Pattern and risk factors of sharp object injuries among health care workers in two tertiary hospitals, Al Taif-Kingdom of Saudi Arabia 2016-2018.

Occupational exposure of healthcare workers to blood and body fluids following skin injury constitutes a risk for transmission of blood-borne pathogens. The risk of exposure is greater as well. The present study aimed to determine the burden and risk factors of sharp object injuries in two tertiary hospitals in the Taif City KSA. Retrospective review of needle stick injury records was included from the two hospital's staff clinics. A Total of 131 health professionals (employees) recorded as exposed to sharp object injuries from both hospitals were enrolled during period 2016-2018. The collected data was cleaned, reviewed and analyzed using Statistical Package of Social Sciences SPSS ver. 25. The result of the study revealed that, the mean age for the 131 enrolled participants was 31 ± 6.6, Male to Female Ratio was 1:3. The most affected age group was 20-30 years (55.7%). Females were more affected 98 out of 131 (74.8%) than male (33out of 131 (25.2%). And there is increasing incidence rates of exposure from 2.89 /10.000 patient/day in 2016 to 3.42/ 10.000 patients'/day in 2017, with highest exposed nationalities; Filipino 42 (32.1%), Saudi 31 (23.7%), and Indians 26 (19.8%), the remaining 24.5% were from 10 mixed nationalities. The frequent affected divisions were: ER, surgical ward, operation room, ICU, laboratory, Medical W, Medical waste facilities (19.8%, 15%, 12.2%, 9.2%, 92% respectively). The most affected HCWs categories were nurses 74(56.5%), doctor 23(17.6%) and housekeeping 18 (13.7%). And the needle prick 104(79.4%) and cut wound 15(11.5%) constitute the highest type of injuries and were during operation 23 (17.6%), waste collection 15 (11.5%), cannulation 12 (9.2%) and giving injection 12 (9.2%). The common devices caused injuries were bore hole needle 63(48.1%), suture needle **(13.7%), cannula and insulin syringe 13 (9.9%) each. This study concluded that, as from 2016-2018, there was an increasing rate of reported accidental exposure to sharp needle injuries amongst HCWs from 3.0 to 3.4/10.000 patient/day, and the younger and nurses were mostly impacted. Workplace, distress, work types and load had influences on injuries rates and types. Fortunately, no exposure among employee with HBV, HCV and HIV seroconversion were documented.

Proteome data set of human gingival crevicular fluid from healthy periodontium sites by multidimensional protein separation and mass spectrometry.

BACKGROUND AND OBJECTIVE: Gingival crevicular fluid has been of major interest for many decades as a valuable body fluid that may serve as a source of biomarkers for both periodontal and systemic diseases. Owing to its very small sample size, submicroliter volumes, identification of its protein composition by classical biochemical methods has been limited. The advent of highly sensitive mass spectrometric technology has permitted large-scale identification of protein components of many biological samples. This technology has been employed to identify the protein composition of gingival crevicular fluid from inflamed and periodontal sites. In this report, we present a proteome data set of gingival crevicular fluid from healthy periodontium sites. MATERIAL AND METHODS: A combination of a periopaper collection method with application of multidimensional protein separation and mass spectrometric technology led to a large-scale documentation of the proteome of gingival crevicular fluid from healthy periodontium sites. RESULTS: The approaches used have culminated in identification of 199 proteins in gingival crevicular fluid of periodontally healthy sites. The present gingival crevicular fluid proteome from healthy sites was compared and contrasted with those proteomes of gingival crevicular fluid from inflamed and periodontal sites, as well as serum. The cross-correlation of the gingival crevicular fluid and plasma proteomes permitted dissociation of the 199 identified gingival crevicular fluid proteins into 105 proteins (57%) that can be identified in plasma and 94 proteins (43%) that are distinct and unique to the gingival crevicular fluid microenvironment. Such analysis also revealed distinctions in protein functional categories between serum proteins and those specific to the gingival crevicular fluid microenvironment. CONCLUSION: Firstly, the data presented herein provide the proteome of gingival crevicular fluid from periodontally healthy sites through establishment of innovative analytical approaches for effective analysis of gingival crevicular fluid from periopapers both at the level of complete elusion and with removal of abundant albumin, which restricts identification of low-abundant proteins. Secondly, it adds significantly to the knowledge of gingival crevicular fluid composition and highlights new groups of proteins specific to the gingival crevicular fluid microenvironment.

