Enrico Gandini

Protein kinase C activity, translocation, and selective isoform subcellular redistribution in the rat cerebral cortex after in vitro ischemia

Protein kinase C (PKC) involvement in ischemia‐induced neuronal damage has been investigated in superfused rat cerebral cortex slices submitted to 15 min of oxygen‐glucose deprivation (OGD) and in primary cultures of rat cortical neurons exposed to 100 μM glutamate (GLU) for 10 min. OGD significantly increased the total PKC activity in the slices, mostly translocated in the particulate fraction. After 1 hr of reperfusion, the total PKC activity was reduced and the translocated fraction dropped by 84% with respect to the control. Western blot analysis of OGD samples showed an increase in total β2 and ϵ PKC isoform levels. After reperfusion, the total levels of α, β1, β2 and γ isoforms were significantly reduced, whereas the ϵ isoform remained at an increased level. Endogenous GLU release from OGD slices increased to about 15 times the basal values after 15 min of oxygen‐glucose deprivation, and to 25 and 35 times the basal level in the presence of the PKC inhibitors staurosporine (0.1 μM) and bisindolylmaleimide (1 μM), respectively. Western blot analysis of GLU‐treated cortical neurons showed a significant decrease only in the total level of β2 isoforms. Cell survival was reduced to 31% in GLU‐treated neuronal cultures; PKC inhibitors were not able to modify this effect. These findings demonstrate that the cell response to OGD and GLU involves PKC in a complex way. The net role played by PKC during OGD may be to reduce GLU release and, consequently, neurotoxicity. The isoforms β2 and ϵ are affected the most and may play a significant role in the mechanisms underlying neurotoxicity/neuroprotection.

Complex translocation t(9;21)(9;22)(q12p13)(q12q11) in the family of a child with 9p trisomy syndrome.

SummaryA case of partial trisomy 9 is described in a mentally retarded and dysmorphic child, confirming that this chromosome unbalance results in a characteristic clinical entity.This trisomy arose through aberrant segregation of translocation chromosome during meiosis in the patients mother, who is a balanced heterozygote for a complex translocation involving chromosomes 9, 21 and 22. The phenotypically normal sister of the propositus is also carrier of the same complex translocation.

Monoclonal antibodies against HLA class I antigens inhibit human lymphocyte proliferation but do not affect mitogen dependent second messenger generation.

Abstract The mechanism through which anti HLA class I monoclonal antibodies inhibit human lymphocyte proliferation induced by the policlonal mitogen phytohemoagglutinin was investigated. Anti HLA class I monoclonal antibodies inhibited mitogen stimulated DNA synthesis even when added several hours after phytohemoagglutinin. The extent of inhibition depended on the duration of the exposure of lymphocytes to the monoclonal antibodies. Anti HLA class I monoclonal Antibodies neither affected the rise in cytosolic fee Ca 2+ concentration nor the production of inositol phosphates induced by phytohemoagglutinin. These results demonstrate that anti HLA class I monoclonal antibodies inhibit lymphocyte proliferation at a step downhill of second messenger generation.

Increased HK1 activity levels in the red cells of a patient with a de novo trisomy 10p: t(Y;10)(p11;p12)

SummaryA male patient with mental retardation and typical clinical features of 10p trisomy syndrome was found to have a duplication of the short arm of chromosome 10 attached to the short arm of the Y chromosome.Quantitative evaluation of nine red cell enzymes showed significantly increased activity levels of HK1 and, to a lesser extent, of PK, PGI, 6PGD, and G6PD. It is suggested that the HK1 locus may be in the 10pter→p12 region. The increased levels of HK1 could affect other erythrocyte metabolic pathways slowing down the physiological rate of cellular senescence and result in increased activity levels of other cell-age-dependent enzymes.

Adaptative Value of a PKC–PKI55 Feedback Loop of Inhibition That Prevents the Kinase’s Deregulation

