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We present the results of a {\it James Webb Space Telescope} NIRCam investigation into the young massive star cluster (YMC) population in the luminous infrared galaxy VV 114. We identify 374 compact YMC candidates with a $S/N \geq 3$, 5, and 5 at F150W, F200W, and F356W respectively. A direct comparison with our {\it HST} cluster catalog reveals that $\sim 20\%$ of these sources are undetected at optical wavelengths. Based on {\it yggdrasil} stellar population models, we identify 17 YMC candidates in our {\it JWST} imaging alone with F150W-F200W and F200W-F356W colors suggesting they are all very young, dusty ($A_{V} = 5 - 15$), and massive ($10^{5.8} < M_{\odot} < 10^{6.1}$). The discovery of these `hidden' sources, many of which are found in the `overlap' region between the two nuclei, quadruples the number of $t < 3$ Myr clusters, and nearly doubles the number of $t < 6$ Myr clusters detected in VV 114. Now extending the cluster age distribution ($dN/d\tau \propto \tau^{\gamma}$) to the youngest ages, we find a slope of $\gamma = -1.30 \pm 0.39$ for $10^{6} < \tau (\mathrm{yr}) < 10^{7}$, which is consistent with the previously determined value from $10^{7} < \tau (\mathrm{yr}) < 10^{8.5}$, and confirms that VV 114 has a steep age distribution slope for all massive star clusters across the entire range of cluster ages observed. Finally, the consistency between our {\it JWST}- and {\it HST}-derived age distribution slopes indicates that the balance between cluster formation and destruction has not been significantly altered in VV 114 over the last 0.5 Gyr., Comment: 13 pages, 4 figures, 1 table, published in the Astrophysical Journal

INTRODUCTION New York-esophageal squamous cell carcinoma (NY-ESO-1) specific adoptive T-cell immunotherapy is an appealing strategy for the treatment of gliomas because it elicits a strong antibody and T-cell response and is downregulated in most normal adult tissues which produces an effective and specific antitumor response. However, NY-ESO-1 is often poorly expressed in GBM, presumably through promoter methylation. Neither the prevalence of NY-ESO-1 downregulation in glioblastoma (GBM) patient tumors nor the presumed mechanism of downregulation by promoter methylation have been formally established. METHODS We characterized baseline CpG methylation of NY-ESO-1 in patient GBM samples using bisulfite sequencing. We induced NY-ESO-1 expression in vitro in human GBM cell lines (U251) and patient-derived gliomaspheres using the demethylating agent decitabine (DAC). We investigated the epigenetic response of DAC treated GBM cell lines with bisulfite sequencing and NY-ESO-1 expression with quantitative real time PCR. Lastly, we performed single cell RNA sequencing on DAC treated and untreated U251 and PGDSC to evaluate tumor subpopulations that upregulate NY-ESO-1 and co-expressed cancer testis antigens after DAC treatment. RESULTS NY-ESO-1 expression is associated with promoter methylation in the majority of GBM. Prolonged low dose of DAC explicitly demethylated the promoter region of NY-ESO-1 and resulted in robust mRNA expression. DAC treatment co-expresses NY-ESO-1 with other cancer testis antigens CTAG2 and MAGEA4 as demonstrated by single cell RNA sequencing. CONCLUSION These results reveal a feasible strategy of cancer testis antigen promoter demethylation which creates an inducible tumor antigen for immunotherapeutic targeting. We speculate that DAC induced upregulation of NY-ESO-1 and co-expressed cancer testis antigens renders tumor recognition by NY-ESO-1 specific T cells.