Proteome of human minor salivary gland secretion.

Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.

Cannula site insertion technique prevents incisional hernia in laparoscopic fundoplication.

INTRODUCTION: Incisional hernia is a common surgical problem encountered after laparotomies. The so-called trocar-site or port-site hernia is a type of incisional one that occurs after laparoscopic procedures. It has an incidence range between 0.1% and 3%. OBJECTIVE: To evaluate our patients who underwent laparoscopic Nissen fundoplication for presence of trocar-site hernia. PATIENTS AND METHODS: This study included 405 patients who underwent laparoscopic Nissen fundoplication in Ankara University, Faculty of Medicine, Department of General Surgery, Turkey. The patients were evaluated by physical examination and anterior abdominal wall ultrasound (US). RESULTS: Trocar-site hernia was not detected in any of our cases either by physical examination or by US. CONCLUSIONS: Trocar-site hernia is a rare complication of laparoscopy. It occurs at the trocar insertion site with a diameter of 10 mm or more in adult patients. Trocar insertion away from the midline can decline the incidence.

Acquired enamel pellicle and its potential role in oral diagnostics.

The acquired enamel pellicle (AEP) is a protein film with unique composition and properties, which is formed by the selective adsorption of a variety of oral fluid-derived proteins onto tooth enamel surfaces. Since events leading to caries and periodontal disease occur in close proximity to the tooth surface, pellicle constituents are likely to contain biomarkers valuable for diagnostic applications. Despite the importance of this oral structure, progress in understanding its formation and composition has been slow because of difficulties in efficient pellicle collection methods and limitations of biochemical techniques for the characterization of microgram amounts of proteins/peptides. Recent developments in both pellicle collection methods and nanoscale sensing technologies have brought the exploitation of pellicle analysis into the realm of point-of-care oral diagnostics.

Routine coagulation screening in children undergoing gastrointestinal endoscopy does not predict those at risk of bleeding.

BACKGROUND AND STUDY AIMS: Routine coagulation screening prior to gastrointestinal endoscopy is performed in many centres in the UK, despite the lack of any evidence to support the practice. The aim of this study was to assess the benefits of routine pre-endoscopy coagulation screening in children and to assess how widespread this practice is in the UK. PATIENTS AND METHODS: We performed a retrospective analysis of the case notes of 250 consecutive patients who had undergone routine coagulation screening prior to endoscopy and biopsy, in accordance with our unit's protocol, looking for evidence of abnormal results or episodes of bleeding. We also performed a telephone survey of the protocols for coagulation screening at other paediatric units in the UK which are known to perform gastrointestinal endoscopy on a routine basis. RESULTS: According to our hospital's laboratory reference ranges, 16.8 % of the children who underwent endoscopy and biopsy had abnormal clotting. This was neither clinically significant nor associated with an increased bleeding risk in any patient. Of the 23 UK paediatric gastroenterology centres surveyed, including our own, five (21.7 %) perform routine coagulation screening before endoscopy. CONCLUSIONS: This study suggests that, although it is a relatively common practice, routine coagulation screening is not indicated in children who are undergoing gastrointestinal endoscopy and biopsy, and that it does not predict those at risk of significant bleeding. We would therefore suggest that if pre-endoscopy screening is to be performed, it should be reserved for those who are potentially at high risk of bleeding.

