Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.     

Evidence for a catabolic role of glucagon during an amino acid load

Despite the strong association between protein catabolic conditions and hyperglucagonemia, and enhanced glucagon secretion by amino acids (AA), glucagon's effects on protein metabolism remain less clear than on glucose metabolism. To clearly define glucagon's catabolic effect on protein metabolism during AA load, we studied the effects of glucagon on circulating AA and protein dynamics in six healthy subjects. Five protocols were performed in each subject using somatostatin to inhibit the secretion of insulin, glucagon, and growth hormone (GH) and selectively replacing these hormones in different protocols. Total AA concentration was the highest when glucagon, insulin, and GH were low. Selective increase of glucagon levels prevented this increment in AA. Addition of high levels of insulin and GH to high glucagon had no effect on total AA levels, although branched chain AA levels declined. Glucagon mostly decreased glucogenic AA and enhanced glucose production. Endogenous leucine flux, reflecting proteolysis, decreased while leucine oxidation increased in protocols where AA were infused and these changes were unaffected by the hormones. Nonoxidative leucine flux reflecting protein synthesis was stimulated by AA, but high glucagon attenuated this effect. Addition of GH and insulin partially reversed the inhibitory effect of glucagon on protein synthesis. We conclude that glucagon is the pivotal hormone in amino acid disposal during an AA load and, by reducing the availability of AA, glucagon inhibits protein synthesis stimulated by AA. These data provide further support for a catabolic role of glucagon at physiological concentrations.     

Degradation kinetics of DMP 777, an elastase inhibitor

PURPOSE: The objective was to evaluate the degradation profile of the elastase inhibitor DMP 777 and lay the foundation for formulation development. METHODS: The pKa was determined by potentiometric titration in mixed-aqueous solvents. The degradation kinetics were studied as a function of pH, buffer concentration, ionic strength, methanol concentration and temperature using a stability-indicating HPLC assay. The degradation products were identified by LC-MS, NMR, and by comparison with authentic samples. RESULTS: The pKa for the protonated piperazine nitrogen was estimated to be 7.04. The pH-rate profile is described by specific acid-, water-, and specific base-catalyzed pathways. The pH of maximum stability is in the range of 4 to 4.5 where water is the principal catalyst in the reaction. Buffer catalysis, primary salt effects and medium effects were observed. The proposed mechanism for acid catalyzed degradation is the rarely observed AAL1 which involves alkyl-nitrogen heterolysis. The driving force for the reaction appears to lie in the stability of the benzylic carbocation. The proposed mechanism for base catalyzed degradation is BAC2 which involves beta-lactam ring opening. The beta-lactam ring of DMP 777, a monolactam, appears to be as reactive as that in benzylpenicillin in the KOH controlled region where a similar mechanism of hydrolysis should be operative. A contributing factor to this increased reactivity may lie in the reduced basicity of the beta-lactam nitrogen making it a good leaving group. CONCLUSIONS: The degradation profile indicates that development of a solution dosage form of DMP 777 with adequate shelf-life stability at room temperature is feasible.     

Diagnosing left lower lobe pneumonia: usefulness of the ''spine sign'' on lateral chest radiographs

