Clinical factors of importance for outcome after lumbar disc herniation surgery: long-term follow-up.

Factors as age, sex, smoking, duration of leg pain, working status, type/level of disc herniation and psychosocial factors have been demonstrated to be of importance for short-term results after lumbar discectomy. There are few studies with long-term follow-up. In this prospective study of lumbar disc herniation patients undergoing surgery, the result was evaluated at 2 and 5-10 (mean 7.3) years after surgery. Predictive factors for satisfaction with treatment and objective outcome were investigated. Out of the included 171 patients undergoing lumbar discectomy, 154 (90%) patients completed the 2-year follow-up and 140 (81%) completed the long-term follow-up. Baseline data and questionnaires about leg- and back pain intensity (VAS), duration of leg pain, disability (Oswestry Disability Index), depression (Zung Depression Scale), sick leave and employment status were obtained preoperatively, at 2-year- and long-term follow-up. Primary outcome included patient satisfaction with treatment (at both time points) and assessment of an independent observer at the 2-year follow-up. Secondary outcomes at 2-year follow-up were improvement of leg and back pain, working capacity and the need for analgesics or sleeping pills. In about 70% of the patients excellent or good overall result was reported at both follow-ups, with subjective outcome measurements. The objective evaluation after 2 years was in agreement with this result. Time on sick leave was found to be a clinically important predictor of the primary outcomes, with a potential of changing the probability of a satisfactory outcome (both objective and subjective) from around 50% (sick leave >3 months) to 80% (sick leave <2 months). Time on sick leave was also an important predictor for several of the secondary outcomes; e.g. working capacity and the need for analgesics.

Amenorrhoea in newly spinal cord injured women: an effect of hyperprolactinaemia?

STUDY DESIGN: Prospective, single centre study. OBJECTIVES: Previous studies have suggested a relationship between stress reaction and elevated levels of prolactine. The aim of the present study was to investigate if there was a relationship between s-prolactine and menstrual cycle status following spinal cord injury (SCI). SETTING: Spinal Cord Injury Unit, Göteborg, Sweden. METHODS: S-prolactine and menstrual cycle status were investigated in 16 consecutive women with SCI, treated at the SCI Unit, Sahlgrens University Hospital, Göteborg, Sweden. Level of injury ranged from C1 to L5, ASIA A-D. Mean age at injury was 45 years (range 20-79). RESULTS: S-Prolactine showed a mean value of 741 mIU/l (standard deviation (s.d.): 625; 95% confidence interval (CI): 435-1788 mIU/l, reference value <400 mIU/l). When dividing the group according to fertility status we found hyperprolactinaemia in the women who were in childbearing age (n=9): mean value 1050 mIU/l (s.d.: 678; 95% CI: 607-1493 mIU/ml), whereas it was normal in the group in menopause (n=7): mean value 343 mIU/l (s.d.: 185, 95% CI: 206-480 mIU/l) (P<0.01 when comparing groups). The group that developed amenorrhoea showed the highest values of s-prolactine. All values but one was normalised 3-6 months later. CONCLUSION: Amenorrhoea following SCI is correlated to level of s-prolactine. We found no correlation between level of s-prolactine and level or degree of injury.

A dual role for the Bacillus subtilis glpD leader and the GlpP protein in the regulated expression of glpD: antitermination and control of mRNA stability.

The Bacillus subtilis glpD gene encodes glycerol-3-phosphate dehydrogenase. This gene is preceded by a leader region containing an inverted repeat which acts as a transcription terminator. Expression of glpD is controlled by antitermination of transcription at the inverted repeat. Antitermination is effected by the glpP gene product in conjunction with glycerol-3-phosphate and, consequently, GlpP mutants fail to grow on glycerol as a sole carbon and energy source. We have isolated a number of glycerol-positive revertants of GlpP mutants. Most of these revertants have mutations in the inverted repeat of the glpD leader and produce glycerol-3-phosphate dehydrogenase constitutively. Unlike wild-type bacteria, they are not sensitive to glucose repression of glpD. A few of the revertants are temperature sensitive, i.e. they grow on glycerol at 32 degrees C but not at 45 degrees C and produce glycerol-3-phosphate dehydrogenase only at 32 degrees C. Northern blot analyses demonstrated that the temperature-sensitive expression of glpD is due to destabilization of glpD mRNA. Furthermore, introduction of the wild-type glpP gene into the revertants stabilized the glpD mRNA. This is probably a result of a direct interaction between the GlpP protein and the leader of glpD mRNA. Besides its function in antitermination of transcription of glpD, it is suggested that GlpP is also involved in controlling glpD mRNA stability. Introduction of the glpP gene into the revertants also restored glucose repression, indicating that this repression is mediated by the GlpP protein.

