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Information on ductal differentiation in the developing rat parotid gland is sparse. Striated and excretory ducts are rich in a number of enzymes related to ion movement. The objective of this investigation was to delineate histochemically the chronology of two of these, ouabain-sensitive Na(+),K(+)-ATPase and NADH-DE, in the developing rat parotid gland. Parotid glands were excised from rats at representative ages from 20 days in utero to 42 days. Enzyme histochemistry was performed on air-dried frozen sections. For Na(+), K(+)-ATPase, some sections also were fixed in phosphate-buffered formalin. Ouabain blocked Na(+),K(+)-ATPase activity, and neither enzyme reacted without substrate. Weak Na(+),K(+)-ATPase reactions were initially seen in unfixed sections at 1 day, and increased steadily to the adult pattern of strong (concentrated basolaterally) in striated ducts and excretory ducts, respectively, and weak to modest (diffuse) in acini and intercalated ducts at 28 days. In fixed sections, localization was sharper but the reaction was somewhat reduced. NADH-DE was modest in terminal buds and ducts before birth, then progressively changed to the adult pattern of weak in acini and intercalated ducts and strong (concentrated basally and luminally) in striated and excretory ducts at 28 days. As demonstrated by enzyme histochemistry of Na(+),K(+)-ATPase and NADH-DE, differentiation of rat parotid striated ducts and excretory ducts occurs mainly between birth and 28 days. Anat Rec 256:72-77, 1999. Published 1999 Wiley-Liss, Inc.
Anat Rec 1999 09 01
PMID:Enzyme histochemical localization of Na(+),K(+)-ATPase and NADH-DE in the developing rat parotid gland. 1045 87

Hypothyroidism was diagnosed in 50 dogs and excluded in 86 dogs suspected of hypothyroidism, on the basis of the results of bovine thyrotropin response tests. Breed, pedigree, sex or neutering status did not significantly influence the likelihood of the dogs being hypothyroid. The hypothyroid dogs were significantly older than the non-hypothyroid dogs referred to the University of Glasgow during the same period. However, when dogs under two years of age were excluded from the statistical analyses there was no significant difference in age between the two groups. The most common clinical characteristics associated with hypothyroidism were metabolic signs (84 per cent of cases), particularly lethargy (76 per cent), obesity or weight gain (44 per cent), and exercise intolerance (24 per cent); and dermatological abnormalities (80 per cent), including alopecia (56 per cent), poor coat quality (30 per cent) and hyperpigmentation (20 per cent). When compared with the laboratory reference limits the most common biochemical and haematological abnormalities were increased concentrations of triglycerides (88 per cent), cholesterol (78 per cent), glucose (49 per cent), and fructosamine (43 per cent), and increased activities of creatine kinase (35 per cent), and decreased concentrations of inorganic phosphate (63 per cent), and a low red blood cell count (40 per cent). When compared with reference limits derived from the euthyroid dogs the most common abnormalities were increased concentrations of gamma-glutamyltransferase (21 per cent), cholesterol (18 per cent), and aspartate aminotransferase (15 per cent) and a decreased red blood cell count (29 per cent), and decreased neutrophils (18 per cent) and decreased activity of creatine kinase (15 per cent). Assessment of cholesterol, creatine kinase, aspartate aminotransferase, gamma-glutamyltransferase, and red blood cell and neutrophil counts may be particularly useful in distinguishing hypothyroid dogs from euthyroid animals with similar clinical signs.
Vet Rec 1999 Oct 23
PMID:Epidemiological, clinical, haematological and biochemical characteristics of canine hypothyroidism. 1059 70

This study examined the continuous cofermentation performance characteristics of a dilute-acid "prehydrolysate-adapted" recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the cofermentation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5 cents/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (Dmax), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted Dmax from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), Dmax was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the Dmax was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the Dmax for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L.h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell.h).
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PMID:Continuous fermentation studies with xylos-utilizing recombinant Zymomonas mobilis. 1084 97

