Initial evaluation and management of the critical burn patient.

The major improvement in burn therapy is likely to focus on the early management of hemodynamic and respiratory failures in combination with an aggressive and early surgical excision and skin grafting for full-thickness burns. Immediate burn care by first care providers is important and can vastly alter outcomes, and it can significantly limit burn progression and depth. The goal of prehospital care should be to cease the burning process as well as prevent future complications and secondary injuries for burn shock. Identifying burn patients appropriate for immediate or subacute transfer is an important step in reducing morbidity and mortality. Delays in transport to Burn Unit should be minimized. The emergency management follows the principles of the Advanced Trauma Life Support Guidelines for assessment and stabilization of airway, breathing, circulation, disability, exposure and environment control. All patients with suspected inhalation injury must be removed from the enclosure as soon as possible, and immediately administer high-flow oxygen. Any patient with stridor, shortness of breath, facial burns, singed nasal hairs, cough, soot in the oral cavity, and history of being in a fire in an enclosed space should be strongly considered for early intubation. Fibroscopy may also be useful if airway damage is suspected and to assess known lung damage. Secondary evaluation following admission to the Burn Unit of a burned patient suffering a severe thermal injury includes continuation of respiratory support and management and treatment of inhalation injury, fluid resuscitation and cardiovascular stabilization, pain control and management of burn wound.

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.

Cloning and promoter analysis of the cotton lipid transfer protein gene Ltp3(1).

A cotton Ltp3 gene and its 5' and 3' flanking regions have been cloned with a PCR-based genomic DNA walking method. The amplified 2.6 kb DNA fragment contains sequences corresponding to GH3 cDNA which has been shown to encode a lipid transfer protein (LTP3). The gene has an intron of 80 bp which is located in the region corresponding to the C-terminus of LTP3. The Ltp3 promoter was systematically analyzed in transgenic tobacco plants by employing the Escherichia coli beta-glucuronidase gene (GUS) as a reporter. The results of histochemical and fluorogenic GUS assays indicate that the 5' flanking region of the Ltp3 gene contains cis-elements conferring the trichome specific activity of Ltp3 promoter.

Differential expression of a lipid transfer protein gene in cotton fiber.

A full-length cDNA clone, GH3, has been isolated from a cotton fiber cDNA library using a differential screening method. The nucleotide and derived amino acid sequence data show that GH3 encodes a lipid transfer protein (LTP) of 120 amino acids. The presence of a transmembrane signal peptide at the N-terminal of the protein would suggest its possible outer cellular location in fiber cells. Northern analysis indicates that the GH3 gene is developmentally regulated.

Cloning and characterization of a cotton lipid transfer protein gene specifically expressed in fiber cells.

A cotton genomic library was screened using a fiber-specific cDNA (GH3) encoding a lipid transfer protein (LTP). One genomic clone (1.7 kb DNA insert) containing the Ltp gene (Ltp6) was sequenced and characterized. The Ltp6 contains an open reading frame of 360 bp, which is interrupted by a single intron (136 bp) located in the region corresponding to the C-terminal of the protein. The derived amino-acid sequence of LTP6 is 64% homologous to that of GH3. Like the GH3 gene, the Ltp6 is specifically expressed in fiber cells in a temporal manner. However, its expression level is lower than that of GH3.

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA.

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.

Nucleotide sequence of the split tRNAleu(UAA) gene from Sorghum bicolor chloroplasts.

The nucleotide (nt) sequence of the split tRNAleu(UAA) gene and 328 nt of its flanking regions from sorghum chloroplasts (cp) has been determined. This gene is located in the BamHI-6 fragment in a map position very similar to that of maize. The exon of sorghum tRNAleu gene has an identical nt sequence to its counterpart in maize. Although the 450 nt of intron in sorghum is 8 nt shorter than that of maize, the nt sequence between them shows 97% homology. Like maize and broad bean, the intron from sorghum cp tRNAleu gene could be folded into a secondary structure which is similar to the postulated structure of the intron from the auto-spliceable rRNA precursor of Tetrahymena. Both introns from sorghum and maize contain open reading frames (ORFs) which are conserved at the N terminus. The putative AUG initiation codon for both ORFs is located in the stem region of a 12-bp secondary structure of highly A + T-rich sequences.

