Clinical longevity of intracoronal restorations made of gold, lithium disilicate, leucite, and indirect resin composite: a systematic review and meta-analysis.

OBJECTIVES: The aim of this systematic review and meta-analysis is to assess the comparative clinical success and survival of intracoronal indirect restorations using gold, lithium disilicate, leucite, and indirect composite materials. MATERIAL AND METHODS: This systematic review and meta-analysis were conducted following the Cochrane Handbook for Systematic Reviews of Interventions and PRISMA guidelines. The protocol for this study was registered in PROSPERO (registration number: CRD42021233185). A comprehensive literature search was conducted across various databases and sources, including PubMed/Medline, Embase, Cochrane Library, Web of Science, ClinicalTrials.gov, and gray literature. A total of 7826 articles were screened on title and abstract. Articles were not excluded based on the vitality of teeth, the language of the study, or the observation period. The risk difference was utilized for the analyses, and a random-effects model was applied. All analyses were conducted with a 95% confidence interval (95% CI). The calculated risk differences were derived from the combined data on restoration survival and failures obtained from each individual article. The presence of heterogeneity was assessed using the I2 statistic, and if present, the heterogeneity of the data in the articles was evaluated using the non-parametric chi-squared statistic (p < 0.05). RESULTS: A total of 12 eligible studies were selected, which included 946 restorations evaluated over a minimum observation period of 1 year and a maximum observation period of 7 years. Results of the meta-analysis indicated that intracoronal indirect resin composite restorations have an 18% higher rate of failure when compared to intracoronal gold restorations over 5-7 years of clinical service (risk difference = - 0.18 [95% CI: - 0.27, - 0.09]; p = .0002; I2 = 0%). The meta-analysis examining the disparity in survival rates between intracoronal gold and leucite restorations could not be carried out due to methodological differences in the studies. CONCLUSIONS: According to the currently available evidence, medium-quality data indicates that lithium disilicate and indirect composite materials demonstrate comparable survival rates in short-term follow-up. Furthermore, intracoronal gold restorations showed significantly higher survival rates, making them a preferred option over intracoronal indirect resin-composite restorations. Besides that, the analysis revealed no statistically significant difference in survival rates between leucite and indirect composite restorations. The short observation period, limited number of eligible articles, and low sample size of the included studies were significant limitations. CLINICAL SIGNIFICANCE: Bearing in mind the limitations of the reviewed literature, this systematic review and meta-analysis help clinicians make evidence-based decisions on how to restore biomechanically compromised posterior teeth.

Clinical survival and performance of premolars restored with direct or indirect cusp-replacing resin composite restorations with a mean follow-up of 14 years.

OBJECTIVES: The objective is to evaluate the long-term clinical survival and performance of direct and indirect resin composite restorations replacing cusps in vital upper premolars. METHODS: Between 2001 and 2007, 176 upper premolars in 157 patients were restored with 92 direct and 84 indirect resin composite restorations as part of an RCT. Inclusion criteria were fracture of the buccal or palatal cusp of vital upper premolars along with a class II cavity or restoration in the same tooth. RESULTS: Forty patients having 23 direct and 22 indirect composite restorations respectively, were lost to follow-up (25.6%). The cumulative Kaplan-Meier survival rates were 63.6% (mean observation time: 15.3 years, SE 5.6%) with an AFR of 2.4% for direct restorations and 54.5% (mean observation time: 13.9 years, SE: 6.4%) with an AFR of 3.3% for indirect restorations. The Cox regression analysis revealed a statistically significant influence of the patient's age at placement on the survival of the restoration (HR 1.036, p = 0.024), the variables gender, type of upper premolar, type of restoration, and which cusp involved in the restoration had no statistically significant influence. Direct composite restorations failed predominantly due to tooth fracture, indirect restorations primarily by adhesive failure (p < 0.05). SIGNIFICANCE: There was no statistically significant difference in survival rates between direct and indirect composite cusp-replacing restorations. Both direct and indirect resin composite cusp-replacing restorations are suitable options to restore compromised premolars. The longer treatment time and higher costs for the indirect restoration argue in favor of the direct technique.

Influence of Preparation Design and Restorative Material on Fatigue and Fracture Strength of Restored Maxillary Premolars.

