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M Melnick

Publications and source records attributed to M Melnick.

At least 19 recordsLinked to original sources
PubMed2004

Sonic hedgehog signaling plays an essential role during embryonic salivary gland epithelial branching morphogenesis.

Gene targeting studies indicate that sonic hedgehog (Shh) signaling plays an essential role during craniofacial development. Because numerous mandibular derivatives (e.g., teeth, tongue, Meckel's cartilage) are absent in Shh null mice and the embryonic submandibular salivary gland (SMG) develops from the mandibular arch, we postulated that Shh signaling is important for embryonic SMG development. To address this question, we first determined the spatiotemporal distribution of Shh; two transmembrane proteins, patched 1 (Ptc) and Smoothened (Smo), which act as a negative or a positive regulator of the Shh signal, respectively; and the Gli 3 transcription factor, which is downstream of the Shh signal. The epithelial localization of Shh, Ptc, Smo, and Gli 3 suggests that Shh signaling may act within the epithelium in a juxtacrine manner. The SMG phenotype in our embryonic day (E) 18.5 Shh null mice can be characterized as "paedomorphic," that is, it fails to progress to ontogenic stages beyond the Early Pseudoglandular ( approximately E14). In a complementary set of experiments, we used organ culture to evaluate the effect of enhanced or abrogated Shh signaling on embryonic SMG development in vitro. Paired E13 (Late Initial Bud stage) or E14 (Pseudoglandular stage) SMGs were cultured in the presence or absence of exogenous Shh peptide supplementation; Shh-supplemented explants exhibit a significant stage-dependent increase in branching morphogenesis compared with control explants. Furthermore, by using cyclopamine, a steroidal alkaloid that specifically disrupts the Shh pathway, to abrogate endogenous Shh signaling in vitro, we found a significant decrease in branching in cyclopamine-treated explants compared with controls, as well as a significant decrease in epithelial cell proliferation. Our results indicate that Shh signaling plays an essential role during embryonic SMG branching morphogenesis. Exogenous FGF8 peptide supplementation in vitro rescues the abnormal SMG phenotype seen in cyclopamine-treated explants, demonstrating that overexpression of a parallel, but related, downstream signaling pathway can compensate for diminished Shh signaling and restore embryonic SMG branching morphogenesis.

Animals↗
PubMed2002

Cleft lip with or without cleft palate and dermatoglyphic asymmetry: evaluation of a Chinese population.

OBJECTIVE: To determine if Chinese individuals with non syndromic cleft lip with or without cleft palate (CL/P) display more dermatoglyphic asymmetry than unaffected relatives or controls. DESIGN: Case-control study with two control groups (genetically related and unrelated). SETTING AND SAMPLE POPULATION: A total of 500 CL/P probands from Shanghai, China, 421 unaffected relatives, and 66 controls of Chinese heritage. METHODS: Finger and palm prints were collected, and pattern frequencies, total ridge counts (TRC), and atd angles were calculated. Asymmetry scores between right and left hands were defined for each of the three dermatoglyphic measures. Probands' asymmetry scores were compared statistically with the scores of unaffected relatives and controls. RESULTS: In general, the probands' asymmetry scores for TRC and atd angle did not differ significantly from the scores of either unaffected relatives or controls. However, probands with a positive family history of clefting showed significantly more asymmetry in their pattern types than either probands without a family history, unaffected relatives or controls. CONCLUSION: These results suggest that a unique genetic mechanism of developmental instability may obtain in CL/P individuals with a positive family history of clefting.

Analysis of Variance↗
PubMed2001

The functional genomic response of developing embryonic submandibular glands to NF-kappa B inhibition.

