RecA gene sequence and Multilocus Sequence Typing for species-level resolution of Burkholderia cepacia complex isolates.

AIM: To identify, by means of recA sequencing and multilocus sequence typing (MLST), Burkholderia cepacia complex (BCC) isolates of environmental and clinical origin, which failed to be identified by recA RFLP and species-specific PCR. METHODS AND RESULTS: By using recA sequence-based identification, 17 out of 26 BCC isolates were resolved at the level of species and lineage (ten Burkholderia cenocepacia IIIB, two Burkholderia arboris and five Burkholderia lata). By using MLST method, 24 BCC isolates were identified. MLST confirmed recA sequence results, and, furthermore, enabled to identify isolates of the BCC5 group, and showed relatedness with Burkholderia contaminans for one of the two isolates not identified. CONCLUSIONS: recA sequence-based identification allowed to resolve, at the level of species and lineage, 65.4%, of the BCC isolates examined, whilst MLST increased this percentage to 88.5%. SIGNIFICANCE AND IMPACT OF THE STUDY: BCC isolates previously not resolved by recA RFLP and species-specific PCR were successfully identified by means of recA sequencing and MLST, which represent the most appropriate methods to identify difficult strains for epidemiological purposes and cystic fibrosis patients management.

Evidence that the bifunctional redox factor / AP endonuclease Ref-1 is an anti-apoptotic protein associated with differentiation in the developing retina.

Retinal cell differentiation leads to resistance to apoptosis induced by inhibition of protein synthesis, suggesting the accumulation of anti-apoptotic proteins. The redox factor/AP endonuclease Ref-1 (APE, APEX, HAP1) affects both DNA repair and the activity of various transcription factors, and controls sensitivity to genotoxic insults. We studied the expression of Ref-1 in the retina and brain of developing rats. Ref-1 immunoreactivity increased progressively within the nucleus of differentiating retinal cells, whereas it decreased in the developing hippocampal formation. During both natural and experimentally-induced cell death, Ref-1 disappeared from the nucleus of apoptotic cells. Degradation of Ref-1 in axotomized ganglion cells preceded the morphological characteristics of apoptosis. The sensitivity to apoptosis triggered by either thapsigargin or okadaic acid was the highest in photoreceptors, that contain the least Ref-1 among differentiated retinal cells. In both these differentiated cell types, inhibition of protein synthesis prevented the loss of Ref-1 and rescued the neurons. The data suggest that Ref-1 is an anti-apoptotic protein associated with cell differentiation in the retina.

Apoptosis in developing retinal tissue.

The mechanisms of apoptosis are strongly dependent on cell-cell interactions typical of organized tissues. Experimental studies of apoptosis using a histotypical preparation of retinal explants are reported in the present article. We found that various characteristics of apoptosis are selectively associated with retinal cell death depending on cell type, stage of maturation, and means of induction of apoptosis. Among these were: (1) the requirements of protein synthesis; (2) the role of cAMP; (3) the expression of certain apoptosis-associated proteins; and (4) the sensitivity to excitotoxicity, modulation of protein phosphatases and calcium mobilization. Dividing cells undergo apoptosis in response to several inducers in specific phases of the cell cycle, and in distinct regions within their pathway of interkinetic nuclear migration. Recent post-mitotic cells are selectively sensitive to apoptosis induced by blockade of protein synthesis, while both proliferating and differentiated cells are more resistant. We also studied the association of several proteins, some of which play critical roles in the cell cycle, with both differentiation and apoptosis in the retinal tissue. Detection of cell cycle markers did not support the hypothesis that retinal cells re-enter the cell cycle on their pathway to apoptosis, although some proteins associated with cell proliferation re-appeared in degenerating cells. The transcription factors c-Jun, c-Fos and c-Myc were found associated with apoptosis in retinal cells, but their sub-cellular location in apoptotic bodies is not consistent with their canonical functions in the control of gene expression. The bifunctional redox factor/AP endonuclease Ref-1 and the transcription factor Max are associated with progressive cell differentiation, and both are down-regulated during cell death in the retina. The data suggest that Ref-1 and Max may normally function as negative modulators of retinal apoptosis. The results indicate that nuclear exclusion of transcription factors and other important control proteins is a hallmark of retinal apoptosis. Histotypical explants may be a choice preparation for the experimental analysis of the mechanisms of apoptosis, in the context both of cell-cell interactions and of the dynamic behavior of developing cells within the organized retinal tissue.