Dehydration is known to significantly reduce both the time required for the first platelet aggregate and the time to full occlusion in photochemically-induced thrombosis, in vivo. Ultrastructural changes that contribute to such events remain unknown. Therefore, the effect of water deprivation for 24 hr, (as a model for dehydration) on the ultrastructure of mouse pial microvessels was investigated. The possible beneficial effect of garlic in preventing such ultrastructural changes was also investigated. Four groups of TO strain: control, control-garlic treated, dehydrated, and dehydrated-garlic treated male mice, 10/group, were used. Dehydration was induced by water deprivation for 24 hr. Garlic solution was i.p. injected at 0.1 ml/10g body weight. In urethane-anesthetized (2 mg/g, i.p.) mice, topical and transvessel bimodal fixation of pial microvessels was done with a phosphate buffered mixture of glutaraldehyde and paraformaldehyde, followed by a conventional electron microscopy procedure. Examination of control cerebral pial microvessels showed no evidence of cellular damage. Membranes of endothelial cells were intact. Within pial microvessels there was no evidence of platelet aggregation. Garlic treatments did not cause any ultrastructure abnormalities in control mice. Compared with control, dehydration caused the appearance of thrombi that consisted of platelet aggregates. Discoid platelets containing granules, spheroid degranulated platelets, and those with large pseudopodia were present in 80% of dehydrated mice. The venular endothelial surface of dehydrated mice revealed dilated profiles of endoplasmic reticulum and variously shaped vacuoles. Swelling of nuclear envelopes and mitochondrial distension were also present in dehydrated mice. Concomitant garlic treatment prevented most of these ultrastructural changes. These findings demonstrated the extent of damage to the pial microvessels in response to water deprivation and demonstrated the beneficial effect of garlic as a possible mean of protection against oncoming vascular pathology.
Anat Rec 2001 05 01
PMID:Garlic prevents ultrastructural alterations caused by dehydration in mouse cerebral microvessels. 1133 74

Ultrastructural examination of 8-day hatched pig blastocysts (large and small), their cultured inner cell mass (ICM), and cultured epiblast tissue (embryonic stem cells) was undertaken to assess the development of epiblast cell junctions and cytoskeletal elements. In small blastocysts, epiblast cells had no desmosomes or tight junction (TJ) connections and few organized microfilament bundles, whereas in large blastocysts the epiblast cells were connected by TJ and desmosomes with associated microfilaments. ICM isolation by immunodissection damaged the endoderm cells beneath the trophectoderm cells but did not appear to damage the epiblast cells or their associated endoderm cells. Epiblast cells in cultured ICMs were similar in character to those in the intact large blastocyst except that perinuclear microfilaments were observed. Isolated pig epiblasts, cultured for approximately 36 hr on STO feeder layers, formed a monolayer whose cells were connected by TJ, adherens junctions and desmosomes with prominent microfilament bundles running parallel to the apical cytoplasmic membranes. Perinuclear microfilaments were a consistent feature in the approximately 36 hr cultured epiblast cells. A feature characteristic of differentiation into notochordal cells, i.e., a solitary cilium, was also observed in the cultured epiblast. Exposure of the cultured epiblast cells to Ca(++)-Mg(++)-free phosphate buffered saline (PBS) for 5-10 min resulted in extensive cell blebbing and lysis. The results may indicate that pig epiblast cells could be more easily dissociated from early blastocysts ( approximately 400 microm in diameter) if immunodissection damage to the ICM can be avoided. It may be difficult, however, to establish them as embryonic stem cell lines because the cultured pig epiblast cells were easily lysed by standard cell-cell dissociation methods.
Anat Rec 2001 09 01
PMID:Ultrastructure of the embryonic stem cells of the 8-day pig blastocyst before and after in vitro manipulation: development of junctional apparatus and the lethal effects of PBS mediated cell-cell dissociation. 1150 76

