Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

Audit of compliance with antenatal protocols.

OBJECTIVE--To assess the implementation of action protocols dictated by antenatal risk factors noted at the initial (booking) antenatal visit. DESIGN--Retrospective study of 2000 women delivered between 1 March 1990 and 29 March 1991. SETTING--Maternity department of a district general hospital supporting a multiethnic population in inner London. MAIN OUTCOME MEASURES--Comparison of clinical actions performed against those dictated by the department''s protocols. Analysis according to clinical importance, gestation at booking, maternal age, parity, birth order, ethnic origin, and certainty of gestational age. RESULTS--Interobserver agreement between the two auditors was good (kappa statistic for risk factors detected, 0.78; for actions generated, 0.80). Of the 15,658 actions dictated by department protocols, 3673 (23.5%) were actually performed by the clinicians. The 63 combinations of risk factors and actions believed by consultants to be of particular clinical importance had an action rate of 28.3% compared with 18.6% for those considered less important (p < 0.001). Mothers who first visited the hospital antenatal clinic at or before 24 weeks'' gestation had 25.2% of relevant protocols fulfilled (p < 0.001). Compliance was significantly improved in women aged 36 or over (32.4%), black women (24.9%), and cases of uncertain gestation (24.5%). Parity and birth order were not associated with an altered action rate. Ethnic origin deemed as "other" (than white, black, Asian, or oriental) or "unknown" was associated with poor compliance (19.3%). CONCLUSIONS--Compliance to a set of agreed protocols was poor even though a computer system was available and a protocol manual had been distributed. Protocols were more likely to be implemented in women who booked early and in some groups of women deemed at high risk including older mothers, black women, and those denoted as having uncertain gestational age.     

Carbonisation of oil palm trunks at moderate temperatures

Thousands of tonnes of dry oil palm trunks will be produced annually in Malaysia after about 1990. A project was initiated to study the feasibility of converting palm trunks into charcoal. Carbonisation was done at terminal temperatures of 400–550°C with holding times of 1–3 h and at two heating rates. From laboratory-scale pyrolysis studies, it was found that holding time does not affect the quantity and quality of the charcoal produced, while heating rate has a minor influence. However, as terminal temperature increases, both yield and volatile content decrease while the fixed carbon content increases. The calorific value and ash contents are independent of the parameters studied and their respective values are 4032 kcal/kg and 37.2%. Since the calorific value is low and the ash content high, it is concluded that oil palm trunks are not suitable for the production of charcoal fuel.     

Lymphocyte activation and serine-esterase induction following recombinant interleukin-2 infusion for lymphomas and acute leukaemias

Summary C57BL mice inoculated with radiation leukemia virus (RadLV) develop preleukemic cells long before the onset of leukemia. These cells are potentially immunogenic but fail to elicit an immune response in the host because of the appearance of virus-specific suppressor T cells. We have studied the effect of polysaccharide K (PSK) on the generation of RadLV-specific cell-mediated immune responses in vitro. Long-term exposure to PSK in culture potentiated the ability of immunized T cells to respond to a RadLV-induced lymphoma. It also abrogated the suppressive activity of suppressor T cells and simultaneously boosted the ability of reactive T cells to respond. The dual immunostimulating activity of PSK resulted in the generation of T cytotoxic lymphocytes that could lyse lymphoma cells in vitro. The results suggest that PSK could be used as a prophylactic immune response modifier in preleukemia.     

Genomic characterization ofCampylobacter jejuni by field inversion gel electrophoresis

The genome ofCampylobacter jejuni was characterized by field inversion gel electrophoresis (FIGE) after digestion with three rare-cutting restriction endonucleases. The restriction enzymesSac II (5-CCGCGG),Sal I (5-GTCGAC), andSma I (5-CCCGGG) were found to produce 13, 5, and 8 fragments respectively from theC. jejuni genome. The fragment sizes ranged from 1.6 kb to 1300 kb, which gaveC. jejuni a genome size of approximately 1900 kb. Furthermore, thegly A and rRNA genes ofC. jejuni were localized to specific fragments by use of Southern analysis, and thegly A gene was shown to be closely linked to one of the three rRNA genes.     

Reaction of pyruvate kinase with the new nucleotide affinity labels 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate

Two new reactive nucleotides have been synthesized and characterized: 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate and 5'-triphosphate (8-BDB-TADP and 8-BDB-TATP). ADP or ATP was converted to 8-thio-ADP (-ATP) via 8-bromo-ADP (-ATP), followed by condensation with 1,4-dibromobutanedione. Rabbit muscle pyruvate kinase is inactivated by both reagents in a biphasic manner with an initial rapid loss of 75% activity, followed by a slow total inactivation. The initial fast reaction with both compounds exhibits nonlinear dependence on reagent concentration, indicating formation of a reversible enzyme-reagent complex prior to covalent attachment. The presence of the gamma-phosphoryl group improves the performance of the affinity label: KI values for the fast phase are similar (about 100 microM), whereas kmax for 8-BDB-TATP is about three times greater than that of 8-BDB-TADP (0.286 min-1 vs 0.0835 min-1). After an 80-min incubation with 175 microM of either reagent, about 2 mol/mol of subunit is incorporated with 76% inactivation caused by 8-BDB-TADP and 97% inactivation by 8-BDB-TATP. Loss of activity is prevented by substrates, with the best protection afforded by a combination of ATP, Mn2+, K+, and phosphoenolpyruvate. Reaction of pyruvate kinase with either compound in the presence of protecting ligands leads to incorporation of about 1 mol of reagent/mol of subunit with only about 15% loss of activity. These results suggest that 8-BDB-TADP and 8-BDB-TATP react with two groups on the enzyme, one of which is at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction and elimination of oscillations in continuous cultures of Saccharomyces cerevisiae

Continuous cultures of Saccharomyces cerevisiae are known to exhibit oscillatory behavior in the oxidative region. Important findings of a series of experiments conducted to identify the causes for initiation of and the means for elimination of oscillations in these cultures are reported in this paper. These oscillations are seen to be connected to the growth kinetics of the microorganism and are induced at very low glucose concentrations and at dissolved oxygen (DO) levels that are neither high nor low (DO values between 20 and 78% air saturation at a dilution rate of 0.2 h(-1) and pH of 5.5 at 30 degrees C). The oscillatory behavior is encountered over a range of dilution rates (0.09-0.25 h(-1) at 30 degrees C for pH = 5.5 and DO = 50% air saturation). The oscillations can be eliminated by raising the DO level above a critical value or by lowering the DO level below a critical value.     

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong.

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.