Germline VHL gene mutations in three Serbian families with von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited cancer predisposition syndrome due to germline mutations in the VHL tumor suppressor gene which is associated with virtually complete penetrance. The VHL syndrome has a highly variable phenotypic expressivity including retinal and CNS haemangioblastomas, pheochromocytomas, renal clear cell carcinomas, and multifocal cysts. In order to establish VHL gene testing, we analyzed three families affected by VHL disease, using SSCP mutation screening and DNA sequencing. Among 18 family members with and without clinical manifestations, eight cases with germline VHL mutations were detected. In family A, a c.490G>T/ p.Gly93Cys substitution was found. In family B, with pheochromocytoma only phenotype, we detected a previously not described c.463G>A/p.Val84Met replacement. Within this family, a prenatal diagnosis was also performed. Affected members of the third family with a VHL type 1 disease carried a c.475T>C/p.Trp88Arg exchange. All these mutations were located in exon 1 of the VHL tumor suppressor gene. Alterations in this hydrophobic region of the core beta domain of the VHL protein are known to have a variety of phenotypic consequences. We observed also intrafamiliar variation in time of onset and severity of the disease.

A kinetic analysis of the distinct regulatory sites on rabbit muscle pyruvate kinase.

Interaction of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with inhibitory and activatory effectors was analyzed in terms of the Yagi-Ozawa equation. Straight-line plots obtained for L-phenylalanine + L-alanine and L-phenylalanine + L-phenylalanyl methyl ester (PheAlaOMe) indicated competition for common sites. Parabolic plots for 2-phosphoglycerate and L-phenylalanine or PheAlaOMe suggested lack of competition between these compounds. Biphasic concave plots were obtained for 2-phosphoglycerate and L-1-amino-2-phenylethyl phosphonic acid (PnPhe), and mixtures of inhibitory (L-phenylalanine, PheAlaOMe) and activatory (PnPhe, L-alanine and L-1-aminoethyl phosphonic acid (PnAla)) amino acid derivatives. The latter non-classical Yagi-Ozawa plots can be interpreted in terms of the existence of distinct regulatory sites for inhibitory and activatory ligands.

Humoral stimulating activities in post-cyclophosphamide rat sera and their purified fractions.

Enhanced humoral, stimulating activities (HSAs) of post-cyclophosphamide (CY) sera and their purified fractions, acting on mitogen activated T-lymphocytes, were detected in Wistar rats after treatment by high single doses of the aplasia producing alkylating cytostatic drug CY. These activities, monitored in vitro, were partially purified from post-CY sera, collected 2, 4, and 7 days after treatment, yielding fractions with higher humoral stimulating activity. The preliminary purification included molecular cutting by Amicon-Diaflo filters (1-30 kDa pore size range) and gel-filtration on Sephadex G-50 and G-75. Results show that post-CY sera partially purified fraction (10-20 kDa), enhances the proliferation of hydrocortisone resistant (HCR) T-lymphocytes up to threefold under microculture conditions (P << 0.001); partially purified post-CY sera fraction (10-20 kDa) increases the proliferative response of T-cells in microcultures to the mitogenic 145-2C11 monoclonal antibody (mAb) up to tenfold (P << 0.001). These results show that the activity of stimulating factors is localized in the molecular weight range of 10-20 kDa.

Distinct spectrum of N-ras mutations in aml and mds patients in yugoslavia.

Mutations that activate ras genes were demonstrated to be associated with certain types of malignancies. Multiple point mutations were predominantly found in the N-ras and occasionally in the K-ras genes. The analysis of 4 MDS, 23 AML and 11 CML patients from Yugoslavia revealed the prevalence of the N-ras mutation (83%) over K-ras mutations (17%). Although the frequencies of the N- and K-ras mutations in these patients were similar to the ones reported for patients from USA and Japan, the N-ras mutational spectra considerably differed. The prevailing type of mutation in patients from Yugoslavia was G-to-T transversion at the first position in the codon 12 of the N-ras gene. This study supports a hypothesis that different geographical and environmental factors may cause the accumulation of different type of point mutations in the same target gene.

