PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer.

Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non-piliated and failed to express pilus-associated phenotypes, and the corresponding genes were designated PilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [3H]-palmitic acid. PilP- as well as PilQ- mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PiIQ were greatly reduced in PilP- mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.

A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro.

Gene rearrangement during the ontogeny of T- and B-cells generates an enormous repertoire of T-cell receptor (TCR) and immunoglobulin (Ig) genes. Because of the error-prone nature of this rearrangement process, two-thirds of rearranged TCR and Ig genes are expected to be out-of-frame and thus contain premature terminations codons (ptcs). We performed sequence analysis of reverse transcriptase-polymerase chain reaction products from fetal and adult thymus and found that newly transcribed TCR-beta pre-mRNAs (intron-bearing) are frequently derived from ptc-bearing genes but such transcripts rarely accumulate as mature (fully spliced) TCR-beta transcripts. Transfection studies in the SL12.4 T-cell line showed that the presence of a ptc in any of several TCR-beta exons triggered a decrease in mRNA levels. Ptc-bearing TCR-beta transcripts were selectively depressed in levels in a cell clone that contained both an in-frame and an out-of-frame gene, thus demonstrating the allelic specificity of this down-regulatory response. Protein synthesis inhibitors with different mechanism of action (anisomysin, cycloheximide, emetine, pactamycin, puromycin, and polio virus) all reversed the down-regulatory response. Ptc-bearing transcripts were induced within 0.5 h after cycloheximide treatment. The reversal by protein synthesis inhibitors was not restricted to lymphoid cells, as shown with TCR-beta and beta-globin constructs transfected in HeLa cells. Collectively, the data suggest that the ptc-mediated mRNA decay pathway requires an unstable protein, a ribosome, or a ribosome-like entity. Protein synthesis inhibitors may be useful tools toward elucidating the molecular mechanism of ptc-mediated mRNA decay, an enigmatic response that can occur in the nuclear fraction of mammalian cells.

Plasma concentrations and hemodynamic changes after repeated interpleural injections of bupivacaine-epinephrine.

BACKGROUND AND OBJECTIVES: The purpose of this investigation was to study the arterial plasma concentrations of bupivacaine and the hemodynamic changes after interpleural injection of 20 ml 0.5% bupivacaine with epinephrine in patients treated by chest drainage for spontaneous pneumothorax. METHODS: Twenty-two patients with spontaneous pneumothorax planned to be treated by chest drainage were randomly allocated to a bupivacaine group and a control group receiving saline. After introduction of the chest drain and evacuation of the pneumothorax, 10 injections through an interpleural catheter were given at 8-hour intervals in a double-blind fashion. Plasma concentrations were determined at 5, 10, 20, 30, 240, and 480 minutes after the first injection and before and at 15 and 30 minutes after the 4th, 7th, and 10th injections. Heart rate and mean arterial pressure was registered before and at 15 and 30 minutes after each injection. RESULTS: Mean Cmax after the first injection was 1.02 +/- 0.24 microgram/ml at 15 minutes, the highest individual Cmax after 20 minutes being 1.56 micrograms/ml. Mean plasma concentrations increased up to and after the 4th injection but did not rise further after the 7th and 10th injections. The highest individual Cmax was 1.92 micrograms/ml at 15 minutes after the 10th injection. The injections were accompanied by a small but statistically significant reduction in mean arterial blood pressure and a rise in heart rate 15 minutes after injection in the bupivacaine group compared with the control group. CONCLUSIONS: Repeated interpleural bolus injections of 20 ml 0.5% bupivacaine-epinephrine at 8-hour intervals are safe regarding plasma concentrations of bupivacaine. Small but statistically significant hemodynamic changes can be noticed, probably the result of beta- adrenergic stimulation by epinephrine.

Interpleural bupivacaine for analgesia during chest drainage treatment for pneumothorax. A randomized double-blind study.

The ability of interpleural analgesia to reduce the pain caused by an indwelling chest drain was evaluated in 22 patients treated for spontaneous pneumothorax. Intermittent 8-hourly bolus injections of 20 ml bupivacaine 0.5% with epinephrine were compared with placebo in a randomized double-blind fashion. Visual analogue pain scale (VAS) scores were registered after the 1st, 2nd, 4th, 7th and 10th injections. The scores were significantly lower in the bupivacaine group at 5, 15, 30 and 60 min after the first injection. No significant differences in pain scores were found after 4 or 8 h. Pain scores in the bupivacaine group were also reduced after the 2nd, 4th, 7th and 10th injections, but compared with placebo the differences were significant only after the 2nd and 7th injections. Parenteral morphine consumption was not significantly lower in the bupivacaine group. Arterial blood gases were unaffected by the treatment in both groups. It is concluded that interpleural analgesia using bupivacaine given as bolus injections at 8-h intervals significantly reduces the pain caused by a chest drain within 5 min of injection, but the duration of pain relief is less than 4 h.

