletion mutant in one of the upstream ORFs (amiA). This mutant (Mad1) showed a constitutively high level of acetamidase expression. We identified four promoters in the upstream region using a beta-galactosidase reporter gene. One of these (P2) was inducible in the wild-type, but was constitutively active in Mad1., Conclusions: These results demonstrate that amiA encodes a negative regulatory protein which interacts with P2. Since amiA has homology to DNA-binding proteins, it is likely that it exerts the regulatory effect by binding to the promoter to prevent transcription.

Gene replacement by homologous recombination (HR) is an invaluable tool in understanding the physiology and the significance of specific genes in the virulence of Mycobacterium tuberculosis. It will also allow for the development of rationally attenuated strains as candidate vaccines to prevent the spread of tuberculosis. Classically, allelic replacement involves the introduction of nonreplicating DNA (suicide plasmids) carrying a mutated copy of the targeted gene, most often disrupted by an antibiotic resistance determinant, into the chromosome. A single recombination event (cross-over) between the two alleles will result in integration of the entire plasmid to generate a single crossover (SCO) strain carrying both wild-type and mutated copies of the gene. If two recombination events occur, a double cross-over (DCO) is generated where the wild-type allele is replaced by the mutant allele. Strains with an SCO can also give rise to DCO strains when a second recombination event takes place (Fig. 1).

Unlabelled: SWTTING: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome., Objective: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential., Design: We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid., Results: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels., Conclusion: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods., (Copyright 2001 Harcourt Publishers Ltd.)

Mycobacterium tuberculosis possesses a homologue of glnE, potentially encoding a regulator of glutamine synthetase activity. We attempted to construct glnE-disrupted mutants using a two-step strategy, whereby a single-crossover strain was first isolated, followed by sacB counterselection to isolate the double-crossover strain. Of 192 sucrose-resistant colonies tested, none were mutants, although the wild-type double crossover could be easily isolated. When a second copy of the wild-type glnE was integrated into the chromosome, we could isolate both wild-type and mutant double-crossover strains. Thus, the chromosomal gene could only be replaced with a disrupted copy when another functional copy of the gene was provided, demonstrating that this gene is essential under the conditions tested.

Progress in the field of mycobacterial research has been hindered by the inability to readily generate defined mutant strains of the slow-growing mycobacteria to investigate the function of specific genes. An efficient method is described that has been used to generate several mutants, including the first double unmarked deletion strain of Mycobacterium tuberculosis. Four mutants were constructed: a marked deletion of the plcABC cluster, which encodes three phospholipases C; separate unmarked deletions in plcABC and tlyA (encoding a haemolysin); and a double unmarked mutant tlyADelta plcABCDelta. To accomplish this, two series of vectors were designed, the first of which, named pNIL, allows manipulation of the target gene sequence at a variety of convenient restriction sites. The second series, named pGOAL, contains marker cassettes flanked by PAC:I restriction enzyme sites. The final suicide plasmid vectors were then obtained by cloning a marker cassette from a pGOAL vector into the single PAC:I site of the pNIL vector with the modified gene of interest. Finally, a two-step strategy was employed whereby single cross-over events were first selected, then screening for the second cross-over was carried out to yield the mutant strains. This technique will now allow the construction of potential vaccine strains without the inclusion of antibiotic resistance markers, the ability to make multiple defined mutations and the possibility of making more subtle defined mutations, such as point mutations.

The use of molecular techniques to study the mycobacteria has advanced greatly since the first genomic libraries of Mycobacterium tuberculosis and M. leprae were constructed in 1985. However, there are still pitfalls for the unwary. Most of the problems associated with the use of molecular techniques to study mycobacteria can be related to one of the following problems: slow growth rate causing problems with contamination; the formation of macroscopic clumps when grown in culture; resistance to standard chemical lysis procedures; the requirement for containment facilities for pathogenic species; the lack of suitable genetic vectors; and the problems of spontaneous antibiotic resistance. Despite these problems, considerable progress has been made and standard techniques have been developed for the preparation of protein, nucleic acids (DNA and RNA) and cell wall components, chemical and transposon mutagenesis and gene replacement methods, the use of reporter genes and expression vectors, and improved detection and drug sensitivity testing.