A 168-bp amplification product was obtained in RT-PCR experiments using a degenerate oligonucleotide designed on a five-amino acid sequence of IN, a 7-kDa protein, previously characterized as a PKC inhibitor. It was included in the coding ORF of the 1530-bp-long IMAGE clone ID 38900 (accession numbers R51337 and R51448) that produces a translation product of 6.5 kDa. The translation of the ORF conceptual reading frame allowed the preparation of the synthetic protein PKI55, which was found to inhibit and degrade both untreated nPKC d isozymes and activated cPKC isozymes. The PKI55 gene is localized in chromosome 2q35. The Repeat Maskers output showed a 533-bp-long LTR32/ERVL segment that included the PKI55 coding sequence and a complete regulatory region. The coding sequence and the structure of PKI55 were detected in a brain cDNA of Macaca fascicularis (diverged from human lineages about 25 Myr ago). Three other human genes with over 60% identities with PKI55 were identified in three different loci (i.e., chromosomes 10, 15, and 20.) Synthesis of PKI55 was stimulated by PKC activation. A feedback loop of inhibition is established. When the PKCs are overactivated, PKI55 induces degradation of the enzyme and prevents the isozyme overexpression implicated in a number of important diseases including cancer, diabetes, and disorders of the immune system. The presence of the PKI55 sequence in Macaca fascicularis as well as in human chromosomes 10, 15, and 20 indicates a selective advantage for the PKI55 sequence and the adaptive value of the feedback mechanism.

Depletion of protein kinase C induced by an anti HLA class I monoclonal antibody in phytohemagglutinin activated human T cells

Anti HLA Class I Monoclonal Antibody depletes Protein Kinase C (PKC) to 20% of control value in PHA activated human T cells. The effect is reversible: in 24 hours the enzymatic activity returns to 58% of control value. Removal of antibody from the culture medium increases the rate of recovery. Implications of this finding for the modulation by HLA Class I antigens of the proliferative response of T cells to lectins are discussed.

Altered proliferative kinetics in PHA‐activated human T‐lymphocytes treated with the anti‐HLA class I monoclonal antibody 01.65

Abstract. Anti‐HLA class I monoclonal antibody (mAb) 01.65 inhibited phyto‐haemagglutinin (PHA)‐induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G1‐phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA‐induced proliferation of human T‐lymphocytes.

Identification of a novel protein kinase C inhibitor in microsomes from phytohaemagglutinin activated human peripheral blood mononuclear cells.

A peptide inhibiting either corpuscolate or purified PKC has been identified from microsomes of PHA‐activated human PBMC but it is not detectable in microsomes of resting PBMC. The peptide was obtained from a microsomal preparation in an oligomeric form that could be transformed into a monomeric form by β‐MSH. The active peptide (IN) was retained on a PC‐11 chromatographic column and could be eluted with Nad. IN is ineffective on PKC‐dependent protamine phosphorylation of protamine and on Ca2+ and phospholipid‐independent activity generated by mild hydrolysis with trypsin of PK‐C. Ca2+ binding is permissive for IN activity. IN inhibits particulate PK.C in PHA‐activated PBMC, but is ineffective after TPA activation. All these data indicate that IN acts at the regulatory domain of PKC.

An anti-HLA class I monoclonal antibody alters the progression in the cell cycle of phytohemagglutinin-activated human T lymphocytes

The monomorphic anti-HLA Class I monoclonal antibody 01.65 inhibits the incorporation of tritiated thymidine ([3H]TdR) in Phytohemagglutinin (PHA)-activated human T lymphocytes. Our data indicate that 01.65 affects the average duration of the cell cycle by increasing the length of the early S subphase. As a consequence of the increase in the doubling time of the cell population, the absolute number of cells at harvesting time was reduced in 01.65-treated cultures compared to that of untreated cultures. The lengthening of the S-phase and the decrease in the cell number can together quantitatively account for the reduction of [3H]TdR incorporation observed in 01.65-treated cultures.

NUCLEAR ACCUMULATION OF C-MYC MRNA IN PHYTOHAEMAGGLUTININ-ACTIVATED T LYMPHOCYTES TREATED WITH ANTI-HLA CLASS I MONOCLONAL ANTIBODY

Anti‐HLA class I monoclonal antibody 01.65 inhibits the proliferative response of PHA‐activated human T lymphocytes from peripheral blood mononuclear cells. The recruitment rate in the cell cycle is slack and the G1 and S phases are prolonged. Among the early events after PHA activation, only the calcium‐dependent PKC activity appears to be modified: particulate PKC is completely depleted while cytosolic residual PKC is reduced by 80% after MAb 01.65 treatment. We have carried out in greater detail the study of c‐myc gene regulation by MAb 01.65 and the results are as follows: (i) c‐myc RNA transcription is normally initiated and finished, suggesting a post‐transcriptional regulation of c‐myc gene expression; (ii) no alteration in c‐myc mRNA stability has been documented; (iii) steady‐state levels of c‐myc mRNA expression by Northern blot analysis and PCR amplification are decreased in the cytoplasmic compartment, while in the nuclear compartment they appear to be increased. Nuclear accumulation of mature mRNA after MAb 01.65 and PKC inhibitor (H7 and StSp) treatment appears to be the most probable mechanism involved. The possible implications of this are discussed.