Isolation, Mapping, and Characterization of Trehalaseless Mutants of Neurospora crassa

Mutant strains of Neurospora crassa that lack trehalase and are unable to grow on trehalose were isolated, and the gene (tre) was positioned on the right arm of linkage group I. Maltase and beta-galactosidase activities are almost identical in tre(-) strains, whereas that of invertase was reduced by more than half and those of acid phosphatase and amylase were somewhat increased. Heterocaryons between standard and trehalaseless strains yield less than one-tenth the activity of the former. In addition, strains with duplications heterozygous for trehalase produce less than 1% of the activity of the standard strain. An inhibitor of trehalase has been found in tre(-) strains; its sensitivity to heat and proteolysis, and its nondialyzability suggest that this substance is a protein. The mig gene, which determines the rate of migration of trehalase on acrylamide gels, has been shown to be less than 1 map unit away from the tre gene.     

Alkaline-phosphatase and adenosine-triphosphatase histochemical reactions in the salivary glands of cat,dog and man,with particular reference to the myoepithelial cells

Summary Salivary myoepithelial cells were demonstrated by alkaline-phosphatase techniques in cat, but not in man or dog, and by an adenosine-triphosphatase technique in man, but not in cat or dog.Electron-microscopical cytochemistry showed that the reaction product from the respective techniques in cat and man was associated with the myoepithelial plasma membrane and that it was most constant and usually strongest at the plasma membrane adjacent to the acinar cells.In the dog, the reaction product from the adenosine-triphosphatase technique was found lining the canaliculi and lumina of the acini of the parotid gland, and of the non-mucous acini of the submandibular and sublingual glands. Alkaline-phosphatase reaction product was found lining the canaliculi and lumina in the sublingual gland.These remarkable species differences indicate that neither technique can be regarded as a universal marker of salivary myoepithelial cells. Inconsistencies in activity were found within the myoepithelium of individual glands and suggest that even the appropriate technique may not be relied upon to demonstrate all the myoepithelial cells present in a tissue section.

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Zusammenfassung Myoepithelzellen der Speicheldrüsen lassen sich bei der Katze — nicht jedoch bei Mensch und Hund — mittels der alkalischen Phosphatase-Reaktion darstellen. Der Nachweis für Adenosintriphosphatase fällt in diesen Zellen beim Menschen, nicht bei Katze und Hund, positiv aus.Elektronenmikroskopisch-cytochemisch zeigt sich, daß das Reaktionsprodukt der jeweiligen Methode bei der Katze und beim Menschen an den Plasmamembranen der Myoepithelzellen auftritt und zwar am regelmäßigsten und meistens am stärksten in der Nachbarschaft der Azinuszellen.Beim Hund fällt in der Ohrspeicheldrüse sowie den nicht-mukösen Acini der Glandulae submandibularis und sublingualis das Reaktionsprodukt der Adenosintriphosphatase-Reaktion an der Begrenzung der Azinuskanälchen und der Lumenoberfläche aus. Alkalische Phosphatase ist in der Glandula sublingualis in den Wänden der Kanälchen und an den Lumina lokalisiert.Diese bemerkenswerten Unterschiede zwischen den verschiedenen Tierarten zeigen, daß keine der verwendeten Methoden zur Universalmarkierung von Myoepithelzellen der Speicheldrüsen geeignet ist. Außerdem ist die Aktivität des Myoepithels bei den einzelnen Drüsen uneinheitlich. Dies legt die Vermutung nahe, daß man sich selbst bei Anwendung der richtigen Methode nicht darauf verlassen kann, alle in einem Gewebsschnitt vorhandenen Myoepithelzellen zu erfassen.

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This work has been supported by a Medical Research Council Grant.     

Survey of the U.S. Gulf Coast for the Presence of Clostridium botulinum

In sediments and animals collected during warm weather months between Key West, Fla., and Brownsville, Tex., Clostridium botulinum, predominantly type E, was demonstrable. Incidence was somewhat higher in the eastern Gulf animals, but the organism was present to the southernmost limits of both Texas and Florida. Types A and F were never detected in warm weather. No bottom type or any single species seemed exclusively vulnerable. In samples collected during colder weather, the east-west incidence differential was minimized in animals but not in sediments, overall incidence was lowered, all known types were present, and type E no longer predominated. Detection by fluorescent-antibody techniques was found to be inadequate.     

Characterization of AAT1: a gene involved in the regulation of amino acid transport in Saccharomyces cerevisiae

A new class of Saccharomyces cerevisiae mutants (aat1 - amino acid transport) has been identified. These mutants are unable to grow on rich medium or on minimal medium supplemented with certain amino acids (isoleucine, methionine, phenylalanine, tyrosine or valine). This phenotype is directly linked to the presence of the leu2 allele in these strains: aat1 LEU2 organisms grow normally on all media tested. Leucine uptake through the leucine-specific permease is inhibited to less than 35% of wild-type levels in aat1 cells preincubated in nonpermissive media, and the activity of the general amino acid permease is also low in these conditions. aat1 cells are therefore unable to grow on rich media because they cannot take up enough leucine to supplement their auxotrophic requirement.     

