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.     

Ciliary receptors associated with the mouth and pharynx of Acoela (Acoelomorpha): a comparative ultrastructural study

The ultrastructure and distribution of receptor cells near the mouth and (where present) the pharynx of Hofstenia miamia, Proporus bermudensis, Conaperta thela, and Convoluta convoluta (Acoela) were investigated by transmission electron microscopy and confocal laser scanning microscopy of specimens stained with a fluorescence marker for actin. Five types of monociliary receptors were identified: (1) non‐collared receptors with a single long and narrow ciliary rootlet; (2) non‐collared receptors with a wide main ciliary rootlet and a smaller posterior rootlet; (3) non‐collared receptors with a single wide and hollow ciliary rootlet with a granulated core; (4) Collar (?) receptors with obliquely radial filament bundles in the cell apex and with a single hollow ciliary rootlet composed of numerous strand‐like elements; and (5) Collar receptors lacking a striated rootlet but with a granular body (swallow's nest rootlet). While H. miamia bears the first two receptor types, P. bermudensis has receptors of type 1, 3 and 5, and Cona. thela and Conv. convoluta have receptors of type 3, 4 and 5. The density of receptors is generally highest at the anterior body tip, regardless of where the mouth is located. Most receptor types occur scattered over the whole body but type 2 receptors of H. miamia are restricted to the pharynx and mouth region. The lack of a common receptor type specific for the mouth and pharynx of the investigated species points to an independent origin of the pharynges in Hofsteniidae and in Proporidae and of the mouth tube in Convolutidae. Moreover, the homology of the so‐called collar receptors in Acoela with typical collar receptors in other invertebrates is questioned.     

Optimum prediction of three-way crosses from single crosses in forage maize (Zea mays L.)

Summary Three-way cross means were predicted with formulae involving linear functions of general (GCA) and specific combining ability (SCA) effects estimated from single-cross factorials between genetically divergent populations. Data from an experiment with 66 single-cross and 66 three-way cross forage maize (Zea mays L.) hybrids was used for comparing the prediction formulae. The genotypic correlation (r) between observed and predicted three-way crosses increased with increasing , the weighting factor of SCA effects, for plant height and ear dry matter (DM) content. It displayed slightly convex curves for total and stover DM yield, ear percentage, and metabolizable energy content of stover. For Jenkins' method B, r was considerably less than 1.0 for all traits, indicating the presence of epistasis. The square root of heritability (h) of the predicted means decreased with increasing , the reduction being small with a greater number of test environments. Using the product r·h as a criterion of efficiency, none of the prediction methods was consistently superior and the differences among them were rather small (< 7.5%) for all traits, irrespective of the number of test environments. We recommend evaluating the GCA of a greater number of lines from each parent population in testcrosses with a small number of elite lines from the opposite population. All possible three-way or double crosses between both sets of lines should be predicted by Jenkins's method C. This procedure allows one to select with a higher intensity among the predicted hybrids and thus should increase the genetic gain.Extended version of a paper (Geiger et al. 1986) read at the sixth meeting of the EUCARPIA Section Biometrics in Plant Breeding held at Birmingham, UK, July 28–August 1, 1986     

Tissue-type plasminogen activator and urokinase: differences in the reaction pattern with the active-site titrant 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride

The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be performed. We therefore studied the kinetics of the interaction of these two plasminogen activators with MUGB. The equilibrium dissociation constant, KS, for the interaction between MUGB and urokinase was 2.9 X 10(-6) M, and for the interaction with t-PA 3.13 X 10(-5) M. However, one main requirement for active-site titration, namely a stable acyl enzyme intermediate (ES'), was only fulfilled for MUGB urokinase but not for MUGB t-PA. Whereas for the reaction of MUGB and urokinase the first-order acylation rate constant k2 was found to be about 10(6)-times higher than the first-order deacylation rate constant k3 (k2 = 3.76 X 10(-1) s-1, k3 = 3.7 X 10(-7) s-1), the k2/k3 ratio for the reaction of MUGB and t-PA (one- and two-chain form) was 0.77 to 3.85. Therefore, urokinase and t-PA differ in their reaction with this fluorogenic substrate and MUGB cannot be used for active-site titration of tPA.     

Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation.

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.     

Triggered efflux of protoberberine alkaloids from cell suspension cultures ofThalictrum rugosum

Cell cultures ofThalictrum rugosum released their protoberberine alkaloids into the medium, when cells were transferred to fresh medium lacking phosphate. The nutritional factors required and the impact of the cells' physiological state for the alkaloid excretion were analyzed. Cell cultures, having released their alkaloids into the medium, continued to grow when the alkaloid containing medium was replaced by fresh growth medium.     

A new type of ultrasensitive bioluminogenic enzyme substrates. I. Enzyme substrates with D-luciferin as leaving group

Derivatives of D-luciferin, D-luciferin methyl ester, D-luciferin O-sulfate, D-luciferin O-phosphate, D-luciferyl-L-N alpha-arginine and D-luciferyl-L-phenylalanine were used as highly sensitive substrates for carboxylic esterase, arylsulfatase, alkaline phosphatase and carboxypeptidases A, B and N. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants have been determined for D-luciferin methyl ester and carboxylic esterase, for D-luciferin O-sulfate and arylsulfatase, for D-luciferin O-phosphate and alkaline phosphatase, for D-luciferyl-L-phenylalanine and carboxypeptidase A, and for carboxypeptidases B and N and D-luciferyl-L-N alpha-arginine. All compounds proved to be highly sensitive substrates for the respective enzymes, permitting a limit of detection for enzymes between 10 and 500 fg per assay.     

A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of ¹⁴C accumulated in the leaf supplied with ¹⁴CO₂ throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO₂ concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.