Expression patterns of mRNAs for ammoniametabolizing enzymes in the developing rat: the ontogenesis of hepatocyte heterogeneity

Summary The expression patterns of the mRNAs for the ammonia-metabolizing enzymes carbamoylphosphate synthetase (CPS), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were studied in developing pre- and neonatal rat liver byin situ hybridization.In the period of 11 to 14 embryonic days (ED) the concentrations of GS and GDH mRNA increases rapidly in the liver, whereas a substantial rise of CPS mRNA in the liver does not occur until ED 18. Hepatocyte heterogeneity related to the vascular architecture can first be observed at ED 18 for GS mRNA, at ED 20 for GDH mRNA and three days after birth for CPS mRNA. The adult phenotype is gradually established during the second neonatal week, i.e. GS mRNA becomes confined to a pericentral compartment of one to two hepatocytes thickness, CPS mRNA to a large periportal compartment being no longer expressed in the pericentral compartment and GDH mRNA is expressed over the entire porto-central distance, decreasing in concentration going from central to portal. Comparison of the observed mRNA distribution patterns in the perinatal liver, with published data on the distribution of the respective proteins, points to the occurrence of posttranslational, in addition to pretranslational control mechanisms in the period of ontogenesis of hepatocyte heterogeneity.Interestingly, during development all three mRNAS are expressed outside the liver to a considerable extent and in a highly specific way, indicating that several organs are involved in the developmentally regulated expression of the mRNAs for the ammonia-metabolizing enzymes, that were hitherto not recognized as such.     

Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli

The gene for ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction. A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21(DE3). Induction of expression in cultures of this transformant led to the accumulation of approx. 25 mg/l per A600 unit of human CNTF. CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn(2+)-affinity chromatography. Purified CNTF contained less than 0.1% contaminating E. coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC). The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A1%(279) = 9.0. Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine. High-purified recombinant human CNTF supported the survival of chick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations. These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.     

Structure-function studies of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a polypeptide that promotes the survival and/or differentiation of a number of neural cell types. Here we present a structural and functional analysis of the human CNTF molecule. Variant proteins were synthesized byEscherichia coli trnsformmed with mutant cDNA constructs, and purified by SDS-polyacrylamide gel electrophoresis and reverse phase high pressure liquid chromatography. Most variant CNTF proteins lacked neurotrophic activity, but two N-and C-terminal deletions (2–14 and 173–200, respectively) actually displayed a several-fold increase in specific activity. Loss of biological activity was accompanied by changes in the alphahelical nature of CNTF as measured by circular dichroism. These data strengthen the proposed similarity between CNTF and the family of hematopoietic cytokines.     

Expression patterns of BMPRs in the developing mouse molar

During development, Bone Morphogenetic Proteins (BMPs) can induce apoptosis, cell growth or differentiation. These different effects are mediated by dimers of two types of BMP–receptors (BMPRs). To identify the responding cells during tooth development and search for possible tissue–or stage–specificities in the receptors involved, the distribution patterns of BMPR–IA, –IB and –II were investigated in the mouse molar, from bud to bell stage. At the bud stage, BMP–2 was suggested to be involved in the formation of an epithelial signaling center, the primary enamel knot (PEK), while BMP–4 would mediate the condensation of the mesenchyme. Immunostaining showed the presence of BMPR–IA and –II in the epithelium instead of BMPR–IB and –II in the mesenchyme. At the cap stage, BMPR–IB was detected in the epithelium but not BMPR–II, suggesting the existence of another type II receptor to form a functional dimer. At the late cap stage in the epithelium, BMP–4, BMPR–IA and –II were restricted to the internal part of the PEK and the stalk: two apoptotic areas. The three proteins were detected in the mesenchyme, showing a strong staining where cusps were about to form. At the late bell stage, BMP–2 or –4 may induce cell differentiation. BMPR–IB and –II were detected in odontoblasts instead of BMPR–IA and –II in ameloblasts. These results provide the first evidence of multiple type I and type II BMP–receptors, expressed in the dental epithelium and mesenchyme at different stages of development, to signal different cellular activities in a time– and tissue–specific way.     

Expression patterns of ADAMs in the developing chicken lens

In the present study the expression patterns of ADAM (a disintegrin and metalloprotease) genes in the chicken developing lens were analyzed. Using in situ hybridization, we found that seven members of the ADAM family including ADAM9, ADAM10, ADAM12, ADAM13, ADAM17, ADAM22, and ADAM23 are expressed in the developing embryonic lens. From embryonic incubation day (E) 2 to E3, most of the ADAMs investigated here are expressed in the lens placode and lens vesicle. From E5 to E7, all seven ADAMs, but predominantly ADAM9 and ADAM10, are throughly expressed in the central epithelium, as well as in the proliferating lens epithelium and the equatorial lens epithelium. From E9 to E14, expression of ADAM9, ADAM10, and ADAM17 decreases moderately in these regions. ADAM12 and ADAM13 are weakly expressed in the central epithelium and the lens epithelium, and are not detectable from E14 onward. ADAM22 and ADAM23 are expressed in the central epithelium, the lens epithelium and the equatorial lens epithelium at E5 and decrease gradually afterwards in the same regions. At E16, only weak ADAM9, ADAM10 and ADAM17 signals are found in the anterior lens epithelium. The changing spatiotemporal expression of the seven ADAMs suggests a regulatory role for these molecules during chicken lens development.     

Characterization of recombinant human brain-derived neurotrophic factor variants

The purpose of this work was the chemical characterization of variants of the recombinant human brain derived neurotrophic factor (rHu-BDNF), expressed in Escherichia coli. This paper also addresses the question of the in vitro activity of these variants. Chemical characterization of the variants employed peptide mapping using Glu-C protease and cyanogen bromide digestion on reduced and alkylated variants followed by the analysis of the digested peptides using mass spectrometry and Edman sequencing. The BDNF variants in this work have been designated by the order of their elution as observed from the high temperature RPLC assay. It was determined that Peaks 1 and 2, which eluted just before the predominant BDNF peak, had methionine sulfoxide instead of methionine at positions 31 and 61, respectively. Peak 4, which is chromatographically a single peak, contained three variants. Two of these variants had norleucine instead of methionine, at positions 61 and 92, respectively, while the third had methionine sulfoxide instead of methionine at position 92. Peak 5 had norleucine at position 31 instead of methionine. All of these variants showed in vitro biological activity consistent with the BDNF standard, suggesting the preservation of the trkB receptor-ligand binding domain of the variants.     

Cloning and expression of human ciliary neurotrophic factor

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure. Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N-terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.     

Brain-derived neurotrophic factor transiently stabilizes silent synapses on developing neuromuscular junctions

A general feature of the developing nervous system is the activity-dependent rearrangement of genetically defined, synaptic connections. A parallel process occurs at the developing neuromuscular junction as activity-dependent synapse withdrawal reduces the initial polyneuronal innervation of individual muscle fibers to a mononeuronal innervation within the first few weeks after birth. Because members of the neurotrophin gene family influence motor neuron differentiation and survival, we examined whether or not they also influence synaptic rearrangements in neonatal muscles. We found that treatment with brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4/5 (NT-4/5) causes the transient retention of multiple synaptic contacts on neonatal myofibers. However, the combination of both electrophysiological and histological assays revealed that the majority of such supernumerary synaptic contacts are functionally inactive or “silent.” There also occurs an increase in the number of retracting axons. Because BDNF mRNA is expressed in developing muscle and the trkB tyrosine kinase receptor for BDNF is expressed by neonatal motor neurons, our results suggest that BDNF may play an endogenous role in the refinement of synaptic connectivity that occurs in skeletal muscles after birth. © 1996 John Wiley & Sons, Inc.     

S100B protein, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor in human milk

Background

Human milk contains a wide variety of nutrients that contribute to the fulfillment of its functions, which include the regulation of newborn development. However, few studies have investigated the concentrations of S100B protein, brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF) in human milk. The associations of the concentrations of S100B protein, BDNF, and GDNF with maternal factors are not well explored.

