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In this case report of autosomal recessive pigmented hypomaturation amelogenesis imperfecta (AI), we identify a novel homozygous missense mutation (g.165151 T>G; c.1317 T>G; p.Leu436 Arg) in SLC24A4, a gene encoding a potassium-dependent sodium-calcium exchanger that is critical for hardening dental enamel during tooth development.

An effective approach to inhibiting the consumption of Cu during multiple reflows of SnAg2.3 solder on Cu is reported. By depositing a very thin layer of solder on Cu, followed by a 10-min reflow, the scallop-type morphology of interfacial Cu 6 Sn 5 intermetallic compounds (IMC) became flat, and the channels between them closed up. When additional solder was deposited on the sample and reflowed again, the consumption of Cu as well as the growth the IMC was retarded.

Enamel formation depends on a triad of tissue-specific matrix proteins (amelogenin, ameloblastin, and enamelin) to help initiate and stabilize progressively elongating, thin mineral ribbons of hydroxyapatite formed during an appositional growth phase. Subsequently, these proteins are eradicated to facilitate lateral expansion of the hydroxyapatite crystallites. The purpose of this study was to investigate changes in enamel mineralization occurring in mice unable to produce kallikrein 4 (Klk4), a proteinase associated with terminal extracellular degradation of matrix proteins during the maturation stage. Mice lacking functional matrix metalloproteinase 20 (Mmp20), a proteinase associated with early cleavage of matrix proteins during the secretory stage, were also analyzed as a frame of reference. The results indicated that mice lacking Klk4 produce enamel that is normal in thickness and overall organization in terms of layers and rod/inter-rod structure, but there is a developmental defect in enamel rods where they first form near the dentinoenamel junction. Mineralization is normal up to early maturation after which the enamel both retains and gains additional proteins and is unable to mature beyond 85% mineral by weight. The outmost enamel is hard, but inner regions are soft and contain much more protein than normal. The rate of mineral acquisition overall is lower by 25%. Mice lacking functional Mmp20 produce enamel that is thin and structurally abnormal. Relatively high amounts of protein remain throughout maturation, but the enamel is able to change from 67 to 75% mineral by weight during maturation. These findings reaffirm the importance of secreted proteinases to enamel mineral acquisition.

Four types of pyramidal sonic nozzles made of silicon crystal were studied experimentally. The throat sizes varied from 38 to 140 μm for type A and D nozzles and from 75 to 188 μm for type B and C nozzles. For each of the nozzle types, the results show that the discharge coefficient is proportional to the throat size, and the critical back pressure ratio for choking is insensitive to Reynolds’ number. In parallel, the flow field of a type B nozzle was investigated by numerical simulation. The effect of heat flux coming from the nozzle body was examined and the flow patterns obtained from Spalart–Allmaras and standard k − ω turbulence models were compared. The simulation results indicate the heat flux does not noticeably change the velocity field and discharge coefficient. Also, the flow downstream of the nozzle throat develops into an under-expanded supersonic jet in which expansion and oblique shock waves appear alternately.

Dynamic events such as machine breakdown and hot jobs may induce problems on the production system such as order delay, increasing machine load, and changing inventory level. Past studies of dynamic events often use traditional design of experiments (DOE) to analyze the effects of dynamic events on system's performance. The shortcoming of this approach is that the number of experimental runs conducted would become exponentially increased as the number of factors increased. This study tries to use frequency domain methodology (FDM) instead so as to detect the higher order effects and rank important factors in a few experimental runs. Spectrum analysis is used to comprehend the effects of different location of machine breakdown and different size of hot jobs on the system's performance of flowshops with different traffic (utilization) and stability (oscillation). This study finds that the important factors identified by the FDM analysis are the same as that of DOE. However, only in some cases can the rankings of important factors be the same for both approaches. The dissimilarity between rankings of important factors found by these two methods is further measured using Kendall tau distance.

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) staining demonstrated expression of beta-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.

Dentin sialophosphoprotein (DSPP) is critical for proper mineralization of tooth dentin, and mutations in DSPP cause inherited dentin defects. Dentin phosphoprotein (DPP) is the C-terminal cleavage product of DSPP that binds collagen and induces intrafibrillar mineralization. We isolated DPP from individual pigs and determined that its N-terminal and C-terminal domains are glycosylated and that DPP averages 155 phosphates per molecule. Porcine DPP is unstable at low pH and high temperatures, and complexing with collagen improves its stability. Surprisingly, we observed DPP size variations on SDS-PAGE for DPP isolated from individual pigs. These variations are not caused by differences in proteolytic processing or degrees of phosphorylation or glycosylation, but rather to allelic variations in Dspp. Characterization of the DPP coding region identified 4 allelic variants. Among the 4 alleles, 27 sequence variations were identified, including 16 length polymorphisms ranging from 3 to 63 nucleotides. None of the length variations shifted the reading frame, and all localized to the highly redundant region of the DPP code. The 4 alleles encode DPP domains having 551, 575, 589, or 594 amino acids and completely explain the DPP size variations. DPP length variations are polymorphic and are not associated with dentin defects.

