Evolutionary Molecular Cytogenetics of Catarrhine Primates: Past, Present and Future. R. Stanyon M. Rocchi(b) F. Bigoni(a) N. Archidiacono(b)

The catarrhine primates were the first group of species studied with comparative molecular cytogenetics. Many of the fundamental techniques and principles of analysis were initially applied to comparisons in these primates, including interspecific chromosome painting, reciprocal chromosome painting and the extensive use of cloned DNA probes for evolutionary analysis. The definition and importance of chromosome syntenies and associations for a correct cladistics analysis of phylogenomic relationships were first applied to catarrhines. These early chromosome painting studies vividly illustrated a striking conservation of the genome between humans and macaques. Contemporarily, it also revealed profound differences between humans and gibbons, a group of species more closely related to humans, making it clear that chromosome evolution did not follow a molecular clock. Chromosome painting has now been applied to more that 60 primate species and the translocation history has been mapped onto the major taxonomic divisions in the tree of primate evolution. In situ hybridization of cloned DNA probes, primarily BAC-FISH, also made it possible to more precisely map breakpoints with spanning and flanking BACs. These studies established marker order and disclosed intrachromosomal rearrangements. When applied comparatively to a range of primate species, they led to the discovery of evolutionary new centromeres as an important new category of chromosome evolution. BAC-FISH studies are intimately connected to genome sequencing, and probes can usually be assigned to a precise location in the genome assembly. This connection ties molecular cytogenetics securely to genome sequencing, assuring that molecular cytogenetics will continue to have a productive future in the multidisciplinary science of phylogenomics.     

Prelamin A is involved in early steps of muscle differentiation

Lamin A is a nuclear lamina constituent implicated in a number of human disorders including Emery-Dreifuss muscular dystrophy. Since increasing evidence suggests a role of the lamin A precursor in nuclear functions, we investigated the processing of prelamin A during differentiation of C2C12 mouse myoblasts. We show that both protein levels and cellular localization of prelamin A are modulated during myoblast activation. Similar changes of lamin A-binding proteins emerin and LAP2α were observed. Furthermore, prelamin A was found in a complex with LAP2α in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affected LAP2α and PCNA amount and increased caveolin 3 mRNA and protein levels, while accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor appeared to inhibit caveolin 3 expression. Our data provide evidence for a critical role of the lamin A precursor in the early steps of muscle cell differentiation.     

A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

Background  

Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct) method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample.     

Structure activity relationship and optimization of N-(3-(2-aminothiazol-4-yl)aryl)benzenesulfonamides as anti-cancer compounds against sensitive and resistant cells

We recently described a new family of bioactive molecules with interesting anti-cancer activities: the N-(4-(3-aminophenyl)thiazol-2-yl)acetamides. The lead compound of the series (1) displays significant anti-proliferative and cytotoxic activities against a panel of cancer cell lines, either sensitive or resistant to standard treatments. This molecule also shows a good pharmacological profile and high in vivo potency towards mice xenografts, without signs of toxicity on the animals. In the present article, we disclose the structure-activity relationships of this lead compound, which have provided clear information about the replacement of the acetamide function and the substitution pattern of the benzenesulfonamide ring. An improved high-yielding synthetic procedure towards these compounds has also been developed. Our drug design resulted in potency enhancement of 1, our new optimized lead compound being 19. These findings are of great interest to further improve this scaffold for the development of future clinical candidates.     

NMES superimposed on movement is equally effective as heavy slow resistance training in patellar tendinopathy

Objective:This study aimed at investigating the effectiveness of an 8-week training protocol, based on neuromuscular electrical stimulation of the quadriceps, which was superimposed onto voluntary exercise (NMES+), in comparison to a traditional heavy slow resistance training (HSRT), in individuals with patellar tendinopathy.Methods:Thirty-two physically active participants, aged: 33.6±10.2 years, were divided into two groups: NMES+ or HSRT. Maximal voluntary isometric contraction (MVIC) of knee extensor and flexor muscles, power during a countermovement jump (CMJ), and VISA-p questionnaire scores were recorded at the start(T0), 2-weeks(T1), 4-weeks(T2), 6-weeks(T3), 8-weeks(T4) and 4-months post-training (T5). Knee pain and rate of perceived exertion (RPE) were recorded at each training session with a 0-10 scale.Results:Knee pain was significantly lower in NMES+ compared to HSRT during all training sessions. No significant between-group differences were found for VISA-p scores and forces recorded during MVICs at T0,T1,T2,T3,T4 and T5. A significant increase of VISA-p and peak forces during MVIC was recorded across-time in both groups. No significant between-group or across-time differences were found for RPE and CMJ parameters.Conclusions:NMES+ and HSRT were equally effective in decreasing tendinopathy symptoms and increasing strength, with NMES+ having the advantage to be a pain-free resistance training modality.     

