Further studies of the β-lactamases ofBacteroides species

The -lactamases of individual strains ofBacteroides fragilis, B. thetaiotaomicron, andB. melaninogenicus were examined to characterize their enzymatic activity and the relation between the periplasmic and cytoplasmic forms of the enzymes. K_m and V_max values indicate that all strains examined were very similar in terms of enzymatic activity with the antibiotics tested. Electrophoretic analysis and treatment with phospholipase D suggest the presence of a cytoplasmic form of the enzyme that is modified upon entry into the periplasmic space.     

Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

Velopharyngeal function following maxillary advancement.

In a series of 40 patients who had maxillary advancements, none developed velopharyngeal incompetence. Unlike the cleft palate patient who is more at risk, there are distinct anatomical characteristics in craniofacial dysostosis which favor maintenance of the integrity of the velopharyngeal mechanism. Hyponasality was eliminated in 5 patients with Crouzon's disease. On cephalometric study, it was observed that after maxillary advancement the nasopharyngeal volume was expanded and the angle formed by the hard and soft palates was increased. On phonating cephalograms, the velopharyngeal contact became more physiological after maxillary advancement in the craniofacial dysostosis patient. The only postoperative articulatory changes after maxillary advancement were in the production of the /s/ sound, which is particularly sensitive to changes in dentoalveolar relationships.     

Penicillinase activity in three strains ofClostridium butyricum

Three strains ofClostridium butyricum exhibited elevated minimal inhibitory concentrations (MICs) to penicillin (64–1,024 g/ml), ampicillin (32–256 g/ml), carbenicillin (128–1,024 g/ml), and oxacillin (32–64 g/ml). Cephalosporin/cephamycin agents were more active than penicillin drugs. All isolates were found to possess a -lactamase. The -lactamases were primarily cell associated during the logarithmic phase of growth. Stationary-phase cells released most of the enzyme into the culture medium. Cephalothin supplementation of broth cultures with concentrations equivalent to one-eighth of the MIC significantly increased the quantity of -lactamase synthesized. The -lactamases produced by these three isolates exhibited greatest activity with penicillin followed by ampicillin>cephaloridine>carbenicillin and oxacillin. No enzymatic activity was observed using cephalothin, cephalexin, cefazolin, cefoxitin, or cephamandole substrates. The -lactamases were inhibited by clavulanic acid and para-chloromurcuribenzoate and not inhibited by cloxacillin. Each enzyme exhibited an isoelectric point of 4.2.     

Evaluation of antibody inhibition assays of melanoma antigens in sera to monitor tumor growth in melanoma patients

Summary It was reported previously that melanoma leukocyte-dependent antibody (LDA) in the sera of melanoma patients was inhibited by small-molecular-weight (small-mol.-wt.) glycoproteins which were similar to cell surface antigens identified in cell membrane extracts of melanoma cells. The present study was to determine whether measurement of the levels of these factors in sera may be a useful monitor of tumor growth in melanoma patients. Small-mol.-wt. fractions were obtained by gel filtration or membrane chromatography of acidified sera and tested for their ability to inhibit LDA in ⁵¹Cr release cytotoxic assays. A panel of LDA was used, consisting of three antisera from melanoma patients, which appeared relatively specific for melanoma, and three non-melanoma antisera against carcinoembryonic antigen, ₂ microglobulin, and fetal antigens. The results showed that in patients with melanoma, approximately 70% had melanoma LDA-inhibitory activity detected in the small-mol.-wt. fractions of their sera when these were tested against the panel of melanoma LDA. The specificity of the inhibitory activity for melanoma LDA was shown by failure of the serum fractions to inhibit non-melanoma LDA and by absence of inhibitory activity in equivalent serum fractions from non-melanoma carcinoma patients for melanoma LDA. The levels of melanoma LDA-inhibitory activity in the serum fractions appeared to correlate with tumor growth, as shown by clearance of the inhibitory activity after surgical removal of melanoma and reappearance in the serum of patients who subsequently developed recurrent melanoma. The 30% false-negative rate indicated that the assays could not be used to reliably exclude melanoma, but the close correlation with tumor growth and the low number of false-positive results suggested that in 70% of patients detection of these small-mol.-wt. antigens would be of value to detect recurrence from melanoma and to monitor the effectiveness of therapy.     

