Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.     

Nucleoside triphosphate pyrophosphatase of rabbit matrix vesicles, a mechanism for the generation of inorganic pyrophosphate in epiphyseal cartilage

Inorganic pyrophosphate (PPi) may be important in the regulation of mineralisation but its origin in epiphyseal cartilage is ill-defined. Nucleoside triphosphate pyrophosphatase is one potential source, as this enzyme catalyses the formation of PPi from nucleoside triphosphates. This enzyme has been identified in matrix vesicles derived from rabbit epiphyseal cartilage and a method developed to measure the activity using ATP as substrate in intact matrix vesicles under relatively physiological conditions. The enzyme had a high affinity for ATP (Km less than 10 microM) and was also active towards GTP, CTP and UTP. Disruption of the matrix vesicle membrane by sonication failed to alter the activity. Treatment of sonicated matrix vesicles with Triton X-100 increased the activity which may indicate a direct effect of the detergent on the enzyme. Activity towards ATP was inhibited substantially by ADP and AMP and by another potential substrate beta,gamma-methyleneadenosine 5'-triphosphate. Dichloromethylene bisphosphonate, an analogue of the product PPi, inhibited the activity to a lesser extent. Two other potential substrates, NADP+ and thymidine 5'-monophosphate p-nitrophenyl ester were only weakly inhibitory as was 1-hydroxyethylidene 1,1-bisphosphonate. These results imply that nucleoside triphosphates are the substrates in vivo and the inhibitory effects of ADP and AMP suggest mechanisms whereby this activity could be regulated.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Mixed-chain phosphatidylcholine analogues modified in the choline moiety: preparation of isomerically pure phospholipids with bulky head groups and one acyl chain twice as long as the other

Diacylphosphatidylcholines were synthesized with widely different acyl chain lengths and bulky head groups. Lysophosphatidylcholine was acylated at room temperature within 6 h with a 10-fold molar excess of fatty acid anhydride in dry, alcohol-free chloroform in the presence of 1.2 equivalents of 4-pyrrolidinopyridine as a catalyst, affording the mixed-acid phosphatidylcholines with widely different chain lengths in more than 90% yield and with less than 1% acyl migration. The syntheses of isomerically pure 1-stearoyl-2-decanoyl- and 1-stearoyl-2-undecenoyl(delta 10)-sn-glycero-3-phosphocholines C(18:0)/C(10:0)-PC and C(18:0)/C(11:1 delta 10)-PC, respectively), followed by conversion to various head-group analogues, are illustrated here. The transition peak widths at half-height of the endotherms obtained by differential scanning calorimetry are consistent with very high isomeric purity. Phospholipase D from Streptomyces chromofuscus was used as a catalyst in the hydrolysis of C(18:0)/C(10:0-PC to give the corresponding phosphatidic acid in quantitative yield. The latter compound was condensed with 10 molar equivalents of various N,N,N-trialkylammonium alkanols (as their p-toluenesulfonate or tetraphenylborate salt) in the presence of trichloroacetonitrile in dry pyridine under nitrogen atmosphere to yield the C(18:0)/C(10:0) phospholipids bearing modified head groups, which were purified by flash chromatography.     

Oxacillin-hydrolysing beta-lactamases. A comparative analysis at nucleotide and amino acid sequence levels

We have extended the sequence of the OXA-2 beta-lactamase which together with S1 mapping has enabled us to identify the promoter site for this gene. This lies in a region that is found upstream from a variety of resistance genes on different plasmids; each gene appears to have been inserted at the same specific site and to be expressed from the same promoter. The ancestral plasmid thus appears to function as a natural expression vector. The sequence of the recombination site at the 5' end of the OXA-2 gene shows a marked similarity with the attP sequence of lambda. DNA-probe analysis confirmed that the OXA-2 and OXA-3 beta-lactamases are related, and indicated no similarity with other beta-lactamase genes. However, a comparison of amino acid sequences demonstrates that the OXA-2, OXA-1 and PSE-2 beta-lactamases show some similarities to the typical class A enzymes, especially in the central helical domain of the latter, which is largely responsible for forming the active site of the enzyme. The three oxacillinases also show marked amino acid sequence similarity with the product of a regulatory gene, blaR1, required for beta-lactamase induction in Bacillus licheniformis.     

Altered hepatic microsomal function and elevated protooncogene expression as residual effects in rats exposed to delta-9-tetrahydrocannabinol

The microsomal activation of the potent hepatocarcinogen aflatoxin B1 (AFB1) and the expression of selected protooncogenes were investigated in the livers of rats exposed to delta 9-tetrahydrocannabinol (THC). At equimolar levels of cytochrome P-450, the microsome-mediated binding of AFB1 to DNA was significantly lower (56% of the controls) in preparations from drug exposed rats. Hepatic expression of the c-k-ras protooncogene was 3-fold higher in THC exposed animals. These results suggest the possible occurrence of long lasting residual effects in the rats exposed to THC.     

Generation of small mutation in large genomic fragments by homologous recombination: description of the technique and examples of its use

We have developed a technique of homologous recombination in bacteria which allows the mutagenesis of large genomic fragments cloned in cosmids. The desired mutation is first introduced into a plasmid clone and is then transferred to the appropriate cosmid clone by the means of double antibiotic selection coupled with phenotypic selection. We describe three different types of construct made by this technique.     

Constancy of synaptic ribbon numbers in the retina of the arctic chary,Salvelinus alpinus

Synopsis At high latitudes, such as in Iceland, the daily photoperiod varies from almost continuous darkness in winter to virtually constant light in summer. Previous studies of detailed retinal structure in vertebrates have shown significant daily and annual effects of photoperiod. We sampled arctic charr in Iceland during the summer, including fish that were both light- and dark-adapted, during both day and night. We observed retinomotor responses characteristic of light- and dark-adaptation, but found no difference in the number of synaptic ribbons in the retina. The morpho-physiological changes, appearing as retinomotor responses, are thus not expressed at the synaptic level.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.