Objectives The aim of this study was to determine the influence of short base lengths and supplemental grooves on surface area and rotational resistance in a simulated‐maxillary premolar. Materials and Methods Trigonometric calculations were done to determine the total surface area with and without supplemental grooves. Additional computations were done to determine the maximum wall angle needed to resist rotation displacement in a premolar‐sized model. Wall heights of 3.0, 4.0, and 5.0 mm were used in the surface area and rotational axis computations. The rotational axis was located on the lingual restoration margin to produce a buccal‐to‐lingual rotational displacement. Results Total surface area decreased with increasing four‐wall taper levels from 2° to 18° and decreasing preparation heights from 5 to 3 mm. Significant surface area improvements were found with the supplemental use of mesial and distal axial grooves compared with the same condition without grooves in all taper levels and preparation height categories. Resistance to rotational displacement was determined to occur at only at very low levels of opposing wall taper angles. The use of supplemental grooves on mesial and distal axial walls significantly improved both total surface area and rotational resistance. Conclusions The vertical wall taper angles, preparation heights, and supplemental grooves play a role in resistance form and restoration stability.

This study examines Mesoamerican influence on kachina ceremonialism, a Southwestern religious institution in which kachina spirits infiltrate pueblo communities for a portion of the year as masked impersonators. Kachina spirits are in fact ancestral spirits who inhabit the underworld. They emerge in cloud-form from mountain tops to bring rain to the fields of the living. Kachina ceremonialism arose in the 13th to 14th centuries during the Pueblo IV period following the Chacoan collapse, representing a social adaptation encouraging integration and stability in newly-established pueblo communities. Whether the kachina cult's genesis is represented by ceramic evidence in the western Little Colorado Basin, or rock art in the eastern Rio Grande, a significant Mesoamerican ideological substrate fed into its origins: the Southwest imported such Mesoamerican commodities as copper bells, Scarlet Macaws, and the Mesoamerican ballgame, the latter two of which left ideological impressions evident in kiva murals and ceramic motifs. Ultimately, it was the Mesoamerican cult of the rain-bringing dead which was modified into the socially-integrative kachina cult in response to Pueblo IV social needs, as well as the Southwest’s arid environment.

Background Unlike most virtual reality (VR) training programs that are targeted at homogenous populations, a set of VR games for rehabilitation purposes targeted at a heterogeneous group of users was developed. The VR games covered physical training, cognitive training (classification and reality orientation), community-living skills training, and relaxing scenery experiences. Special considerations for local older adults and people with disabilities were made in terms of hardware choice and software design. Objective This study aimed to evaluate the feasibility, acceptance, and efficacy of VR training among users with varying abilities. Methods A single-arm pretest-posttest evaluation study was conducted. The participants of the evaluation study were encouraged to undergo 30-minute VR training three times a week for 6 weeks. The 30-minute session consisted of 10 minutes of upper-limb motion games, 10 minutes of lower-limb motion games, and 10 minutes of cognitive games/community-living skills training/relaxing scenery experiences, as appropriate. On completion of each session, usage statistics were documented via the built-in VR software, whereas feedback on the experience of the VR games and adverse events was collected via self-reports and staff observations. Feasibility was reflected by usage statistics, and acceptance was reflected by positive feedback. In addition, health outcomes, including upper-limb dexterity, functional mobility, cognitive function, and happiness, were assessed at baseline, as well as 6 weeks and 3 months after baseline. The primary outcomes were upper-limb dexterity and acceptance of playing VR games. Results A total of 135 participants with a mean age of 62.7 years (SD 21.5) were recruited from May 2019 to January 2020, and 124 (91.9%) completed at least one follow-up. Additionally, 76.3% (103/135) of the participants could attend at least 70% of the proposed 18 sessions, and 72.5% (1382/1906) of the sessions had a training time of at least 20 minutes. Linear mixed effect models showed statistically significant effects in terms of upper-limb dexterity (small effect) and cognitive function (moderate effect). Among the 135 participants, 88 provided positive comments. Additionally, 10.4% (14/135) reported mild discomfort, such as dizziness, and none reported severe discomfort. Conclusions A set of VR training games for rehabilitation could be applied to users with heterogeneous abilities. Our VR games were acceptable to local older adults and those with different disabilities. Benefits in upper-limb dexterity and cognitive function were observed despite partial compliance to the training protocol. Service providers could refer to our experiences when developing VR training systems for their clients.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