Germline mutation analysis of BRCA1 and BRCA2 genes in Yugoslav breast/ovarian cancer families.

The frequency of germline BRCA1 and BRCA2 mutations was tested in Yugoslav breast and breast/ovarian cancer families using combined heteroduplex/single-strand conformation polymorphism analysis for the entire coding region of both genes. Three different recurrent BRCA1 mutations (one 185delAG, one 3447del4 and two 5382insC) were identified in 4 of 12 families (33%), whereas no definite disease-causing alterations of BRCA2 was detected. Genotype analysis revealed a possible common founder effect for each 185delAG and 5382insC. The relatively high frequency of germline BRCA1 mutations determined in this panel of families confirms the important role of BRCA1 in disease predisposition in the Yugoslav population, while the lack of population specific founder and/or unique mutations show the need of further analysis of samples from this yet unexamined region of Europe.

Hematological toxicity associated with tiazofurin-influence on erythropoiesis.

In this study hematological toxicity was analyzed after the single and repeated applications of tiazofurin (TZF). Cellularity of bone marrow, spleen and peripheral blood was examined, spanning the period of fifty days after the initial application. Analysis of hematological parameters was performed by slightly modified conventional techniques. The fraction of erythroid series was monitored during the experiment. Presented data describe kinetics of damage and recovery of hemopoietic tissue. Our results indicate that the effect of tiazofurin on cellularity of bone marrow and spleen and on erythropoiesis is reversible and dose dependent within tested dose range and therapeutic regimes. Twenty days after the application normal function of hemopoietic tissues was restored. This approach and results can be useful in defining the timing for sequencing and combination therapy with tiazofurin.

Bone formation within porous hydroxylapatite implants in human periodontal defects.

Tissue samples from three subjects who had periodontal defects treated with a porous hydroxylapatite implant were investigated using light microscopy and scanning electron microscopy. The 3-month specimen showed connective tissue infiltration through the pores and a narrow zone of bone formation present along the walls of the pores. At 4 months, continued evidence of bone deposition was present with osteocytes, osteoblasts and organization of collagen fibers apparent throughout the implant. The 6-month implant had further evidence of continued bone formation with lamellar bone being the major component within the pores.

Histologic study of healing of human periodontal defects after placement of porous hydroxylapatite implants.

Two intrabony pockets on teeth that were to be extracted for prosthetic reasons, in two patients, were treated by means of papilla preservation flaps and implantation with porous hydroxylapatite. The teeth, with a portion of their periodontium, were extracted 5 and 6 months after treatment and processed histologically. Both cases showed the implants invaded by connective tissue, which was actively forming bone. There was no inflammatory reaction triggered by the implant material, although there was some recurrence of inflammation in the vicinity of the pocket wall. The junctional epithelium in both cases extended slightly beyond the coronal level of the implant. One of the cases had an abundant new cementum formation with fibrous tissue next to the implant, that appeared to be a rebuilt attachment of collagen fibers. Although some multinucleated giant cells were found in the pores and next to the newly formed bone, the implanted material did not appear to be resorbed in the period of time studied.

The use of a porous hydroxylapatite implant in periodontal defects. I. Clinical results after six months.

Twenty-five patients with advanced periodontal destruction were used in the study. Following initial therapy, two angular interproximal defects were selected in each patient. During flap surgery a porous hydroxylapatite implant shaped to fit the periodontal defect was placed in one defect, the other defect was used as nonimplanted control. The material used for implantation was a hydroxylapatite replicate of coral from the genus Porites, with a pore size of 190 to 220 micron. Clinical parameters were measured prior to flap surgery for each of the defects. An occlusal acrylic stent was used to give a stable reference point for pocket depth, attachment level and gingival margin height measurements. Also gingival fluid, gingival inflammation, plaque index and tooth mobility were recorded. Periapical radiographs using a standardized positioning device were also taken. At the time of surgery, the depth of the osseous defect and the height of the alveolar crest were recorded. After 6 months the clinical measurements were repeated and a re-entry surgery was carried out in 15 selected sites. Results showed that the porous implant produced statistically significant reduction in pocket depth, in the depth of osseous lesion, and a statistically significant gain in attachment level, as compared to control areas.

