Farnesal-loaded pH-sensitive polymeric micelles provided effective prevention and treatment on dental caries.

BACKGROUND: Farnesol is a sesquiterpene from propolis and citrus fruit that shows promising anti-bacterial activity for caries treatment and prevention, but its hydrophobicity limits the clinical application. We aimed to develop the novel polymeric micelles (PMs) containing a kind of derivative of farnesol and a ligand of pyrophosphate (PPi) that mediated PMs to adhere tightly with the tooth enamel. RESULTS: Farnesal (Far) was derived from farnesol and successfully linked to PEG via an acid-labile hydrazone bond to form PEG-hyd-Far, which was then conjugated to PPi and loaded into PMs to form the aimed novel drug delivery system, PPi-Far-PMs. The in vitro test about the binding of PPi-Far-PMs to hydroxyapatite showed that PPi-Far-PMs could bind rapidly to hydroxyapatite and quickly release Far under the acidic conditions. Results from the mechanical testing and the micro-computed tomography indicated that PPi-Far-PMs could restore the microarchitecture of teeth with caries. Moreover, PPi-Far-PMs diminished the incidence and severity of smooth and sulcal surface caries in rats that were infected with Streptococcus mutans while being fed with a high-sucrose diet. The anti-caries efficacy of free Far can be improved significantly by PPi-Far-PMs through the effective binding of it with tooth enamel via PPi. CONCLUSIONS: This novel drug-delivery system may be useful for the treatment and prevention of dental caries as well as the targeting therapy of anti-bacterial drugs in the oral disease.

Oral Supplementation with Sucrosomial Ferric Pyrophosphate Plus L-Ascorbic Acid to Ameliorate the Martial Status: A Randomized Controlled Trial.

Altered martial indices before orthopedic surgery are associated with higher rates of complications and greatly affect the patient's functional ability. Oral supplements can optimize the preoperative martial status, with clinical efficacy and the patient's tolerability being highly dependent on the pharmaceutical formula. Patients undergoing elective hip/knee arthroplasty were randomized to be supplemented with a 30-day oral therapy of sucrosomial ferric pyrophosphate plus L-ascorbic acid. The tolerability was 2.7% among treated patients. Adjustments for confounding factors, such as iron absorption influencers, showed a relevant response limited to older patients (≥ 65 years old), whose uncharacterized Hb loss was averted upon treatment with iron formula. Older patients with no support lost -2.8 ± 5.1%, while the intervention group gained +0.7 ± 4.6% of circulating hemoglobin from baseline (p = 0.019). Gastrointestinal diseases, medications, and possible dietary factors could affect the efficacy of iron supplements. Future opportunities may consider to couple ferric pyrophosphate with other nutrients, to pay attention in avoiding absorption disruptors, or to implement interventions to obtain an earlier martial status optimization at the population level.

High Bioavailability from Ferric Pyrophosphate-Fortified Bouillon Cubes in Meals is Not Increased by Sodium Pyrophosphate: a Stable Iron Isotope Study in Young Nigerian Women.

BACKGROUND: It is challenging to find an iron compound that combines good bioavailability with minimal sensory changes when added to seasonings or condiments. Ferric pyrophosphate (FePP) is currently used to fortify bouillon cubes, but its bioavailability is generally low. Previously, the addition of a stabilizer, sodium pyrophosphate (NaPP), improved iron bioavailability from a bouillon drink. OBJECTIVE: We assessed whether there is a dose-response effect of added NaPP on iron bioavailability from local meals prepared with intrinsically labeled FePP-fortified bouillon cubes in young Nigerian women using iron stable isotope techniques. METHODS: In a double-blind, randomized, cross-over trial, women (n = 24; aged 18-40 y; mean BMI 20.5 kg/m2) consumed a Nigerian breakfast and lunch for 5 d prepared with bouillon cubes containing 2.5 mg 57Fe (as FePP) and 3 different molar ratios of NaPP: 57Fe (0:1, 3:1, and 6:1). Iron bioavailability was assessed by measuring 57Fe incorporation into erythrocytes 16 d after each 5 d NaPP: 57Fe feeding period. Data were analyzed using a linear regression model of log iron absorption on NaPP ratio, with body weight and baseline body iron stores as covariates and subject as a random intercept. RESULTS: Of the women included, 46% were anemic and 26% were iron deficient. Iron bioavailability was 10.8, 9.8, and 11.0% for the 0:1, 3:1, and 6:1 NaPP:57Fe treatments, respectively. There was no dose-response effect of an increasing NaPP:57Fe ratio (ß ± SE: 0.003 ± 0.028, P = 0.45). CONCLUSIONS: In this study, the addition of NaPP did not increase iron bioavailability from FePP-fortified bouillon cubes. However, iron bioavailability from the Nigerian meals prepared with FePP-fortified bouillon cubes was higher than expected. These results are encouraging for the potential of bouillon cubes as a fortification vehicle. Further studies are needed to assess the effect of FePP-fortified bouillon cubes on improving iron status in low-income populations. This trial was registered at clinicaltrials.gov as NCT02815449.

