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.     

Body water content over a range of ambient salinities in the sheepshead minnow

Body water content was determined on groups of Cyprinodon variegatus after sequential acclimation to ambient salinities in the range from fresh water to 100%. A multiple linear regression model applied to the data included body mass ( M ) of individuals and acclimation salinity ( S ) as independent variables, and body water content ( W ) as the dependent variable. The model that described the relationship was W (g) = 0·059 + 0·723 M (g)–0001 S (%). Converting values of body water content to percent of wet body mass produced a range of values from 74·5% in fresh water to 72·0% at 100%, only a 2·5% difference in body water content over this wide range of ambient salinities.     

Distribution and quantification of alpha1-integrin subunit in rat organs

Summary The ₁₁-integrin is known to be a receptor for collagen and laminin mediating cell-matrix interactions. A monoclonal antibody, 33.4, which specifically inhibits the ₁-integrin-mediated in vitro cell-collagen binding of rat hepatocytes and hepatoma-derived A-cells (Löster et al., 1994), was used to purify by immunoaffinity chromatography the ₁-integrin subunit from rat liver in large quantities for inducing a polyclonal antiserum. In immunoblot analysis on membrane extracts of several rat organs this polyclonal antiserum recognized only a 190 kDa-band, suggesting that it is highly specific for the ₁-integrin subunit. A sandwich-ELISA with monoclonal antibody 33.4 and the polyclonal antiserum against the ₁-integrin subunit, respectively, enabled the quantitative expression pattern of the ₁-integrin subunit to be studied in different rat organs. With the exceptions of brain (not detectable) and muscle (low concentration), the ₁-integrin subunit was detectable in almost all organs of the digestive, respiratory and urogenital system as well as in lymphatic organs. The highest relative concentrations of ₁-integrin subunit were found in uterus, lung and spleen, whereas in seminal vesicle, stomach, parotid gland, epididymis, kidney and liver only modest concentrations were evident. The organ distribution and localization of ₁-integrin subunit were studied by immunohistochemistry with monoclonal and polyclonal antibodies. Immunoreactivity was present in the plasma membranes of all smooth muscle cells, vascular endothelial cells of many organs and fibrocyte-fibroblast sheaths in the heart and kidney. Since these cells are in close contact with collagen-containing basal membranes as well as reticular fibrils, strong evidence exists that in rat tissues the ₁-integrin subunit is expressed at sites where collagen is present and might be involved in vivo in cell—ollagen binding.Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

Dobutamine as a countermeasure for reduced exercise performance of rats exposed to simulated microgravity

Tipton, Charles M., and Lisa A. Sebastian. Dobutamineas a countermeasure for reduced exercise performance of rats exposed tosimulated microgravity. J. Appl.Physiol. 82(5): 1607-1615, 1997.Post-spaceflightresults and findings from humans and rodents after conditions of bedrest or simulated microgravity indicate maximum exercise performance issignificantly compromised. However, the chronic administration ofdobutamine (a synthetic adrenomimetic) to humans in relevantexperiments improves exercise performance by mechanisms that preventthe decline in peak O₂ consumption (O_{2 peak}) and reducethe concentration of lactic acid measured in the blood. Althoughdobutamine restores maximumO₂values in animals participating in simulated microgravitystudies, it is unknown whether injections of this₁-,₁-, and₂-adrenoceptor agonist in ratswill enhance exercise performance. To investigate this, adult male ratswere assigned to three experimental groups: caged control receivingsaline; head-down, tail-suspended (HDS) receiving saline (HDS-S); andan HDS group receiving dobutamine hydrochloride injections (1.8 mg/kgtwice daily per rat). Treadmill tests were performed before suspension,at 14 days, and after 21 days.O_{2 peak}, run time,and the rate of rise in colonic temperature (heating index) wereevaluated after 14 days, whereas at 21 days, hemodynamic responses(heart rate, systolic blood pressure, and double product) weredetermined during submaximal exercise with blood pH, blood gases, andlactic acid concentration values obtained during maximal exercise. Incontrast to the results for the HDS-S rats, dobutamine administrationdid restore O_{2 peak} and "normalized" lactic acid concentrations during maximalexercise. However, daily injections were unable to enhance exerciseperformance aspects associated with treadmill run time, the mechanicalefficiency of running, the heating index, or the retention of muscleand body mass. These simulated microgravity findings suggest that dobutamine's potential value as a countermeasure for postflight maximal performance or for egress emergencies is limited and that othercountermeasures must be considered.