STATEMENT OF PROBLEM: Extensive carious lesions and/or large preexisting restorations possibly contribute to crack formation, ultimately resulting in a fracture that may lead to the loss of a tooth cusp. Hence, preparation design strategy in conjunction with the restorative material selected could be influential in the occurrence of a cuspal fracture. PURPOSE: The purpose of this in vitro study was to evaluate the fatigue behavior and fracture strength of maxillary premolars restored with direct composite and indirect ceramic inlays and overlays, with different preparation depths in the presence or absence of cuspal coverage, and analyze their failure types. METHODS AND MATERIALS: Sound maxillary premolars (N=90; n=10) were divided into nine groups: group C: control; group DCI3: direct composite inlay 3 mm; group DCI5: direct composite inlay 5 mm; group ICI3: indirect ceramic inlay 3 mm; group ICI5: indirect ceramic inlay 5 mm; group DCO3: direct composite overlay 3 mm; group DCO5: direct composite overlay 5 mm; group ICO3: indirect ceramic overlay 3 mm; group ICO5: indirect ceramic overlay 5 mm. In indirect ceramic, lithium disilicate restoration groups, immediate dentin sealing was applied. After restoration, all specimens were tested in fatigue (1,200,000 cycles, 50 N, 1.7 Hz). Samples were critically appraised, and the specimens without failure were subjected to a load to failure test. Failure types were classified and the data analyzed. RESULTS: Zero failures were observed in the fatigue testing. The following mean load to failure strengths (N) were recorded: group ICO5: 858 N; group DCI3: 829 N; group ICO3: 816 N; group C: 804 N; group ICI3: 681 N; group DCO5: 635 N; group DCI5: 528 N; group DCO3: 507 N; group ICI5: 482 N. Zero interaction was found between design-depth-material (p=0.468). However, significant interactions were found for the design-depth (p=0.012) and design-material (p=0.006). Within restorations at preparation depth of 3 mm, direct composite overlays obtained a significantly lower fracture strength in comparison to indirect ceramic onlays (p=0.013) and direct composite inlays (p=0.028). In restorations at depth 5 mm, significantly higher fracture load values were observed in indirect ceramic overlays compared with the inlays (p=0.018). Indirect ceramic overlays on 3 mm were significantly stronger than the deep inlays in ceramic (p=0.002) and tended to be stronger than the deep direct composite inlays. Severe, nonreparable fractures were observed with preparation depth of 5 mm within ceramic groups. CONCLUSIONS: The preparation depth significantly affected the fracture strength of tooth when restored with either composite or ceramic materials. Upon deep cavity preparations, cuspal coverage proved to be beneficial when a glass ceramic was used as the restorative material. Upon shallow cavity preparations, a minimally invasive approach regarding preparation design used in conjunction with a direct composite material was favorable.

The Influence of Dentin Wall Thickness and Adhesive Surface in Post and Core Crown and Endocrown Restorations on Central and Lateral Incisors.

CLINICAL RELEVANCE: Post and core crowns and endocrowns perform similarly on fracture strength, but endocrowns have more repairable fractures.

Copine-III interacts with ErbB2 and promotes tumor cell migration.

ErbB2 amplification and overexpression in breast cancer correlates with aggressive disease and poor prognosis. To find novel ErbB2-interacting proteins, we used stable isotope labeling of amino acids in cell culture followed by peptide affinity pull-downs and identified specific binders using relative quantification by mass spectrometry. Copine-III, a member of a Ca(2+)-dependent phospholipid-binding protein family, was identified as binding to phosphorylated Tyr1248 of ErbB2. In breast cancer cells, Copine-III requires Ca(2+) for binding to the plasma membrane, where it interacts with ErbB2 upon receptor stimulation, an interaction that is dependent on receptor activity. Copine-III also binds receptor of activated C kinase 1 and colocalizes with phosphorylated focal adhesion kinase at the leading edge of migrating cells. Importantly, knockdown of Copine-III in T47D breast cancer cells causes a decrease in Src kinase activation and ErbB2-dependent wound healing. Our data suggest that Copine-III is a novel player in the regulation of ErbB2-dependent cancer cell motility. In primary breast tumors, high CPNE3 RNA levels significantly correlate with ERBB2 amplification. Moreover, in an in situ tissue microarray analysis, we detected differential protein expression of Copine-III in normal versus breast, prostate and ovarian tumors, suggesting a more general role for Copine-III in carcinogenesis.

Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity.

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.

Identification of tyrosine phosphorylation sites on 3-phosphoinositide-dependent protein kinase-1 and their role in regulating kinase activity.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in signal transduction pathways that activate phosphoinositide 3-kinase. Despite its key role as an upstream activator of enzymes such as protein kinase B and p70 ribosomal protein S6 kinase, the regulatory mechanisms controlling PDK1 activity are poorly understood. PDK1 has been reported to be constitutively active in resting cells and not further activated by growth factor stimulation (Casamayor, A., Morrice, N. A., and Alessi, D. R. (1999) Biochem. J. 342, 287-292). Here, we report that PDK1 becomes tyrosine-phosphorylated and translocates to the plasma membrane in response to pervanadate and insulin. Following pervanadate treatment, PDK1 kinase activity increased 1.5- to 3-fold whereas the activity of PDK1 associated with the plasma membrane increased approximately 6-fold. The activity of PDK1 localized to the plasma membrane was also increased by insulin treatment. Three tyrosine phosphorylation sites of PDK1 (Tyr-9 and Tyr-373/376) were identified using in vivo labeling and mass spectrometry. Using site-directed mutants, we show that, although phosphorylation on Tyr-373/376 is important for PDK1 activity, phosphorylation on Tyr-9 has no effect on the activity of the kinase. Both of these residues can be phosphorylated by v-Src tyrosine kinase in vitro, and co-expression of v-Src leads to tyrosine phosphorylation and activation of PDK1. Thus, these data suggest that PDK1 activity is regulated by reversible phosphorylation, possibly by a member of the Src kinase family.