The use of autogenous periosteal grafts as barriers for the treatment of intrabony defects in humans.

The purpose of this study was to evaluate the clinical effectiveness of a connective tissue graft including periosteum used as a barrier for guided periodontal tissue regeneration in interproximal bony defects. An open flap debridement of a comparable interproximal bony defect in the same patient was used as a control. This study was performed at 2 different clinical centers. Six paired defects were treated at one center, and 16 paired defects at the other. Reentry surgeries were performed at 6 months. Preoperative comparisons of control and experimental sites with respect to clinical parameters and osseous measurements were similar. Post-surgical experimental sites produced more gain in clinical attachment (1.25 mm on buccal and 1.25 mm on lingual sites at center A and 1.26 mm on buccal and 1.18 mm on lingual sites at center B) and osseous defect fill (1.84 mm on buccal and 2.00 mm on lingual sites at center A and 1.66 mm on buccal and 2.04 mm on lingual sites at center B) when compared to control sites. The results of this trial indicate that clinical resolution of interproximal periodontal defects can be obtained with periosteal grafts used as barriers.

Preservation of alveolar bone in extraction sockets using bioabsorbable membranes.

The purpose of this study was to evaluate the clinical effectiveness of a bioabsorbable membrane made of glycolide and lactide polymers in preserving alveolar ridges following tooth extraction using a surgical technique based on the principles of guided bone regeneration. Sixteen patients requiring extractions of 2 anterior teeth or bicuspids participated in the study (split-mouth design). Following elevation of buccal and lingual full-thickness flaps and extraction of teeth, experimental sites were covered with bioabsorbable membranes; control sites did not receive any membrane. Titanium pins served as fixed reference points for measurements. Flaps were advanced in order to achieve primary closure of the surgical wound. No membrane became exposed in the course of healing. Reentry surgeries were performed at 6 months. Results showed that experimental sites presented with significantly less loss of alveolar bone height, more internal socket bone fill, and less horizontal resorption of the alveolar bone ridge. This study suggests that treatment of extraction sockets with membranes made of glycolide and lactide polymers is valuable in preserving alveolar bone in extraction sockets and preventing alveolar ridge defects.

Localization of recognition site between transforming growth factor-beta1 (TGF-beta1) and TGF beta receptor type II: possible implications in breast cancer.

Although overexpression of TGF-beta1 protein has been demonstrated in advanced breast cancer (BC) patients, as well as in other solid tumours, the molecular mechanism of this process remains obscure. This paper proposes that a genetic/epigenetic alteration might occur in the TGF-beta1 gene, within the region coding for the recognition site with TGFbeta receptor type II, leading to a disruption of the ligand-receptor interaction and triggering the TGF-beta1 cascade-related BC progression. To establish the operational framework for this hypothesis, in the present study, this recognition site was identified by the Informational Spectrum Method (ISM) to comprise two TGF-beta1 peptides (positions 47-66 aa and 83-112 aa) and one receptor peptide at positions 112-151 aa of the extracellular domain of the receptor (TbetaRIIM). The TbetaRIIM locus was further evaluated by ISM-derived deletion analysis of the TbetaRII sequences. To provide experimental support for the proposed model, a pilot study of plasma TGF-beta1 analysis was performed in advanced BC patients (n = 8). Two commercial ELISA assays, one with specific alphaTGF-beta1 MAb (MAb) and other with TbetaRIIM as the immobilized phase, revealed pronounced differences in the pattern of plasma TGF-beta1 elevation. In MAb-profile, the TGF-beta1 increase was detected in 7 of 8 patients, whereas analogous TbetaRIIM-profile revealed the elevation in 3 of 8 patients, taking a 50% of maximal elevation as the cut-off value. These findings are consistent with the proposed aberration of TGF-beta1 ligand within the TbetaRII recognition site. Summarizing, this model system is a good starting point for further genetic studies, particularly on genetic/epigenetic alterations of sequences involved in TGF-beta1 and TbetaRIIM interaction, with putative prognostic value for breast cancer.