Percutaneous cardiopulmonary assist devices (PCPS) have become available in interventional cardiology within recent years. These tools offer the opportunity of performing percutaneous transluminal coronary angioplasty (PTCA) in high-risk patients characterized by significant stenoses of several coronary arteries and a poor left ventricular function. It is unclear for which patients PCPS are necessary and which patients will profit by PTCA as compared to coronary artery bypass grafting (CABG). Therefore, the anticipated risk of CABG and of PTCA without assist devices was calculated according to risk scores and compared with our results of assisted PTCA. In addition the long-term survival rate was investigated. In 35 patients (mean 65.5 years of age, 12 females, 23 males), we performed PTCA concomitant with the use of cardiac assist devices. The indications for the use of a cardiac assist device were severely impaired LV function (EF 30% +/- 8.9%) in combination with significant coronary artery disease (2.7 +/- 0.3 vessels) and a significant supply area of the vessel to be dilated. In 6 patients, PCPS was started before coronary angioplasty because of hemodynamic instability. In 21 cases, PCPS was on a standby basis without being connected to the patient's circulation. In 8 patients, a left heart assist device, the 14F-Hemopump, was inserted percutaneously. The patients were analyzed using risk scores of angioplasty and of coronary bypass graft surgery. The calculated risk of hemodynamic compromise during PTCA according to the risk scores was more than 50%. The anticipated risk of a fatal outcome following CABG would have been 19.8%. PTCA was performed on an average of 2.0 coronary arteries per patient and was successful in 85%. We observed a decline in angina pectoris classification (CCS) from 3.5 to 1.6. An average reduction of 1.1 NYHA class was achieved. The in-hospital mortality was 8.6% (3 patients: 1 x sepsis, 1 x early reocclusion, 1 x cerebral embolism). At 24 months follow-up, a re-PTCA was necessary in four cases because of restenosis. In the remainder, NYHA and CCS class were stable during the follow-up period. An additional five patients died during the first year and two patients in the second year. We conclude that PTCA with the use of a cardiac assist device shows favorable short-term results in a subset of patients with extended coronary artery disease and severely impaired LV function who are not suitable for nonsupported PTCA or CABG due to their risk profile. However, the long term results are not satisfying and stress the need for complete revascularisation with CABG once the patient's condition is stabilized by means of supported PTCA.     

Bone mineral density and bone markers in relation to vitamin D receptor gene polymorphisms in Chinese men and women

Whether vitamin D receptor gene (VDRG) polymorphism can be used as a predictor for bone turnover rate or bone mass remains controversial. Its role within various ethnic populations are also unsettled. We examined VDRG polymorphism using restrictive enzymes Bsm-I, Apa-I, and Taq-I in 155 men aged 22-88 and 113 premenopausal women aged 40-53. The bone mineral density (BMD) of the vertebrae (L2-4), proximal femur, and total body bone mineral content (tb-BMC) (women only), as well as urinary N-terminal crosslinked fragment of type I collagen (NTX), serum osteocalcin, bone isozyme of alkaline phosphatase, and caboxyterminal propeptide of type I procollagen levels were measured. Chinese men and women exhibited a low prevalence for B (absence of Bsm-I restriction site) phenotypes than white and Japanese. Within the tested samples there were 0.4% BB homozygotes, 6.7% Bb heterozygotes, and 93% bb homozygotes. The distributions of Apa-I polymorphism (9.0% AA, 42.5% Aa, and 48.5% aa) also differed from those reported for the white populations. Most of the Chinese men and women were TT homozygous (96.6%). A comparison of actual values and values adjusted for age and weight of tb-BMC and BMD at the lumbar spine, Trochanter, Ward's triangle, and femoral neck showed no significant difference among three subgroups in each of the three sets of polymorphism. Furthermore, the actual values and adjusted values (adjusted for age) of the four bone markers, respectively, showed no significant differences. We conclude that given the very low prevalence of the suspected high risk genotypes (B, A, and t), and the lack of difference among the polymorphic subgroups, VDRG polymorphism may not be an important determinant of the bone turnover rate and bone mass of Chinese men and women.     

Type I collagen and procollagen fragments in patients with metabolic bone diseases

The purpose of the present study was to evaluate the serum levels of two new markers, and to compare their clinical usefulness with two conventional markers. Healthy women and patients with aberrant bone metabolism were evaluated for serum or urine levels of different bone markers. We measured serum levels of the pyridinoline cross-linked telopeptide domain of type I collagen (S-ICTP) and carboxy-terminal propeptide of type I procollagen (S-PICP) as markers of bone resorption and formation, respectively. These levels were compared to the concentrations of serum bone gamma-carboxyglutamic acid protein (S-BGP) and total urinary pyridinium cross-links (U-PYD). The control group included 222 premenopausal and postmenopausal women, and the disease groups consisted of 61 individuals with malignancy-associated hypercalcemia, Graves' thyrotoxicosis or primary hyperparathyroidism. Both S-PICP and S-BGP reflected higher bone turnover in postmenopausal than premenopausal women. All patient groups had significantly higher S-ICTP and U-PYD than the controls. Increased S-PICP was seen in malignancy-associated hypercalcemia and Graves' thyrotoxicosis, but not in primary hyperparathyroidism, while higher S-BGP was seen in Graves' thyrotoxicosis and primary hyperparathyroidism, but not in malignancy-associated hypercalcemia. Discrepancy between S-PICP and S-BGP in malignancy-associated hypercalcemia and primary hyperparathyroidism was noted. S-ICTP and U-PYD had higher sensitivity and specificity in discriminating patients from controls. We conclude that S-ICTP is superior to U-PYD as an index of bone resorption in aberrant bone metabolism. S-PICP may also be a useful bone turnover marker but discrepancies between it and S-BGP in malignancy-associated hypercalcemia and primary hyperparathyroidism need further investigation.     

