Sequential development of flagellar defects in spermatids and epididymal spermatozoa of selenium-deficient rats

In this study cauda epididymal spermatozoa of rats maintained on a selenium-deficient diet for 5 and 7 months exhibited an array of flagellar defects. Spermatids and spermatozoa were analyzed by light and electron microscopy to define the appearance of flagellar abnormalities during spermiogenesis and post-testicular sperm development. Late spermatids of selenium-deficient rats displayed normal structural organization of the flagellar plasma membrane, axoneme, outer dense fibers, fibrous sheath and annulus, but they exhibited a premature termination of the mitochondrial sheath. A comparison of late spermatids and caput epididymal spermatozoa revealed that a late step in flagellar differentiation was the structural remodeling of the annulus and its accompanying fusion with both the fibrous sheath and the mitochondrial sheath. In selenium-deficient animals, however, the annulus failed to fuse with the mitochondrial sheath, generating an apparent weak point in the flagellum. After epididymal passage, cauda epididymal spermatozoa of selenium-deficient animals also exhibited extensive flagellar disorganization resulting from the apparent sliding and extrusion of specific outer dense fiber-doublet microtubule complexes from the proximal and the distal ends of the mitochondrial sheath and the accompanying loss of the midpiece plasma membrane. Only fiber complex number 4 was extruded proximally, whereas fibers 4, 5, 6 and 7 were extruded from the mitochondrial sheath-deficient posterior midpiece. Axonemal fibers 8, 9, 1, 2 and 3 retained their normal geometric relationships. These data suggest that the known loss of male fertility in selenium deficiency results from the sequential development of sperm defects expressed during both spermiogenesis and maturation in the epididymis.     

Spermatotoxic effect of aflatoxin B1 in rat: extrusion of outer dense fibres and associated axonemal microtubule doublets of sperm flagellum

Male Wistar rats were treated with aflatoxin B1 (AFB1). Live as well as methanol-fixed cauda epididymal spermatozoa were stained with acridine orange (AO) and ethidium bromide (EB) and observed under a fluorescence microscope. Giemsa-stained smears were observed in a bright field microscope. Unstained smears were observed with phase contrast illumination. The axoneme of more than 10% of the spermatozoa of treated rats had the outer dense fibres (ODFs), in varying numbers, and the associated axonemal microtubule doublets of the flagellum extruded either at midpiece-principal piece junction or connecting piece. This could be perceived in all light microscopic preparations, but AO-EB staining offered an advantage of the assessment of the viability as well. TEM observation of sections of the testis and cauda epididymidis also revealed ODF extrusion, as seen in the transverse sections of sperm flagella missing one or more ODFs and the associated axonemal microtubule doublets. In a few such sections, the extruded elements were seen in the cytoplasm, outside the mitochondrial sheath or peripheral sheath. Marginal to severe mitochondrial pathologies were observed in the spermatozoa and elongated spermatids, suggesting a link between AFB1-induced sperm mitochondrial pathology and extrusion of ODFs. However, the possibility that AFB1 treatment would disrupt the cytoskeletal proteins of the flagellum, resulting in the extrusion of ODFs, cannot be excluded. This sperm abnormality is reported for the first time as produced by a dietary toxin. Dietary aflatoxins, therefore, could also be contributory factors for the deterioration of the reproductive health of men.     

PAR-1 and the microtubule-associated proteins CLASP2 and dynactin-p50 have specific localisation on mouse meiotic and first mitotic spindles

The site of second meiotic division, marked by the second polar body, is an important reference point in the early mouse embryo. To study its formation, we look at the highly asymmetric meiotic divisions. For extrusion of the small polar bodies during meiosis, the spindles must be located cortically. The positioning of meiotic spindles is known to involve the actin cytoskeleton, but whether microtubules are also involved is not clear. In this study we investigated the patterns of localisation of microtubule regulatory proteins in mouse oocytes. PAR-1 is a member of the PAR (partitioning-defective) family with known roles in regulation of microtubule stability and spindle positioning in other model systems. Here we show its specific localisation on mouse meiotic and first mitotic spindles. In addition, the microtubule-associated proteins CLASP2 (a CLIP associating protein) and dynactin-p50 are found on kinetochores and a subset of microtubule-organising centres. Thus we show specific localisation of microtubule regulatory proteins in mouse oocytes, which could indicate roles in meiotic spindle organisation.     