The hemX gene of the Bacillus subtilis hemAXCDBL operon encodes a membrane protein, negatively affecting the steady-state cellular concentration of HemA (glutamyl-tRNA reductase).

The Bacillus subtilis hemAXCDBL operon encodes enzymes for the biosynthesis of uroporphyrinogen III from glutamyl-tRNA. The function of the hemX gene product was studied in this work. The deduced amino acid sequence suggests HemX to be an integral 32 kDa membrane protein. This was confirmed by experiments using Escherichia coli minicells and hemX-phoA gene fusions. Deletion of the hemX gene from the Bacillus subtilis chromosome demonstrated that this gene is not required for haem synthesis. However, the deletion strain was found to overexpress the hemA gene product, glutamyl-tRNA reductase. A combination of results obtained with B. subtilis hemA and hemX in Escherichia coli and Bacillus subtilis shows that HemX negatively affects the steady-state cellular concentration of HemA protein. The mechanism by which HemX affects the HemA concentration is unclear.

The glpP and glpF genes of the glycerol regulon in Bacillus subtilis.

The Bacillus subtilis glpPFKD region contains genes essential for growth on glycerol or glycerol 3-phosphate (G3P). The nucleotide sequence of glpP encoding a regulatory protein and the previously unidentified glpF encoding the glycerol uptake facilitator was determined. glpF is located immediately upstream of glpK and the two genes were shown to constitute one operon which is transcribed separately from glpP. A sigma A-type promoter and the transcriptional start point for glpFK were identified. In the 5' untranslated leader sequence (UTL) of glpFK mRNA a conserved inverted repeat is found. The repeat is believed to be involved in the control of expression of glpFK by termination/antitermination of transcription, a control mechanism previously suggested for the regulation of glpD encoding G3P dehydrogenase. Expression of glpFK and glpD requires the inducer G3P and the glpP gene product. A 2.9 kb B. subtilis chromosomal DNA fragment containing the glpP open reading frame was cloned to give plasmid pLUM7. pLUM7 contains a functional glpP gene as shown by its ability to complement various glpP mutants. Immediately upstream of glpP an open reading frame is found (ORF1). Disrupting ORF1 by plasmid integration in the B. subtilis chromosome does not affect the ability to grow on glycerol as sole carbon and energy source. With the present report all B. subtilis glp genes located at 75 degrees on the chromosomal map have been identified.

Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis.

Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system. In spite of this, B. subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source. Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase. Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes. Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon. Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B. subtilis chromosomal map. This region contains the glpP, the glpFK and the glpD operons. The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK). The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation. Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B. subtilis.

An inverted repeat preceding the Bacillus subtilis glpD gene is a conditional terminator of transcription.

The Bacillus subtilis glpD gene, encoding glycerol-3-phosphate (G3P) dehydrogenase, is preceded by a promoter and an inverted repeat which is located between the promoter and the glpD coding region. The inverted repeat acts as a transcriptional terminator in vitro. Expression of glpD is induced by G3P in the presence of the glpP gene product. Full-length glpD transcripts can be detected only in glycerol-induced cells. The major glpD transcript is initiated from the glpD promoter but minor amounts of larger transcripts, possibly initiated at upstream glp promoters, can also be found. In uninduced cells short transcripts are present, corresponding to initiation at the glpD promoter and termination at the inverted repeat. Upon induction, these short transcripts disappear and are replaced by full-length glpD transcripts. The 3'-ends of full-length glpD transcripts were mapped to an inverted repeat located immediately downstream of glpD. These results show that glpD of B. subtilis is regulated by termination/antitermination of transcription.

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schröder, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.

Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD).

The glpPKD region of the Bacillus subtilis chromosome was cloned in its natural host in plasmid pHP13. The glpPKD region contains genes required for glycerol catabolism: glpK coding for glycerol kinase, glpD coding for glycerol-3-phosphate (G3P) dehydrogenase and glpP, proposed to code for a positively acting regulatory protein. The cloned 7 kb fragment carries wild-type alleles of glpK, glpD and glpP. It can also complement a strain deleted for the entire glpPKD region. The wild-type alleles were mapped to different subfragments, establishing the gene order glpP-glpK-glpD. The nucleotide sequence of glpK and glpD was determined. Immediately upstream of glpK, an additional open reading frame was found, possibly being part of the same operon. Putative transcription terminators were found in the region between glpK and glpD and downstream of glpD. In a coupled in vitro transcription/translation system, two proteins were found, corresponding in size to those predicted from the deduced amino acid sequences of glycerol kinase and G3P dehydrogenase (54 kDa and 63 kDa, respectively).