BACKGROUND: The proper balance between epithelial cell proliferation, quiescence, and apoptosis during development is mediated by the specific temporal and spatial appearance of transcription factors, growth factors, cytokines, caspases, etc. Since our prior studies suggest the importance of transcription factor NF-kappaB during embryonic submandibular salivary gland (SMG) development, we attempted to delineate the emergent dynamics of a cognate signaling network by studying the molecular patterns and phenotypic outcomes of interrupted NF-kappaB signaling in embryonic SMG explants. RESULTS: SN50-mediated inhibition of NF-kappaB nuclear translocation in E15 SMG explants cultured for 2 days results in a highly significant increase in apoptosis and decrease in cell proliferation. Probabilistic Neural Network (PNN) analyses of transcriptomic and proteomic assays identify specific transcripts and proteins with altered expression that best discriminate control from SN50-treated SMGs. These include PCNA, GR, BMP1, BMP3b, Chk1, Caspase 6, E2F1, c-Raf, ERK1/2 and JNK-1, as well as several others of lesser importance. Increased expression of signaling pathway components is not necessarily probative of pathway activity; however, as confirmation we found a significant increase in activated (phosphorylated/cleaved) ERK 1/2, Caspase 3, and PARP in SN50-treated explants. This increased activity of proapoptotic (caspase3/PARP) and compensatory antiapoptotic (ERK1/2) pathways is consistent with the dramatic cell death seen in SN50-treated SMGs. CONCLUSIONS: Our morphological and functional genomic analyses indicate that the primary and secondary effects of NF-kappaB-mediated transcription are critical to embryonic SMG developmental homeostasis. Relative to understanding complex genetic networks and organogenesis, our results illustrate the importance of evaluating the gene, protein, and activated protein expression of multiple components from multiple pathways within broad functional categories.

Animals↗
PubMed2001

Developmental expression of survivin during embryonic submandibular salivary gland development.

BACKGROUND: The regulation of programmed cell death is critical to developmental homeostasis and normal morphogenesis of embryonic tissues. Survivin, a member of the inhibitors of apoptosis protein (IAP) family primarily expressed in embryonic cells, is both an anti-apoptosis and a pro-survival factor. Since our previous studies have demonstrated the importance of apoptosis during embryonic submandibular salivary gland (SMG) development, we postulated that survivin is a likely mediator of SMG epithelial cell survival. RESULTS: We investigated the developmental expression of survivin in Pseudoglandular (approximately E14), Canalicular (approximately E15) and Terminal Bud (approximately E17) Stage SMGs. We report a significant 26% increase in transcript levels between the Canalicular and Terminal Bud Stages. Immunohistochemical studies demonstrate nuclear-localized survivin protein in epithelial cells bounding forming lumina in Canalicular and Terminal Bud Stage SMGs. CONCLUSIONS: Survivin is known to be a pro-survival and anti-apoptotic factor. Given that survivin translocation into the nucleus is required for the induction of entry into the cell cycle and the inhibition of apoptosis, our demonstration of nuclear-localized survivin protein in presumptive ductal and proacinar lumen-bounding cells suggests that survivin may be a key mediator of embryonic SMG epithelial cell survival.

Amino Acid Sequence↗
PubMed2001

Embryonic mouse submandibular salivary gland morphogenesis and the TNF/TNF-R1 signal transduction pathway.

TNF is a pleiotropic cytokine that modulates cell proliferation and apoptosis. The objective of the present study was to investigate the possible function(s) of the TNF/TNF-R1 signaling pathway in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro experiments to investigate the phenotypic outcomes of enhanced and deficient ligand. The results of these experiments indicate that the TNF/TNF-R1 signal transduction pathway plays an important role in balancing cell proliferation and apoptosis during SMG duct and presumptive acini formation.

Animals↗
PubMed2001

An alternatively spliced Muc10 glycoprotein ligand for putative L-selectin binding during mouse embryonic submandibular gland morphogenesis.

Late-gestation (embryonic day 18; E18) mouse submandibular glands (SMG) comprise a network of large and small ducts that terminate in lumen-containing, presumptive acini (terminal buds) expressing unique, cell membrane-associated embryonic mucin. The objective here was to clone and sequence embryonic low molecular-weight SMG mucin, predict its secondary structure, and begin to investigate its possible role in SMG development. Evidence was found that: (1) embryonic low molecular-weight mucin is an alternatively spliced Muc10 gene product, 220 amino acids in size (approximately 25 kDa), rich in potential O-glycosylation sites, and variably glycosylated (approximately 40 and 68 kDa); (2) consensus secondary-structure prediction for embryonic low molecular-weight mucin is consistent with a molecule that is anchored to the plasma membrane, directly or indirectly (via a glycolipid), and has a protein core that serves as a scaffold for carbohydrate presentation; (3) embryonic L-selectin is immunolocalized to the plasma membrane region of terminal-bud epithelial cells in a pattern similar to that seen for embryonic mucin; (4) embryonic, but not adult, mucin is able to bind L-selectin and does so endogenously in E18 SMG. As the primary role of L-selectin is to mediate cell adhesion and its ligands are mucin-like glycoproteins, it is suggested that this embryonic low molecular-weight mucin be termed MucCAM.

Age Factors↗
PubMed2001

Interleukin-6 signaling and embryonic mouse submandibular salivary gland morphogenesis.

Interleukin-6 (IL-6) is a multifunctional cytokine that mediates cell growth, differentiation, and survival. It was the objective of the present study to investigate the possible function(s) of IL-6 signaling in embryonic mouse submandibular salivary gland (SMG) morphogenesis. After characterizing in vivo mRNA and protein expression of various constituents of this pathway, we utilized in vitro strategies to investigate the phenotypic outcomes of enhanced IL-6-induced signaling and immunoperturbation of IL-6 binding to cognate receptors. These experiments demonstrate: (1) there is a significant increase of IL-6 mRNA with progressive SMG development, and that this is highly correlated with TNF transcript levels; (2) IL-6 and its cognate receptors are immunolocalized in SMG branching epithelia from the canalicular stage to the late terminal bud stage, as are other constituents of the IL-6 pathway; (3) as compared to controls, IL-6-supplemented explants exhibit a substantial increase in overall size and in the number of ductal branches and terminal buds, as well as a highly significant increase in epithelial cell proliferation; (4) SMG explants cultured in the presence of anti-IL-6 neutralizing antibodies exhibit a marked decrease in epithelial ducts and terminal buds, concomitant with a significant decline in cell proliferation and a highly significant increase in apoptosis. Taken together, our experimental results indicate that IL-6 signaling is important to SMG developmental homeostasis.

Animals↗
PubMed2000

Thrombospondin-2 gene expression and protein localization during embryonic mouse palate development.

The mammalian palate develops from projections of the paired maxillary processes termed palatal shelves. Shelf growth is an essential in normal palatal morphogenesis. Mesenchymal proliferation in the palatal shelves is modulated by transforming growth factor-1 (TGF-1), among other growth factors. Several pathways effect TGF-beta activation, including one which utilizes thrombospondin (TSP). TSP-1 is a major activator of TGF-beta in vivo and has been localized in head mesenchyme, including palates. TSP-2 appears to inhibit TSP-1 activation of latent TGF-beta by competitively binding the latent TGF-beta. Here the TSP-2 mRNA transcript and the immunolocalization of TSP-2 protein with progressive palatogenesis were quantified. There was a significant (p < 0.05) decline of TSP-2 transcript with palatal maturation; there was no evidence correlating the TSP-2 transcription with the amount of activated TGF-beta. At the vertical shelf stage of palatogenesis, TSP-2 protein was found throughout the extracellular matrix of shelf mesenchyme. By the horizontal shelf stage, TSP-2 protein was principally localized to the ossification centres of the developing maxilla, both in extracellular matrix and bone; far less was seen in palatal shelves proper. These results suggest that TSP-2 is multifunctional during embryonic palate formation.

Animals↗
PubMed2000

Mouse submandibular gland morphogenesis: a paradigm for embryonic signal processing.

Signal processing is the sine qua non of embryogenesis. At its core, any single signal transduction pathway may be understood as classic Information Theory, adapted as an open system such that, because of networking, the "receiver" is presented with more information than was initially signaled by the "source". Over 40 years ago, Waddington presented his "Epigenetic Landscape" as a metaphor for the hierarchical nature of embryogenesis. Mathematically, Waddington's landscape may be modeled as a neural net. The "black box" of the neural net is an interacting network of signal transduction pathways (using hormones, growth factors, cytokines, neurotransmitters, and others) which inform the Boolean logic gates. An emerging theme in developmental biology is that defined sets of epigenetic circuits are used in multiple places, at multiple times, for similar and sometimes different purposes during organogenesis. As we show here, submandibular gland embryonic and fetal development is a splendid paradigm of these epigenetic circuits and their phenotypic outcomes, such as branching and lumen formation.

Animals↗
PubMed2000

Descriptive epidemiology of nonsyndromic cleft lip with or without cleft palate in Shanghai, China, from 1980 to 1989.