Differences in receptor binding of LDL subfractions

Differences in low density lipoprotein (LDL) receptor-binding affinity among LDL particles of different size were examined in competitive binding assays in human skin fibroblasts and LDL (d = 1.020 to 1.050 g/mL) from subjects with a predominance of large (> or = 272 A), medium (259 to 271 A), and small (< or = 257 A) LDL. Among 57 normolipidemic subjects with LDL cholesterol (-C) levels < 160 mg/dL, binding affinity was reduced by 16% in those with predominantly large LDL and by 14% in those with small LDL compared with most subjects who had a predominance of medium-size LDL and in all LDL size subgroups in 66 subjects with LDL-C > or = 160 mg/dL. Differences in LDL receptor-binding affinity were further investigated by using LDL density subfractions (I, d = 1.026 to 1.032 g/mL; II, d = 1.032 to 1.038 g/mL; and III, d = 1.038 to 1.050 g/mL) from three subjects with predominantly large (pattern A) and small (pattern B) LDL particles. The binding affinity (Kd) of LDL-II was similar for patterns A and B (9.2 +/- 1.4 and 9.4 +/- 0.7, respectively) and 30% lower in LDL-III from both groups (P < .05). The binding affinity of LDL-I in pattern A (12.6 +/- 1.5 micrograms/mg) was lower (P < .05) than that in LDL-II and LDL-I from pattern B (8.0 +/- 2.4 micrograms/mg). After incubation with a monoclonal antibody that specifically blocked the LDL receptor-binding domain of apoE, LDL-I from two pattern B subjects showed substantially lower binding affinity (Kd = 20.0 and 19.2 micrograms/mg) than in pattern A (Kd = 13.2 and 14.2 micrograms/mg), a result consistent with our finding of a higher apoE content in pattern B LDL-I (P < .001). Thus, factors associated with variations in particle size and apoE content in LDL subclasses in normolipidemic subjects contribute to the differences in LDL receptor binding that may result in differing metabolic behavior in vivo.     

Effects of sleeping in a chemical protective mask on sleep quality and cognitive performance

PURPOSE: We wanted to determine whether sleep is disrupted when soldiers sleep in a new chemical protective mask, the M40. Sleep quantity and quality, extent of protection provided by the mask during sleep, and next day performance were assessed. METHOD: After several days of training, 9 male soldiers slept with and without the M40 mask on four occasions. RESULTS: Soldiers were able to tolerate the mask for most or all of the night. However, sleep, as assessed by wrist-worn activity monitors, was significantly disturbed. Minutes (mean +/- SEM) of waking significantly increased, from 25 +/- 2.1 to 86 +/- 8.5 per night (p < 0.001), and number of awakenings rose from 8 +/- 0.6 to 20 +/- 0.9 (p < 0.0001). Soldiers reported that it took longer and was more difficult to fall asleep when wearing the mask. Errors on a choice reaction time task increased significantly and subjects reported greater fatigue and sleepiness the day after sleeping in the mask. Protection provided by the masks varied substantially among subjects and declined over the course of the study. Some soldiers were protected throughout the night but others were only protected intermittently. CONCLUSION: We conclude that sleeping in the chemical protective mask should only be done when necessary, given the adverse effects on sleep and daytime function, as well as the variability of protection, of the mask.     

Mimosine blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells

We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-gamma from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with IC50 of less than 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12-14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, whereas doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after preincubation with a panel of recombinant cytokines. IFN-gamma primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-gamma during infection of SCID mice with Listeria inhibited TNF-alpha mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous Il-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage.