Nicotinamide–adenine dinucleotide pyrophosphatase in the growing and aging mosquito

1. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds as demonstrated by the formation of NMN and AMP from NAD(+). This reaction was also demonstrated by the loss in the coenzyme functioning property of NAD(+) (yeast alcohol dehydrogenase reaction) without a concomitant loss in reactivity towards cyanide. Transglycosidase activity was not observed in the mosquito homogenates, and low concentrations of nicotinamide did not inhibit the NAD(+) splitting activity of these homogenates. These observations are all in accord with the presence in these homogenates of a NAD(+) pyrophosphatase rather than a NADase. 2. The NAD(+) pyrophosphatase is destroyed by boiling, is not heat-activated, and has a pH optimum at pH8.75. In addition to NAD(+), other dinucleotides such as NADP(+), the 3-acetylpyridine and thionicotinamide analogues of NAD(+) and the thionicotinamide analogue of NADP(+), function as substrates in the hydrolysis catalysed by the pyrophosphatase. 3. A decrease in the specific activity of NAD(+) pyrophosphatase was observed during larval development, and a barely detectable activity was found in the pupa and adult. 4. Enzyme activity per organism increased in the larva but decreased to a very low value in the pupa and adult. These results indicate that the decrease in specific activity was due to a decrease in enzyme concentration rather than an increase in amounts of protein.     

Hemodynamic basis for cocaine-induced pulmonary edema in dogs.

We tested the hypothesis that cocaine-induced impairment of left ventricular function results in cardiogenic pulmonary edema. Mongrel dogs, anesthetized with alpha-chloralose, were injected with two doses of cocaine (5 mg/kg iv) 27 min apart. Cocaine produced transient decreases in aortic and left ventricular systolic pressures that were followed by increases exceeding control. As aortic pressure recovered, left ventricular end-diastolic, left atrial (Pla), pulmonary arterial (Ppa), and central venous pressures rose. Cardiac output and stroke volume were reduced when measured 4-5 min after cocaine administration. Peak Ppa and Pla were 31 +/- 5 (SE) mmHg (range 17-51 mmHg) and 26 +/- 5 mmHg (range 12-47 mmHg), respectively. Increases in extravascular lung water content (4.10 to 6.24 g H2O/g dry lung wt) developed in four animals in which Pla exceeded 30 mmHg. Analysis of left ventricular function curves revealed that cocaine depressed the inotropic state of the left ventricle. Cocaine-induced changes in hemodynamics spontaneously recovered and could be elicited again by the second dose of the drug. Our results show that cocaine-induced pulmonary hypertension, associated with decreased left ventricular function, produces pulmonary edema if pulmonary vascular pressures rise sufficiently.     

L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene.

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by lambda plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.     

Chronic Exposure of NG 108-15 Cells to Opiate Agonists Does Not Alter the Amount of the Guanine Nucleotide-Binding Proteins Gi and GO

We have characterized the pertussis toxin substrate in NG 108-15 cell membranes using site-specific antisera and ADP-ribosylation. Cell membranes contain two pertussis toxin-sensitive guanine nucleotide-binding protein alpha-subunits (G alpha) whose Rf values in gel electrophoresis coincide with those of G alpha o and G alpha i2. The total quantity of Gi and Go immunoreactivity amounted to 24.3 +/- 2.8 pmol/mg, whereas only 1.5 +/- 0.2 pmol/mg are capable of undergoing ADP-ribosylation catalyzed by pertussis toxin. Pretreatment of cells with the agonist [D-Ala2,D-Leu2]-enkephalin (DADLE) for 24 h and DADLE or morphine for 72 h did not alter the incorporation of ADP-ribose or the immunoreactive amount of Gi and Go subunits. However, pretreatment for 72 h with naloxone increased the incorporation of ADP-ribose without an apparent change in affinity or in the immunochemically determined protein levels of Gi and Go. This indicates that the process of down-regulation and desensitization of the delta-opioid receptor neither requires quantitative alterations in the levels of Gi and Go nor changes in the degree of coupling among their subunits. In contrast, chronic exposure to antagonists seems to alter the degree of precoupling between alpha- and beta-subunits of Gi and/or Go.     

