Biomedical subjects
Publications and source records attributed to L Sanz.
The HLA-Cw*1801 specificity, a Cw7/Cw4 hybrid allele, has recently been described in association with B*8101 (formerly B"DT"). In this study, the new Cw*1802 variant, differing from Cw*1801 at exon 5, is found associated with B*5703 in Bubi individuals from Equatorial Guinea. Confirmatory complete coding regions of B*5703 and B*3910 are also reported.
We determined the HLA frequency distribution in a sample of 100 Bubi individuals born on the island of Bioko (Equatorial Guinea). HLA-A, -B and -C typing was performed by serology and PCR-SSP. DRB1/3/4/5, DQB1 and DQA1 alleles were determined by PCR-SSOP. The HLA allele distribution of this population group resembles those found in other Bantu-speaking groups; however, the higher frequency of A30, A32, B44, DRB1*1301 in the Bubi with respect to other Bantu groups and the absence of DR4 deserve special mention. The cloning and sequencing of class I and II genes in this population allowed the description of five new allelic variants: B*4407, Cw*0706, Cw*1801, Cw*1802 and DQB1*0612 and five confirmatory sequences: B*3910, B*5703, B*8101, Cw*1203 and Cw*1701. The following new HLA-C,B haplotypes have been found in Bubi: Cw*08-B*57, Cw*18-B*57, Cw*0302-B*53, Cw*07-B*53 and Cw*1601-B*63. The most frequent seven-locus haplotype is: A*30-Cw*17-B*42-DRB1*1102-DRB3*0202-DQA1*05-DQB1 *0301. In terms of genetic distance, the Bubi are closer to other Bantu groups than to West African populations.
Recent studies have documented the involvement of the atypical protein kinase C (aPKC) isoforms in important cellular functions such as cell proliferation and survival. Exposure of cells to a genotoxic stimulus that induces apoptosis, such as UV irradiation, leads to a profound inhibition of the atypical PKC activity in vivo. In this study, we addressed the relationship between this phenomenon and different proteins involved in the apoptotic response. We show that (i) the inhibition of the aPKC activity precedes UV-induced apoptosis; (ii) UV-induced aPKC inhibition and apoptosis are independent of p53; (iii) Bcl-2 proteins are potent modulators of aPKC activity; and (iv) the aPKCs are located upstream of the interleukin-converting enzyme-like protease system, which is required for the induction of apoptosis by both Par-4 (a selective aPKC inhibitor) and UV irradiation. We also demonstrate here that inhibition of aPKC activity leads to a decrease in mitogen-activated protein (MAP) kinase activity and simultaneously an increase in p38 activity. Both effects are critical for the induction of apoptosis in response to Par-4 expression and UV irradiation. Collectively, these results clarify the position of the aPKCs in the UV-induced apoptotic pathway and strongly suggest that MAP kinases play a role in this signaling cascade.
The primary-structure of HSP-7, a 14-kDa protein isolated from stallion seminal plasma, has been determined, HSP-7 belongs to the spermadhesin protein family, shares 98% sequence identity with the boar seminal plasma protein AWN, and, like its boar homolog, displays zona-pellucida-binding activity. Despite these conserved structural and functional features, the equine and porcine spermadhesins differ in their topography on spermatozoa.
BSP-30K is a major acidic glycoprotein of bovine seminal plasma. It displays heparin-, gelatin-, and phospholipid-binding activities. BSP-30K binds to spermatozoa upon ejaculation and is thought to play a role in sperm capacitation. We have determined its amino acid sequence, disulfide bonds, and 0-glycosylation sites. BSP-30K consists of 158 amino acids arranged in a mosaic structure. BSP-30K has a unique 48-residue N-terminal extension which includes three 7-8- amino acid repeats and the six O-glycosylated threonine residues. The polypeptide stretch 49-71 is homologous to type 'A' domains found in heparin-binding proteins from other mammalian species. The C-terminal portion of BSP-30K is organized in a tandem of 40-44-residue domains each sharing the consensus pattern of the gelatin-binding fibronectin type II module. The mosaic structure of BSP-30K suggests that this glycoprotein might be a factor contributing to the different sperm-capacitating effects exerted by heparin in different mammalian species.
The atypical PKCs are involved in a number of important cellular functions, including cell proliferation. We report here that the product of the par-4 gene specifically interacts with the regulatory domains of zeta PKC and lambda/LPKC, which dramatically inhibits their enzymatic activity. This is particularly challenging, because expression of par-4 has been shown to correlate with growth inhibition and apoptosis. Results are shown here demonstrating that the expression of par-4 in NIH-3T3 cells induces morphological changes typical of apoptosis, which are abrogated by cotransfection of either wild-type zeta PKC or lambda/LPKC, but not by their respective kinase-inactive mutants. These findings support a role for the atypical PKC subspecies in the control of cell growth and survival.