Bone sialoprotein-coated femoral implants are osteoinductive but mechanically compromised.

Aseptic loosening of femoral implants in total hip replacement remains an unsolved orthopaedic problem. This paper investigates the potential role of bone sialoprotein (BSP) in enhancing bone-implant adherence. As BSP is osteoinductive in rat calvarial models, we investigated whether BSP is similarly osteoinductive when coated onto intramedullary femoral implants. BSP-coated titanium implants were implanted into the femur of female 'Wistar' rats (average weight 215 g) that were sacrificed at days 10, 20 and 30. Harvested femoral implants were subjected to pullout testing and then examined histologically. BSP-coated implants demonstrate osteoinduction when examined histologically. Plugging the femoral canal with BSP prior to inserting the implant neither increased implant pullout strengths nor further increased osteoblastic activity. This study has demonstrated for the first time that BSP is osteoinductive when coated onto femoral implants and inserted into bones subjected to mechanical loading. However, we found that pullout strengths are a function of implant surface topographical characteristics and are not affected by BSP coating or histological osteoinduction.

Selective adhesion of osteoblastic cells to different integrin ligands induces osteopontin gene expression.

Skeletal homeostasis is partly regulated by the mechanical environment and specific signals generated by a cell's adhesion to the matrix. Previous studies demonstrated that osteopontin (OPN) expression is stimulated in response to both cellular adhesion and mechanical stimulation. The present studies examine if specific integrin ligands mediate osteoblast selective adhesion and whether opn mRNA expression is induced in response to these same ligands. Embryonic chicken calvaria osteoblastic cells were plated on bacteriological dishes coated with fibronectin (FN), collagen type I (Col1), denatured collagen/gelatin (G), OPN, vitronectin (VN), laminin (LN) or albumin (BSA). Osteoblastic cells were shown to selectively adhere to FN, Col1, G and LN, yet not to VN, OPN or BSA. Opn mRNA expression was induced by adhesion to Col1, FN, LN and G, but neither OPN nor VN induced this expression. Examination of the activation of the protein kinases A and C second signaling systems showed that only adhesion to FN induced protein kinase A and protein kinase C (PKC) activity while adherence to Col1 induced PKC. Evaluation of the intracellular distribution of focal adhesion kinase (FAK) and p-tyrosine within cells after adherence to FN, VN or BSA demonstrated that adherence to FN stimulated FAK translocation from the nucleus to the cytoplasm and high levels of p-tyrosine localization at the cell surface. However, cell adherence to VN or BSA did not show these morphological changes. These data illustrate that osteoblast selective adhesion is mediated by specific integrin ligands, and induction of intracellular second signal kinase activity is related to the nature of the ligand.

Phosphorylation of calsenilin at Ser63 regulates its cleavage by caspase-3.

Calsenilin is a member of the neuronal calcium sensor (NCS) family of proteins that interacts with the presenilins. Calsenilin has been found to act as a Kv4alpha channel interactor and as a transcriptional repressor. We have recently shown that calsenilin can be cleaved by caspase-3 and that its cleavage separates the conserved calcium-binding domain from the variable N-terminal domain. Here, we demonstrate that calsenilin can be phosphorylated by casein kinase I and that its phosphorylation can be regulated by intracellular calcium. In addition, phosphorylated calsenilin is a substrate for serine/threonine protein phosphatase (PP) 1 and/or 2A. Phosphorylation within the N-terminal domain at Ser63, the major phosphorylation site of calsenilin, inhibits cleavage of the molecule by caspase-3. Given that the N-terminal domain of calsenilin is not conserved in the larger NCS family including other KChIP/CALP proteins, phosphorylation of calsenilin may regulate a functional role that is unique to this member of the superfamily.

[Mineralization of the dental pulp: contributions of tissue engineering to tomorrow's therapeutics in odontology]. / Minéralisation de la pulpe dentaire: apports de l'ingénierie tissulaire aux thérapeutiques de demain en odontologie.