Lateral bias in feeding and brachiation inHylobates

Lateralized hand use in gibbons was assessed for both food reaching and leading limb in brachiation. Sex and age effects were found in hand preference for food reaching. Adult females were all very strongly right hand preferent, whereas adult males had no across group consistent preference. Within the female group there was a strong correlation between age and strength of right handedness. When compared in terms of absolute strength of hand preference, females were found to be more strongly lateralized than males. Leading limb preference in brachiation was scored into vocal and non-vocal categories. Three subjects had a shift in preferred leading limb from the non-vocal brachiation condition to the vocal brachiation condition. This shift may be influenced by the arousal effects of species typical vocalization. The results of this study underline the importance of consideration of such factors as sex and age when interpreting behavioral lateralization data. The exploration of laterality in many different response measures is important to the achievement of a complete understanding of behavioral lateralization in primates.     

Effect of Halide Ions on t-[35S]Butylbicyclophosphorothionate Binding

The binding of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to a site on the GABAA receptor complex is ion dependent. This study was conducted to determine the effects of ion species and concentration on the time course, affinity, and number of sites of [35S]TBPS binding. At a concentration of 200 mM ion, the time to equilibrium for [35S]TBPS binding was shortest for I-, followed by Br- less than Cl- less than F-. A similar rank order was observed for the concentration of ion required to produce half-maximal [35S]TBPS binding. Saturation binding experiments were conducted to evaluate the effect of increasing ion concentration on the KD and Bmax of [35S]TBPS binding. The Bmax was independent of both ion species and concentration. The receptor affinity, however, increased with increasing concentration for each ion. Calculated maximal affinity values were not different between ions; however, the EC50 to produce those values was different among ions and ranked in the same order as that for time course and maximal binding data. Association and dissociation rates for [35S]TBPS binding were greater in I- than in Cl-. These data emphasize the importance of ion selection and incubation times on [35S]TBPS binding.     

1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast. Five independent sequential connectivity pathways via one-, two-, and three-bond heteronuclear J couplings are exploited to obtain unambiguous sequential assignments. Specifically, CO(i)-N(i + 1),NH(i + 1) correlations are observed in the HNCO experiment, the C alpha H(i), C alpha (i)-N(i + 1) correlations in the HCA(CO)N experiment, the C alpha(i)-N(i + 1),NH(i + 1) correlations in the HNCA and HN(CO)CA experiments, the C alpha H(i)-N(i + 1),NH(i + 1) correlations in the H(CA)NH and HN(CO)HB experiments, and the C beta H(i)-N(i + 1),NH(i + 1) correlations in the HN(CO)HB experiments. The backbone intraresidue C alpha H(i)-15N(i)-NH(i) correlations are provided by the 15N-edited Hartmann-Hahn (HOHAHA) and H(CA)NH experiments, the C beta H(i)-15N(i)-NH(i) correlations by the 15N-edited HOHAHA and HNHB experiments, the 13C alpha(i)-15N(i)-NH(i) correlations by the HNCA experiment, and the C alpha H(i)-13C alpha(i)-13CO(i) correlations by the HCACO experiment. Aliphatic side-chain spin systems are assigned by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and total correlated (HCCH-TOCSY) spectroscopy. Because of the high resolution afforded by these experiments, as well as the availability of multiple sequential connectivity pathways, ambiguities associated with the limited chemical shift dispersion associated with helical proteins are readily resolved. Further, in the majority of cases (88%), four or more sequential correlations are observed between successive residues. Consequently, the interpretation of these experiments readily lends itself to semiautomated analysis which significantly simplifies and speeds up the assignment process. The assignments presented in this paper provide the essential basis for studies aimed at determining the high-resolution three-dimensional structure of IL-4 in solution.     

cAMP inhibits bud growth in a yeast strain compromised for Ca2+ influx into the Golgi

Biochemical and physiological studies have implicated cAMP and cAMP-dependent protein kinase (PKA) in a plethora of essential cellular processes. Here we show that yeast cells partially depleted of PKA activity (due to atpk ^w mutation) and bearing a lesion in a Golgi-localized Ca²⁺ pump (Pmr1), arrest division with a small bud. The bud morphology of the arrestedtpk1 ^w pmr1 mutant cells is characteristic of cells in S phase; however, the terminal phenotype of processes such as DNA replication and nuclear division suggests arrest at the G2/M boundary. This small bud, G2-arrest phenotype is similar to that of strains with a defect in cell wall biosynthesis (pkc1) or membrane biogenesis (och1); however, the biochemical defect may be different since thetpk1 ^w pmr1 double mutants retain viability. The growth defect of thetpk1 ^w pmr1 mutant can be alleviated by preventing the increase in cellular cAMP levels that is known to be associated with a decrease in PKA activity, or by supplementing the medium with millimolar amounts of Ca²⁺. Although the biochemical consequences of this increase in cAMP concentration are not known, the small-bud phenotype of the double mutant and the known protein processing defect of thepmr1 lesion suggest that the localization or function of some membrane component might be compromised and susceptible to perturbations in cellular cAMP levels. One candidate for such a protein is the cAMP-binding membrane ectoprotein recently described in yeast.