Dynamics of the thiol status of rat spermatozoa during maturation: analysis with the fluorescent labeling agent monobromobimane

Mammalian spermatozoa undergo maturation as they pass through the epididymis. Maturation is accompanied by the oxidation of thiols to disulfides. Disulfides are probably involved in sperm chromatin condensation and tail structure stabilization. In this work, we used the fluorescent thiol-labeling agent monobromobimane to determine the changes occurring in thiols and disulfides in rat sperm heads and tails during maturation. Spermatozoa were obtained from testis, epididymis (caput, corpus, cauda, and vas deferens), and ejaculate. Intact spermatozoa were labeled with monobromobimane, with or without pretreatment with dithiothreitol. Labeling was evaluated microscopically, and quantitative analysis was carried out spectrofluorimetrically with labeled globin used as a standard. Samples were also analyzed by gel electrophoresis. The total amount of thiols and disulfides remained the same during the entire period of sperm maturation (26 +/- 0.5 nmoles thiols + disulfides/10(6) spermatozoa). However, the reactive thiols decreased markedly between the corpus and the cauda (from greater than 90% of total in testis and 75% in corpus to about 25% in cauda), with little or no further change in vas deferens and ejaculated sperm. Trypsin treatment followed by sucrose gradient was used to separate the heads from the tails. Thiols comprised 84% of the total SH + SS in the heads and 74% in the tails of caput spermatozoa, decreasing to 14% and 45%, respectively, in cauda sperm. Thus, the decrease in reactive thiols involved both heads and tails-oxidation to disulfides being very marked in the head. Electrophoresis revealed that oxidation of thiols to disulfides occurred in many protein fractions during maturation in the epididymis.     

Surface areas,lengths and volumes of Picea abies (L.) Karst. needles: determination,biological variability and effect of environmental factors

Summary A method for the rapid determination of the lengths and surface areas of very large samples of needles of Picea abies (L.) Karst. using a computer-aided image analysis system was developed. Two independent methods for measuring non-destructively the volumes of individual needles and of all needles attached to a twig were devised. The surface areas and lengths of about 38000 needles sampled from the three youngest needle age-classes (1986, 1985, 1984) of 48 trees approximately 130 years old at four sites in the Fichtelgebirge mountains (N. E. Bavaria, FRG) were measured. The frequency distributions of lengths and areas for each site and age-class are given. Variability of needle size was fairly large. Even though the sites differed in climate, soil, and air pollution levels no consistent effect of these factors on needle size could be detected. Needle lengths and surface areas did not correlate with either the total chlorophyll content of the needles or the degree of crown thinning. The needle surface area (in mm²) of fully developed P. abies needles can be estimated by the empirical equation surface area = 4.440 x needle length -24.8 (r = 0.937), and the needle volume (in mm³) by needle volume = 0.208 x projected needle area ^1.353 (r = 0.969).     

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : III. The Purification of Erythroid Cells by pH-Induced Density Changes

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.     

The unusual social organization of the antPachycondyla tridentata (Formicidae,ponerinae)

Colonies of the ponerine antPachycondyla tridentata from Malaysia occur with and without queens. In a total of 7 colonies we found more than 80% of the workers to be mated, irrespective of the presence or absence of queens. This is a hitherto unknown social organisation in ants. Queens and workers competed equally for reproduction. In the colonies investigated several ants were laying eggs. Behavioral observations revealed persistent dominance interactions between colony members. A few ants, but not necessarily a queen, occupied top positions. Removal of the most dominant ants led to a new hierarchy in which subordinate ants with developed ovaries were attacked significantly more frequently than non-reproductive ants. On the average, callows were more aggressive than older subordinate ants, displacing most of the older laying workers in one colony. Nestmate recognition tests revealed that non-reproductive ants were much more aggressive towards foreign ants than were ants with developed ovaries.     