Methodology/Principal Findings

To investigate the concentrations of S100B protein, BDNF, and GDNF in human milk and characterize the maternal factors associated with their levels in human milk, human milk samples were collected at days 3, 10, 30, and 90 after parturition. Levels of S100B protein, BDNF, and GDNF, and their mRNAs in the samples were detected. Then, these concentrations were compared with lactation and other maternal factors. S100B protein levels in human milk samples collected at 3, 10, 30, and 90 d after parturition were 1249.79±398.10, 1345.05±539.16, 1481.83±573.30, and 1414.39±621.31 ng/L, respectively. On the other hand, the BDNF concentrations in human milk samples were 10.99±4.55, 13.01±5.88, 13.35±6.43, and 2.83±5.47 µg/L, while those of GDNF were 10.90±1.65, 11.38±1., 11.29±3.10, and 11.40±2.21 g/L for the same time periods. Maternal post-pregnancy body mass index was positively associated with S100B levels in human milk (r = 0.335, P = 0.030<0.05). In addition, there was a significant correlation between the levels of S100B protein and BDNF (z = 2.09, P = 0.037<0.05). Delivery modes were negatively associated with the concentration of GDNF in human milk.

Conclusions

S100B protein, BDNF, and GDNF are present in all samples of human milk, and they may be responsible for the long term effects of breast feeding.     

Expression patterns of the ADAMs in early developing chicken cochlea

Members of the ADAM (a disintegrin and metalloprotease) family are type I transmembrane proteins involved in biological processes of proteolysis, cell adhesion, cell–matrix interaction, as well as in the intracellular signaling transduction. In the present study, expression patterns of seven members of the ADAM family were investigated at the early stages of the developing cochlea by in situ hybridization. The results show that each individual ADAM is expressed and regulated in the early developing cochlea. ADAM9, ADAM10, ADAM17, and ADAM23 are initially and widely expressed in the otic vesicle at embryonic day 2.5 (E2.5) and in the differential elements of the cochlear duct at E9, while ADAM12 is expressed in acoustic ganglion cells at E7. ADAM22 is detectable in cochlear ganglion cells as early as from E4 and in the basilar papilla from E7. Therefore, the present study extends our previous results and suggests that ADAMs also play a role in the early cochlear development.     

Initial formation of cellular intrinsic fiber cementum in developing human teeth

Summary The present study describes the formative process of the initiation of cellular intrinsic fiber cementum (CIFC) in still growing human teeth. From 29 premolars and molars with incomplete roots developed to 60–90% of their final length, 8 premolars (with roots formed to three quarters of their final length) were selected for electron-microscopic investigation. All teeth were clinically intact and prefixed in Karnovsky's fixative immediately after extraction. Most of them were decalcified in ethylene diaminetetraacetic acid (EDTA), and the apical part of the roots was divided axially into mesial and distal portions that were subdivided in about 5 slices each. Following osmication and embedding in Epon, these blocks were cut for light- and electron-microscopic examination. In addition, 5 teeth with incomplete roots were freed from organic material and processed for scanning electron microscopy. It was found that CIFC-initiation commenced very close to the advancing root edge and resulted in a rapid cementum thickening. Thereafter, appositional growth continued on the already established cementum surface. Large, basophilic and rough endoplasmic reticulum-rich cementoblasts, some of which became cementocytes, were responsible for both fast and slow CIFC-formation. The CIFC-matrix was free of Sharpey's fibers and composed of more or less organized intrinsic collagen fibrils, in part fibril bundles, that ran roughly parallel to the root surface. Initially, the cementum fibrils intermingled with those of the dentinal collagen fibrils, which were not yet mineralized. This boundary subsequently underwent calcification. The development of collagen fibril bundles and their extracellular arrangement were associated with cytoplasmic processes probably involved in fibril formation and fibril assembly. Many cementoblasts contained intracytoplasmic, membrane-bounded collagen fibrils, which probably were related to fibril formation rather than degradation.     

