Use of Fmoc-N-(2-hydroxy-4-methoxybenzyl) amino acids in peptide synthesis

The use of N, O-bisFmoc-N-(2-hydroxy-4-methoxybenzyl) amino acid derivatives in the synthesis of peptides with difficult sequences has already been described. With these amino acid derivatives the reversible protecting group 2-hydroxy-4-methoxybenzyl (Hmb) for the backbone amide bonds of peptide chains is introduced, and thus the aggregation due to hydrogen-bond interchain association is inhibited. This paper describes the synthesis and use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives as an alternative means of introducing Hmb backbone protection. These new monoFmoc derivatives were obtained in higher yield than the bisFmoc derivatives. Coupling yields to the amino peptide resin were the same as those obtained with bisFmoc derivatives, under the TBTU/HOBt/DIEA conditions. We also compared different syntheses of a difficult peptide with the Fmoc approach [triple coupling, capping, use of chaotropic agents, backbone protection using monoFmoc (Hmb)Ala] and with optimized Boc chemistry. Both the backbone protection and optimized Boc chemistry approaches gave the desired product in excellent yield and purity. © Munksgaard 1997.     

Stability of subtilisins and related proteinases (subtilases)

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10^?3-10^?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca²⁺ in the second (third in the case of thermitase) binding site increases the T_m, values by 11-21 °C and the δG_D(H₂O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca²⁺ concentration of 2 × 10^?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca²⁺. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.     

Computer-Assisted Design of an Implantable, Intrathoracic Artificial Lung

Abstract: A semiempirical mathematical model of convective oxygen transport is used to design a new, low pressure loss, implantable artificial lung that could be used as a bridge to lung transplantation in patients with advanced respiratory failure. The mass transfer and flow friction relations pertinent to the design of a cross–flow hollow fiber membrane lung are described. The artificial lung is designed to transfer over 200 ml/min of oxygen at blood flow rates up to 5 L/min. A compact design and a blood-side pressure loss of <15 mm Hg allows the device to be implanted in the left chest without the need for a prosthetic blood pump. Surgical implantation of the artificial lung would require the creation of inflow and outflow anastomoses. Oxygen would be supplied via an external source. Blood properties, operating conditions, and empirically determined mass transfer and flow properties are all specified and input into a computer program that numerically solves the design equations. Computer–generated values for the device frontal area, blood path length, and fiber surface area are thereby obtained. The use of this computer–assisted design minimizes the need for extensive trial–and–error testing of prototype devices. Results from in vitro tests of a prototype implantable lung indicate that the mathematical model we describe is an accurate and useful tool in the design of hollow fiber artificial lungs.     

Analysis of the Intracellular Transport Properties of Recombinant La Crosse Virus Glycoproteins

The G1 and G2 glycoproteins of La Crosse virus, a member of theBunyavirusgenus of the Bunyaviridae, are encoded as a single open reading frame (ORF) in the viral middle-sized RNA segment. The primary product from this ORF is processed, either cotranslationally or shortly after translation, into the two glycoproteins and a nonstructural protein, NSm, of unknown function. We have expressed La Crosse glycoproteins using vaccinia vectors and studied their processing and localization. When expressed in the native G2-NSm-G1 configuration, both G1 and G2 targeted to the Golgi apparatus as shown by their colocalization with wheat germ agglutinin and acquired resistance to endoglycosidase H. When expressed independently, G2 was targeted to the Golgi apparatus but G1 was retained in the endoplasmic reticulum, indicating that a G1–G2 association is required for Golgi targeting of G1. In contrast to results with other members of the Bunyaviridae, we found that expression of G1 and G2 from separate vectors did not lead to the transport of the G1–G2 complex to the Golgi. However, disruption of the NSm region with a foreign sequence did not interfere with transport of the complex. When a portion of the β-galactosidase gene was inserted in frame into NSm, the glycoproteins derived from this construct were processed and targeted properly and were capable of mediating cell-to-cell fusion.     

A 90-Day Chloroform Inhalation Study in F-344 Rats: Profile of Toxicity and Relevance to Cancer Studies

