Clinical correlation of a skin antisepsis model.

The use of pigskin as a test substrate for evaluating topical antimicrobial activity has been developed. Simulated handwashing protocols with this in vitro model in parallel with in vivo studies have been evaluated, based on an ASTM method for the clinical evaluation of a healthcare personnel handwash. Using Serratia marcescens as the test organism, similar log reductions were observed using the in vitro model when compared to in vivo efficacy. Results suggest that this model can be used as a reliable indicator of antiseptic efficacy on the skin. The use of sterilized skin simplifies the use of this model for both efficacy and skin-pathogen interaction studies.

Growth factors (EGF, IGF-I) modulate the morphological differentiation of adult marmoset (Callithrix jacchus) Sertoli cells in vitro.

The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on mature marmoset (Callithrix jacchus) Sertoli cells in vitro were investigated using light and electron microscopic and histochemical means. The morphological data were substantiated by morphometric analysis at the electron microscopic level. In a bicameral chamber system, cultured Sertoli cells displayed a high degree of ultrastructural differentiation and exhibited a polarized appearance. Basally located tight junctions joined adjacent cells. Germ cells of early stages of development were regularly seen. Under the influence of IGF-I, cells developed extensive cell-cell contacts. The surface density of smooth endoplasmic reticulum was increased. In contrast, the volume density of lipid inclusions was decreased. The morphological integrity of enclosed germ cells was maintained for a longer period. With EGF, cells were arranged in loose aggregates. Intercellular spaces were widened. The volume density of lipid inclusions was increased. Germ cells exhibited profound signs of degeneration early in culture, paralleled by increased development of phagolysosomes and high acid-beta-galactosidase activity. Under the influence of either growth factor, mitochondria displayed a shift from the crista to the tubulo-vesicular type. Mitochondrial dimensions and the volume density of mitochondrial compartment were increased. In comparison with control cultures all documented changes were statistically significant. Our findings indicate that marmoset Sertoli cells are target cells for EGF and IGF-I. Moreover, the dynamics of intercellular contacts, germ cell survival, and morphological indices of lipid and/or steroid metabolism seem to be differentially modulated.

Activity assays for characterizing the thrombolytic protein fibrolase.

Characterization of the thrombolytic agent fibrolase was accomplished employing specific proteolytic and thrombolytic assays. This paper describes a method to measure enzyme proteolytic activity using the oxidized beta-chain of insulin as a substrate. Advantages of this method include a short incubation time for substrate cleavage followed by an isocratic HPLC method with a retention time of approx. 5 min. Proteolytic activity can be rapidly and easily quantitated with this procedure. An azocasein assay was also used to quantitate proteolytic activity. This method was optimized with respect to substrate concentration and incubation time allowing for the rapid quantitation of fibrolase activity. A thrombolytic assay is described which employs fibrin plate clearance and has the advantage of rapid and accurate quantitation compared with previously described methods. It also allows for the standardization of fibrolase in plasmin-equivalent units.

Fibrolase. A fibrinolytic protein from snake venom.