A precise imaging technique to evaluate osteogenesis, osteodifferentiation, and osseointegration following peri-implant surgery is in high clinical demand. Herein, we report the generation of two new, near-infrared (NIR) fluorescent probes for use in the molecular imaging of bone repair. The first probe aims to monitor the in vitro differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. A NIR fluorochrome was conjugated to a cyclic peptide that binds to integrin α5β1, a factor that promotes osteogenesis in MSCs and therefore functioned as an osteoblast-specific marker. The second probe aims to monitor osteogenesis, and was generated by conjugating the drug pamidronate to a NIR fluorescent gold nanocluster. Pamidronate specifically binds to hydroxyapatite (HA), a mineral present in bone that is produced by osteoblasts, and therefore provides a functional marker for new bone formation. Our results show that both probes bind to their specific targets in vitro-differentiated osteoblasts, and not to undifferentiated MSCs, and emit NIR fluorescence for functional detection. This in vitro work demonstrates the ability of these probes to bind to active osteoblasts and their mineral deposits and highlight their potential utility as clinical tools for the imaging of the osseointegration process at the molecular level.

Crowns or abutments of fixed partial denture tooth preparations to restore natural teeth or implant-supported restorations require tooth or abutment reduction in a tapered format. Low angulations of axial wall taper to the long axis of the tooth or abutment have been shown to increase the restoration’s stability during masticatory function with large occlusal loads. The tooth preparation and luting agent stabilize the restoration during these loading cycles to prevent dislodgement. Restoration dislodgement represents a catastrophic failure of the prosthesis in function. The preparation attributes contribute to prevent displacement failure and reduce luting agent tensile and shear stresses. Low axial wall taper levels increase resistance to rotational displacement. Increasing axial wall angulations produces negative consequences including reduced rotational resistance, reduced surface area and increased volume of remaining tooth structure. These three considerations have been shown to involve three variables: preparation vertical height, horizontal base width from rotational axis and rotational axis vertical height compared to the opposing axial resisting wall. Natural tooth preparations are completed by a restorative dentist with a hand-held high-speed handpiece with cutting instruments within the oral cavity; while implant-supported custom abutments are prepared in a similar manner but machined outside the oral cavity. As a result, the machined abutment is more controlled for ideal taper in inanimate materials compared to the intraoral tooth preparation on human vital tissues. This experimental study will analyze the preparation of simulated-vital tooth premolars and molars for volume loss at ideal and non-ideal axial wall angulations with variation of vertical heights and vital pulpal tissue volumes.

Digital impression procedures increasingly have been utilized to capture final impressions of fixed prosthodontic preparations in the natural tooth and single-implant restorations as well as for fixed partial dentures in both conditions. The literature related to restoration resistance to rotational displacement has been reviewed. Many digital camera systems have "open architecture" with the generation of generic standard tessellation language (STL) files. These STL files can be analyzed by software to determine preparation attribute-compliance with evidence-based standards. This literature review presents an overview of the knowledge base and survey data of US certified dental technicians (CDTs) and Canadian registered dental technicians (RDTs). The technician data reveal opinions about the level of clinician compliance with standards from the literature and possible future developments for additional applications of this emerging technology.