Sucrosomial® Iron Supplementation in Mice: Effects on Blood Parameters, Hepcidin, and Inflammation.

Sucrosomial® Iron is a recently developed formulation to treat iron deficiency based on ferric pyrophosphate covered by a matrix of phospholipids plus sucrose esters of fatty acids. Previous data indicated that Sucrosomial® Iron is efficiently absorbed by iron-deficient subjects, even at low dosage, and without side effects. Its structural properties may suggest that it is absorbed by an intestinal pathway which is different to the one used by ionic iron. Although, studies in vitro showed that Sucrosomial® Iron is readily absorbed, no animal models have been established to study this important aspect. To this aim, we induced iron deficient anemia in mice by feeding them with a low-iron diet, and then we treated them with either Sucrosomial® Iron or sulfate iron by gavage for up to two weeks. Both iron formulations corrected anemia and restored iron stores in a two-week period, but with different kinetics. Ferrous Sulfate was more efficient during the first week and Sucrosomial® Iron in the second week. Of note, when given at the same concentrations, Ferrous Sulfate induced the expression of hepcidin and four different inflammatory markers (Socs3, Saa1, IL6 and CRP), while Sucrosomial® Iron did not. We conclude that anemic mice are interesting models to study the absorption of oral iron, and that Sucrosomial® Iron is to be preferred over Ferrous Sulfate because of similar absorption but without inducing an inflammatory response.

Drug therapy targeting pyrophosphate slows the ossification of spinal ligaments in twy mice.

The lack of an effective drug therapy against ossification of spinal ligament (OSL) warrants investigation into the therapeutic target of this disease. An endogenous inhibitor of biomineralization, pyrophosphate (PPi) is a potential therapy for ectopic ossification; however, exogenous PPi is rapidly hydrolyzed by tissue non-specific alkaline phosphatase (TNAP) present in body fluids. In this study, we examined whether a drug therapy targeting PPi is efficacious for the treatment of OSL using the Enpp1ttw/ttw (twy) mouse model. Twenty male twy mice were randomized into four groups: (i) vehicle (Control); (ii) alkaline phosphatase inhibitor levamisole (5 mg/kg/day sc continuously); (iii) levamisole + exogenous PPi (160 µmol/kg/day sc continuously); and (iv) nuclear retinoic acid receptor-γ (RARγ) agonist (6 µg/kg sc daily). The RARγ agonist, which is a proven inhibitor of ectopic endochondral ossification, was used as a positive control. Treatments commenced when the mice were 5 weeks of age and continued for 4 weeks. Longitudinal micro-computed tomography and postmortem histological analysis were performed. Administration of levamisole alone and in combination with PPi increased serum PPi concentration by 17% and 52%, respectively, compared to that in vehicle-treated mice. The development of OSL in twy mice was suppressed by levamisole + PPi and RARγ agonist treatments, but not by levamisole alone. The levamisole + PPi therapy did not cause osteoporosis, whereas RARγ agonist-treated mice developed osteoporosis. Treatment of twy mice with levamisole in combination with exogenous PPi increased serum PPi level, which slowed the progression of OSL without producing adverse effect on bone. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1256-1261, 2018.

Sodium pyrophosphate enhances iron bioavailability from bouillon cubes fortified with ferric pyrophosphate.