Changes in the stability of specific mRNA species in response to growth stage in Bacillus subtilis.

In this study we compared the cellular concentrations and stability of the mRNA transcribed from the aprE (subtilisin) gene (a gene preferentially expressed in stationary growth phase) with those of a vegetative mRNA, succinate dehydrogenase (SDH) mRNA. The subtilisin transcript was shown to be at least 3 times more stable in early stationary phase than it is 2 hr further into stationary phase. When cells were shifted from maximum expression of the subtilisin transcript in stationary phase to physiological conditions, which allowed for the resumption of vegetative growth, the cellular concentration of the subtilisin mRNA decreased rapidly. We conclude that mRNA degradation is one of the means by which the cellular concentrations of the SDH and subtilisin transcripts are adjusted in response to growth stage.

The importance of the 5'-region in regulating the stability of sdh mRNA in Bacillus subtilis.

The decay of the polycistronic Bacillus subtilis sdh mRNA was analysed using probes specific for each of the component cistrons, sdhC, sdhA and sdhB. In exponentially growing cells, the entire sdh mRNA seems to decay with an 'all or nothing' mechanism and with a uniform half-life of 2-3 min for all cistrons. In stationary-phase cells, the half-life of the 5'-part had dropped to about 0.6 min whereas that of the 3'-part was about 1.2 min. Decay of sdh mRNA was also measured in exponentially growing cells containing a 'down-mutation' in the ribosomal binding site preceding sdhC which decreases the expression of sdhC by about 90%. The mutation has a moderate effect on expression of the downstream cistron sdhA. In this mutant, the half-life of the 5'-part of sdh mRNA was about 0.5 min (i.e. the same as in stationary phase wild-type cells) and the half-life of the 3'-part about 1.3 min. Also, analysis of the decay of an sdh-cat fusion transcript revealed that the sdh (5') part decayed more rapidly than the cat part and this difference was more pronounced in stationary-phase cells compared to exponentially growing cells. The results of these experiments demonstrate the importance of the 5'-segment of sdh mRNA in controlling the stability of the transcript under different growth conditions.

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.

The structural gene for aspartokinase II in Bacillus subtilis is closely linked to the sdh operon.

The aecA and aecB loci map at 250 and 290 degrees, respectively, on the Bacillus subtilis chromosomal genetic map. The aecB locus has been proposed as the structural gene for aspartokinase II. From DNA sequence analyses and comparisons to the sequence of the aspartokinase II gene, it can be concluded that the structural gene for aspartokinase II is located close to sdh at 250 degrees and cannot be aecB. A detailed map over 7 kbp in the 250 degree region is presented.

Transcriptional and posttranscriptional control of the Bacillus subtilis succinate dehydrogenase operon.

The amount of succinate dehydrogenase (SDH) in Bacillus subtilis varies with growth conditions. In this work we studied the steady-state level and the rate of decay of B. subtilis sdh mRNA under different growth conditions. In exponentially growing cells, the steady-state level of sdh mRNA was severalfold lower when glucose was present compared with growth without glucose, whereas the rate of decay of sdh mRNA was the same with and without glucose. Thus, glucose repression seems to act by decreasing sdh mRNA synthesis. When the bacteria entered the stationary phase, the steady-state level of sdh mRNA dropped about sixfold. At the same time, sdh mRNA half-life decreased from 2.6 to 0.4 min. This result indicates that transcription of the sdh operon is initiated at the same rate in exponentially growing and in stationary-phase cells. The start point of the sdh transcripts, as measured by primer extension, was the same under all conditions studied, suggesting that the sdh operon is solely controlled by the previously identified sigma 43-like promoter. The increase of SDH activity in stationary phase may be explained by reduced dilution of the SDH proteins as a result of the retarded growth rate. We suggest that enhanced degradation of the sdh transcript is a means by which the bacteria adjust expression to the demands of stationary phase.

Genetic and biochemical characterization of Bacillus subtilis mutants defective in expression and function of cytochrome b-558.

