Ribosomal protein L29/HIP deficiency delays osteogenesis and increases fragility of adult bone in mice

Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29‐null mice is due to delayed entry of chondro‐osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro‐computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender‐specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender‐matched controls in three‐point bending tests, and smaller post‐yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29‐deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub‐efficient protein translation increased fracture risk in HIP/RPL29‐null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:28–35, 2009     

核糖体蛋白L26基因的克隆及其在肝癌中的表达

目的 克隆和分析与肝细胞癌发生发展相关的基因。方法 应用差异显示技术获得24例肝癌及其癌旁组织表达差异的目标基因片段,对其进行克隆、测序,并在基因数据库中检索进行同源性比较,应用印迹法（Northern)杂交技术研究其正常组织分布和肝癌中表达的变化。结果 肝癌中获得一目标基因片段经测序证明其来源于一已知基因核糖体蛋白L26（RPL26）,Northern杂交分析表明该基因在各种组织中广泛表达,83.3%原发性肝癌患者癌组织中RPL26表达显著高于癌旁组织。结论 RLP26可能是肝癌进展的有用的生物学标志。     

ERp29蛋白在BALB／c小鼠附睾头部和尾部精子中的鉴定和定位

目的 对在BALB/c小鼠附睾精子中内质网蛋白29(endoplasmic reticulum protein 29,ERp29)进行鉴定以及定位.方法 应用免疫印迹方法鉴定BALB/c小鼠附睾头、尾部精子ERp29蛋白,同时利用激光共聚焦显微镜和间接免疫荧光定位的方法研究ERp29在附睾头、尾部精子上的定位.结果 证实ERp29蛋白存在于BALB/c小鼠附睾头部和尾部精子中,且其在尾部精子中含量较头部明显增高.ERp29蛋白主要定位于BALB/c小鼠附睾头部精子的头部前端,而在附睾尾部精子则主要定位于头部和尾部主段.结论 通过对ERp29蛋白在BALB/c小鼠附睾头部和尾部精子上的鉴定和定位,可以帮助进一步研究该蛋白对小鼠精子的作用.     

Ultrastructural features and pathogenesis of decapitated spermatozoa in a boar

Summary. A boar with decapitated spermatozoa was examined. The growth rate and libido were normal. The right testis was in the scrotum, but the left one was intra-abdominal. Ejaculated spermatozoa and tissue specimen from both testes were observed by light and electron microscopy. Only tailless heads and headless tails of spermatozoa were observed in the ejaculate. The ratio of heads to tails was about 1:4. Motility of the headless tails was about 25%. The proximal extremity in the tail was occupied by the proximal centriole. The basal plate could not be found in the head or in the tail. Pathogenesis of decapitated spermatozoa in the present case was analysed by investigating spermiogenesis in the scrotal testis. Since the pair of centrioles failed to approach the nucleus, mechanical connection between the proximal centriole and the nucleus did not seem to be established. In addition, the basal plate was not formed on the nuclear membrane. Since the ratio of tailless heads to headless tails was also 1:4 in the testis, it was concluded that the heads had already detached the tails in the testis.     

Capacitation-associated changes in protein tyrosine phosphorylation, hyperactivation and acrosome reaction in hamster spermatozoa

The aim of this study was to evaluate the effects of Ca2+, BSA, NaHCO3 and PVA on the capacitation-associated time-dependent increase in protein tyrosine phosphorylation, hyperactivation, and acrosome reaction in hamster spermatozoa. Hamster spermatozoa when incubated in TALP, a medium that assists capacitation, showed a time-dependent increase in protein tyrosine phosphorylation that correlated with the capacitated state of the spermatozoa. An absence of Ca2+ or NaHCO3 in the capacitation medium delayed the phosphorylation of the proteins, but without both there was a significant decrease in the phosphorylation of the proteins throughout the period of capacitation. An absence of bovine serum albumin also caused a decrease in the phosphorylation of the proteins but this did not occur if polyvinyl alcohol was substituted for it in the medium. The percentage hyperactivation was not affected in the absence of bovine serum albumin if the medium contained polyvinyl alcohol. However, it was delayed in the absence of NaHCO3 and inhibited in the absence of Ca2+. The absence of NaHCO3 or bovine serum albumin had no effect on the acrosome reaction. These results show that hamster spermatozoa undergo capacitation-associated protein tyrosine phosphorylation similar to that of the spermatozoa of other mammals. However, hamster spermatozoa are unique in that the capacitation-associated protein tyrosine phosphorylation is not absolutely dependent on the presence of Ca2+ and NaHCO3. As far as we know, this study is the first to provide evidence that capacitation-associated protein tyrosine phosphorylation is linked to hyperactivation in hamster spermatozoa.     