The ability to generate mutants of Mycobacterium tuberculosis will be important if we are to understand the biology of this major pathogen. However, allelic replacement methods have only recently achieved success. We have developed a reproducible method for generating defined mutants of M. tuberculosis using homologous recombination. The transforming DNA was used following pre-treatment either with UV light or alkali denaturation in order to stimulate homologous recombination and abolish illegitimate recombination. Suicide vectors carrying one of nine amino acid biosynthesis genes were electroporated into M. tuberculosis, and homologous recombinants were obtained in all nine genes; eight resulted from single-crossover events (SCOs) and one from a double-crossover event (DCO) (in the metB gene). The remaining colonies were spontaneous hygromycin-resistant mutants; no products of illegitimate recombination were observed. To more efficiently distinguish spontaneous mutants, the lacZ gene was cloned into five vectors (two containing genes not previously tested), and the transformations were repeated. SCO mutants were identified by screening for blue colonies on indicator plates. White transformants were tested for auxotrophy and trpD, hisD and proC auxotrophic mutants were obtained. Only blue SCOs were obtained for argF and glnE. Thus, using this methodology we have obtained homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is possible to generate targeted mutants in a reproducible way.

Allelic replacement will be a vital tool for understanding gene function in mycobacteria. Disruption of the chromosomal hisD gene of Mycobacterium smegmatis by standard gene replacement methods was surprisingly difficult, with most products being caused by illegitimate recombination (IR) events. A recombination assay was therefore developed and used to optimize conditions for homologous recombination (HR) in M. smegmatis. Treatment of competent cells with UV, hydrogen peroxide or mitomycin C did not improve the frequency of HR; however, treatment of the DNA with alkali or UV enhanced recombination frequency, while boiling did not. Applying these observations to allele replacement, UV and alkali treatment of transforming DNA increased HR events with pyrF and hisD, while the level of IR was unchanged. The introduction of ss phagemid DNA improved the level of HR and abolished IR. In Mycobacterium intracellulare the use of alkali-denatured DNA increased the numbers of recombinants obtained with an inactivated 19Ag gene, while in Mycobacterium tuberculosis, inactivation of a putative haemolysin gene, tlyA, was achieved using both UV-irradiated DNA and ss phagemid DNA. Significantly, IR, which has been reported to be a problem in this species, was not observed. Thus, four genes in three species were successfully knocked-out using non-replicating DNA pretreated with alkali, UV or in an ss form. The use of these methods to enhance HR will greatly facilitate experiments to inactivate other genes in these important species.

A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.

There is still an urgent requirement for more sensitive, cost-effective methods for detection and susceptibility testing of mycobacteria in clinical samples. We have been investigating a simple bacteriophage-based system which could be used for both purposes. As this depends upon the detection of phages which have successfully infected cells, a key step is the efficient removal or inactivation of phages remaining free in the culture medium. We demonstrate here the use of ferrous ammonium sulphate as an effective agent for the inactivation of mycobacteriophage D29 without impairing phage replication in previously infected host bacteria. Using this property, we report the detection of viable Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis using simple low-cost technology. The method is highly sensitive, since it is able to detect 10 colony-forming units of M. smegmatis. It is also rapid, with the detection of M. tuberculosis in sputum specimens within 48 h.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.

In the present study, high school students who received training in either behavioral relaxation or progressive muscle relaxation demonstrated significantly lower state anxiety scores than did those who had not received such training. No significant differences were found on trait anxiety scores. There was no significant effect of gender and also no significant interaction effects between the three groups and gender for either state or trait anxiety. Implications of these findings for high school counselors who work with students dealing with anxiety-producing problems are presented.

A bacteriophage infection mutant (strain LIMP7) of Mycobacterium smegmatis was isolated following transposon mutagenesis. The mutant showed an unusual phenotype, in that all phages tested produced larger plaques on this strain compared to the parent strain. Other phenotypic characteristics of the mutant were slower growth, increased clumping in liquid culture, increased resistance to chloramphenicol and erythromycin, and increased sensitivity to isoniazid and several beta-lactam antibiotics. Permeability studies showed decreases in the accumulation of lipophilic molecules (norfloxacin and chenodeoxycholate) and a small increase with hydrophilic molecules (cephaloridine); taken together, these characteristics indicate an altered cell envelope. The DNA adjacent to the transposon in LIMP7 was cloned and was shown to be highly similar to genes encoding bacterial and mammalian inositol monophosphate phosphatases. Inositol is important in mycobacteria as a component of the major thiol mycothiol and also in the cell wall, with phosphatidylinositol anchoring lipoarabinomannan (LAM) in the cell envelope. In LIMP7, levels of phosphatidylinositol dimannoside, the precursor of LAM, were less than half of those in the wild-type strain, confirming that the mutation had affected the synthesis of inositol-containing molecules. The impA gene is located within the histidine biosynthesis operon in both M. smegmatis and Mycobacterium tuberculosis, lying between the hisA and hisF genes.