OBJECTIVE: The purpose of this study was to characterize nonsyndromic cleft lip with or without cleft palate (CL+/- CP) in an Asian population. DESIGN: Birth prevalence was assessed in a large birth series in Shanghai, China. A 1:3 sex-age-hospital matched case-control design was used to assess the effects of parental ages and pregnancy history on risk of CL+/- CP. PARTICIPANTS: Records of live births from 1980 to 1989 in 22 hospitals in Shanghai, China, were reviewed, comprising 541,504 consecutive births, which is by far the largest such Chinese sample ever investigated. The case-control study included 528 (308 male, 220 female) nonsyndromic CL+/- CP cases and 1,563 (912 male, 651 female) controls. RESULTS: From 1980 to 1989, the overall birth prevalence was 1.2 per 1,000 live births with statistically significant seasonal variation (more CL+/- CP births in January to July). The overall male:female ratio was 1.40:1. For males, statistically significant associations were identified with maternal age for the most severe clefts (bilateral overall, and also bilateral CL+CP subgroup). For females, statistically significant association was shown for pregnancy age with birth order (overall and in most subgroups). CONCLUSIONS: The birth prevalence of CL+/- CP in this Asian population was similar to published Caucasian rates. The observed seasonal variation would be consistent with possible environmental factors. Significant associations with maternal age, pregnancy age, and birth order warrant additional study of pregnancy history in Asian CL+/- CP.

Birth Order↗
PubMed1999

Submandibular gland morphogenesis: stage-specific expression of TGF-alpha/EGF, IGF, TGF-beta, TNF, and IL-6 signal transduction in normal embryonic mice and the phenotypic effects of TGF-beta2, TGF-beta3, and EGF-r null mutations.

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell-cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF-alpha/EGF/EGF-R; IGF-II/IGF-IR/IGF-IIR; TGF-betas and cognate receptors; TNF, IL-6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF-beta2, TGF-beta(3), EGF-R). Among many observations, the following seem of particular importance: (1) TGF-alpha and EGF-R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF-R1 signalling; (3) TGF-beta2 and TGF-beta3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF-R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies.

Animals↗
PubMed1998

Insulin-like growth factor II receptor, transforming growth factor-beta, and Cdk4 expression and the developmental epigenetics of mouse palate morphogenesis and dysmorphogenesis.

The B10/B10.A congenic mouse pair serves as a model for identifying specific genes related to morphogenesis and dysmorphogenesis of the embryonic palate and other organs. The present report describes our initial investigation of the Fraser-Juriloff paradigm, which proposes that susceptibility to malformation results from genetically determined differences in normal developmental patterns. Specifically, we evaluated the relationship between Igf2r gene expression, transforming growth factor-beta (TGF-beta) activation, and cdk4 gene expression. By using in situ hybridization, RNase protection assays, indirect immunofluorescence, Western blots, and bioassays, we show 1) the presence of insulin-like growth factor II (IGF-II), IGF-II receptor (IGF-IIR), IGF-IR, TGF-beta, plasminogen, plasminogen activators [urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA)], and Cdk4 in developing palates; 2) on embryonic day 14 (E14), which is a critical day for palatal growth, B10.A embryos have 82% greater IGF-IIR mRNA than B10; 3) on E14, B10.A embryonic palates have a 57% greater level of active TGF-beta2 than B10, although the total TGF-beta2 is nearly identical; and 4) on E14, B10 embryonic palates have a 52% greater level of Cdk4 mRNA than B10.A palates, a measure of cell cycle progression. Because cellular activation of latent TGF-beta appears to require binding to the mannose-6-phosphate (M6P) binding site of the IGF-IIR and is plasmin and plasminogen activator dependent, the positive correlation of IGF-IIR levels and active TGF-beta2 levels seems to be key. Thus, the strain variation of TGF-beta2/IGF-IIR-mediated growth inhibition in late G1 phase would appear to account for the slower growth and development of B10.A palates relative to B10. Elevated corticosteroid (CORT) exposure in E14 B10.A embryos significantly increases TGF-beta levels, 87% of which is TGF-beta2, as well as the levels of active TGF-beta, 64% of which is TGF-beta2. Without exogenous CORT, B10.A embryos do not have clefts; hence, we present an outline of pathogenesis: slower growing B10.A embryos have an up-regulation of IGF-IIR, which serves to sequester IGF-II from the growth-promoting IGF-IR and to bind more CORT-up-regulated, latent TGF-beta2 for subsequent plasmin-dependent activation; higher levels of TGF-beta2 signaling down-regulate Cdk4 and result in greater palatal growth inhibition at a critical stage of palatogenesis and, thus, cleft palate. We present an epigenetic model of information processing related to cell proliferation. The model is a dynamical network that uses continuous logic to learn its rules from changing conditions.

Animals↗
PubMed1998

Mouse submandibular gland mucin: embryo-specific mRNA and protein species.

Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein.

Animals↗
PubMed1998

Dual function of Ras in Raf activation.