Platelet-derived growth factor (PDGF) induces the expression of human stromelysin-1, a matrix metalloproteinase involved in tumor invasion and metastasis. Here it is shown that stromelysin-1 gene induction by PDGF depends on Ras and involves three previously identified promoter elements (the stromelysin-1 PDGF-responsive element (SPRE) site, the two head-to-head polyomavirus enhancer A-binding protein-3 (PEA3) sites, and the activator protein-1 (AP-1) binding site). During mitogenic induction, these responsive elements appear to be organized in two independent transcriptional units, SPRE-AP-1 and PEA3-AP-1, which result from specific element cross-talking. Interestingly, expression of a dominant negative mutant of Raf-1 significantly interfered with the induction through PEA3-AP-1 but not with that operating through SPRE-AP-1. Conversely, only the induction operating through SPRE-AP-1 was affected significantly by the expression of a dominant negative mutant of the atypical lambda/iota protein kinase C (lambda/iotaPKC). These data strongly suggest that the signal triggered by PDGF flows through Ras and bifurcates toward two distinct pathways, one operating through Raf and involving PEA3-AP-1 and the other one Raf-independent, operating through lambda/iotaPKC and SPRE-AP-1. Furthermore, we present evidence suggesting that the novel SPRE-binding transcription factor SPBP cross-couples with c-Jun to transactivate the SPRE site.
Although bone marrow biopsy is considered the best procedure to detect bone marrow involvement by Hodgkin's disease (HD), in recent years several studies have emphasized the value of magnetic resonance imaging (MRI). We present the case of a patient with HD apparently localized in a laterocervical lymph node, who also referred disestasiae at a region corresponding to D10 metamera. Bone marrow biopsy, vertebral TC and 67-Ga scintigraphy were all normal. However, a node of 1 cm in diameter was detected by MRI in the tenth dorsal vertebra. Because of the topographic coincidence between the patient's symptomatology and the MRI findings, the HD was considered to be in advanced stage and CMOPP/ABV chemotherapy was administered, this resulting in a rapid improvement of symptoms and disappearance of the MRI abnormalities. Since in the present case, the MRI determined a change in disease stage and treatment, the role of MRI as a complementary exploration of bone marrow biopsy to detect marrow involvement by HD is reviewed.
Boar spermadhesin PSP-I/PSP-II (M(r) 29 000-30 000), a non-covalent heterodimer of two CUB domains, was crystallized in two crystal forms. Complete diffraction data sets for hexagonal (space group P6(1,5)22) and trigonal (space group P3(1,2)21) crystals have been collected up to 2.9 and 2.5 angstrom resolution, respectively. Cell constants of the hexagonal and trigonal crystal forms are a=b=87.2 angstrom, c=152.4 angstrom, and a=b=96.2 angstrom, c=70.8 angstrom, respectively. The calculated packing parameters (Vm) are 2.8 and 3.2 angstrom(3)/DA for the hexagonal and trigonal crystal forms, respectively, indicating that, in both cases, the asymmetric unit is constituted by one PSP-I/PSP-II heterodimer. This paper reports the first crystals of a protein built up by a CUB domain architecture.
Boar spermadhesins are a group of seminal plasma, heparin-binding proteins which appear to be involved in sperm capacitation and gamete interaction. Using a proteolytic protection assay we have identified regions of AQN-1, AQN-3, PSP-I and AWN which remain attached to a heparin-Sepharose column following in-column digestion of bound spermadhesins with chymotrypsin and elastase. In addition, the complete amino acid sequence of spermadhesin AWN was synthesized as overlapping peptides, and their ability to bind to a heparin-Sepharose column and to inhibit the interaction of soluble heparin with purified ELISA plate-coated AWN was tested. Both approaches gave similar results and as a whole showed that different regions of AWN may converge in its tertiary structure to form a composite heparin-binding site. The conformational heparin-binding surface resides on the GFCC'C'' face of the proposed structural model for AWN and is in an opposite location to the carbohydrate-binding region of the spermadhesin.
A method for the stepwise purification of bovine seminal plasma proteins aSFP, PDC-109, TIMP-2, RNAse, and BSP-30K/PDC-109 complex is presented. It is based on the use of nondenaturing, classical chromatographic procedures. The isolation protocol is reproducible, inexpensive, easy to carry out in a common laboratory, and yields large amounts of pure proteins for biochemical and structural studies.