Composition of diet may influence growth, diseases, tumor rates,and responses to chemical treatment. Since 1980 the NIH–07open formula nonpurified diet has been the selected diet forthe National Toxicology Program (NTP) toxicity and carcinogenicitystudies in rodents. Studies with nonpurified experimental dietswith lower protein and higher fat and fiber than the NIH-07diet indicated that the diet for Fischer-344 (F344) rats inlong-term studies could be modified to decrease the severityof chronic diseases and to decrease/delay the development ofspontaneous tumors. Based on the results of these studies anew open formula nonpurified diet designated as NTP-2000 wasformulated to contain 14.5% protein, 8.5% fat, and 9.5% fiber.Corn, wheat, and wheat middlings contribute to about 60% ofthe ingredients; soybean meal, fish meal, and alfalfa meal arethe additional sources of protein; purified cellulose, oat hulls,and alfalfa meal are the major sources of fiber; and soy oiland corn oil are the major sources of fat in the NTP-2000 diet.The Ca:P ratio and mineral and vitamin concentrations were reformulatedbased on AIN-93 and NRC-95 recommendations. The NIH-07 and theNTP-2000 diets were fed to groups of 6-week-old F344 rats for13 weeks and evaluated for growth patterns, food and water consumptions,hematology and clinical chemistry parameters, and organ weightsand pathological changes. Growth patterns and body weights weresimilar for both diets. Food consumptions were slightly higherand water consumptions were slightly lower for the groups fedNTP-2000 diet. There were no differences in hematological parametersbetween the groups fed the above diets. Serum levels of cholesterol,alkaline phosphatase, and 5' nucleotidase were slightly higherin groups fed the NTP–2000 diet possibly due to higherfat content of this diet. However, the serum triglyceride levelswere slightly lower in groups fed the NTP–2000 diet andit may be related to higher fiber content of the NTP–2000diet. The liver and kidney weights of the groups fed NTP–2000diet were significantly lower possibly due to lower proteincontent of this diet and lower protein consumption associatedchanges in Phase I and Phase II drug metabolizing enzyme systems.The adrenal weights were also lower in groups fed the new diet.The NTP–2000 diet prevented nephrocalcinosis and decreasedthe severity of nephropathy and cardiomyopathy, the common lesionsof F344 rats in 13–week studies. These results indicatethat the NTP–2000 diet is adequate for growth and maintenance of rats and appears to prevent or decrease the severityof diet-associated lesions.     

Chronic Dietary Toxicity/Oncogenicity Studies on 2,4-Dichlorophenoxyacetic Acid in Rodents

Forms of 2,4-dichlorophenoxyacetic acid (collectively knownas 2,4-D) are herbicides used to control a wide variety of broadleafand woody plants. Doses in the 2-year chronic/oncogenicity ratstudy were 0, 5, 75, and 150 mg/kg/day. The chronic toxicityparalleled subchronic findings, and a NOEL of 5 mg/kg/day wasestablished. A slight increase in astrocytomas observed (inmales only) at 45 mg/kg/day in a previously conducted chronicrat study was not confirmed in the present study at the highdose of 150 mg/kg/day. Doses in the 2-year mouse oncogenicitystudies were 0, 5, 150, and 300 mg/kg/day for females and 0,5, 62.5, and 125 mg/kg/day for males. No oncogenic effect wasnoted in the study. In summary, the findings of these studiesindicate low chronic toxicity of 2,4-D and the lack of oncogenicresponse to 2,4-D following chronic dietary exposure of 2,4-Din the rat and mouse.     

AA genotype of IL‐8 −251A/T is associated with low PaO2/FiO2 in critically ill patients and with increased IL‐8 expression

Background and Objective: Interleukin‐8 (IL‐8) is a central chemokine in acute respiratory distress syndrome (ARDS), and the IL‐8 gene contains a functional single nucleotide polymorphism (SNP) ?251A/T in its promoter region. We hypothesized that IL‐8 ?251A/T SNP is associated with PaO₂/FiO₂ in critically ill patients. Methods: We conducted genetic‐association studies in intensive care units at academic teaching centres using a derivation septic shock cohort (vasopressin and septic shock trial (VASST), n = 467) and a validation post‐cardiopulmonary bypass surgery cohort (CPB, n = 739) of Caucasian patients. Patients in both cohorts were genotyped for IL‐8 ?251A/T. The primary outcome variable in both cohorts was the fraction of patients who had a PaO₂/FiO₂ < 200. IL‐8 mRNA expression was measured in genotyped lymphoblastoid cells in vitro. Results: The frequency of the patients with PaO₂/FiO₂ <200 was significantly greater in patients who had the AA genotype of ?251A/T than in patients who had the AT or TT genotypes in both VASST (AA = 60.8% vs AT and TT = 53.8% and 48.0%, P = 0.038) and the CPB cohort (AA = 37.0% vs AT and TT = 27.0% and 26.0%, P = 0.039). Patients having the AA genotype had a higher probability to remain on mechanical ventilation (P = 0.047) in the first 14 days. Lymphoblastoid cells having the AA genotype had significantly higher IL‐8 mRNA expression than cells having the AT or TT genotype (P = 0.022). Conclusions: Critically ill Caucasian patients who had the AA genotype of IL‐8 ?251A/T had an increased risk of PaO₂/FiO₂ <200. The AA genotype was associated with greater IL‐8 mRNA expression than the AT or TT genotypes.     

Percutaneous Myocardial Laser Revascularization

Transmyocardial laser revascularization (TMR) is an experimental procedure for intractable angina that has demonstrated clinical benefit. The majority of patients who had TMR have significant reduction of angina. TMR has been performed exclusively by thoracic and cardiac surgeons using a handhold CO₂ laser device. The development of a prototype percutaneous device that delivers mid-infrared laser energy (Ho:YAG) through a 9Fr coaxial catheter system recently has been reported. The long-term benefits of this percutaneous approach is being defined in the context of the randomized PACIFIC Trial. The initial follow-up suggested significant relief of angina in the majority of treated patients, and the results are likely to be equal or similar to those of surgical TMR. The safety data of percutaneous myocardial revascularization (PMR) with the CardioGenesis system showed exceedingly low morbidity and mortality.