Cartilage has limited self-repair ability. The purpose of this study was to investigate the effects of different species of collagen-engineered neocartilage for the treatment of critical-size defects in the articular joint in a rabbit model. Type II and I collagen obtained from rabbits and rats was mixed to form a scaffold. The type II/I collagen scaffold was then mixed with rabbit chondrocytes to biofabricate neocartilage constructs using a rotating cell culture system [three-dimensional (3D)-bioreactor]. The rabbit chondrocytes were mixed with rabbit collagen scaffold and rat collagen scaffold to form neoRBT (neo-rabbit cartilage) and neoRAT (neo-rat cartilage) constructs, respectively. The neocartilage matrix constructs were implanted into surgically created defects in rabbit knee chondyles, and histological examinations were performed after 2 and 3 months. Cartilage-like lacunae formation surrounding the chondrocytes was noted in the cell cultures. After 3 months, both the neoRBT and neoRAT groups showed cartilage-like repair tissue covering the 5-mm circular, 4-mm-deep defects that were created in the rabbit condyle and filled with neocartilage plugs. Reparative chondrocytes were aligned as apparent clusters in both the neoRAT and neoRBT groups. Both neoRBT and neoRAT cartilage repair demonstrated integration with healthy adjacent tissue; however, more integration was obtained using the neoRAT cartilage. Our data indicate that different species of type II/I collagen matrix and 3D bioreactor cultivation can facilitate cartilage engineering in vitro for the repair of critical-size defect.

The Ca 2+ -dependent tissue transglutaminase is widely distributed in various tissues and has been reported to participate in many cellular growth and differentiation processes. In the past decade, tissue transglutaminase is also identified as a G protein, G αh , for intercellular signaling. To further characterize testicular transglutaminase, the rat testicular transglutaminase was purified by ammonium sulfate precipitation, DEAE ion-exchange, heparin–agarose, and GTP–agarose affinity chromatographies. This purification protocol resulted in a 8400-fold enrichment of the enzyme with a reproducible 15% yield. The purified enzyme showed as a single band of 78 kDa on SDS-polyacrylamide gel. Western blot analysis using anti-liver tissue transglutaminase monoclonal antibody also recognized the enzyme, indicating it is a t-TGase in nature. The K m values of purified testicular transglutaminase for putrescine and N , N -dimethylcasein were determined to be 35 and 17 μ M , respectively. Its transglutaminase cross-linking activity was strongly inhibited by EGTA, GTP, polyamines, and cystamine, as well as moderately by ATP and NaCl. The enzyme exhibited a magnesium-dependent GTP-hydrolyzing capacity, but its GTP-binding activity did not require magnesium. Furthermore, the enzyme activity was found to be closely related with the first wave of spermatogenesis. Thus, testicular transglutaminase is speculated to participate in the event of spermatogenesis. In conclusion, the purified testicular transglutaminase displays property of either the tissue-type transglutaminase, or the GTP-binding and hydrolyzing characteristics. The activity of testicular transglutaminase is age-dependent, greatly stimulated during the first wave of spermatogenesis.

Mesenchymal stem cells (MSCs) are currently thought to transdifferentiate into neural lineages under specific microenvironments. Studies have reported that the tenascin family members, tenascin-C (TnC) and tenascin-R (TnR), regulate differentiation and migration, in addition to neurite outgrowth and survival in numerous types of neurons and mesenchymal progenitor cells. However, the mechanisms by which TnC and TnR affect neuronal differentiation are not well understood. In this study, we hypothesized that different forms of tenascin might regulate the neural transdifferentiation of human bone marrow-derived mesenchymal stem cells. Human MSCs were cultured in media incorporated with soluble tenascins, or on precoated tenascins. In a qualitative polymerase chain reaction analysis, adding a soluble TnC and TnR mixture to the medium significantly enhanced the expression of neuronal and glial markers, whereas no synaptic markers were expressed. Conversely, in groups of cells treated with coated TnC, hMSCs showed neurite outgrowth and synaptic marker expression. After being treated with coated TnR, hMSCs exhibited neuronal differentiation; however, it inhibited neurite outgrowth and synaptic marker expression. A combination of TnC and TnR significantly promoted hMSC differentiation in neurons or oligodendrocytes, induced neurite formation, and inhibited differentiation into astrocytes. Furthermore, the effect of the tenascin mixture showed dose-dependent effects, and a mixture ratio of 1:1 to 1:2 (TnC:TnR) provided the most obvious differentiation of neurons and oligodendrocytes. In a functional blocking study, integrin α7 and α9β1-blocking antibodies inhibited, respectively, 80% and 20% of mRNA expression by hMSCs in the coated tenascin mixture. In summary, the coated combination of TnC and TnR appeared to regulate neural differentiation signaling through integrin α7 and α9β1 in bone marrow-derived hMSCs. Our findings demonstrate novel mechanisms by which tenascin regulates neural differentiation, and enable the use of cell therapy to treat neurodegenerative diseases.