Randomized, controlled study evaluating effects of branched chain amino acids and alpha-ketoisocaproate on protein metabolism after surgery.

The effect of nutritional supplementation with branched chain amino acids or the ketoacid alpha-ketoisocaproate on protein metabolism after surgery was studied in 24 patients with gastrointestinal cancer. They were randomized to receive one of three nutritional regimens. All patients received a balanced amino acid solution and group 1 (n = 8) received no further supplements, group 2 (n = 8) received supplementation with alpha-ketoisocaproate (17 g day-1) and group 3 (n = 8) received a branched chain solution including leucine, isoleucine and valine, corresponding to 3.3 g nitrogen day-1. Plasma albumin, prealbumin, fibronectin and serum urea concentrations, nitrogen balance and 3-methylhistidine release from the leg and its excretion in the urine were measured. Albumin and prealbumin concentrations fell after surgery in all groups, and fibronectin levels fell in group 2 (P less than 0.001). In group 2 there was also a significant increase in serum urea concentration after operation (P less than 0.05). Cumulative nitrogen balance after 3 days was -5.6 g (group 1), -3.8 g (group 2) and -1.7 g (group 3). The release of 3-methylhistidine (nmol 100 g-1 min-1) from the leg after operation, following an overnight fast, was -0.42 (group 1), -0.51 (group 2) and -0.66 (group 3). During infusion the release was -0.56, -0.99 and -0.81, respectively. A balanced amino acid solution with an adequate energy supply has an optimal nitrogen-sparing effect. Branched chain amino acids or alpha-ketoisocaproate did not improve nitrogen balance or reduce protein degradation.

Rebreathing during oxygen treatment with face mask. The effect of oxygen flow rates on ventilation.

The influence of different oxygen flow rates on ventilation and arterial blood gases was investigated in ten healthy volunteers during oxygen treatment with the Hudson mask. Respiratory parameters were calculated using inductive plethysmography calibrated against pneumotachography. The minute ventilation was greater when using the mask with oxygen flow rates less than 5 l/min compared to when no mask was used. With an oxygen flow rate of 3 l/min, minute ventilation was about 140% of minute ventilation without face mask. With 0 l/min, minute ventilation increased to about 160%. The increase in minute ventilation was mainly due to an increase in tidal volume. No change was seen, however, in PaCO2 with different oxygen flow rates. Secondary objective signs following an increase in respiratory work (changes in heart rate, systolic blood pressure and oxygen saturation) were not seen. We recommend 5 l/min as the lowest oxygen flow rate to be used during oxygen therapy with the Hudson mask, in order to avoid rebreathing and excessive respiratory work.

The effect of carnitine supplemented total parenteral nutrition on lipid, energy and nitrogen metabolism in severely ill patients.

To analyse the effects of L-carnitine supplemented TPN on lipid, energy and nitrogen metabolism, 16 severely injured patients were studied during the first 8 days after trauma. An L-carnitine solution (3g = 18.6mmol) was added to the fat emulsion and infused over 16h in a blind randomised fashion to half of the patients. Plasma triglyceride, free fatty acid and 3-OH-butyrate concentrations increased during the fat infusion, and fell to pre-infusion concentrations within 24h. There were no differences in plasma levels before, during or after infusion between the groups. ATP and phosphocreatine in muscle tissue were not influenced by carnitine supplementation. Glycogen, however, remained unchanged in the carnitine group and fell in the non-carnitine group. A cumulative N-balance measured from day 2 to day 8 was equally negative in both groups. Plasma carnitine levels were significantly higher in the supplemented group from day 3. The mean daily urinary carnitine excretion was increased 15-fold in the supplemented group. Muscle carnitine, however, remained unchanged in both groups and did not differ between them. The present results do not demonstrate any beneficial effects of parenterally administered L-carnitine on lipid, energy or nitrogen metabolism except for maintaining normal muscle glycogen levels in critically ill patients receiving TPN during the early phase after trauma.

