Characterization of staphylococci

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.     

Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

Keratinocyte growth-promoting activity from human placenta

Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25-500 micrograms/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth-promoting activity in bovine hypothalamic extract, the activity is non-dialyzable and is destroyed at 100 degrees C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth-promoting properties for epithelium.     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid     

Location of the isoleucine arrest point in CHO and 3T3 cells

An isoleucine arrest point in G1 was determined by two methods for CHO and 3T3 cells. In the first method the fraction of cells entering S after isoleucine deprivation was assessed by [³H]thymidine labelling and autoradiography. In the second method cells entering S after isoleucine deprivation were identified by double-label autoradiography using [³H] and [¹⁴C]thymidine. From the fraction of cells entering S, determined by the two methods, the arrest point in G1 (and entry into G0) is located within the last 40 min of G1.