Modulation of the expression of the transcription factor Max in rat retinal ganglion cells by a recombinant adeno-associated viral vector.

Exclusion of the transcription factor Max from the nucleus of retinal ganglion cells is an early, caspase-independent event of programmed cell death following damage to the optic axons. To test whether the loss of nuclear Max leads to a reduction in neuroprotection, we developed a procedure to overexpress Max protein in rat retinal tissue in vivo. A recombinant adeno-associated viral vector (rAAV) containing the max gene was constructed, and its efficiency was confirmed by transduction of HEK-293 cells. Retinal ganglion cells were accessed in vivo through intravitreal injections of the vector in rats. Overexpression of Max in ganglion cells was detected by immunohistochemistry at 2 weeks following rAAV injection. In retinal explants, the preparation of which causes damage to the optic axons, Max immunoreactivity was increased after 30 h in vitro, and correlated with the preservation of a healthy morphology in ganglion cells. The data show that the rAAV vector efficiently expresses Max in mammalian retinal ganglion cells, and support the hypothesis that the Max protein plays a protective role for retinal neurons.

Carcinogen metabolism, DNA damage repair and oral head and neck squamocellular carcinoma (HNSCC). A review.

Head and neck squamocellular carcinoma (HNSCC) has now become the 6th most common cancer among men in the developed world and affects the oral cavity, salivary glands, larynx and pharynx. Tobacco chewing, alcohol consumption and last but not least, smoking seem to be the most important risk factors. In particular in non-drinkers, smoke increases the relative risk (RR) of developing HNSCC of the oral cavity and pharynx from 2 to 20 fold; especially in the oral cavity, the association between alcohol and smoke could have a multiplier effect. Cancer arises from damage to DNA of genes located at various points of the short (p) and long (q) arms of a number of chromosomes, caused by exposure to various carcinogens. Thus, the carcinogenic process requires continuous exposure to environmental carcinogens (i.e., longstanding history of smoking and drinking), an increased susceptibility to carcinogens (induced by xenobiotic metabolizing enzyme polymorphism) and an impaired DNA repair capacity (both inherited and acquired). Our purpose in this paper is to review advances in the understanding of the role of the European or Caucasian genetic aberrations that affect carcinogen metabolism and DNA repair genes in oral HNSCC development: we consider that those abnormalities will be useful in assessing individuals at risk.

Molecular characterization of rhizosphere and clinical isolates of Burkholderia cepacia.

Four Burkholderia cepacia strains isolated from the rhizosphere and pathological samples of infected human patients were characterized at the molecular level by different methodologies, including the determination of 16S ribosomal rDNA sequence, restriction endonuclease analysis of total DNA, random amplified polymorphic DNA fingerprinting and Southern hybridization with gene probes for nitrogen fixation and siderophore synthesis. The results indicate that the four strains cluster together within genus Burkholderia, but differ from one another. The DNA from the four strains hybridized to the nifA gene probe from Klebsiella pneumoniae, and an appreciable homology with the nifHDK structural genes of Azospirillum brasilense was demonstrated for one rhizosphere strain. Although the four isolates produced an ornibactin-like siderophore, they did not give hybridization with the pvdA probe for hydroxamate biosynthesis from Pseudomonas aeruginosa.

Protein kinases selectively modulate apoptosis in the developing retina in vitro.

