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L T Smith

Publications and source records attributed to L T Smith.

At least 91 records · Page 5Linked to original sources
PubMed1988

Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti.

Intracellular accumulation of glycine betaine has been shown to confer an enhanced level of osmotic stress tolerance in Rhizobium meliloti. In this study, we used a physiological approach to investigate the mechanism by which glycine betaine is accumulated in osmotically stressed R. meliloti. Results from growth experiments, 14C labeling of intermediates, and enzyme activity assays are presented. The results provide evidence for the pathway of biosynthesis and degradation of glycine betaine and the osmotic effects on this pathway. High osmolarity in the medium decreased the activities of the enzymes involved in the degradation of glycine betaine but not those of enzymes that lead to its biosynthesis from choline. Thus, the concentration of the osmoprotectant glycine betaine is increased in stressed cells. This report demonstrates the ability of the osmolarity of the growth medium to regulate the use of glycine betaine as a carbon and nitrogen source or as an osmoprotectant. The mechanisms of osmoregulation in R. meliloti and Escherichia coli are compared.

Alcohol Oxidoreductases↗
PubMed1986

Collagen types I, III, and V in human embryonic and fetal skin.

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult-like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger-diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermal-epidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno-SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70-75%, type III collagen was 18-21%, and type V was 6-8% of the total of these collagens at all gestational ages tested, compared to 85-90% type I, 8-11% type III, and 2-4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel- and nerve-associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.

Collagen↗
PubMed1986

Embryogenesis of the dermis in human skin.

The process of human dermal development has been examined from the stage of the embryonic mesenchymal dermis (5-8 weeks of gestation) through the formation of the fibrous dermal connective (end of the first trimester), and during the growth of the fetal dermis (second and third trimesters) until birth. The elaboration of dermal organization and the overall increase in dermal thickness, largely consequences of extracellular, fibrous matrix accumulation, parallel the development and growth of the fetus as a whole. Although the mechanisms that determine the formation of the connective tissue architecture are not fully understood, the structural, histochemical, and biochemical changes that occur throughout development suggest processes that may be important in human dermal embryogenesis.

Embryonic and Fetal Development↗
PubMed1985

Age-related changes in collagenase expression in cultured embryonic and fetal human skin fibroblasts.

Since skin collagenase is required for initiation of the degradation of types I and III collagens, the major collagens of the human dermis, we examined its expression during embryonic and fetal development. When using skin fibroblasts cultured from human embryos and fetuses, immunoreactive collagenase concentrations were strongly correlated with estimated gestational age (p less than 0.001), with levels at 7-8 weeks of gestation that were about one-twentieth of those in the 29-week cell cultures. In crude culture medium, the apparent catalytic efficiency (activity per unit immunoreactive protein) was variable, an observation attributable in part to variable expression of a collagenase-inhibitory protein. Following chromatographic purification, four of ten fetal collagenases were found to have greater than or equal to 4-fold decrease in specific activity, suggesting that these particular fetal collagenases may be structurally and/or catalytically altered. Since the decreased levels of immunoreactive protein suggested that decreased enzyme synthesis was the major mechanism, we examined collagenase synthesis in a cell-free translation system. Here, we quantitated collagenase expression in the culture medium of intact cells prior to harvesting mRNA. Compared with the intact adult cells, the fetal cells had 3-17 times less collagenase activity in the medium, while in cell-free translation there was a 2- to 3-fold decrease in collagenase synthesis. These data suggest that decreased in vitro expression is correlated with decreased levels of translatable collagenase mRNA but that other factors, such as the collagenase inhibitor and altered specific activity of the enzyme, may be important in modulating collagenase activity.

Aging↗
PubMed1985

Recombinant plasmid conferring proline overproduction and osmotic tolerance.

A recombinant plasmid carrying the proBA (pro-74) mutant allele which governs osmotic tolerance and proline overproduction was constructed by using the broad-host-range plasmid vector pQSR49. The physiological, biochemical, and genetic properties of strains carrying the pQSR49 derivatives pMJ101 and pMJ1, mutant and wild type, respectively, were investigated. pMJ101 conferred enhanced osmotolerance compared with strains carrying the wild type, pMJ1. These results are in contrast to those obtained previously with strains carrying recombinant plasmids based on pBR322 that failed to confer the osmotic tolerance phenotype. gamma-Glutamyl kinase (first step in proline biosynthesis) from strains carrying pMJ101 was 200-fold less sensitive to feedback inhibition than was the wild-type enzyme. As expected, the intracellular proline levels of strains carrying pMJ101 were more than an order of magnitude higher than those of the wild type. An analysis of copy number revealed that the pQSR49 constructs were present in the cell at a level six- to eightfold lower than those of the pBR322 recombinants, which may account for the difference in phenotype. We found that the genetic stability of the pQSR49 derivative in a variety of gram-negative bacteria was dependent on the insert orientation and the presence of foreign DNA on the plasmid. These factors may be significant in future studies aimed at expanding the osmotolerance phenotype to a broad range of gram-negative bacteria.

DNA Transposable Elements↗
PubMed1985

Characterization of a gamma-glutamyl kinase from Escherichia coli that confers proline overproduction and osmotic tolerance.

Mutation(s) in the proBA operon of Escherichia coli confers proline overproduction and enhanced osmotic tolerance in enteric bacteria (L. N. Csonka, Mol. Gen. Genet. 182:82-86, 1981; M. J. Mahan and L. N. Csonka, J. Bacteriol. 156:1249-1262, 1983). A glutamate-dependent ATPase assay was developed and used to determine proB-encoded gamma-glutamyl kinase activity in the absence of glutamate-gamma-semialdehyde dehydrogenase. This assay indicated that the feedback insensitivity of mutant gamma-glutamyl kinase was independent of glutamate-gamma-semialdehyde dehydrogenase. However, the capacity of glutamate-gamma-semialdehyde dehydrogenase from the osmotolerant mutant to interact with the kinase was altered in thermal stability, suggesting that mutations in both proB and proA may be required for osmotolerance.