High-level soluble production and characterization of porcine ribonuclease inhibitor.

Ribonucleases can be cytotoxic if they retain their ribonucleolytic activity in the cytosol. The cytosolic ribonucleolytic activity of ribonuclease A (RNase A) and other pancreatic-type ribonucleases is limited by the presence of excess ribonuclease inhibitor (RI). RI is a 50-kDa cytosolic scavenger of pancreatic-type ribonucleases that competitively inhibits their ribonucleolytic activity. RI had been overproduced as inclusion bodies, but its folding in vitro is inefficient. Here, porcine RI (pRI) was overproduced in Escherichia coli using the trp promoter and minimal medium. This expression system maintains pRI in the soluble fraction of the cytosol. pRI was purified by affinity chromatography using immobilized RNase A and by anion-exchange chromatography. The resulting yield of 15 mg of purified RI per liter of culture represents a 60-fold increase relative to previously reported recombinant DNA systems. Differential scanning calorimetry was used to study the thermal denaturation of pRI, RNase A, and the pRI-RNase A complex. The conformational stability of the complex is greater than that of the individual components.

Substrate specificity and antifungal activity of recombinant tobacco class I chitinases.

Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.

Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry.

O-Fucosylation is an unusual posttranslational modification present in several proteins that play important roles in physiological processes such as coagulation, cell signaling and metastasis. Although the exact function of the modification is still unclear, the number of proteins found to be modified is increasing, and there is a need for further structural and functional analyses. Here we report on a rapid and straightforward approach in the analysis of glycosylation status and determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight (nano-ESI Q-TOF) mass spectrometry. In a single measurement of previously chemically untreated O-fucosylated peptides originating from the thrombospondin-1 repeats, we were able to determine the glycosylation status of the analyzed peptide, the glycosylation site, and the glycan structure. The abundance of glycosylated peptide fragment ions in MS(2) spectra suggests that nano-ESI Q-TOF mass spectrometry can be used as a general approach in structural studies of O-fucosylation in proteins.

Requirement of the Lec35 gene for all known classes of monosaccharide-P-dolichol-dependent glycosyltransferase reactions in mammals.

The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose-beta-1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.

C-mannosylation and O-fucosylation of the thrombospondin type 1 module.

Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 repeats (TSRs), modules of approximately 60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an alpha-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is well described in epidermal growth factor-like modules. Both C- and O-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.

Properdin, the positive regulator of complement, is highly C-mannosylated.

Properdin is the positive regulator of the alternative pathway of complement activation. The 53-kDa protein is essentially composed of six thrombospondin type 1 repeats, all of which contain the WXXW motif, the recognition sequence for C-mannosylation. C-Mannosylation is a post-translational modification of tryptophan residues in which, in contrast to the well known N- and O-glycosylation, the carbohydrate is attached via a C-C bond to C-2 of the indole moiety of tryptophan. C-Mannosylation was first found in human RNase 2 and interleukin-12. The terminal complement proteins C6-C9 also carry this modification as part of their thrombospondin type 1 repeats. We studied the C-mannosylation pattern of human properdin by mass spectrometry and Edman degradation. Properdin contains 20 tryptophans of which 17 are part of a WXXW motif. Fourteen tryptophans were found to be modified 100%. This is the first example of a protein in which the majority of tryptophan residues occurs in the C-mannosylated form. These results show that C-mannosylated proteins occur at several steps along the complement activation cascade. Therefore, this system would be ideal to investigate the function of C-mannosylation.

Stat5a serine phosphorylation. Serine 779 is constitutively phosphorylated in the mammary gland, and serine 725 phosphorylation influences prolactin-stimulated in vitro DNA binding activity.

