Secretion of eukaryotic growth hormones in Escherichia coli is influenced by the sequence of the mature proteins.

We report the construction of secretion plasmids expressing the fusion proteins, OmpA::pGH (pSpGH.01) and OmpA::hGH (phGH.01), and compare the secretion of mature porcine growth hormone (pGH) and human growth hormone (hGH) employing Escherichia coli. E. coli [phGH.01] secreted 10-15 micrograms hGH/ml/A600 cells into the periplasmic space, representing 30% of total periplasmic proteins. E. coli [pSpGH.01], however, secreted 30-fold less mature pGH. On the basis that both pSpGH.01 and phGH.01 are stably maintained in E. coli and in vitro transcription/translation data showed equivalent expression of OmpA::pGH and OmpA::hGH precursors, we attribute the higher secretion of hGH to the translocation-competent OmpA::hGH protein configuration. Two OmpA::GHF (growth hormone fusion) precursors, OmpA::GHF.02 and OmpA::GHF.03, both with hGH helix 3/helix 4 together instead of the pGH equivalent, secreted mature proteins as efficiently as OmpA::hGH. We propose that hGH helices 3 and 4 in these OmpA::GHF precursors play a major role in the folding of the precursor to a translocation-competent state, mimicking the translocation-competent nature of the OmpA::hGH precursor.

Inactivation of the Escherichia coli B41 (O101:K99/F41) rfb gene encoding an 80-kDa polypeptide results in the synthesis of an antigenically altered lipopolysaccharide in E. coli K-12.

The genetic organisation of the rfb region from Escherichia coli B41 (O101:K99/F41) which determines the biosynthesis of the O101 O-antigen of the lipopolysaccharide (LPS) has been examined in E. coli K-12. Maxicell analysis of the plasmid-encoded proteins facilitated the construction of a physical map of the rfb region, consisting of six proteins, designated A (87 kDa), B (80 kDa), C (49 kDa), D (38 kDa), E (36.5 kDa) and F (27 kDa). Proteins E and F are not required for O-antigen biosynthesis. The introduction of frameshift mutations within the region encoding protein B resulted in the synthesis of an antigenically altered LPS which is shorter than the wild-type LPS, as assessed by reaction to antisera in colony and Western immunoblots, and by silver staining of LPS separated on sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The results demonstrate that protein B has a novel role in O-antigen biosynthesis associated with both the control of LPS chain length and antigenic structure. The nucleotide sequence of the rfb gene encoding protein B has been determined, confirming it to be a 697-amino acid protein of 78.9 kDa predicted to be located in the cytoplasmic membrane.

Molecular cloning and genetic analysis of the rfb region from Shigella flexneri type 6 in Escherichia coli K-12.

The rfb gene cluster which determines the biosynthesis of the Shigella flexneri serotype 6 O-antigen specificity has been cloned in pHC79, generating plasmids pPM3115 and pPM3116. These plasmids mediate expression, in Escherichia coli K-12, of lipopolysaccharides (LPS) immunologically similar to the S. flexneri type 6 LPS as judged by SDS-PAGE and Western-immunoblot analysis using S. flexneri type 6 specific antisera. Thus, unlike other S. flexneri serotypes, no additional loci are required for serotype specificity. This expression is independent of E. coli K-12 rfb genes. Southern-hybridization analysis using the 16.2-kb BglII probe from S. flexneri type 6 rfb region detected very little sequence homology in S. flexneri serotypes 1-5, however, some homology was detected with E. coli O2 and O18, but not in E. coli 0101 strains, Salmonella and Vibrio cholerae.

Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5.

The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined. Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3. The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes. ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids. The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI. Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1. The conservation of traX sequences suggests a functional importance for that gene and/or its product.

Genetic analysis of the rfb region of Shigella flexneri encoding the Y serotype O-antigen specificity.

The gene cluster (rfb region) which determines the biosynthesis of the Shigella flexneri O-antigen of the Y serotype specificity was cloned from a S. flexneri serotype 2a strain. Two plasmids, pPM2212 and pPM2213, which conferred O-antigen biosynthesis were generated from separate cosmid clones by deletion with Clal. These plasmids expressed O-antigen in Escherichia coli K12 like that of the parental strain, as assessed by reactions to antisera in colony and Western immunoblots, sensitivity to bacteriophage Sf6, and by silver staining of lipopolysaccharides separated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. These plasmids also mediated O-antigen expression in an E. coli K12 rfb-delete background, indicating that all the necessary genes have been cloned. A detailed restriction map of the region has been constructed and analysis of various subclones has allowed the limits of the coding region for O-antigen biosynthesis to be defined to a maximum of 11 kb. Expression of these plasmids demonstrates a novel phenotype associated with control of lipopolysaccharide chain length. The gene(s) responsible maps adjacent to, but separate from, those associated with the biosynthesis of the O-antigen unit. Analysis of plasmid-encoded proteins in minicells and maxicells has facilitated the construction of a physical map. Finally, plasmid pPM-2212 was used to probe a collection of S. flexneri serotypes by Southern hybridization. With the exception of serotype 6, which appears to be unrelated, a similar pattern was found in all serotypes.

