Construction of a sequencedClostridium perfringens-Escherichia coli shuttle plasmid

A newClostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from theC. perfringens replicon pIP404 and theE. coli vector pUC18. The multiple cloning site and lacZ gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants inE. coli. Both chloramphenicol and erythromycin resistance can be selected inC. perfringens andE. coli since pJIR418 carries theC. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes fromC. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.     

Use of Bioluminescence Markers To Detect Pseudomonas spp. in the Rhizosphere

The use of bioluminescence as a sensitive marker for detection of Pseudomonas spp. in the rhizosphere was investigated. Continuous expression of the luxCDABE genes, required for bioluminescence, was not detectable in the rhizosphere. However, when either a naphthalene-inducible luxCDABE construct or a constitutive luxAB construct (coding only for the luciferase) was introduced into the Pseudomonas cells, light emission could be initiated just prior to measurement by the addition of naphthalene or the substrate for luciferase, n-decyl aldehyde, respectively. These Pseudomonas cells could successfully be detected in the rhizosphere by using autophotography or optical fiber light measurement techniques. Detection required the presence of 10³ to 10⁴ CFU/cm of root, showing that the bioluminescence technique is at least 1,000-fold more sensitive than β-galactosidase-based systems.     

Effect of inserted oxysterols on phospholipid packing in normal and sickle red blood cell membranes

Fourier transform infrared (FTIR) spectroscopy was used to examine the effect of oxysterol insertion into normal and sickle RBC membranes and the total lipid extracts of the membranes. Examination of the FTIR C-H stretch and fingerprint regions reveal that the insertion of 7 alpha- and 7 beta-hydroxycholesterol has the greatest effect on the fluidity of RBC membranes and lipid extracts. The results confirm the observation that sterol molecules are oriented in the membrane so that the 7 position is located in the phospholipid head group region at the lipid/water interface. The substitution of a keto for a hydroxy group at the number seven position decreases the effect of the sterol on membrane packing.     

Variation at the apolipoprotein (apo) AI-CIII-AIV gene cluster and apo B gene loci is associated with lipoprotein and apolipoprotein levels in Italian children.

We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasmid lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group II (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AI-CIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo AI, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo AI levels. In the total sample of 209 children 7.4% of the sample variance in apo AI levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo AI levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo AI levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo AI levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children.     

Release of Rhizobium spp. from Tropical Soils and Recovery for Immunofluorescence Enumeration

Limitations associated with immunofluorescence enumeration of bacteria in soil derive largely from the efficiency with which cells can be separated from soil particles and collected on membrane filters for staining. Many tropical soils fix added bacteria tightly, resulting in low recoveries. Eight soils, representative of three of the major soil orders found in the tropics (oxisols, vertisols, and inceptisols), were tested for recovery of added Rhizobium strains. All except one Hawaiian andept (Typic Eutrandept) yielded recoveries ranging from <1 to 13%. Recovery from the andept was 100%. In soil-sand mixtures, addition of only a small amount of soil caused a dramatic decrease in recovery of added rhizobia. Increasing the soil content of the mixture from 0% (10 g of sand) to 50% (5 g of soil-5 g of sand) reduced recoveries from >90 to <1%. Varying the ionic strength and pH of the extracting solution did not cause marked increases in recovery. Protein solutions, ethylenediaminetetraacetate, and NaHCO₃, on the other hand, improved release of bacteria. We report a modification to the usual membrane filter immunofluorescence procedure which yielded consistently high and reproducible recovery (coefficient of variation, 30%) of rhizobia from several tropical soils. In the modified procedure, partially hydrolyzed gelatin, diluted in ammonium phosphate, was used to suspend the soil. This caused dispersion of the soil and release of the bacteria from soil flocs. The efficiency of recovery of Rhizobium spp. from several tropical and two temperate soils remained high as the content of these soils in soil-sand mixtures was increased from 0 to 100%. The modified membrane filter immunofluorescence procedure was used to follow the growth of a strain of chickpea (Cicer arietinum) Rhizobium in a sterilized oxisol. The results showed a close agreement with viable counts at different stages during the growth cycle. Diluent for the hydrolyzed gelatin also had a marked effect on recovery. The efficiency of release of Rhizobium spp. from an oxisol was in the following order for the diluents used: 0.1 M (NH₄)₂HPO₄ > 0.1 M Na₂HPO₄ = 0.1 M sodium-phosphate-buffered saline (pH 7.2) > 0.2 M NH₄Cl > 0.2 KCl > NaCl = LiCl > water.     

Methionine degradation inCandida utilis

Incubation of cell-free extracts ofC. utilis and its ethionine-resistant mutants with methionine, aspartate and alanine, and with various acceptor oxo-acids showed the presence of several transamination activities (Glu-Asp, Ala-Glu, Met-Glu, Met-Ala, Ala-Asp, but not Met-Asp). The lack of utilization of methionine by mec mutants appears to be due to a transport lesion.     

Sucrose:sucrose fructosyltransferase and fructan:fructan fructosyltransferase from allium cepa

Sucrose: sucrose fructosyltransferase and fructan:fructan fructosyltransferase were isolated from the inner leaf bases of bulbing onion plants (Allium cepa) and separated by gel filtration on Bio-Gel P-150. Sucrose:sucrose fructosyltransferase produced only one trisaccharide, 1^F-fructosylsucrose, from sucrose. Fructan:fructan fructosyltransferase produced tetrasaccharide and higher polymers from trisaccharide. The trisaccharide found in the greatest concentration in onion, 6^G-fructosylsucrose, was produced from 1^F-fructosylsucrose by fructan:fructan fructosyltransferase and was not a product of sucrose:sucrose fructosyltransferase.     

On the effect of glucono-δ-lactone on the yeast Pichia polymorpha

Investigation has been made into the action of glucono--lactone on living cells of Pichia polymorpha in relation to the uptake of D-(U-¹⁴C) glucose, and the incorporation of (2-¹⁴C) uracil and L-(U-¹⁴C)-threonine into RNA and protein respectively. Other factors such as the action of glucono--lactone on cell morphology and on enzymic synthesis have also been studied. The action of this compound on -glucanase has been found to take place in the hydrolytic power and not in the synthesis.     

Synthesis of β-glucanase and other extracellular proteins by cells and protoplasts of the yeast Pichia polymorpha

The synthesis of β-glucanase either by cells or by protoplasts of the yeast Pichia polymorpha has been found to occur in the presence of 2-deoxy-d-glucose in the growth medium. On the other hand, the synthesis of typical extracellular proteins such as invertase and acid phosphatase is strongly affected by the presence of the drug. The degree of inhibition is, however, directly related to the 2-deoxy-d-glucose concentration.     

Preliminary note on anti-insulin antibody formation in diabetics treated with porcine insulin

In the present study, we have tested the sera of sixty five diabetic patients treated with insulin, researching the action of some physiologic and therapeutic factors (sex, age, insulin dose and time of treatment), to production of anti-insulin antibodies. Our results have shown that an important percentage of diabetic patients treated by porcine insulin produce antibodies: 72% of studied patients, concerning chiefly all the women and patients under-fourty years old. However our results have not pointed out any relation between the administered insulin dose versus the anti-insulin antibodies production, in spite of the early production of these antibodies in a great part of the patients.