Quantitative neutron capture radiography for studying the biodistribution of tumor-seeking boron-containing compounds.

Biodistribution of two compounds presently considered for use in neutron capture therapy has been studied in mice carrying a transplantable Harding-Passey melanoma. A method is described by which quantitative assessment can be made of the boron distribution in whole-body sections of such animals. An alpha-particle-sensitive film is placed in close contact with a freeze-dried section of an animal and exposed to neutrons. The tracks visible after etching are analyzed optoelectronically in fields of 0.6 X 0.6 mm2 and compared to standards of boron homogeneously distributed in liver homogenates. The dynamic range of this method is about two orders of magnitude in concentration, with a lower detection limit of 0.1 to 0.01 ppm 10B, depending on the rate of induction of spurious tracks by fast neutrons present in the neutron beam chosen. In a transplantable Harding-Passey melanoma in mice, it was found that the sulfhydryl boron hydride Na2B12H11SH presently used for therapy of glioblastoma clears blood, muscle, and brain very rapidly. Its accumulation in tumors was persistent for more than three days. A higher tumor accumulation was observed with its disulfide, which has been suggested for neutron capture therapy. For both compounds, a marked heterogeneity of boron distribution within one tumor was found.

Implications for in vitro studies of the autoxidation of ferrous ion and the iron-catalyzed autoxidation of dithiothreitol.

The influences of buffers and iron chelators on the rate of autoxidation of Fe2+ were examined in the pH range 6.0-7.4. The catalysis by Fe2+ and Fe3+ of the autoxidation of dithiothreitol was also investigated. In buffers which are non- or poor chelators of iron, 0.25 mM Fe2+, and 0.3 mM dithiothreitol when present with iron, oxidize within minutes at pH 7.4 and 30 degrees C. The stability of each increases as the pH is decreased and more than 90% of each remains after 1 h at pH 6.0. In the presence of buffers or oxy-ligands which preferentially and strongly chelate Fe3+ over Fe2+, Fe2+ autoxidizes rapidly in the pH range 6.0-7.4 while dithiothreitol is protected. Ligands which preferentially bind strongly to Fe2+ stabilize both Fe2+ and dithiothreitol at pH 7.4. Dithiothreitol readily reduces Fe3+ in non-chelating buffers or in the presence of strong chelators of Fe2+, however, the ferrous ions produced are prone to reoxidation at higher pH values. These results show that Fe2+ and dithiothreitol are very susceptible to autoxidation in the neutral pH range, and that the rates are strongly influenced by the presence of chelators of Fe2+ and Fe3+. The rapid autoxidations of these species need to be taken into account when designing and interpreting experiments involving Fe2+ or both dithiothreitol and iron.

Structural changes in base-paired region 28 in 16 S rRNA close to the decoding region of the 30 S ribosomal subunit are correlated to changes in tRNA binding.

Escherichia coli 30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association. In the inactive form, 16 S rRNA base-pairs (921-922).(1395-1396) and (923-925).(1391-1393), which are part of region 28, are unstable and an alternate arrangement, (921-923).(1532-1534), is detected by psoralen photochemical crosslinking. Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit. 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit. On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions. When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929. Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926. Mutations were made to insert or delete a nucleotide at position 920. The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926. We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding. Molecular models of region 28 were made using the program MC-SYM. Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity.

Fiber types and fiber diameters in canine respiratory muscles.

In the present study, we measured fiber types and fiber diameters in canine respiratory muscles and examined regional variation within the diaphragm. Samples of eight diaphragm regions, internal intercostals, external intercostals, transversus abdominis, and triceps brachii were removed from eight adult mongrel dogs, frozen, and histochemically processed for standard fiber type and fiber diameter determinations. The respiratory muscles were composed of types I and IIa fibers; no IIb fibers were identified. Fiber composition differed between muscles (P less than 0.0001). Normal type I percent (+/- SE) were: diaphragm 46 +/- 2, external intercostal 85 +/- 6, internal intercostals 48 +/- 3, transversus abdominis 53 +/- 1, and triceps 33 +/- 7. The diaphragm also contained a type I subtype [6 +/- 1% (SE)] previously thought only to occur in developing muscle. Fiber composition varied between diaphragm regions (P less than 0.01). Most notably, left medial crus contained 64% type I fibers. Fiber size also varied systematically among muscles (P less than 0.025) and diaphragm regions (P less than 0.0005). External intercostal fiber diameter was largest (47-50 microns) and diaphragm was smallest (34 microns). Within diaphragm, crural fibers were larger than costal (P less than 0.05). We conclude that there are systematic differences in fiber composition and fiber diameter of the canine respiratory muscles.

Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneus.

After ingestion of a specimen of the crab Zosimus aeneus (Xanthidae), an East Timorese adult male died within several hours. Xanthid crabs are known to harbour paralytic shellfish toxins (PSTs), tetrodotoxin and palytoxin. A post-mortem examination did not find any obvious pathological abnormalities. This absence of pathologies is more often associated with PSTs and tetrodotoxin intoxication. A second, yet uneaten specimen of Z. aeneus from the same meal, contained a significant amount of PSTs and these same toxins were identified in the gut contents, blood, liver and urine of the victim. Metabolism of the PSTs occurred with the ingested crab harbouring gonyautoxin 2, gonyautoxin 3 and saxitoxin (STX) whereas neoSTX, decarbamoylSTX and STX dominated the PSTs in the victim's urine. The PST composition in the gut contents, in both their identity and proportion, was intermediate between the eaten crab and the urine suggesting that toxin conversion commenced in the victim's gut. The dose consumed by the victim was calculated to be between 1 and 2 microg STX equivalents/kg based upon the concentration in the remains of the cooked crab. The victim's meal did not consist solely of the toxic crab eaten and the possibility of other food items acting in a synergistic manner with the consumed PSTs cannot be discounted.

DNA adduct formation in northern pike (Esox lucius) exposed to a mixture of benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c, g]carbazole: time-course and dose-response studies.

The time-course and dose dependent formation of DNA adducts in juvenile northern pike (Esox lucius) following a single exposure to a mixture of benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazole (DBC) were investigated by use of the (32)P-postlabelling assay. A complex adduct pattern was detected in liver and intestine of exposed fish. For the time-course studies fish were exposed either by oral administration or by intraperitoneal (i.p.) injection. Following a single i.p. injection of the mixture (40micromole/kg body weight of each substance) significantly elevated DNA adduct levels were detected in the liver after 1 day. Adduct levels were higher in liver than in intestine, in which significant elevation were detected from day 3 to 12. Following exposure via food (80micromole/kg body weight of each substance), adduct levels were detected in both liver and intestine 1 day after exposure, and continued to increase until day 3 in liver and day 6 in intestine. Calculation of a binding index, which compensates for differences in dosage, resulted in much higher adduct formation (five times in liver and 22 times in intestine) following oral exposure. Pikes receiving single oral doses of 12.5, 50, 100 or 200micromole/kg body weight of each substance exhibited significantly higher adduct levels in both liver and intestine compared to controls. Hepatic adduct levels were also higher in fish given 100 and 200micromole/kg compared to 12.5micromole/kg. Results from this study show that DNA adducts are rapidly formed in juvenile northern pike following both i.p. injection and feeding of a mixture of BaP, BkF and DBC. A maximum level was reached within a few days, which then persisted at approximately the same level for at least 9-12 days. The results also shows that higher levels of adducts were obtained following oral administration compared to i.p. injection, particularly in the intestine.

DNA adduct formation and persistence in liver and extrahepatic tissues of northern pike (Esox lucius) following oral exposure to benzo[a]pyrene, benzo[k]fluoranthene and 7H-dibenzo[c,g]carbazole.