In the retina of newborn rats there is evidence for two mechanisms of programmed cell death. Apoptosis of ganglion cells (RGCs) following axotomy depends on protein synthesis. In contrast, inhibition of protein synthesis leads to apoptosis in the neuroblastic layer (NBL). The induction of apoptosis following translational arrest suggests that post-translational modifications of apoptosis-associated proteins may be crucial to the cell death programs in the developing retina. We investigated the possible role of protein kinases upon apoptosis in retinal explants in vitro. An increase in the intracellular concentration of cAMP produced either by the adenylyl-cyclase activator forskolin (10 microM) or by 8-Br-cAMP (1 mM), prevented apoptosis induced in the NBL by inhibition of protein synthesis, but had no statistically significant effect upon RGC death. In contrast, neither 8-Br-cGMP (1 mM) nor the specific cGMP-phosphodiesterase inhibitor zaprinast (10-100 microM) had significant effects on apoptosis in the retina. The cAMP-phosphodiesterase inhibitors isobutylmethylxantine (IBMX, 0.1-1 mM) and Ro-201724 (50-200 microM) also prevented apoptosis in the NBL. The isoquinolinesulfonamide H89 (20 microM), a specific cAMP-dependent protein kinase inhibitor, partially reverted the protective effect of either forskolin or IBMX within the NBL. Neither 12-O-tetradecanoyl phorbol-13-acetate (TPA, 10 nM) nor bisindolylmaleimide (0.2-0.5 microM), respectively an activator and an inhibitor of protein kinase C had significant effects upon the retinal explants. The protein kinase inhibitor 2-aminopurine (2-AP, 10 mM) prevented apoptosis of axotomized ganglion cells and induced apoptosis in the NBL. Forskolin prevented the apoptosis induced by 2-AP in the NBL, whereas TPA had no effect. The effects of 2-AP were, however, not dependent on inhibition of protein synthesis. The data indicate that modulation of the activity of both cAMP-dependent protein kinase and several protein kinases sensitive to 2-aminopurine selectively affect apoptosis in distinct cell layers of the developing retina.

Tissue biology of apoptosis. Ref-1 and cell differentiation in the developing retina.

Programmed cell death by apoptosis plays a major role in neurogenesis. The sensitivity to apoptosis in developing nervous tissue is strongly dependent on cell interactions taking place within a highly structured environment, composed of various cell types at distinct stages of differentiation. In this article, we review evidence gathered both in vivo and in a histotypical retinal explant preparation in vitro that the bifunctional AP endonuclease/redox factor Ref-1 (HAP1, APE, APEX) may be an anti-apoptotic protein associated with cell differentiation in the developing retina.

Bias Caused by Using Different Isolation Media for Assessing the Genetic Diversity of a Natural Microbial Population.

The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium.

Soil Type and Maize Cultivar Affect the Genetic Diversity of Maize Root-Associated Burkholderia cepacia Populations.

Abstract Burkholderia cepacia populations associated with the Zea mays root system were investigated to assess the influence of soil type, maize cultivar, and root localization on the degree of their genetic diversity. A total of 180 B. cepacia isolates were identified by restriction analysis of the amplified 16S rDNA (ARDRA technique). The genetic diversity among B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique, using the 10-mer primer AP5. The analysis of molecular variance (AMOVA) method was applied to estimate the variance components for the RAPD patterns. The results indicated that, among the factors studied, the soil was clearly the dominant one in affecting the genetic diversity of maize root-associated B. cepacia populations. In fact, the percentage of variation among populations was significantly higher between B. cepacia populations recovered from maize planted in different soils than between B. cepacia populations isolated from different maize cultivars and from distinct root compartments such as rhizoplane and rhizosphere. The analysis of the genetic relationships among B. cepacia isolates resulted in dendrograms showing bacterial populations with frequent recombinations and a nonclonal genetic structure. The dendrograms were also in agreement with the AMOVA results. We were able to group strains obtained from distinct soils on the basis of their origin, confirming that soil type had the major effect on the degree of genetic diversity of the maize root-associated B. cepacia populations analyzed. On the other hand, strains isolated from distinct root compartments exhibited a random distribution which confirmed that the rhizosphere and rhizoplane populations analyzed did not significantly differ in their genetic structure.http://link.springer-ny.com/link/service/journals/00248/bibs/38n3p273.html

Nuclear exclusion of transcription factors associated with apoptosis in developing nervous tissue.

