Crystallization and preliminary X-ray investigation of a ubiquitin carrier protein (E2) from Arabidopsis thaliana.

Crystals of a recombinant ubiquitin carrier protein from Arabidopsis thaliana have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 41.8(1) A, b = 44.9(1) A and c = 83.2(1) A. The crystals are quite stable to X-rays and diffract beyond 2.1 A resolution. There is one molecule in the asymmetric unit.     

Occurrence and performance of the aspen blotch miner,Phyllonorycter salicifoliella,on three host-tree species

Summary Larvae of the aspen blotch miner, Phyllonorycter salicifoliella Chambers (Lepidoptera: Gracillariidae), feed within leaves of three host-tree species in north-central Minnesota, USA. Far more individuals occur on Populus tremuloides than on P. balsamifera or P. grandidentata. We tested whether this pattern of host use reflected variable performance among alternative hosts by examining survivorship, sources of mortality, pupal mass, feeding efficiency, and development time of miners on each tree species. We also determined foliar water, nitrogen, condensed tannin, and phenolic glycoside content of host trees to test if host-tree chemical attributes were responsible for differences in performance. There was no significant difference in egg-to-adult survival among miners on different hosts, although dominant sources of mortality did vary. Miners on P. grandidentata suffered less parasitism and more predation than those on the other hosts, even though most parasitoid species attacked miners on all hosts. The other performance parameters varied among host species, but not in a consistent pattern. Pupal mass was greatest on P. tremuloides and P. balsamifera, the hosts with comparatively high foliar nitrogen and low phenolic glycoside concentrations. However, feeding efficiency was greatest and development time shortest for miners on P. grandidentata. Thus, pupal mass was the only index of performance maximized on P. tremuloides, the most commonly used host. Infrequent occurrence of Phyllonorycter salicifoliella on P. grandidentata results in part from phenological differences between this and the other host species. Low oviposition rates on P. balsamifera are correlated with low abundance of this host at the study site and a phenolic glycoside profile different from that of the other host species.     

Estradiol pulses induce progestin receptors selectively in substance P-immunoreactive neurons in the ventrolateral hypothalamus of female guinea pigs

Low doses of estradiol, administered as pulses, are as effective as higher doses for priming ovariectomized (OVX) guinea pigs to display progesterone-facilitated lordosis. High doses of estradiol, administered by constant-release implants, induce progestin receptors in many substance P-immunoreactive (SP-IR) neurons in the ventrolateral hypothalamus (VLH), a site at which estradiol primes OVX guinea pigs to respond behaviorally to progesterone. To test the hypothesis that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P-containing neurons in the VLH, OVX females received estradiol implants 1 week prior to perfusion, or two pulses of estradiol- 17β, injected 39 and 11 h before perfusion. Colchicine was administered intracerebroventricularly prior to perfusion. No significant differences were observed in the total number of progestin receptor-immunoreactive (PR-IR) or substance P-immunoreactive cells in the VLH and VLH/ventromedial hypothalamus (VMH), respectively, of females receiving the two estradiol treatments. However, the percentage of PR-IR cells in the VLH also immunoreactive for SP was significantly higher in the estradiol pulse-treated (53%), than in the estradiol capsule-implanted animals (36%). These data suggest that behaviorally effective estradiol pulses induce progestin receptors selectively in substance P-containing neurons in the VLH and are consistent with the hypothesis that substance P is involved in progesterone-facilitated lordosis in guinea pigs.     

Differential plating medium for quantitative detection of histamine-producing bacteria.

A histidine-containing agar medium has been devised for quantitative detection of histamine-producing bacteria that are alleged to be associated with scombroid fish poisoning outbreaks. The responsible bacteria produce a marked pH change in the agar, with attendant color change of pH indicator adjacent to the colonies, thus facilitating their recognition. Proteus morganii and Klebsiella pneumoniae were the two most common histidine-decarboxylating species isolated from scombroid fish and mahi mahi.     

The gelatinolytic activity of human skin fibroblast collagenase

The gelatinolytic activity of human skin fibroblast collagenase was examined on denatured collagen types I-V. All denatured substrates were cleaved, including types IV and V, which are resistant to collagenase in native form. Interestingly, the earliest major cleavage in denatured collagen types I-III occurred at a 3/4-1/4 locus, resulting in products electrophoretically identical with TCA and TCB fragments of mammalian collagenase action on these native collagens. However, in the denatured substrates, multiple additional proteolytic cleavages followed. The propensity for cleavage at a 3/4-1/4 site in denatured collagen, where sequence is the major specifier of enzymatic action, would seem to indicate that the most favorable amino acid sequence of gamma chains for catalysis is located in this region. The peptide bond specificity of human fibroblast collagenase on gelatin was examined by amino acid sequencing of extensively cleaved denatured type I collagen. Analysis of the NH2-terminal amino acid residues from the resultant gelatin peptides showed sequences of "-H2N-Ile-Y-Gly" and "H2N-Leu-Y-Gly" only (where Y indicates that any amino acid can be found in that position), indicating that Gly-Ile and Gly-Leu bonds are the only sites of collagenase cleavage in this substrate. Whereas the gamma1 chains of denatured collagen types I-III were cleaved at similar rates, fibroblast collagenase was a much better gamma2-gelatinase than gamm1-gelatinase on denatured type 1 collagen. This preference for the cleavage of gamma2(I) was the result of both a higher kcat (750 versus 230 h-1) and lower Km (3.7 versus 7.0 microM) than for a gamma1(1), resulting in an overall selectivity (kcat/Km) of greater than 6-fold. Compared to such kinetic parameters on native collagen, these values indicate that gelatinolysis is somewhat slower than collagenolysis.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

Human plasma-derived immunosuppressive factors having anti-lymphoma activity

Summary Extraction of Cohn IV-1, an -globulin enriched fraction of human plasma, with a high-salt, low-pH solution, followed by sequential ultrafiltration steps yielded an immunosuppressive preparation (UM05R) of mol.wt. 500–10,000. UM05R inhibited antibody formation in the mouse in vivo and transformation in vitro of lymphocytes treated with either T-or B-cell stimulants. Suppression of lymphocyte transformation, indicated by inhibition of ³H-thymidine incorporation into DNA, was confirmed by inhibition of blast cell formation. From dose-response curves the UM05R concentration to produce 50% suppression of lymphocyte blast transformation was 15–50 g protein/ml. Selectivity for lymphoid cells was suggested by growth inhibition in vitro of L1210 and P1798 leukemias but not murine neuroblastoma or human fetal fibroblasts. This observation also rules out the presence of an agent which is broadly cytotoxic. Fractionation of UM05R on Sephadex G-25 in 10% acetic acid yielded an early-emerging fraction, mol. wt. 5,000–10,000, containing B-cell inhibitor, and a late fraction, mol. wt. 1,400, inhibitory for both T- and B-cell transformation and growth of L1210. The inhibitory activity for B cells was removed from the other two activities by 5% trichloroacetic acid (TCA). The possibility is raised that the inhibitory activity for T cells and L1210 may reside in the same molecule. Sensitivity of the early-emerging B-cell inhibitor to carboxypeptidase B suggests that it is a polypeptide, but resistance of the T-cell inhibitor to various treatments leaves its nature uncertain. The properties of these factors suggest consideration of them as lymphocyte chalones occurring in plasma complexed to high-molecular-weight components.