Cloning and determination of the nucleotide sequence of the Mn-containing superoxide dismutase gene from Halobacterium halobium.

A group of synthetic 17-mer oligodeoxynucleotides (oligos) was constructed to correspond to a sequence of amino acids situated near the N terminus of the manganese-containing superoxide dismutase (Mn-SOD) purified from the halophilic bacterium, Halobacterium halobium. A cosmid library of a Sau3AI partial digest of halobium DNA, cloned into the BamHI site of pHC79, was probed with the radiolabeled oligos. Cosmid DNA was purified from the clone that showed hybridization at the highest stringency. A 1.8-kb PstI fragment of this DNA which hybridized the probes was subcloned into bacteriophage M13 and transfected into Escherichia coli JM101. The entire insert containing a 600-bp sequence coding for Mn-SOD and its 5'- and 3'-flanking regions was sequenced. The derived amino acid sequence of the structural gene showed a similarity to other manganese and iron-containing SODs in normally conserved regions.

Nucleotide sequences of two regions of the human genome containing tRNAAsn genes.

The primary structures of two human tRNAAsn genes and 600-700 nucleotides of their flanking regions have been determined from two separate isolates of a fetal DNA library in phage lambda vector. The tRNA gene from one clone differs from the major mammalian tRNAAsn by a single base substitution at position 47, with an A replacing a G, while the tRNAAsn gene from the second clone has base substitutions at positions 17 and 65, with a G replacing a C and a T replacing a C, respectively. The sequences of the noncoding 5'- and 3'-flanking regions of both clones are over 90% homologous. As with other mammalian tRNA genes, these two human tRNAAsn genes contain CTTTTPu, which might act as a transcription termination signal, 11 bp 3' to the structural gene. In vitro transcription experiments in a HeLa cell extract demonstrate that both cloned tRNAAsn genes can be transcribed and processed to mature-sized tRNAs.

Nucleotide sequence of cloned nad4 (urf4) gene from Chlamydomonas reinhardtii mitochondrial DNA.

Chlamydomonas reinhardtii mitochondrial (mt)DNA was digested with ClaI + HpaI and shotgun cloned into the M13mp19 vector cleaved with AccI + SmaI. One of the recombinant clones, with a 1.8-kb DNA insert, was completely sequenced using the dideoxy chain-termination method. Besides containing part of the cytochrome b (COB)-encoding gene (cob), this DNA fragment encodes subunit 4 of NADH dehydrogenase (NAD4). The deduced amino acid sequence and hydrophilicity plot indicate that NAD4 is highly hydrophobic. The nad4 gene shows a unique preference for certain codons which are also found in other C. reinhardtii mt proteins. Both the genes encoding NAD4 and COB are shown to be transcriptionally active by Northern hybridization. These closely linked genes suggest that RNA-processing events found in vertebrate mt are present in Chlamydomonas mt as well.

Amplification and characterization of an inverted repeat from the Chlamydomonas reinhardtii mitochondrial genome.

Based on the nucleotide (nt) sequences of cob and L2a, two oligodeoxyribonucleotides (oligos) were synthesized and used in the polymerase chain reaction (PCR) to amplify the termini of the Chlamydomonas reinhardtii mitochondrial (mt) genome. A 0.8-kb PCR product was detected by agarose-gel electrophoresis when using unligated mt DNA as the template for PCR. This may have indicated the presence of a naturally occurring circular mt DNA molecule that acted as the PCR template. The 0.8-kb DNA could also be amplified from the linear mt DNA via an intramolecular jump during PCR. The sequence data from the 0.8-kb PCR product, and the right 0.6-kb and left 1-kb terminal fragments of the linear mt DNA, along with Southern hybridization analysis, indicated that a 0.49-kb inverted repeat (IR) sequence is present at the right and left termini of the linear mt DNA. The IR contains A+T-rich clusters, as well as numerous short direct repeats (DR) and IR, and might be involved in the recombination, replication and expression of the C. reinhardtii mt genome.

Nucleotide sequence of the gene coding for a 130-kDa mosquitocidal protein of Bacillus thuringiensis israelensis.