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases.

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation.

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.

Expression and processing of human rhinovirus type 14 polypeptide precursors in Escherichia coli maxicells.

Human rhinovirus serotype-14 (HRV-14) cDNA, encompassing 87.9% of the coding region, was subcloned in an Escherichia coli expression vector, generating plasmid pKCC101. HRV-14 polypeptides encoded by pKCC101 were synthesized in E. coli maxicells. Pulse-chase experiments with pKCC110, a smaller derivative of pKCC101 containing the protease 3C coding region, have clearly demonstrated the proteolysis of a 55-kDa precursor to several polypeptides, including a doublet with the expected size of protease 3C (20 kDa). The proteolysis of the 55-kDa precursor polypeptide was prevented by ZnCl2, a known inhibitor of picornavirus 3C proteases. Results with a derivative of pKCC110 (pKCC115) which is partially deleted for the protease 3C sequence, support the idea that the doublet proteins are specified by the protease 3C coding region. Taken together, our investigations indicate that the precursor form of protease 3C must be responsible for its own cleavage.

Pain insensitivity in schizophrenic patients. A surgical dilemma.

Some schizophrenic patients have decreased pain perception while others have decreased pain expression. These factors frequently lead to difficulties in the diagnosis of acute intra-abdominal surgical emergencies. Increasingly large numbers of schizophrenic patients are being cared for in the community. It is therefore imperative that surgeons be acutely aware of the diagnostic dilemmas presented by this group of patients so that misdiagnosis is avoided and appropriate surgical therapy is instituted in a timely manner.

Surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12. Determination of the nucleotide sequence and promoter and terminator activities.

The DNA encoding the surface exclusion genes traS and traT of the F sex factor of Escherichia coli K-12 has been sequenced and the biological activity of the various terminators and promoters determined. The data show that traS encodes a 16,861 Mr protein with no apparent signal sequence, as expected for its cytoplasmic membrane location. The protein is extremely hydrophobic. traS has its own promoter and a weak terminator region follows the gene. After the traS termination loop there is a small intergenic region before the traT promoter. The traT gene encodes a 25,932 Mr precursor for the 23,709 Mr mature protein. The amino-terminal signal peptide is 21 amino acid residues, consistent with it being an outer membrane lipoprotein. A very strong termination loop follows the gene and adjacent to this a further loop can be predicted from the sequence. These secondary structures would be expected to enhance the stability of the mRNA in the presence of 3' specific ribonucleases accounting for the apparent long half-life of the messenger. The amino acid sequence of the mature product of traT of F differs from that of R100 by only a single amino acid substitution (Gly for Ala at position 119), whereas that of pED208 (Folac) differs at 40 positions. traT lies in a region of heteroduplex homology between F and R100, and the nucleotide sequence confirms this and demonstrates that this homology breaks down immediately preceding and following the coding region. Sequence analysis shows that this is also so for pED208. Thus the entire traS of F, R100 and pED208 are very different at the DNA level. An open reading frame, preceded by a typical promoter sequence and a weak and poorly located Shine-Dalgarno sequence, follows traT and corresponds to the start of traD. Alone, this promoter appears to be inactive.

finO sequences on conjugally repressed and derepressed F-like plasmids.

DNA-DNA hybridization studies have demonstrated the physical relatedness of the fertility inhibition gene, finO, among both FinO+ and FinO- F-like conjugative plasmids, viz. ColV2-K94, R100-1,R1drd19,R1,R6-5, UCR123, R386, p307, R453, R773, and pIP162-1. Furthermore, the data indicate that finO sequences on the FinO- plasmid ColV2-K94 map downstream of the transfer region, within 93.6-95.3 ColV2-K94.

The F plasmid carries an IS3 insertion within finO.

DNA-DNA hybridization studies support the conclusion that coding sequences of the fertility inhibition gene, finO, are present on the F plasmid and map downstream of the transfer region, entirely within 99.19-2.0 F. In addition, the results indicate that the IS3a element at 100/0-1.26 F is inserted within the F finO coding region. This insertion may have inactivated the finO gene on the F plasmid.

