Cellular and metabolic characteristics of pre-leukemic hematopoietic progenitors with GATA2 haploinsufficiency.

Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as Gata2 heterozygote mice do not develop hematologic malignancies. Here we show that two different germline Gata2 mutations (TgErg/Gata2het and TgErg/Gata2L359V) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of Erg/Gata2het fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring GATA2 aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline GATA2 mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline Gata2 genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with GATA2 germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem.

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

Broomrape can acquire viruses from its hosts.

Broomrapes (Phelipanche, formerly Orobanche) are parasitic plants that physically connect with the vascular systems of their hosts through haustorial structures. In this study, we found that Cucumber mosaic virus (CMV), Tomato mosaic virus (ToMV), Potato virus Y (PVY), and Tomato yellow leaf curl virus (TYLCV) translocate from infected host plants to Phelipanche aegyptiaca. In order to examine whether these viruses, and specifically CMV, replicate in the parasite, we tested several replication parameters. We detected accumulation of both plus and minus strands of CMV genomic RNA and CMV-derived siRNAs in the shoots of Phelipanche grown on CMV-infected tobacco and tomato plants. We purified CMV particles from Phelipanche grown on CMV-infected plants. These particles were present in amounts comparable to those found in the hosts' leaves. These data indicate that CMV replicates in Phelipanche tissues. In addition, viable ToMV and PVY were observed, and the plus and minus strand RNAs of ToMV were detected in Phelipanche shoots grown on infected hosts. However, we found only low levels of ToMV coat protein and did not detect any PVY coat protein. We also detected genomic TYLCV DNA in shoots of Phelipanche grown on TYLCV-infected tomato. Thus, for the first time, we demonstrate that broomrape is a host for at least one plant virus CMV, and possibly various other viruses.

Identification of an Arabidopsis unknown small membrane protein targeted to mitochondria, chloroplasts, and peroxisomes.

In a functional genomic screen performed by combining an Arabidopsis-yellow fluorescent protein (YFP)-fused complementary DNA (cDNA) library, rat fibroblasts as host and automatic microscopy, we found a short protein with a predictable trans-membrane domain encoded on chromosome 2. In rat fibroblasts, its pattern of distribution was to various organelle-like structures. From the databases, we learned that it has another family member in Arabidopsis and homologs in several other plants, Chlamydomonas and fungi, with a highly conserved N-terminal region. We named this protein from Arabidopsis short membrane protein (SMP) 2. No SMP homologs were found in mammalian sequence databases. When the full-length cDNAs of SMP2 was fused to YFP under the 35S promoter, comparable distribution was observed in Nicotiana benthamiana leaves, suggesting an unknown, evolutionarily conserved localization signal. Similar localization was observed when SMP2 was expressed in N. benthamiana leaves under the control of its own 5' regulatory sequences. Colocalization studies with green fluorescent protein and red fluorescent protein chimeras revealed its colocalization with chloroplasts, peroxisomes, and mitochondria. No localization of SMP2 was observed in the Golgi. Immunostaining with specific antibodies corroborated the SMP2 localization to the three organelles.

MRI evidence of white matter damage in a mouse model of Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a genomic instability disease caused by hypomorphic mutations in the NBS1 gene encoding the Nbs1 (nibrin) protein. Nbs1 is a component of the Mre11/Rad50/Nbs1 (MRN) complex that acts as a sensor of double strand breaks (DSBs) in the DNA and is critical for proper activation of the broad cellular response to DSBs. Conditional disruption of the murine ortholog of NBS1, Nbn, in the CNS of mice was previously reported to cause microcephaly, severe cerebellar atrophy and ataxia. In this study we used MRI to study the brain morphology and organization of Nbn deleted mice. Using conventional T(2)-weighted magnetic resonance, we found that the brains of the mutant mice (Nbs1-CNS-del) were significantly smaller than those of the wild-type animals, with marked mal-development of the cerebellum. Region of interest analysis of the T(2) maps revealed significant T(2) increase in the areas of white matter (corpus callosum, internal capsule and midbrain), with minor changes, if any, in gray matter. Diffusion tensor imaging (DTI) data confirmed that fractional anisotropy values were significantly reduced in these areas, mainly due to increased radial diffusivity (water diffusion perpendicular to neuronal fibers). Biochemical analysis showed low and dispersed staining for MBP and GalC in Nbs1-CNS-del brains, indicating defects in myelin formation and oligodendrocyte development. Myelin index and protein levels were significantly reduced in these brains. Our results point to a novel function of Nbs1 in the development and organization of the white matter.

Comparative spermatogenesis, spermatocytogenesis, and spermatozeugmata formation in males of viviparous species of clinid fishes (Teleostei: Clinidae, Blennioidei).

