Novel Technique for Measuring Tissue Firmness within Tomato (Lycopersicon esculentum Mill.) Fruit

Developmental changes of tomato (Lycopersicon esculentum) fruit tissues during maturation were analyzed by a physically defined method (stress-relaxation analysis). The tip of a conical probe connected to a load sensor was positioned on the cut surface of a sliced tomato fruit, and the decay of the imposed stress was monitored. Stress-relaxation data thus obtained were used for the calculation of three stress-relaxation parameters. Different zones within tomato fruit harvested at six different ripening stages were analyzed. One of the stress-relaxation parameters, minimum stress-relaxation time (T₀), decreased as the fruits matured. The decrease in T₀ was first found in the core of the carpel junction within the endopericarp at the blossom end during the breaker stage. The decrease in T₀ progressed from the blossom end, through the equatorial region and finally throughout the shoulder, as the fruit matured. In mature green fruit, T₀ values within the placenta and the proximal carpel junction were lower than those by other parts of the fruit. For all measurements the maximum stress-relaxation time was not substantially changed during maturation, nor were their changes observed in different regions of the fruit. The observed relaxation rate was therefore correlated with softening. The results indicate that fruit softening may be physically associated with the stress-relaxation parameter, T₀, and the extent of softening is a function of position within the fruit. Decreases in T₀ value appear to be correlated with the reported regional variation in the appearance of polygalacturonase.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

Developmental Regulation of the (1,3)-beta-Glucan (Callose) Synthase from Tomato : Possible Role of Endogenous Phospholipases

Activity levels of UDP-glucose: (1,3)-β-glucan (callose) synthase in microsomal membranes of pericarp tissue from tomato fruit (Lycoperisicon esculentum Mill, cv Rutgers) were determined during development and ripening. Addition of the phospholipase inhibitors O-phosphorylcholine and glycerol-1-phosphate to homogenization buffers was necessary to preserve enzyme activity during homogenization and membrane isolation. Enzyme activity declined 90% from the immature green to the red ripe stage. The polypeptide composition of the membranes did not change significantly during ripening. The enzyme from immature fruit was inactivated by exogenously added phospholipases A₂, C, and D. These results suggest that the decline in callose synthase activity during ontogeny may be a secondary effect of endogenous lipase action.     

A 150 Kilodalton Cell Surface Protein Is Induced by Salt in the Halotolerant Green Alga Dunaliella salina

Dunaliella salina is an extremely halotolerant, unicellular, green alga lacking a rigid cell wall. Osmotic adaptation to high salinities is based on the accumulation of glycerol. To uncover other functions responsible for halotolerance, protein profiles of algae continuously grown in different salinities were compared. A 150 kilodalton protein (p 150) increased in amount with salt concentration. Furthermore, when the cells were subjected to drastic hyperosmotic shocks, p150 started to rise long after completion of the osmotic response but coincident with reinitiation of cell proliferation. Cells with an initially higher level of p150 resumed growth faster than cells with a lower level of the protein. Addition of cycloheximide early after hyperosmotic shock prevented the rise in p150, indicating this rise was due to de novo synthesis of the protein. These observations suggest that p150 is a saltinduced protein required for proliferation of the cells in saline media. p150 was purified to homogeneity and found to be a detergent-soluble glycoprotein. Polyclonal antibodies against p150 recognized a single protein component in D. salina crude extracts. A high M_r cross-reacting protein was also observed in another Dunaliella strain, D. bardawil. Immunoelectron microscopy localized p150 to the cell surface.     

Ultrastructural analysis of sperm flagella in two clitellates (Annelida), plasma membrane and periaxonemal area

The region between axonemes and plasma membrane in the sperm tails of the tubincid oligochaetes Tubifex tubifex and Monopylephorus limosus has been studied by means of thin sections of conventionally and tannic acid fixed material, and of freeze-fracture replicas. The main portion of the flagellum in both species showed prominent, regularly repeating bridges connecting doublets to plasma membrane. In correspondence to the doublets, characteristic double rows of intramembrane particles are present, with an arrangement reminiscent of the "zipper lines" described in other species. A well-developed cortical web with a honeycomb appearance underlies the plasma membrane. Glycogen granules are regularly arranged within the cells. An outstanding difference between the two species is to be found in the presence only in Monopylephorus of complex muff-like structures apparently formed by membrane particles and series of teeth embedded in the cortical web. Nothing similar has been found in Tubifex. This difference may be related to the fact that spermatozoa in Monopylephorus are not enclosed in spermatozeugmata as are those of Tubifex.     

The effects of recombinant bovine interferon-alpha on fertility in ewes

Recombinant bovine interferon-alpha(I)1 (rBolFN-alpha) may be useful for enhancing fertility in sheep because it has extensive sequence homology with ovine trophoblast protein-1. To test the effectiveness of rBolFN-alpha, several experiments were performed in which bred females were given intramuscular injections of rBolFN-alpha around the time of maintenance of the corpus luteum. Treatment with rBolFN-alpha enhanced the fertility of ewes that were bred via natural service or embryo transfer of whole or demi-embryos. Interferon treatment was successful in enhancing lambing rate if injections were given twice daily from Days 11 to 18, 12 to 14, 12 to 15 or 12 to 16. Overall, the lambing rate for ewes bred via natural service was 94/126 (74.6%) for control ewes and 101/126 (80.2%) for rBolFN-alpha treated ewes. Litter size was not affected by treatment. Interferon treatment was not successful in increasing the lambing rate if given as a single injection on Day 12 or as a series of once-daily injections from Days 11 to 16. These results demonstrate that rBolFN-alpha can increase the lambing rate in ewes.     

The use of the probit model to predict pregnancy status of buffalo based on physio-chemical properties of estrual mucus

Improper timing of artificial insemination with respect to ovulation is one of the major factors hampering the conception rate in buffalo. The present study was an attempt to relate physio-chemical changes in estrual mucus to subsequent pregnancy status in order to find their optimal values for determining the time for artificial insemination (AI). Serum estradiol, total protein and dry matter contents of estrual mucus were evaluated to predict the subsequent pregnancy in 36 buffalo during October 1988 to February 1989. Serum estradiol was determined by radioimmunoassay (RIA); spinnbarkeit, dry matter and total protein were determined by standard methods. Multivariate probit analyses were carried out to relate these variables to subsequent pregnancy status. Elasticity and protein concentration were significantly related to prediction probability of pregnancy status, and they predicted the pregnancy status 86% of the times correctly (P < 0.05). The probability of pregnant animals being correctly classified was 0.76, whereas the corresponding value for non-pregnant animals was 0.95. The present study demonstrated the possibility of using such a statistical model on mucus characteristics for determining proper AI time for better conception rates in Nili-Ravi water buffalo.     

Testicular development in Chinese Meishan boars

The developmental process of the testis and age-related changes in the morphology of rete testicular spermatozoa were investigated in Meishan boars at 1 to 364 days of age. Testicular weight and the diameter of seminiferous tubules increased rapidly until 150 to 180 days of age. Leptotene stage spermatocytes, round spermatids and spermatozoa were first found in the section of seminiferous tubules at 30 to 45, 60 and 75 days of age, respectively. However, after 105 to 120 days of age, most rete testicular spermatozoa were morphologically normal. These results indicate that Meishan boars reach puberty as early as 75 days of age, though the testes acquire the ability to produce morphologically normal spermatozoa at about 120 days.