When bioactive molecules such as bone sialoprotein (BSP), bone morphogenetic protein-7 (BMP-7, also termed OP-1) and chondrogenic Inducing Agents (CIA, A+4 and A-4) were implanted in the pulp of the first upper molars, mineralizations were induced. They were either limited to the formation of a reparative dentinal bridge closing the pulpal wound (CIA A+4), or filled the mesial part of the coronal pulp (BSP), or filled totally the pulp located in the root canal (BMP-7 and CIA A-4). Consequently, these molecules may change in the next future the every day practice in dentistry.

Osteogenic proteins (bone sialoprotein and bone morphogenetic protein-7) and dental pulp mineralization.

Bone sialoprotein (BSP) cross-linked to collagen/gelatin was implanted in the pulp of rat's upper molars. Comparison was carried out with a sham group (non implanted), with a group of rats receiving the carrier alone, and a group of molars where the perforated pulps were capped with calcium hydroxide. The cavities were occluded with a glass-ionomer cement (GIC). After 8, 14 and 30 days respectively the rats were killed by intracardiac perfusion of the fixative and processed for light microscopy. Dentin and predentin debris pushed into the pulp during the preparation enhanced self-repair processes, with large pulp remnants. The carrier alone induced slight inflammation, and calcium hydroxide the formation of a reparative dentin bridge. BSP stimulated the recruitment of cells which produced an homogeneous atubular dentin-like structure, filling after one month the mesial third of the crown pulp. Osteogenic protein (OP-1) used in the same experimental conditions induced the formation of osteodentin in the coronal pulp and the radicular part of the pulp was totally filled by a mineralized material. The differences reported here suggest two possible different therapeutic approaches with the two osteogenic proteins, BSP inducing pulp mineralization in the crown part, and OP-1 occluding the root part of the pulp.

Application of bioactive molecules in pulp-capping situations.

To evaluate the effects of bioactive molecules in pulpal wound healing, we carried out experiments using the rat upper molars as an in vivo model. Cavities were prepared on the mesial aspect, and pulp perforation was accomplished by the application of pressure with the tip of a steel probe. After the pulp-capping procedure, the cavities were filled with a glass-ionomer cement. Comparison was made between and among: (1) sham-operated controls with dentin and predentin fragments implanted in the pulp during perforation after 8, 14, and 28 days; (2) carrier without bioactive substance; (3) calcium hydroxide; (4) Bone Sialoprotein (BSP); (5) different concentrations of Bone Morphogenetic Protein-7 (BMP-7), also termed Osteogenic Protein-1 (OP-1); and (6) N-Acetyl Cysteine (NAC), an anti-oxidant agent preventing glutathione depletion. Histologic and morphometric comparison, carried out among the first 4 groups on demineralized tissue sections, indicated that, at 28 days after implantation, BSP was the most efficient bioactive molecule, inducing homogeneous and well-mineralized reparative dentin. BMP-7 gave reparative dentin of the osteodentin type in the coronal part of the pulp, and generated the formation of a homogeneous mineralized structure in the root canal. These findings indicate that the crown and radicular parts of the pulp bear their own specificity. Both BSP and BMP-7 were superior to calcium hydroxide in their mineralization-inducing properties, and displayed larger areas of mineralization containing fewer pulp tissue inclusions. The overall mineralization process to these molecules appeared to proceed by mechanisms that involved the recruitment of cells which differentiate into osteoblast-like cells, producing a mineralizing extracellular matrix. We also provide preliminary evidence that NAC induces reparative dentin formation in the rat molar model. Pulp-capping with bioactive molecules provides new prospects for dental therapy.

An investigation into the role of oxygen free radical scavengers in preventing polymethylmethacrylate-induced necrosis in an osteoblast cell culture.