An alphoid DNA sequence conserved in all human and great ape chromosomes: evidence for ancient centromeric sequences at human chromosomal regions 2q21 and 9q13

Using vector-CENP-B box polymerase chain reaction (PCR) we isolated and cloned from a human chromosome 21-specific plasmid library, a 1 kb DNA sequence, named pH21. In in situ hybridization experiments, pH21 hybridized, under high stringency conditions, to the centromeric region of all the human, chimpanzee, gorilla and orangutan chromosomes. On human chromosomes pH21 also identified non-centromeric sequences at 2q21 (locus D2F33S1) and 9q13 (locus D9F33S2). The possible derivation of these sequences from ancestral centromeres is discussed. Sequence analysis confirmed the alphoid nature of the whole pH21 insert.GenBank accession number, M64321     

Identification of the Human βA2 Crystallin Gene (CRYBA2): Localization of the Gene on Human Chromosome 2 and of the Homologous Gene on Mouse Chromosome 1

By using primers synthesized on the basis of the bovine βA2 crystallin gene sequence, we amplified exons 5 and 6 of the human gene (CRYBA2). CRYBA2 was assigned to human chromosome 2 by concordance analysis in human × rodent somatic cell hybrids using the amplified PCR products as probe. Regional localization to 2q34-q36 was established by hybridizing the CRYBA2 probe to microcell and radiation hybrids containing defined fragments of chromosome 2 as the only human contribution. The CRYBA2 probe was also used to localize, by interspecific backcross mapping, the mouse gene (Cryba2) to the central portion of chromosome 1 in a region of known human chromosome 2 homology. Finally, we demonstrate that in both species the βA2 crystallin gene is linked but separable from the γA crystallin gene. The βA2 crystallin gene is a candidate gene for human and mouse hereditary cataract.     

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production

Six Thr1 (O-glyco)-derivatives of the "phagocytosis stimulating peptide" tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(beta-D-GlcNAc)-Ser-Thr-OH, which correspond to the "tuftsin-region" at the Fc-domain of immunoglobulin G (amino acid residues 289-299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.     

Molecular characterization of porphyrias in Italy: a diagnostic flow-chart.

The porphyrias are disorders associated with inherited or acquired enzyme deficiencies in the heme biosynthetic pathway. The differential diagnosis is often difficult since the phenotype is very similar in some forms and the biochemical tests are not commonly available. Here we provide an update on the molecular diagnosis of porphyrias in Italy and a flow-chart to facilitate the identification of mutations in heme biosynthetic genes. The molecular analysis has allowed us to identify the molecular defect underlying the disease in 66 probands with different porphyrias [acute intermittent porphyria (AIP), variegate porphyria (VP), porphyria cutanea tarda (PCT), erythropoietic protoporphyria (EPP)]. No Italian patients with defects in coproporphyrinogen oxidise (CPOX) gene, responsible for hereditary coproporphyria (HCP), have been detected. The molecular characterization has been extended to 115 relatives with the identification of 55 asymptomatic mutation carriers and 60 normal subjects. We have so far identified 50 different mutations among 4 genes associated with the most common porphyrias showing a high molecular heterogeneity: 22 in the hydroxymethylbilane synthase (HMBS) gene (AIP), 7 in the protoporphyrinogen oxidase (PPOX) gene (VP), 16 in the uroporphyrinogen decarboxylase (UROD) gene (PCT) and 5 in the ferrochelatase (FECH) gene (EPP). Among the 50 molecular defects, 29 seem to be restricted to the Italian population.