Effects of additives on the stability of Humicola lanuginosa lipase during freeze-drying and storage in the dried solid.

The effects of various classes of additives on the stability of a protein with a relatively hydrophobic surface, Humicola lanuginosa lipase (HLL), during lyophilization and storage in the dried solid, were investigated. Prior to lyophilization, it was found that 1 M trehalose or 1% (wt/vol) Tween 20 caused the protein to precipitate. Infrared spectroscopy indicated that trehalose "salted-out" native HLL, whereas Tween 20 induced non-native aggregates. Optimal recovery of native protein in the initial dried solid was obtained in the presence of additives which formed an amorphous phase and which had the capacity to hydrogen bond to the dried protein (e.g., trehalose and sucrose). Additives which crystallized during lyophilization (e.g., mannitol) or which remained amorphous, but were unable to hydrogen bond to the dried protein (e.g., dextran), afforded less stabilization relative to that seen in the absence of additives. Optimal storage stability in the dried solid required that both protein unfolding during lyophilization was minimized and that the formulation was stored at a temperature below its Tg value. Crystallization of sucrose during storage greatly reduced the storage stability of HLL. This was attributed to the increased moisture content and the reduced Tg value in the remaining amorphous phase containing the protein. Sucrose crystallization and the resulting damage to the protein were inhibited by decreasing the mass ratio of sucrose:protein.

Effects of additives on the stability of recombinant human factor XIII during freeze-drying and storage in the dried solid.

Freeze-drying is often used to improve storage stability of therapeutic proteins. In order to obtain a product with optimal storage stability it is important to understand the mechanisms by which solutes protect the protein against freeze-drying-induced stresses and also against damage induced during subsequent storage. The objective of the current study was to examine the importance of various mechanisms proposed to account for acute and long-term storage stability using recombinant human Factor XIII (rFXIII)4 as a model protein. Initially, for acute stability during freeze-drying, it was found that solutes which formed an amorphous phase stabilized rFXIII to a greater degree than solutes which crystallized during freeze-drying. However, only amorphous solutes which were able to hydrogen bond to the protein, and thus preserve the native protein structure in the dried solid, provided optimal acute stability. Thus, in addition to forming an amorphous phase, it was also important to possess the ability to hydrogen bond to the protein. Long-term storage stability was found to be optimal in the presence of solutes which formed and maintained amorphous phases with Tg values above the storage temperature and which also preserved the native protein structure during freeze-drying. Solute crystallization during storage compromised storage stability.

Effect of Tween 20 on freeze-thawing- and agitation-induced aggregation of recombinant human factor XIII.

Agitation- and freeze-thawing-induced aggregation of recombinant human factor XIII (rFXIII) is due to interfacial adsorption and denaturation at the air-liquid and ice-liquid interfaces. The aggregation pathway proceeds through soluble aggregates to formation of insoluble aggregates regardless of the denaturing stimuli. A nonionic surfactant, polyoxyethylene sorbitan monolaurate (Tween 20), greatly reduces the rate of formation of insoluble aggregates as a function of surfactant concentration, thereby stabilizing native rFXIII. Maximum protection occurs at concentrations close to the critical micelle concentration (cmc), independent of initial protein concentration. To study the mechanistic aspects of the surfactant-induced stabilization, a series of spectroscopic studies were conducted. Electron paramagnetic resonance spectroscopy indicates that binding is not occurring between Tween 20 and either the native state or a folding intermediate state of rFXIII. Further, circular dichroism spectroscopy suggests that Tween 20 does not prevent the secondary structural changes induced upon guanidinium hydrochloride-induced unfolding. Taken together, these results imply that Tween 20 protects rFXIII against freeze-thawing- and agitation-induced aggregation primarily by competing with stress-induced soluble aggregates for interfaces, inhibiting subsequent transition to insoluble aggregates.

Cefotetan disodium-induced hemolytic anemia.

OBJECTIVE: To report a case of cefotetan disodium-induced hemolytic anemia. DATA SOURCES: Original research articles and case reports. DATA SYNTHESIS: A 46-year-old woman developed fulminant hemolytic anemia following a second exposure to intravenous cefotetan disodium for postoperative prophylaxis. She developed respiratory failure requiring intubation and acute renal failure requiring hemodialysis. The hemolysis was successfully treated with plasmapheresis, but the patient died on the 25th postoperative day. Positive Coomb's tests have been reported in less than three percent of patients receiving cefotetan. To our knowledge, this is the first case of fulminant hemolytic anemia associated with intravenous cefotetan disodium therapy. CONCLUSIONS: Cefotetan should be added to the list of drugs known to cause hemolytic anemia. Monitoring for hemolysis should be considered for patients who receive multiple courses of therapy.

Compression of foods during freeze-drying: water plasticization at the ice-dry layer interface.

The compressive mechanical properties of freeze-dried green beans show a pronounced decrease in rigidity when moisture content and/or temperature are increased. There exist several temperature and moisture combinations which give common values for mechanical properties. These combinations also give a common compressive behavior. Using this information on mechanical properties, it is possible to predict a stress-strain relationship, if given either a temperature, a moisture content, or the value of a pertinent mechanical property. It is shown that the moisture contents and temperatures that exist in the dry layer during freeze-drying result in mechanical properties that are suitable for compression of the dry layer. From studies on compression behavior during freeze-drying, it is shown that applied compressive pressure is the main determinant of final degree of compression. Increasing the compressive pressure gave a higher compression effect and gave a more rapid drying, presumably due to improved heat transfer in the compressed dry layer. From the above information, a simple method to predict compression behavior during freeze-drying was developed.

