The Potential of a Bioactive, Pre-reacted, Glass-Ionomer Filler Resin Composite to Inhibit the Demineralization of Enamel in Vitro.

CLINICAL RELEVANCE: A prereacted, glass-ionomer filler fluoride-containing resin composite had lower remineralization potential than glass-ionomer cements but was able to inhibit enamel demineralization; thus, it may be an option for restoring dental surfaces for patients at high risk of caries. SUMMARY: Evidence is lacking on the use of surface prereacted glass-ionomer filler resin composites to inhibit demineralization and that simulate real clinical conditions. The present laboratory study evaluated the potential of such composites to prevent demineralization and quantified fluoride (F) and other ions released from restorative materials after a dynamic pH-cycling regimen applied to the tooth material interface in vitro. The pH-cycling regimen was assessed by measuring surface hardness (SH) along with energy dispersive X-ray spectroscopy (EDX). METHODS AND MATERIALS: Ninety blocks of bovine enamel were subjected to composition analysis with EDX, and were further categorized based on SH. The blocks were randomly divided into 6 treatment groups (n=15 each): F IX (Fuji IX Extra; GC Corporation); IZ (Ion Z, FGM); F II (Fuji II LC, GC Corporation); B II (Beautifil II, Shofu); F250 (Filtek Z250 XT, 3M ESPE); and NT (control, no treatment). The blocks were subjected to a dynamic pH-cycling regimen at 37°C for 7 days concurrently with daily alternations of immersion in demineralizing/remineralizing solutions. EDX was conducted and a final SH was determined at standard distances from the restorative materials (150, 300, and 400 µm). RESULTS: The EDX findings revealed a significant increase in F concentration and a decrease in Ca2+ in the enamel blocks of group B II after the pH-cycling regimen (p<0.05). SH values for groups F IX, IZ, and F II were greater than those for groups B II, F250, and NT at all distances from the materials. CONCLUSIONS: The results suggest that each of 3 restorative materials, F IX, IZ, and F II, partially inhibited enamel demineralization under a dynamic pH-cycling regimen.

Perspective on the evolution of cell-based bone tissue engineering strategies.

Despite the compelling clinical needs in enhancing bone regeneration and the potential offered by the field of tissue engineering, the adoption of cell-based bone graft substitutes in clinical practice is limited to date. In fact, no study has yet convincingly demonstrated reproducible clinical performance of tissue-engineered implants and at least equivalent cost-effectiveness compared to the current treatment standards. Here, we propose and discuss how tissue engineering strategies could be evolved towards more efficient solutions, depicting three different experimental paradigms: (i) bioreactor-based production; (ii) intraoperative manufacturing, and (iii) developmental engineering. The described approaches reflect the need to streamline graft manufacturing processes while maintaining the potency of osteoprogenitors and recapitulating the sequence of biological steps occurring during bone development, including vascularization. The need to combine the assessment of efficacy of the different strategies with the understanding of their mechanisms of action in the target regenerative processes is highlighted. This will be crucial to identify the necessary and sufficient set of signals that need to be delivered at the injury or defect site and should thus form the basis to define release criteria for reproducibly effective engineered bone graft substitutes.

Blood exposure has a negative effect on engineered cartilage.

PURPOSE: The aim of this study was to investigate the in vitro effect of different concentrations of blood on the morphological and biochemical properties of engineered cartilage. Previous studies have demonstrated a negative effect of blood on native cartilage; however, the effect of the contact of blood on engineered cartilage is unclear. METHODS: Articular chondrocytes were isolated from swine joints, expanded in monolayer culture, and seeded onto collagen membranes. The seeded membranes were cultured for 3 days in the presence of different concentrations of peripheral blood. Some samples were retrieved at the end of the blood contact, others after 21 additional days of standard culture conditions, in order to investigate the "long-term effect" of the blood contact. RESULTS: All seeded samples showed an increase in the weight and an evident cartilage-like matrix production. A concentration-dependent reduction in the mitochondrial activity due to blood contact was shown at the earlier culture time, followed by a partial recover at the longer culture time. CONCLUSION: A blood contact of 3 days affected the chondrocytes' activity and determined a delay in the maturation of the engineered cartilage. These findings have clinical relevance, as autologous chondrocytes seeded onto biological scaffolds has become an established surgical method for articular cartilage repair. Therefore, further investigation into material sciences should be encouraged for the development of scaffold protecting the reparative cells from the blood insult.