Fe fortification of centrally manufactured and frequently consumed condiments such as bouillon cubes could help prevent Fe deficiency in developing countries. However, Fe compounds that do not cause sensory changes in the fortified product, such as ferric pyrophosphate (FePP), exhibit low absorption in humans. Tetra sodium pyrophosphate (NaPP) can form soluble complexes with Fe, which could increase Fe bioavailability. Therefore, the aim of this study was to investigate Fe bioavailability from bouillon cubes fortified with either FePP only, FePP+NaPP, ferrous sulphate (FeSO4) only, or FeSO4+NaPP. We first conducted in vitro studies using a protocol of simulated digestion to assess the dialysable and ionic Fe, and the cellular ferritin response in a Caco-2 cell model. Second, Fe absorption from bouillon prepared from intrinsically labelled cubes (2·5 mg stable Fe isotopes/cube) was assessed in twenty-four Fe-deficient women, by measuring Fe incorporation into erythrocytes 2 weeks after consumption. Fe bioavailability in humans increased by 46 % (P<0·005) when comparing bouillons fortified with FePP only (4·4 %) and bouillons fortified with FePP+NaPP (6·4 %). Fe absorption from bouillons fortified with FeSO4 only and with FeSO4+NaPP was 33·8 and 27·8 %, respectively (NS). The outcome from the human study is in agreement with the dialysable Fe from the in vitro experiments. Our findings suggest that the addition of NaPP could be a promising strategy to increase Fe absorption from FePP-fortified bouillon cubes, and if confirmed by further research, for other fortified foods with complex food matrices as well.

Targeting tumour hypoxia to improve outcome of stereotactic radiotherapy.

BACKGROUND: Hypoxia is a characteristic feature of solid tumours that significantly reduces the efficacy of conventional radiation therapy. In this study we investigated the role of hypoxia in a stereotactic radiation schedule by using a variety of hypoxic modifiers in a preclinical tumour model. MATERIAL AND METHODS: C3H mammary carcinomas were irradiated with 3 × 15 Gy during a one-week period, followed three days later by a clamped top-up dose to produce a dose response curve; the endpoint was tumour control. The hypoxic modifiers were nimorazole (200 mg/kg), nicotinamide (120 mg/kg) and carbogen (95% O2 + 5% CO2) breathing, OXi4503 (10 mg/kg), and hyperthermia (41.5°C; 1 h). RESULTS: The radiation dose controlling 50% of clamped tumours (TCD50) following 3 × 15 Gy was 30 Gy. Giving nimorazole or nicotinamide+ carbogen prior to the final 15 Gy fraction non-significantly (χ(2)-test; p < 0.05) reduced this TCD50 to 20-23 Gy; when administered with each 3 × 15 Gy fraction these values were significantly reduced to ≤ 2.5 Gy. Injecting OXi4503 or heating after irradiating significantly reduced the TCD50 to 9-12 Gy regardless of whether administered with one or all three 15 Gy fractions. Combining OXi4503 and heat with the final 15 Gy had a significantly larger effect (TCD50 = 2 Gy). CONCLUSIONS: Clinically relevant modifiers of hypoxia effectively enhanced an equivalent stereotactic radiation treatment confirming the importance of hypoxia in such schedules.

Extracellular matrix mineralization promotes E11/gp38 glycoprotein expression and drives osteocytic differentiation.

Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype.

Efficacy of a microencapsulated iron pyrophosphate-fortified fruit juice: a randomised, double-blind, placebo-controlled study in Spanish iron-deficient women.

Fe-deficiency anaemia is a worldwide health problem. We studied the influence of consuming an Fe-fortified fruit juice on Fe status in menstruating women. A randomised, double-blind, placebo-controlled study of 16 weeks of duration was performed. Subjects were randomised into two groups: the P group (n 58) or the F group (n 64), and consumed, as a supplement to their usual diet, 500 ml/d of a placebo fruit juice or an Fe-fortified fruit juice, respectively. The Fe-fortified fruit juice, containing microencapsulated iron pyrophosphate, provided 18 mg Fe/d (100 % of the RDA). At baseline and monthly, dietary intake, body weight and Fe parameters were determined: total erythrocytes, haematocrit, mean corpuscular volume (MCV), red blood cell distribution width (RDW), Hb, serum Fe, serum ferritin, serum transferrin, transferrin saturation, soluble transferrin receptor (sTfR) and zinc protoporphyrin (ZnPP). The fruit juice consumption involved increased intake of carbohydrates and vitamin C, and increased BMI within normal limits. Ferritin was higher in the F group after week 4 (P < 0·05) and became 80 % higher than in the P group after week 16 (P < 0·001), and transferrin decreased in the F group compared with the P group after week 4 (P < 0·001). RDW was higher at weeks 4 and 8 in the F group compared with the P group (P < 0·05). Transferrin saturation increased after week 8, and haematocrit, MCV and Hb increased after week 12, in the F group compared with the P group. Serum Fe did not change. sTfR and ZnPP decreased in the F group at week 16 (P < 0·05). Iron pyrophosphate-fortified fruit juice improves Fe status and may be used to prevent Fe-deficiency anaemia.

Control of pyrimidine nucleotide formation in Pseudomonas fulva.

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 50-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.

