Mary Anne Berberich

Molecular embryology of the lung: then, now, and in the future

Complementary molecular and genetic approaches are yielding information about gain- versus loss-of-function phenotypes of specific genes and gene families in the embryonic, fetal, neonatal, and adult lungs. New insights are being derived from the conservation of function between genes regulating branching morphogenesis of the respiratory organs in Drosophila and in the mammalian lung. The function of specific morphogenetic genes in the lung are now placed in context with pattern-forming functions in other, better understood morphogenetic fields such as the limb bud. Initiation of lung morphogenesis from the floor of the primitive foregut requires coordinated transcriptional activation and repression involving hepatocyte nuclear factor-3β, Sonic hedgehog, patched, Gli2, and Gli3 as well as Nkx2.1. Subsequent inductive events require epithelial-mesenchymal interaction mediated by specific fibroblast growth factor ligand-receptor signaling as well as modulation by other peptide growth factors including epidermal growth factor, platelet-derived growth factor-A and transforming growth factor-β and by extracellular matrix components. A scientific rationale for developing new therapeutic approaches to urgent questions of human pulmonary health such as bronchopulmonary dysplasia is beginning to emerge from work in this field.

[147] Enzymes and intermediates of histidine biosynthesis in Salmonella typhimurium

Publisher Summary This chapter focuses on enzymes and intermediates of histidine biosynthesis in Salmonella typhimurium. A series of 10 enzymes in Salmonella typhimurium mediates the conversion of phosphoribosyl-pyrophosphate and ATP to histidine. Mutants lacking all 10 enzymes require only histidine for growth; therefore this pathway has no branches giving rise to other essential metabolites. The intermediates of the pathway have been isolated and characterized and assays for all 10 enzymes have been devised. The PR-AIC is converted back to ATP by way of the purine biosynthetic pathway. The histidine operon on the S. typhimurium (and probably on the Escherichia coli ) chromosome is a cluster of the structural genes for the histidine biosynthetic enzymes. The enzymes are always purified either from cells grown under conditions of derepression or from strains containing a constitutive mutation. Several of the assays for the histidine biosynthetic enzymes are followed spectrophotometrically. These assays generally have a total volume of 0.3 ml in cuvettes of 1-cm light path and a total capacity of 1 ml. Purification of the dehydratase-phosphatase complex has proved to be exceedingly difficult because the enzyme appears to undergo multiple and still unpredictable aggregation disaggregation reactions.

A glutamate-dependent phenotype in E., coli K12: The result of two mutations

Abstract Phenotypic expression of a glutamate growth requirement depends on the presence of two independent mutations. The enzymes affected are glutamate dehydrogenase and glutamate synthase. Studies demonstrating the absence of linkage between the genes for these two enzymes are presented.

Studies on the size of the messenger-RNA transcribed from the histidine operon during simultaneous and sequential derepression

The techniques of double isotope labeling and specific hybridization have been used to determine the size of the mRNA transcribed from the histidine operon in Salmonella typhimurium under conditions of derepression. The results obtained by both techniques agree with the previous results of Martin3 which demonstrated that, in a constitutive mutant, the histidine mRNA is polycistronic. Previous studies revealed that derepression of the enzymes for histidine biosynthesis may proceed by either of two modes1,2. One mode is characterized by simultaneous derepression of all the enzymes, whereas the other mode is characterized by the derepression of the enzymes in a temporal sequence which corresponds with the positional sequence of genes in the histidine operon. Since the present studies show that the mRNA transcribed from the histidine operon is always polycistronic, regardless of the mode of derepression, we may rule out the possibility that the difference between the two modes reflects any difference in the size of the histidine mRNA. Rather, the difference must lie in the manner in which the polycistronic histidine mRNA is translated.

Catabolism and nitrogen control in Escherichia coli

It would appear from these studies that nitrogen control reflects the catabolic capacity of the cell and that utilizable nitrogen sources and some carbon sources are, to some extent, in competition for this capacity. The series of catabolic events initiated by addition of D-amino acids or by growth on aldol sugars, in the presence of ammonia nitrogen in the growth medium, provide an opportunity for study of the positive aspect of nitrogen control under conditions where negative control predominates. This approach may eventually clarify the apparent interactions between the modification cascade components, PII and UT/UR, with the nitrogen regulatory gene, glnG. The utilization of nutrients by E. coli seems less a matter of energy than of expeditious use of whatever is offered in the diet. A comparison of the rate of increase of GS on cultural downshift with the rate of increase following D-glutamate addition would suggest that control by nitrogen limitation is about eight times more effective than positive activation by D-glutamate in the presence of ammonia nitrogen. This observation is consistent with the finding of an additive effect for the D-amino acids which can function as positive activators in GS regulation. It has been demonstrated for the wild-type organism that the increase in GS level generated by a mixture of D-glutamate, D-lysine, D-threonine, and glycine approximates the increase in GS level observed during step-down of the culture from an ammonia-sufficient to an ammonia-limited condition. This observation further supports the physiologic relevance of the effect of D-amino acids in nitrogen control and suggests that the apparent derepression of GS observed upon exhaustion of the ammonia nitrogen supply represents a composite of positive activation generated as alternative catabolic functions assume a greater importance. As might be expected, addition of D-glutamate to cells at the point of ammonia exhaustion had no additional positive effect. Following a downshift from glucose-ammonia-glutamine to glucose-glutamine cultural conditions, only the level of GS increases during the initial 60-minute observation period. This finding suggests that glutamine catabolism may, like D-threonine, D-lysine, and glycine, bypass the positive activation of GDH and GAT controls. The likely possibility in that the increases observed for GAT and GDH depend on D-glutamate as specific inducer. There are several instances where D-amino acids function as inducers of L-amino acid dehydrogenases and where amino acid racemase activity is directly coupled to flavoprotein dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)