Sunhwa Lee

Candidate Gene Strategy Reveals ENAM Mutations

Amelogenesis imperfecta (AI) is a genetically and phenotypically heterogeneous genetic disorder affecting tooth enamel without other non-oral syndromic conditions. Based on a review of the literature, the authors constructed a candidate-gene-based mutational analysis strategy. To test the strategy, they identified two Turkish families with hypoplastic enamel without any other non-oral syndromic phenotype. The authors analyzed all exons and exon/intron boundaries of the enamelin (ENAM) gene for family 1 and the DLX3 and ENAM genes for family 2, to identify the underlying genetic etiology. The analysis revealed 2 ENAM mutations (autosomal-dominant g.14917delT and autosomal-recessive g.13185–13186insAG mutations). A single T deletion in exon 10 is a novel deletional mutation (g.14917delT, c.2991delT), which is predicted to result in a frameshift with a premature termination codon (p.L998fsX1062). This result supports the use of a candidate-gene-based strategy to study the genetic basis for AI.

Novel WDR72 Mutation and Cytoplasmic Localization

The proven candidate genes for amelogenesis imperfecta (AI) are AMELX, ENAM, MMP20, KLK4, FAM83H, and WDR72. We performed mutation analyses on seven families with hypomaturation AI. A novel WDR72 dinucleotide deletion mutation (g.57,426_57,427delAT; c.1467_ 1468delAT; p.V491fsX497) was identified in both alleles of probands from Mexico and Turkey. Haplotype analyses showed that the mutations arose independently in the two families. The disease perfectly segregated with the genotype. Only persons with both copies of the mutant allele were affected. Their hypomineralized enamel suffered attrition and orange-brown staining following eruption. Expression of WDR72 fused to green fluorescent protein showed a cytoplasmic localization exclusively and was absent from the nucleus. We conclude that WDR72 is a cytoplasmic protein that is critical for dental enamel formation.

High-Dose Versus Conventional-Dose Continuous Venovenous Hemodiafiltration and Patient and Kidney Survival and Cytokine Removal in Sepsis-Associated Acute Kidney Injury: A Randomized Controlled Trial

BACKGROUND Soluble inflammatory mediators are known to exacerbate sepsis-induced acute kidney injury (AKI). Continuous renal replacement therapy (CRRT) has been suggested to play a part in immunomodulation by cytokine removal. However, the effect of continuous venovenous hemodiafiltration (CVVHDF) dose on inflammatory cytokine removal and its influence on patient outcomes are not yet clear. STUDY DESIGN Prospective, randomized, controlled, open-label trial. SETTING & PARTICIPANTS Septic patients with AKI receiving CVVHDF for AKI. INTERVENTION Conventional (40mL/kg/h) and high (80mL/kg/h) doses of CVVHDF for the duration of CRRT. OUTCOMES Patient and kidney survival at 28 and 90 days, circulating cytokine levels. RESULTS 212 patients were randomly assigned into 2 groups. Mean age was 62.1 years, and 138 (65.1%) were men. Mean intervention durations were 5.4 and 6.2 days for the conventional- and high-dose groups, respectively. There were no differences in 28-day mortality (HR, 1.02; 95% CI, 0.73-1.43; P=0.9) or 28-day kidney survival (HR, 0.96; 95% CI, 0.48-1.93; P=0.9) between groups. High-dose CVVHDF, but not the conventional dose, significantly reduced interleukin 6 (IL-6), IL-8, IL-1b, and IL-10 levels. There were no differences in the development of electrolyte disturbances between the conventional- and high-dose groups. LIMITATIONS Small sample size. Only the predilution CVVHDF method was used and initiation criteria were not controlled. CONCLUSIONS High CVVHDF dose did not improve patient outcomes despite its significant influence on inflammatory cytokine removal. CRRT-induced immunomodulation may not be sufficient to influence clinical end points.

Prenatal development of myoepithelial cell of human submandibular gland observed by immunohistochemistry of smooth muscle actin and rhodamine-phalloidin fluorescence.

