Stimulation of gibberellin activity in winter wheat by metribuzin

Sublethal doses of metribuzin applied to wheat plants at the stage of ear emergence increased endogenous gibberellin levels in the ears. The activation of hormonal systems in connection with “chemical stress” is briefly discussed.     

Sodium channels in planar lipid bilayers. Channel gating kinetics of purified sodium channels modified by batrachotoxin

Single channel currents of sodium channels purified from rat brain and reconstituted into planar lipid bilayers were recorded. The kinetics of channel gating were investigated in the presence of batrachotoxin to eliminate inactivation and an analysis was conducted on membranes with a single active channel at any given time. Channel opening is favored by depolarization and is strongly voltage dependent. Probability density analysis of dwell times in the closed and open states of the channel indicates the occurrence of one open state and several distinct closed states in the voltage (V) range-120 mV less than or equal to V less than or equal to +120 mV. For V less than or equal to 0, the transition rates between stages are exponentially dependent on the applied voltage, as described in mouse neuroblastoma cells (Huang, L. M., N. Moran, and G. Ehrenstein. 1984. Biophysical Journal. 45:313-322). In contrast, for V greater than or equal to 0, the transition rates are virtually voltage independent. Autocorrelation analysis (Labarca, P., J. Rice, D. Fredkin, and M. Montal. 1985. Biophysical Journal. 47:469-478) shows that there is no correlation in the durations of successive open or closing events. Several kinetic schemes that are consistent with the experimental data are considered. This approach may provide information about the mechanism underlying the voltage dependence of channel activation.     

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.     

Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.     

Are Chaoborus larvae more abundant in acidified than in non-acidified lakes in Central Canada?

Eriksson et al (1980) hypothesized that the abundance of certain macroinvertebrate predators, such as larvae of the phantom midge Chaoborus , should increase in acidified lakes because of the elimination of fish. To examine the influence of pH and presence of fish on Chaoborus abundance, we surveyed Chaoborus populations in 33 lakes in Ontario, Canada which ranged in pH from 4.5 to 7.4. Chaoborus larvae were not more abundant in the acidified lakes that were devoid of fish than in the remaining lakes. Therefore, we concluded that pH and presence of fish are not prime determinants of total Chaoborus abundance in Canadian Shield lakes. We hypothesized that significant increases in Chaoborus abundance should only be anticipated when fish populations are eliminated by acidification of relatively nutrient rich lakes.