Programmed cell death in the form of apoptosis involves a network of metabolic events and may be triggered by a variety of stimuli in distinct cells. The nervous system contains several neuron and glial cell types, and developmental events are strongly dependent on selective cell interactions. Retinal explants have been used as a model to investigate apoptosis in nervous tissue. This preparation maintains the structural complexity and cell interactions similar to the retina in situ, and contains cells in all stages of development. We review the finding of nuclear exclusion of several transcription factors during apoptosis in retinal cells. The data reviewed in this paper suggest a link between apoptosis and a failure in the nucleo-cytoplasmic partition of transcription factors. It is argued that the nuclear exclusion of transcription factors may be an integral component of apoptosis both in the nervous system and in other types of cells and tissues.

In vitro collagen synthesis by liver connective tissue cells isolated from schistosomal granulomas.

Hepatic injury elicits an excessive deposition of extracellular matrix probably due to a loss of control mechanisms in mesenchymal cells in fibrotic lesions, or a local activity of growth factors. To study collagen synthesis in an in vitro model of fibrotic lesions, we isolated liver connective tissue cells (LCTC) from murine schistosomal granulomas in C3H/HeN mice. Collagen was quantified in culture supernatants using a sirius red dye assay. LCTC and skin fibroblasts (SF) secreted similar amounts of collagen per cell and secretion was inversely proportional to the cell density. Cells cultured at low density (10,000 cells/cm2) secreted two- to three-times more collagen per cell when compared to cells grown in high-density cultures (60,000 cells/cm2). Collagen secretion was stimulated by transforming growth factor-beta (TGF-beta) in both cell lines, but the response of LCTC was detected from 1 ng/ml on, while SF responded only to higher concentrations (2.5 and 5 ng/ml). These data do not support the hypothesis that cells from fibrotic livers have lost the normal control mechanisms and suggest that their control is disturbed locally by the presence of peptide growth factors during the development of fibrosis.

Simultaneous bimaxillary alveolar ridge augmentation by a single free fibular transfer: a case report.

Class VI atrophy according to Cawood still represents a major challenge in pre-prosthetic surgery. Reconstruction of mandibular and maxillary bony defects using microvascular techniques is safe and reliable. The fibula, due to its morphological properties, is ideal for alveolar ridge augmentation and its donor site morbidity is the lowest among vascularized bone flaps. In this paper, we report the first case, to our knowledge, of extreme atrophy of both jaws, successfully treated by simultaneous bony augmentation of the maxillary and mandibular alveolar ridges with just one free fibula flap. Pre-operative planning, surgical technique and prosthetic restoration are discussed in detail.

Functional rehabilitation of the atrophic mandible and maxilla with fibula flaps and implant-supported prosthesis.

Historically, nonvascularized bone grafts have been the standard treatment for severe mandibular and maxillary atrophy, followed by immediate or delayed implant placement. Extreme atrophy is an unfavorable biological and mechanical location for nonvascularized autologous bone transplants. The authors present the results of a multidisciplinary treatment protocol for rehabilitation of extreme mandibular and maxillary atrophy by use of the vascularized fibular flap. This protocol includes bone augmentation, implant surgery, soft-tissue management, and prosthetic restoration. Since 1993, 18 patients with a mean age of 47.5 years presented with extreme mandibular and/or maxillary atrophy and underwent alveolar crest augmentation with vascularized fibular flaps. Bone healing was achieved in 17 of the 18 patients. Seventy-three osteointegrated implants were inserted in 12 of 17 fibular flaps. Altogether, 62 implants were loaded and 11 dental prostheses were made. Average follow-up of the loaded implants was 41 months. The success rate of loaded implants was 100 percent. The authors strongly recommend the use of the fibular bone flap when dealing with extreme atrophy of the mandible and maxilla and suggest the protocol outlined in this review.

Intracranial spread of a giant frontal mucocele: case report.