An F-derived conjugative cosmid: analysis of tra polypeptides in cosmid-infected cells.

The genes involved in the conjugational transfer of F plasmid DNA are organized into three closely linked operons spanning an overall length of approximately 33 kilobase pairs of F. The entire transfer (tra) region comprising all three operons has been cloned into the cosmid vector pHC79 by in vitro recombination and packaging techniques. The transfer-proficient chimeric cosmid pRS2405 was packaged into lambda capsids, and uv-irradiated E. coli cells were infected with these DNA-filled particles. A number of polypeptides programmed by the infecting DNA were identified as tra-specified products; a traJ90 mutation on pRS2405 resulted in the significant reduction of synthesis of all detectable pRS2405-specified tra polypeptides, with the exception of TraTp.

Expression of F plasmid traT: independence of traY----Z promoter and traJ control.

We have shown, using an F-derived Tra+ cosmid in conjunction with the infected-cell translational system and a time-course study, that one of the surface exclusion genes, traT, can be expressed independently of the promoter of the traY----Z operon, PYZ, and in the absence of a normal quantity of traJ gene product. Studies with deleted derivatives of the cosmid pRS2405 confirmed this independence and also indicated that expression of traD can be independent of PYZ. We propose that the expression of traT by these deleted plasmids is directed from a traJ-independent promoter, PT, located adjacent to traT.

Cloning and molecular analysis of the finO region from the antibiotic-resistance plasmid R6-5.

The cloning of the finO region from the antibiotic-resistance plasmid R6-5 is reported. On the basis of DNA deletion analysis and Tn5 transposon insertional mutagenesis of finO+ chimeric plasmids, finO has been located within the coordinates 94.0-94.85 on the R6-5 map. A 32,000-Da polypeptide (32K), which is encoded within 92.75-94.25R6-5, has been identified and shown not to be associated with the FinO phenotype.

The prevention of postappendicectomy sepsis by metronidazole and cotrimoxazole: a controlled double blind trial.

A double-blind, randomized controlled trial was carried out in University Hospital, Kuala Lumpur to study the effect of metronidazole and cotrimoxazole on the incidence of wound infection following appendicectomy from November 1978 to January 1980. Patients were allocated at random into one of four groups: cotrimoxazole injection and placebo suppository, metronidazole suppository and cotrimoxazole injection, metronidazole suppository and placebo injection or placebo suppository and placebo injection. Treatment was started 30 min before operation and continued for 72h. All patients were followed up for 2 weeks and thereafter for one month. A total of 283 patients was finally accepted into the study. Sepsis rates were found to be 27% for the untreated group, 9% for the group receiving metronidazole only, 8% for the group receiving cotrimoxazole injection only and 2.7% for the group receiving both drugs. The study showed that a combination of metronidazole and cotrimoxazole is a regime highly effective for prophylaxis against wound infection following appendicectomy.

Psychiatric findings in the population of a geriatric evaluation unit: implications.

Psychiatric evaluation was performed routinely in 262 patients newly admitted to a Medical Geriatric Evaluation Unit (GEU). The study was conducted in a medical facility that provides excellent medical and surgical care for acute illnesses. The psychiatric disorders found far exceeded those one might expect in a comparable general population, and most were not recognized prior to the patient's transfer. For example, in the GEU, the incidence of organic brain syndrome was 65.3 percent, and of dysphoria-depression 31.3 percent. The data indicate a need to recognize psychiatric problems in order to ensure appropriate care, and suggest that medical care of the elderly with acute illness will be inadequate if it is based upon the approach used for younger populations. This situation apparently exists in most hospitals, including leading medical centers. The needs of the elderly with acute illnesses are quite different from those of younger patients. Recognition of factors that potentially influence outcomes and overall future health will meet public health's primary and secondary prevention goals.

Geriatric evaluation unit of a medical service: role of a geropsychiatrist.

Psychiatric evaluation as a part of the complete geriatric workup was done on 143 consecutive patients transferred to a Medical Geriatric Evaluation Unit. The patients' age ranged from 48 to 94 years. The findings were: free of psychiatric problems--19.1%; organic brain syndrome--58.8%; dysphoria-depression--36.8%; paranoid--3.7%; alcohol abuse--8.1%; marital maladjustment 18.3% (of marrieds). The Geropsychiatrist diagnoses, participates in psychiatric management, consults, and supervises psychiatric evaluation by other team members. He is an esential member of the Geriatric team since proper recognition and treatment of psychiatric problems is necessary to complete treatment and to make optimum disposition.