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam+/- mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam-/- mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam-/- mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.

Amelogenesis imperfecta (AI) is a collection of diverse inherited disorders featuring dental-enamel defects in the absence of significant nondental symptoms. AI phenotypes vary and are categorized as hypoplastic, hypocalcified, and hypomaturation types. Phenotypic specificity to enamel has focused research on genes encoding enamel-matrix proteins. We studied two families with autosomal-dominant hypocalcified AI and have identified nonsense mutations (R325X and Q398X) in the FAM83H gene on chromosome 8q24.3. The mutations perfectly cosegregate with the disease phenotype and demonstrate that FAM83H is required for proper dental-enamel calcification.

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper tooth dentin formation. During dentinogenesis, DSPP is proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps, and failure to make these cleavages is a potential cause of developmental tooth defects. We tested the hypothesis that dentin-resident matrix metalloproteinases catalyze the cleavages that process DSPP. We defined the exact DSPP cleavages that are catalyzed by proteases during crown formation by isolating DSPP-derived proteins from developing porcine molars and characterizing their N-terminal sequences and apparent size on SDS-PAGE and Western blots. The in vivo DSPP cleavage sites were on the N-terminal sides of Thr(200), Ser(330), Val(353), Leu(360), Ile(362), Ser(377), Ser(408), and Asp(458). The initial DSPP cleavage is between dentin glycoprotein (DGP) and dentin phosphoprotein (DPP), generating dentin sialoprotein (DSP)/DGP and DPP. Gelatin and casein zymograms identified MMP-2, MMP-20, and KLK4 in the dentin extracts. MMP-2 and MMP-20 were purified from over 150 g of porcine dentin powder and incubated with DSP-DGP and DPP. These enzymes show no activity in further cleaving DPP. MMP-20 cleaves DSP-DGP to generate DSP and DGP. MMP-20 also cleaves DSP at multiple sites, releasing N-terminal DSP cleavage products ranging in size from 25 to 38 kDa. MMP-2 makes multiple cleavages near the DSP C terminus, releasing larger forms of DGP, or "extended DGPs." Exact correspondence between DSPP cleavage sites that occur in vivo and those generated in vitro demonstrates that MMP-2 and MMP-20 process DSPP into smaller subunits in the dentin matrix during odontogenesis.

The measurement uncertainty of a T-shaped vortex flowmeter was examined with a gas flow calibration facility, for Reynolds numbers, Re d , in a range of 2.56×104–1.56×105. The total uncertainty interval of the Strouhal values deduced from a Fourier spectral analysis was ±0.745%, and the linearity of the data was ±1.78%. Meanwhile, a scheme based on pulse counting in reference to the analog signals measured gave the total uncertainty interval of the meter factor as ±0.568%, with the linearity ±1.89%. Further, the flowmeter was tested in a fully developed water pipe flow facility, for Re d of 5.6×103–5.29×104. The vortex shedding frequencies were obtained with the techniques of Fourier transform and autocorrelation, respectively. At low flow rates, the autocorrelation technique gave a smaller uncertainty interval than the Fourier analysis did; at higher flow rates the situation was reversed. The linearity of the St values obtained in the water pipe flow experiment was ±0.391%, noticeably smaller than that obtained in the gas flow facility.

