Radioiodination and characterization of the plasma membrane of sea urchin sperm

Two methods were used to radioiodinate sea urchin sperm: lactoperoxidase-glucose oxidase and Iodo-Gen. Following iodination the sperm are viable, they undergo the acrosome reaction, and they fertilize eggs. Of the radioactivity associated with the labeled sperm, 28–50% is presumed to be free ¹²⁵I^?, 37–47% is incorporated in lipid, and 8–15% is in trypsin-digestible material believed to be protein. Digestion of the labeled, living sperm with trypsin removes 95.6–99.5% of the macromolecular label (the cells are alive after digestion) suggesting that almost all the protein label is on the external surface of the cell. Thin-layer chromatography of the lipid fraction shows that the major membrane phospholipids and cholesterol are labeled. SDS-PAGE analysis shows the protein-incorporated ¹²⁵I is distributed among four glycoproteins of >250K, 84K, 64K, and 52K dalton apparent molecular weight. Twenty-eight percent of the total protein (trypsin-digestible) label is in the 84K component and 46% in the 64K band. Although both molecules contain much of the label, they are relatively minor components of the TX-100 extract of sperm. The methods outlined will be useful in determining the role of sperm surface components in fertilization.     

Intrinsic vs. extrinsic influences on life history expression: metabolism and parentally induced temperature influences on embryo development rate

Intrinsic processes are assumed to underlie life history expression and trade‐offs, but extrinsic inputs are theorised to shift trait expression and mask trade‐offs within species. Here, we explore application of this theory across species. We do this based on parentally induced embryo temperature as an extrinsic input, and mass‐specific embryo metabolism as an intrinsic process, underlying embryonic development rate. We found that embryonic metabolism followed intrinsic allometry rules among 49 songbird species from temperate and tropical sites. Extrinsic inputs via parentally induced temperatures explained the majority of variation in development rates and masked a relationship with metabolism; metabolism explained a minor proportion of the variation in development rates among species, and only after accounting for temperature effects. We discuss evidence that temperature further obscures the expected interspecific trade‐off between development rate and offspring quality. These results demonstrate the importance of considering extrinsic inputs to trait expression and trade‐offs across species.     

Frost resistance of winter rape leaves as related to the changes in water potential and growth capability

Differential thermal analysis indicated that the frost resistance of winter rape leaves ( Brassica napus L. var. oleifera L. cv. Gòrczanski), collected from plants grown in the cold (5/2°C), relies mainly on their ability to supercool to −9 to −11°C, i.e. consists in freezing avoidance. Initiation of ice formation in the cold-acclimated leaves resulted in the death of more than 50% of the cells as determined with a conductivity method. The development of freezing tolerance appeared to be an attribute of the second stage of plant hardening and was induced by the exposure of plants to a slightly subzero temperature (−5°C) for 18 h. Such a treatment brought about a sudden and persistent water potential decrease in the leaves, despite the fact that they had reabsorbed water from the medium prior to water potential measurements. Water potential changes were associated with a higher growth capability of the leaves as checked by determinations of disk area increments. It is suggested that the increased frost tolerance of the cold-grown winter rape leaves, subjected to subfreezing temperature, is related to the decreased water potential of the tissue caused by changes in turgor and/or in osmotic pressures of the cells.     

Effects of cold on CO2 exchange in winter rape leaves

A viable wheat hybrid intermediate of the same height as the parents was obtained by crossing the female parent of tall variety NP4 with the male parent of the dwarf variety HD2160. Seeds of the hybrid and its parents were germinated and their growth pattern as well as the activities of peroxidase, indolyl-3-acetic acid oxidase and amylase in extracts made from them were studied at the early seedling stages i.e. up to 96 h.
A positive correlation existed between the length of the axis at the early seedling stage and at mature plant height as far as the parental varieties are concerned but no such correlation was observed with the hybrid. Growth of the hybrid seedlings was less than of its parents. Light appeared to stimulate the longitudinal growth of the axis to different extents in the parents and hybrid. Higher activities of peroxidase, indolyl-3-acetic acid oxidase and amylase were observed in the hybrid as compared to both of its parents. Lethal wheat hybrid also exhibits increased activities of amylase, indolyl-3-acetic acid oxidase and peroxidase. Therefore, it appears that seedling growth and enzyme activities at the seedling stage are not always correlated with hybrid vigour.     

