Inconsistency in the species concept for yeasts due to mutations during vegetative growth

Summary The generic classification of yeasts is based mainly on morphological characteristics whereas the definition of a species depends predominantly on physiological properties such as the utilization of carbon and nitrogen sources. Classification procedures are routinely done on agar slants, and in negative tests single colonies are often noticed. These colonies are spontaneous mutations and can be idetified as such after transfer onto adequate media and appropriate genetic tests. It is sometimes possible after selection steps to obtain a completely different species. This means that in many cases the classification depends only on single gene differences, where the differences in DNA base homology is almost certainly less than 1%. Since it is rather difficult to justify a new species on the basis of a single biochemical gene mutation, it is necessary in practice to perform at regular intervals an extended series of physiological tests in order to avoid confusion in nomenclature.     

Additional evidence for separable responses to auxin in soybean hypocotyl

Additional evidence for two separable responses to auxin is presented. The average of 24 control experiments indicated lag times of 12.4 and 35.4 min, and maximum rates of 0.57 and 0.54 mm hr⁻¹, for the first and second response, respectively. The auxin analog 4-azido-2-chlorophenoxyacetic acid increased the lag time of the second response (but not the first), resulting in the temporal separation of the two responses. Plots of elongation rates against time, taken from the literature, allowed the characterization of the two responses in monocotyls and dicotyls. Study of published rate-time elongation curves showed that the maximum rate of the first response is frequently greater than the maximum rate of the second response; however, the maximum rate of the second response has not yet been shown to exceed the maximum rate of the first response.     

Two elongation responses to auxin respond differently to protein synthesis inhibition

The first and second responses to auxin react differently to the inhibition of protein synthesis by cycloheximide. It was determined that the protein with the shortest half-life, among the several necessary for the first response, is different from its counterpart among the several necessary for the second response. Specifically, the protein half-lives are 28 minutes and 11 minutes for the first and second responses, respectively.     

Clearance of lysosomal hydrolases following intravenous infusion. Kinetic and competition experiments with beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Clearance experiments with highly purified lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, following intravenous infusion revealed widely varying clearance profiles which depended on the tissue source of the enzyme. Normal rat serum β-glucuronidase and epididymal N-acetyl-β-d-glucosaminidase were cleared slowly from the circulation when compared with rat preputial gland β-glucuronidase, liver lysosomal β-glucuronidase, and liver lysosomal N-acetyl-β-d-glucosaminidase, respectively, which were cleared rapidly. Experiments comparing the catalytic properties and molecular dimensions of the enzymes revealed no differences between rapid and slow clearance forms. Kinetic analysis using the rapid clearance forms of β-glucuronidase has allowed the resolution of at least two components, rapid and slow. Clearance of the rapid component is saturable and appears to reflect binding or uptake by a limited number of sites. By contrast, the clearance rate of the slow component increased linearly with respect to dose and may be due to nonspecific or low-affinity binding. Competition experiments with β-glucuronidase-free lysosomal extract and highly purified lysosomal enzymes, but not serum glycoproteins or colloidal silver, suggest that one lysosomal enzyme inhibits clearance of others and that a common mechanism may be involved in their binding.     

Clearance of lysosomal hydrolases following intravenous infusion. The role of liver in the clearance of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase.

Certain highly purified forms of rat lysosomal glycosidases, β-glucuronidase and N-acetyl-β-d-glucosaminidase, are rapidly cleared from the circulation following intravenous infusion. Several lines of evidence are presented which indicate that the primary site of enzyme uptake is the liver. Clearance of the two enzymes was unaffected by nephrectomy, whereas it was abolished by evisceration. Tissue distribution experiments with native and [¹²⁵I]β-glucuronidase indicate the liver as the major, if not exclusive, site of enzyme uptake. Experiments with the isolated perfused liver showed clearance of certain enzyme preparations but not others. Those enzymes cleared by the isolated perfused liver were likewise cleared in vivo. Liver fractionation studies following infusion of large doses of β-glucuronidase revealed a rapid, short-lived increase in microsomal β-glucuronidase and a slower but larger increase in lysosomal β-glucuronidase. The results indicate that β-glucuronidase, N-acetyl-β-d-glucosaminidase, and probably other glycosidases are rapidly incorporated into the lysosomal compartment of liver.     

Interaction of a new fluorescent reagent with sulfhydryl groups of the (Na+ + K+)-stimulate ATPase.

The (Na+ + K+)-ATPase enzyme of rat brain microsomes can be reversibly inhibited by a new fluorescent sulfhydryl (SH) probe, dimethylaminoaphthalenecysteine-Hg+ (Dn-cys-Hg+). This reagent is more reactive than N-ethylamaleimide (MalNEt) toward membrane sulfhydryl groups. A procedure using the two SH reagents sequentially seems to permit a more selective labelling of the SH groups involved in the (Na+ + K+)-ATPase than is possible by using MalNEt alone. Brain microsomes treated by this method incorporate the fluorescent label within or near the active site of the enzyme. When the probe was bound a blue shift of its fluorescence emission maximum (from 540 to 495 nm) and a 20-fold increase in relative fluorescence occurred. This indicates that the Dn moiety is within a very non-polar region of the membrane.     

Purification and characterization of rat liver microsomal beta-glucuronidase.

Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.     

Histochemical localization of potassium-stimulated P-nitrophenylphosphatase activity in the somatosensory cortex of the rat.

Potassium-stimulated p-nitrophenylphosphatase (K+-pNPPase) activity was investigated in rat somatosensory cortex where 64-88% of enzymatic activity survived 5-10 min of fixation with 3% formaldehyde in 0.1 M cacodylate buffer, pH 7.4. Potassium-stimulated activity was inhibited by 1-10 mM ouabain. Levamisole (1.7 mM) inhibited brain alkaline phosphatase activity, facilitating the detection of K+-pNPPase activity. Strontium (10-20 mM) inhibited enzymatic activity by 38-75%. In parallel histochemical studies reaction product was found in strata, with cortical layers 2, 3, 4 and the outer portion of 5 containing the heaviest deposits. Highly reactive, vertically oriented, large diameter fibers were seen as groups between the outer portion of layer 5 and the pail surface. These fibers apparently arborize in the superficial layers. Smaller fibers were also positive and were oriented in various planes. The highest density of smaller, positive fibers occurred in layers 2 through 5. All positive fibers appeared to be axons or dendrites. Reaction product was not heavily concentrated in neuron perikarya or in glial elements. Sections did not contain reaction product when incubated in media lacking K+ or containing ouabain. The convergence of data from parallel histochemical and biochemical approaches supports the conclusion that the reactivity localized in the cerebral cortex represented the site of K+-pNPPase, a known component of the Na+,K+-adenosine triphosphatase complex. Neuronal processes demonstrated the highest enzymatic activity and may be most important in the active transport of Na+ and K+ in somatosensory cortex.