UDP-Galactose 4′-Epimerase Activities toward UDP-Gal and UDP-GalNAc Play Different Roles in the Development of Drosophila melanogaster

In both humans and Drosophila melanogaster, UDP-galactose 4′-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans

PC-based multi-terminal message mailbox specially designed for the DLSU College of Engineering Faculty Room

This thesis is designed for use in the De La Salle University College of Engineering Faculty Room. Two personal computers are to be installed. One of these computers would be installed outside the faculty room to be used by students while another computer will be installed inside the faculty room. The terminal inside the faculty room is primarily used for faculty-to-student feedback, announcement purposes, and other faculty uses such as changes in schedules, etc. These computers are interconnected and programmed with a software with database that could be used to transmit messages typed in by various users (e.g., students, visitors, etc.). A digital circuit is interfaced with these computers to act as a transmitting and routing device to send the message (s) to any faculty member. Transmission of signals is all via transmission lines. A battery-operated receiver is located on the table of each faculty member. This consists of an alphanumeric LCD incorporated with a ceramic transducer and indicator LEDs to notify the faculty of an incoming message. In the event that a faculty is absent or presently not in his/her table, the message is then stored and could then be viewed by the faculty later through the display

Oxidative stress contributes to outcome severity in a Drosophila melanogaster model of classic galactosemia

SUMMARY Classic galactosemia is a genetic disorder that results from profound loss of galactose-1P-uridylyltransferase (GALT). Affected infants experience a rapid escalation of potentially lethal acute symptoms following exposure to milk. Dietary restriction of galactose prevents or resolves the acute sequelae; however, many patients experience profound long-term complications. Despite decades of research, the mechanisms that underlie pathophysiology in classic galactosemia remain unclear. Recently, we developed a Drosophila melanogaster model of classic galactosemia and demonstrated that, like patients, GALT-null Drosophila succumb in development if exposed to galactose but live if maintained on a galactose-restricted diet. Prior models of experimental galactosemia have implicated a possible association between galactose exposure and oxidative stress. Here we describe application of our fly genetic model of galactosemia to the question of whether oxidative stress contributes to the acute galactose sensitivity of GALT-null animals. Our first approach tested the impact of pro- and antioxidant food supplements on the survival of GALT-null and control larvae. We observed a clear pattern: the oxidants paraquat and DMSO each had a negative impact on the survival of mutant but not control animals exposed to galactose, and the antioxidants vitamin C and α-mangostin each had the opposite effect. Biochemical markers also confirmed that galactose and paraquat synergistically increased oxidative stress on all cohorts tested but, interestingly, the mutant animals showed a decreased response relative to controls. Finally, we tested the expression levels of two transcripts responsive to oxidative stress, GSTD6 and GSTE7, in mutant and control larvae exposed to galactose and found that both genes were induced, one by more than 40-fold. Combined, these results implicate oxidative stress and response as contributing factors in the acute galactose sensitivity of GALT-null Drosophila and, by extension, suggest that reactive oxygen species might also contribute to the acute pathophysiology in classic galactosemia

Differentially impaired fecundity of flies lacking different GALE activities.

<p>(A) Diagram of the method used to achieve expression of different <i>GALE</i> transgenes in the background of <i>dGALE</i> knockdown animals. The timing of knockdown and concurrent transgene expression was controlled by switching flies from the permissive temperature (18°C) to the restrictive temperature (28–29°C), as indicated. (B) Knockdown efficiency in male and female animals switched to the restrictive temperature as early to mid-stage pupa and harvested for biochemical analysis as newly eclosed adults or three days after eclosion. Of note, GALT activity was completely normal in all samples tested and apparently unaffected by the <i>dGALE</i> knockdown (data not shown). (C) Each box represents the outcome of flies switched from 18°C to 28°C at the stage indicated in the column on the left. The number of days the flies developed at 18°C to reach each stage is shown in parentheses.</p

The Leloir pathway of galactose metabolism.

<p>UDP-galactose 4′-epimerase, the third enzyme in the pathway, also interconverts UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc) in humans, <i>Drosophila</i>, and other metazoans tested.</p

Enzyme activities of flies expressing different <i>GALE</i>

<p><b>transgenes.</b> Assays for all genotypes were performed on flies with <i>dGALE</i> knockdown (KD) driven by the <i>Act5C-GAL4</i> driver with the exception of flies labeled “no knockdown”; those flies carried the same <i>UAS-RNAi^dGALE</i> and <i>GAL80^ts</i> alleles, but were balanced over TSTL, and thus lacked the driver. In addition to RNAⁱ knockdown of <i>dGALE</i>, <i>Act5C-GAL4</i> also drives expression of the specified transgenes in these animals. Panel A: GALE activity using UDP-gal as substrate. Panel B: GALE activity using UDP-galNAc as substrate.</p

Flies lacking GALE activity toward UDP-gal/UDP-glc have a shortened life span when exposed to galactose as adults.

<p>The life spans of male (A and B) and female (C and D) flies reared on molasses food and then tapped as newly eclosed adults to food containing either 555 mM glucose only (A and C), or 555 mM glucose plus 175 mM galactose (B and D), is illustrated. As indicated by the key, these cohorts of flies included controls expressing endogenous <i>dGALE</i> as well as animals that expressed endogenous <i>dGALE</i> early in development but then were subjected late in development to <i>dGALE</i> knockdown coupled with induced expression of either no <i>GALE</i> transgene, or <i>wbgU</i>, <i>eGALE</i>, <i>hGALE</i>, or both <i>wbgU</i> and <i>eGALE</i> in combination. Based on Log rank and Wilcoxon tests for significance, the life spans of knockdown animals expressing either no transgene or expressing only <i>wbgU</i> were significantly decreased on food containing galactose compared with food containing only glucose (p<0.0001).</p

Metabolite profiles of <i>Drosophila</i> exposed to galactose.

<p>Metabolites were extracted from cohorts of larvae raised on food containing either 555 mM glucose or 555 mM glucose+175 mM galactose. Animals were shifted from the permissive temperature (18°C) to the restrictive temperature (28°C) as first instar larvae and allowed to develop for four days before harvest. Accumulated metabolite values for gal-1P (A), UDP-gal (B), and UDP-galNAc (C) are shown on food containing glucose and glucose+galactose. To demonstrate the impact of diet on metabolite levels, values for gal-1P (D), UDP-gal (E), and UDP-galNAc (F) are shown as ratios of the amount of each metabolite accumulated by animals on food containing galactose over that accumulated by animals of the same genotype on food containing only glucose. Error bars show the 95% confidence interval for each ratio.</p

Crosses to test rescue of <i>wbgU</i> and <i>eGALE</i> transgenes individually and in combination.

<p>Crosses to test rescue of <i>wbgU</i> and <i>eGALE</i> transgenes individually and in combination.</p