An outbreak of nitrogen dioxide-induced respiratory illness among ice hockey players.

During February 1987 an outbreak of nitrogen dioxide-induced respiratory illness occurred among players and spectators of two high school hockey games played at an indoor ice arena in Minnesota. The source of the nitrogen dioxide was the malfunctioning engine of the ice resurfacer. Case patients experienced acute onset of cough, hemoptysis, and/or dyspnea during, or within 48 hours of attending, a hockey game. One hundred sixteen cases were identified among hockey players, cheerleaders, and band members who attended the two games. Members of two hockey teams had spirometry performed at 10 days and 2 months after exposure; no significant compromise in lung function was documented. Nitrogen dioxide exposure in indoor ice arenas may be more common than currently is recognized; only three states require routine monitoring of air quality in ice arenas, and the respiratory symptoms caused by exposure to nitrogen dioxide are nonspecific and easily misdiagnosed.

Triple hybridization with cultivated barley (Hordeum vulgare L.).

A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. 'Gull', 'Golden Promise' and 'Vada'. Generally, cv 'Gull' had high crossability in crosses with related species in the primary hybrid. It is suggested that 'Gull' has a genetic factor for crossability not present in cv 'Vada' and cv 'Golden Promise'. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv 'Vada' but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x 'Gull' (2n=7-30) and (H. jubatum x H. lechleri) x 'Gull' (2n=20-22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19-22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.

Complex interspecific hybridization in barley (Hordeum vulgare L.) and the possible occurrence of apomixis.

Several complex hybrids were produced from the combination [(Hordeum lechleri, 6x xH. procerum, 6 x) × H. vulgare, 2 x]. Crosses with six diploid barley lines resulted in triple hybrids, most of which had a full complement of barley chromosomes (no. 1-7), but were mixoploid with respect to alien chromosomes (19-22). In one combination, chromosome no. 7 was duplicated. Meiosis in triple hybrids showed low, but variable pairing (1.3-5.5 chiasmata per cell). The syndesis probably did not include the barley chromosomes. Direct back-crosses to di- and tetraploid barley lines were unsuccessful. Chromosome doubling of the triple hybrid based on cv 'Pallas' resulted in a plant with 2n = 53-56, which had an increased fertility. Backcrosses to one di- and one tetraploid barley line resulted in offspring. The cross made with the tetraploid line ('Haisa II'), produced a 28-chromosomic plant in which the male parental genome was absent. We suspect that this plant may have arisen through parthenogenetic development of a reduced female gamete. The other cross with a diploid line ('9208/9') resulted in plant with 2n = 51-53. The most likely explanation for this second plant is that an unreduced gamete from the amphiploid was fertilized by a normal gamete from the backcross parent, and during early embryo development, some chromosomes were eliminated.

Selective effect of microembolization on pulmonary removal of biogenic amines.

Pulmonary amine extraction (E) was measured by triple-indicator dilution techniques from bolus injections of trace amounts of 5-hydroxy[14C]tryptamine ([14C]HT)m [3H]norepinephrine ([3H]NE), indocyanine green dye before and after glass-bead embolization in 23 anesthetized dogs. Control E(5-[14C]-HT) was 89.7 +/- 1.7%; 10 min after embolization (which approximately doubled pulmonary artery pressure and pulmonary vascular resistance), E(5-[14C]HT) was significantly reduced to 65.9 +/- 3.0% (kappa +/- SE; n = 10) (P less than 0.01). Control E([3H]NE) (40.1 +/- 4.5%) was unaffected by embolization. Imipramine (8 mg/kg) depressed control E(5-[14C]HT) to 38.7 +/- 1.5% and control E([3H]NE)d to 35.0 +/- 3.9% (P less than 0.05; n = 4). In these animals, pulmonary hemodynamic changes secondary to embolization were comparable to those in non-drug-treated dogs, but E(5-[14C]HT) and E([3H]NE) were not further depressed. Progressive pulmonary lobar artery ligation (n = 5) did not affect amine extraction until perfusion was limited to one lobe. The selectivity of the effect of embolization on E(5-[14C]HT), the lack of an effect on imipramine-insensitive E(5-[14C]HT) extraction, and the much smaller changes after progressive lobar ligation indicate that, although derecruitment of vascular surface area secondary to mechanical obstruction may contribute to postembolization depression of E(5-[14C]HT), additional mechanisms such as local saturation of 5-HT uptake or selective damage to endothelial cell transport of 5-HT may underlie these observations.

Design of an artificial skin. Part III. Control of pore structure.

Several methods are compared for preparing collagen-glycosaminoglycan (GAG) membranes of high or low porosity. Collagen-GAG membranes have been used to cover satisfactorily large experimental full-thickness skin wounds in guinea pigs over the past few years. Methods studied as means for controlling pore size are confined to purely physical processes which do not require use of additives or chemical reagents to form the porous membrane. We find that membranes, initially swollen in distilled water or saline, shrink linearly to no less than 94% of original dimension after freeze drying; to 75% after critical point drying (from CO2, following water-ethanol exchange); and to 41% of original dimension following air drying from the swollen state. Scanning electron microscopic study of the pore structure resulting from eah drying procedure confirms our major conclusion: A carefully designed freeze drying process, two variants of which are described in detail, yields membranes with the highest mean pore size, as measured by quantitative stereological procedures. Critical point drying gave significantly more shrinkage and a lower mean pore size than either one of the two freeze drying procedures used.