The nucleotide sequence of pVB131 containing the gene coding for a 130-kDa Bacillus thuringiensis israelensis (B.t.isr) mosquitocidal protein was determined. The pVB131 plasmid was constructed by Sekar and Carlton [Gene 33 (1985) 151-158]. Our sequencing revealed only one open reading frame large enough to code for a protein of 130 kDa. The translation start site was determined by sequencing the protein isolated from B.t.isr. The amino acid sequence of the protein was deduced from the nucleotide sequence, and its Mr was determined as 128,505. Immunological and biochemical analyses of B.t.isr mosquitocidal proteins indicated that the 130-kDa protein coded by pVB131 was indeed expressed in B.t.isr. Comparing the peptide sequence of the 130-kDa B.t.isr toxin with the sequences of other B.t. toxins having activities specific to lepidopteran species showed that several domains were highly homologous. This suggests that they are evolutionarily related to each other, and in the evolutionary process the sequences in the homologous domains that are important to the insecticidal activity have been conserved.

Molecular analysis of the Actinobacillus pleuropneumoniae RTX toxin-III gene cluster.

Actinobacillus pleuropneumonia strains that secrete three different exotoxins (ApxI, ApxII, and ApxIII) have been implicated in the etiology of porcine pleuropneumonia. To understand the role of these toxins in the pathogenesis of this disease, we have previously reported the cloning of the hemolysin gene (apxII) (Chang et al., 1989a), which encodes a 110-kD polypeptide with hemolytic and cytotoxic activity. To clone the third toxin gene (apxIII), a new genomic library using A. pleuropneumoniae serotype 2 chromosomal DNA was constructed. A series of five overlapping recombinant phage clones carrying the gene (apxIII) for this 120-kD antigen were identified using a DNA probe containing sequences from the Pasteurella haemolytica lktBD genes. Sequence analysis of a region of the cloned DNA reveals four open reading frames encoding proteins with predicted masses of 20.4, 112.5, 80.3, and 54.7 kD. These genes, designated apxIIC, apxIIIA, apxIIIB, and apxIIID, respectively, are similar in sequence to the RTX (repeat of toxin) toxin family. The toxin produced by the cloned gene kills BL-3 cells and is not hemolytic in vitro.

Cloning, sequencing and expression of a Pasteurella haemolytica A1 gene encoding a PurK-like protein.

A membrane protein antigen of Pasteurella haemolytica A1 encoded on the recombinant plasmid pYFC13 is isolated and characterized. Nucleotide sequence analysis of the insert DNA in pYFC13 identified the gene mpa1, which codes a protein of approximately 45 kDa without signal sequence. The deduced amino acids from the DNA sequence are homologous to Bacillus subtilis PurK by 29.4%; to Schizosaccharomyces pombe Pur6 by 29.34%, to Saccharomyces cerevisiae Pur6 by 25.867%; and to E. coli PurK by 25.223% identity, respectively. The purK and pur6 from these organisms are responsible for the activity of 5'-phosphoribosyl- 5-amino-4-imidazole carboxylase which is involved in de novo purine biosynthesis. The protein was over-expressed in E. coli by its own promoter. The antigen we designated as Mpa1, could be localized to the cytoplasmic membrane of both P. haemolytica A1 and E. coli TB1 harbored pYFC13. The Mpa1 was antigenic in rabbit and in cattle since both animals produced antibody against this protein.

Cloning and expressionanalysis of two cotton (Gossypium hirsutum L.) genes encodingcell wall proline-rich proteins.

Two cotton (Gossypium hirsutum L.) genes, ghprp1 and ghprp2, encoding cell wall proline-rich proteins (PRPs) have been cloned and characterized. The ghprpl gene has an open reading frame (ORF) that encodes a PRP of 299 amino acids (aa), whereas the ghprp2 gene contains an ORF that codes for a 310-aa PRP. The GhPRP1 has an 80% identity in aa sequence with that of GhPRP2. Like other plant cell wall PRPs, both cotton PRPs have a hydrophobic signal peptide at their N-termini, followed by repeating peptide units. Northern blot analyses showed that the ghprpl gene is predominantly expressed in the fiber during the elongation stage of fiber development. Reverse transcription (RT)-PCR analysis showed that ghprpl is expressed in both fiber and root tissues, whereas ghprp2 is in roots only. The ghprpl gene was shown to be present in the A1, A2, D1 and D5 genomes of Gossypium by PCR amplification, whereas the ghprp2 gene is only present in the A1 and A2 genomes. The ghprpl gene was over-expressed in the yeast Pichia pastoris, and the expressed GhPRP1 protein was used as an antigen to raise polyclonal antibodies (anti-GhPRP1). Western analysis using the anti-GhPRP1 probe detected a major protein band (50 kDa) in 5-31-day postanthesis (DPA) fibers. However, the 50 kDa protein was absent in other cotton tissues.