Research on the mechanism of action of coenzyme B12, adenosylcobalamin, as a graduate student introduced the author to the field of organic free radicals in enzymology. Twenty years later, related work on S-adenosylmethionine (SAM) as a "poor man's coenzyme B12" was initiated in a detailed analysis of the mechanism of action of lysine 2,3-aminomutase (LAM). The interconversion of L-lysine and L-beta-lysine is catalyzed by LAM, which requires SAM, pyridoxal-5'-phosphate (PLP), and a [4Fe-4S] cluster as coenzymes. The mechanism of this reaction has been delineated as a radical isomerization, in which radical formation is initiated by the [4Fe-4S]-dependent cleavage of the SAM into methionine and the 5'-deoxyadenosyl radical. The mechanism of this process is discussed, together with the role of this radical in hydrogen abstraction from lysine to initiate the substrate radical isomerization. The chemistry underlying the functions of SAM, PLP, and [4Fe-4S] in the action of LAM is novel in all respects, except for the formation of a lysine-PLP aldimine at the active site. Of the four free radicals in the mechanism, three have been characterized by EPR spectroscopy. In the suicide inactivation of adenosylcobalamin-dependent dioldehydrase (DDH) by glycolaldehyde, the formation of cob(II)alamin and 5'-deoxyadenosine is accompanied by the conversion of glycolaldehyde to cis-ethanesemidione radical at the active site. The cis-ethanesemidione radical has been characterized by EPR spectroscopy. Its exceptional stability at the active site is the basis for the inactivation of DDH by glycolaldehyde.
Chem Rec 2001
PMID:The role of radicals in enzymatic processes. 1189 68

The studies reported here have established the biosynthetic origin of the mC7N units of acarbose and validamycin from sedo-heptulose 7-phosphate, and have identified 2-epi-5-epi-valiolone as the initial cyclization product. The deoxyhexose moiety of acarbose arises from glucose with deoxythymidyl-diphospho-4-keto-6-deoxy-D-glucose (dTDP-4-keto-6-deoxy-D-glucose) as a proximate intermediate. However, despite the identical origin of the aminocyclitol moieties in acarbose and validamycin A, the pathways of their formation seem to be substantially different. Validamycin A formation involves a number of discrete ketocyclitol intermediates, 5-epi-valiolone, valienone, and validone, whereas no free intermediates have been identified on the pathway from 2-epi-5-epi-valiolone to the pseudodisaccharide moiety of acarbose. The stage is now set for unraveling the mechanism or mechanisms by which the two components of the pseudodisaccharide moieties of acarbose and validamycin are uniquely coupled to each other via a nitrogen bridge.
Chem Rec 2001
PMID:The biosynthesis of acarbose and validamycin. 1189 70

We have studied microbial secondary metabolism in a simulated microgravity (SMG) environment provided by NASA rotating-wall bioreactors (RWBs). These reactors were designed to simulate some aspects of actual microgravity that occur in space. Growth and product formation were observed in SMG in all cases studied, i.e., Bacillus brevis produced gramicidin S (GS), Streptomyces clavuligerus made beta-lactam antibiotics, Streptomyces hygroscopicus produced rapamycin, and Escherichia coli produced microcin B17 (MccB17). Of these processes, only GS production was unaffected by SMG; production of the other three products was inhibited. This was determined by comparison with performance in an RWB positioned in a different mode to provide a normal gravity (NG) environment. Carbon source repression by glycerol of the GS process, as observed in shaken flasks, was not observed in the RWBs, whether operated in the SMG or NG mode. The same phenomenon occurred in the case of MccB17 production, with respect to glucose repression. Thus, the negative effects of carbon source on GS and beta-lactam formation are presumably dependent on shear, turbulence, and/or vessel geometry, but not on gravity. Stimulatory effects of phosphate and the precursor L-lysine on beta-lactam antibiotic production, as observed in flasks, also occurred in SMG. An almost complete shift in the localization of produced MccB17 from cells to extracellular medium was observed when E. coli was grown in the RWB under SMG or NG. If a plastic bead was placed in the RWB, accumulation became cellular, as it is in shaken flasks, indicating that sheer stress favors a cellular location. In the case of rapamycin, the same type of shift was observed, but it was less dramatic, i.e., growth in the RWB under SMG shifted the distribution of produced rapamycin from 2/3 cellular:1/3 extracellular to 1/3 cellular:2/3 extracellular. Stress has been shown to induce or promote secondary metabolism in a number of other microbial systems. RWBs provide a low stress SMG environment, which, however, supports only poor production of MccB17, as compared to production in shaken flasks. We wondered whether the poor production in RWBs under SMG is due to the low level of stress, and whether increasing stress in the RWBs would raise the amount of MccB17 formed. We found that increasing shear stress by adding a single Teflon bead to the RWB improved MccB17 production. Although shear stress seems to have a marked positive effect on MccB17 production in SMG, addition of various concentrations of ethanol to RWBs (or to shaken flasks) failed to increase MccB17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the RWB were much more resistant to the growth- and production-inhibitory effects of ethanol than cells growing in shaken flasks. With respect to S. hygroscopicus, addition of Teflon beads to the RWB reversed the inhibition of growth, but rapamycin production was still markedly inhibited, and the distribution did not revert back to a preferential cellular site.
Chem Rec 2001
PMID:Secondary metabolism in simulated microgravity. 1189 73

