Sterilization of Regenerated Collagen Sutures with β-Propiolactone

Data are presented to show that β-propiolactone when properly applied is a very effective agent for sterilization of regenerated collagen sutures. The chemical sterilization is accomplished with little or none of the loss in strength encountered with heat sterilization. The finished sterile suture is obtained without any harmful residue that might be detrimental to the patient.     

New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally

We have established and characterized a spontaneously immortalized, nontumorigenic mouse mammary cell line, designated IM-2. IM-2 cells synthesize large amounts of the milk protein beta-casein upon addition of lactogenic hormones. The induction of beta-casein occurs rapidly and does not require any exogenous extracellular matrix components. The IM-2 cell line is morphologically heterogeneous and could be separated into cell clones with epithelial and fibroblastic characteristics. In monoculture, none of the epithelial clones could be induced to synthesize caseins. Coculture of epithelial and fibroblastic clones, however, rendered the epithelial cells competent to differentiate functionally; the addition of lactogenic hormones to these cocultures resulted in the synthesis of beta-casein in amounts comparable to that seen with the original IM-2 line. Using this unique cell system, we have investigated the interrelationships between different steps in differentiation leading to hormone-induced casein production. Independent of hormones, epithelial-fibroblastic cell contacts led to the formation of characteristic structures showing the deposition of laminin. We found that the epithelial cells located in these structures also exhibited significantly increased levels of cytokeratin intermediate filament polypeptides. Double immunofluorescence revealed that the cells inducible by hormones to synthesize casein, colocalized exactly with the areas of laminin deposition and with the cells showing greatly intensified cytokeratin expression. These results suggest that hormone-independent differentiation events take place in response to intercellular epithelial-mesenchymal contacts. These events in turn bring about a state of competence for functional differentiation after lactogenic hormonal stimulation.     

Na pump and plasma membrane structure in L-cell fibroblasts expressing rat liver fatty acid binding protein.

Although the intracellular fatty acid binding proteins have been investigated for nearly two decades and purified proteins are now available, little is known regarding the function of these proteins in intact cells. Therefore, L-cell fibroblasts transfected with cDNA encoding for rat liver fatty acid binding protein (L-FABP) were examined as to whether L-FABP expression in intact cells modifies plasma membrane enzyme activities, fluidity, and lipids. Plasma membrane Na/K-ATPase activity was 65.9 +/- 18.7 and 38.6 +/- 22.8 (P less than 0.001) nmol/mg protein x min for control and high-expression transfected cells, respectively. Consistent with this observation, [3H] ouabain binding to whole cells was significantly decreased from 3.7 +/- 0.3 to 2.0 +/- 0.8 pmol ouabain bound/mg cell protein in control and high-expression cells, respectively, whereas the cell's affinity for ouabain was not significantly altered. Unexpectedly, Western blot analysis indicated that transfected cells had higher levels of Na+, K(+)-ATPase protein; in contrast, the activities of 5'-nucleotidase and Mg-ATPase were unaltered. The effects of L-FABP expression on plasma membrane Na/K-ATPase function appeared to be mediated through alterations in plasma membrane lipids and/or structure. The plasma membrane cholesterol/phospholipid ratio decreased and the bulk plasma membrane fluidity increased in the high-expression cells. In conclusion, plasma membrane Na/K-ATPase activity in L cells may be regulated in part through expression of cytosolic L-FABP.     

Activation of glycogenolysis by stimulation of the hepatic nerves in perfused livers of guinea pig and tree shrew as compared to rat: differences in the mode of action

A study on the metabolic and hemodynamic actions of hepatic nerve stimulation in the perfused liver of guinea pig and tree shrew as compared to rat was performed, since the density of liver innervation was reported to be different. 1) Nerve stimulation resulted in an increase in glucose release and decrease in lactate uptake or in a shift to output as well as a decrease in portal flow in all three species. The change in glucose output was very similar, that in lactate balance and flow was smaller in tree shrew than in guinea pig and rat. Apparently, the metabolic and hemodynamic changes did not reflect the different densities of liver innervation. 2) The overflow of the neurotransmitter noradrenaline into the hepatic vein differed very clearly in the three animals. In the guinea pig and tree shrew the maximal increase in noradrenaline concentration measured in the effluent was about 6-7-fold higher than in the rat. 3) The content of noradrenaline in the liver in vivo was about five-fold higher in the guinea pig and again another four-fold higher in the tree shrew than in the rat. The contents of adrenaline and dopamine were very low in comparison to those of noradrenaline. The different hepatic noradrenaline contents of the three species investigated are in line with the anatomical findings on the different innervation density. 4) Inhibitors of eicosanoid synthesis reduced the nerve stimulation-dependent metabolic and hemodynamic alterations in guinea pig liver as in rat liver indicating a similar mechanism in these species. Apparently, prostaglandins might be involved as mediators or modulators of nerve actions also in the more densely innervated guinea pig liver and not only in the less densely innervated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate of disappearance of glycerol from plasma of fetal and newborn sheep