Effects of calpain and Rho-kinase inhibitors on the acrosome reaction and motility of fowl spermatozoa in vitro

At the avian body temperature of 40 degrees C, intact fowl spermatozoa require Ca(2+) for the initiation of motility and a combination of both Ca(2+) and homogenized inner perivitelline layer (IPVL) together to induce the acrosome reaction. Within the range of 1-100 micromol/l, neither PD 150606 (a Ca(2+)-dependent calpain inhibitor) nor Y-27632 (an inhibitor of Ca(2+)-dependent Rho-kinase) were able to inhibit the acrosome reaction induced by the presence of Ca(2+) and IPVL. However, PD 150606, although not Y-27632, was able to inhibit sperm motility initiated by Ca(2+), as well as motility initiated by calyculin A -- a specific inhibitor of protein phosphatases, which also initiates sperm motility at 40 degrees C. The addition of PD 150606 did not reduce the ATP concentrations of intact spermatozoa, nor the motility of demembranated spermatozoa. Immunoblot analysis of sperm extract using a polyclonal antibody against calpain 12 revealed a cross-reacting protein of approximately 80 kDa. These results suggest that Rho-kinase is not involved in the regulation of the acrosome reaction or of motility in fowl spermatozoa. In contrast, calpain appears to be involved in the regulation of flagellar movement, but not izn that of the acrosome reaction. Furthermore, it seems that endogenous calpain is present in the cytoplasmic matrix and/or the plasma membrane, but not retained in the axoneme and/or accessory cytoskeletal components.     

Cytoplasmic microtubule organization in fission yeast

During the cell cycle of the fission yeast Schizosaccharomyces pombe, striking changes in the organization of the cytoplasmic microtubule cytoskeleton take place. These may serve as a model for understanding the different modes of microtubule organization that are often characteristic of differentiated higher eukaryotic cells. In the last few years, considerable progress has been made in our understanding of the organization and behaviour of fission yeast cytoplasmic microtubules, not only in the identification of the genes and proteins involved but also in the physiological analysis of function using fluorescently-tagged proteins in vivo. In this review we discuss the state of our knowledge in three areas: microtubule nucleation, regulation of microtubule dynamics and the organization and polarity of microtubule bundles. Advances in these areas provide a solid framework for a more detailed understanding of cytoplasmic microtubule organization.     

Hyperactivation of mammalian spermatozoa: function and regulation

Hyperactivation is a movement pattern observed in spermatozoa at the site and time of fertilization in mammals. It may be critical to the success of fertilization, because it enhances the ability of spermatozoa to detach from the wall of the oviduct, to move around in the labyrinthine lumen of the oviduct, to penetrate mucous substances and, finally, to penetrate the zona pellucida of the oocyte. The movement of hyperactivated spermatozoa appears different under different physical conditions and in different species, but basically it involves an increase in flagellar bend amplitude and beat asymmetry. Presumably, there is a signal or signals in the oviduct to initiate hyperactivation at the appropriate time; however, none has yet been identified. There is evidence that the source of the signal is follicular fluid, yet spermatozoa are known to hyperactivate before ovulation would release the fluid into the oviduct. Although the signal transduction cascade regulating hyperactivation remains to be described completely, it is clear that calcium ions interact with the axoneme of the flagellum to switch on hyperactivation. The process may also involve increases in intracellular cAMP, which at least is required to support motility in general. Although hyperactivation usually occurs during capacitation, the two events are regulated by different pathways.     

