Breast lesion sizing by B-mode imaging and sonoelastography in comparison to histopathological sizing--a prospective study.

PURPOSE: This prospective study evaluates whether sonoelastography can improve B-mode ultrasonographic sizing of breast tumors. Precise measuring is important for effective therapy planning for breast cancer patients. MATERIALS AND METHODS: The size of 100 surgically excised breast lesions (92 patients: 77 malignant, 23 benign) was compared to preoperative measurements. Lesions were imaged with both ultrasonographic techniques in identical planes. The largest sizes measured with each modality were compared to the largest histopathological measurements. The interobserver variability was also computed from measurements made by two examiners assessing identical planes. RESULTS: Both ultrasonographic measuring techniques underestimate lesion size. The sonoelastography measurements were within ± 5 mm of the histological size in 70.1 % of malignant lesions, and the B-mode measurements in 57.1 % of cases. Sonoelastography leads to more accurate measurements of 13.0 % of cases (statistically not significant). A total of 22 lesions were also imaged by a second examiner. Sonoelastography had 27.3 % less interobserver variability (examiners agreed in 36.4 % of sonoelastography and in 9.1 % of B-mode results). CONCLUSION: In this study there is no significant advantage of sonoelastography, although a tendency is apparent. The low interobserver variability also favors sonoelastography for preoperative diagnostics, since it may be less dependent on the observer than conventional B-mode imaging. The results of this prospective study require validation in a prospective multicenter study with larger case numbers.

Sonoelastography in the diagnosis of malignant and benign breast lesions: initial clinical experiences.

PURPOSE: This prospective study aimed to compare sonoelastography, B-mode ultrasonography, and mammography in terms of their ability to distinguish benign from malignant breast lesions. We also assessed how the diagnostic value of sonoelastography differs between palpable and clinically occult lesions. MATERIALS AND METHODS: Evaluation revealed a total of 97 lesions (66 benign; 31 malignant) without histological confirmation at the time of the initial examination. The sensitivity, specificity, positive (PPV) and negative predictive value (NPV) as well as efficiency were calculated. These parameters were separately assessed for palpable lesions and for non-palpable lesions. We subsequently compared these results. RESULTS: Sonography had a sensitivity of 97% and a specificity of 82% (PPV: 71 %, NPV: 98%, efficiency: 87%). For mammography, the respective figures were 84% and 89% (PPV: 79%, NPV: 92%, efficiency: 88%). Sonoelastography had a sensitivity of 71% and a specificity of 48% (PPV: 39%, NPV: 78%, efficiency: 56%). The combination of sonography and sonoelastography yielded a sensitivity of 100% and a specificity of 38% (PPV: 43%, NPV: 100%, efficiency: 58%). The sensitivity and specificity were not statistically different between the groups of palpable and non-palpable lesions. CONCLUSION: Sonoelastography is easily performed and not very time-consuming. Used by itself, the method is not more efficacious than alternative techniques. When used in conjunction with B-mode ultrasonography, the latter's sensitivity was increased, albeit at the expense of specificity.

Tree-based modeling of complex interactions of phosphorus loadings and environmental factors.

Phosphorus (P) enrichment has been observed in the historic oligotrophic Greater Everglades in Florida mainly due to P influx from upstream, agriculturally dominated, low relief drainage basins of the Everglades Agricultural Area (EAA). Our specific objectives were to: (1) investigate relationships between various environmental factors and P loads in 10 farm basins within the EAA, (2) identify those environmental factors that impart major effects on P loads using three different tree-based modeling approaches, and (3) evaluate predictive models to assess P loads. We assembled thirteen environmental variable sets for all 10 sub-basins characterizing water level management, cropping practices, soils, hydrology, and farm-specific properties. Drainage flow and P concentrations were measured at each sub-basin outlet from 1992-2002 and aggregated to derive monthly P loads. We used three different tree-based models including single regression trees (ST), committee trees in Bagging (CTb) and ARCing (CTa) modes and ten-fold cross-validation to test prediction performances. The monthly P loads (MPL) during the monitoring period showed a maximum of 2528 kg (mean: 103 kg) and maximum monthly unit area P loads (UAL) of 4.88 kg P ha(-1) (mean: 0.16 kg P ha(-1)). Our results suggest that hydrologic/water management properties are the major controlling variables to predict MPL and UAL in the EAA. Tree-based modeling was successful in identifying relationships between P loads and environmental predictor variables on 10 farms in the EAA indicated by high R(2) (>0.80) and low prediction errors. Committee trees in ARCing mode generated the best performing models to predict P loads and P loads per unit area. Tree-based models had the ability to analyze complex, non-linear relationships between P loads and multiple variables describing hydrologic/water management, cropping practices, soil and farm-specific properties within the EAA.