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A novel HLA-Cw*15, B7 haplotype has been found in Caucasians. Molecular cloning studies demonstrate that this haplotype is constituted by the new alleles Cw*15052 and B*0706, which seem to be intermediate steps in the diversification of their respective allelic families. A pathway for the evolution of Cw*15 alleles is proposed.
Nucleotide sequence analysis of the HLA-C alleles of the GB92 cell line, heterozygous for B*8101 and B*4407, revealed the existence of two new allelic variants: Cw*1801 and Cw*0706. The former allele, initially detected as a PCR-SSP variant, displays a hybrid aspect, sharing sequence motifs with Cw*07 at exons 1 and 2, and with Cw*04 at distal exons. In serological assays, Cw*1801 is only recognized by some cross-reactive sera. Cw*0706 shows a primary structure closely related to previously known Cw7 alleles, but carries new sequence motifs at its 3'-end. Preliminary data indicate that Cw*1801 is associated to B*8101 and that Cw*0706, B*4407 could account for a part of the Cw7, B44 haplotypes observed in African populations.
The increase in cancer incidence in renal transplant recipients is a well recognized fact, which has been related to post-transplant immunosuppressive therapy. Solid tumors, skin cancer and non-Hodgkin's lymphomas account for most of the neoplasms in these patients, whereas chronic myeloproliferative disorders are infrequent. A patient is reported who developed chronic myelogenous leukemia (CML) six years after renal transplantation, which was followed by immunosuppressive with azathioprine, and the published data on such an association are reviewed. In all 10 cases reported azathioprine had been administered after transplantation. The amount and type of post-transplant immunosuppressive therapy seems to be the most important risk factor for the development of secondary CML in these patients, since no cases of CML in patients receiving cyclosporine A have been reported.
Protein kinase C zeta (zeta PKC) is critically involved in the control of a number of cell functions, including proliferation and nuclear factor kappa B (NF-kappa B) activation. Previous studies indicate that zeta PKC is an important step downstream of Ras in the mitogenic cascade. The stimulation of Ras initiates a kinase cascade that culminates in the activation of MAP kinase (MAPK), which is required for cell growth. MAPK is activated by phosphorylation by another kinase named MAPK kinase (MEK), which is the substrate of a number of Ras-activated serine/threonine kinases such as c-Raf-1 and B-Raf. We show here that MAPK and MEK are activated in vivo by an active mutant of zeta PKC, and that a kinase-defective dominant negative mutant of zeta PKC dramatically impairs the activation of both MEK and MAPK by serum and tumour necrosis factor (TNF alpha). The stimulation of other kinases, such as stress-activated protein kinase (SAPK) or p70S6K, is shown here to be independent of zeta PKC. The importance of MEK/MAPK in the signalling mechanisms activated by zeta PKC was addressed by using the activation of a kappa B-dependent promoter as a biological read-out of zeta PKC.
Boar and stallion seminal plasmas were fractionated using affinity chromatography on heparin-Sepharose. In both species, among other proteins, the heparin-binding (H+) and non-heparin-binding (H-) fractions each contained glycoforms of either porcine PSP-I or equine HSP-1 and HSP-2. However, porcine H+/PSP-I eluted as a monomeric protein, whereas H-/PSP-I formed a heterodimer with PSP-II, another major seminal plasma protein. On the other hand, the stallion proteins H+/HSP-1 and H+/HSP-2 eluted together as an aggregate of relative molecular mass (M(r)) 90,000, whereas H-/HSP-1 and H-/HSP-2 eluted as monomers (15,000). Remarkably, when PSP-I and PSP-II from the H- fraction were separated, both proteins bound to heparin. Altogether these data show that glycosylation has an indirect effect on the heparin-binding ability of PSP-I, HSP-1 and HSP-2 through modulation of their aggregation state.
We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (BSP-A1/A2) and BSP-A3. However, unlike the bovine proteins which bind quantitatively to a heparin-Sepharose column, stallion HSP-1 was recovered in both the flow-through and the heparin-bound fractions. Structural analysis showed that the two HSP-1 forms contain identical polypeptide chains which are differently glycosylated. Moreover, size-exclusion chromatography showed that heparin-bound HSP-1 associates with HSP-2, another major seminal plasma protein, into a 90 kDa product, whereas the non-heparin-bound glycoform of HSP-1 is eluted as a monomeric (14 kDa) protein. This suggests that glycosylation may have an indirect effect on the heparin-binding ability of HSP-1 through modulation of its aggregation state. On the other hand, both glycoforms of HSP-1 displayed gelatin-binding activity, indicating that the molecular determinants for binding heparin and gelatin are different.