This study examined the effect of polymethylmethacrylate (PMMA) on osteocytic necrosis and the role of free radical scavengers in minimizing this damage. Bovine osteoblast cells with a characteristic phenotype were seeded at a density of 4x10(4) cells/cm2 and cultured in a DMEM supplemented with 10% fetal calf serum. A transwell insert with 2 cc of PMMA was suspended above the culture, and a time log response curve was established following elusion of free radicals around the osteoblast media. Chemiluminescence was used to determine quantitative free radical release. Using a Student's two-tailed t test there was a significant difference in the amount of hydroxyl radical released at 1-6 hours compared with controls (P=.028). Using histologic markers, there was a significant correlation between the use of PMMA and osteoblast cell necrosis. Transwell plates were coated with varying concentrations of mannitol, a known hydroxyl radical scavenger. A log dose response curve was established. There was a clear statistical association between a 10% mannitol solution and a reduction in the free radical release from PMMA (P=.03). Similarly, using Trypan blue histologic staining, there was a significant reduction in PMMA-induced cell necrosis when 10% mannitol was used as a scavenger (P=.01). A Rockwell superficial hardness test was used to determine whether mannitol had any effect on the surface hardness of the polymer. No statistical difference could be found between those treated with mannitol and controls at a depth of up to 1 mm. These results demonstrate hydroxyl radical is released from the polymerization reaction of PMMA. These radicals cause cell death in an osteoblast culture medium. This has been addressed using a 10% mannitol solution, which reduced cell necrosis.

Bone sialoprotein-induced reparative dentinogenesis in the pulp of rat's molar.

Bone sialoprotein (BSP), an osteogenic protein (OP), mixed with a carrier, was implanted in the pulp of rat first upper molars (OP group). Cavities were prepared with dental burs and pulp perforation was carried out by pressure with the tip of a steel probe. After 8, 14, and 30 days, the rats were killed and the pulps of the OP group were compared with (1) a sham group (S group), (2) a group where the carrier was implanted alone (C group), and (3) capping with calcium hydroxide (Ca group). After 8 days, a few inflammatory cells were seen, mostly located at the pulp surface near the perforation. In the Ca group, a dentin bridge started to form, in contrast to the other groups. After 15 days, globular structures were seen in the pulps of the S and C groups. A reparative osteodentin bridge isolated the pulp from the cavity in the Ca group. Variable reactions were seen in the OP group, with some evidence of cell and matrix alignments or plugs of osteodentin in continuity with an inner layer of reparative dentin. After 30 days, irregular osteodentin formation was observed in the pulps of the S and C groups, with a tendency for globular structures to merge, but with interglobular spaces filled by pulp remnants. In the Ca group, osteodentin was observed in the mesial part of the pulp chamber. In the BSP-implanted group, the osteogenic protein stimulated the formation of a homogeneous dentin-like deposit occupying most of the mesial part of the pulp. Apparently, BSP stimulates the differentiation of cells which secrete an organized extracellular matrix more efficiently than any other capping material used so far. Altogether, the results reported here support that bone sialoprotein displays novel bioactive properties and is capable of stimulating in 1 month's time the development of a thick reparative dentinal tissue in the pulp, occluding the perforation and filling the mesial third of the pulp chamber.

Expression of bone microsomal casein kinase II, bone sialoprotein, and osteopontin during the repair of calvarial defects.

