Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Morphological and cytochemical studies of circulating Hodgkin's and Reed-Sternberg cells

Morphological and cytochemical studies of circulating neoplastic cells were carried out in a patient who presented a preterminal leukaemic phase of Hodgkin's disease (HD). Three types of abnormal cells were found in the peripheral blood: abnormal mononuclear cells, Hodgkin's cells and Reed-Sternberg cells. All neoplastic cells were cytochemically negative to Sudan black B, peroxidase and alkaline phosphatase. Some neoplastic cells were positive to PAS and all were positive to acid phosphatase, alpha-naphthylacetate esterase and beta-glucuronidase. The origin of the neoplastic population in HD is discussed.     

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.     

Effects of weight loss on erythrocyte membrane composition and fluidity in overweight and moderately obese women

A previous study showed chemical and physical impairment of the erythrocyte membrane of overweight and moderately obese women. The present study investigated the effects of a low-calorie diet (800 kcal/day deficit for 8 weeks) on erythrocyte membrane properties in 70 overweight and moderately obese (body mass index, 25-33 kg/m²) normotensive, nondiabetic women. At the end of dietary intervention, 24.3% of women dropped out, 45.7% lost less than 5% of their initial weight (Group I) and only 30% of patients lost at least 5% of their initial body weight (Group II). Group I showed no significant changes in erythrocyte membrane composition and function. The erythrocyte membranes of Group II showed significant reductions in malondialdehyde, lipofuscin, cholesterol, sphingomyelin, palmitic acid and nervonic acid and an increase in di-homo-γ-linolenic acid, arachidonic acid and membrane fluidity. Moreover, Group II showed an improvement in total cholesterol, low-density lipoprotein cholesterol, glycemia and insulin resistance. These changes in erythrocyte membrane composition could reflect a virtuous cycle resulting from the reduction in insulin resistance associated with increased membrane fluidity that, in turn, results in a sequence of metabolic events that concur to further improve membrane fluidity.     

From symmetrical to asymmetrical nitrido phosphino-thiol complexes: a new class of neutral mixed-ligand (99m)Tc compounds as potential brain imaging agents

A general procedure is presented for the preparation of a new class of nitrido asymmetrical Tc-99m complexes containing two different bidentate ligands bound to the same [Tc(N)]2+ core that could be used to design either essential or target specific imaging agents. This procedure is based on the chemical properties of a new monosubstituted [Tc(N)(R2PS)Cl(PPh3)] species composed of a TcN multiple bond and an ancillary phosphine thiol ligand (R2PSH). This intermediate readily reacts with bidentate mononegative ligands (S--Y) containing soft pi-donor coordinating atoms to give neutral pentacoordinate asymmetrical complexes of the type [Tc(N)(R2PS)(S--Y)]. The ability of several bidentate ligands containing different combination of heteroatoms (S, N, O) to form complexes with the [Tc(N)(R2PS)]+ building block was investigated. It was found that mononegative dithiocarbamate (DTC) or cysteine carboxyl derivate ligands promptly react with the monosubstituted species to form the final mixed compound in high yield. Preliminary biodistribution data in rats of some representative [Tc(N)(R2PS)(DTC)] compounds revealed an interesting initial brain uptake (in the range 0.20 +/- 0.01% ID/g and 0.91 +/- 0.06% ID/g), indicating their ability to cross in and out of the intact BBB. In these complexes the dithiocarbamate, or more generally the bidentate ligand (S--Y), can be designed to carry a functional group or a bioactive molecule, which could be involved in a trapping mechanism to increase brain retention for longer time intervals. These results could be conveniently utilized to devise a new procedure for the production of a novel class of brain perfusion and/or brain receptor imaging agents.     

The AQEM multimetric system for the southern Italian Apennines: assessing the impact of water quality and habitat degradation on pool macroinvertebrates in Mediterranean rivers