Histochemical observations of protein-bound amino groups in human developing deciduous teeth

Summary Human developing deciduous teeth were studied by histochemical methods for protein groups; dinitrofluorobenzene (DNFB) H-acid stain for tyrosine histidine and tryptophan, coupled tetrazonium reaction with pre-iodination for histidine, diazotization and Millon's reaction for tyrosine revealed strong staining in the granular precursor of ameloblastic cytoplasma, pre- and young enamel layers. But only alloxan-Schiff stain showed a more intense stainability in the dentin matrix and pre-enamel than the young enamel matrix. The reaction of sulfhydryl and disulfide groups was observed in the matrix-forming ameloblast and Tomes' process with some variation of staining in the enamel matrix. The present histochemical results of protein groups in the enamel matrix were similar to those of the cornified layer of skin.Ameloblasts moderately stained with DNFB H-acid, coupled tetrazonium and alloxanschiff reaction were similarly stained in both matrix-forming stage and reduced stage. Increasing stainability of odontoblasts was limited in the matrix-forming stage.Pronounced changes of staining intensity in the enamel matrix for protein groups were found especially in the intra-rod organic substance with progress of mineralization and growth of deposited mineral crystallites, starting from the region of apex of cusp, parallel to the dentino-enamel junction.The stainability of the dentin matrix for protein groups increases following decalcification, and non-decalcified dentin was weakly stained for the alloxan-schiff method, coupled tetrazonium reaction, sulfhydryl and disulfide method.     

Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle

Changes in theactivity and in the expression of adenylyl cyclase (AC) were examinedin mouse skeletal muscle after denervation and during development. Fourisoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blotanalysis in gastrocnemius muscle, AC9 being the most abundant. Afterdenervation, the levels of AC2 and AC9 mRNA decreased, whereas those ofAC6 and AC7 increased. AC activity in response to severalneurotransmitters was increased after denervation. During development,AC activity was high in fetus and neonate and declined in the adult;the sensitivity of AC activity to various neurotransmitters was thehighest on the third postnatal day. The levels of AC6 and AC7 mRNAswere high on the third postnatal day and then decreased in adult,paralleling the decline in AC activity. All the characteristics of ACexpression and activity in fetus and neonate resembled those observedin denervated adult muscle. These results indicate that changes in ACactivity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscleatrophy.

     

Genetic variation in brain-derived neurotrophic factor and human fear conditioning

Brain-derived neurotrophic factor (BDNF) has been implicated in hippocampal-dependent learning processes, and carriers of the Met allele of the Val66Met BDNF genotype are characterized by reduced hippocampal structure and function. Recent nonhuman animal work suggests that BDNF is also crucial for amygdala-dependent associative learning. The present study sought to examine fear conditioning as a function of the BDNF polymorphism. Fifty-seven participants were genotyped for the BDNF polymorphism and took part in a differential-conditioning paradigm. Participants were shocked following a particular conditioned stimulus (CS+) and were also presented with stimuli that ranged in perceptual similarity to the CS+ (20, 40 or 60% smaller or larger than the CS+). The eye blink component of the startle response was measured to quantify fear conditioning; post-task shock likelihood ratings for each stimulus were also obtained. All participants reported that shock likelihood varied with perceptual similarity to the CS+ and showed potentiated startle in response to CS ± 20% stimuli. However, only the Val/Val group had potentiated startle responses to the CS+. Met allele carrying individuals were characterized by deficient fear conditioning – evidenced by an attenuated startle response to CS+ stimuli. Variation in the BDNF genotype appears related to abnormal fear conditioning, consistent with nonhuman animal work on the importance of BDNF in amygdala-dependent associative learning. The relation between genetic variation in BDNF and amygdala-dependent associative learning deficits is discussed in terms of potential mechanisms of risk for psychopathology.     

Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.     

Cryopreservation of developing teeth

By using a slow rate of cooling, successful storage of developing teeth has been performed in liquid nitrogen. Dye exclusion tests indicate that 78 ± 8% of cells remain viable and transplantation to isologous hosts demonstrates that 86% of the stored developing teeth commence to grow and produce normal dental hard and soft tissue elements. The length of storage at −196 °C, for periods up to 3 months, appears to have no detrimental effect upon subsequent survival and development of tooth germs.