Nitrogen requirements in severely injured patients.

The study was designed to evaluate nitrogen needs in severely injured patients during the first week after trauma. Thirty-nine patients aged from 18 to 65 years with a burn or fractures of more than two long bones were studied. Energy requirements were given parenterally as fat and glucose in isocaloric amounts. The patients were randomized into five groups receiving different amounts of nitrogen from zero to 0.3 g kg body-weight-1 24 h-1. Daily and cumulative nitrogen balance, urinary 3-methylhistidine excretion and nitrogen retention were calculated on days 2-8 after trauma. With no nitrogen, the mean(s.e.m.) daily nitrogen balance after the trauma was -13.8(0.5) gN. The balance improved markedly in groups with a nitrogen intake of up to 0.2 g kg body-weight-1 (P less than 0.001) compared with the no-nitrogen group. The 3-methylhistidine excretion increased because of the trauma in all groups with no statistically significant difference between the groups. Nitrogen retention decreased with increase in nitrogen supply and with time after injury. It is suggested that a nitrogen supply of 0.20 kg bodyweight-1 24 h-1 is optimal for severely injured patients during the first week after trauma.

Relationship between intracellularly and extracellularly generated oxygen metabolites from primed polymorphonuclear leukocytes differs from that obtained from nonprimed cells.

The ability of primed human polymorphonuclear leukocytes (PMNLs) to respond metabolically to stimulation with formylmethionyl-leucyl-phenylalanine (FMLP) was investigated. Cells isolated from an aseptic inflammatory reaction and from patients with a severe bacterial infection as well as cells that had been treated with a bacterial lipopolysaccharide were investigated. When these cells were compared to peripheral blood cells isolated from healthy controls, they were found to be metabolically primed, i.e., the cells gave rise to an increased chemiluminescence response to subsequent stimulation with the peptide. It was also shown that proportionally more of the activity generated from the primed PMNL was of an intracellular origin compared with that obtained from nonprimed cells. The biological effects induced by radicals produced extracellularly and intracellularly are discussed.

Human neutrophil chemiluminescence and f-Meth-Leu-Phe receptor exposure in bacterial infections.

Polymorphonuclear leukocytes were isolated from 12 patients with acute bacterial infections and the ability of the chemoattractant formylmethionyl-leucyl-phenylalanine (FMLP) to bind and induce a metabolic response in these cells was investigated. Cells isolated from the patients showed a significantly increased metabolic response in a luminol enhanced chemiluminescence assay compared to cells, isolated and analyzed in parallel, from healthy controls i.e. the patient cells were primed. The primed state was, as calculated by Scatchard analysis, accompanied by a significantly increased number of FMLP receptors exposed on the cell surface while the receptor binding affinity remained unchanged. There was, however, no correlation between the degree of priming and the degree of receptor upregulation. Furthermore, it was found that stimulation also with phorbol myristate acetate (PMA), a substance lacking specific cell surface receptors on the PMNL, gave rise to an increased metabolic response in the primed cells. These results indicate that the priming activity induced by a bacterial infection can only partly be explained by receptor modulation and that other mechanisms must also be considered. This study was approved by the Ethics Committee of the Medical Faculty, University of Linköping.

Percutaneous, tunneled silicone elastomer central venous catheters for total parenteral nutrition: low sepsis and thrombosis rate. A prospective study of 315 catheters.

Silastic catheters were inserted by the percutaneous route, and tunneled subcutaneously, in 315 patients who needed venous access for total parenteral nutrition. The catheters were managed with a daily program that included heat sterilization of the metal hub with an electrical soldering iron. This study aimed to evaluate prospectively the incidence of catheter-related sepsis and thrombosis. There was one case of pneumothorax. All catheters were x-rayed post-insertion: eight catheters were malpositioned initially. The median catheter duration was 18 days with a range of 2-138 days. The total duration was 240 catheter-months. Twenty-seven catheters were removed due to mechanical problems. Nine were removed because of suspected sepsis; six patients had negative blood and catheter cultures, while three grew pathogens. The sepsis rate was thus 0.95%. There were no clinical signs of thrombosis. Pull-out venography was performed in 93 patients. Fibrin sleeves were seen in the majority of cases. Two patients had wall-adherent, non-occlusive thrombus masses (2%); they both had proximal catheter positions. We conclude that there is a low risk of catheter-related sepsis and thrombosis with this technique.