Healing of meniscal tissue by cellular fibrin glue: an in vivo study.

Menisci represent fundamental structures for the maintenance of knee homeostasis, playing a key role in knee biomechanics. However, their intrinsic regenerative potential is poor. As a consequence, when a lesion occurs and the meniscus is partially removed by surgery, knee mechanics is subject to dramatic changes. These have been demonstrated to lead often to the development of early osteoarthritis. Therefore, menisci should be repaired whenever possible. In the last decades, tissue engineering approaches have been advocated to improve the reparative processes of joint tissues. In this study, the bonding capacity of an articular chondrocytes-fibrin glue hydrogel was tested as a biologic glue to improve the bonding between two swine meniscal slices in a nude mouse model. The composites were wrapped with acellular fibrin glue and implanted in subcutaneous pouches of nude mice for 4 weeks. Upon retrieval, a firm gross bonding was observed in the experimental samples while none of the control samples, prepared with acellular fibrin glue at the interface, presented any sign of bonding. This was consistent with the histological and scanning electron microscope findings. In particular, a fibrocartilaginous tissue was found at the interface between the meniscal slices, partially penetrating the native meniscus tissue. In order to overcome the lack of regenerative properties of the meniscus, the rationale of using cellular fibrin glue is that fibrin provides immediate stability while carrying cells in the site of lesion. Moreover, fibrin gel is recognized as an optimal scaffold for cell embedding and for promoting fibrocartilaginous differentiation of the cells which synthesize matrix having healing property. These results demonstrated the potential of this model for improving the meniscal bonding. However, further orthotopic studies in a large animal model are needed to evaluate its potential for clinical application.

Mammary-type myofibroblastoma of popliteal fossa.

Mammary-type myofibroblastoma is a very rare, benign, spindle cell lesion, arising mainly in the inguinal region. This clinical entity strictly duplicates the features of its breast counterpart. To our knowledge, this is the first report of this particular lesion occurring in the popliteal fossa. We discuss the clinical, radiological and histopathological features of this case, emphasizing the role of incisional biopsy in such an unusual neoplasia.

Prosthetic joint replacement for long bone metastases: analysis of 154 cases.

INTRODUCTION: Metastatic bone disease is the most common cause of malignancies to the skeleton in adults. The treatment of bone metastases is frequently palliative aiming to achieve a satisfactory control of pain and to prevent or to treat pathological fractures. In selected cases the resection of a single bone metastasis may improve the survival of the patients. Our experience with bone metastases located in the appendicular skeleton, between 1992 and 2004, is retrospectively reviewed here. MATERIALS AND METHODS: We report a series of 154 patients (95 females and 59 males) treated with prosthesis for metastatic bone disease. Lower limb localization was more frequent with 117 cases, while upper limb was affected in 37 cases. Metastatic breast and renal carcinoma predominated and accounted for 66% of the lesions. Indications to surgery were reported, oncologic outcome was evaluated and functional results were obtained by the Musculoskeletal Tumor Society scoring system. RESULTS: Follow up ranged from 6 months to 12 years (median 26 months). One-year survival was 69.5%, 2-years survival was 44.8%, 5-years survival was 19.5%; and 5 (3.2%) died in the early post surgical period. Functional results were good or higher in 73.8% of patients for the proximal femur, in 50% of patients for the knee and 30.6% of patients for the proximal humerus. CONCLUSION: In this series, satisfactory results were achieved with few complications. We emphasized the importance of giving the patient a definitive treatment and preventing pathological fractures as they determine disability and a spreading of the tumor in the soft tissues, leading to an increased probability of local recurrence. Prosthetic replacement contributes to an improved quality of life and limb functionality and, in selected cases; this radical surgical approach is indicated as it may improve patient's life expectancy.

Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization.

AIMS: To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. METHODS AND RESULTS: Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). CONCLUSIONS: Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. SIGNIFICANCE AND IMPACT OF THE STUDY: Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.

Modular prostheses in the treatment of proximal humerus metastases: review of 40 cases.