Insulin resistance and lipodystrophy in mice lacking ribosomal S6 kinase 2

The p90 ribosomal S6 kinase 2 (RSK2) is a serine/threonine kinase with high expression levels in adipose tissue. Numerous in vitro studies show that RSK2 is activated by a broad number of cellular stimuli and suggest that RSK2 is involved in the regulation of a variety of cellular processes. However, the physiological role of RSK2 still remains elusive. We therefore generated rsk2 knockout (KO) mice to better understand the function of RSK2 in vivo. Birth weights of RSK2 KO mice are normal, but the body weight is reduced with age, as compared with wild-type littermates. We found that the difference in body weight was largely caused by a specific loss of white adipose tissue that is accompanied by reduced serum levels of the adipocyte-derived peptide, leptin. KO mice also have impaired glucose tolerance and elevated fasting insulin and glucose levels that are restored following administration of low amounts of leptin, which do not affect food intake. We conclude that RSK2 plays a novel and an important role in regulation of adipose mass in mice and speculate that the reduction in fat tissue may negatively affect insulin sensitivity, as observed in human lipodystrophy, through reduced levels of adipocyte-derived factors, such as leptin.     

GM1 dynamics as a marker for membrane changes associated with the process of capacitation in murine and bovine spermatozoa

We previously showed that in live murine and bovine sperm heads, the ganglioside G(M1) localizes to the sterol-rich plasma membrane overlying the acrosome (APM). Labeling G(M1) using the pentameric cholera toxin subunit B (CTB) induced a dramatic redistribution of signal from the APM to the sterol-poor postacrosomal plasma membrane (PAPM) upon sperm death. We now show a similar phenomenon in the flagellum where CTB induces G(M1) redistribution to sterol-poor membrane subdomains of the annulus and flagellar zipper. Because sterol efflux from the plasma membrane is required for capacitation, we examined whether G(M1) localization might be useful to detect membrane changes associated with capacitation and/or acrosomal exocytosis. First, incubation of murine and bovine sperm with their respective stimuli for capacitation did not change G(M1) distribution in live cells. However, incubation of sperm of both species with specific stimuli for capacitation, followed by the use of specific fixation conditions, induced reproducible, stimulus-specific patterns of G(M1) distribution. By assessing changes in G(M1) distribution in response to progesterone-induced AE, we show that these patterns reflect the response of murine sperm populations to capacitating stimuli. These data suggest that G(M1) localization can be used as a diagnostic tool for evaluating sperm response to stimuli for capacitation and/or AE. Such information could be useful when deciding between technologies of assisted reproduction or when screening for male fertility. Furthermore, stimulus-specific changes in G(M1) distribution showed that sperm could respond to NaHCO(3) or mediators of sterol efflux independently, thereby refining existing models of capacitation.     

Endogenous protein carboxyl methylation in hamster spermatozoa: changes associated with capacitation in vitro

Protein carboxyl methylase (PCM) and its substrate(s), methyl acceptor protein(s) (MAP), are present in the spermatozoa of rat, rabbit and man. In the present study, PCM activity and MAP capacity were measured in homogenates of hamster testes and isolated spermatozoa, and found to be similar to those of other species. Using solubilized preparations of spermatozoa from the cauda epididymis of hamster, PCM activity was twice as high in sperm tails as in heads while the reverse was true for MAP capacity. Since in this and in previous studies this enzyme reaction has been measured in broken cells, we thought it pertinent to measure methylation under physiological conditions (i.e., in motile spermatozoa). To accomplish this, an assay was developed which depends upon the conversion of [3H-methyl]methionine to S-adenoxyl-L [methyl-3H]methionine which, in turn, serves as a methyl donor for PCM. In sperm that had been labelled with [3H]methionine the MAP for the endogenous methylation reaction was not solubilized with Triton X-100, and was found primarily in sperm tails. When hamster spermatozoa were incubated in medium containing taurine, epinephrine and bovine serum albumin to induce capacitation, endogenous methylation was stimulated 8- to 9-fold; when taurine was omitted from this medium, methylation was stimulated 14-fold. Taurine, however, was essential for in vitro fertilization as the number of eggs fertilized declined from 92% in complete medium to 0% in medium minus taurine. The decrease in fertilization rate was also associated with a decrease in sperm motility. In previous studies we have demonstrated an association between PCM and sperm motility, and have suggested that this enzyme system could be involved in other functions such as the acrosome reaction. From the results in the present study we conclude that the conditions that lead to capacitation in vitro are associated with a marked change in endogenous protein carboxyl methylation.     