Expression from a 2.3 kb region upstream of the inducible acetamidase gene from Mycobacterium smegmatis was shown to be upregulated by acetamide. A DNA fragment containing the start of the M. smegmatis hisD gene was cloned in front of the promoter, such that the antisense message was produced. When this construct was induced in vivo, the bacteria became phenotypically histidine auxotrophs; this auxotrophy was restored by histidine supplementation. Auxotrophy was not observed under non-induced conditions. Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes, and an inducible system such as that described would allow the study of essential genes.

Certain monoclonal antibodies (MAbs) directed against CD4 can efficiently block HIV-1 replication in vitro. To explore CD4-directed passive immunotherapy for prevention or treatment of AIDS virus infection, we previously examined the biological activity of a nondepleting CD4-specific murine MAb, mu5A8. This MAb, specific for domain 2 of CD4, blocks HIV-1 replication at a post-gp120-CD4 binding step. When administered to normal rhesus monkeys, all CD4+ target cells were coated with antibody, yet no cell clearance or measurable immunosuppression occurred. However, strong anti-mouse Ig responses rapidly developed in all monkeys. In the present study, we report a successfully humanized form of mu5A8 (hu5A8) that retains binding to both human and monkey CD4 and anti-AIDS virus activity. When administered intravenously to normal rhesus monkeys, hu5A8 bound to all target CD4+ cells without depletion and showed a significantly longer plasma half-life than mu5A8. Nevertheless, an anti-hu5A8 response directed predominantly against V region determinants did eventually appear within 2 to 4 weeks in most animals. However, when hu5A8 was administered to rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques, anti-hu5A8 antibodies were not detected. Repeated administration of hu5A8 in these animals resulted in sustained plasma levels and CD4+ cell coating with humanized antibody for 6 weeks. These studies demonstrate the feasibility of chronic administration of CD4-specific MAb as a potential means of treating or preventing HIV-1 infection.

The inducible acetamidase of Mycobacterium smegmatis NCTC 8159 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. After induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.

The purpose of this study was to determine whether rugae patterns change with age and to compare the number and pattern of rugae in Australian Aborigines with those of Caucasians. For the longitudinal part of the study, serial dental casts of ten Aborigines, from 6 to 20 years of age, were examined and rugae patterns were recorded. To enable comparisons to be made between different ethnic groups an additional 100 dental casts of Australian Aborigines and 200 casts of caucasians, ranging in age from 13 to 17 years, were examined. Characteristics observed were number, length, shape, direction and unification of rugae. The length of rugae increased significantly with age but the total number of rugae remained constant. Thirty-two per cent of rugae showed changes in shape, while 28 per cent displayed a change in orientation. In contrast to studies suggesting that rugae move forward with age, the majority of Aboriginal rugae that changed direction moved posteriorly. Changes in rugae patterns have been assumed to result from palatal growth but alterations in pattern were observed in the Aboriginal sample even after palatal growth had ceased. The mean number of primary rugae in Aborigines was higher than in Caucasians, although more primary rugae in Caucasians exceeded 10 mm in length than in Aborigines. The most common shapes in both ethnic groups were wavy and curved forms, whereas straight and circular types were least common. There was a statistically significant association between rugae forms and ethnicity, straight forms being more common in Caucasians whereas wavy forms were more common Aborigines.

In this study 128 college students were surveyed regarding many of their perceptions concerning themselves and their family members (i.e., self-concepts, evaluations of parents, how they interact with their parents, and how their parents interact with each other). Nearly all of the respondents' perceptions of these individuals and how they interacted with one another were found to be significantly correlated. Such findings suggest that family members need to be more attentive to the messages they convey through their actions as well as their attitudes, and strive to be as positive as possible if they truly wish to benefit all concerned.

Research has generally supported the view that parents' attitudes and practices predispose children to act in certain ways. Bell (1968), however, proposed an alternate theory which suggested that children and adolescents often mold the way their parents act. Parish (1980) subsequently reported support for Bell's position in that parents were, indeed, found to parent like one another, possibly in response to their children's actions. The present study sought to further examine the Bell (1968) theory by seeking to determine if parents are consistent with each other or with themselves in their parenting attitudes and practices. Parents were perceived to act like one another (at least to a moderately significant degree), but fathers were much more likely to parent in particular ways (i.e., if they were restrictive they were also more likely to be warm, or if they were permissive they were also more likely to be hostile), independent of how their wives were perceived to act. The implications of these findings are discussed.