BACKGROUND: The humerus is the second most common site of metastatic bone disease involving long bones. Tumors which have a predilection for dissemination to bone are those of breast, prostate, thyroid, lung and kidney. The rationale for surgical treatment of these lesions is to prevent or treat pathological fractures in order to relieve pain and improve function. MATERIALS AND METHODS: Forty patients who had resection of the proximal humerus for metastatic bone disease and reconstruction with a modular prosthesis were retrospectively reviewed. RESULTS: Mean functional outcome was 73.1% (Enneking score) and better results were achieved when a reverse prosthesis was implanted. Overall survival was 70% at 1 year, 42.5% at 2 years and 20% at 5 years. Local recurrence occurred in 4 patients, each of whom had initially been treated for a pathological fracture. CONCLUSIONS: It is important to follow rational guidelines, like those of Capanna and Mirels, in order to prevent pathological fractures and to give the patient a definitive treatment, as the advances in the management of cancer prolong the survival of these patients. In this series, satisfactory results were obtained, giving the patients an acceptable quality of life.

A tissue engineered osteochondral plug: an in vitro morphological evaluation.

Articular cartilage lesions have a poor intrinsic healing potential. The repair tissue is often fibrous, having insufficient biomechanical properties, which could frequently lead to the development of early osteoarthritis. In the last decade, tissue engineering approaches addressed this topic in order to restore joint function with a differentiated and functional tissue. Many biomaterials and techniques have been proposed and some of them applied in clinical practice, even though several concerns have been raised on the quality of the engineered tissue and on its integration in the host joint. In this study, we focused on engineering in vitro a biphasic composite made of cellular fibrin glue and a calcium-phosphate scaffold. Biphasic composites are the latest products of tissue engineering applied to articular cartilage and they seem to allow a more efficient integration of the engineered tissue with the host. However, a firm in vitro bonding between the two components of the composite is a necessary condition to validate this model. Our study demonstrated a gross and microscopic integration of the two components and a cartilage-like quality of the newly formed matrix. Moreover, we noticed an improvement of this integration and GAGs production during the in vitro culture.

Effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage.

The use of autologous chondrocytes seeded onto a biological scaffold represents a current valid tool for cartilage repair. However, the effect of the contact of blood to the engineered construct is unknown. The aim of this work was to investigate in vitro the effect of blood on the morphological, biochemical and biomechanical properties of engineered cartilage. Articular chondrocytes were enzymatically isolated from swine joints, expanded in monolayer culture and seeded onto collagen membranes for 2 weeks. Then, the seeded membranes were placed for 3 days in contact with peripheral blood, which was obtained from animals of the same species and diluted with a standard medium. As controls, some samples were left in the standard medium. After the 3 days' contact, some samples were retrieved for analysis; others were returned to standard culture conditions for 21 additional days, in order to investigate the "long-term effect" of the blood contact. Upon retrieval, all seeded samples showed increasing sizes and weights over time. However, the samples exposed to blood presented lower values with respect to the controls. Biochemical evaluation demonstrated a reduction in the mitochondrial activity due to blood contact at the early culture time (3 days post blood contact), followed by a partial recovery at the longer culture time (21 days post blood contact). Histological evaluation demonstrated evident cartilage-like matrix production for both groups. Biomechanical data showed a reduction of the values, followed by stabilization, regardless of the presence of blood. Based on the data obtained in this study, we can conclude that blood contact affects the chondrocyte activity and determines a delay in the dimensional growth of the engineered cartilage; however, at the experimental times utilized in this study, this delay did not affect the histological pattern and the biomechanical properties of the construct.

Preliminary observations on haemodynamics during physiological stress conditions following 'double-orifice' mitral valve repair.

OBJECTIVE: The 'double-orifice' (DO) technique has been proposed to simplify mitral valve repair (MVRep) in particular settings of mitral insufficiency. However, the haemodynamic effects of such a redesigned valve are poorly documented, particularly during stress conditions. Thus, we sought to evaluate the haemodynamic changes during exercise conditions after the DO procedure. METHODS: Twenty-seven selected patients were enrolled for this study. Mean age was 60 +/- 14 years (range 31--80 years). All patients had had severe mitral regurgitation and normal LV function preoperatively, and had undergone DO as isolated procedure for MVRep. Annular remodelling was associated in 24 cases (Carpentier classic ring in 13 patients and autologous pericardium in 11 patients, respectively). Postoperatively, haemodynamic data were recorded at baseline and during supine exercise test at submaximal workloads by means of transthoracic echocardiography. A logistic regression analysis was applied to evaluate the association between the observed haemodynamic changes and surgical technique. RESULTS: Mean follow-up was 47 +/- 20 months. Significant residual mitral insufficiency (grade three over four) was found in five patients at baseline assessment, and in six patients at peak exercise. Transmitral pressure gradient showed a significant (P < 0.001) increase in both peak and mean values at peak exercise (from 7 +/- 4 to 17 +/- 10 and from 3 +/- 2 to 8 +/- 6 mmHg, respectively). Pulmonary hypertension was observed in six patients (moderate in all cases) at rest and in 13 patients (moderate in seven cases and severe in five cases) at peak exercise. We did not find any correlation between the haemodynamic data and surgical factors. CONCLUSIONS: This study indicates that though effective mitral valve competence is achieved in the majority of operated patients, DO repair may induce impaired diastolic mitral dynamism in some cases, particularly during exercise conditions. Further investigations are required to thoroughly elucidate the overall mechanics of a DO valve, especially at strenuous cardiocirculatory states.

Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils.

We analyzed the genetic diversity of 531 Sinorhizobium meliloti strains isolated from nodules of Medicago sativa cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of S. meliloti. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.

Production of endogenous nitric oxide in chronic obstructive pulmonary disease and patients with cor pulmonale. Correlates with echo-Doppler assessment.

Exhaled nitric oxide (NO) production in stable chronic obstructive pulmonary disease (COPD) has been loosely related to the severity of illness, being significantly reduced in the most severe cases. Pulmonary hypertension is associated with lower NO output from the lung. In this study expired NO was measured in patients with severe stable COPD with or without cor pulmonale (CP). Echocardiographic estimates of right heart function, lung function, diffusion capacity, respiratory muscle strength, and arterial blood gases were obtained in 34 consecutive patients with stable COPD (mean age, 68 +/- 7 yr). Expired NO was measured by chemiluminiscence to obtain fractional exhaled concentrations at peak (FENOp) and at plateau (FENOpl) points of the single-breath curve and resting NO output (V NO). All measurements of expired NO output, FENOp, FENOpl and V NO showed a negative correlation with both systolic pulmonary artery pressure (Pspa) (r = -0.51, -0.63, and -0.63, respectively, p < 0.01 for all) and right ventricle wall dimension (r = -0.41, -0.59, and -0.43, respectively, p < 0.05 for all), but not with any measurement of lung function. When the patients were divided according to the Pspa using a cutoff limit of 35 mm Hg, those subjects with CP showed lower FENOp (13.2 +/- 4.0 versus 36.7 +/- 30.8 ppb, p < 0.05), FENOpl (5.7 +/- 1.9 versus 8.9 +/- 4.7 ppb, p < 0.05), and V NO (69. 2 +/- 5.6 versus 107.6 +/- 14.6 nl/ min, p = 0.02) than did those with a normal resting Pspa. NO production from the airways was significantly lower and inversely related to development of CP in patients with severe COPD. Impaired endothelial release may account for the reduced levels of expired NO.

The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

Biological versus prosthetic ring in mitral-valve repair: enhancement of mitral annulus dynamics and left-ventricular function with pericardial annuloplasty at long term.

OBJECTIVE: The effects of different annuloplasty rings on mitral annulus dynamics and left-ventricular (LV) function after mitral-valve repair (MVR) are still controversial. This study sought to compare biological versus prosthetic rigid rings for annular remodelling in MVR at long term. METHODS: Forty-four consecutive patients were retrospectively enrolled. All patients had isolated posterior-leaflet prolapse and underwent identical surgical mitral-valve reconstruction (quadrangular resection of the posterior leaflet associated with annuloplasty). Twenty-three patients underwent mitral annuloplasty with an autologous pericardial ring (group I), whereas 21 patients had MVR with a Carpentier-Edwards rigid ring (group II). No differences existed between the groups in terms of pre-operative patient profile. Post-operative LV systolic indices have been assessed by two-dimensional echocardiography at rest and during supine bicycle exercise. Mitral annular motion has been examined by means of the extent of mitral annulus systolic excursion (MASE), as measured in four longitudinal LV segments (anterior, inferior, septal and lateral). Mean and peak trans-mitral flow velocities (TMFV) have been also evaluated by continuous-wave Doppler. RESULTS: The mean follow-up did not differ between the groups, those being 41+/-12 months in group I (range17-65 months) and 46+/-15 months in group II (range 23-83 months), respectively. Post-operative echocardiographic study did not show significant mitral regurgitation at rest or at peak exercise in any patient. ANOVA analysis for repeated measures showed a significant interaction in peak TMFV (F((1,42))=5.23; P=0.03), and in left-ventricular ejection fraction (LVEF; F((1,42))=7.61, P=0.01). The analysis of contrasts showed a significant increase in TMFV in both groups (group I from 1.22+/-0.22 to 1.79+/-0.32 m/s, t=-8.8, P<0.0001; and group II from 1.19+/-0.17 to 1.96+/-0.33 m/s, t=-12.8, P<0.0001). Recruitment of LVEF reserve during exercise was observed only in group I (from 59.5+/-6 to 65.8+/-6%, t=-3.95, P<0.005), whereas no substantial change occurred in LV performance in group II. A trend towards better MASE at all the studied longitudinal segments at rest and during exercise was observed in group I. No minor or major calcifications have been observed on pericardial rings. CONCLUSIONS: The autologous pericardium seems to be superior to rigid prosthetic rings for annuloplasty in MVR since it provides more favourable mitral annulus dynamics and preserves LV function during stress conditions. Effective and durable annular remodelling with the autologous pericardium is achieved up to 6 years from surgery, with no echocardiographic sign of degeneration in the long term. Further studies are required to compare biological versus flexible prosthetic rings in MVR.