The formation and persistence of DNA adducts in liver, intestinal mucosa, gills and brain of juvenile northern pike (Esox lucius) following oral exposure to benzo[a]pyrene (BaP), benzo[k]fluoranthene (BkF) and 7H-dibenzo[c,g]carbazol (DBC) were analysed by 32P-postlabelling. The dosage was 25 micromol/kg body weight of each substance, administered on 5 occasions with an interval of 12-14 days. Sampling was carried out 9 days after the second treatment, and 9, 16, 33 and 78 days after the fifth treatment. Pikes were also fed with the substances singly for comparison of adduct patterns. A complex pattern of adducts was detected in all examined tissues from fish treated with the mixture. Total adduct levels were highest in intestine (347+/-17.4 nmol adducts/mol nucleotides, mean+/-SE), followed by liver (110+/-9.3), gills (69+/-6) and brain (14+/-4.2). In pike treated with BaP alone, one major adduct was detected in all examined tissues. This BaP-adduct made up approximately 50% of the total amount of adducts in the brain. Corresponding values in liver, intestine and gills were 23, 31 and 34%, respectively. One relatively weak BkF-adduct and at least 10 different DBC-adducts were detected in all analysed tissues. Total adduct level in the intestine declined to 29.4% of the maximum value 78 days after the last exposure, while there was no significant decline in adduct levels in liver, gills or brain. The results suggest that intestine is more susceptible to adduct formation than liver after oral exposure, and that adduct levels in the intestine represent ongoing or relatively recent exposure. DNA adducts in the other investigated tissues were much more persistent and may therefore accumulate during long-term exposure.

DNA adducts as indicators of genotoxic exposure in indigenous and transplanted mussels, Mytilus edulis L. from Icelandic coastal sites.

Indigenous mussels, Mytilus edulis, were collected at sites with supposed different amounts of pollution; Reykjavík harbour, Keflavík harbour, Grafarvogur and Hvalfjördur (reference), along the south-western coast of Iceland in March 2000. Mussels from Hvalfjördur and Reykjavík harbour were also collected in August the same year. Additionally, mussels were transplanted from the reference site to Reykjavík harbour for 6 weeks during both winter and summer for comparison. DNA adducts were analysed by 32P-post-labelling in gills and digestive gland. Highest adduct levels were found in gill tissue from indigenous mussels collected in Reykjavík harbour. Adduct levels in both tissues from mussels collected at the reference site were below or very close to the detection limit during winter, but seemed to increase a little during summer. Mussels from sites with supposed intermediate pollution had intermediate levels of DNA adducts in gills but did not differ from Reykjavík harbour in digestive gland. No increase in adduct levels was observed in mussels transplanted from the reference site to Reykjavík harbour, except for a slight increase in digestive gland during winter. This study shows that 32P-post-labelling analysis of DNA adducts is sensitive enough to be used on indigenous mussels from relatively pristine areas and that adduct levels are increased in harbours/urban sites. However, transplantation of mussels from a clean site to the harbour for 6 weeks did not result in increased adduct levels in gills, the tissue with the highest adduct levels. The results also indicate that seasonal variation in adduct levels may occur.

Tissue differences, dose-response relationship and persistence of DNA adducts in blue mussels (Mytilus edulis L) exposed to benzo[a]pyrene.

Baltic Sea blue mussels (Mytilus edulis) were experimentally exposed to the genotoxic model substance benzo[a]pyrene (B[a]P) to study DNA adduct formation. The specific aims were (a). to examine where in the mussels the DNA adducts were formed, in gills or digestive gland; (b). to study the dose-response relationship between B[a]P exposure and DNA adduct formation; and (c). to examine the persistence of the formed adducts. A Scope for growth (SFG) study was also run to compare physiological responses of the mussels with the degree of DNA adduct formation. In an initial dose-response experiment, the mussels were exposed to 0, 5, 50, and 100 microg/l of tritium labelled B[a]P under semi-static conditions for 4 days, and thereafter the bioaccumulation of B[a]P and DNA adduct formation in different tissues was determined using liquid scintillation counting and 32P-postlabelling analysis, respectively. In a following exposure-depuration experiment, mussels were exposed to 17 microg/l of radiolabelled B[a]P under semi-static conditions for 6 days. B[a]P accumulation and DNA adduct formation were determined during the exposure, and B[a]P elimination and persistence of DNA adducts were studied during 28 days of depuration in uncontaminated water. The results revealed large tissue differences in DNA adduct formation. DNA adduct levels were not elevated in the digestive gland of the mussels at any exposure concentration (0-100 microg/l), even though the highest B[a]P tissue concentrations were found in the digestive gland (1.0+/-0.1 mg B[a]P/g tissue dry wt at 100 microg/l, mean+/-SE, n=12). DNA adducts were on the other hand formed in the gills, with the highest levels found in mussels exposed to 50 and 100 microg B[a]P/l, and a dose dependent increase in adduct levels (from 1.6 to 5.9 nmol adducts/mol nucleotides) from 0 to 50 microg B[a]P/l. In gills, DNA adduct levels increased with time during the 6-day exposure period in the exposure-depuration experiment, and then persisted for at least 2 weeks after exposure cessation while B[a]P tissue levels exhibited a rapid decrease (half-life of 8 days). No significant differences were observed in SFG between the control and exposed groups. Since DNA adducts exhibited a relatively high persistence in gills compared to B[a]P tissue concentrations, they seem to be a more integrated measure of genotoxic exposure than only chemical analysis of the contaminant bioaccumulation. The results also suggest that if using analysis of DNA adducts in M. edulis for monitoring purposes, analysis of gills in addition to the more commonly used digestive gland should be taken into consideration.

Use of reverse transcription to determine the exact locations of psoralen photochemical crosslinks in RNA.

We have studied the properties of avian myeloblastosis virus reverse transcriptase on Escherichia coli 16S ribosomal RNA that was known to contain a psoralen crosslink in a restricted region around residue 920. These crosslinked RNA molecules were purified on the basis of loop size by gel electrophoresis. Reverse transcription stopped at specific positions in the crosslinked molecules but not in control linear molecules. With the particular crosslink that was studied, at the earliest time of reverse transcription, the most frequent stopping site was G925. At later times two nearly equal stops at G925 and C924 were seen. The crosslinked site was an absolute stop since even at long times of incubation the reverse transcriptase was not able to proceed beyond G925/C924. An independent determination of the crosslinked site by directly sequencing a section of the crosslinked RNA indicated that C924 was the sole nucleotide involved in the crosslink. Therefore, reverse transcriptase was able to synthesize cDNA all the way up to and including the nucleotide that contained the psoralen crosslink. Thus, reverse transcription can be a rapid and precise method for determining psoralen crosslinking sites when prefractionated, crosslinked RNA templates are used.

Cross-sectional study of patients treated with removable partial dentures with special reference to the caries situation.

Thirty-four patients provided with removable partial dentures (RPDs) were reexamined after 3 yr. Caries that had developed during the period were analyzed with regard to the following potential caries risk factors: Lactobacilli, Streptococcus mutans, flow rate and buffer pH of paraffin stimulated saliva, oral hygiene and daily sucrose intake. Of 436 initially intact surfaces 31 (7.1%) had decayed and/or were restored during the 3-yr period. Caries recurred in 26 (6.2%) out of 422 initially restored surfaces during the same period. The development of new or recurrent caries had no correlation to whether or not the surfaces affected were in contact with the RPDs. No single caries risk factor seemed to be sufficiently closely correlated to the number of caries lesions developed to be used alone in the selection of patients at risk. When the sum of assumed negative factors was used a correlation was found for the group as a whole between the number of negative factors and the development of caries. However predicting the development of caries in any individual case seems to be more complicated.

DNA adducts in perch (Perca fluviatilis) living in coastal water polluted with bleached pulp mill effluents.

DNA adducts were analyzed by (32)P-postlabeling in liver and intestine of perch (Perca fluviatilis). Fish were collected in the receiving water of a bleached kraft pulp mill at Norrsundet, Sweden, on the coast of the Bothnian Sea, and in three different reference areas. Sampling was carried out the last week in September 1993, 1995, and 1997. Since 1984/1985 the pulping process at Norrsundet has been successively modified and an external waste-water treatment has been installed, resulting in lowered and altered effluent discharges. Nevertheless, perch captured in the water area closest to the mill (2 km) had significantly elevated levels of aromatic/hydrophobic DNA adducts in both liver and intestine when compared to perch captured 8 km away and in the reference areas. Autoradiograms indicated a diagonal zone consisting of multiple overlapping adducts, a pattern typical of exposure to a complex mixture of aromatic/hydrophobic genotoxic substances. No significant difference in adduct levels was detected between the years. Results from this study reveal that fish in the area closest to the mill are exposed to substances with a genotoxic potential.