The small guanine nucleotide binding protein p21(Ras) plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21(Ras )in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21(Ras) (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.

Animals↗
PubMed1996

Bis tertiary amide inhibitors of the HIV-1 protease generated via protein structure-based iterative design.

A series of potent nonpeptide inhibitors of the HIV protease have been identified. Using the structure of compound 3 bound to the HIV protease, bis tertiary amide inhibitor 9 was designed and prepared. Compound 9 was found to be about 17 times more potent than 3, and the structure of the protein-ligand complex of 9 revealed the inhibitor binds in an inverted binding mode relative to 3. Examination of the protein-ligand complex of 9 suggested several modifications in the P1 and P1' pockets. Through these modifications it was possible to improve the activity of the inhibitors another 100-fold, highlighting the utility of crystallographic feedback in inhibitor design. These compounds were found to have good antiviral activity in cell culture, were selective for the HIV protease, and were orally available in three animal models.

Amides↗
PubMed1996

Structure-based design and synthesis of substituted 2-butanols as nonpeptidic inhibitors of HIV protease: secondary amide series.

The design, synthesis, and crystallographic analysis of protein-inhibitor complexes is described for a novel series of nonpeptidic HIV protease (HIV Pr)inhibitors. Beginning with a cocrystal structure of a Phe-Pro peptidomimetic bound to the HIV Pr, design was initiated that resulted in the substituted 2-butanol compound 8 as the lead compound (Ki = 24.5 microM, racemic mixture). Modifications on the initial compound were then made on the basis of its cocrystal structure with HIV Pr and inhibition data, resulting in compounds with enhanced potency against the enzyme (compound 18, Ki = 0.48 microM). These inhibitors were found to bind to the enzyme essentially as predicted on the basis of the original design hypothesis. Stereospecific synthesis of individual enantiomers confirmed the prediction of a binding preference for the S alcohol stereochemistry. Modest antiviral activity was demonstrated for several of the more potent HIV Pr inhibitors in a HIV-1 infected CEM-SS cell line.

Amides↗
PubMed1996

Developmental expression and CORT-regulation of TGF-beta and EGF receptor mRNA during mouse palatal morphogenesis: correlation between CORT-induced cleft palate and TGF-beta 2 mRNA expression.

Glucocorticoids (CORT) have been shown to induce cleft palate in mice. Although the pathogenetic pathway of CORT-induced cleft palate has been investigated for several decades, the molecular details remain to be elucidated. Since growth factors have been shown to regulate palate morphogenesis, and the expression of several growth factors or their receptors, e.g. TGF-beta, EGF receptor (EGF-R), are known to be modulated by CORT, we postulate that CORT modulation of growth factor (or receptor) gene expression is a key mechanism involved in CORT-induced cleft palate. To test this hypothesis, we analyzed the steady-state levels (Northern and RNase protection) and developmental expression (in situ hybridization) of four CORT-responsive genes--TGF-Beta 1, TGF- beta 2, TGF-beta 3, and EGF receptor (EGF-R)--in developing mouse palates in the presence or absence of exogenous CORT. Pregnant B10.A dams were injected on day 12 of gestation with CORT or sham-injected and embryonic palates were collected at 1, 2, and 3 days postinjection (E13-E15). During mouse palate development, significant increases in TGF-beta 1 and TGF-beta 3 mRNA levels, as well as significant decrease in TGF-beta 2 mRNA levels, are detected; no significant difference in EGF-R transcript level is observed with progressive development. In CORT-exposed palates, we demonstrate no significant differences in the direction or magnitude of change with time in TGF-beta 1, TGF-beta 3, and EGF-R mRNA levels compared to controls. However, CORT delays by 1 day the down-regulation of palatal TGF-beta 2 transcript normally seen on day 14 of gestation. TGF-beta 2 is known to inhibit cell proliferation. The level of TGF-beta 2 mRNA, the only isoform primarily expressed in the palatal mesenchyme, significantly decreases with progressive palatal development; this down-regulation of TGF-beta 2 expression is associated with increased mesenchymal cell proliferation and palatal shelf growth. CORT, at a critical stage of palatogenesis, induces a delay in the normal down-regulation of TGF-beta 2 gene expression. Given that CORT is known to inhibit mesenchymal cell proliferation and palatal shelf growth, we conclude that the CORT-induced delay in the normal down-regulation of TGF-beta 2 gene expression is probably key event in the pathogenesis of CORT-induced cleft palate.

Animals↗