Background: Progressive ventricular remodeling after myocardial damage is associated with a poor prognosis. Optimal prevention of the histopathological processes involved in remodeling requires a more complete understanding of the mechanisms involved in initiating and maintaining these structural changes. Since the sympathetic nervous system and the renin-angiotensin system may be involved in the remodeling process, the structural effects of pharmacological inhibitors have been evaluated in a canine model of localized myocardial injury resulting from transmyocardial DC shock., Methods and Results: The study is comprised of two protocols run in series. In protocol 1, zofenopril (Z), a converting enzyme inhibitor (CEI), prevented the increase in left ventricular mass (LVM) and end-diastolic volume (LVV) observed in the control group (C) at 16 weeks (Z: LVM, 69.8 +/- 3.4 to 65.4 +/- 2.6 g, P = NS; LVV, 45.4 +/- 2.7 to 51.6 +/- 2.7 mL, P = NS; C: LVM, 68.4 +/- 3.2 to 91.4 +/- 2.9 g, P = .0001; LVV, 56.6 +/- 3.0 to 71.9 +/- 2.4 mL, P = .0003). Terazosin, an alpha 1-adrenoceptor antagonist, failed to prevent remodeling at 16 weeks despite continued receptor blockade. In protocol 2, the antiremodeling effect of full-dose CEI therapy with ramipril was confirmed. Low-dose ramipril that exerted no hemodynamic effect failed to prevent remodeling (LVM, 89.7 +/- 4.6 to 105.7 +/- 3.4 g, P = .01; LVV, 61.8 +/- 3.8 to 76.8 +/- 3.3 mL, P = .002). An angiotensin II subtype 1 receptor blocker also failed to prevent the increase in LVM or LVV (LVM, 89.0 +/- 4.6 to 109.7 +/- 5.3 g, P = .0001; LVV, 66.0 +/- 1.9 to 78.4 +/- 3.6 mL, P = .007)., Conclusions: High-dose CEI therapy can prevent progressive structural changes resulting from localized myocardial damage induced by DC shock. the failure of alpha 1-adrenoceptor blockade and angiotensin II subtype 1 blockade to attenuate remodeling argues against an important direct role for norepinephrine acting through alpha 1-receptors or angiotensin II acting through the type 1 receptor in the remodeling process in this model.

This study surveyed 188 middle school children in order to ascertain their perceptions of their parents' actions toward one another. In addition, they evaluated themselves and their parents. These data were subsequently correlated with each other. It was found that ratings of fathers generally varied with how loving they were toward their wives and how loving their mothers were toward their husbands. Ratings of mothers only varied with how loving they were toward their husbands, and not vice versa, but only for their daughters--not for their sons. Self-concepts also generally varied for both sex groups with how loving their fathers were toward their wives, but how loving mothers were toward their husbands was only directly related to how their daughters--not their sons--rated themselves. Finally, how fathers acted toward their wives was significantly correlated with how their wives acted toward them. Some implications of these findings are discussed.

This paper describes the construction of pSG10, the first mycobacterial promoter probe shuttle vector to use the structural gene of a bacterial luciferase as a reporter gene. To examine the utility of using bacterial luciferase to measure gene expression in mycobacteria, the authors have used this vector to monitor the induction of the acetamidase gene promoter of Mycobacterium smegmatis. Luciferase proved to be a rapid, sensitive and easily assayable reporter of changes in gene activity in response to environment in mycobacteria.

In this study 212 high school students from different familial configurations were compared regarding their descriptions of themselves and their parents, as well as how their parents interacted with each other. Findings revealed that fathers' ratings or evaluations, but not mothers' ratings or the students' self-ratings, suffered in the wake of divorce. Regarding perceived "loving" actions by parents, however, students from intact families clearly had an advantage over their counterparts from reconstituted, divorced, and single-parent families. Implications of the findings are discussed.

This paper reports changes in the incidence and manifestations of decompression illness in amateur scuba divers in the United Kingdom (UK) between 1981 and 1993, a period during which the popularity of the sport increased. Since 1981, there has been a trend to increased annual incidence of decompression illness, but the large yearly fluctuations reflect a considerable annual variation in the numbers of dives. The need for recompression facilities to treat decompression illness in amateur scuba divers in the UK should take account of this greater public participation in the sport, and should also allow for large annual fluctuations related to meteorological and financial factors.

In the present study, 64 college students described how their parents acted toward one another and also evaluated their families and various family members. Many significant correlations were found. Perceptions of parents' actions toward one another were associated with respondents' ratings of their families and individual family members. Interestingly, for females, but not males, there was a strong relationship between perceptions of how their fathers interacted with their mothers and how their mothers interacted with their fathers. Such findings lend support to the notion that ongoing parental interactions, and not just traumatic events such as parental death or divorce, are related to how youth and young adults subsequently view their families and the individuals within them.