In accordance with the aims of the E.U. funded AQEM Project, an assessment system module based on aquatic macroinvertebrates was developed for small sized rivers in the southern Apennines (south Italy). Eleven stream sites, impacted to a greater or a lesser extent by organic pollution and/or habitat impairment and chosen to cover the whole degradation gradient present in the geographical area were sampled in three seasons. The samples were collected following a proportional, multihabitat procedure, afterwards considering separately the replicates collected in the depositional (pool) and transport (riffle) areas for the analysis. A PCA multivariate analysis was performed to extract the main axes of variation of the biological community, which resulted in the first axis being strongly correlated to ecological quality. The final assessment module is based on a multimetric system, structured by selecting the best metrics in simulating the first axis gradient. The system considers a total of 15 different metrics, mainly providing information concerning tolerance to pollution, taxa richness, habitat features and trophic structure of the community. In accordance with the WFD requirements, some of these metrics are based on abundance classes of taxa. Depositional and transport units, due to the observed dissimilarity in the structure of their benthic communities, were kept separate during the development of the assessment system to retain this potentially useful information and to clear interpretation of the results. Both `riffle' and `pool' invertebrate data showed clear differences in ecological quality between sites. Nevertheless, the final assessment module is based on the macroinvertebrates inhabiting depositional areas of rivers only, because the metrics for these river units showed a better performance than those examined for the transport river units. The application of the assessment module requires 10 replicates to be quantitatively collected, for a total area of 0.5 m². In terms of sampling and identification effort, the assessment module shows a good comparability with the standard Italian method presently in use and might thus be easily applied for river sites classification according to the Water Framework Directive in southern Italy. The site classification obtained with the proposed multimetric index shows a very good correspondence with the post-classification based on multivariate analysis.     

The STAR common metrics approach to the WFD intercalibration process: Full application for small, lowland rivers in three European countries

Class boundaries of three European assessment systems based on macroinvertebrates were compared and harmonized. Three different approaches to comparison, one based on regression analysis and the other two on statistical testing, were described and used, however only one was considered useful for the harmonization of boundaries. In all cases, the calculations were based on a set of six Intercalibration Common Metrics, combined into a simple multimetric index (ICMi). The ICMi was calculated for three test datasets from Italy, Poland and the UK, all belonging to the same stream type (small lowland siliceous sand rivers). For comparison, a regression model was employed to convert national assessment boundary values into ICMi values. The ICMi was also calculated on samples included in a strictly WFD-compliant benchmark dataset. The values of the ICMi obtained for the quality classes Good and High for the test and benchmark datasets were statistically compared. When significant differences were observed in the harmonization phase, the boundaries of the national method were refined until no further differences were observed. For the test datasets and assessment systems of Italy (IBE index) and Poland (Polish BMWP index) small refinements of the boundaries between High/Good and Good/Moderate classes were sufficient to remove the differences from the benchmark dataset. After harmonization, in the studied stream type, the percentage of samples requiring restoration to Good quality increased by 22 and 6% for Italy and Poland, respectively. For the UK dataset (EQI ASPT) the comparison to benchmark dataset showed no significant differences, thus no harmonization was proposed. A general discussion of the options used to compare boundaries based on the ICMi and their potential for harmonization is provided. Lastly, the option of harmonizing class boundaries through comparison to an external, benchmarking dataset and then re-setting them until no differences are found is supported.     

Juvenile hemochromatosis locus maps to chromosome 1q

Juvenile hemochromatosis (JH) is an autosomal recessive disorder that leads to severe iron loading in the 2d to 3d decade of life. Affected members in families with JH do not show linkage to chromosome 6p and do not have mutations in the HFE gene that lead to the common hereditary hemochromatosis. In this study we performed a genomewide search to map the JH locus in nine families: six consanguineous and three with multiple affected patients. This strategy allowed us to identify the JH locus on the long arm of chromosome 1. A maximum LOD score of 5.75 at a recombination fraction of 0 was detected with marker D1S498, and a LOD score of 5. 16 at a recombination fraction of 0 was detected for marker D1S2344. Homozygosity mapping in consanguineous families defined the limits of the candidate region in an approximately 4-cM interval between markers D1S442 and D1S2347. Analysis of genes mapped in this interval excluded obvious candidates. The JH locus does not correspond to the chromosomal localization of any known gene involved in iron metabolism. These findings provide a means to recognize, at an early age, patients in affected families. They also provide a starting point for the identification of the affected gene by positional cloning.     

Demonstration of lysozyme in primary ovarian tumors using the immunoperoxidase technique

The presence of lysozyme in ovarian tumors has been detected for the first time using the immunoperoxidase technique. A study of the distribution of this enzyme in 50 primary ovarian neoplasms revealed its presence exclusively in mucinous and mixed (serous-mucinous) cystadenomas and cystadenocarcinomas. The secretion of lysozyme seems to be correlated to the production of strongly acidic mucins.