Influence of bioactive glass on changes in alveolar process dimensions after exodontia.

BACKGROUND: Preservation of the alveolar process after tooth extraction is desirable because it facilitates placement of endosseous implants and minimizes adverse esthetic results associated with fixed partial dentures. The purpose of this study was to evaluate the clinical effectiveness of bioactive glass used as a graft material combined with calcium sulfate used in the form of a mechanical barrier in preserving alveolar ridges after tooth extraction. METHODS: Sixteen patients who required extraction of 2 anterior teeth or bicuspids participated in the study (split mouth design). After tooth extraction and elevation of a buccal full-thickness flap, experimental sockets were filled with bioactive glass, which in turn was covered with a layer of calcium sulfate. Control sites did not receive any graft or calcium sulfate. Titanium pins served as fixed reference points for measurements. No attempt was made to advance the flap to cover the socket areas on control or experimental sites (open socket approach). Reentry surgeries were performed at 6 months. RESULTS: Reentry surgeries showed that experimental sites presented with (1) significantly more internal socket bone fill (6.43 +/- 2.78 mm vs 4.00 +/- 2.33 mm on control sites), (2) less (although not statistically significantly less) resorption of alveolar bone height (0.38 +/- 3.18 mm vs 1.00 +/- 2. 25 mm on control sites), and (3) similar degree of horizontal resorption of the alveolar bony ridge as compared with controls (3. 48 +/- 2.68 mm vs 3.06 +/- 2.41 mm on control sites). CONCLUSIONS: This study suggests that treatment of extraction sockets with a combination of bioactive glass and calcium sulfate is of some benefit in preserving alveolar ridge dimensions after tooth extraction.

Prevalence of human cytomegalovirus and Epstein-Barr virus in subgingival plaque at peri-implantitis, mucositis and healthy sites. A pilot study.

This study evaluated the prevalence of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in peri-implantitis and mucositis sites and the correlation between herpesvirus and clinical parameters. Fifty-six dental implants (mean time of loading, 4.27±1.6 years) were evaluated (20 peri-implantitis, 18 mucositis, 18 healthy peri-implant sites.) The clinical parameters assessed were: visible plaque index (PI), bleeding on probing (BOP), suppuration (SUP), probing depth (PD). A polymerase chain reaction assay identified HCMV and EBV in subgingival plaque samples. The percent of sites with plaque and BOP was significantly higher around mucositis and peri-implantitis compared with healthy implants (p<0.05). The mean PD around the implants was significantly higher in peri-implantitis, followed by mucositis and healthy implants (p<0.05). HCMV was detected in 13 (65%) and EBV in 9 (45%) of the 20 peri-implantitis sites. HCMV was found in 1 of the 18 (6%) healthy periodontal sites and EBV in 2 (11%). A statistically significant correlation was found between presence of HCMV and EBV subgingivally and clinical parameters of peri-implantitis and healthy sites. These results confirm the high prevalence of HCMV and EBV in subgingival plaque of peri-implantitis sites and suggest the viruses have a possible active pathogenic role in peri-implantitis.

Correlation between different genotypes of human cytomegalovirus and Epstein-Barr virus and peri-implant tissue status.

BACKGROUND: The purpose of this study was to estimate the prevalence of different genotypes of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in peri-implantitis and mucositis sites, and to evaluate the correlation between herpesvirus presence and clinical parameters. METHODS: A total of 80 dental implants (mean time of loading, 4.16 ± 1.8 years) were evaluated during the course of the study (30 peri-implantitis, 25 mucositis and 25 healthy peri-implant sites). The following clinical parameters were assessed: visible plaque index, bleeding on probing, suppuration and probing depth. A polymerase chain reaction (PCR) assay was used to identify the presence of different HCMV and EBV genotypes in peri-implant tissue plaque samples. RESULTS: HCMV-2 was detected in 53.3% and EBV-1 in 46.6% of the 30 peri-implantitis sites evaluated. By contrast, HCMV-2 was not detected in healthy periodontal sites and EBV-1 was detected in one healthy site. A statistically significant correlation was found between the presence of HCMV-2 and EBV-1 genotypes and clinical parameters of peri-implantitis. CONCLUSIONS: The results from the present study confirmed the high prevalence of HCMV-2 and EBV-1 in the peri-implant tissue plaque of peri-implantitis sites and suggests a possible active pathogenic role of the viruses in peri-implantitis.

