Coadministration of mefloquine and chloroqine: use of a pharmacokinetic-based approach to reduce toxicity.

A child with malaria from a chloroquine-resistant area received an accidental overdose of chloroquine administered by a parent. Application of pharmacokinetics permitted definitive treatment with mefloquine in a safe and effective manner.

Characterization of deletion and frameshift mutants of satellite tobacco mosaic virus.

A series of frameshift and deletion mutations was created in the genome of satellite tobacco mosaic virus (STMV) by modifying full-length cDNA clones of the type strain, from which biologically active transcripts could be synthesized in vitro. Deletions and frameshift mutations in the 5' open reading frame had no effect, compared to wild-type STMV, on RNA accumulation, systemic movement, or the symptoms induced by STMV in Nicotiana tabacum co-inoculated with tobacco mild green mosaic tobamovirus (TMGMV). This implies that the protein encoded by this reading frame is not necessary for biological activity. Deletions and frameshift mutations in the coat protein open reading frame resulted in decreased accumulation of STMV RNA in N. tabacum, although these mutants were still capable of systemic movement, presumably in a nonencapsidated or free RNA form. Furthermore, the mild symptoms induced in tobacco by co-inoculations of wild-type STMV/TMGMV or infection with TMGMV alone were altered to severe systemic necrosis when plants were co-inoculated with these STMV coat protein mutants and TMGMV. Mutants within the 3' untranslated region were much less able to accumulate in TMGMV-infected plants than was wild-type STMV, and under some growth conditions did not accumulate to detectable levels.

Pediatric firearm injuries, Kansas City, 1992: a population-based study.

OBJECTIVE: To determine the morbidity, mortality, and epidemiologic features of pediatric powder-firearm injuries in a defined urban population. DESIGN AND SETTING: A population-based, descriptive epidemiologic study was conducted of firearm injuries to children in a mid-size urban community (total population: 435,178) in 1992. The population was 56% white and 39% black. Data from prehospital care providers, all city and adjacent community hospitals, and medical examiner and police records were searched for cases of firearm injury. The 1990 United States census provided denominator data. CASE DEFINITION: Subjects were all 0- to 16-year-old residents of Kansas City, Missouri who sought medical treatment at a hospital for a powder-firearm injury or who presented to the medical examiner with a fatal firearm injury in calendar year 1992. RESULTS: Seventy-two children met the case definition, for an incidence of 70 per 100,000 persons per year. There were 12 (16.7%) fatalities, for a mortality rate of 11.7 per 100,000 persons per year. Almost 10% of the patients sustained permanent disability. Mean and median ages of the patients were 14.9 years and 15.8 years, respectively; 79% were male and 82% were black. The majority of the children (63%) lived in census tracts with a high proportion of families in poverty. Black males had the highest rates of firearm injury, with a 1-year incidence of 233 per 100,000 persons per year. At younger than 12 years, the rates were equal among the races; however, for those 12 years and older, black adolescents had 13 times the risk of white adolescents (541 compared to 42 per 100,000 persons per year). The majority (71%) of injuries were due to assaults, with drive-by shootings the most frequent circumstance. The majority of unintentional injuries occurred to adolescents as the result of an unplanned discharge of a handgun as it was being placed in or removed from concealment. Among the patients, 39% were admitted to the hospital and 26% required surgery. CONCLUSIONS: 1) Black male adolescents had the highest risk of firearm injury or fatality. 2) The majority of victims lived in census tracts characterized by poverty. 3) injuries were alarmingly severe. 4) Interpersonal violence was the leading contributor to fatal and nonfatal injuries. 5) Unintentional injuries characteristically occurred during the process of weapon concealment. 6) The leading contributor to injury and death was the interaction of adolescents and guns, particularly handguns. The main implication for firearm-injury prevention in this population is the limiting of access to guns by adolescents. In addition, measures aimed at preventing violent behavior, such as education in nonviolent methods of conflict resolution, should be explored.

The interpretation of urogenital findings in children with straddle injuries.