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper dentin formation. DSPP is believed to be processed into only two structural/functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here we report the isolation and characterization of a third domain of DSPP, designated dentin glycoprotein (DGP). DGP was isolated from a guanidine/EDTA extract of porcine tooth dentin by ion exchange, hydroxyapatite affinity, size exclusion, and RP-HPL chromatography. Endoproteinase lysine C digestion products of DGP were characterized by Edman sequencing and mass spectrometry. The porcine DGP backbone is the 81-amino acid segment of DSPP (Ser392 to Gly472) between the DSP and DPP domains. DGP has four phosphorylated serine residues (Ser453, Ser455, Ser457, and Ser462) and one glycosylated asparagine (Asn397). There are no other post-translational modifications. DGP is a stains-all positive protein with an apparent molecular mass on SDS-PAGE of 19 kDa, which is reduced by glycopeptidase A digestion to 16 kDa. A variety of glycans can be linked to Asn397. All are complex biantennary structures with a common N-linked pentasaccharide core (mannose3-N-acetylglucosamine2), most with a fucosyl residue on the innermost N-acetylglucosamine. The α1–3 and α1–6 arms are always galactose β1–4 N-acetylglucosamine β1–2 mannose, and either or both arms can be unsialidated or monosialidated. The calculated monoisotopic molecular masses of the different glycosylated forms of the DGP phosphoprotein are: unsialidated 10,523 and 10,670, monosialidated 10,815 and 10,961, and disialidated 11,106, and 11,252 Da, with the disialidated forms being the most abundant.

Experiments were made with a vortex meter situated downstream of two elbows out-of-plane, at Reynolds numbers between 7.8×10 4 and 2.1×10 5 . Analysis on the signals obtained from a piezo-electric sensor embedded in the vortex shedder shows that the signal quality, concerned with the vortex shedding frequency component, deduced in the vicinity of 15 pipe diameters downstream is superior to that at other locations, and is remarkably better than that found in the region of fully-developed turbulent pipe flow. The vortex shedding frequencies measured in this region, in terms of the Strouhal value, fall within 0.129±0.001, that 0.129 is the reference value reduced in the fully-developed turbulent pipe flow region. Also noted, the uncertainty interval associated with the vortex shedding frequency measured in this region is smaller than that reduced in the region of fully-developed turbulent pipe flow.

Experiments were performed to study the response of a vortex flowmeter to structural vibrations due to impulsive forces applied on the pipe. Vortex-shedding signals obtained by a piezoelectric sensor embedded in a vortex shedder were examined. Major findings are described as follows. First, by improving the design of the piezoelectric sensor, the sensor sensitivity to structural vibrations could be reduced. Specifically speaking, the noise component due to impulsive force with level up to 13.8 kN could be removed effectively from the output. Second, by applying repetitive impulsive forces on the pipe, characterized by a frequency greater than the vortex-shedding frequency, the quality of vortex-shedding signals measured was degraded substantially. This is explained as being due to suppression of vortex shedding, not a problem in conjunction with the characteristics of the sensor.

Although the Mr values of the coat proteins (CPs) of several cucumoviruses have been calculated from their deduced amino acid sequences to be approximately 24,000, the experimentally determined M(r) values using the Laemmli SDS-PAGE system were 30,000-31,000. Examination of the amino acid composition revealed that these CPs are neither highly acidic nor highly basic. Post-translational glycosylation or phosphorylation were also ruled out as contributing factors to the observed anomalous electrophoretic mobility because the products of in vitro translation of cucumovirus RNA 4 and in vivo bacterial expression of the cloned CP gene co-migrated with authentic cucumovirus CPs. Comparison of the hydropathy profiles of the CPs revealed the presence in each of a strikingly similar, highly hydrophilic N-terminal domain of 30-32 amino acid residues that contains a cluster of basic amino acids, mainly arginine. Selective chemical cleavage at tryptophan residues in the CPs of cucumoviruses, known to contain single tryptophan residues, yielded two peptides; an N-terminal peptide that contained the conserved hydrophilic domain and a C-terminal peptide. SDS-PAGE analysis showed that the N-terminal, but not the C-terminal, peptide exhibited the anomalous electrophoretic mobility.

Using four different protein antigens, two different strains of mice, and various immunization protocols, we have studied production in mice of immunological enhancement antibodies that specifically suppress induction of delayed hypersensitivity. Primary assay of these antibodies was in vivo , because no in vitro test used detected them dependably. Any antigen priming that favored initiation of humoral antibody responses prepared mice to make these contrasensitizing antibodies vigorously following appropriate boosting. The method of boosting usually was more important than that of priming, high titers regularly developing only when primed mice were boosted with much antigen in a short time and were bled a few days later. The presence or absence of delayed hypersensitivity was immaterial. CAF 1 mice made these antibodies better than CF-1 mice, and antigen effectiveness correlated with propensity to induce humoral antibody formation in mice, decreasing from ovalbumin through human serum albumin and bovine serum albumin to methylated human serum albumin. In certain antigenmouse combinations (e.g., ovalbumin in CAF 1 mice) immunosuppressive antibody production was vigorous and prolonged; in others (e.g., bovine serum albumin in CF-1 mice) it was moderate and brief. From our results one can predict what conditions should induce formation of strongly enhancing/contrasensitizing antisera, and speculate that these conditions also should elicit strong, active immunologic tolerance for averting induction of delayed hypersensitivity.