To compare the modulatory effects of different prostaglandins on collagen gene expression in human chondrocytes, PGE(2), PGE(1), misoprostol (PGE(1) analog), and PGF(2alpha) (10, 50 and 100 ng ml(minus sign1)) were added to human chondrocytes with or without interleukin-1beta (IL-1beta) in the presence of indomethacin to inhibit endogenous prostaglandin synthesis and the effects evaluated on chondrocyte morphology, collagen synthesis, and procollagen mRNA levels. The effects of prostaglandins on the expression of collagen gene regulatory sequences were examined using transient transfection assays of reporter gene constructs in human chondrocytes and BALB/c3T3 fibroblasts, PGE(1), misoprostol, and PGF(2alpha), similar to PGE(2), inhibited type I collagen gene expression in fibroblasts and promoted type II collagen gene expression in chondrocytes. PGE(2), the major inflammatory prostaglandin produced by IL-1-activated chondrocytes and fibroblasts, and PGF(2alpha) were somewhat more potent than the anti-inflammatory prostaglandins PGE(1) and misoprostol in counteracting the IL-1-induced suppression of type II collagen gene expression by chondrocytes and stimulation of type I collagen gene expression by fibroblasts. Rather than promoting degradation of the cartilage matrix in joint diseases, prostaglandins may be somewhat protective, suppressing fibrosis, and maintaining or promoting appropriate cartilage repair.

The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.

The remediation of neurodegeneration and cognitive decline in Alzheimer's Disease (AD) remains a challenge to basic scientists and clinicians. It has been suggested that adult bone marrow stem cells can transdifferentiate into different neuronal phenotypes. Here we demonstrate that the alpha-secretase-cleaved fragment of the amyloid precursor protein (sAPPalpha), a potent neurotrophic factor, potentiates the nerve growth factor (NGF)/retinoic acid (RA) induced transdifferentiation of bone marrow-derived adult progenitor cells (MAPCs) into neural progenitor cells and, more specifically, enhances their terminal differentiation into a cholinergic-like neuronal phenotype. The addition of sAPPalpha to NGF/RA-stimulated MAPCs resulted in their conversion to neuronal-like cells as evidenced by the extension of neurites and the appearance of immature synaptic complexes. MAPCs differentiated in the presence of sAPPalpha and NGF/RA exhibited a 40% to as much as 75% increase in neuronal proteins including NeuN, beta-tubulin III, NFM, and synaptophysin, compared to MAPCs differentiated by NGF/RA alone. This process was accompanied by an increase in the levels of choline acetyltransferase, a marker of cholinergic neurons, compared to those of GABAergic and dopaminergic neuronal subtypes. MAPCs immunopositive for sAPPalpha were identified within the septohippocampal system of transgenic PS/APP mice injected intravenously with sAPPalpha-transfected MAPCs and found in close proximity to the cerebral vasculature. Given that in AD cholinergic neurons are severely vulnerable to neurodegeneration and that the levels of sAPPalpha are significantly reduced, these findings suggest the combined use of sAPPalpha and MAPCs offers a new and potentially powerful therapeutic strategy for AD treatment.

Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.