The retinoblastoma gene product RB stimulates Sp1-mediated transcription by liberating Sp1 from a negative regulator.

Studies have demonstrated that the retinoblastoma susceptibility gene product, RB, can either positively or negatively regulate expression of several genes through cis-acting elements in a cell-type-dependent manner. The nucleotide sequence of the retinoblastoma control element (RCE) motif, GCCACC or CCACCC, and the Sp1 consensus binding sequence, CCGCCC, can confer equal responsiveness to RB. Here, we report that RB activates transcription of the c-jun gene through the Sp1-binding site within the c-jun promoter. Preincubation of crude nuclear extracts with monoclonal antibodies to RB results in reduction of Sp1 complexes in a mobility shift assay, while addition of recombinant RB in mobility shift assay mixtures with CCL64 cell extracts leads to an enhancement of DNA-binding activity of SP1. These results suggest that RB is directly or indirectly involved in Sp1-DNA binding activity. A mechanism by which RB regulates transactivation is indicated by our detection of a heat-labile and protease-sensitive Sp1 negative regulator(s) (Sp1-I) that specifically inhibits Sp1 binding to a c-jun Sp1 site. This inhibition is reversed by addition of recombinant RB proteins, suggesting that RB stimulates Sp1-mediated transactivation by liberating Sp1 from Sp1-I. Additional evidence for Sp1-I involvement in Sp1-mediated transactivation was demonstrated by cotransfection of RB, GAL4-Sp1, and a GAL4-responsive template into CV-1 cells. Finally, we have identified Sp1-I, a approximately 20-kDa protein(s) that inhibits the Sp1 complexes from binding to DNA and that is also an RB-associated protein. These findings provide evidence for a functional link between two distinct classes of oncoproteins, RB and c-Jun, that are involved in the control of cell growth, and also define a novel mechanism for the regulation of c-jun expression.

Non-palpable breast lesions in asymptomatic women: diagnostic value of initial ultrasonography and comparison with mammography.

AIM: This prospective double-blind study was designed to assess (i) if primary breast screening by ultrasonography is capable of detecting breast cancer independent of tissue density and (ii) if the rate of unnecessary biopsies remains acceptable when diagnostics are based on ultrasonography. PATIENTS AND METHODS: Bilateral breast ultrasonography was performed in 448 asymptomatic women as the initial diagnostic method. Sonograms were interpreted using a set of standardized diagnostic criteria. Subsequently, mammograms were obtained. The radiologists reading the mammograms were blinded to the sonographic results. RESULTS: Overall, 3 non-palpable breast cancers were detected by ultrasound and mammography. All 3 ultrasonographically detected breast cancers were smaller than 1 cm (0.7, 0.7, 0.6 cm). All 3 carcinomas were correctly detected by both methods. For ultrasonography, the false positive rate was 1.1% (n=5) and for mammography 0.6% (n=3). When both methods were combined, the rate of unnecessary open biopsies was 1.6% (n=7). The ratio of benign to malignant lesions was 3.7/1. CONCLUSION: Without prior mammography, primary high-resolution breast ultrasonography is capable of detecting non-palpable breast carcinomas in asymptomatic women at an early stage. The rate of unnecessary open biopsies is low and the ratio of benign to malignant biopsies acceptable.