Bacillus subtilis succinate dehydrogenase is bound to the cytoplasmic membrane by cytochrome b-558, a 23-kDa transmembrane protein which also functions as electron acceptor to the dehydrogenase. The structural gene for the apocytochrome, sdhC, has previously been cloned and sequenced. In this work the structure and translation of cytochrome b-558 was studied in different sdhC mutants. Mutant cytochrome was analyzed both in B. subtilis and after amplification in Escherichia coli. It is concluded that amino acid substitutions in the C-terminal half of the cytochrome can prevent the binding of succinate dehydrogenase without affecting membrane binding of the cytochrome protein or heme ligation. Mutagenesis of His-113 excludes this residue as an axial heme ligand. A base-pair exchange of G to A in the ribosome-binding sequence of sdhC was found to reduce cytochrome b-558 translation about tenfold in B. subtilis, whereas the mutation had no effect on translation in E. coli. Translation of the two succinate dehydrogenase genes from the sdhCAB polycistronic transcript does not seem to be coupled to translation of sdhC. Less than 10% of the wild-type amount of membrane-bound succinate dehydrogenase in B. subtilis still allows growth on non-fermentable substrate, but makes the dehydrogenase a limiting enzyme in the tricarboxylic acid cycle and leads to succinate accumulation.

Identification of the promoter of the Bacillus subtilis sdh operon.

The Bacillus subtilis sdhCAB operon contains the structural genes for the three subunits of the membrane bound succinate dehydrogenase complex. An sdh-specific transcript of about 3,450 nucleotides was detected in vegetative bacteria. S1 nuclease mapping experiments showed that the sdh operon is transcribed from a sigma-43 promoter; the transcript starts at a guanosine residue 90 base pairs upstream from the first gene of the operon, sdhC. No sdh transcript was found in B. subtilis carrying the sdh-115 mutation, which decreases expression of the sdh operon by more than 99%. The sdh-115 mutation is a G-to-A transition in the -35 region of the sigma-43 promoter. The sdh operon is sensitive to glucose repression. When the sdh promoter region was used to drive transcription of the cat-86 gene this gene also became glucose repressed.

Nucleotide sequence encoding the flavoprotein and iron-sulfur protein subunits of the Bacillus subtilis PY79 succinate dehydrogenase complex.

The nucleotide sequence of a 2.7-kilobase segment of DNA containing the sdhA and sdhB genes encoding the flavoprotein (Fp, sdhA) and iron-sulfur protein (Ip, sdhB) subunits of the succinate dehydrogenase of Bacillus subtilis was determined. This sequence extends the previously reported sequence encoding the cytochrome b558 subunit (sdhC) and completes the sequence of the sdh operon, sdhCAB. The predicted molecular weights for the Fp and Ip subunits, 65,186 (585 amino acids) and 28,285 (252 amino acids), agreed with the values determined independently for the labeled Fp and Ip antigens, although it appeared that the B. subtilis Fp was not functional after expression of the sdhA gene in Escherichia coli. Both subunits closely resembled the corresponding Fp and Ip subunits of the succinate dehydrogenase (SDH) and fumarate reductase of E. coli in size, composition, and amino acid sequence. The sequence homologies further indicated that the B. subtilis SDH subunits are equally related to the SDH and fumarate reductase subunits of E. coli but are less closely related than are the corresponding pairs of E. coli subunits. The regions of highest sequence conservation were identifiable as the catalytically significant flavin adenine dinucleotide-binding sites and cysteine clusters of the iron-sulfur centers.

Nucleotide sequence of the gene for cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex.

The nucleotide sequence was determined for the first part of the Bacillus subtilis sdh operon. An open reading frame corresponding to the structural gene, sdhA, for cytochrome b558 was identified. The predicted molecular weight of the cytochrome (excluding the N-terminal methionine) is 22,770. It is a very hydrophobic protein with five probable membrane-spanning segments. There is little homology between the B. subtilis cytochrome b558 and cytochrome b of mitochondrial complex III from different organisms or between cytochrome b558 and the hydrophobic sdhC and sdhD peptides of the Escherichia coli sdh operon. About 30 bases downstream of the sdhA stop codon, a new open reading frame starts. The nucleotide sequence predicts the presence of a typical flavin-binding peptide which identifies this reading frame as part of the sdhB gene. Seven bases upstream of the sdhA initiation codon ATG there is a typical B. subtilis ribosome binding site (free energy of interaction, -63 kJ), and further upstream, tentative sigma 55 and sigma 32 promoter sequences were found. The upstream region also contains two 12-base-long direct repeats; their significance is unknown.