Effects of Phosfon-D and of low temperature on the morphology of cell protoplasts

Summary The adventitious roots of onion (Allium cepa L.) or onion bulb scales were treated either with growth retardant Phosfon-D (2,4-dichlorobenzyltributylphosfonium chloride), or with low temperature (5° C). Low temperature, likewise Phosfon-D treatment, resulted in the appearance of membranous structures within the cytoplasm of the root cells fixed in chromaceto-formalin (CrAF) and in the occurrence of minute vacuoles in the living onion bulb scale cells. The cytochemical analysis revealed the membrane-like elements to contain basic and acid proteins as well as nucleic acids.It seems that the observed effect of Phosfon-D and that of low temperature were due to the local accumulation of protein resulting in changes of the physical properties of the cytoplasm. The fact that membranous structures do contain nucleic acid may suggest their important, metabolic role especially in the process of cold acclimation of plant tissue.This work was partly supported by Grant No. FG-PO-225 from U.S. Department of Agriculture, Agricultural Research Program, Public Law 480.     

Histochemische Untersuchungen über den Involutionsmechanismus der Milchdrüse

Zusammenfassung Es wurden mit histochemischen Methoden Milchdrüsen von Meerschweinchen in physiologischer und durch Kastration während der Laktationsperiode hervorgerufener Involution untersucht. Nachgewiesen wurden alkalische und saure Phosphatase (APh und SPh), unspezifische Esterase, Sukzinodehydrase, SH-Gruppen, Nukleinsäuren und Lipide. — Die Aktivität der SPh nimmt während der Involution zu. Die Bedeutung dieses Enzyms für den Rückbildungsprozeß des Drüsengewebes wird diskutiert. — Das Vorkommen der APh-Aktivität in Drüsenendstücken im Involutionsstadium kann auf die Beteiligung dieses Enzyms an der Rückresorption der mit der Milch ausgeschiedenen Substanzen hinweisen. — Zwischen der Aktivität der unspezifischen Esterase und dem Gehalt sowie der Lokalisation von Lipiden besteht eine Abhängigkeit. — Es konnten keine Unterschiede in der Sukzinodehydraseaktivität und dem DNS-Gehalt aufgezeigt werden. — Die Verminderung von SH-Gruppen und RNS hängt mit dem Aufhören der Sekretionsproduktion durch die Zellen der Drüsenendstücke der Milchdrüsen zusammen.

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Histochemical studies on the involution mechanism of the mammary gland

Summary Histochemical methods were used to study mammary glands of guinea pigs in the course of physiologic involution and that induced by castration during lactation. Alkaline and acid phosphatases, unspecific esterase, succinic dehydrogenase, SH groups, nucleic acids and lipids were determined. Acid phosphatase activity was found to be increased in mammary glands, subject to involution. The participation of the enzyme in the involutionary process of the gland tissue is discussed. The distribution of alkaline phosphatase in the secretory sections of the gland during involution would suggest the participation of the enzyme in the reabsorption of the substance secreted with milk. A correlation existing between the activity of unspecific esterase, the level and distribution of lipids in the mammary gland could be established. No differences were detected in the activity of succinic dehydrogenase and DNA level. A decrease in SH groups and RNA content is related to cessation of milk secretion.

     

Freezing pattern in apple seeds as affected by the temperature of fruit storage

Storage of apple fruits ( Pyrus malus L. cv. Golden Delicious) at different temperatures (0, 12 and 35°C) markedly altered the pattern of water freezing in the seeds. Higher temperatures of storage brought about a shift from the sequential to the discontinuous type of freezing in seeds, the low temperature exotherm (LTE) being much more pronounced than the high temperature one (HTE). The occurrence of LTE was highly correlated with embryo death. Fruit storage at higher temperatures also caused a decrease in the threshold super cooling temperature of seeds and of naked embryos, removed from the seed coat and endosperm. The decrease was less pronounced in naked embryos than in intact seeds. The results presented show that the frost resistance of apple seeds relies mainly on the supercooling ability of the embryos and is increased by the presence of seed coat and endosperm. The broad peak of the LTE indicates that, in contrast with other seeds and many super cooling organs, massive ice nucleation in apple seeds occurs within the embryo tissues and that extra organ freezing seems to be of less importance. Therefore, the increased super cooling ability of apple seeds, isolated from fruits stored at higher temperatures, seems to rely on those seed properties that protect embryo cells against heterogeneous ice nucleation.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.