The temporal expression of bone microsomal casein kinase II, osteopontin, bone sialoprotein, alkaline phosphatase, and the accumulation of a solid calcium-inorganic orthophosphate mineral phase, have been charted from day 2 to day 21 during the repair of calvarial defects in rats induced by the implantation of decalcified rat bone matrix. Unlike the sequence of events that occur when the same decalcified bone matrix is implanted subcutaneously or intramuscularly, in which cases the first tissue to form in response to the implant is cartilage that subsequently calcifies and is later resorbed and replaced by bone, the repair of cranial defects is quite different. In the latter case, the first cells induced are undifferentiated mesenchymal cells and early fibroblasts followed by osteoblastic direct bone formation. Somewhat later a few small islands of cartilage are formed, widely separated and spatially distinct from the newly formed bone matrix. All of the cartilage and most of the implanted decalcified bone matrix are later resorbed and replaced by new bone by day 21. This in vivo model of the repair of a bone defect by direct bone formation has provided an excellent system to follow specific biochemical and physicochemical events. The total accumulation and rate of accumulation of the mineral and the two noncollagenous phosphoproteins (bone sialoprotein and osteopontin), as well as the activities of alkaline phosphatase, and for the first time either in vivo or in cell culture, the activity of microsomal casein kinase II, the major enzyme that phosphorylates the bone phosphoproteins, have been determined as a function of healing time in vivo. The overall general pattern of accumulation of the phosphoproteins and calcium-phosphate mineral phase and their relationships are similar to those reported in osteoblast cell cultures also monitored as a function of time.

Isolation of a novel bone glycosylated phosphoprotein with disulphide cross-links to osteonectin.

An 80 kDa protein was purified from calf bone by HCl-demineralization followed by 0.5 M EDTA/1.0 M NaCl extraction and sequential chromatography on DE-52, hydroxyapatite, and TSK-gel G3000SW HPLC columns. From the DE-52 column the protein was eluted at three different fractions, of which one further separated into two fractions on the hydroxyapatite column, indicating that the protein is present in four different molecular forms designated as 80 k-I-1, k-I-2, k-II, k-III. The N-terminal sequence analysis of all four forms gave the same sequence, SEQYNQEPNNV. Several tryptic internal peptides were also generated, purified and sequenced, leading to the identification of several repeat sequences, IFLGXXEI. Homology searching of the N-terminal and internal sequences indicates that this is a novel protein. Both 80 k-I-2 and k-III had similar amino acid composition with high contents of Asx, Glx and Leu and contained 7 and 16 phosphoserines per 1000 total amino acids, respectively. The 80 k-I-1 and 80 k-II forms were stained with Rhodamine B specific for phosphoproteins. The four forms contained different contents of neutral sugars ranging from 5.5 to 26% (w/w protein) and approximately 1.7% sialic acid. These data indicated that the 80 kDa protein exists in four isomeric forms, at least based on the different post-translational modifications. The evaluation of the 80 kDa glycosylated phosphoprotein under alkylating, reducing and non-reducing conditions indicated that this protein undergoes polymerization through intermolecular disulphide bonds. Furthermore, the 80 kDa protein and osteonectin (ON), both of which are cysteine-rich proteins, can cross-link with each other via disulphide bonds, and this process can be induced to take place in vitro under experimental conditions. The occurrence of such a phenomenon in vivo was confirmed from the presence of similar high Mr components containing both 80 kDa and ON in the same SDS/PAGE bands, detected by the respective antibody reactions in crude bone extracts which were extracted in the presence of alkylating agent.

Enamel specific protein kinases and state of phosphorylation of purified amelogenins.

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear + plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of 32P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca2+ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca2+ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca2+, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly CaZ+ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of 14C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, 14C-mercaptoethanol, 0.64 moles 14C-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by 14C-mercaptoethanol, providing 0.46 and 0.88 moles 14C-incorporated/mole respectively. Further studies of the 14C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.

Identification of the phosphorylated sites of metabolically 32P-labeled osteopontin from cultured chicken osteoblasts.