Immunostaining of monoclonal antibody (MoAb) of smooth muscle actin in paraffin sections and fluorescence of actin-specific phalloidin in cryostat sections were utilized to demonstrate the myoepithelial cells in prenatal and adult salivary glands of humans. In the early developmental stage (10-18 weeks) MoAb actin was weakly positive in the basal cells of the gland epithelium, and the positivity gradually accentuated at the basal portions of the terminal ducts and acini as the gestational period advanced. In the early intermediate developmental stage (19-24 weeks) the polyhedral myoepithelial cells were arranged in the basal portions of the acini and intercalated ducts. At this stage the myoepithelial cells produced phalloidin-positive spindle cytoplasmic processes. In the late intermediate developmental stage (25-32 weeks) the myoepithelial cells became flattened and formed dendritic processes to surround the acini and intercalated ducts. In the late developmental stage (33-40 weeks) numerous myoepithelial cells with well developed dendritic processes were demonstrable in the acini and intercalated ducts. In conclusion, it was found that the myoepithelial cells began to develop at 15-16 weeks of gestation when the acinar cells were still immature. The primitive myoepithelial cells were polyhedral in shape to form compact basal layer beneath the developing acinar cells during 19-24 weeks of gestation. In late gestational period the myoepithelial cells almost matured like the dendritic ones of adult salivary glands. However, the myoepithelial cells were never demonstrated in the striated and excretory ducts of the fetal salivary glands as opposed to its normal presence in the adult salivary glands. A possible aging process of myoepithelial cells was discussed in accordance with the histogenesis of transformed myoepithelial cells of salivary gland tumors.

Prenatal growth pattern of the human maxilla.

Regarding maxillofacial morphogenesis there has been a long debate on the growth of the maxillary structure. Using 120 normal fetal maxillae of gestational ages from 16 to 41 weeks, palatal radiograms and frontal histologic sections were made. We have observed two pairs of accentuated growth areas in the fetal maxillae and named them primary growth centers to formulate the maxillary trapezoid (MT) by radiologic image. The MT is formed by four primary growth centers that are best demonstrated by palatal radiograms of the fetal maxilla as well as by frontal histologic sections. The dimensional increase in the MT during the fetal period is documented and statistically analyzed. From this series of results, we have suggested that the growth centers which demarcate the MT are the basic structures of the developing human maxilla. It was also found that the four primary growth centers are the most active sites for maxilla formation until 20 weeks of gestation and thereafter the growth of the maxilla is enhanced by the participation of the intramembranous bone formation along the periphery. This was in contrast to the central primary growth centers that have already finished maturation in the early fetal period and remain only as a peripherally radiating arrangement of thick trabecular bones.

Expression of tenascin in hamster buccal pouch mucosa during experimental carcinogenesis

Experimental carcinogenesis by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) in hamster buccal pouch mucosa was evaluated for expression of tenascin, an extracellular matrix glycoprotein expressed at the epithelial-mesenchymal interface during embryonic and fetal development, wound healing and in the stroma of various neoplastic lesions, by using immunohistochemical methods. The buccal pouch mucosa in normal hamsters showed immunoreactive tenascin either as a linear delicate band or without reactivity at the immediate vicinity of the basement membrane. During carcinogenesis, in the second to fourth week of application of DMBA, the hyalinous changes in the submucosal connective tissue had a weak but diffuse immunoreactivity for tenascin. The hyperkeratinised and hyperplastic mucosa following 5 weeks of application of DMBA showed focal areas of enhanced expression in the vicinity of the basement membrane. Subsequently, specimens showing hyperplasia, dysplasia, carcinoma in situ and invasive carcinomas had comparatively more widespread stromal immunoreactivity where the extent of enhanced reactivity positively correlated with the advancing lesion. These results compared with the results of expression in human normal mucosa, leukoplakia and squamous cell carcinoma of the oral cavity (Shrestha et al., Oral Oncol, Eur J Cancer 1994, 30, 132-137) suggest that the expression of tenascin in experimental carcinogenesis of hamster buccal pouch mucosa, as a model, faithfully mimics the same in human oral mucosa.

N1-guanyl-1,7,-diamineoheptane, an inhibitor of deoxyhypusine synthase, suppresses differentiation and induces apoptosis via mitochondrial and AMPK pathways in immortalized and malignant human oral keratinocytes