Cartilage preserves limited regenerative capacity and results in insufficient healing after joint injury. Previous researches showed that biocompatible materials and growth factors enable to facilitate cartilage repair; however, the mechanisms of cartilage regeneration are still not completely understood. The purpose of this study was to determine the effects of extracellular matrix and growth factor on chondrocyte migration and phenotype during cartilage regeneration. Neocartilage, 35 mm in diameter and 1 mm in thickness, was fabricated by using rabbit primary chondrocytes embedded in an in vitro three-dimensional collagen matrix. Five mm diameter defects were made. The effects of collagen matrices and basic-FGF on three-dimensional cartilage wound healing were determined. Results showed that both type I and type II collagen matrices (1 mg/ml) facilitated migration of chondrocytes from surrounding cartilage into the defect area in the presence of 10 ng/ml basic-FGF. The average of migration speed was 91.5micron/day for type I, and 88.1micron/day for type II collagen matrix in the period of 26 days. Incorporation with basic-FGF, chondrocytes increased approximately 1.5 times motility in the both collagen matrices. However, the cartilaginous phenotype was noted only when embedded in type II collagen matrix. The results indicate that type II collagen may trigger the redifferentiation during the migration of chondrocytes. Extracellular matrix such as type I and type II collagen, in addition to cytokines such as basic-FGF, play an important role in the process of cartilage wound healing in a three-dimensional defect model.

In a conventional matte smelting or slag resistance furnace electrical conduction currents flowing in a molten slag are used to generate the required process heat. Such furnaces generally involve a 6-in line electrode geometry, with the electrodes generally being in excess of 1m diameter and the electrode currents being on the order of 100 kA. One of the principal electrical design problems is the calculation of the power and current distribution. These parameters directly influence the furnace operating practice and the design of the furnace electrical equipment and refractories. An additional design problem is that of refractory and electrode erosion due to the slag motion. The electromagnetic body forces are among the principle forces which drive the recirculatory slag flow. A finite-element numerical model, which determines the distribution of the furnace power and the electromagnetic body forces as a function of the furnace operating parameters is described. The advantages of this method over existing methods are described.

Tissue-transglutaminase (t-TGase) is a family of calcium-dependent enzymes. A Ca2+-independent soluble enzyme, in addition to t-TGase, capable of incorporating polyamines into proteins was demonstrated in rat intestinal mucosa. The Ca2+-independent enzyme was stimulated 2- to 5-fold by Fe2+ and Co2+ ions but inhibited by Cu2+ and Zn2+ ions. The Ca2+-stimulated t-TGase activity was inhibited by divalent ions in the following order: Zn2+, Fe2+>Co2+>Cu2+. The opposite effects of EGTA, Fe2+ and Co2+ on these two enzyme activities indicate that they are two distinct classes of enzymes. Competition studies demonstrated differential preferences of the two enzymes for substrates. The Ca2+-dependent enzyme preferred putrescine, monodansylcadaverine>cadaverine, spermidine, spermine>1,10-diaminodecane>triethylbutylamine. On the other hand, the Ca2+-independent enzyme preferred putrescine>cadaverine>spermine, 1,10-diaminodecane>spermidine>monodansylcadaverine>triethylbutylamine. Further studies with divalent ions excluded the possible association of this novel Ca2+-independent enzyme with diamine oxidase. Finally, the Ca2+-independent enzyme had a higher affinity for putrescine (Km=0.02 mM) than did Ca2+-dependent t-TGase (0.2 mM). As judged by gel filtration on HiPrep Sephacryl 200 column, the Ca2+-independent enzyme had a molecular weight of ∼48 kDa, the intestinal Ca2+-dependent t-TGase was about 188 kDa while that of testicular t-TGase was about 96 kDa. In conclusion, the Ca2+-independent enzyme is stimulated by cobalt or ferric ions, and selectively incorporates aliphatic diamines or polyamines with symmetric amino groups. The observed Ca2+-independent enzyme activity is not related to diamine oxidase or its products. With a 10 times greater affinity for putrescine, the calcium-independent, 48-kDa intestinal enzyme may mediate polyamine function better than calcium dependent, 188-kDa intestinal tissue transglutaminase in the intestinal mucosa.

The kinetics of wetting during the process of making tablet granulations were studied; the process progresses through an intermediate (overwetted) nonequilibrium granule to a final, equilibrated granule. In systematic formulation changes, there is an optimum composition from the point of view of hardness and content of equilibrium granules.