An RNA secondary structure switch between the inactive and active conformations of the Escherichia coli 30 S ribosomal subunit.

Psoralen cross-linking was used to produce intramolecular cross-links in the Escherichia coli 16 S ribosomal RNA in the inactive and active forms of the 30 S subunit. A number of psoralen cross-links were made in the inactive form that were not made in the active form. The most frequent of these cross-links was sequenced by a series of techniques and identified as C-924 to U-1532. In this region, a three-base complementary, (921-923).(1532-1534), forms a site where psoralen can stack and produce a cross-link between C-924 and U-1532. When psoralen monoadducts were placed on inactive subunits and the conformation was switched to the active form before cross-linking, a new cross-link involving U-1393 was detected. U-1393 is part of the complementarity, (923-925).(1391-1393), that has previously been proposed as being an element of the functional secondary structure on the basis of sequence comparison. The complementarity between (921-923).(1532-1534) occurs in most nonmitochondrial small subunit RNAs; however, there are several counter examples in which it does not occur. This suggests that this alternate secondary structure interaction is not necessary for the function of the 30 S subunit.

Cross-sectional study of the periodontal status of removable partial denture patients.

Thirty-four patients, 18 men and 16 women with a mean age of 62 years, provided with removable partial dentures were reexamined after 3 years. Approximately 92% of the dentures were bilateral distal-extension prostheses. The periodontal parameters registered were (1) oral hygiene, (2) gingival inflammation, (3) pocket depth, and (4) tooth mobility. Fourteen of the patients had followed the recommendation to visit a dentist for regular checkups at least once a year. For all periodontal parameters, the results were somewhat better for those patients who had regular checkups than for those who had not. The results for the regularly checked patients were similar to those of the patients in an earlier longitudinal study from the same clinic after a corresponding period of time. Thus the good results obtained in the longitudinal study were duplicated 10 years later in this cross-sectional study.

Cross-sectional study of patients fitted with fixed partial dentures with special reference to the caries situation.

The caries situation among 30 patients with fixed partial dentures was examined 3 yr after cementation of the constructions. The fit of the crowns on the abutment teeth, the size of the interproximal areas and the relation between the crown margin and the gingival margin were registered. Risk factors for caries development, such as salivary factors, microbiologic factors, oral hygiene and diet were evaluated for each patient. During the 3-yr period 54 caries lesions occurred in 25 patients. Eleven lesions were found on abutment teeth. Two were located on the root surfaces unconnected with the crown margin (greater than 2mm from the gingival margin), three were recurrent lesions, where the crown margin was located above the gingival margin but closer than 2 mm and six were recurrent lesions where the crown margin was located under the gingival margin. No single caries risk factor seemed to be so closely correlated to the number of caries lesions developed that it could be used alone to select of patients at risk. When the sum of the assumed negative factors was used, more caries was found among the patients with three or more factors with negative values in the group as a whole. However, predicting the development of caries in an individual case with a high degree of probability seems to be complicated. Generally speaking there was no indication that caries disease development was caused by the presence of the fixed partial denture per se.

Morphological and histochemical organization of the flexor carpi radialis muscle in the cat.

Most studies concerning the structure and function of skeletal muscle have utilized the hind limb of the experimental animal. However, it has been shown that the number of behavioral tasks performed by the cat's forelimb is greater than that of the hind limb. In addition, the forelimb muscles exhibit a functional complexity not observed in hind-limb musculature. The purpose of this study was to investigate the distribution of fast-twitch and slow-twitch muscle fibers and muscle spindles in the flexor carpi radialis muscle (FCR) and to correlate the distributional patterns in these structures with muscle tendon architecture and muscle function. It was found that the FCR, a wrist flexor, contains 37% slow-twitch fibers and 63% fast-twitch fibers. However, the slow-twitch fibers were concentrated in the deep region located between the tendons of origin and insertion, while the fast-twitch-glycolytic fibers were concentrated more peripherally. Muscle spindles were associated with the slow-twitch region and were never found in the region containing high concentrations of fast-twitch-glycolytic fibers. Fast-twitch-oxidative-glycolytic fibers were uniformly distributed throughout the muscle. It is proposed that the association of muscle spindles with slow-twitch fibers and the differential distribution of muscle fibers into slow-twitch and fast-twitch regions might allow these regions to function independently of one another when called upon to perform complex behavioral tasks.

