The effect of Ca2+ on the thermal stability of trypsin in phosphate-buffered saline solution used for harvesting of human embryonic lung fibroblast cultures

The stabilizing effect of Ca2+ (264.9 mg CaCl2 X 2H2O ml-1) on a 0.5% solution of twice-crystallized bovine trypsin in phosphate-buffered saline (used for harvesting human embryonic lung fibroblasts) was studied at 7-37 degrees C and at -20 and -70 degrees C. It can be concluded that storage of the enzyme in the buffer (with or without Ca2+) is not advisable at temperatures greater than or equal to 20 degrees C. At 7 degrees C, on the other hand, trypsin can be stored for some weeks in the calcium-containing phosphate-buffered saline if a moderate loss of activity is acceptable. At -20 degrees C and -70 degrees C the stability of the enzyme was good. In the presence of Ca2+ about 90% of the activity remained after 18 weeks. Without Ca2+ the activity was approximately 10% lower.     

An abnormal terminal X-Y interchange accounts for most but not all cases of human XX maleness

To determine if human XX maleness results from an abnormal chromosomal X-Y interchange, we studied the inheritance of the paternal pseudoautosomal region in nine patients. Those six patients in whom Y-specific DNA was found (Y(+)) inherited the entire pseudoautosomal region from the paternal Y chromosome and lost that of the paternal X chromosome. Moreover, in three Y(+) cases, we observed the deletion of a paternal Xp locus tightly linked to the pseudoautosomal region. These results definitively show that an abnormal and terminal X-Y interchange during paternal meiosis causes Y(+)XX maleness. In contrast, no abnormal X-Y interchange was observed in any of the three Y(-) cases analyzed, suggesting that maleness can occur in the absence of any Y-specific DNA.     

Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium woodii

Growth characteristics of Eubacterium limosum and Acetobacterium woodii during one-carbon-compound utilization were investigated. E. limosum RF grew with formate as the sole energy source. Formate also replaced a requirement for CO2 during growth with methanol. Growth with methanol required either rumen fluid, yeast extract, or acetate, but their effects were not additive. Cultures were adapted to grow in concentrations of methanol of up to 494 mM. Growth occurred with methanol in the presence of elevated levels of Na+ (576 mM). The pH optima for growth with methanol, H2-CO2, and carbon monoxide were similar (7.0 to 7.2). Growth occurred with glucose at a pH of 4.7, but not at 4.0. The apparent Km values for methanol and hydrogen were 2.7 and 0.34 mM, respectively. The apparent Vmax values for methanol and hydrogen were 1.7 and 0.11 mumol/mg of protein X min-1, respectively. The Ks value for CO was estimated to be less than 75 microM. Cellular growth yields were 70.5, 7.1, 3.38, and 0.84 g (dry weight) per mol utilized for glucose, methanol, CO, and hydrogen (in H2-CO2), respectively. E. limosum was also able to grow with methoxylated aromatic compounds as energy sources. Glucose apparently repressed the ability of E. limosum to use methanol, hydrogen, or isoleucine but not CO. Growth with mixtures of methanol, H2, CO, or isoleucine was not diauxic. The results, especially the relatively high apparent Km values for H2 and methanol, may indicate why E. limosum does not usually compete with rumen methanogens for these energy sources.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attachment as a factor in the protection of Enterobacter cloacae from chlorination

Enterobacter cloacae attached to drinking water distribution particles was subjected to chlorination. Attachment resulted in the protection of these organisms from disinfection. This effect was found to be dependent upon both the level of chlorine in the system and attachment time. The results obtained in this study indicate that attached organisms may play an important role in coliform outbreaks.     

Preparation of scirpentriol and triacetoxyscirpenol in good yield from cultures of Fusarium sambucinum NRRL 13495

Crude extracts of filtrates of cultures of Fusarium sambucinum NRRL 13495 were acetylated or hydrolyzed. After chromatography on cartridge columns of silica gel and recrystallization three times from mixtures of ethyl acetate and hexane, 3,4,15-triacetoxyscirpenol (435 +/- 10 mg/liter of filtrate; mean +/- standard error [n = 3]) and the parent alcohol scirpentriol were isolated (261 +/- 29 mg/liter of filtrate; mean +/- standard error [n = 3]) in 68 and 53% yield for a 130- and 14-fold improvement, respectively, over prior reports.     

Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-iodobenzoate by Alcaligenes denitrificans NTB-1

Alcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and 2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source. During growth, stoichiometric amounts of halide were released. Experiments with whole cells and cell extracts revealed that 4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate. The initial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobic organisms, involving inducible reductive dechlorination to 4-chlorobenzoate. Under conditions of low and controlled oxygen concentrations, A. denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate.     

Construction of DNA-probes and their immunochemical detection using nonradioactive hybridization tests

Simple and efficient chemical approaches to preparation of DNA probes carrying 2,4-dinitrophenyl, dansyl or biotin residues were developed. The residues were introduced using following DNA derivatization procedures: a) transamination of cytidine residues with O-(4-aminobutyl)hydroxylamine; b) mercuration of pyrimidine residues followed by beta-mercaptoethanol modification. It was shown that 2,4-dinitrophenyl-containing DNA probes can be used for nonradioactive hybridization detection of nucleic acids. DNP-DNA: DNA complexes were detected using mouse antibodies specific to 2,4-dinitrophenyl groups, which were developed with peroxidase-conjugated antimouse immunoglobulins. Peroxidase-catalyzed chemoluminescent reaction of luminol oxidation with hydrogen peroxide allowed to detect 10 picograms of the dinitrophenylated single-stranded DNA probe.     

Determination of interproton distances in peptides by two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Conformation of a cyclic analog of substance P in a solution

Quantitative method is developed for evaluation interproton distances in peptides in solution. The method is based on the measurement of the relative intensities of the cross-peaks in the pure-phase absorption NOESY spectra. The ratios of the cross-peak intensities IN alpha/I alpha N and INN/I alpha N enable to determine the corresponding interproton distances dN alpha, d alpha N and dNN for several amino acid residues. These distances can be used to estimate other distances with cross-peaks in NOESY spectra. As example, the interproton distances are determined in a cyclic hexapeptide, namely cyclic analogue of substance P: cyclo [H-Glu-Phe-Phe-Gly-Leu-Met-NH(CH2)3-NH-]. The spatial structure of the molecule in dimethylsulphoxide solution is established.     

Purification of the parasporal crystals ofBacillus thuringiensis subsp.israelensis and development of a novel bioassay technique

Summary A spore-free parasporal crystal suspension was prepared fromBacillus thuringiensis subsp.israelensis by an aqueous biphasic separation technique and the waste fractions quantified with regard to spore numbers and insecticidal potencies. The technique proved efficient in selectively removing spores from the spore-crystal mixture. The final crystal suspension was used to develop a novel bioassay system which allowed rapid determination of crystal effectiveness in 1/8 to 1/6 of the time required in the conventional mortality versus concentration bioassay.     

A new species of Bradyidius (Copepoda, Calanoida) from the Gulf of Carpentaria, Australia

A new species of copepod belonging to the family Aetideidae,Bradyidius slyliformis sp. nov. is described and figured. Therelationship of this species to the other 12 members of thegenus is discussed. ¹Present address: 121 Jalan Athinahapan Dua, Taman Tun Dr Ismail,60000 Kuala Lumpur, Malaysia