Because urogenital trauma frequently raises the question of sexual abuse, it is important to be able to relate the mechanism of injury to expected examination findings. This study was undertaken to characterize the trauma that results from straddling and correlate such injuries with the history, examination, and patient characteristics. The charts of 100 patients examined in an urban pediatric emergency department were reviewed; their conditions met the criteria of straddle injury--a blow to the perineum as a result of falling or striking a surface or an object with the force of one's own body weight. Ages ranged from 9 to 187 months (mean, 77.9; median, 67.2); 72% were female. Most injuries were minor lacerations and abrasions of the genitalia. Eleven percent had injury to the posterior fourchette. Hymenal and vaginal injuries were primarily caused by penetrating mechanisms. Five patients who presented with a history of straddling subsequently received the diagnosis of sexual assault based on disclosure by the patient or a witness and inconsistency of physical findings. There were no urethral or perianal injuries resulting from nonpenetrating straddle mechanisms. Straddle injuries include a variety of mostly minor urogenital injuries. Perianal, hymenal, or vaginal trauma suggests a penetrating mechanism, either unintentional or from sexual assault. An investigation for sexual assault should be initiated in the following cases: infants younger than 9 months of age; perianal, hymenal, or vaginal injury; extensive or severe injury; concurrent nonurogenital injuries; and whenever there is lack of correlation between history and physical findings.

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening.

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.

Factors affecting efficient infection of tobacco with in vitro RNA transcripts from cloned cDNAs of satellite tobacco mosaic virus.

Recombinant cDNA clones of the complete satellite tobacco mosaic virus (STMV) genome (1059 ribonucleotides) were constructed with unique Xbal and HindIII or Pstl restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of T7 or SP6 phage promoters. Genome-sense RNAs transcribed in vitro from the T7 promoter were biologically active, while negative-sense RNAs transcribed in vitro from the SP6 promoter were not. Constructs that were identical to STMV and two other constructs in which there were two or six specific nucleotide differences in the 3' noncoding region yielded RNAs that were infectious. Sequence analysis of the progeny RNA derived from infections with transcripts containing nucleotide differences between nucleotides 682 and 753 revealed that these changes in sequence were maintained. In contrast, differences in the nucleotide sequence between nucleotides 989 and 1059 were not maintained in progeny RNA; one mutant reverted to the wild-type sequence, and the other generated a new sequence during infection.

TAC use and absorption of cocaine in a pediatric emergency department.

The topical anesthetic TAC (tetracaine 0.5%, adrenaline 0.05%, cocaine 11.8%) has been reported to be effective in pain control for local procedures. However, it has the potential for cocaine toxicity by absorption through an open wound. A study was undertaken to assess the systemic absorption of cocaine and its metabolites when TAC is used as a local anesthetic. Fifty-one children, 1 to 14 years of age, were enrolled in the study. Plasma for cocaine and/or its metabolite levels was available from 46 children and obtained 20 to 40 minutes after the topical anesthetic was applied. No plasma sample had detectable parent cocaine levels; however, 26 (56.5%) had cocaine metabolite levels. Ecgonine methylester levels were detected in plasma from six children and ranged from 59 to 985 ng/mL. Benzoylecgonine levels were detected in none of 19 specimens not preserved with sodium fluoride, and in 23 of 27 specimens to which sodium fluoride had been added. Benzoylecgonine levels ranged from 40 to more than 600 ng/mL. No clinical sign of cocaine toxicity was observed in any child.

Analyses of the splenic mRNA expressed by rabbits of different immunoglobulin kappa-light chain allotypes: conserved sequences in the 3' untranslated region and allotype-specific probes.

The allelism of the structural genes for the complex rabbit b allotypes of immunoglobulin kappa-light chains has been questioned because of observations of unexpected phenotypic expression of "latent" allotypes. We find that the coding sequences of the b4 and b5 "alleles" are only 80% homologous for the last 60 nucleotides but there is a high degree of homology (96%) in the 3' untranslated region (3'DT). The high conservation of 3' DT region sequences enabled us to detect kappa-light chain mRNAs from rabbits of different genetic types (b4, b5, b9 and bbas) on northern blots and dot blots. We can distinguish mRNA encoding b9 and b5 allotypes on dot blots with b5 fragment-probes of known sequence and detect mRNA produced by unstimulated cultured splenic lymphocytes. Analyses of mRNA from cultured cells manipulated to enhance mRNA synthesis and production of unexpected or "latent" b allotypes can now be conducted.

Use of Trypanosoma equiperdum infected rabbits as a source of splenic mRNA; construction of cDNA clones and identification of a rabbit mu heavy chain clone.