【原创论文】小鼠杀菌渗透增强性蛋白起源于睾丸及附睾表达并定位于精子中

杀菌渗透增强性蛋白（BPI）是具有抗革兰氏阴性菌活性的内源性杀菌蛋白。在本研究中,我们通过自行制备的多克隆抗体,检测了BPI蛋白在小鼠出生后睾丸及附睾组织中的表达,以及在附睾精子头部的亚细胞定位。实验结果表明,睾丸和附睾均独立表达BPI基因。在附睾中,自起始段至尾部,BPI蛋白的表达水平递减,并逐步特异性富集于亮细胞的胞质中。在顶体反应前的顶体基质内可见BPI蛋白,应起源于睾丸表达;顶体反应后,可见BPI蛋白分布于整个精子头部质膜表面,尤其是赤道板区域,可能有睾丸或附睾表达的两种起源。我们的研究结果提示,BPI蛋白可能参与顶体反应前后精子质膜结构的调控,并参与后续的精卵融合过程。     

Ultrastructural study of anomalies of the acrosome in spermatozoa with irregular heads]

The study of the ultrastructure of spermatozoa having an irregular head, encountered in large number in semen of men supposed to be sterile, shows that the acrosome is often modified in its shape or its texture. In several samples the spermatozoa have either no acrosome or a small one, at a certain distance from the nucleus. In other cases the ratio between the segments of the acrosome is modified and the parallelism between the edges is not conserved. These acrosomes are often covered with a cytoplasmic velum the post acrosomal cape is missing. Finally the acrosome can be deformed by local expansions of the subacrosomal space, or by large gaps. Other anomalies which are less common have been observed. The elimination of possible artefacts and the comparison between these human anomalies and similar acrosomal anomalies of mammalian semen, are discussed in this paper.     

Ultrastructural changes in the hepatocytes following massive hepatectomy in rats

This study was carried out to investigate the ultrastructural changes of the hepatocytes of the remnant liver after 70% hepatectomy in rats. The remnant liver regained the preoperative weight one week after hepatectomy. During this early posthepatectomy period, the volume of the each hepatocytes increased to a peak 48 hours after hepatectomy, then returned to normal within one week. Electronmicroscopically mitochondria of the hepatocytes became swollen and the cristae were shortened until 3 hours after hepatectomy. Dividing mitochondria were also observed in the initial 2 hours. The volume density of the mitochondria measured as a percentage of the mitochondrial volume per the cytoplasmic volume of hepatocytes. It increased and reached a peak 3 hours after hepatectomy (p less than 0.05) and thereafter returned to the same value as the control within 6 hours. Then it gradually decreased until 7th day (p less than 0.001). The volume density of lipid droplets increased to a peak 48 hours after hepatectomy (p less than 0.01). These significant ultrastructural changes of the hepatocytes indicate an important roles of mitochondria especially in the energy metabolism in the early posthepatectomy period.     

Ultrastructural changes in the nerve elements in Crohn's disease.

Electron microscopic investigations were carried out to study the nerve elements in the wall of the small intestine in Crohn's disease, comparing it with the control. In the ileum of Crohn's disease only a few synapses were found. The number of nerve terminals were decreased, as well as that of the vesicle population in the remaining nerve terminals. Some of the nerve processes were observed in degeneration. The number of the lysosomes in the nerve cell bodies increased. Inflammatory cells as lymphocytes, plasma cells, mast cells were noted in the tola submucosa and in the mucous membrane, their number was also increased. It is suggested that the immunological effector cells and their products could be responsible for changing the neuronal elements.     

Foraminal changes with distraction and compression of the L4/5 and L5/S1 segments

The width of the foramen in the lumbar spine is directly related to the position of the vertebrae. In an MRI study the measurements of the cross-sectional area of the neuroforamen of L4/5 and L5/S1 in neutral position, segmental distraction and compression were calculated. Nine cadaver specimens were investigated and the foraminal width of L4/5 and L5/S1 was measured. In both segments of all specimens the foraminal space significantly enlarged under distraction and decreased under compression. In the L4/5 segment the average relative difference between distraction and compression was 27%.     

Ultrastructural changes in human skeletal muscles following tendon and nerve injuries. I. Ultrastructural changes following tendon injuries]

During reconstructive procedures performed 4-16 weeks after the tendon lesion the specimens obtained from the injured muscle have been examined by the authors. It was found that after the tendon injury inactivity atrophy develops and a condition of equilibrium could be observed at this time. The most important changes in the fine structure were seen in the contractile elements: these were atrophied, homogenized, fragmentated and ragged independently from the functional unities. The number of the mitochondria was considerably decreased, the sarcoplasmic reticulum was increased, and the difference between the originally red and white muscular fibres was indistinct. The glycogen content of the musculature was decreased, or it disappeared completely. No pathologic changes have been observed in the sarcolemma, the cell nuclei and the motor nerve end-organs.