Cell cycle-dependent localization and possible roles of the small GTPase Ran in mouse oocyte maturation, fertilization and early cleavage

The small GTPase Ran controls numerous cellular processes of the mitotic cell cycle. In this experiment, we investigated the localization and possible roles of Ran during mouse oocyte meiotic maturation, fertilization and early cleavage by using confocal laser scanning microscopy, antibody microinjection and microtubule disturbance. The results showed that Ran was localized mainly in the nucleus (except for the nucleolus) in the oocyte, zygote and early embryo. At pro-metaphase of meiosis I, Ran distributed throughout the cell, but predominantly concentrated around the condensed chromosomes. During the completion of meiosis I and meiosis II, it concentrated to the meiotic spindle microtubules except for the midbody region. After sperm penetration, Ran dispersed with the extrusion of the second polar body and gradually concentrated in the male and female pronuclei thereafter. Ran was also observed to exist diffusely in the cytoplasm in prophase; it concentrated at the mitotic spindle, and migrated to the nucleus during early cleavage. Ran's concentration around the spindle disappeared when microtubule assembly was inhibited by colchicine, while it was concentrated around the chromosomes after microtubule stabilization with taxol treatment. Ran did not display any role in cytokinesis during division when pseudo-cleavage of germinal vesicle-intact oocytes was induced. Anti-Ran antibody microinjection decreased the germinal vesicle breakdown and the first polar body extrusion, and distorted spindle organization and chromosome alignment. Our results indicate that Ran has a cell cycle-dependent localization and may have regulatory roles in cell cycle progression and microtubule organization in mouse oocytes, fertilized eggs and early embryos.     

Requirement for an intact cytoskeleton for volume regulation in boar spermatozoa

Osmotically induced cell swelling triggers a chain of events leading to a net loss of major cell ions and water, resulting in cell volume recovery, a process known as regulatory volume decrease (RVD). In many cell types, there is an evidence that the cytoskeleton may play a role in the initial sensing and transduction of the signal of volume change. In this study, we tested the hypothesis that an intact microfilament and microtubule network is required for volume response and RVD in boar sperm before and after capacitation treatment and whether addition of cytochalasin D and colchicine to the capacitation medium would affect volumetric behaviour. Capacitation is a series of cellular and molecular alterations that enable the spermatozoon to fertilize an oocyte. Cell volume measurements of washed sperm suspensions were performed electronically in Hepes-buffered saline solutions of 300 and 180 mosmol/kg. After exposure to hypoosmotic conditions, boar sperm showed initial swelling (up to 150% of initial volume within 5 min), which was subsequently partially reversed (to about 120-130% after 20 min). Treatment with cytochalasin D led to reduced initial swelling (1 micromol/l) and loss of RVD in washed sperm (1-10 micromol/l) and at the beginning of incubation under capacitating conditions (5 micromol/l). Short treatment with 500 micromol/l colchicine affected the volume regulatory ability in sperm under capacitating conditions but not in washed sperm. No significant differences in cell volume response were observed after subsequent addition of cytochalasin D and colchicine to the suspensions of sperm incubated for 3 h under capacitating conditions. However, the incubation under capacitating conditions in the presence of cytochalasin D led to improved volume regulation at the end of the incubation period (23%). The microfilament network appears to be important for volume regulation in washed boar spermatozoa while intact microtubules do not seem to be necessary for osmotically induced RVD. The changes in cytoskeleton microfilament organization during capacitation, possibly affecting the osmotically induced volume response, appear to occur at the later stages of capacitation, whereas changes in microtubules, related to volume regulatory ability, may be programmed within the first stages of capacitation.     

Texture and rehydration properties of texturised soy protein: analysis based on soybean 7S and 11S proteins

Soy protein, one of the most commonly used raw materials for texturised vegetable protein, has an important influence on texturised soy protein (TSP) with its 7S and 11S fractions. In this study, soy 7S and 11S proteins were extracted from soybean isolate and added back to the raw material to prepare TSP and analyse the effect of both on the physical properties of TSP. The results showed that the addition of 5% soy 7s or 11s protein increased the water-holding capacity (up to 9.04%) and rehydration rate (up to 25.71%) of TSP. Compared with adding soy 11s protein, adding soy 7s protein has a faster rehydration rate at a lower temperature (30 and 45 °C). After extrusion, the content of free sulphhydryl groups, total sulphhydryl groups, and disulphide bonds was significantly reduced (P < 0.05). The extrusion treatment caused degradation of the protein chains, and the proteins mainly formed insoluble polymers. Electrophoretic analysis revealed that the sodium dodecyl-sulphate (SDS) reducing the extractable rate of the precipitate after SDS non-reduction extraction of the TSP added with 5% soy 7S and 11S proteins were lower than that of the control. The proportion of different soybean protein components in TSP could change its texture, water-holding, and rehydration characteristics of it, which provides a new method for the characteristics design of TSP.     

