The interaction of α-chymotrypsin with isosteric substrates of different charge type

1. The synthesis of three substrates of alpha-chymotrypsin of closely similar steric requirements but different charge type is reported. 2. The interaction of these compounds [SS-dimethyl-(l-3-carboxymethyl-3-acetamido)propyl sulphonium iodide, l-2-acetamido-5-methylhexanoic acid methyl ester and N-acetyl-l-glutamic acid alpha-methyl ester] with alpha-chymotrypsin has been studied. 3. For the charged substrates, values of k(0) are two orders of magnitude smaller than, and values of K(m) two orders of magnitude larger than, the corresponding values for the uncharged isostere. 4. The results are interpreted in terms of the known specificity of the enzyme, and the relationship between binding and kinetic specificities is discussed.     

Expression vectors for human adenosine deaminase gene therapy

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.     

Altered expression of fibronectin gene in cells infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) induces morphological changes in infected cells that are remarkably similar to those seen in oncogenically transformed cells. The molecular bases of these phenotypic alterations are not known but their occurrence in some transformed cells can be associated with abnormal fibronectin (FN) expression. In this report, we have compared FN levels in normal and HCMV-infected cells. In these studies, the HCMV-infected fibroblasts exhibited a progressive loss of cellular FN. Northern (RNA) blot analysis revealed that the decrease in FN levels resulted from a lowering of FN mRNA levels in HCMV-infected cells. We detected an initial decrease in FN mRNA of 25 to 30% at immediate-early and early times, whereas at late times after infection the levels of FN mRNA were lowered by greater than 80%. These results indicated that the HCMV-induced decrease in FN expression is due to a decrease in the quantity of FN mRNA and suggested that HCMV-encoded and/or -induced functions may be involved in producing these alterations.     

Chromosomal band assignments of the genes encoding human FKBP12 and FKBP13.

Human FKBP12 and FKBP13 are encoded by distinct genes designated FKBP1 and FKBP2, respectively. Human FKBP1 was previously characterized. The characterization of human FKBP2 is described. FKBP2 is three kb in length and contains six exons. Fluorescence in situ hybridization of FKBP1 and FKBP2 genomic probes to metaphase chromosomes localized FKBP1 to human chromosome 20 band p13 and FKBP2 to human chromosome 11 band q13.1-q13.3.     

The effects of brain-stem section on the breathing and behavioural response to morphine in the fetal sheep

In the unanesthetized fetal sheep the administration of morphine causes initial apnoea followed by hyperpnoea. We thought that a section of the brain at midcollicular level might separate these two effects. Therefore we sectioned the brain stem of five fetuses at 132 +/- 1 (SEM) days of gestation and compared their responses to morphine (17 experiments) with that observed in seven intact fetuses at similar gestational ages (15 experiments). Brain stem sections were confirmed morphologically and histologically. Morphine, 1 mg/kg was injected in the fetal jugular vein during low-voltage electrocortical activity (ECoG). We measured ECoG, eye movements, diaphragmatic activity, blood pressure and amniotic pressure. Sectioned fetuses before the administration of morphine had a complete dissociation between ECoG and breathing activity. With the administration of morphine we found: (i) the length of the apnoea was 139.8 +/- 15.5 min in sectioned fetuses and 17.0 +/- 5.8 min in intact fetuses (P less than 0.01); and (ii) there was no hyperpneic response in the sectioned fetus whereas the length of hyperpnoea in the intact group was 99.1 +/- 11.8 min (P less than 0.001). The results support the idea of two central distinct areas of action of morphine in the fetal brain. The absence of hyperpnoea in the sectioned fetuses suggests that neurons inhibiting the 'respiratory neurons' are located rostrally to the mid-collicular line.     

Hyperammonaemia causes many of the changes found after portacaval shunting.

1. Portacaval shunting in rats results in several metabolic alterations similar to those seen in patients with hepatic encephalopathy. The characteristic changes include: (a) diminution of cerebral function; (b) raised plasma ammonia and brain glutamine levels; (c) increased neutral amino acid transport across the blood-brain barrier; (d) altered brain and plasma amino acid levels; and (e) changes in brain neurotransmitter content. The aetiology of these abnormalities remains unknown. 2. To study the degree to which ammonia could be responsible, rats were made hyperammonaemic by administering 40 units of urease/kg body weight every 12 h and killing the rats 48 h after the first injection. 3. The changes observed in the urease-treated rats were: (a) whole-brain glucose use was significantly depressed, whereas the levels of high-energy phosphates remained unchanged; (b) the permeability of the blood-brain to barrier to two large neutral amino acids, tryptophan and leucine, was increased; (c) blood-brain barrier integrity was maintained, as indicated by the unchanged permeability-to-surface-area product for acetate; (d) plasma and brain amino acid concentrations were altered; and (e) dopamine, 5-hydroxytryptamine (serotonin) and noradrenaline levels in brain were unchanged, but 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-hydroxytryptamine, was elevated. 4. The depressed brain glucose use, increased tryptophan permeability-to-surface-area product, elevated brain tryptophan content and rise in the level of cerebral 5-HIAA were closely correlated with the observed rise in brain glutamine content. 5. These results suggest that many of the metabolic alterations seen in rats with portacaval shunts could be due to elevated ammonia levels. Furthermore, the synthesis or accumulation of glutamine may be closely linked to cerebral dysfunction in hyperammonaemia.     

Volume-sensitive taurine efflux from mammary tissue is not obliged to utilize volume-activated anion channels

Cell-swelling, induced by a hyposmotic shock, activates the release of taurine from lactating rat mammary tissue expiants. The degree of stimulation of taurine efflux was dependent upon the extent of cell-swelling. Volume-sensitive taurine release was attenuated by the anion transport inhibitors NPPB, DIOA, DIDS, niflumate, flufenamate, mefenamate and diiodosalicylate but not by salicylate. Cell-swelling, following a hyposmotic challenge, did not increase the unidirectional efflux of radiolabelled I^– or D-asparate from mammary tissue expiants. The results suggest that although mammary tissue expresses a volume-sensitive amino acid transport system which is inhibited by anion transport blockers the pathway has no identity with volume-activated anion channels.     

Stochastic dynamics of the nerve growth cone and its microtubules during neurite outgrowth

The controlled extension of neurites is essential not only for nervous system development, but also for effective nerve regeneration after injury. This process is critically dependent on microtubule assembly since axons fail to elongate in the presence of drugs which disrupt normal assembly dynamics. For this reason, neurite outgrowth is potentially controllable by manipulation of the assembly state of the intracellular array of microtubules. Therefore, understanding how microtubule assembly dynamics and neurite outgrowth are coupled, in the absence of drugs, can lend valuable insight into the control and guidance of the outgrowth process. In the present study we characterized the stochastic dynamics of neurite outgrowth and its corresponding microtubule array, which advances concomitantly with the advance of the nerve growth cone, the highly motile structure at the terminus of the growing neurite, using reported fluorescent microscopic image sequences (Tanaka and Kirschner, 1991, J. Cell Biol. 115:345-363). Although previously modeled as an uncorrelated random walk, the stochastic advance of the growth cone was found to be anticorrelated over a time scale of approximately 4 min, meaning that growth cone advances tended to be followed by growth cone retractions approximately 4 min later. The observed anticorrelation most likely reflects the periodic stops and starts of neurite outgrowth that have been reported anecdotally. A strikingly similar pattern of anticorrelation was also identified in the advance of the growth cone's microtubule array. Cross-correlation analysis showed that growth cone dynamics tended to precede microtubule dynamics on a time scale of approximately 0-2 min, while microtubules tended to precede growth cone dynamics on a approximately 0-20-s time scale, indicating a close temporal coupling between microtubule and growth cone dynamics. Finally, the scaling of the mean-squared displacements with time for both the growth cone and microtubules suggested a fractional Brownian motion model which accounts for the observed anticorrelation of growth cone and microtubule advance. (c) 1996 John Wiley & Sons, Inc.     

Inheritance and expression patterns of BN28, a low temperature induced gene in Brassica napus, throughout the Brassicaceae.

Molecular genetics is becoming an important tool in the breeding and selection of agronomically important traits. BN28 is a low temperature induced gene in Brassicaceae species. PCR and Southern blot analysis indicate that BN28 is polymorphic in the three diploid genomes: Brassica rapa (AA), Brassica nigra (BB), and Brassica oleracea (CC). Of the allotetraploids, Brassica napus (AACC) is the only species to have inherited homologous genes from both parental genomes. Brassica juncea (AABB) and Brassica carinata (BBCC) have inherited homologues from the AA and CC genomes, respectively, while Sinapsis arvensis (SS) contains a single homologue from the BB genome and Sinapsis alba (dd) appears to be different from all the diploid parents. All species show message induction when exposed to low temperature. However, differences in expression were noticed at the protein level, with silencing occurring in the BB genome at the level of translation. Results suggest that silencing is occurring in diploid species where duplication may not have occurred. Molecular characterization and inheritance of BN28 homologues in the Brassicaceae may play an important role in determining their quantitative function during exposure to low temperature. Key words : Brassicaceae, BN28, inheritance, polymorphism.