Sequence analysis of leukotoxin secretion determinants from a Pasteurella haemolytica-like organism.

The pHLBD genes encoding the secretion functions for the 105 kDa RTX leukotoxin of Pasteurella haemolytica-like (PHL) organism has been cloned and sequenced. Like analogous genes from other RTX determinants, the pHLBD genes lie immediately downstream from the leukotoxin structural gene, pHLA. Although isolated from a diverse group of gram-negative organisms, the pHLBD genes and the characterized RTX BD genes from other organisms exhibit a high degree of homology at both the DNA and predicted amino acid sequence levels. We have previously reported the cloning of the leukotoxin gene (pHLCA) (Chang et al., Infect. Immun. 61:2089-2095), which encodes a 105-kda polypeptide with cytotoxic activity. DNA sequence analysis of the pHLBD genes shows 83.93% and 86.05% homologous to that of P. haemolytica IktBD genes, respectively.

Characterization of plasmids with antimicrobial resistant genes in Pasteurella haemolytica A1.

Two R plasmids, pYFC1 and pYFC2, from Pasteurella haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFC1 consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, RSF1010, pGS05, pFM739, pHD148 and pGS03B. pYFC1 encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which is homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range IncQ plasmid RSF1010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 is similar to the plasmid from P. haemolytica LNPB51 isolated in France by partial restriction enzyme mapping. pYFC1 and pYFC2 have the same size of 4.2 kbp.

Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone.

OBJECTIVE: To develop a method to experimentally induce Borrelia burgdorferi infection in young adult dogs. ANIMALS: 22 healthy Beagles. PROCEDURE: All dogs were verified to be free of borreliosis. Twenty 6-month-old dogs were exposed to Borrelia burgdorferi-infected adult ticks and treated with dexamethasone for 5 consecutive days. Two dogs not exposed to ticks were treated with dexamethasone and served as negative-control dogs. Clinical signs, results of microbial culture and polymerase chain reaction (PCR) testing, immunologic responses, and gross and histologic lesions were evaluated 9 months after tick exposure. RESULTS: Predominant clinical signs were episodic pyrexia and lameness in 12 of 20 dogs. Infection with B burgdorferi was detected in microbial cultures of skin biopsy specimens and various tissues obtained during necropsy in 19 of 20 dogs and in all 20 dogs by use of a PCR assay. All 20 exposed dogs seroconverted and developed chronic nonsuppurative arthritis. Three dogs also developed mild focal meningitis, 1 dog developed mild focal encephalitis, and 18 dogs developed perineuritis or rare neuritis. Control dogs were seronegative, had negative results for microbial culture and PCR testing, and did not develop lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Use of this technique successfully induced borreliosis in young dogs. Dogs with experimentally induced borreliosis may be useful in evaluating vaccines, chemotherapeutic agents, and the pathogenesis of borreliosis-induced arthritis.

Insertion/deletion mutations in the Zea chloroplast genome.

The chloroplast (cp) genomes of Zea species are distinguished by at least four restriction fragment length (insertion/deletion) mutations. All four mutations occur in the large unique sequence region of the genome. Restriction fragments containing three of these mutations were cloned. The large and small forms of two of the mutated fragments were sequenced. This revealed 80 and 83 bp insertion/deletions. The inserted/deleted segments are not parts of tandem repeats nor were they flanked by direct repeats. Two other insertion/deletion mutations were not sequenced, but their sizes were estimated to be 150 and 250 bp by size fractionation on agarose gels. Use of Tripsacum pilosum and Sorghum bicolor as outgroups suggests that three of the fragment length mutations arose via deletions. The fourth could not be polarized. The three species of section Luxuriantes of Zea were identical to one another for each of the four length mutations, and they were consistently distinguished from the taxa of section Zea by these mutations. These data support the division of Zea into the above named sections.