Zidovudine-induced experimental myopathy: dual mechanism of mitochondrial damage.

Myopathy often complicates Zidovudine (AZT) treatment in patients with acquired immunodeficiency syndrome (AIDS). The pathogenesis of the myopathy is controversial, since clinical phenomena intrinsic to AIDS may interfere per se with the onset of the myopathy. In the present work we investigated the in vivo effect of AZT in an animal model species (rat) not susceptible to HIV infection. Histochemical and electron microscopic analyses demonstrated that, under the experimental conditions used, the in vivo treatment with AZT does not cause in skeletal muscle true dystrophic lesions, but rather mitochondrial alterations confined to the fast fibers. In the same animal models, the biochemical analysis confirmed that mitochondria are the target of AZT toxicity in muscles. The effects of AZT on mitochondria energy transducing mechanisms were investigated in isolated mitochondria both in vivo and in vitro. Membrane potential abnormalities, due to a partial impairment of the respiratory chain capability observed in muscle mitochondria from AZT-treated rats, closely resemble those of control mitochondria in the presence of externally added AZT. mtDNA deletion analysis by PCR amplification and Southern blot analysis did not show any relevant deletion, while mtDNA depletion analysis demonstrated a significant decrease in mtDNA in AZT-treated rats. The present findings show that AZT causes damage to mitochondria by two mechanisms: a short-term mechanism that affects directly the respiratory chain, and a long-term mechanism that alters the mitochondrial DNA thus impairing the mitochondrial protein synthesis. In addition, the ultrastructural observations indicate that the fiber types are differently affected upon AZT treatment, which poses a number of questions as to the pathogenesis of this myopathy.

Influence of plant genotype on the selection of nodulating Sinorhizobium meliloti strains by Medicago sativa.

We analysed the genetic diversity of 270 Sinorhizobium meliloti strains isolated from nodules of three different Medicago sativa varieties, planted in three different Italian soils, combining the Analysis of Molecular Variance (AMOVA) with the Random Amplified Polymorphic DNA (RAPD) technique to estimate variance among RAPD patterns with the aim to draw an objective description of the population genetic structure. Results indicated that a general intraspecific genetic diversity was globally distributed among all the population, however a very high level of diversity was found among strains nodulating different Medicago sativa varieties. Moreover the distribution of the RAPD haplotypes among the plant varieties also showed to be non-random. The overall data indicated that the plant genotype is a major factor in shaping the genetic structure of this natural Rhizobium population.

Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnmT genes involved in doxorubicin (adriamycin) biosynthesis.

Sequence analysis of a 3.4-kb region Streptomyces peucetius daunorubicin (DNR) gene cluster established the presence of the dnrH and dnmT genes. In dnrH mutants, DNR production increased 8.5-fold, compared with that in the wild-type strain, while dnmT mutants accumulated epsilon-rhodomycinone (RHO), which normally becomes glycosylated in daunorubicin biosynthesis. Hence, dnmT may be involved in the biosynthesis or attachment of daunosamine to RHO or in the regulation of this process. Since the DnrH protein is similar to known glycosyl transferases, this protein may catalyze the conversion of DNR to its polyglycosylated forms, known as baumycins. Overexpression of dnmT in the wild-type and dnrH mutant strains resulted in a major decrease in RHO accumulation and increase in DNR production.

The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae.

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.

Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.

We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym.