Structure and function of a mitochondrial late embryogenesis abundant protein are revealed by desiccation

Few organisms are able to withstand desiccation stress; however, desiccation tolerance is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. These hydrophilic proteins are prominent in plant seeds but also found in desiccation-tolerant organisms. In spite of many theories and observations, LEA protein function remains unclear. Here, we show that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. Structural modeling revealed an analogy with class A amphipathic helices of apolipoproteins that coat low-density lipoprotein particles in mammals. LEAM appears spontaneously modified by deamidation and oxidation of several residues that contribute to its structural features. LEAM interacts with membranes in the dry state and protects liposomes subjected to drying. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. According to sequence analyses of several homologous proteins from various desiccation-tolerant organisms, a similar protection mechanism likely acts with other types of cellular membranes.     

Farnesylated RhoB inhibits radiation-induced mitotic cell death and controls radiation-induced centrosome overduplication

Our previous results demonstrated that expressing the GTPase ras homolog gene family, member B (RhoB) in radiosensitive NIH3T3 cells increases their survival following 2 Gy irradiation (SF2). We have first demonstrated here that RhoB expression inhibits radiation-induced mitotic cell death. RhoB is present in both a farnesylated and a geranylgeranylated form in vivo. By expressing RhoB mutants encoding for farnesylated (RhoB-F cells), geranylgeranylated or the CAAX deleted form of RhoB, we have then shown that only RhoB-F expression was able to increase the SF2 value by reducing the sensitivity of these cells to radiation-induced mitotic cell death. Moreover, RhoB-F cells showed an increased G2 arrest and an inhibition of centrosome overduplication following irradiation mediated by the Rho-kinase, strongly suggesting that RhoB-F may control centrosome overduplication during the G2 arrest after irradiation. Overall, our results for the first time clearly implicate farnesylated RhoB as a crucial protein in mediating cellular resistance to radiation-induced nonapoptotic cell death.     

Micromorphology,structural and ultrastructural changes during somatic embryogenesis of a Tunisian oat variety (<Emphasis Type="Italic">Avena sativa</Emphasis> L. var ‘Meliane’)

To better understand micromorphological and structural changes, histological sections provide additional insight into cellular process and developmental pathways occurring in oat somatic embryogenesis. Environmental scanning electron microscopy (ESEM) and transmission electron microscopy (TEM) were also used to follow the ultrastructural modifications during this system. Histological observations allowed following the events leading to the development of mature somatic embryos. The scheme includes the following steps: cell reactivation, the first organized cell division in diads, triads, tetrads as well as octant stages, the observation of an extracellular matrix (ECM) as a fibrillar material that bounded the surface of individualized proembryos. The transition from proembryo stage to an early globular somatic embryo was noted, where the embryogenic cortex is surrounded by the protoderm. The late globular stage was marked by bipolarity. The early and late transitional stages, the coleoptilar, mature and germinated stages were also described. The ESEM allowed us to follow some rearrangements, related to the morphology and surfaces involved in somatic embryos development. These events are proembryo formation, transition from proembryo to globular stage, marked by protoderm formation, scutellum and coleoptile development and finally somatic embryos germination. The TEM showed that embryogenic cells were very rich in organelles; mitochondria, rough endoplasmic reticulum, Golgi apparatus and ribosomes. Cells of proembryos, globular and late somatic embryos showed more vacuoles and differentiated organelles. The ECM was also detected by TEM as fibrillar material coating the cell walls. These results on structural and ultrastructural changes provided new insights and findings on oat somatic embryogenesis.     

Ring chromosome 3 in a mentally retarded adult dwarf

A 46-year-old man with mental retardation, growth failure and some dysmorphic features is found to have a 46,XX,r(3)(p26q29) karyotype in 93% of his peripheral lymphocytes. This observation is compared with previously cases of ring 3. The existence of a "ring syndrome" is considered.     

Cardiac two-dimensional imaging and reference values for blood flow velocities in the ovine fetus.

Since the ovine model is the most commonly used for foetal haemodynamic investigation it was felt important 1) to investigate the technical difficulties involved with ultrasound fetal cardiac imaging in this species and 2) to establish normal reference values for ovine cardiac and umbilical blood flow velocity measurements. Both two-dimensional and pulsed Doppler techniques were used for this assessment in 25 unsedated ewes. All morphological features described in human features to identify the ventricular cavities could be found in the ovine fetus with the two-dimensional echocardiogram. Specific differences included a flatten thorax as visualized from the lateral position, the mesocardial position of the heart, and a large left azygos vein behind the left atrium draining blood into a dilated coronary sinus. The mean peak velocities (cm/sec) of the early diastolic wave (E) and the atrial wave (A), along with their calculated ratio, were not statistically different between the two atrio-ventricular valves (E: 30.6 +/- 6.6, 31.2 +/- 6.1; A: 43.0 +/- 8.3, 41.6 +/- 8.0; E/A: .72, .76 for mitral and tricuspid valves respectively). A significant difference was observed between the acceleration times of the blood ejected into the aorta and the pulmonary artery, with the time interval being shorter in the pulmonary artery (Aorta: 0.052 +/- 0.011; Pulmonary artery: 0.037 +/- 0.009 s). A mean pulsatility index of the umbilical artery of 0.89 was recorded. The data recorded in this study should serve as a reference base for further non-invasive studies of the ovine foetal circulatory system using the ultrasound technique.     

Mucopolysaccharidesis Type VII resulting from beta-glucuronidase deficiency. Report of one family]

The patient is a north african female, fourth born child in a family with consanguinity. Facial dysmorphia, clubfeet, swollen extremities and heel borne ponctuate calcifications are observed soon after birth. beta glucuronidase activity is very low in serum, leukocytes and skin fibroblasts. At 18 months, gorwth and psychomotor development are normal. Flat facies and dorsolumbar cyphosis are striking. There is no clinical sign of storage disease, neither ocular or cytologic (blood, bone-marrow) abnormalities. Squeletal abnormalities are predominant on cervical and lumbar column and pelvis. Urinary excretion of chondroitin 4 sulfate and chondroitin 6 sulfate is increased. A 4 year old sister is affected. Facial dysmorphia, mild squeletal abnormalities are observed. Again, growth is normal and there is no symptom of storage disease. Enzymic expression of parent heterozygotism is marked in serum studies, but less marked in leukocytes and fibroblasts. The last two children are heterozygotes. At the time of a 5th pregnancy, enzymic activity studies of amniotic fluid and amniotic cells have shown that the foetus was an inaffected female. This child was normal at birth.     

Effect of rearing method on the storage of fats by adipose tissue in sheep

Adipocyte synthesis de novo and lipoprotein lipase activity have been used simultaneously to measure the lipogenic activity of adipose tissue in sheep. Acetate and glucose were used as precursors of fatty acid synthesis. The sheep were raised either outdoors or in a sheepfold. They were slaughtered by lots at mean weights of 24 and 32.5 kg. Compared to lipoprotein lipase activity, de novo synthesis of fatty acids was the main way of constituting lipid depositions. Raising the sheep outdoors favored the use of glucose as precursor of lipid synthesis at the first slaughter stage at 24 kg. Later at 32.5 kg, glucose utilization was practically zero compared to acetate, whatever the mode of rearing. The NADPH production needed for fatty acid synthesis was almost entirely due to NADP isocitrate dehydrogenase activity. Variations in both de novo synthesis and in lipoprotein lipase activity in relation with rearing method and slaughter weight were especially evident in the group raised outdoors.     

Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp³⁴² and Asn³⁴³. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.