Regular consumption of a complementary food fortified with ascorbic acid and ferrous fumarate or ferric pyrophosphate is as useful as ferrous sulfate in maintaining hemoglobin concentrations >105 g/L in young Bangladeshi children.

BACKGROUND: Non-water-soluble iron compounds have been reported to be less well absorbed than ferrous sulfate in young children, and concern has been raised about their usefulness as food fortificants. OBJECTIVE: The objective was to evaluate the usefulness of ferrous fumarate and ferric pyrophosphate, compared with ferrous sulfate, in maintaining hemoglobin concentrations >105 g/L in Bangladeshi children. DESIGN: Two hundred thirty-five children aged 7-24 mo (hemoglobin >105 g/L) were randomly assigned in a double-blind study to receive an infant cereal fortified with ferrous fumarate, ferric pyrophosphate, or ferrous sulfate. One serving of cereal (9.3 mg Fe; molar ratio of ascorbic acid to iron of 3:1) was consumed per day, 6 d/wk, for 9 mo. Blood samples were drawn at 4.5 and 9 mo. RESULTS: Raw data were reformatted, and a "time to event" was calculated that corresponded to reaching the following thresholds: hemoglobin <105 g/L, plasma ferritin <12 microg/L, or plasma C-reactive protein >10 mg/L at baseline, 4.5 mo, or 9 mo. Data were censored when children did not reach the threshold or were lost to follow-up. A Kaplan-Meier approach was used to compare the 3 groups. No statistically significant differences were observed for hemoglobin <105 g/L (P = 0.943), plasma ferritin <12 microg/L (P = 0.601), or plasma C-reactive protein >10 mg/L (P = 0.508). CONCLUSIONS: Contrary to earlier concerns, these results do not indicate differences in usefulness between water-soluble and non-water-soluble iron compounds in maintaining hemoglobin concentrations and preventing iron deficiency. These data will be important in the development of food-fortification strategies to combat anemia and iron deficiency in highly vulnerable population groups.

Phytate inhibits bovine pericardium calcification in vitro.

OBJECTIVE: The present study examined the inhibitory effects of pyrophosphate, etidronate, and phytate on bovine pericardium calcification in vitro. METHODS: Bovine pericardium was glutaraldehyde fixed and then placed in a flow chamber in the presence of a synthetic physiological fluid alone (control) or the fluid plus various concentrations of pyrophosphate, etidronate, or phytate. Following a 96-h incubation, fragments were removed and assayed for calcification by measuring calcium and phosphorus levels. RESULTS: The data indicated that both pyrophosphate and etidronate at 1 mg/l (5.75 and 4.95 microM, respectively) inhibited bovine pericardium calcification, whereas neither agent had an effect at 0.5 mg/l (2.87 and 2.47 microM, respectively). Phytate was the most potent inhibitor of calcification, and the effects of this agent were apparent at levels as low as 0.25 mg/l (0.39 microM). CONCLUSIONS: While pyrophosphate, etidronate, and phytate were all able to inhibit bovine pericardium calcification in vitro, phytate was found to be the most effective.

Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate.

Extracellular nucleotides, signaling through P2 receptors, may act as local regulators of bone cell function. We investigated the effects of nucleotide agonists [ATP, ADP, uridine triphosphate (UTP), and uridine diphosphate] and pyrophosphate (PPi, a key physiological inhibitor of mineralization) on the deposition and mineralization of collagenous matrix by primary osteoblasts derived from rat calvariae. Our results show that extracellular ATP, UTP, and PPi strongly and selectively blocked the mineralization of matrix nodules; ADP and uridine diphosphate were without effect. Significant inhibition of mineralization occurred in the presence of relatively low concentrations of ATP, UTP, or PPi (1-10 microm), without affecting production of fibrillar or soluble collagen. In cultures treated with 10 microm ATP or UTP, the expression and activity of alkaline phosphatase, which promotes mineralization by hydrolyzing PPi, was inhibited. The potent inhibitory actions of ATP and UTP on bone mineralization are consistent pharmacologically with mediation by the P2Y(2) receptor, which is strongly expressed by mature osteoblasts. In support of this notion, we found 9-17% increases in bone mineral content of hindlimbs of P2Y(2)-deficient mice. We also found that osteoblasts express ectonucleotide phosphodiesterase/pyrophosphatase-1, an ectonucleotidase that hydrolyzes nucleotide triphosphates to yield PPi, and that addition of 10 microm ATP or UTP to osteoblast cultures generated 2 microm PPi within 10 min. Thus, a component of the profound inhibitory action of ATP and UTP on bone mineralization could be mediated directly by PPi, independently of P2 receptors.

