A unique variant of streptococcal group O-antigen (C-polysaccharide) that lacks phosphocholine.

Streptococcus mitis strain SK598, which represents a subgroup of biovar 1, possesses a unique variant of the C-polysaccharide found in the cell wall of all strains of Streptococcus pneumoniae and in some strains of S. mitis. This new variant lacks the choline methyl groups in contrast to the previously characterized forms of C-polysaccharide, which all contain one or two choline residues per repeat. The following structure of the repeating unit of the SK598 polysaccharide was established: where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This structure is identical to the double choline-substituted form of C-polysaccharide, except that it is substituted with ethanolamine instead of choline. This extends the number of recognized C-polysaccharide variants to four.     

Thermal Inactivation of Nonproteolytic Clostridium botulinum Type E Spores in Model Fish Media and in Vacuum-Packaged Hot-Smoked Fish Products

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10³. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10⁶ spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

An approach to integrated antibody production: Coupling of fluidized bed cultivation and fluidized bed adsorption

Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.The help of H. Schmitz, A. Bader, J. Gätgens and M. Halfar during the experiments is gratefully acknowledged. This work was partially funded by the ministry of science and research of the Federal Republic of Germany within the project Stoffumwandlung mit Biokatalysatoren.     

Davydov Soliton Dynamics in Proteins: II. The General Case

We performed long time simulations using the |D₁> approximation for the solution of the Davydov Hamiltonian. In addition we computed expectation values of the relevant operators with the state (_D/J)|D₁> and the deviation |> from the exact solution over long times, namely 10 ns. We found that in the very long time scale the |D₁> ansatz is very close to an exact solution, showing expectation values of the relevant physical observables in the state (_D/J)|D₁> being about 5-6 orders of magnitudes larger than in the deviation state |>. In the intermediate time scale of the ps range such errors, as known from our previous work, are somewhat larger, but still more or less negligibly. Thus we also report results from an investigation of the very short time (in the range 0-0.4 ps) behaviour of the |D₁> state compared with that of an expansion of the exact solution in powers of time t. This expansion is reliable for about 0.12 ps for special cases as shown in the previous paper. However, the accuracy of the exactly known value of the norm and the expectation value of the Hamiltonian finally indicates up to what time a given expansion is valid, as also shown in the preceding paper. The comparison of the expectation values of the operators representing the relevant physical observables, formed with the third order wave function and with the corresponding results of |D₁> simulations has shown, that our expansion is valid up to a time of roughly 0.10-0.15 ps. Within this time the second and third order corrections turned out to be not very important. This is due to the fact that our first order state contains already some terms of the expansion, summed up to inifinite order. Further we found good agreement of the results obtained with our expansion and those from the corresponding |D₁> simulations within the time of about 0.10 ps. At later times, the factors with explicit powers of t in second and third order become dominant, making the expansion meaningless. Possibilities for the use of such expansions for larger times are described. Alltogether we have shown (together with previous work on medium times), that the |D₁> state, although of approximative nature, is very close to an exact solution of the Davydov model on time scales from some femtoseconds up to nanoseconds. Especially the very small time region is of importance, because in this time a possible soliton formation from the initial excitation would start.     

Serum complex of trypsin 2 and alpha 1 antitrypsin as diagnostic and prognostic marker of acute pancreatitis: clinical study in consecutive patients.

OBJECTIVE--To estimate the usefulness of serum concentrations of the complex of trypsin 2 and alpha 1 antitrypsin in diagnosing and assessing the severity of acute pancreatitis in comparison with serum C reactive protein, amylase, and trypsinogen 2 concentrations (reference markers). DESIGN--Markers were measured in consecutive patients admitted with acute abdominal pain that was either due to pancreatitis or to other disease unrelated to the pancreas (controls). SETTING--Department of surgery of a teaching hospital in Helsinki. SUBJECTS--110 patients with acute pancreatitis and 66 with acute abdominal diseases of extrapancreatic origin. On the basis of the clinical course, acute pancreatitis was classified as mild (82 patients) or severe (28 patients). MAIN OUTCOME MEASURES--Clinical diagnosis of acute pancreatitis and severity of the disease. RESULTS--At admission all patients with acute pancreatitis had clearly raised concentrations of trypsin 2-alpha 1 antitrypsin complex (32 micrograms/l), whereas only three of the controls had such values. Of the markers studied, trypsin 2-alpha 1 antitrypsin complex had the largest area under the receiver operating curve, both in differentiating acute pancreatitis from extrapancreatic disease and in differentiating mild from severe disease. CONCLUSIONS--Of the markers studied, trypsin 2-alpha 1 antitrypsin complex was the most accurate in differentiating between acute pancreatitis and extrapancreatic disease and in predicting a severe course for acute pancreatitis.     

Intra-pancreatic release of bradykinin during the course experimental pancreatitis in rat

Kallikrein/Kininogn activation is an important pathophysiological event in acute pancreatitis, leading to microcirculatory changes within the gland. Hitherto, only indirect measurements of pancreatic bradykinin formation have been performed, monitoring the peptide in the circulation and in the peritoneal exudate. In the present study, intra-pancreatic bradykinin release was assessed using microdialysis during experimental acute pancreatitis in rat. In mild, oedematous pancreatitis, induced by caerulein hyperstimulation, the levels of bradykinin within the gland were not elevated compared with those of control rats. However, in necrotic pancreatitis, induced by retrograde injection of taurocholate into the pancreatic duct, significantly elevated levels of intraglandular bradykinin were seen. Several rats in this group died whilst in a state of circulatory shock.