We have studied the stereospecificities of various pyridoxal 5'-phosphate dependent enzymes for the hydrogen transfer between the C-4' of a bound coenzyme and the C-2 of a substrate in the transamination catalyzed by the enzymes. Prior to our studies, pyridoxal enzymes so far studied were reported to catalyze the hydrogen transfer only on the si-face of the planar imine intermediate formed from substrate and coenzyme. This finding had been considered as the evidence that pyridoxal enzymes have evolved divergently from a common ancestral protein, because identity in the stereospecificity reflects the similarity in the active-site structure, in particular in the geometrical relationship between the coenzyme and the active site base participating in the hydrogen transfer. However, we found that D-amino acid aminotransferase, branched-chain L-amino acid aminotransferase, and 4-amino-4-deoxychorismate lyase catalyze the re-face specific hydrogen transfer, and that amino acid racemases catalyze the nonstereospecific hydrogen transfer. These findings suggest the convergent evolution of pyridoxal enzymes. Crystallographical studies have shown that the stereospecificity reflects the active-site structure of the enzymes, and that the enzymes with the same fold exhibit the same stereospecificity. The active site structure with the catalytic base being situated on the specific face of the cofactor has been conserved during the evolution among the pyridoxal enzymes of the same family.
Chem Rec 2001
PMID:Stereospecificity for the hydrogen transfer of pyridoxal enzyme reactions. 1193 44

The pyridoxal-5'-phosphate (vitamin B(6))-dependent enzymes that act on amino acid substrates have multiple evolutionary origins. Thus, the common mechanistic features of B(6) enzymes are not accidental historical traits but reflect evolutionary or chemical necessities. The B(6) enzymes belong to four independent evolutionary lineages of paralogous proteins, of which the alpha family (with aspartate aminotransferase as the prototype enzyme) is by far the largest and most diverse. The considerably smaller beta family (tryptophan synthase beta as the prototype enzyme) is structurally and functionally more homogenous. Both the D-alanine aminotransferase family and the alanine racemase family consist of only a few enzymes. The primordial pyridoxal-5'-phosphate-dependent protein catalysts apparently first diverged into reaction-specific protoenzymes, which then diverged further by specializing for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages, providing examples of convergent enzyme evolution. The functional specialization of most B(6) enzymes seems to have already occurred in the universal ancestor cell before the divergence of eukaryotes, archebacteria, and eubacteria 1500 million years ago. Pyridoxal-5'-phosphate must have emerged very early in biological evolution; conceivably, metal ions and organic cofactors were the first biological catalysts. To simulate particular steps of molecular evolution, both the substrate and reaction specificity of existent B(6) enzymes were changed by substitution of active-site residues, and monoclonal pyridoxal-5'-phosphate-dependent catalytic antibodies were produced with selection criteria that might have been operative in the evolution of protein-assisted pyridoxal catalysis.
Chem Rec 2001
PMID:From cofactor to enzymes. The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes. 1193 50


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