In the present study, 130 college students voluntarily described the support system failures they encountered during their childhood and/or adolescence through their responses on the Personal History Inventory and their ratings of self and family on the Personal Attribute Inventory. Significant direct relationships were found between the level of support system failures and the negativeness expressed in their ratings of both self and family. Notably, however, these significant relationships applied only to individuals from intact families, and not those from families divided either by parental divorce or death of a parent. Possible explanations are offered to account for these findings.

The present study assessed the self-concepts and evaluations of parents of a sample of high school students. Findings indicated that students' evaluations of parents, but not self-concepts, were significantly correlated with the perceived "loving" actions engaged in by their parents toward one another. These findings are in marked contrast to the results reported by Parish (1988), who surveyed college students, and suggest that more research is needed to determine if these findings are artifacts of the samples surveyed, or are indicative of developmental shifts that occur as individuals move from adolescence to adulthood.

In the present study, 123 college students were surveyed in order to assess their self-concepts, evaluations of parents, and perceptions of their parents' parenting styles. Notably, the students' self-concepts were found to vary directly with perceived level of parental warmth, but did not vary as a function of their parents' level of restrictiveness. Fathers and mothers were found to be rated more highly if they were perceived as being warm and permissive rather than hostile and restrictive. Finally, opposite-sex parents' level of warmth also correlated with how each parent was evaluated. Some explanations for these findings are offered.

This study examined differences between high school students who were at risk for school failure and a control group of peers. Statistically significant differences were found with respect to locus of control, self-concept, and personal styles of learning. Implications focused upon approaches and suggestions regarding the use of such knowledge in facilitating improved adjustment and achievement in at-risk students.

This study examined the differential effects of familial configuration and support system failures in a sample of 256 college students. Regarding familial configuration, the results indicated that individuals who had experienced parental divorce were significantly more likely to have encountered parental hostility and/or lack of care, inadequate supervision when not in school, lack of concern by teachers, and more financial hardship. In turn, lower self-concepts and/or social skills were found to be associated with these kinds of life experiences (i.e., support system failures).

In the present study, 648 youths from across the state of Kansas voluntarily evaluated themselves and their parents using the Personal Attribute Inventory for Children. Self-concept was found to be significantly higher for those from intact families in comparison with those from divorced remarried families. Evaluations of mothers were significantly higher for those from intact and divorced nonremarried families as compared with those from divorced remarried families. The ratings of fathers by youths from intact families were significantly more favorable than the ratings by those from either divorced nonremarried or divorced remarried families. Interestingly, gender by family status two-way interaction effects were also found for self-concept and ratings of fathers. Possible explanations for these findings, and their implications, are discussed.

Background: Severe plaque-type psoriasis has been successfully treated with orally administered cyclosporine, but there has been no comparative, controlled evaluation of various dosages and their efficacy and side effects., Methods: In a 16-week, double-blind trial, we randomly assigned 85 patients with severe psoriasis to receive 3, 5, or 7.5 mg of cyclosporine per kilogram of body weight per day or a placebo consisting of the vehicle for the drug. After eight weeks the dose could be adjusted to improve safety or efficacy while maintaining blinding., Results: The psoriasis improved in a dose-dependent fashion. After eight weeks of fixed-dose therapy, 36, 65, and 80 percent of the patients receiving 3, 5, and 7.5 mg of cyclosporine per kilogram per day, respectively, were rated as being clear or almost clear of psoriasis; each group had significant improvement (P less than 0.0001) as compared with the group receiving vehicle, in which none of the patients were rated as clear or almost clear. The patients who received 5 mg per kilogram were the least likely to require dosage adjustments because of side effects or a lack of efficacy. The glomerular filtration rate, measured in a subgroup of 34 patients receiving cyclosporine, decreased by a median of 16 percent. Higher doses of cyclosporine had greater adverse effects on systolic blood pressure, glomerular filtration rate, and serum levels of creatinine, uric acid, bilirubin, and cholesterol. Delayed-type hypersensitivity reactions to skin-test antigens were reduced by cyclosporine administration. Cyclosporine appears to become concentrated in skin., Conclusions: Cyclosporine therapy leads to a rapid and thorough clearing of psoriasis; an initial dose of 5 mg per kilogram per day seems to be appropriate. However, the safety of cyclosporine for the long-term treatment of psoriasis remains to be determined.