Adenosine Triphosphatases↗
PubMed1984

Molecular biology of osmoregulation.

The drought of 1983 resulted in some 10 billion dollars in agricultural losses and has focused attention on the vulnerability of our major crops to this devastating form of environmental stress. This article is concerned with the molecular biology of a new class of genes, called osm (osmotic tolerance) genes, that protect bacteria like Escherichia coli against osmotic stress and may work in a similar manner in plants and animals. Osm genes govern the production of a class of molecules, such as betaine and proline, that protect the cell and its constituents against dehydration. These osmoprotectant molecules have been known for many years to accumulate in plants but have only recently been shown to have potent antistress activity for bacteria.

Adaptation, Physiological↗
PubMed1984

The appearance of four basement membrane zone antigens in developing human fetal skin.

In order to study the ontogeny of various structural and antigenic components of the basement membrane zone of human skin, we have examined skin specimens from 20 aborted fetuses ranging in gestational ages from 6 to 25 weeks, utilizing light microscopy, transmission electron microscopy, and indirect immunofluorescence with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and to the antigen defined by KF-1 monoclonal antibody. Both laminin and type IV collagen were detectable as early as 6 weeks of gestational age. In contrast, bullous pemphigoid antigen and the antigen defined by KF-1 antibody were not detectable before 10 weeks and 16 weeks, respectively. The appearance of bullous pemphigoid antigen correlated with stratification of the epidermis and the formation of hemidesmosomes and anchoring fibrils at the basement membrane zone. KF-1 antigen is first expressed when the epidermis is further stratified, hemidesmosomes and anchoring fibrils are present in greater numbers and with increased frequency at the dermal-epidermal junction, and hair follicles have begun to bud downward from the basal layer of the epidermis. Our findings suggest an orderly sequence to the appearance of these basement membrane zone components within human skin.

Antibodies, Monoclonal↗
PubMed1983

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase.

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.

ATP Synthetase Complexes↗
PubMed1983

Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate.

Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.

Gas Chromatography-Mass Spectrometry↗
PubMed1982

Structure of the dermal matrix during development and in the adult.

To describe a normal adult dermis is a seemingly simple task considering the diverse microscopic methods available for examination of the tissue, staining procedures to delineate the fibrous and cellular components, and immunolabeling techniques to identify precisely the various fibrous elements. Yet it is not simple because the range of normal in any of the dermal components has never been surveyed. There are well recognized age-related changes in the dermis; the tissue can be modified by environmental insults (e.g., actinic damage) and alterations can occur in tissue of individuals with inherited disorders of connective tissue metabolism, other metabolic diseases (e.g., diabetes) and in those receiving topically applied or systemic medication. From our own experience there is also marked individual variability (at any age) in the connective tissue architecture and its fibrous components. Thus, we are describing the structure of a normal dermis without demonstrating the range of normal in any one of its elements. Reference will occasionally be made to abnormal conditions of the matrix since through these deviations it is possible to understand more about the normal. Structural and biochemical properties of the dermal connective tissue in human embryos and fetuses have been described in a number of studies, but in only a few instances was the goal of the research focused on this problem; instead, fetal tissue was used for comparative purposes in aging studies, or a certain characteristic of the fetal dermis was pointed out along with the description of another structure (e.g., hair follicle). In the few instances where a sequential study was carried out on one matrix component during development, an animal (pig, chick) was selected for the work.

Aging↗
PubMed1982

Development of dermal connective tissue in human embryonic and fetal skin.

The primordial dermis begins as a cellular mesenchyme and transforms into a fibrous, sparsely cellular network. At six weeks gestation a fine meshwork of collagen fibrils underlies the dermal-epidermal junction and adheres to the dermal mesenchymal cell surfaces. With increasing gestational age, the amount of extracellular collagen matrix increases, fibrils associate into collagen fiber bundles, cells are spaced further apart and their elongated axes tend to orient parallel to the plane of the skin. The fine collagen meshwork persists throughout development at the dermal-epidermal junctions and ensheathes epidermal appendages as they extend into the dermis. A similar network encases developing vessels. The overall thickness of the dermis increases from 0.1 to 0.15 mm at 7 weeks to greater that 0.7 mm at 20 weeks. By 16 weeks gestation the epidermal appendages extend deeply into the dermis and the dermis is organized into two regions: an upper, finely fibrillar papillary region and a deep reticular region that contains larger collagen fiber bundles. A vascular plexus separates the dermis from the hypodermis. The dermis of the 20-week fetus is adult-like in overall appearance, although it is still smaller in total thickness and size of collagen fibrils and fiber bundles than in the newborn skin.

Connective Tissue↗
PubMed1981

Dose effects of secobarbital in a Sternberg memory scanning task.

Mean reaction times obtained in a Sternberg memory-scanning task were examined for the effects of secobarbital at two doses (1.47 mg/kg and 2.94 mg/kg) spanning the dose range commonly used in clinical practice. Both doses slowed reaction time significantly, with a more pronounced effect at the higher dose. The discriminability of the probe stimulus interacted with the barbiturate, producing a hyperadditive effect on reaction time, but only at the high dose. There were no other significant interaction effects involving the drug. These data are interpreted as additional support for an hypothesis localizing the effects of secobarbital to stimulus-encoding stages in the reaction process.

Adult↗