Osteopontin (OPN) is one of the major secretory phosphoproteins in both calcifying and non-calcifying tissues. Evidence has accumulated for the biological importance of the phosphoproteins and, in particular, the phosphate groups in bone formation, resorption, and calcification. The precise locations of the phosphate groups in the OPN molecule were determined by metabolically labeling OPN with 32P in cultured chicken osteoblasts, followed by purification to homogeneity. N-terminal sequencing showed a single sequence of WPVSKRQHAISA, consistent with that deduced from both cDNA, and previous amino acid sequencing of the protein isolated from chicken bone. Three 32P-labeled peptides were isolated by reverse-phase high performance liquid chromatography of thrombin-digested, 32P-labeled OPN. The N-terminal sequencing of each of these thrombin fragments gave single sequences as follows: WPVSKSRQHAIS, SHHTHRYHQDHVD, and ASKLRKAARKL, with approximate molecular masses of 5, 30, and 20 kDa. These data demonstrate that 32P was incorporated throughout the N- to C-terminal sequence of the protein. Thrombin specifically cleaved chicken OPN at two sites: between Arg-22 and Ser-23, which generated the 5-kDa N-terminal end fragment, and another between Lys-138 and Ala-139, which generated the 30- and 20-kDa fragments. To further define the exact locations of the phosphorylated amino acids and the surrounding amino acid sequences, OPN was digested with trypsin, which generated seven major 32P-labeled peptides whose amino acid sequences were determined. The phosphorylated peptide regions of osteopontin were identified as amino acids 8-18 (QHAIS*AS*S*EEK), 39-54 (LASQQTHYS*S*EENAD), 150-171 (LIEDDAT*AEVGDSQLAGLWLPK), 179-191 (ELAQHQSVENDSR), 194-205 (FDS*PEVGGDSK), 214-219 (ES*LASR), and 239-248 (HSIENNEVTR). The phosphorylated amino acid sites are followed by an asterisk (*). Of the seven identified phosphorylated peptide regions, three were localized on the N-terminal end of the osteopontin molecule (with five phosphorylated serines) and contained the sequence motifs that were phosphorylated by casein kinase II type(s), whereas the remaining four peptides are concentrated toward the C-terminal half of the molecule (with five phosphorylated residues) and contained recognition motifs for other kinases as well as casein kinase II.

Protein kinases of cultured osteoblasts: selectivity for the extracellular matrix proteins of bone and their catalytic competence for osteopontin.

The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with M(r)'s of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, M(r)'s 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.

Phosphorylation of purified bovine bone sialoprotein and osteopontin by protein kinases.

The large number of covalently bound phosphates on the extracellular phosphoproteins osteopontin (OPN) and bone sialoprotein (BSP) have been implicated in biological functions such as mineral deposition and osteoclast binding. In the present study the state of phosphorylation of BSP and OPN was evaluated by in vitro 32P labeling using a series of protein kinases and quantification. Both the purified bovine BSP and OPN were radiolabeled by [32P]ATP and factor-independent protein kinase. Quantification of 32P radioactivity incorporated on dephosphorylated BSP and OPN provided 6.6 and 8.9 mol of phosphate incorporated/mol, respectively. Native OPN incorporated 1.07 and BSP 2.46 mol of phosphate/mol by factor-independent protein kinase. These data led to calculations that OPN and BSP, respectively, contain 7.83 and 4.14 mol of phosphate/mol in their natural state. Thrombin digests of 32P-labeled BSP showed radioactivity to be associated with fragment of approximately molecular mass values 30 kDa (N-terminal half), with no observable radioactivity associated with the 40-kDa fragment (C-terminal half). Similar experiments with 32P-labeled OPN provided two radiolabeled thrombin fragments, with molecular mass 30 kDa (N-terminal half) and 20 kDa (C-terminal half), both were radioactive. The major phosphorylation was associated with the N-terminal half containing 7.0 mol of phosphate, and 1.9 mol of phosphate were associated with the C-terminal half. Additional experiments of in vitro phosphorylation of OPN and BSP by several other known protein kinases were carried out. cAMP-dependent protein kinase showed no phosphorylation of OPN or BSP, while protein kinase C and cGMP-dependent protein kinase led to minor phosphorylation, each of the latter introduced about 1 mol of phosphate/mol of OPN and BSP molecule.