挤压组织化对芝麻蛋白理化性质及结构的影响

以亚临界芝麻蛋白粉为主要原料,利用双螺杆挤压技术研究挤压组织化处理对蛋白原料理化性质及结构的影响,探讨组织化蛋白形成机理。结果表明,挤压组织化对蛋白原料理化性质及结构都造成了一定的影响。挤压后蛋白原料持水性增加了14.16%,吸油性减少了14.52%,蛋白消化率增加了11.18%,游离巯基含量减少了11.55%,氨基酸各组分含量均有一定损失,挤压组织化过程中没有新的肽键生成,有大量的二硫键生成,挤压得到的组织化蛋白结构主要是由二硫键和非共价键共同作用维持。挤压组织化并未完全破坏蛋白质二级结构,只有部分不稳定的α-螺旋和无规则卷曲向相对稳定的β-折叠和β-转角转化,蛋白原料由无序变为有序,并产生了大量的纤维结构,本文为后续深入开展组织化蛋白形成机理研究、开发组织化芝麻蛋白产品提供理论基础。     

Role of Tea1p, Tea3p and Pom1p in the determination of cell ends in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells are rod-shaped and grow along a single axis from their two ends. Microtubules extend from the cell centre terminating at the cell ends. The ERM(ezrin/radixin/moesin)-like proteins Tea1p and Tea3p, and the Dyrk-like kinase Pom1p are cell end markers involved in the regulation of growth and microtubular dynamics at the cell ends. We have analysed the relative contribution of these three proteins to the determination of cell ends as sites both for cell growth and for microtubular termination. Pom1Delta, in combination with Tea1Delta or Tea3Delta, has the greatest difficulty in relocalizing actin to the cell ends following actin depolymerization and generates the most defective growth pattern. Tea1Delta, in combination with Pom1Delta or Tea3Delta, displays the highest number of microtubules bending round the cell ends. Tea1DeltaPom1Delta, which has the most defective growth pattern and microtubules, also displays the highest number of branched cells. We show that Tea1p, Tea3p and Pom1p all contribute, to different extents, to the determination of cell ends, as sites for both cell growth and microtubular termination. We also show that the fission yeast cell relies on both the positioning of landmarks and a properly organized microtubule cytoskeleton to direct cell growth.     

Extrusion of food proteins.

Protein extrusion has frustrated earlier predictions regarding its impact in the development of food products. The main reason for this disappointing performance has been its failure to yield fabricated food products with textural quality close enough to that of natural products at competitive prices. Texturized soya protein by extrusion is presently the only commercial success in this area, being incorporated into several convenience products, increasing their protein content and quality and conferring them some desirable sensory properties. Technological and scientific gaps in the extrusion texturization are still to be bridged if this technique is to be applied for upgrading unconventional protein. The precise mechanisms responsible for protein texturization through extrusion are still unclear. Proteins show a very wide range of extrusion behavior that is probably related to large differences in their association properties. New peptide bonds, formed by free amino and carboxylic groups of the protein, were postulated as being responsible for the cross-linking that takes place in protein extrusion. However, disulfide bonds and electrostatic and hydrophobic interactions are regarded presently as the texturization mechanism in this process. The recently suggested suspension (or filled "melt") model for biopolymer extrusion offered a new framework for testing extrusion of novel proteins. According to this view, the large differences between the association properties of proteins produce different types of aggregates. Some of them can be insoluble under extrusion conditions and act as a dispersed phase within the melt phase. The extrusion performance of a protein will thus depend on the amount of insoluble aggregate produced inside the extruder and on protein-protein interactions that occur after the superheated molten mass leaves it.     