Assessment of the spatial distribution of soil properties in a northern Everglades marsh.

Florida Everglades restoration plans are aimed at maintaining and restoring characteristic landscape features such as soil, vegetation, and hydrologic patterns. This study presents the results from an exhaustive spatial sampling of key soil properties in Water Conservation Area 1 (WCA 1), which is part of the northern Everglades. Three soil strata were sampled: floc, upper 0- to 10-cm soil layer, and 10- to 20-cm soil layer. A variety of properties were measured including bulk density (BD), loss on ignition (LOI), total phosphorus (TP), total inorganic phosphorus (TIP), total nitrogen (TN), total carbon (TC), total iron (TFe), total magnesium (TMg), total aluminum (TAl), and total calcium (TCa). Interpolated maps and model prediction uncertainties of properties were generated using geostatistical methods. We found that the uncertainty associated with spatial predictions of floc, particularly floc BD, was highest, whereas spatial predictions of soil chemical properties such as soil Ca were more accurate. The resultant spatial patterns for these soil properties identified three predominant features in WCA 1: (i) a north to south gradient in soil properties associated with the predominant hydrological gradient, (ii) areas of considerable soil nutrient enrichment along the western canal of WCA 1, and (iii) areas of considerable Fe enrichment along the eastern canal. By using geostatistical techniques we were able to describe the spatial dynamics of soil variables and express these predictions with an acceptable level of uncertainty.

Spatial patterns of labile forms of phosphorus in a subtropical wetland.

Phosphorus (P) has been identified as the key constituent defining wetland productivity, structure, and function. Our goal was to investigate the spatial patterns of total P and three labile forms of P (labile organic, inorganic, and microbial biomass P) across a subtropical wetland located in east-central Florida, the Blue Cypress Marsh Conservation Area (BCMCA), and link spatial patterns to ecosystem processes. The wetland received a continual input of nutrients primarily from the south and intermittently from the west and east, respectively, which ceased in the mid-1990s. Since then the marsh system has been undergoing natural succession. We used (i) ordinary kriging to characterize the spatial patterns of total P and labile P forms across the wetland, (ii) local, moving spatial correlations to investigate relationships between total P and labile P forms, and (iii) a clustering technique to link the identified spatial patterns to biogeochemical processes. The spatially explicit analyses revealed patterns of total P and labile P forms as well as changing relationships between variables across the marsh. We were able to distinguish P-enriched areas from unaffected ("natural") areas and intermediate zones that are currently undergoing change as P is mobilized and translocated. We also identified areas that are at risk, showing a shift toward a more P-enriched status. Our results improve our understanding of P and its labile components within a spatially explicit context.

Reduced methyl group acceptance of 1-beta-D-arabinofuranosylcytosine-containing DNA polymers.

Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.

Isolation and characterization of DNA cytosine 5-methyltransferase from human placenta.

DNA cytosine 5-methyltransferase has been extensively purified (about 2600-fold) from the soft tissue of human placenta by chromatography on DEAE-cellulose and hydroxyapatite, and by an affinity step on agarose-immobilized S-adenosylhomocysteine. The isolated enzyme has a molecular weight of 135,000 and methylates DNA from various sources in native and heat-denatured forms. The synthetic copolymer poly(dG-dC) . poly(dG-dC) is methylated in B- and Z-conformation to about the same extent. DNA containing hemimethylated sites was isolated from P815 cells grown in the presence of 5-azacytidine. This P815 DNA was used to measure the "maintenance' DNA methylase activity, whereas 5-methylcytosine-free procaryotic DNA served as a substrate for the "de novo' DNA methylase activity in our enzyme preparation. The crude extract as well as the highly purified DNA methylase are capable of transferring methyl groups to these two types of substrate. The fact that both types of activity co-chromatograph during the isolation procedure suggests that one enzyme molecule may exercise both the "maintenance' and "de novo' activity.

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA.

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200-300 micrograms/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-L-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT), but not poly(dI-dC).poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the 'de novo' activity of the enzyme.