Preoperative nutrition.

There are studies indicating a positive effect of preoperative nutritional support in patients with gastrointestinal malignancy, and that this nutritional support must be given for at least 7-10 days to achieve this effect. Intensive preoperative nutritional support should be used only in malnourished patients undergoing major surgical procedures and in patients receiving preoperative radio- and/or chemotherapy, irrespective of nutritional status.

Skeletal muscle function and metabolism in obese women.

A possible mechanism to regulate body weight during a high calorie intake may be an increased metabolic rate in skeletal muscle. To approach this hypothesis the energy metabolites, ATP, phosphocreatine, creatine, glycogen and lactate were measured in biopsies from the quadriceps femoris muscle. Concomitantly the function of the adductor pollicis muscle was studied as assessed after electrical stimulation of the ulnar nerve. The muscle function variables were, force of contraction at 5, 10, 20, and 50 Hz of stimulation, relaxation rate, and endurance. Eight obese women were studied before gastroplastic surgery and 6 months postoperatively and a weight loss of 19.4 +/- 3.4% (mean +/- SEM). Preoperatively ATP, phosphocreatine, glycogen, and lactate were significantly decreased and the same pattern was found postoperatively. These findings can be related to a low production of energy-rich phosphates or a high energy utilization. Both pre- and postoperatively there was, a decreased force of contraction at 10 Hz of stimulation (p less than 0.001), a faster relaxation rate (p less than 0.01) and a normal endurance. These functional results indicate a high metabolic rate. At admission a decreased serum insulin level indicated a moderate insulin resistance which was normalized after the weight loss. The triiodothyronine concentration was normal before and after operation. In conclusion our findings of changed muscle energy metabolite concentrations and altered muscle function indicate a high metabolic rate in skeletal muscle in obese women. This may be an adaptation in skeletal muscle energy metabolism to a high body weight.

Carnitine levels in skeletal muscle of malnourished patients before and after total parenteral nutrition.

Carnitine is necessary for the transport of long-chain fatty acids across the mitochondrial membrane. Carnitine is derived from the diet and from endogenous synthesis from lysine and methionine. About 98% of the body's carnitine pool is located in skeletal muscle tissue. Skeletal muscle carnitine levels were determined in two groups of malnourished patients, eight patients with anorexia nervosa with a weight loss of 32.4% +/- 1.8 (mean +/- SEM) and six surgical patients with major gastrointestinal disorders and a weight loss of 15.2% +/- 2.7. Their hepatic and kidney functions were normal. On admission, the muscle carnitine levels were 16.9 +/- 4.0 mumol/g dry weight (mean +/- SD) for the surgical patients and 20.8 +/- 5.0 mumol/g dry weight for the anorexia nervosa patients, which corresponded to carnitine levels seen in healthy subjects. No statistical significance was found between the two groups. Total parenteral nutrition was given to the surgical patients for 2 weeks and to the anorexia nervosa patients for 3-5 weeks. No statistical difference in muscle carnitine levels was found in either group after nutritional support. These malnourished patients had no decreased muscle carnitine levels on admission and maintained them during several weeks of total parenteral nutrition.

The effect of starvation on skeletal muscle function in man.

Measuring skeletal muscle function may be a diagnostic aid to detect malnutrition. To evaluate this hypothesis 11 lean women were studied before and after 5 days of total starvation. Skeletal muscle function was assessed as hand grip strength and as adductor pollicis muscle function after electrical stimulation of the ulnar nerve. The muscle function variables were force of contraction at 5, 10, 20 and 50 Hz stimulation, relaxation rate and endurance. In addition some currently used anthropometric and biochemical nutritional indices were serially determined. Hand grip strength was decreased (p < 0.05) and the function of the adductor pollicis muscle was altered with a higher contraction force at 10 Hz (p < 0.01), a slower relaxation rate (p < 0.05) and a reduced endurance (p < 0.05). These changes were all within the normal ranges. Triceps skin fold and arm muscle circumference were not changed after a mean weight loss of 3.3 kg (p < 0.05). However blood glucose, insulin and triiodothyronine all decreased (p < 0.01) indicating a metabolic adaptation to starvation. Prealbumin (p < 0.05) and fibronectin (p < 0.01) were also lowered. In conclusion, skeletal muscle function was significantly altered after five days of total starvation. However, to identify short term nutritional depletion in clinical practice the changes in muscle function were too small to be diagnostically useful.