Four Holstein cows with ruminal and duodenal cannulas were assigned to a 4 x 4 Latin square at each of four stages of lactation (peak, wk 4; early, wk 8 to 12; mid, wk 17 to 21; and late, wk 27 to 31). Treatments were duodenal infusions of 1) Met, 2) Lys, 3) Met plus Lys, and 4) casein; periods were 7 to 10 d. Quantities of DL-Met, L-Lys, and casein infused at the four stages of lactation were 12, 30, and 400; 12, 30, and 400; 10, 25, and 333; and 8, 20, and 266 g/d, respectively. Rations were composed of corn and grass-legume silages, corn meal, wheat middlings, soybean meal, and distillers dried grains with solubles. Intake of CP (percentage of NRC) and percentage of total CP from corn sources were (peak) 87, 56; (early) 90, 71; (mid) 98, 73; and (late) 114, 77. Using content and yield of milk protein as primary response criteria, Lys appeared to be first-limiting and Met second-limiting at peak lactation, their infusion together resulted in the same production of milk (40 kg/d) and milk protein (1135 g/d) as did casein. Lysine was first-limiting in early lactation, but whether Met was second-limiting was questionable. The two AA were colimiting in midlactation. There appeared to be no AA deficiencies in late lactation. Amounts of Lys and Met (percentage of total essential AA) in duodenal digesta during peak (12.4, 3.7), early (12.6, 3.5), and midlactation (14.9, 3.9) were not adequate for optimal AA utilization.

No abstract

Amelogenesis imperfecta is a broad classification of hereditary enamel defects, exhibiting both genetic and clinical diversity. Most amelogenesis imperfecta cases are autosomal dominant disorders, yet only the local hypoplastic form has been mapped to human chromosome 4q between D4S242 1 and the albumin gene. An enamel protein cDNA, termed ameloblastin (also known as amelin and sheathlin), has been isolated from rat, mouse and pig. Its human homolog has been mapped to chromosome 4q21 between markers D4S409 and D4S400, flanking the local hypoplastic amelogenesis imperfecta critical region. Therefore, ameloblastin is a strong candidate gene for this form of amelogenesis imperfecta. To facilitate genetic studies related to this dental disease, we isolated and characterized a human ameloblastin cDNA. A human third molar cDNA library was screened and two ameloblastin clones identified. Nucleotide sequencing of these cDNAs indicated alternative splicing of the putative open reading frame, use of different polyadenylation signals, and a high degree of similarity to reported rat, mouse and porcine cDNAs. Immunohistochemistry studies on embryonic human teeth using an antibody to recombinant ameloblastin indicated ameloblastin expression by ameloblasts with localization in the enamel matrix associated with the sheath structures.

Phenotypes associated with Msx1 mutations have established the prominent role of this divergent homeogene in skeletal patterning. Previous studies have been achieved during antenatal development in relation with the early death of null mutant mice. Therefore, the present study is devoted to Msx1 homeogene in the postnatal craniofacial, axial, and appendicular skeleton. A knock-in transgenic mouse line was studied from the first postnatal week until 15 months. Whole-mount ␤-galactosidase enzymology identified Msx1 protein expression pattern. Maintained expression of Msx1 was observed in growing and adult mice, specifically in the sites where Msx1 plays an early morphogenetic role during initial skeletal patterning. These included the craniofacial sutures, autopodium, mandible, and alveolar bone. Furthermore, active membranous and endochondral bone formation involved Msx1 in the entire skeleton. Histologic sections showed that progenitor as well as differentiating and differentiated cells of all the bone cell lineages could express the Msx1 protein (chondrocytes, osteoblasts, tartrate-resistant acid phosphatase positive osteoclasts and chondroclasts). Recent developments in the genetic and developmental biology of skeletal morphogenesis demonstrate that genes critical for development are jointly expressed in discrete embryonic signalling and growth centers, the enamel knot in teeth, the cranial suture in skull morphogenesis, and the progress zone in the limb buds. The present study suggests that these signalling pathways are jointly important throughout the entire lifetime with an exquisite site-specificity spatially related to early patterning.

The present study is devoted to Msx1 distribution and function from birth to 15 months, events and periods still unexplored in vivo using Msx1 knock in transgenic mice. The study is focused on the mandible, as an exemplary model system for Msx1-dependent neural crest-derived skeletal unit. The transgenic line enabled study of morphological abnormalities in Msx1 null mutation mice and Msx1 protein expression in Msx1+/- heterozygous mice. In Msx1 null mutation, the most striking feature was an inhibition of the mandibular basal convexity, the absence of teeth and alveolar bone processes, and absence of endochondral ossification in the mandibular condyle. At birth, in Msx1+/- heterozygous animals, we identified for the first time a double Msx1 aboral-oral and disto-proximal gradient field developmental pattern located in the low border of the mandibular bone in relation with this bone segment modeling. Msx1 expression involved both osteoblast and osteoclast cells. A distinct pattern characterized bone surfaces: Periosteum osteoblast differentiation was related to Msx1 down-regulation, while in the endosteum both differentiated osteoblasts and osteoclasts expressed the homeoprotein. In postnatal stages, Msx1 expression was maintained in the alveolar bone processes and dento-alveolar cells in relation with tooth function. Our data suggest that Msx1 play a role in a site-specific manner not only in early patterning but also in skeletal growth and modeling by acting on heterogenous bone cell populations.

No abstract

Rickets is associated with site-specific disorders of enamel and dentin formation, which may reflect the impact of vitamin D on a morphogenetic pathway. This study is devoted to potential cross-talk between vitamin D and Msx/Dlx transcription factors. We raised the question of a potential link between tooth defects seen in mice with rickets and Msx2 gene misexpression, using mutant mice lacking the nuclear vitamin D receptor as an animal model. Our data showed a modulation of Msx2 expression. In order to search for a functional impact of this Msx2 misexpression secondary to rickets, we focused our attention on osteocalcin as a target gene for both vitamin D and Msx2. Combining Msx2 overexpression and vitamin D addition in vitro, we showed an inhibitory effect on osteocalcin expression in immortalized MO6-G3 odontoblasts. Finally, in the same cells, such combinations appeared to modulate VDR expression outlining the existence of complex cross-regulations between vitamin D and Msx/Dix pathways.

Changes of activities of liver enzymes and of metabolism during pregnancy and lactation have been documented in studies with rodents. Therefore, a study of activities of ruminant liver enzymes and metabolism was undertaken. Several changes of activities of enzymes involved in lipogenesis and gluconeogenesis were observed. However, magnitudes of enzyme change were less than expected from major changes in liver function that accompany lactation. Also, a number of enzymatic changes expected from rodent data were absent. Effects of breed and diet on enzyme activities in cow livers were minimal or absent. Changes in rates of glucose metabolism from lactation were greater than implied by enzyme adaptations. Analysis of kinetic data according to Michaelis-Menten kinetics indicated that apparent maximal velocities of conversion of lactate and propionate to glucose by liver slices from cattle ranged from 10 to 30 mumoles/(g.h) and apparent coefficients were 1.8 to 2.0- mM. Rates of gluconeogenesis with lactate plus propionate were not additive at saturating concentrations. The background data on enzymatic activities, glucose metabolism, lipogenesis, and gluconeogenesis in ruminant liver indicate that sophisticated studies with isolated hepatocytes from ruminants will be required to clarify metabolic adaptations, metabolic patterns, and regulatory mechanism in this tissue.