Structure of synthetic unmethylated 16S ribosomal RNA as purified RNA and in reconstituted 30S ribosomal subunits.

16S ribosomal RNA was made by in vitro transcription of a cloned gene, and its structure was compared to authentic 16S ribosomal RNA. The comparison was made by subjecting the two types of 16S rRNA to chemical reagents that react specifically with unpaired bases and determining the extent of reaction by reverse transcription and gel electrophoresis of the cDNA. In solution, the rRNAs were indistinguishable in their pattern of reactivity, except for a difference at A1408 and many differences in the region between residues 470 and 562. When the 16S rRNAs were reconstituted into 30S ribosomal subunits, these reactivity differences were absent. When the synthetic 16S rRNA was reconstituted into 30S subunits and then extracted and tested in solution, its pattern of chemical reactivity in the 470-562 region was the same as authentic 16S rRNA, but differences in the 1408-1410 region persisted. This study indicates that the synthetic 16S rRNA has a secondary structure in solution similar to a native secondary structure except in two regions, one apparently incorrectly folded during synthesis and the other in which nucleotides which are normally methylated in authentic 16S rRNA may be responsible for a structural difference.

An experimental model for the study of exercise-induced skeletal muscle hypertrophy.

A procedure for training laboratory cats to perform weight-lifting exercise has been developed. This program consists of operantly conditioning adult cats to move a bar a specific distance with their right forelimb to receive a food reward. Weights attached to the bar via a pully are lifted as the bar is moved. The cat is then exercised at the same load for 5 days before the weight is increased. A linear potentiometer, attached to the hinged bar, produces an analog voltage proportional to the bar movement. This voltage is then monitored by a general purpose computer using a real-time behavioral program. In this way, the numbber of times the cat moves the bar, the time required to move the bar, and the time between bar movements are all recorded. The total physical work accomplished and the average power expended by the cat during the weight-lifting exercise regimen can then be calculated. This procedure has the advantage of inducing significant gross muscle hypertrophy (from 7 to 34%) and muscle fiber hypertrophy in one limb, while the muscles of the opposite limb can be utilized for comparative studies. The striking morphological and histochemical transformations that occur during physiological hypertrophy are now available for experimental investigation using this model.

Skeletal muscle fiber splitting induced by weight-lifting exercise in cats.

Adult skeletal muscle hypertrophy induced by exercise has been thought to be exclusively related to an increase in cross-section area of individual muscle fibers and not to an increase in the number of muscle fibers. Recent experiments using surgical intervention to cause muscle overload have induced an increase in fiber numbers; however, the muscle also exhibited pathological alterations. The purpose of this study was to determine if an exercise regimen also induced hyperplasia. Cats were trained to lift weights with their right forelimb to receive a food reward. After 19-46 weeks of training, the flexor carpi radialis muscle (FCR) was removed and prepared for histochemical examination. The total number of muscle fibers of the right exercised FCR increased significantly (19.3%) when compared with that of the unexercised left FCR (p less than 0.05). This increase was found to be due to muscle fiber splitting.

A 2-year follow-up study of titanium crowns.

In 1986, 149 patients were provided with titanium crowns. The method used for fabrication of the titanium copings involved two principles: machine duplication of models and electric discharge machining. For the veneering of the copings, Isosit was used for the first 90% of the crowns in the series, and the Dentacolor-Silicoater technique for the last 10%. Of 205 individual crowns cemented in 1986, 167 could be examined after 2 years. The crowns were rated by four independent examiners using the CDA quality evaluation system. Bleeding Index and Margin Index were also used. The Margin Integrity score was recorded as satisfactory for all crowns examined over the period studied. A vast majority of the margins were rated as excellent. Isosit (n = 145) disclosed shortcomings including fractures and substantial deteriorations of Surface and Color and of Anatomic Form. With Dentacolor as veneering material (n = 18) the results with the factors Surface and Color and Anatomic Form were still rated satisfactory after 2 years, and no fractures of the veneering material were registered. Bleeding Index and Margin Index showed comparatively small changes after 2 years.