[Prosthetic reconstruction of the secondary deformed maxillary arch in a patient with congenital cleft]. / Proteticko zbrinjavanje sekundarne deformacije maksilarnog luka u osobe sa urodenim rascepom.

A case history of a bilateral cleft lip and palate male patient is presented. He was operated late at the age of his twelve, without any previous orthodontic treatment. Surgical reconstruction included, lip, hard and soft palate, removal of the premaxilla, and later on the pharingoplasty. At the age of twenty years he was prosthetically rehabilitated. Nine inner telescope crowns were made in "Palador" silver alloy, and nine outer in chrom-cobalt splinted together and connected with the skeletal frame. Intermaxillary relations were recorded and established in a manner used in the construction of complete dentures, and occlusion of the teeth, too. A complete obturation of the oronasal communication in the palatomaxillary section was achieved. The patient was followed up throughout next twenty years.

[Evaluation of condyle fracture with PA X-rays by computer measurements]. / Procena dislokacije i repozicije preloma kondila na anteroposteriornim RDG snimcima merenjem na racunaru.

The degrees of dislocation after treatment and reduction of the condylar fractures is empirically evaluated with standard X-rays of the lower jaw. The aim of this paper is to investigate the possibilities a more precise evaluation of the degree of dislocation and reduction of the shorter fragment with PA X-rays of the lower jaw. Forty X-rays of 20 patients with unilateral condylar fracture were used. X-rays were put into the memory of a compatible IBM PC with a graphic table. On the graphical presentation of these X-rays the following parameters were determinated: axis of the individual symmetry and condyle axis; angles between the condyle axis and the horizontal line were measured: D--in dislocated condyle, IR--in "ideally" reduced condyle and SR--actually reduced condyle. The angles between the axis of the undamaged condyles and the horizontal line were measured on the X-rays before and after the completion of the treatment. The difference between them was presented am an X-ray error. The obtained results are encouraging.

[Immediate prosthetic reconstruction after resection of the upper jaw]. / Imedijatna proteticka rekonstrukcija posle resekcija gornje vilice.

In order to determine the right time when appliance should be applied to the patient after partial, subtotal or total resection of the maxilla, represent cases treated during a six year period at the Clinic of Maxillofacial Surgery of the Stomatology Faculty in Belgrade. In 11 operated patients the appliance was applied immediately after surgery and tamponade of postresection activity cavity. In 46 patients operated at other clinics it was applied more than month after surgery. Thus, 57 patients received immediate prosthetic reconstructive treatment. According to criteria of adaptability considering new appliances major differences could be observed among different groups of patients. 57 patients recovery was normal, while in 4 adaptability was slower. In 12 referral cases not a single case of normal adaptability was registered. 4 patients demonstrated slower adaptability, while in 8 patients severe adaptability problems were registered.

Mismatch-specific DNA breakdown in nuclear extract from tobacco (Nicotiana tabacum) callus.

Mismatch-specific enzymatic activity was sought for in nuclei from normal and transformed plant cells originating from tobacco (Nicotiana tabacum) callus and crown gall tumor induced by Agrobacterium tumefaciens. The specific enzymatic activity was assayed with substrates derived from synthetic oligonucleotides (19-mer sequences corresponding to the human K-ras gene). Single-base changes in the middle of the sequence were the basis for creating heteroduplexes with all eight mismatches. Homo- and heteroduplexes were ligated in a size ladder and used as substrates. We detected mismatch-specific DNA breakdown and determined basic requirements for the reactions. Kinetic analysis indicates the following reactivity order of preference: C:A=C:C=C:T greater than G:T approximately A:A approximately G:A approximately G:G approximately T:T much greater than G:C. It can be said now that specific mismatch recognition and repair activities have been detected in all kingdoms of living species.