Angiogenesis and vascular endothelial growth factor expression in the equine corpus luteum

The hydrodynamic basis for the accumulation of spermatozoa at surfaces has been investigated. The general conclusion is that when spermatozoa arrive at a surface, they will remain there if the vector of the time-averaged thrust is directed towards that surface. This can arise in two basic ways. First, consider spermatozoa that maintain a three-dimensional waveform and roll (spin) as they progress: in this case, it is argued that the conical (rather than cylindrical) shape of the flagellar envelope will establish the direction-of-thrust necessary for capture by the surface. (Additional findings, for spermatozoa of this type, are that the swim-trajectory is curved and that the direction of its curvature reveals the roll-direction of the cell.) Second, consider spermatozoa that maintain a strictly two-dimensional waveform at the surface: in this case, spermatozoa can be captured because the plane-of-flattening of the sperm head is tilted slightly relative to the plane of the flagellar beat. The sperm head is acting as a hydrofoil and, in one orientation only, it comes to exert a pressure against the surface. (This pressure may possibly, in vivo, aid the penetration of the zona pellucida.) The hydrofoil action of sperm heads may explain any bias in the circling direction of spermatozoa that execute two-dimensional waves at surfaces. Finally, a more complex phenomenon is where interaction of the spermatozoa with the surface appears to induce a three-dimensional to two-dimensional conversion of the flagellar wave (thus permitting the hydrofoil effect described). This is characteristic of sperm with 'twisted planar' rather than helical waves. In mammalian spermatozoa, approximately half the beat cycle is planar and the other half generates a pattern of torque causing the head to roll clockwise (seen from ahead), producing a torsion of the neck region of the flagellum. It is the gradual suppression of this torsion, by either impedance at the solid boundary or by raised viscosity, that converts the 'twisted planar' shape into a planar wave.     

Proteolysis of Sorghum Endosperm Proteins when Mashing with Raw Grain Plus Exogenous Protease and Potassium Metabisulphite

With the aim of improving free amino nitrogen (FAN) production when mashing with raw sorghum grain and exogenous enzymes, the effect of mashing with the addition of the reducing agent potassium metabisulphite (KMS) on the sorghum endosperm proteins was studied. When mashing was conducted at low temperature (40°C) over an extended period (7 h) with 0.1% KMS (sorghum basis) in addition to exogenous protease, FAN increased by approx. 6 fold to approx. 91 mg/100 g sorghum, as opposed to 5 fold to approx. 75 mg/100 g sorghum with the exogenous enzyme only. Confocal laser scanning microscopy revealed that the exogenous protease caused the endosperm protein matrix that surrounds the starch granules to break up on cooking. Transmission electron microscopy showed that the exogenous protease predominantly hydrolysed the glutelin matrix protein surrounding the kafirin protein bodies. In the presence of KMS there was also substantial breakdown of the protein bodies. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis indicated that KMS had the effect of reducing kafirin polymers and oligomers into monomers. It appears that the addition of KMS in a sorghum grain mashing system significantly improves the rate of sorghum protein hydrolysis because of the reduction of intermolecular disulphide bonds in the kafirin protein, which allows better access of the protease, resulting in improved FAN production.     

Protein-Protein Interactions in the Extrusion of Soya at Various Temperatures and Moisture Contents

Soya protein isolate extruded at various temperatures and moisture contents was solubilized before and after processing in buffers containing urea, sodium disulphite, 2-mercaptoethanol, sodium dodecyl sulphate, alone or in combination to elucidate stabilizing mechanisms for their three dimensional structure. The main mechanisms were disulphide linkages, hydrophobic and electrostatic interactions. All extrudates resisted retorting in water at 120°C but complete disruption occurred in media containing reducing agents plus sodium dodecyl sulphate. Little change in amino acid occurred in extrusion except for aspartic and glutamic acids, which decreased in extrusion at 140°C. Infrared spectra showed the presence of β-sheet anti parallel structures. Peptide bonds were of negligible importance in extrusion texturization of soya protein. If formed they only contributed to an insoluble phase in the melt.     

Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 degrees C. In the presence of 2 mmol CaCl(2)/l at 40 degrees C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca(2+), motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 degrees C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10-1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca(2+). These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca(2+), was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca(2+) and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca(2+) plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.     

Isolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula)

The flagellum of a mammalian spermatozoon consists of a central axoneme surrounded by two cytoskeletal structures, the outer dense fibres and the fibrous sheath, which may aid in sperm motility or stability. In this study the outer dense fibres and fibrous sheath were isolated and partially characterized in a marsupial species, the brushtail possum (Trichosurus vulpecula). Spermatozoa from the cauda epididymidis were decapitated by sonication, and the head and tail fractions were separated by centrifugation over a 20, 40 and 60% (w/v) sucrose density gradient. After confirming sperm tail purity by Nomarski microscopy, the tails were incubated in either SDS-dithiothreitol to isolate the outer dense fibres or urea-dithiothreitol to isolate the fibrous sheaths. Purified outer dense fibres and fibrous sheaths were solubilized in SDS and beta-mercaptoethanol and proteins were separated by one-dimensional PAGE. Coomassie blue staining showed that the outer dense fibres were composed of seven major proteins (molecular masses: 73, 58, 55, 54, 52, 41 and 16 kDa), and the fibrous sheath was composed of 12 major proteins (molecular masses: 106, 76, 66, 62, 55, 53, 52, 46, 40, 30, 28 and 16 kDa). A polyclonal antibody to the fibrous sheath proteins showed strong crossreactivity with those of fibrous sheath from spermatozoa of several other marsupial species, as well as those from laboratory rats. Subsequent western blotting identified the immunoreactive 76 and 62 kDa proteins from all species, thus indicating their high conservation between species. No crossreactivity of the fibrous sheath antibody to any other cytoskeletal structures, including the outer dense fibres, mid-piece fibre network or connecting laminae, or to the acrosome or underlying subacrosomal material, was evident, indicating that the fibrous sheath proteins are localized to this structure alone. Further work is in progress to determine the extent of homology of these proteins to those in eutherian mammals.     

Gel-Forming Characteristics of Milk Proteins, 2. Roles of Sulfhydryl Groups and Disulfide Bonds

The gel-forming characteristics of milk proteins were investigated by employing skim milk either nonpreheated or preheated at 80°C, and coagulating them at 70 or 80°C with glucono-delta-lactone. The solubility of each gel in the phosphate buffer (pH 7.0) containing urea and 2- mercaptoethanol was examined. The disulfide bonds played a more important role in the gel coagulated at 80°C from skim milk preheated at 80°C than in the gel coagulated at 70°C from nonpreheated skim milk.The effect of the reduction treatment with 2-mercaptoethanol was more pronounced on preheated skim milk than on nonpreheated skim milk. Sulfhydryl groups and disulfide bonds, which were buried in the molecules of whey proteins in their native state, were rendered accessible following heat treatment at 80°C.The gel prepared from skim milk pretreated with oxidizing agent, hydrogen peroxide (i.e., skim milk has no accessible sulfhydryl groups as a result), or the gel prepared from skim milk pretreated with reducing agent, 2-mercaptoethanol, (i.e., skim milk has few disulfide bonds as a result), displayed weak gel formation. But the gel prepared from the mixture of these skim milks with appropriate ratio displayed higher gel firmness. These findings suggest that intermolecular disulfide bonds are formed by the exchange reaction between sulfhydryl groups and disulfide bonds during gel formation.     

The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse

There is some evidence suggesting that Ca2+ is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+ takes part in its regulation. However, the precise roles of Ca2+ in spermatogenesis remain to be elucidated. Calpains are a family of Ca(2+)-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca(2+)-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain's ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.     

非热加工技术消减食物过敏原研究进展

主要介绍了低温等离子体技术、高压处理、超声波处理、辐照处理等非热物理加工技术及其优缺点,这些技术均能够通过暴露或掩盖食物中过敏原的抗原表位,改变过敏原的二级结构,破坏维持三、四级结构的非共价键如氢键、疏水相互作用等方式来改变致敏蛋白的空间结构,从而消减其致敏性。