Branched-chain alpha-keto acid dehydrogenase is a multienzyme complex consisting of three catalytic components, i.e. branched-chain alpha-keto acid decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3). In this report the E2 component of highly purified branched-chain alpha-keto acid dehydrogenase from bovine kidney and liver was characterized with an independent radiochemical assay for this component. The assay uses the model reaction: R-14CO-S-CoA + Lip-(SH)2 in equilibrium R-14CO-S-Lip-SH + CoA-SH, which is similar to that catalyzed by the transacetylase component of the pyruvate dehydrogenase complex. In this reaction, exogenous dihydrolipoamide substitutes for the protein (E2)-bound dihydrolipoyl moiety, and [1-14C]acyl-CoA synthesized enzymatically is the acyl-CoA substrate. The thioester structure of the reaction product, S-acyldihydrolipoamide, was identified by mass spectrometry, its characteristic absorption at 232-245 nm and by formation of hydroxamate with hydroxylamine. Rates of the E2-catalyzed transacylation reaction with various [1-14C]acyl-CoAs are in the order of [1-14C]isobutyryl-CoA greater than [1-14C] isovaleryl-CoA greater than [1-14C]acetyl-CoA. The activity with acetyl-CoA is 15% of that with isobutyryl-CoA. The E2 activity is strongly inhibited by arsenite. Modification of the covalently bound lipoyl moiety through reductive acylation in the presence of N-ethylmaleimide is without effect on the transacylation reaction. These data, along with results of initial velocity and product inhibition suggest that the model reaction proceeds via a random Bi Bi mechanism. Limited proteolysis of purified bovine liver branched-chain alpha-keto acid dehydrogenase with trypsin results in complete loss of the overall activity catalyzed by the complex. Nonetheless the activity of the E2 component is not affected. The tryptic digestion cleaves E2 subunits (Mr = 52,600) into a major fragment of Mr = 25,700. By contrast, E1 alpha and E1 beta subunits of the complex are relatively resistant to proteolysis with trypsin. The results indicate that structural properties of the E2 component of branched-chain alpha-keto acid dehydrogenase are similar but not identical to those of the transacetylase component of the pyruvate dehydrogenase complex.

This paper describes experiments on the nature of humoral antibodies which suppress induction of delayed hypersensitivity in mice to purified proteins, i.e., contrasensitizing antibodies. These antibodies may be analogous to those responsible for afferent or early central immunosuppression in immunological enhancement phenomena and for similar forms of immunosuppression sometimes referred to as immune deviation, split tolerance, or preimmunization tolerance. CF-1 mice were sensitized with either human serum albumin or ovalbumin in incomplete Freund adjuvant, were injected with contrasensitizer-rich mouse antisera or fractions thereof, and subsequently were skin-tested to measure the relative capacities of antisera or their components to interfere with sensitization. Agar gel zone electrophoresis located Contrasensitizing activity among the γ 1 globulins, and this identification was confirmed by DEAE cellulose chromatography. Fractionation on Sephadex G-200 dextran columns showed the inhibitory activity to be among molecules of approximately 170,000 molecular weight, and a combination of chromatographic procedures further indicated that this activity is due to specific antibodies closely associated with mouse IgF or 7S γ 1 globulin. Contrasensitizing antibodies also were found to be heat-stable, and although precipitin and passive hemagglutination antiserum titers often correlated well with immunosuppressive activity, neither was equivalent to it.

Enhancing antibody (referred to here as "contrasensitizer"), produced in hyperimmunized mice and injected daily into normal mice during the first 5 days of their exposure to purified protein antigens in incomplete Freund adjuvant, prevents these mice from developing delayed (tuberculin-type) hypersensitivity by specifically blocking antigen-host cell interactions in the earliest stages of sensitization. An antigen inoculum is permanently inactivated by such a series of contrasensitizer injections; but treated mice are not made immunologically unresponsive, for they can respond normally to a second inoculation not neutralized by a second course of contrasensitizer injections. The inhibitory antibody, apparently a 7S gamma-1 globulin and not a precipitin, cannot prevent activation of an anamnestic response; but its administration can be delayed as much as 4 days after original antigen inoculation and still prevent primary induction of delayed hypersensitivity. Much less effectively, it also suppresses induction of Arthus hypersensitivity. In terms of retained inhibitory activity, it is estimated to have a half-life of only 10 to 12 hours.