Accumulation of hexoses in leaf vacuoles: Studies with transgenic tobacco plants expressing yeast-derived invertase in the cytosol,vacuole or apoplasm

The subcellular distribution of hexoses, sucrose and amino acids among the stromal, cytosolic and vacuolar compartments was analysed by a nonaqueous fractionation technique in leaves of tobacco (Nicotiana tabaccum L.) wild-type and transgenic plants expressing a yeast-derived invertase in the cytosolic, vacuolar or apoplasmic compartment. In the wild-type plants the amino acids were found to be located in the stroma and in the cytosol, sucrose mainly in the cytosol and up to 98% of the hexoses in the vacuole. In the leaves of the various transformants, where the contents of hexoses were greater than in wild-type plants, again 97–98% of these hexoses were found in the vacuoles. It is concluded that leaf vacuoles contain transporters for the active uptake of glucose and fructose against a high concentration gradient. A comparison of estimated metabolite concentrations in the subcellular compartments of wild-type and transformant plants indicated that the decreased photosynthetic capacity of the transformants is not due to an osmotic effect on photosynthesis, as was shown earlier to be the case in transformed potato leaves, but is the result of a long-term dedifferentiation of tobacco leaf cells to heterotrophic cells.Abbreviations apo-inv tobacco plant with yeast invertase in the apoplasm - Chl chlorophyll - cy-inv tobacco plant with yeast invertase in the cytosol - vac-inv tobacco plant with yeast invertase in the vacuole - WT wild-type tobacco plant The authors thank A. Großpietsch for her able technical assistance. This work has been supported by the Bundesminister für Forschung und Technologie.     

Spontaneous generation of adriamycin semiquinone radicals at physiologic pH.

Adriamycin semiquinone radicals are spontaneously generated by adriamycin solutions at physiologic pH. Rate of radical formation and equilibrium-state radical yield increase with increasing pH from 7.4 to 8.85. The radicals are oxygen sensitive, but the mechanism of radical formation is oxygen independent and associated with proton removal from the dihydroquinone of adriamycin. The less cardiotoxic and non-mutagenic (Ames test) anthracycline 5-iminodaunorubicin does not form semiquinone radicals spontaneously at physiologic pH.     

The N‐Terminal Sequences of Four Major Hydrogenosomal Proteins Are Not Essential for Import into Hydrogenosomes of Trichomonas vaginalis

The human pathogen Trichomonas vaginalis harbors hydrogenosomes, organelles of mitochondrial origin that generate ATP through hydrogen‐producing fermentations. They contain neither genome nor translation machinery, but approximately 500 proteins that are imported from the cytosol. In contrast to well‐studied organelles like Saccharomyces mitochondria, very little is known about how proteins are transported across the two membranes enclosing the hydrogenosomal matrix. Recent studies indicate that—in addition to N‐terminal transit peptides—internal targeting signals might be more common in hydrogenosomes than in mitochondria. To further characterize the extent to which N‐terminal and internal motifs mediate hydrogenosomal protein targeting, we transfected Trichomonas with 24 hemagglutinin (HA) tag fusion constructs, encompassing 13 different hydrogenosomal and cytosolic proteins of the parasite. Hydrogenosomal targeting of these proteins was analyzed by subcellular fractionation and independently by immunofluorescent localization. The investigated proteins include some of the most abundant hydrogenosomal proteins, such as pyruvate ferredoxin oxidoreductase (PFO), which possesses an amino‐terminal targeting signal that is processed on import into hydrogenosomes, but is shown here not to be required for import into hydrogenosomes. Our results demonstrate that the deletion of N‐terminal signals of hydrogenosomal precursors generally has little, if any, influence upon import into hydrogenosomes. Although the necessary and sufficient signals for hydrogenosomal import recognition appear complex, targeting to the organelle is still highly specific, as demonstrated by the finding that six HA‐tagged glycolytic enzymes, highly expressed under the same promoter as other constructs studied here, localized exclusively to the cytosol and did not associate with hydrogenosomes.     

A bicistronic baculovirus vector for transient and stable protein expression in mammalian cells

Baculoviruses are widely used for protein production in insect cells, and their potential for gene transfer to mammalian cells is increasingly being recognized. Here we describe a baculovirus vector with a bicistronic mammalian expression cassette and demonstrate its suitability for efficient transient and stable protein expression in human glioblastoma cells. Bicistronic baculovirus vectors are safe, cost efficient, and easy to produce; thus, they represent an excellent gene transfer system for mammalian cells.