Extruded rice fortified with micronized ground ferric pyrophosphate reduces iron deficiency in Indian schoolchildren: a double-blind randomized controlled trial.

BACKGROUND: Iron fortification of rice could be an effective strategy for reducing iron deficiency anemia in South Asia. OBJECTIVE: We aimed to determine whether extruded rice grains fortified with micronized ground ferric pyrophosphate (MGFP) would increase body iron stores in children. DESIGN: In a double-blind, 7-mo, school-based feeding trial in Bangalore, India, iron-depleted, 6-13-y-old children (n = 184) were randomly assigned to receive either a rice-based lunch meal fortified with 20 mg Fe as MGFP or an identical but unfortified control meal. The meals were consumed under direct supervision, and daily leftovers were weighed. All children were dewormed at baseline and at 3.5 mo. Iron status and hemoglobin were measured at baseline, 3.5 mo, and 7 mo. RESULTS: At baseline, the prevalences of iron deficiency and iron deficiency anemia in the total sample were 78% and 29%, respectively. After 7 mo of feeding, there was a significant increase in body iron stores in both study groups (P < 0.001), with a greater increase in the iron group than in the control group (P < 0.05). There was a significant time x treatment interaction for iron deficiency, which fell from 78% to 25% in the dewormed iron group and from 79% to 49% in the dewormed control group. Iron deficiency anemia decreased from 30% to 15% (NS) in the iron group but remained virtually unchanged in the control group (28% and 27%). In sensory tests, the MGFP-fortified rice (fortified at 3 and 5 mg Fe/100 g) was indistinguishable from natural rice, in both cooked and uncooked form. CONCLUSIONS: Extruded rice fortified with MGFP has excellent sensory characteristics. Fed in a school lunch meal, it increases iron stores and reduces the prevalence of iron deficiency in Indian children.

Potentiation of antigen-stimulated V gamma 9V delta 2 T cell cytokine production by immature dendritic cells (DC) and reciprocal effect on DC maturation.

Vgamma9Vdelta2 T cells, a major gammadelta PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vgamma9Vdelta2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vgamma9Vdelta2 T cell clones and ex vivo memory Vgamma9Vdelta2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vgamma9Vdelta2 production of inflammatory cytokines required for their own maturation suggested that Vgamma9Vdelta2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vgamma9Vdelta2 cells induced accelerated maturation of Ag-expressing iDC but not "bystander" DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vgamma9Vdelta2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vgamma9Vdelta2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vgamma9Vdelta2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.

Effects of two types of emulsifying salts on the functionality of nonfat pasta filata cheese.

Effects of 2 types of emulsifying salts (ES) on the functionality of nonfat pasta filata cheese were examined. Nonfat pasta filata cheese was made from skim milk by direct acidification. Trisodium citrate (TSC) and tetrasodium pyrophosphate (TSPP) were added to curds (at 1, 3, and 5%, wt/wt) at the dry-salting step, together with glucono-delta-lactone to maintain a constant pH. When TSC was added, there were no significant compositional differences, although insoluble Ca and P contents significantly decreased with the addition of TSC. When TSPP was added, fat content was not significantly different, but protein content decreased with increasing concentrations of TSPP. Both insoluble Ca and P contents increased with the addition of 1% TSPP. The addition of ES affected textural and functional properties. With increasing concentrations of TSC, meltability increased, whereas increasing the TSPP content decreased meltability. Cheese made with 1% TSC had better stretchability compared with control cheese. However, the addition of more than 3% TSC decreased stretchability. Addition of TSPP caused a considerable decrease in stretchabilty. Scanning electron microscopy revealed that the size and number of serum pockets decreased and protein appeared more hydrated with the addition of both ES. These results suggested that TSC and TSPP influenced the functionality of nonfat pasta filata cheese differently; that is, the effects of TSC were probably caused by a decrease in the number of colloidal calcium phosphate cross-links and an increase in electrostatic repulsion, whereas the effects of TSPP may have been related to the formation of new TSPP-induced casein-casein interactions.

Structural and functional characterization of rabbit and human L-gulonate 3-dehydrogenase.

L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH.

Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1.

Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.

Regulation of the DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol and growth phase. Inhibition of DGPP phosphatase activity by CDP-diacylglyceron and activation of phosphatidylserine synthase activity by DGPP.

The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Delta, ino2Delta, and ino4Delta phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K(m) and a decrease in V(max). DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Delta mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.