Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Further evidence for coupling of sodium and proton movements in dog red blood cells

Using 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and tributyltin the sodium transport pathway activated by shrinkage in dog red blood cells is shown to behave as expected for an electroneutral Na+/H+ exchanger. When the driving forces for sodium and protons are equal, flow through the pathway stops. Amiloride inhibits the shrinkage-induced Na+/H+ exchange.     

AIDS, gays, and state coercion

Mohr argues that coercive government policies in response to the AIDS crisis are unjustified and pose a serious threat to the rights of homosexuals. AIDS presently can be transmitted only to those whose actions place them at risk. Paternalistic state coercion is warranted only when a person has diminished capacity or when necessary to prevent someone from ceasing to be an independent agent. Furthermore, an individual may regard sex as a central value. Finally, using public health as a rationale for closing bathhouses or otherwise regulating sexual behavior is a step toward totalitarianism, because coercion is unwarranted when protection from a disease can be achieved by individual action and because the public good served by reducing the size of the pool of AIDS-exposed people is outweighed by the unjust discrimination involved in using coercive measures that affect only certain individuals.     

Effects of pre-treatment with aflatoxin on a second aflatoxin treatment in guinea pigs

Two-hundred guinea pigs, weighing approximately 500 grams each, were placed in 8 groups, 4 of which received 20 g/kg/day of partially purified aflatoxin for 7 days, followed by a 7 day recovery period. Paired groups then received 0,20,35 or 50 g/kg/day of partially purified aflatoxin for 21 days. Animals were sacrificed periodically from all groups and blood was drawn for chemical and immunologic analysis. Weight gains were recorded and histopathologic studies were done on all animals. Pretreatment did not protect guinea pigs from a second exposure, and in fact enhanced mortality and liver toxicity as determined by histopathology. Serum chemistries and immunologic parameters of guinea pigs dosed twice were less conclusive, as neither high nor low doses differed from guinea pigs treated once. Glycocholic acid concentrations were more sensitive than traditional enzymes (aspartate and alanine amino transferase, alkaline phosphatase) for indicating hepatotoxicity.     

Regression of blood vessels precedes cartilage differentiation during chick limb development

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.     

Purification and separation of two soluble fucosyltransferase activities of small intestinal mucosa

1. Rat small intestinal soluble fucosyltransferase is purified more than 2000-fold using chromatographic procedures with DEAE-cellulose, CM-cellulose, GDP-Sepharose and Concanavalin A-Sepharose. 2. Chromatography on Sephadex G15 of the final enzymatic fraction clearly separates two activities: a first peak incorporates fucose on asialoserotransferrin and a second peak on asialofetuin. 3. The use of small saccharidic acceptors (phenylgalactose, lactose, lacto-N-fucopentaose I) and the analysis of fucosylated asialoglycoproteins indicate that the first activity corresponds to an alpha-(3/4)-fucosyltransferase and the second one to an alpha-(1-2)-fucosyltransferase. 4. Protein analysis by polyacrylamide gel electrophoresis in the presence of SDS for each enzyme shows two bands corresponding to a mol. wt of about 65,000 and 70,000. The two enzymes have the same sensitivity to the action of N-ethylmaleimide.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

A toxin fromPasteurella multocida serogroup D enhances swine herpesvirus 1 replication/lethality in vitro and in vivo

In swine, the nasal turbinate epithelium is both a site of swine herpesvirus 1 (pseudorabies virus, PRV) replication and a tissue affected by toxin fromPasteurella multocida serogroup D. We examined the effects of exposure to PRV and exposure to toxin in mice, swine, and nasal turbinate cell cultures. Increased mortality in mice was observed when nonlethal doses of PRV (1000 or 100 plaque-forming units, PFU) were administered along with nonlethal doses (60–200 ng/kg) of toxin. In swine, clinical disease and death in adult pigs was observed after an intradermal injection of toxin (20 ng/kg) and intranasal exposure to 1000 PFU/kg of PRV. Nasal turbinate cell cultures incubated with toxin and PRV had increased protein synthesis, DNA synthesis, and increased recovery of virus particles. These findings indicate that a toxin fromP. multocida serogroup D enhances swine herpesvirus 1 replication and lethality in cell cultures and animal models.     

Isolation ofAcholeplasma oculi from human amniotic fluid in early pregnancy

Acholeplasmas have been isolated from a variety of animals, insects, and plants, but onlyAcholeplasma laidlawii has previously been found in humans. We have isolatedAcholeplasma oculi in pure culture from the amniotic fluid of a woman at 19 weeks of gestation. The organism was positively identified by growth inhibition, epi-immunofluorescence, and arbutin hydrolysis. Demonstration of organisms directly in amniotic fluid by DNA fluorochrome and immunofluorescence staining provided additional evidence that the isolate was genuine and not a medium contaminant. The remainder of the pregnancy was unremarkable, and a full-term male infant was delivered without complications. Even though there is some evidence possibly associatingA. oculi with various diseases in livestock, the prevalence and significance ofA. oculi in humans has not been determined.