Retinal hemodynamics in proliferative diabetic retinopathy. A laser Doppler velocimetry study.

PURPOSE: This study investigated retinal hemodynamic changes associated with different pathologic features observed on fundus color and fluorescein angiography in patients with proliferative diabetic retinopathy. METHODS: Retinal circulatory characteristics were investigated in 25 eyes of 23 diabetic patients with proliferative retinopathy using a combination of bidirectional laser Doppler velocimetry and monochromatic fundus photography. RESULTS: Eyes with severe capillary nonperfusion had 32% less average volumetric blood flow rate (Q) than eyes with less severe nonperfusion (P = 0.0005). In addition, eyes with severe vessel staining with fluorescein had 20% less average Q than eyes without staining (P = 0.0508). Eyes with severe fluorescein leakage in the macula had a 17% larger total venous cross-section than eyes with milder leakage (P = 0.027). Eyes with clinically significant macular edema had 11% larger average venous diameter than eyes without this feature (P = 0.0085). CONCLUSIONS: Severe capillary nonperfusion and vessel staining with fluorescein are associated with decreased retinal blood flow rates. Vasodilatation may be an important factor for increased vascular permeability and macular edema in proliferative diabetic retinopathy.

Strict control of glycaemia: effects on blood flow in the large retinal vessels and in the macular microcirculation.

AIMS: The purpose of this study was to investigate the effect of instituting strict diabetic glycaemic control on the retinal macular microcirculation and to compare this effect with that observed in the main retinal veins. METHODS: In 28 insulin dependent diabetic patients with poor glycaemic control a regimen of strict diabetic control, consisting of four daily insulin injections was instituted and maintained for 6 months. Retinal haemodynamics were investigated in the macular microcirculation by the blue field simulation technique and in the major retinal veins by a combination of bidirectional laser Doppler velocimetry and monochromatic fundus photography. Progression of diabetic retinopathy was assessed from fundus photographs taken at baseline and at the end of the study. RESULTS: Institution of strict diabetic control resulted in a significant increase in leucocyte velocity in the macular circulation (p = 0.013). No significant difference in this increase was observed between eyes that showed progression (n = 8) and no progression (n = 20) of retinopathy during the study. Significant correlations were found between relative changes over time of blood flow measured in the main retinal veins and relative changes of leucocyte velocity determined in the macular microcirculation at 2 months (p = 0.008) and 6 months (p = 0.001) but not at 5 days (p = 0.49). In the eight eyes that showed progression of retinopathy, the product of leucocyte velocity and density at baseline was significantly higher than normal (p < 0.05). During the length of this study, this product was also significantly higher in the eight eyes that showed retinopathy progression than in the 20 eyes that did not show progression (p = 0.005). CONCLUSION: Our results suggest that increased flow in the macular microcirculation may be associated with progression of retinopathy, thus supporting the hypothesis that increased blood flow may play a role in the development of diabetic microangiopathy. Although there are correlations between the changes detected in the macular microcirculation and those measured in the main retinal vessels, there are also differences which need to be further investigated in order to better understand pathogenetic mechanisms.

Microbial associations in gut systems of wood- and bark-inhabiting longhorned beetles [Coleoptera: Cerambycidae].

Using fluorescence in situ hybridization (FISH) techniques and PCR-based rDNA sequencing, gut microflora in the larvae of bark- and wood-inhabiting cerambycid beetles (Rhagium inquisitor, Tetropium castaneum, Plagionotus arcuatus and Leptura rubra [Coleoptera: Cerambycidae]) was investigated. A total of 12 novel ascomycetous yeast strains were isolated from the gut content. Panfungal and strain-specific oligonucleotide probes identified two yeast strains as Candida rhagii and Candida shehatae, which were colonizing specialized organs (mycetomes) adhering to the gut of R. inquisitor and L. rubra larvae, respectively. Fragments containing these organisms were constantly being released from the mycetomes into the gut lumen. Whereas the mycetome symbiont of T. castaneum could not be identified, all larvae of this species harbored an additional bacterial endocytobiont in their gut epithelium. This novel gammaproteobacterium belonged to the Sodalis clade of insect symbionts, which includes the secondary endosymbiont of tsetse flies (Sodalis glossinidius) and the Sitophilus oryzae primary endosymbiont (SOPE). Extracellular gut flora of the investigated cerambycid larvae was comprised of Alpha-, Beta-, and Gammaproteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia and Acidobacteria. However, the individual composition among investigated larvae was highly variable and supposedly depended on individual host nutrition.