A giant mucocele eroded both the anterior and posterior wall of the frontal sinus and infiltrated the dura mater. Its extracranial growth caused a frontal bony prominence. The tumour and part of the dura were resected. A 12 x 6cm defect in the dura was repaired with a freeze-dried patch. A split-thickness bone graft from the right parietal region was used to repair the anterior frontal bony defect. The result one year later was satisfactory. Spiral computed tomography with thr ee-dimensional reconstructions excluded any recurrence of the tumour and showed good integration of bone grafts.

Combined effect of vestibular and craniomandibular disorders on postural behaviour.

A correlation has been reported in the dental literature between temporomandibular disorders and musculoskeletal abnormalities, however, the question whether they modify body postural sway remains controversial. In the present investigation, the Craniomandibular Index was used to evaluate the clinical extension of temporomandibular joint dysfunction and related problems in 40 patients with normal vestibular function and in 42 patients with peripheral vestibular disorders. Balance function was assessed by static posturography and body sway area was measured in two conditions: i) eye open, and g) eye closed. Data were compared to those of 40 healthy subjects. Postural control showed a significantly different behaviour between groups with an increase in average body sway in patients with craniomandibular disorders as opposed to controls (p < 0.005). Although the involvement of the stomatognathic apparatus was not quantitatively different in the two groups of patients, those also presenting a peripheral vestibular disorder exhibited greater average body sway than patients with only craniomandibular disorders (p < 0.005). The latter showed a greater average body sway than controls only in the trial with eyes closed (p < 0.05). The results demonstrated that craniomandibular alterations could produce moderate postural instability in patients with a normal vestibular function. Conversely, their association with peripheral vestibular disorders becomes a real challenge to the upright quiet stance probably due to a negative effect of somatosensory origin on the vestibulo-spinal reflex impairment.

[Treatment of the patient with a coagulation defect in oral and maxillofacial surgery. III. The management of patients in a hypocoagulative state because of a hemophilic-type primary pathology]. / Il trattamento del paziente con difetto di coagulazione in chirurgia orale e maxillo-facciale. Nota III: Il management dei pazienti in stato ipocoagulativo a causa di una patologia primaria di tipo emofilico.

Hemophilia plays a particularly important role among the diseases caused by abnormal coagulation. Defective blood-clotting factor diseases have a particular importance between coagulopathies: hemophilia, among these hematic disorders, plays a principal role. In this paper the authors present the results of scientific research on hemophilic disease carried out to obtain a correct clinical and therapeutic approach for clinical and surgical Odontostomatology. The authors, after having presented in short the physiopathologic function of coagulation factors, illustrate the clinical and therapeutic aspects of Hemophilia A and Hemophilia B. The correct Odontostomatological and Maxillo-Facial Surgical approach is presented as the result of the authors' research. Also von Willebrand's disease is illustrated even if it is not exactly a hemophilic disease. This is because all hemophilias must produce a gynephoric inheritance pattern. Nevertheless clinical, therapeutic and molecular biology appearance suggests the illustration of von Willebrand's disease together with hemophilias. Von Willebrand's disease can be divided into three nosologic groups and to each one corresponds a particular clinical and therapeutic management. Such cases are illustrated and examined from an Odontostomatologic point of view. The results obtained suggest the necessity of keeping to the management that was described. Actually a low percentage of accidents occurred only when the above-mentioned clinical processes were completely performed.

[Management of patients with coagulation disorder in oral and maxillofacial surgery. I. Management of patients with hypocoagulation caused by primary thrombocytopathy]. / Il trattamento del paziente con difetto di coagulazione in chirurgia orale e maxillo-facciale. Nota II. Il management dei pazienti in stato ipocoagulativo a causa di una patologia primaria trombocitopatica.

Any oral and maxillo-facial surgical treatment, however urgent it may be, must not include pathological states in which the patient's life may be particularly at risk as, for example, with Disseminated Intravascular Coagulation (DIC) or throm-botic thrombocytopenic purpura. In this article the authors present the result of studies carried out on the nosology of thrombocytopathy from an odontostomatological point of view. Thrombocytopathy can be divided into two groups: the first including the pathologies with a predominant defective number of thrombocytes (i.e.: thrombocytopenia, thrombocythemia, thrombocyto-sis), the second including forms with predominant qualitative defects (commonly known as thrombocytopathies). The authors, after having presented in short the physiopathologic functions of thrombocytes, illustrate the clinical and therapeutic aspects of the most important thrombocytopathies. Morbus Maculosus Werhofii, Glanzmann's disease, Bernard-Soulier syndrome, thrombocytopathies from defective reaction of release, Thrombocytopathies from defective procoagulant activity of blood plaques, thrombocytopathies in linkage to other genetic anomalies, von Willebrand's pseudodisease and a lot of acquired thrombocytopathies are identified. In the last part the authors illustrate the most opportune clinical steps corresponding to the most important thrombocytopathies. The results obtained suggest the necessity of keeping to the management that was described, Actually a low percentage of accidents occurred only when the above-mentioned clinical processes were completely performed.

[Management of patients with coagulation defect in oral and maxillofacial surgery. I. Management of patients with drug-induced hypocoagulation]. / Il trattamento del paziente con difetto di coagulazione in chirurgia orale e maxillo-facciale. Nota I: Il management dei pazienti in stato ipocoagulativo farmacoindotto.

Odontoiatric problems, clinical and surgical, connected with defective coagulation, are very frequent. Such cases can be divided into two groups: in the first we find patients with iatrogenic coagulopathy while in the second we find patients with hypocoagulative diseases. In this article the authors present the result of several years of research carried out to obtain a correct clinical and therapeutic approach for clinical and surgical Odontostomatology. After an introduction on clinical pharmacology and the use of anticoagulants, the principal clinical cases are discussed. Various laboratory tests evaluating patients with pharmacological coagulopathy are examined. The most specific and significant tests are illustrated following up the authors experiences. In the last part the authors illustrate cases corresponding to the two serious and frequent complications that can be found in patients with iatrogenic coagulopathy: hematorrhea and thromboembolism. These matters were dealt with from an Odontostomatologic point of view. The results obtained suggest the necessity of keeping to the management that was described. Actually a low percentage of accidents occurred only when the above-mentioned clinical processes were completely performed.

[Precancerous conditions of the oral cavity. Note I. General considerations. Critical review of the literature]. / Stati cancerizzabili del cavo orale. Nota I. Considerazioni generali. Rassegna critica della letteratura.

The authors deal with the current topic of precancerous states of the oral cavity, considering various aspects such as etiopathology, diagnosis, the clinical picture and therefore, nosological classifications. The region of the Air Passages and Superior Digestive Tract (APSDT) should be considered as a single system and it is affected by a set of genetic and environmental conditions common to the various anatomical regions of which it consists. The significance of several irritating agents, which are also habitually used (e.g. tobacco), has been demonstrated for some time, whereas, the actual harmful effects of other factors such as immunodepression in particular, are only now being evaluated and detected. The discussion concerning precancerous lesions of the oral cavity, should actually refer to lesions that do not present the histologic, biological and clinical characteristics of malignant neoplasms, but that have the objective possibility of developing them. Some lesions that traditionally belong to this pathological grouping often present dysplastic aspects, if not the actual characteristics of "carcinoma in situ". Therefore, the availability of a set of indexes is of primary importance and it should be capable of providing an orientation for diagnosis and clinical practices in a precise, standardized manner. The authors hold these indexes to be divisible into three groups as follows. The first group is composed of the "parameters of cellular kinetics" and includes the percentage of cells in phase S (LI), phase S time (Ts), cell cycle duration (Tc), and the growth fraction (GF). The second group consists in the "parameters of cellular morphology", including the nuclear content in DNA, the value of the nuclear surface and the ploidy. The third group is more specific for the existence of a pathologic mass. However, given the uncertainties of the borderlines traditionally attributed to precancerous pathology, this group is definitely useful. It is composed of the potential doubling time (Tpot) and the tumor volume doubling time (Td). Moreover, biopsy is held to be an indispensable tool and the procedure should be conducted in the various manners possible according to well-defined conditions. A correct evaluation of these parameters allows for a correct approach to precancerous pathology and the prevention of the clinical risks of "rapid proliferation" even in the diagnostic biopsy phase.