Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

Allocation of Photosynthate to Individual Tubers of Solanum tuberosum L.: I. RELATIONSHIP BETWEEN TUBER GROWTH RATE AND ENZYME ACTIVITIES OF THE STARCH METABOLISM

Potato plants (Solanum tuberosum L.) were grown in water culturein a controlled environment. Cooling (+8°C) of individualtubers decreased their growth rates and increased the growthrates of non-cooled tubers of the same plant. The carbohydrateconcentration in non-cooled and cooled tubers did not differsignificantly, but ¹⁴C-import from labelled photosynthate waslower in cooled than in non-cooled tubers. The markedly lowerconversion rate of ethanol-soluble ¹⁴C to starch in cooled,in comparison to non-cooled tubers, was not associated withsignificant differences in the in vitro activities of starchsynthase, ADPG-pyrophosphorylase and starch phosphorylase understandard assay conditions (+30°C). However, the Q₁₀-valuesof the enzymes differed in vitro in the temperature range between30°C and 8°C, leading to a marked decrease in the activityratio of ADPG-pyrophosphorylase/starch phosphorylase in cooledtubers. In tubers differing in growth rates without manipulation, 14d after tuber initiation significant positive correlations werefound between ¹⁴C-concentration of tuber tissue and the in vitroactivities of starch synthase and ADPG-pyrophosphorylase anda significant negative correlation between ¹⁴C-concentrationand starch phosphorylase. In contrast, in tubers which wereanalysed 5 d after initiation, there were only small differencesbetween tubers in growth rate, ¹⁴C import and the activity ratioADPG-pyrophosphorylase/starch phosphorylase. From various directand indirect evidence it is concluded that the growth rate ofindividual tubers, and thus the sink strength, is at least inpart controlled by the activity of starch synthesizing enzymes. Key words: Potato tuber, cooling, starch synthesizing enzymes     

Experimentelle analyse der infektion und des entwicklungszyklus vonMalamoeba scolyti inDryocoetes autographus [Coleoptera: scolytidae]

Malamoeba scolyti (Purrini), ein Parasit im Verdauungstrakt des BorkenkäfersDryocoetes autographus (Ratz.), schmarotzt intrazellulär in bestimmten Abschnitten des Mitteldarmepithels und im Epithel der Malpighischen Gefä\e. Sie zerstört die befallenen Epithelzellen, wobei sie sich bis zum zystenstadium entwickeln kann. in umfangreichen Infektionsexperimenten wird gezeigt, daß die Infektion in den vordersten Krypten des Mitteldarms beginnt und sich in mehreren Infecktionswellen bis zu den hintersten Darmdivertikeln und den Malpighischen Gefäßen ausbreitet. Bei 20° Dauertemperatur nimmt die Erstinfektion etwa 6 Tage, der gesamte Krankheitsverlauf bis zum Exitus des Wirtes 5 bis 7 Wochen in Anspruch. Im Gegensatz zu anderen Beschreibungen konnte nur eine einzige Form von Trophozoiten beobachtet werden. Auf die mögliche Bedeutung dieses Umstandes für die taxonomische Identität der Amöbe wird hingewiesen.     

Sympathetic secretory response to hypercapnic acidosis in swine

We studied the effect of graded acute hypercapnic acidosis (HA) on sympathetic neural activation in 15 juvenile farm swine in vivo. In seven animals with acute HA, plasma norepinephrine (NE) concentration increased progressively from 189 +/- 34 to 483 +/- 80 pg/ml (P less than 0.04) as arterial CO2 partial pressure (PaCO2) increased in steps from 40 to 80 Torr (pH 7.17 +/- 0.01). Plasma epinephrine (EPI) concentration increased from 30 +/- 15 to 125 +/- 66 pg/ml (P = NS) over the same change in PaCO2. At PaCO2 of 110 Torr, plasma NE increased 3.4-fold above maximal basal concentrations; plasma EPI was 1.8-fold greater than basal under the same conditions. With HA, systemic vascular resistance (SVR) decreased from 1,748 +/- 110 to 1,392 +/- 145 dyn.s.cm-5 (P less than 0.0002), cardiac output (CO) increased from 3.4 +/- 0.3 to 4.3 +/- 0.3 l/min (P less than 0.01), and heart rate (HR) increased from 117 +/- 11 to 154 +/- 17 beats/min (P less than 0.03). To demonstrate that catecholamine secretion was related directly to acidosis caused by an increase in PaCO2, HCO3- was infused in eight other swine to buffer extracellular acute HA (pH 7.37 +/- 0.01 at PaCO2 of 80 Torr). Buffering attenuated the increase in plasma NE, which remained within the normal range at PaCO2 of 80 Torr. The decrease in SVR and increases in CO and HR also were also attenuated by HCO3- buffering of HA. We demonstrate the effects of graded acute HA on endogenous secretion of catecholamine and on the associated hemodynamic responses in swine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

The rad10, rad16, rad20, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as rad16, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RAD1 protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.