Cecilia Garrè

Identification of an import signal for, and the nuclear localization of, human lactoferrin

Many different unique functions have been attributed to lactoferrin (Lf), including DNA and RNA binding, and transport into the nucleus, where Lf binds to specific sequences and activates transcription. A pentapeptide, Gly‐Arg‐Arg‐Arg‐Arg, corresponding to a region of the N‐terminal portion of human Lf rich in basic amino acids, was synthesized and its intracellular localization was investigated. Peptide internalization was assayed using the rhodaminated form of the same molecule. This N‐terminal peptide sequence is able to be internalized within less than 10 min at concentration as low as 1 μM, and its intracellular localization is nuclear, mainly nucleolar. Similar behaviour was observed using peptides composed of either all l or d amino acids, the last one being a retro‐inverse peptide. The internalization process does not involve an endocytotic pathway, since no inhibition of the uptake was observed at 4 °C. The kinetics of peptide internalization was also evaluated. The internalization properties of such a short Lf pentapeptide have been assayed for its ability to transport peptide nucleic acids (PNAs) inside cells in order to improve their efficacy. The abundant transmembrane transport and nuclear localization of the proposed peptide, deriving from hLf and, for the first time, identified as a nuclear localization signal, could be used as an alternative strategy to tackle the unsolved problem of intracellular accumulation of antisense and antigene drugs and for the development of new pharmacological tools.

Mutation Analysis of CCM1, CCM2 and CCM3 Genes in a Cohort of Italian Patients with Cerebral Cavernous Malformation

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K‐Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three‐gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at‐risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified, nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255‐4delGTA; c.1681‐1682delTA in CCM1; c.48A > G; c.82‐83insAG in CCM2; and c.396G > A in CCM3 genes. The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at‐risk relatives.

A SOD1 gene mutation in a patient with slowly progressing familial ALS

Article abstract We report a new missense mutation (Gly12Ala) in exon 1 of the Cu/Zn superoxide dismutase (SOD1) gene in a 67-year-old patient with familial ALS (FALS). The clinical course showed an unusually slow progression. The enzymatic activity of the mutated SOD1 was 80% of normal. At the molecular level, the Gly12Ala mutation occurs in a region outside the active site and may lead to local distortion strain in the protein structure.

Coincidence between fragile site expression and interstitial deletion of chromosome 11 in a case of myelofibrosis

SummaryCytogenetic examination of multiple peripheral blood cultures of a patient with myelofibrosis with myeloid metaplasia revealed the presence of an interstitial deletion of the long arm of chromosome 11, del(11)(q13q21). A folic acid dependent fragile site fra(11)(q13) was found in about 12% of the cells. The possible correlation between constitutional fragile site and acquired chromosomal alteration is discussed briefly.

Unusual Ph translocations in CML: Four new cases

Four variants of the Ph chromosome translocation in chronic myelogenous leukemia (CML) patients are described. Two had an unusual simple translocation involving chromosomes #7 and #17. In two cases, the translocation, aside from involving #9 and #22, involved a third chromosome, chromosome #6 and chromosome #11, respectively. Three cases showed also karyotypic evolution during the blastic phase of the disease: in two cases, a new reciprocal translocation was found that involved a chromosome #9 at band q34. The clinical and cytogenetic significance of these results is briefly discussed.

Search for loss of heterozygosity and mutation analysis of KRIT1 gene in CCM patients

Cerebral cavernous malformation (CCM) is a vascular disorder of the brain characterized by dilated capillary-like sinusoid chambers lined by a single layer of endothelium and thin fibrous adventitia but lacking other vessel wall components and intervening neural structures [Clatterbuck et al., 2001]. Spinal, cutaneous, retinal, and vertebral cavernous angiomas have also been observed. Both sporadic and familial forms have been identified. The familial form exhibits autosomal dominant inheritance with variable clinical expression and incomplete penetrance with three known loci on chromosomes 7q21.2 (CCM1), 7p15-p13 (CCM2), and 3q25.2-q27 (CCM3). The first gene identified on CCM1 locus is KRIT1 encoding KREV-1/RAP1 interaction trapped 1 protein (KRIT1), which is responsible for 40% or more of familial cases. Recently a novel gene MGC4607 has been isolated on the CCM2 locus [Liquori et al., 2003]. The KRIT1 gene has been reported to include 16 coding exons [Sahoo et al., 2001]. KRIT1 is a protein of 736 aminoacids and a molecular mass of 81 kDa containing N-terminal ankyrin repeats, one FERM domain, one trans-membrane domain, and a region at the C-terminal interacting with RAP1A [Sahoo et al., 2001; Marini et al., 2003]. The combina-

Identification of a novel KRIT1 mutation in an Italian family with cerebral cavernous malformation by the protein truncation test

Familial cerebral cavernous malformation (CCM) exhibits autosomal dominant inheritance and is characterized by vascular disorders of the brain, which can lead to seizures, focal neurological deficits, hemorrhagic stroke, and migraine. Three CCM loci have been mapped, but the gene for only one locus--KRIT1 coding for Krev-1/rap1 interaction trapped 1 (KRIT1) protein, which is responsible for more than 40% of familial cases--has been identified. To date, a total of 72 mutations have been described, with one founder effect in the Mexican/Hispanic community. We report the case of an Italian family with CCM that has a novel KRIT1 gene mutation leading to a truncated KRIT1 protein. The protein truncation test (PTT) has been used as a rapid method of identifying germline mutations in the KRIT1 gene.

An Italian dominant FALS Leu144Phe SOD1 mutation: Genotype-phenotype correlation

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease. Mutations of the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of autosomal dominant familial ALS (FALS). RESULTS: We examined the clinical features of the first Italian FALS with the Leu144Phe SOD1 mutation. Seven affected members were identified in a six‐generation pedigree. A slowly progressive course (20.4±14.6 years) was observed in five patients. One patient died of cardiac failure two years after the onset of the disease. The propositus is still alive. Neurological manifestations began in the legs in all patients, while bulbar signs were absent or appeared late in the course of the disease. DISCUSSION: There is evidence of a correlation between this mutation and a slowly progressive phenotype of ALS. Moreover this rare mutation might derive from a common ancestor.

c-Rel and p65 subunits bind to an upstream NF-κB site in human granulocyte macrophage-colony stimulating factor promoter involved in phorbol ester response in 5637 cells

To further clarify the complex transcriptional regulation of the human GM‐CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF‐κB like site in the 5637 non‐lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM‐CSF. This sequence, named the A element, has an active role on GM‐CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF‐κB subunits (c‐Rel and p65) which is identical to the one obtained using the HIV‐LTR‐κB site as recognition sequence and different from the one (c‐Rel and p50) observed with nuclear extracts from Mo T‐lymphoid HTLV‐II infected cells.

Electrophoretic difference between fetal and adult acetylcholinesterase of human red cell membranes

The membrane bound enzyme acetylcholinesterase (AChE) of human erythrocytes shows a lower level of activity in the newborn than in the adult. To evaluate if the decreased activity is correlated with changes in other properties of the enzyme, an electrophoretic method, recently devised to detect AChE activity, was used. Our results revealed an electrophoretic difference between adult and fetal enzymes. Sialic acid removal through neuraminidase treatment reduced the electrophoretic mobility of both forms rendering them identical in migration without, however, altering the activity.