Radial scar/complex sclerosing lesion of the breast--value of ultrasound.

PURPOSE: Although benign, radial scar/complex sclerosing adenosis is a lesion which histopathologically resembles tubular carcinoma. On physical examination, it is difficult to distinguish radial scar from a malignant tumour. Mammography cannot differentiate radial scar from malignancy. This clinical study aims to delineate the role of preoperative ultrasonography with emphasis on the question whether ultrasonography could lower the number of false-positive readings and therefore the number of open biopsies required. MATERIALS AND METHODS: In this examination, we present the clinical, mammographic, ultrasonographic, and histopathological features of 6 cases of radial scars. RESULTS: Although most authors describe radial scars as non-palpable, 2 of 6 lesions were indeed palpable. On mammograms, radial scars have a spiculated appearance, a feature observed in all of our cases. Numerous ultrasonographic characteristics are listed in the literature, but ultrasonography is not reported to have clear-cut advantages. CONCLUSION: Although this study did not elucidate any unique ultrasonographic features to characterise these lesions, the analysis of all ultrasonographic results made us recognise a set of "nearly specific ultrasonographic features" of radial scars. Current B-mode imaging does not appear to lead to the desirable reduction of the rate of unnecessary open biopsies.

Ultrasound and mammography guided wire marking of non-palpable breast lesions: analysis of 741 cases.

PURPOSE: Aim of the study were to evaluate the success of ultrasound and mammography guided wire marking of non-palpable breast lesions and the results of specimen mammography/ultrasonography, completeness of resection, and number of secondary resections (during the initial surgical session and as a separate intervention) were analysed. MATERIALS AND METHODS: Between May 1994 and December 2004, 668 women with 741 non-palpable breast lesions underwent surgery at the Greifswald University Department of Gynaecology and Obstetrics. Ultrasound directed wire marking was used in 418, mammography directed marking in 284 cases. In 39 lesions, both techniques were combined. RESULTS: Out of all lesions approached with ultrasound directed wire marking, 88 (21.1 %) were malignant. Among lesions marked during mammography, 52 (19.3 %) were malignant. Specimen ultrasonography indicated that 90.9 % of lesions were resected completely. Specimen mammography demonstrated complete resection in 89.1 %. On histological examination, 19.5 % of the malignant lesions marked with sonographic guiding and 36.5 % of the malignant lesions marked with mammographic guiding did not have clear margins. Secondary resections (during the first procedure) for incomplete specimens were needed in 10 patients in whom sonographic localisation had been used, and in 25 patients in whom mammographic localisation had been employed. A second surgical session for secondary resection was required in 5.5 % of lesions marked with ultrasound and in 12.3 % of lesions marked with mammography guidance. CONCLUSION: Sonography directed wire localisation appears to be superior to the respective mammographic method. Ultrasound guided wire marking should be considered the preferred method for all mammographic lesions with an ultrasonographic equivalent and no micro-calcifications.

Parenchymal leiomyoma of the breast--clinical, sonographic, mammographic and histological features.