BACKGROUND Although N(1)-guanyl-1,7,-diamineoheptane (GC7), an inhibitor of deoxyhypusine synthase, has been shown to inhibit cell growth, the mechanism of its action is not completely understood. In this study, we investigated the mechanisms of the effects of GC7 on cell growth, differentiation and apoptosis in relation to adenosine monophosphate-activated protein kinase (AMPK) activation, as AMPK is known to be a possible target for cancer treatment. METHODS The effects of GC7 on the growth of immortalized human oral keratinocytes (IHOK) and primary oral cancer cells (HN4), was investigated using MTT assay, Western blotting, cell cycle analysis, DNA fragmentation and expression of apoptotic pathway proteins. RESULTS N(1)-guanyl-1,7,-diamineoheptane inhibited cell proliferation in a time- and dose-dependent manner in IHOK and HN4 cells. GC7 treatment decreased the expression of differentiation markers, such as involucrin, CK13 and CK19. The major mechanism of growth inhibition by GC7 treatment was induction of apoptosis, which is supported by sub-G(1) phase arrest, annexin V-FITC staining and DNA fragmentation analysis. GC7 treatment increased the cytosolic level of cytochrome c and resulted in caspase-3 activation. GC7 treatment also resulted in a strong activation of AMPK. Furthermore, specific AMPK activator blocked the GC7-induced growth inhibition effect, as well as apoptosis. CONCLUSION These results demonstrate that GC7 blocks immortalized and malignant keratinocyte cell proliferation and differentiation by inducing apoptosis through the mitochondrial and AMPK pathways. On the basis of these observations, we propose that a strategy combining GC7 and AMPK inhibition could be developed into a novel chemotherapeutic modality in oral cancer.

Expression of Transglutaminase C during the Prenatal Development of Human Submandibular Glands

The involvement of transglutaminase C (TGase C) in morphogenesis and cytodifferentiation during glandular tubule formation was addressed by immunolocalization of the protein at different stages of prenatal human submandibular gland development in 100 fetuses and 20 adult salivary glands. Immunocytochemical detection was carried out using a monospecific antibody to TGase C. The results showed TGase C reactivity in both acini and ducts early in development (from 10 to 14 weeks), followed by a marked increase in ductal activity and a decline in acinar activity up to 32 weeks. During the peak of reactivity at 25 to 32 weeks, staining was concentrated in the apical ends of the columnar cells. In the adult, staining was weakly and diffusely distributed in the striated and excretory ducts. Western blot analysis of the cellular extracts of pooled samples from various stages of salivary gland development showed a single strong band at 76 kDa early in development. This band became weaker after 32 weeks of prenatal development and in the adult. These findings of transient high expression of TGase C, which coincide with the development of tubulo-alveolar structure, suggest that TGase C may play a role in morphogenesis in human salivary gland development.

Immunohistochemical distribution of MAM-3 and MAM-6 antigens in developing salivary glands of the human fetus

SummaryThe immunohistochemical expression of MAM-3 and MAM-6 antigens was studied in developing human fetal salivary gland removed at autopsy of 22 normal fetuses of varying maturity (10–40 weeks of gestation). The onset of functional maturation in the fetal gland was seen at 21 weeks of gestational maturity. The acini and ducts then underwent distinct alterations in antigen expression with growth and maturation until the late developmental stage (33–40 weeks of gestation) when they resemble the adult salivary gland. The role of maturing duct cells in histogenesis of salivary gland tumours is discussed.

Lower serum potassium associated with increased mortality in dialysis patients: A nationwide prospective observational cohort study in Korea

Background Abnormal serum potassium concentration has been suggested as a risk factor for mortality in patients undergoing dialysis patients. We investigated the impact of serum potassium levels on survival according to dialysis modality. Methods A nationwide, prospective, observational cohort study for end stage renal disease patients has been ongoing in Korea since August 2008. Our analysis included patients whose records contained data regarding serum potassium levels. The relationship between serum potassium and mortality was analyzed using competing risk regression. Results A total of 3,230 patients undergoing hemodialysis (HD, 64.3%) or peritoneal dialysis (PD, 35.7%) were included. The serum potassium level was significantly lower (P < 0.001) in PD (median, 4.5 mmol/L; interquartile range, 4.0–4.9 mmol/L) than in HD patients (median, 4.9 mmol/L; interquartile range, 4.5–5.4 mmol/L). During 4.4 ± 1.7 years of follow-up, 751 patients (23.3%) died, mainly from cardiovascular events (n = 179) and infection (n = 120). In overall, lower serum potassium level less than 4.5 mmol/L was an independent risk factor for mortality after adjusting for age, comorbidities, and nutritional status (sub-distribution hazard ratio, 1.30; 95% confidence interval 1.10–1.53; P = 0.002). HD patients showed a U-shaped survival pattern, suggesting that both lower and higher potassium levels were deleterious, although insignificant. However, in PD patients, only lower serum potassium level (<4.5 mmol/L) was an independent predictor of mortality (sub-distribution hazard ratio, 1.35; 95% confidence interval 1.00–1.80; P = 0.048). Conclusion Lower serum potassium levels (<4.5 mmol/L) occur more commonly in PD than in HD patients. It represents an independent predictor of survival in overall dialysis, especially in PD patients. Therefore, management of dialysis patients should focus especially on reducing the risk of hypokalemia, not only that of hyperkalemia.