Partial versus productive immunoglobulin heavy locus rearrangements in chronic lymphocytic leukemia: implications for B-cell receptor stereotypy

The frequent occurrence of stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences among unrelated cases with chronic lymphocytic leukemia (CLL) is widely taken as evidence for antigen selection. Stereotyped VH CDR3 sequences are often defined by the selective association of certain immunoglobulin heavy diversity (IGHD) genes in specific reading frames with certain immunoglobulin heavy joining (IGHJ ) genes. To gain insight into the mechanisms underlying VH CDR3 restrictions and also determine the developmental stage when restrictions in VH CDR3 are imposed, we analyzed partial IGHD-IGHJ rearrangements (D-J) in 829 CLL cases and compared the productively rearranged D-J joints (that is, in-frame junctions without junctional stop codons) to (a) the productive immunoglobulin heavy variable (IGHV )-IGHD-IGHJ rearrangements (V-D-J) from the same cases and (b) 174 D-J rearrangements from 160 precursor B-cell acute lymphoblastic leukemia cases (pre-B acute lymphoblastic leukemia [ALL]). Partial D-J rearrangements were detected in 272/829 CLL cases (32.8%). Sequence analysis was feasible in 238 of 272 D-J rearrangements; 198 of 238 (83.2%) were productively rearranged. The D-J joints in CLL did not differ significantly from those in pre-B ALL, except for higher frequency of the IGHD7-27 and IGHJ6 genes in the latter. Among CLL carrying productively rearranged D-J, comparison of the IGHD gene repertoire in productive V-D-J versus D-J revealed the following: (a) overuse of IGHD reading frames encoding hydrophilic peptides among V-D-J and (b) selection of the IGHD3-3 and IGHD6-19 genes in V-D-J junctions. These results document that the IGHD and IGHJ gene biases in the CLL expressed VH CDR3 repertoire are not stochastic but are directed by selection operating at the immunoglobulin protein level.     

Hexadecenoic Fatty Acid Isomers in Human Blood Lipids and Their Relevance for the Interpretation of Lipidomic Profiles

Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles.     

Chromosomal Location and Properties of Radiation Sensitivity Mutations in Bacillus subtilis

Transformation and transduction crosses involving recA1, recB2, and urv-1 mutations have shown that these mutations belong to three distinct unlinked genetic loci. The precise position of these loci on the Bacillus subtilis chromosome map has been determined. The behavior of recB2 strains in transformation studies suggested a dominance of recB2(+) function over recB2 and an early expression of this phenotype during transformation. Strains bearing two ultraviolet sensitivity markers possess a phenotype characteristic of the marker with the most adverse effect on recombination. The possibility that the effects of the two mutations are additive was also considered. Results are also presented which show that a phage-induced enzyme is not responsible for the high transducibility of recA1 strains.     

Defining synchrony of cell cultures

A method was developed for the statistical analysis of growth data from synchronized growth experiments. The analysis provided a firm basis for the recognition of synchrony and the objective graphical presentation of the growth pattern of a synchronized culture. The latter could then supply reliably the parameters required for the calculation of a synchronization index, i.e. for the synchrony evaluation.     

Genetic Studies Relating to the Production of Transformed Clones Diploid in the Tryptophan Region of the Bacillus subtilis Genome

Transformation experiments with Bacillus subtilis strains carrying trpE26 (the marker responsible for the detection of merodiploid clones after transformation or transduction) have established the precise position of this marker on the "aromatic region" of the chromosome, at the distal end of the anthranilate synthetase locus. Integration efficiency of the mutant allele (trpE26) seems to be very low. Co-transfer of markers situated on either side of it is almost nil when both donor and recipient carry this mutation. The "exclusion" of trpE26 does not, however, affect recombination frequencies for nearby markers. To explain these facts we considered the hypothesis of a preferential breakage of the deoxyribonucleic acid (DNA) at the trpE26 site or that of an insertion mutation. These studies have also demonstrated the establishment of physical linkage of a marker from the exogenote (hisH2) to a resident marker (tyrA1) in stable and unstable merodiploid clones, thus confirming integration of the donor DNA segment into a genetic structure of the recipient. Furthermore, duplication was shown in merodiploid clones (through reversion and transformation) for a locus of the recipient (tyrA) which was not involved in the initial transformation. This suggests that the diploid condition in this region extends beyond the transformed area. Interpretation of the genetic constitution of these partial diploids calls for postulation of the existence of long duplications, a second (incomplete) chromosome, or an episome-like element.     

Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity.

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.     

Heterotopic bone formation: An apparently benign complication of median sternotomy

Two cases are reported that involve heterotopic bone formation in midline sternotomy scars. The authors relate similar complications associated with abdominal incisions and discuss possible causes.     

In-vivo sensitivity of canine myocardium to acute depression of serum ionized calcium

Recent technologic advances make it possible to measure reliably serum or plasma ionized calcium with a high degree of accuracy. In this study, with the use of 16 mongrel dogs, the quantitation of the hemodynamic response to acute changes in ionized calcium was attempted. It was found that normal cardiac function required a normal level of ionized calcium. In intact animals, severe depression of myocardium is masked by pressor reflexes designed to maintain homeostasis. In their absence, myocardial depression is readily apparent. Animals that are hypercalcemic at the beginning of infusion respond in essentially the same manner. To a degree previously unsuspected, myocardial function depends upon a constant level of ionized calcium.     

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.