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.     

树鼩(Tupaia belangeri chinensis)的脑底动脉环

用25只树鼩,从升主动脉灌注带色的橡胶乳液,在解剖显微镜下进行解剖观察,用目测微尺进行测量。大多数树鼩(22只)有完整的脑底动脉环。由左、右大脑前动脉向内侧各发一前交通动脉组成大脑前总动脉。前交通动脉口径为大脑前动脉的75～85％。后交通动脉口径与大脑后动脉相近,连于颈内动脉与大脑后动脉(基底动脉的分支)之间。测量了组成脑底动脉环有关动脉的口径。由于后交通动脉足够粗大,只有中断左、右颈总动脉和左、右椎动脉,才能造成全脑缺血。     

Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells.

Herpes simplex virus type 1 (HSV-1) plasmid vectors have promise for genetic intervention in the brain, but several problems caused by the helper virus have compromised their utility. To develop a helper virus-free packaging system for these vectors, the DNA cleavage/packaging signals were deleted from a set of cosmids that represents the HSV-1 genome. Following cotransfection into cells, this modified cosmid set supported replication and packaging of vector DNA. However, in the absence of the DNA cleavage/packaging signals, the HSV-1 genome was not packaged, and consequently vector stocks were free of detectable helper virus. In the absence of helper virus, the vectors efficiently infected rat neural cells in culture or in the brain with minimal cytopathic effects. beta-galactosidase-positive cells were observed for at least 1 month in vivo, and vector DNA persisted for this period. This system may facilitate studies on neuronal physiology and potential therapeutic applications.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

Vascular Development and Sap Flow in Apple Pedicels

Xylem and phloem tissues of the pedicel of apple fruit increasein cross-sectional area throughout development. The increasein phloem is similar in the two cultivars examined (Cox's OrangePippin and Royal Gala) and reflects a steadily increasing phloemsap flow to the fruit. The increase in xylem tissue is due toa proliferation of non-conducting, structural, components sinceclose examination reveals no increase in the number of vesselelements from just after flowering onwards. The greater number,and the larger diameter, of the vessels in Cox's explains theinitially higher xylem conductance found in this cultivar. In vitro measurements of xylem exudation reveal a decline duringthe growing season in the xylem conductance of both cultivarsand an increasing proportion of fruit (particularly in Cox's)in which the xylem comes to be totally non-conducting. Thisobservation is in line with previously reported measurementsof xylem sap flow in vivo. The straightforward techniques used in this study offer a feasiblealternative to more arduous methods of assessing xylem and phloemsap flows and their balance during growth.Copyright 1994, 1999Academic Press Apple, xylem, phloem, vascular development, sap flow, Malus domestica Borkh     

Machado-Joseph Disease in Pedigrees of Azorean descent is Linked to Chromosome 14

A locus for Machado-Joseph disease (MJD) has recently been mapped to a 30-cM region of chromosome 14q in five pedigrees of Japanese descent. MJD is a clinically pleomorphic neurodegenerative disease that was originally described in subjects of Azorean descent. In light of the nonallelic heterogeneity in other inherited spinocere-bellar ataxias, we were interested to determine if the MJD phenotype in Japanese and Azorean pedigrees arose from mutations at the same locus. We provide evidence that MJD in five pedigrees of Azorean descent is also linked to chromosome 14q in an 18-cM region between the markers D14S67 and AACT (multipoint lod score +7.00 near D14S81). We also report molecular evidence for homozy-gosity at the MJD locus in an MJD-affected subject with severe, early-onset symptoms. These observations confirm the initial report of linkage of MJD to chromosome 14; suggest that MJD in Japanese and Azorean subjects may represent allelic or identical mutations at the same locus; and provide one possible explanation (MJD gene dosage) for the observed phenotypic heterogeneity in this disease.