Rabbits were infected by Trypanosoma equiperdum and the splenic mRNA was isolated. In vitro translation of this RNA and immunoprecipitation with anti-light chain, anti-heavy chain, anti-mu and anti-VH antibodies demonstrated that T. equiperdum infection elicits large quantities of splenic mRNA encoding mu and kappa chains. The mu and gamma heavy chains and the kappa light chains synthesized in the cell-free translation system were specifically immunoprecipitated by antisera to heavy chain VHa and light chain kappa b allotypes. In vitro labeling of spleen cells from trypanosome-infected animals demonstrated that the biosynthetically labeled IgM has a mu chain of higher molecular weight than the mu chain synthesized by in vitro translation, a difference that is largely abolished when cellular glycosylation is blocked with the antibiotic tunicamycin. Enrichment for heavy chain or light chain mRNA was achieved by fractionating mRNA from trypanosome-infected animals on a sucrose gradient. cDNA clones carrying mu heavy chain sequences were produced using a 'one tube' protocol and identified by cross species hybridization and hybridization selection. Infection of rabbits with T. equiperdum followed by sucrose gradient enrichment of splenic mRNA has provided sufficient quantities of mRNA encoding mu heavy chain suitable for cDNA cloning.

Construction of a partial rabbit spleen cDNA library and identification of immunoglobulin clones.

A partial cDNA library was constructed from total poly A(+)-RNA isolated from the spleen of a rabbit (kappa allotype b5; heavy chain allotypes a3d12e15) that had been hyperimmunized with Streptococcus pneumoniae (type III). In spite of the absence of either specific DNA probes for rabbit immunoglobulin (Ig) sequences or cross-hybridizing mouse Ig DNA probes, recombinant clones containing cDNA sequences of rabbit gamma heavy chain and kappa light chains were identified by a combination of screening techniques: (a) colony hybridization using labeled mRNA; (b) mRNA hybridization selection and translation and (c) hybridization to electrophoretically fractionated poly A(+)-RNA ("Northern" analysis). Sequencing of three kappa light chain recombinant DNA sequences, including part of the 3' untranslated (UT) region, has confirmed the fact that recombinant DNA for kappa light chain mRNA has been identified. An unexpectedly high degree of homology between the 3' UT region sequence of this DNA from a rabbit of b5 allotype and the published 3' UT sequence from a b4 rabbit was found. It appears that 3' UT sequences from b4 and b5 alleles have diverged less than the coding sequences for the constant regions. The functional significance of this conservation of 3' UT sequences remains to be elucidated.

Messenger RNA from allotype-defined rabbits directing the cell-free synthesis of immunoglobulin heavy and light chains.

In vitro synthesis studies were performed utilizing polyA(+)-RNA and lymphocytes from the spleens of rabbits hyperimmunized with Micrococcus luteus or Streptococcus pneumoniae (type III). PolyA(+)-RNA isolated after 4 M guanidinium thiocyanate extraction and oligo(dT)-selection appeared to be undegraded on CH3HgOH-agarose gel electrophoresis and demonstrated biological activity when translated in a rabbit reticulocyte cell-free system. The electrophoretic patterns of the specifically immunoprecipitated cell-free products were compared with those of Nonidet-P40 extracts (lysates) and secretions (supernatants) from rabbit spleen lymphocyte cultures and serum proteins. Kappa light chains with specific b allotypes, as well as immunoglobulin heavy chains, were identified. The efficient translation of mRNA of defined allotypes was a necessary prerequisite for production of characterized cDNA clones and identification of genomic sequences for rabbit immunoglobulin heavy and light chains.

Mouse immunoglobulin D: construction and characterization of a cloned delta chain cDNA.

TEPC 1017 is a BALB/c plasmacytoma that synthesizes IgD in large enough amounts to permit the isolation of mRNA for mouse delta chains. cDNA has been prepared from this mRNA, and an 880-base-pair fragment of it has been cloned by recombinant DNA techniques. The hybridization selection technique has been used to show that this cDNA clone specifically binds only mRNA that can be translated into immunoprecipitable delta chains. the sequence of a portion of this clone has been determined and, when translated, shows homology with the C delta 3 of a human myeloma protein. Using this cDNA clone as a probe, we have found that several different-sized delta RNAs are present in TEPC 1017 and in another IgD-secreting plasmacytoma, TEPC 1033.