     

Regulation of guinea pig macrophage collagenase production by dexamethasone and colchicine

Previous studies have demonstrated that exposure of guinea pig macrophages to a primary signal, such as lipopolysaccharide (LPS), stimulates the synthesis of prostaglandin E2 (PGE2) which, in turn, elevates cAMP levels resulting in the production of the enzyme, collagenase. The potential of regulating the biochemical events in this activation sequence was examined with the anti-inflammatory agents dexamethasone and colchicine, which suppress the destructive sequelae in chronic inflammatory lesions associated with the degradation of connective tissue. The addition of dexamethasone with LPS to macrophage cultures resulted in a dose-dependent inhibition of PGE2 and collagenase production, which was reversed by the exogenous addition of phospholipase A2. Collagenase production was also restored in dexamethasone-treated cultures by the addition of products normally produced as a result of phospholipase action, such as arachidonic acid, PGE2 or dibutyryl-cAMP. Since the effect of dexamethasone was thus linked to phospholipase A2 inhibition, mepacrine, a phospholipase inhibitor, was also tested. Mepacrine, like dexamethasone, caused a dose-dependent inhibition of PGE2 and collagenase. In addition to corticosteroid inhibition, colchicine was also found to block collagenase production. However, this anti-inflammatory agent had no effect on PGE2 synthesis. Colchicine was effective only when added at the onset of culture and not 24 h later, implicating a role for microtubules in the transmission of the activation signal rather than enzyme secretion. The failure of lumicolchicine to inhibit collagenase activity provided additional evidence that microtubules are involved in the activation of macrophages. These findings demonstrate that dexamethasone and colchicine act at specific steps in the activation sequence of guinea pig macrophages to regulate collagenase production.     

Pulse fluorimetry study in polarized light of DNA-ethidium bromide complexes

In previous works, a quantitative analysis of the fluorescence anisotropy decay, based on a comparison of the experimental measurements with a Monte Carlo simulation of the excitation energy migration, has been shown to provide the value of the unwinding angle of the DNA helix, induced by an ethidium bromide (E.B.) molecule intercalation. In the present work some of the characteristics of the model used in the computation are reexamined: namely the influence of the direction of the E.B. electronic moment, and the influence of the dye distribution along the DNA helix are studied. The computations are compared with experimental results obtained with new experiments performed with calf thymus and micrococcus lysodeikticus DNA-E.B. complexes. It is found that the difference in base composition of these DNA does not influence the fluorescence properties of their E.B. complexes. Our study confirms the validity of the dye distribution obtained with the single adjacent excluded site principle. Reasonable values of the unwinding angle are obtained by assuming that the transition moment direction lies along the great axis of the E.B. molecule. The value of this unwinding angle is compared with other values proposed in the literature.     

Time resolved spectroscopy of the tryptophyl fluorescence of the E. coli LAC repressor.

A new crystal form of a mitogenic lectin from pea seeds ($\text{Pisum sativum}$) has been obtained which is suitable for high resolution structural work. The crystals are orthorhombic, space group P2₁2₁2₁, with unit cell dimensions: $\text{a}$ = 64.2Å, $\text{b}$ = 72. 7Å, $\text{c}$ = 108. 3Å. The asymmetric unit contains one protein molecule.     

Pulse fluorimetry study of energy transfers between tryptophan residues and NADPH in beef liver glutamate dehydrogenase complexes

A method is proposed to determine the rates of singlet energy transfers in an array of chromophores containing a finite number of donors and fluorescent acceptors. This method is based on measurements of transfer efficiency coupled with pulse fluorimetry. Three classes of donors can be distinguished which differ in their energy transfer rate. The rates of the first, the second and the third class are respectively greater than, of the order of, and smaller than the emission rate. The method is applied to the study of the energy transfers from tryptophan residues to NADPH, in ternary and quaternary glutamate dehydrogenase complexes. Practically, all these tryptophan residues belong to the first class. They can be divided into two subclasses having different transfer rate values. The distances between these residues and the NADPH site are of the order of 2.5 nm. In addition, the ligand binding induces a protein conformational change, leading to a fluorescence quenching of the tryptophanyl emission.