Natural Dermatophilus congolensis infection in camels (Camelus dromedarius) from Kenya.

Natural Dermatophilus congolensis infection is found in many species of livestock and wild animals. It is, however, rarely described in camels and there are no details of bacterial isolation. In an investigation of both arid and semi-arid areas in Kenya, an outbreak of dermatophilosis was observed in camel calves being reared on a commercial farm in a semi-arid area. Histopathology and bacterial isolation were used to diagnose the diseases. The potential impact of the disease in camels is discussed.

Individual animal susceptibility and its relationship to induced adaptation or tolerance in sheep to Galega officinalis L.

Oral administration of Galega officinalis L to sheep demonstrated a marked variation in individual animal susceptibility to the toxic effects of the plant. As little as 5 g/kg of dried ground plant induced moderate tracheal frothing in 1 ewe while nearly 5 times that amount failed to elicit any recognizable toxic effects such as frothing, pulmonary edema or hydrothorax in others. Ten g/kg induced severe effects in 3 ewes. Ewes administered levels of plant between 5 and 24 g/kg had toxic effects whose severity was often unrelated to level administered. There was no apparent difference in average severity of clinical signs of toxicity nor pathologic lesions to challenge doses of 24 g/kg of the plant between groups of ewes with an immediate previous history of increasing doses of the plant and others with no history of ingesting the plant. Previously reported apparent induced adaptation or tolerance to G officinalis L in some animals is more likely to have been a result of the extreme variation in individual animals susceptibility.

Toxicosis from and possible adaptation to Galega officinalis in sheep and the relationship to Verbesina encelioides toxicosis.

Galega officinalis L (goatsrue), a plant introduced from Europe and found in abundance in northern Utah, was tested for toxicity in ewes (Ovis aries). Clinical signs of poisoning induced by doses as small as 0.8 g of dried plant/kg body weight/day included dyspnea, anoxia, and foaming nasal discharge. Pathologic signs in animals that died following overdose of the plant included severe hydrothorax, generalized lung congestion, foamy exudate in bronchioles and trachea, epicardial and endocardial petechiation, and pericardial effusion in severely affected ewes. Microscopic lesions included a severe diffuse alveolar and interlobular edema. No significant alterations in blood constituents were observed in treated animals. Ewes administered the plant on several consecutive days developed an apparent adaptation to the toxin of the plant and were thereby able subsequently to tolerate levels 5-10 times the pre-treatment lethal dose with no discernible adverse clinical or pathological effects.

Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. II. Irreversible transport inhibition is induced by photolysis.

In the dark, phloretinyl-3'-benzylazide (PBAz), at a nominal concentration of 10 microM, will inhibit the transport of D-glucose in human erythrocytes by more than 90%. This inhibition can be completely reversed by percolating the cell suspension through a small column of Sephadex G-10; cells recovered after this treatment, and then loaded with 100 mM D-glucose, possess a transport capacity (glucose efflux) equal to untreated cells. The Sephadex matrix completely removes non-covalently bound inhibitor even though, under these conditions (subdued light, 0.2% hematocrit, 0 degrees C, pH 6.2 or 7.8), from 70 to 80% of the PBAz added is bound to the cells (mostly non-specifically to hemoglobin). However, when erythrocytes exposed to 10 microM inhibitor are irradiated with long wavelength ultraviolet light, the glucose transporter is irreversibly inhibited; after 1 min irradiation, about 50% of transporter activity cannot be restored by Sephadex treatment. Under identical conditions, control cells (no PBAz, but irradiated and treated with Sephadex) retain over 90% of carrier activity. The photolytic conversion of the inhibition to an irreversible form is directly dependent on PBAz concentration. The results reaffirm our earlier conclusions that PBAz is a potentially useful photoaffinity labeling agent for the glucose transporter in erythrocyte membranes.

Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. I. Hexose transport inhibition and photolabeling of mutarotase.