The disappearance of glycerol from plasma was studied after a single intravenous injection to estimate its volume of distribution (Vdist), plasma clearance rate, and rate constant for irreversible loss (kd). Studies were repeated before and after birth of the lamb to test whether loss of the placenta could account for rapidly increasing plasma concentrations in the newborn. The disappearance of glycerol was closely described by a double-exponential model in each instance. In fetal sheep Vdist averaged 0.41 +/- 0.15 (SD) 1/kg fetal wt (n = 15). This volume decreased to 0.33 +/- 0.11 l/kg (n = 8) soon after functionally removing the placenta (by snaring the umbilical cord and maintaining the fetus with intrauterine ventilation), but the change was not significant. In newborn lambs 1-3 days of age, Vdist averaged 0.45 +/- 0.11 l/kg (n = 5, NS). Plasma clearance rate also did not change significantly, averaging 7.9 +/- 2.9, 7.9 +/- 3.8, and 9.0 +/- 5.9 ml.min-1.kg-1 in the fetus, after simulated birth, and in the newborn lamb, respectively, kd also was not altered measurably and averaged 0.020 +/- 0.006, 0.024 +/- 0.007, and 0.019 +/- 0.007 min-1 during the same time periods. Similar results were obtained by using three widely different amounts of infused glycerol. The results indicate that removal of glycerol does not depend on placental function to an appreciable extent. It is concluded that plasma glycerol concentration reflects principally glycerol turnover and, hence, lipolysis before and after birth.     

Early Administration of 17β-Estradiol Partially Masculinizes Song Control Regions and α2-Adrenergic Receptor Distribution in European Starlings (Sturnus vulgaris)

The vocal control system in many songbird species is a sexually dimorphic neural circuit that mediates learning and production of song. The mechanism by which this system is sexually differentiated has been investigated in only one species, the zebra finch (Taeniopygia guttata). Estradiol may be involved in the sexual differentiation of this system, as female zebra finches treated with estradiol as nestlings develop a male-like song system; however, blocking estradiol action in embryonic and nestling male zebra finches does not demasculinize the song system. Therefore, the role of estradiol in song system development is unclear. The role of estradiol in song system sexual differentiation was assessed in European starlings (Sturnus vulgaris). This species is of potential interest because it is less extreme in the degree of sexual dimorphism of the song system and song behavior than zebra finches. While in the field, starling nestlings were implanted with 500 μg of estradiol at 3 days of age. These birds were brought into the laboratory at Day 11 and hand-reared. In females, estradiol produces significant increases in the volumes of song control regions defined by Nissl stain, as well as by autoradiography for α₂-adrenergic receptors; however, these estradiol-treated females have song systems that more closely resemble those of control females than control males. Estradiol-treated males exhibit significant hypermasculinization at 210 days of age, but this effect is transient and hypermasculinization is no longer evident at Day 345. The role of estradiol in sexual differentiation of the neural circuit mediating song behavior remains enigmatic.     

Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1.

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.     

Inactivation of Mycobacterium paratuberculosis in cows' milk at pasteurization temperatures.

The thermal inactivation of 11 strains of Mycobacterium paratuberculosis at pasteurization temperatures was investigated. Cows' milk inoculated with M. paratuberculosis at two levels (10(7) and 10(4) CFU/ml) was pasteurized in the laboratory by (i) a standard holder method (63.5 degrees C for 30 min) and (ii) a high-temperature, short-time (HTST) method (71.7 degrees C for 15 s). Additional heating times of 5, 10, 15, 20, and 40 min at 63.5 degrees C were included to enable the construction of a thermal death curve for the organism. Viability after pasteurization was assessed by culture on Herrold's egg yolk medium containing mycobactin J (HEYM) and in BACTEC Middlebrook 12B radiometric medium supplemented with mycobactin J and sterile egg yolk emulsion. Confirmation of acid-fast survivors of pasteurization as viable M. paratuberculosis cells was achieved by subculture on HEYM to indicate viability coupled with PCR using M. paratuberculosis-specific 1S900 primers. When milk was initially inoculated with 10(6) to 10(7) CFU of M. paratuberculosis per ml, M. paratuberculosis cells were isolated from 27 of 28 (96%) and 29 of 34 (85%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. Correspondingly, when 10(3) to 10(4) CFU of M. paratuberculosis per ml of milk were present before heat treatment, M. paratuberculosis cells were isolated from 14 of 28 (50%) and 19 of 33 (58%) pasteurized milk samples heat treated by the holder and HTST methods, respectively. The thermal death curve for M. paratuberculosis was concave in shape, exhibiting a rapid initial death rate followed by significant "tailing." Results indicate that when large numbers of M. paratuberculosis cells are present in milk, the organism may not be completely inactivated by heat treatments simulating holder and HTST pasteurization under laboratory conditions.