Display Specificity and Reproductive Isolation in the Fiddler Crabs,Uca panacea and U. pugilator

The factors responsible for reproductive isolation between two sibling species of fiddler crabs (Uca pugilator and U. panacea) were studied under both laboratory and field conditions. ♂♂ showed species differences in their visual and acoustical displays. These differences were exaggerated in the overlap zone, where U. pugilator showed character displacement of its acoustic signals. ♀♀ of U. pugilator confined with ♂♂ of U. panacea produced fewer clutches of young. Occasionally, forced matings took place in the laboratory, resulting in hybrids suffering greater mortality through development. The data indicate that both premating (behavioral) as well as postmating (higher larval mortality) barriers act to prevent interbreeding.     

Chemical modification of Bacillus thuringiensis subsp. thuringiensis (HD-524) trypsin-activated endotoxin: implication of tyrosine residues in lepidopteran cell lysis

A purified protein fraction from a solubilized and trypsin-digested extract of Bacillus thuringiensis subsp. thuringiensis (HD-524) fermentation powder was lytic to cells from several lepidopteran lines. Maximum yield was obtained by alkaline carbonate-thiocyanate solubilization of washed powder followed by trypsin digestion and Sephacryl (S-300) chromatography. The alkaline carbonate-solubilized fraction consisted predominantly of two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with MW of 144 +/- 0.9 kDa and 134 +/- 1.4 kDa. After trypsin treatment and column chromatography, the cytolytic fraction consisted of a major band with a MW of 60.0 +/- 1.8 kDa and a minor band of 69 +/- 0.9 kDa. Cells from Trichoplusia ni (TN368) were most susceptible to lysis with 50% of cells lysed at 3 micrograms/ml, followed by Spodoptera frugiperda cells (SF21AE) exhibiting 50% cell lysis at 5 micrograms/ml and Lymantria dispar cells (Ld652Y) showing 40% lysis at 10 micrograms/ml. Chemical modification of the polypeptides was performed to determine the role of certain amino acid residues in the cytolytic activity. The group-specific reagent tetranitromethane was used to nitrate and oxidize tyrosine and cysteine residues, respectively. Cysteine residues alone were also modified with p-hydroxymercuribenzoic acid. Lysine residues were modified with O-methylisourea. Of the three types of amino acid residues, only the modification of tyrosine resulted in reduced cell lysis.     

Analysis of two different tandem repetitive elements within the human apolipoprotein B gene.

A large number of copies of the sequence (dTG-dAC)n, where n is between 10 and 60, exist in the human genome, and many are useful as polymorphic markers. One of these sequences occurs about 3 kilobases 5' of the human apolipoprotein (apo) B gene as seven distinguishable alleles containing from (TG)12 to (TG)18. This repeat is also present in the DNA of other primates. A second alternating purine-pyrimidine sequence with nine dinucleotide repeats and located in intron 4 is not polymorphic. Together with the apoB hypervariable repeat immediately 3' of the gene, the (TG)n sequence will provide a useful haplotype marker capable of distinguishing a large number of human apoB alleles, some of which may be associated with disease states.     

Modulation of Ca2+ channels by atrial natriuretic peptide in the bovine adrenal glomerulosa cell.

In the bovine adrenal glomerulosa cell, calcium influx through voltage-dependent calcium channels is critical to maintaining an aldosterone secretory response. In patch clamp, atrial natriuretic peptide (ANP) inhibits T-type calcium channel current yet stimulates L-type calcium channel current. In the present study the channel effects of ANP observed in the patch-clamp configuration were extended and related to populations of cells. We observed the following. (i) The effect of ANP on T-channel current resulted in the reduction in the open state probability. ANP decreased the mean open state duration from 14.2 to 1.8 ms/sweep. (ii) In the weakly depolarized cell stimulated by 8 mM K+, ANP reduced the level of aequorin luminescence (a measure of cytosolic calcium) and completely inhibited the stimulated rate of aldosterone secretion, returning it to prestimulation values. These effects are consistent with a decrease in net calcium channel influx and the reported inhibition of T-channel current. In contrast, the calcium channel blocker, nitrendipine, which at low dose selectively blocks L-type calcium channel flux, only slightly reduced luminescence, and partially inhibited the sustained secretory response. (iii) In the strongly depolarized cell, stimulated by 60 mM K+, ANP increased the level of aequorin luminescence consistent with an increase in net calcium channel influx and the reported stimulation of L-channel current. These results indicate that under physiological conditions the inhibition of T-type calcium channels may be involved in the inhibition of the aldosterone secretion induced by ANP.