Fibrolase is an active fibrinolytic agent and possesses potential for use in thrombolytic therapy. Its mode of action had been characterized, both in vitro and in vivo. Possessing three disulfide bonds, native fibrolase is nonglycosylated and binds an intrinsic zinc atom. The zinc is essential for retention of activity and structural integrity. In solution, fibrolase is sensitive to changes in pH and temperature (Pretzer et al., 1991). At neutral to basic pH (pH 5-9), the solubility and stability of fibrolase is nearly constant. Little structural variation can be detected by CD spectroscopy. However, decrease in pH below 5 leads to a pronounced reduction in both the solubility and activity of fibrolase. At pH 3 and below, the solubility of fibrolase returns but the activity does not. This solubility profile is unusual in that the minimal solubility is well removed from the pI (which is 6.7). It is proposed that the behavior of fibrolase with variation in pH can be understood in terms of capacity to bind zinc. At pH 5 to 9, the protein binds zinc and the structure and activity are preserved. Near pH 5, the histidine residues which serve as ligands for the zinc become protonated and zinc binding is lost. Loss of zinc leads to local unfolding of a helical segment of fibrolase, exposing hydrophobic groups which allow the protein to rapidly aggregate. At lower pH values (1-3), the protein again adopts a more globular structure, similar to molten globule states, and the solubility increases. However, without the zinc, fibrolase remains inactive. Changes in pH also affect thermal stability. The Tm for fibrolase moves from 50 degrees C at pH 8 to 43 degrees C at pH 5. Increases in temperature also lead to removal of the zinc ion, again producing a partially denatured protein with a marked tendency to aggregate. In both cases (decrease in pH and increase in temperature), analysis of the CD spectra indicates that the protein has primarily lost alpha-helical secondary structure. A major change in structure can also be observed using NMR spectroscopy. At temperatures below 35 degrees C, the globular structure of fibrolase remains intact, although some increase in chain mobility can be noted with increased temperature. Upon melting, numerous signals collapse as the protein unfolds. Transition temperatures (Tm) as measured by CD and NMR are in good agreement. Similar structural changes can be induced by adding zinc chelators such as EDTA and DTT. This leads to complete loss of activity at EDTA concentrations above 1.0 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrastructure of adult and juvenile marmoset (Callithrix jacchus) Sertoli cells in vivo and in vitro.

The morphologic differentiation of Sertoli cells isolated from adult and juvenile marmosets and cultured on different extracellular matrices was evaluated by light and electron microscopy and compared to cells in vivo. Both cell types could be maintained in culture for at least 6 days. The degree of cellular differentiation, shape, ultrastructural appearance, and polarity seemed to benefit from laminin-coated substrata, compared with collagen-, fibronectin-, serum-, and heparan sulfate-coated substrata. With the former two substrates, a difference in behavior between juvenile and adult cells was evident. Whereas juvenile cells displayed a lesser degree of differentiation, adult cells exhibited identical morphologic characteristics in culture and in vivo. Cyclicity of morphologic features was not found, neither in vivo nor in vitro. The results indicate that: (1) laminin plays a unique role for marmoset Sertoli cell differentiation in vitro compared with other extracellular components; (2) a greater similarity between cells in vivo and in vitro is evident with adult Sertoli cells; and (3) the adult marmoset monkey could provide a primate model for mature Sertoli cells in culture, since there is a close similarity to human adult Sertoli cells in vitro and in vivo.

Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom.

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an alpha-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an alpha-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.

Intercellular communication between cultured granulosa cells of the marmoset (Callithrix jacchus).

The influence of follicle-stimulating hormone, forskolin, insulin-like growth factor type I, epidermal growth factor, and 12-tetradecanoyl-phorbol-13-acetate on marmoset granulosa cell communication via gap junctions was investigated by morphological means and microinjection of carboxyfluorescein. Gap junctions between neighbouring granulosa cells were present in all groups. The number, but not length, of gap junctions between marmoset granulosa cells increased when the cells had been treated with follicle-stimulating hormone, insulin-like growth factor type I, and follicle-stimulating hormone plus insulin-like growth factor type I. No effect on gap junctions was seen, after exposure of the cells to the other three substances. Carboxyfluorescein and counting of the surrounding labelled cells showed that supplementation with follicle-stimulating hormone, forskolin, insulin-like growth factor type I and epidermal growth factor from the beginning of cultivation led to an increase in stained cells after 48 h. When treatment was started in 48 h cultures the substances reached their maximal activity within 30 min (forskolin and epidermal growth factor) or 3 h (follicle-stimulating hormone and insulin-like growth factor type I). Spreading of the fluorescent dye was inhibited when the medium was supplemented with 12-tetradecanoyl-phorbol-13-acetate. This effect was maximal after 30 min. Additive effects regarding the coupling of the cells were seen by combining of epidermal growth factor with follicle-stimulating hormone, but not with insulin-like growth factor type I or forskolin plus follicle-stimulating hormone.

Stability of the thrombolytic protein fibrolase: effect of temperature and pH on activity and conformation.

The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (approximately 22 degrees C). At 37 degrees C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alpha-helical structure was lost in a cooperative transition (Tm of 50 degrees C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the Tm determined by NMR was 46 degrees C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43 degrees C at pH 5. Protein concentrations determined over the pH range of 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.