BACKGROUND: Intraparenchymal leiomyomas of the breast are quite rare. Areolar lesions are distinguished from intraparenchymal leiomyomas, which are less frequent. Clinically, leiomyomas appear as nodules; mammographically, they show up as round lesions. Reports on sonographic criteria are rare, and the criteria are nonspecific. Based on our case of an intraparenchymal leiomyoma, we describe additional sonographic features. The clinical, mammographic and sonographic characteristics of an intraparenchymal leiomyoma of the breast were evaluated. After surgery, the diagnosis was confirmed histologically. RESULTS: The clinical presentation of our patient with deep-seated leiomyoma of the breast included skin dimpling and a reduction in tissue mobility, differing from more commonly reported characteristics. Mammographically, the lesion was dense and only partly demarcated clearly, corresponding to other reports. On breast ultrasonography, the leiomyoma appeared as a hypodense, well demarcated, inhomogeneous lesion with posterior acoustic shadowing. A central tumour vessel was visible on Doppler imaging, and Cooper's ligaments were discontinuous. Acoustic shadowing, the hypodense character, hyperechoic border and the central tumour vessel are therefore additional ultrasonographic characteristics of an intraparenchymal leiomyoma of the breast. This type of lesion is usually described as isodense to hyperdense and homogeneous, possibly containing semicystic components. Previous reports have only described posterior acoustic enhancement, but not acoustic shadowing. CONCLUSION: On breast ultrasonography, an intraparenchymal leiomyoma of the breast can present with posterior acoustic shadowing, hypodense echogenicity, a hyperechoic border and a central tumour vessel. Neither imaging studies nor palpation allow distinction between benign and malignant lesions.

Real-time elastography--an advanced method of ultrasound: First results in 108 patients with breast lesions.

OBJECTIVES: To evaluate whether real-time elastography, a new, non-invasive method for the diagnosis of breast cancer, improves the differentiation and characterization of benign and malignant breast lesions. METHODS: Real-time elastography was carried out in 108 potential breast tumor patients with cytologically or histologically confirmed focal breast lesions (59 benign, 49 malignant; median age, 53.9 years; range, 16-84 years). Tumor and healthy tissue were differentiated by measurement of elasticity based on the correlation between tissue properties and elasticity modulus. Evaluation was performed using the three-dimensional (3D) finite element method, in which the information is color-coded and superimposed on the B-mode ultrasound image. A second observer evaluated the elastography images, in order to improve the objectivity of the method. The results of B-mode scan and elastography were compared with those of histology and previous sonographic findings. Sensitivities and specificities were calculated, taking histology as the gold standard. RESULTS: B-mode ultrasound had a sensitivity of 91.8% and a specificity of 78%, compared with sensitivities of 77.6% and 79.6% and specificities of 91.5% and 84.7%, respectively, for the two observers evaluating elastography. Agreement between B-mode ultrasound and elastography was good, yielding a weighted kappa of 0.67. CONCLUSIONS: Our initial clinical results suggest that real-time elastography improves the specificity of breast lesion diagnosis and is a promising new approach for the diagnosis of breast cancer. Elastography provides additional information for differentiating malignant BI-RADS (breast imaging reporting and data system) category IV lesions.

DNA-cytosine-5-methyltransferase from P815 mouse mastocytoma cells: "maintenance" and "de novo" activities are carried out by the same enzyme molecule.

DNA-5-methyltransferase has been purified (about 1400-fold) from rapidly proliferating mouse P815 mastocytoma cells by chromatographies on DEAE cellulose, hydroxyapatite and a heparine-agarose affinity step. The isolated enzyme has an isoelectric point of 7.3 and in neutral 10-30% glycerol gradient it bands in an area corresponding to molecular weight of 135,000 dalton. During the enzymatic reaction, the enzyme first interacts with DNA and then accomplishes a series of methyl group transfers without being detached. The formation of the initial DNA-enzyme complexes is probably random and independent of the cofactor, S-adenosyl-L-methionine, as well as the sequences recognized as methylation sites. The "maintenance" and "de novo" types of activity have been monitored using hemimethylated and completely unmethylated DNA as methyl group accepting polymers. Both these activities copurify in three different chromatographic procedures. This, together with the fact that the enzyme purified near to homogeneity possesses both types of activities suggests that "de novo" and "maintenance" DNA methyltransferase activities are exercised by the same enzyme molecule.

NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum.

Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state.