A new phloretin derivative, phloretinyl-3'-benzylazide (PBAz), has been synthesized and compared with phloretin for its ability to inhibit the hexose transporter in human erythrocyte membranes in subdued light. Transport measurements were made using the light scattering (Orskov optical) method and a Millipore filtration technique with isotopically labeled sugars. Initial rates of sugar flux were measured under four different conditions to test for inhibition asymmetry. In each experimental condition, PBAz is from 6-20-times more potent than phloretin, making it one of the most effective reversible inhibitors known. Although both agents penetrate the cell membrane, they apparently fail to reach inhibitory levels at the inner surface over the time course of our nonequilibrated experiments, because of extensive binding to hemoglobin. The mechanism by which PBAz and its parent phloretin inhibit transport is pure competition with hexose for the carrier which faces the exterior of the membrane. If given time to equilibrate with the cells, the inhibition by both agents converts to a mixed type, i.e., both competitive and noncompetitive. The noncompetitive component could be due to inhibition of those transporter units oriented internally. Alternatively pre-equilibration with the inhibitors may cause them to attain high levels in the lipid membrane and produce nonspecific effects. PBAz and its precursor amine, phloretinyl-3'-benzylamine (PBA), compete with glucose for the sugar binding site on mutarotase at least as well as phloretin. When exposed to long wavelength ultraviolet radiation, PBAz is converted to a reactive intermediate which becomes covalently bound to the enzyme. Both irreversible ligand attachment and mutarotase inhibition are related to dose of the azide and irradiation time, but inactivation is from 5 to 6-times greater than label incorporation. We conclude that PBAz is a potentially useful photoaffinity labeling agent capable of covalently interacting with the transporter site facing the exterior of the red cell.

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase.

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin.

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.

Evidence that the coproporphyrinogen oxidase activity of rat liver is situated in the intermembrane space of mitochondria.

Preferential rupture of the outer membrane of mitochondria from rat liver releases coproporphyrinogen oxidase in parallel with components of the intermembrane space. Coproporphyrinogen III enters the mitochondrion through the freely-permeable outer membrane. Either protoporphyrinogen IX or protoporphyrin IX must then cross the inner membrane before haem synthesis can be completed.

A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver.

[14C2]Coproporphyrin III, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.

Factors determining the sequence of oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III by coproporphyrinogen oxidase in rat liver.

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyses the oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III to form protoporphyrinogen IX. A 4-propionate-substituted porphyrinogen, harderoporphyrinogen, which is also a substrate for coproporphyrinogen oxidase, is formed during the reaction. Synthetic [(14)C]coproporphyrinogens III, specifically labelled in the carboxyl carbon atoms of either the 2- or 4-propionate substituents, were used to measure the rate of decarboxylation of each substituent by rat liver coproporphyrinogen oxidase. The experimental results, together with the recognition that in all known substrates of coproporphyrinogen oxidase only those propionate groups flanked by a specific arrangement of substituents are decarboxylated, indicate that the 4-propionate group of coproporphyrinogen III cannot be attacked until the 2-propionate group has been decarboxylated. Production of (14)CO(2) from the substrate labelled in the 2-propionate group therefore measures the formation of harderoporphyrinogen, whereas (14)CO(2) from the 4-propionate-labelled substrate measures protoporphyrinogen IX formation. The rate of harderoporphyrinogen formation is about twice that of protoporphyrinogen, and this ratio is unchanged by varying the concentration of coproporphyrinogen III or by competitive inhibition of the enzyme. When coproporphyrinogen III is present in an excess, two fractions of harderoporphyrinogen can be distinguished. One accumulates during the reaction, and the other, which is destined to become protoporphyrinogen IX, does not equilibrate with added harderoporphyrinogen. It is suggested that both decarboxylations take place at the same active centre, which becomes temporarily inaccessible to coproporphyrinogen III and added harderoporphyrinogen, and that the molecule rotates after the first decarboxylation to allow the second to take place.

The primary enzyme defect in hereditary coproporphyria.

The activity of coproporphyrinogen oxidase (E.C. 1.3.3.3) in cultured skin fibroblasts from three patients with hereditary coproporphyria (H.C.) was approximately half that in fibroblasts from normal subjects and patients with other types of porphyria. It is suggested that this is the primary defect in H.C., which is inherited as an autosomal dominant, and that the same abnormality is present in the liver. Consideration of the probable relative activities of the enzymes of haem biosynthesis in the liver in H.C. suggests that the acute attacks of porphyria which are its major clinical manifestation occur when the activity of uroporphyrinogen-I-synthase (E.C. 4.3.1.8) becomes rate-limiting for haem synthesis.

The effect of the porphyrogenic compound, hexachlorobenzene, on the activity of hepatic uroporphyrinogen decarboxylase in the rat.

1. A new method for the measurement of uroporphyrinogen decarboxylase (EC 4. 1.1.37) in rat liver homogenates, with 5- carboxyl porphyrinogen as substrate, is described. 2. The administration of a diet containing 0-3% (w/w) hexachlorobenzene produces porphyria in female Wistar rats after a delay of at least 4 weeks. The development of porphyria is accompanied by a progressive fall in hepatic uroporphyrinogen decarboxylase activity to 18% of control values after 11 weeks. The features of hexachlorobenzene prophyria are consequences of this enzyme defect. 3. Feeding with hexachlorobenzene did not lead to the accumulation of iron in the liver. It is suggested that hexachlorobenzene or a metabolite acts directly to decrease the activity of the enzyme.