The activity of transcription factors of the Stat family is controlled by phosphorylation of a conserved, carboxyl-terminal tyrosine residue. Tyrosine phosphorylation is essential for Stat dimerization, nuclear translocation, DNA binding, and transcriptional activation. Phosphorylation of Stats on specific serine residues has also been described. We have previously shown that in HC11 mammary epithelial cells Stat5a is phosphorylated on Tyr(694) in a prolactin-sensitive manner, whereas serine phosphorylation is constitutive (Wartmann, M., Cella, N., Hofer, P., Groner, B., Xiuwen, L., Hennighausen, L., and Hynes, N. E. (1996) J. Biol. Chem. 271, 31863-31868). By using mass spectrometry and site-directed mutagenesis, we have now identified Ser(779), located in a unique Stat5a SP motif, as the site of serine phosphorylation. By using phospho-Ser(779)-specific antiserum, we have determined that Ser(779) is constitutively phosphorylated in mammary glands taken from different developmental stages. Stat5a isolated from spleen, heart, brain, and lung was also found to be phosphorylated on Ser(779). Ser(725) in Stat5a has also been identified as a phosphorylation site (Yamashita, H., Xu, J., Erwin, R. A., Farrar, W. L., Kirken, R. A., and Rui, H. (1998) J. Biol. Chem. 273, 30218-30224). Here we show that mutagenesis of Ser(725), Ser(779), or a combination of Ser(725/779) to an Ala had no effect on prolactin-induced transcriptional activation of a beta-casein reporter construct. However, following prolactin induction the Ser(725) mutant displayed sustained DNA binding activity compared with that of wild type Stat5a. The results suggest that Ser(725) phosphorylation has an impact on signal duration.

Protein C-mannosylation: facts and questions.

Among the posttranslational modifications of proteins, glycosylation is probably the most abundant one. Two main types of protein glycosylation have been known for several years, namely N-glycosylation and O-glycosylation. Their biochemical properties, structure and biosynthesis, have been described extensively. Their biological functions are also known for a number of proteins, although in many cases the function remains speculative despite continuous efforts. A few years ago, a new type of protein glycosylation was found, which is different from the above-mentioned ones. It was called C-glycosylation, since the sugar is linked to the protein through a carbon-carbon bond. This article reviews the biochemistry of C-glycosylation, the biosynthetic pathway and structural requirements. Possible biological functions of this modification are also discussed.

The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.

C-Mannosylation is a unique form of protein glycosylation, involving the C-glycosidic attachment of a mannosyl residue to the indole moiety of Trp. In the two examples found so far, human RNase 2 and interleukin-12, only the first Trp in the recognition motif WXXW is specifically C-mannosylated. To establish the generality of protein C-mannosylation, and to learn more about its mechanism, the terminal components of the human complement system (C6, C7, C8,and C9), which contain multiple and complex recognition motifs, were examined. Together with C5b they form the cytolytic agent, the membrane attack complex. These are the first proteins that are C-mannosylated on more than one Trp residue as follows: six in C6, four in C7, C8alpha, and C8beta, and two in C9. Thus, from the 113 Trp residues in the complete membrane attack complex, 50 were found to undergo C-mannosylation. The other important finding is that in C6, C7, C8, and C9 Trp residues without a second Trp (or another aromatic residue) at the +3 position can be C-mannosylated. This shows that they must contain an additional C-mannosylation signal. Whether this is encoded in the primary or tertiary structure is presently unknown. Finally, all modified Trp residues are part of the highly conserved core of the thrombospondin type 1 repeats present in these proteins. Since this module has been found in a large number of other proteins, the results suggest further candidates for C-mannosylation.

Characterization of the adenylation site in the RNA 3'-terminal phosphate cyclase from Escherichia coli.

RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human and Escherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA, yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3'-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation.

Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms.

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.

Recombinant human interleukin-12 is the second example of a C-mannosylated protein.

The beta-chain of human interleukin 12 (IL-12) contains at position 319-322, the sequence Trp-x-x-Trp. In human RNase 2 this is the recognition motif for a new, recently discovered posttranslational modification, i.e., the C-glycosidic attachment of a mannosyl residue to the side chain of tryptophan. Analysis of C-terminal peptides of recombinant IL-12 (rHuIL-12) by mass spectrometry and NMR spectroscopy revealed that Trp-319beta is (partially) C-mannosylated. This finding was extended by in vitro mannosylation experiments, using a synthetic peptide derived from the same region of the protein as an acceptor. Furthermore, human B-lymphoblastoid cells, which secrete IL-12, were found to contain an enzyme that carries out the C-mannosylation reaction. This shows that nonrecombinant IL-12 is potentially C-mannosylated as well. This is only the second report on a C-mannosylated protein. However, the occurrence of the C-mannosyltransferase activity in a variety of cells and tissues, and the presence of the recognition motif in many proteins indicate that more C-mannosylated proteins may be found.

Ribonuclease 4, an evolutionarily highly conserved member of the superfamily.

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a Kd of 4 x 10(-15) M.