Spermatogenesis and spermatocytogenesis in 16 species of viviparous clinid fishes (Clinidae, Blennioidei) from various localities were followed for the first time by means of light and electron microscopy. The testes of the studied species are of the lobular type, with germinal stem cells situated at the apical ends of the lobules and a vas efferens along the internal margin. Maturation of the spermatides takes place in spermatocysts formed by Sertoli cells around the B-spermatogonia. The gradual condensation and relocation of the chromosomes along the nuclei membranes are highly prominent in this process, which can be divided into several stages. Anisodiametric and slightly flattened sperm heads are eventually formed, 0.4-0.5 microm in diameter and 7.5 +/- 1 microm long, bearing 80 +/- 15 microm long flagella. The sperms are packed into spermatozeugmata within the spermatocysts, enveloped and penetrated by the mucotic material of the Sertoli cells. With division of the germ cells and maturation of the spermatids, the spermatocyst dimensions increase, attaining 40 +/- 8 microm in diameter in the smaller species of Heteroclinus, and up to 90 +/- 10 microm in the larger males of Clinus superciliosus and C. cottoides. Accordingly, the volume of the maturing spermatocysts attains ca. 1,300 +/- 100 microm(3) in the smaller species, and ca. 6,500 +/- 300 microm(3) in the larger ones. As sperm head volume is ca. 2.24 microm(3), the number of sperm in the smallest mature spermatocysts reaches ca. 440 and in the largest over 2,900. Upon release from the cysts, the spermatozeugmata are transported along the sperm ducts to the posterior ampullae where they are stored in the epididymis. During copulation, the sperms are transported from there to the female via the intromittent organ. The sperm formation parameters and their structure and numbers are discussed.

Comparative morphology and cytology of the male sperm-transmission organs in viviparous species of clinid fishes (Clinidae: Teleostei, Perciformes).

This work comprises the first comparative study of the morphology and cytology of the sperm transmission organs in males of 14 species of viviparous clinid fishes (Clinidae, Blennioidei, Teleostei). The form and dimensions of these organs differ among the various species studied. The organs are composed of intra-abdominal ampullae, into which the sperm ducts and urinary bladder anchor, and an external protruding intromittent papilla used for insemination. The form of the ampullae differs among the various species, from pear-shaped to horseshoe-shaped. It increases in dimensions with increasing length of the male. In all the species this organ is covered by a connective-tissue tunic that encompasses both circular and longitudinal striated muscle bundles. The lumina of the ampullae harbor the epididymis, a strongly convoluted and plicated duct, which becomes filled with spermatozeugmata during reproduction. From here, the epididymis continues into the protruding intromittent papillae, where its folds gradually straighten at the apical part of the intromittent organ. The form and dimensions of this copulatory organ also differ in the various species. Papillae bearing taste buds are found on the apical parts of the intromittent organ, and it is probable that these, together with the difference in forms of the organ, help to prevent interspecific copulation.

Axonal damage is reduced following glatiramer acetate treatment in C57/bl mice with chronic-induced experimental autoimmune encephalomyelitis.

Glatiramer acetate (GA) is efficacious in reducing demyelinating-associated exacerbations in patients with relapsing-remitting multiple sclerosis (RRMS) and in several experimental autoimmune encephalomyelitis (EAE) models. Here we report that GA reduced the clinical and pathological signs of mice in chronic EAE induced by myelin oligodendrocyte glycoprotein (MOG). GA-treated mice demonstrated only mild focal inflammation, and less demyelination, compared with controls. Moreover, we also found minimal axonal disruption, as assessed by silver staining, antibodies against amyloid precursor protein (APP) and non-phosphorylated neurofilaments (SMI-32), in the GA-treated group. In conclusion, our study demonstrated for the first time that axonal damage is reduced following GA treatment in C57/bl mice with chronic MOG-induced EAE.

Up-regulation of semaphorin expression in retina of glaucomatous rabbits.

BACKGROUND: Glaucoma is a term encompassing a variety of diseases that end in the death of retinal ganglion cells (RGC). Although a variety of factors can initiate the disease onset, increased intraocular pressure (IOP) is one of the major risk factors. In our previous study we found that semaphorins were causally involved in RGC death following axotomy. Since a common feature of all retinal neuropathies is axonal damage, we hypothesized that semaphorins are involved in glaucoma-induced RGC death. The purpose of this study was to analyze the effect of increased IOP on RGC viability and to analyze semaphorin expression pattern in glaucomatous retinas. METHODS: Utilizing retrograde-labeled dye (4-Di-10-Asp) and hematoxylin-eosin staining, we investigated the effect of elevated levels of IOP on RGC viability. In addition, immunohistochemical analysis and western blotting were used to study the pattern of semaphorin expression in retinas of rabbits with genetically developed increased IOP and subsequently glaucoma. RESULTS: Using specific anti-semaphorin antibodies, the expression of a single protein with the size of a semaphorin protein, 110 kDa, was detected; its expression was up-regulated in glaucomatous rabbits compared with controls. Time-course analysis revealed that semaphorin expression peaked between 2 and 6 months of age and declined thereafter. Immunohistochemical analysis revealed that semaphorin expression was up-regulated specifically in the ganglion cell layer, which is a structure that is highly affected in glaucoma. CONCLUSION: Deciphering the molecular mechanisms of glaucoma-induced death and its mediators is a crucial step towards designing new therapeutic strategies to treat this incurable disease.

Anti-semaphorin 3A antibodies rescue retinal ganglion cells from cell death following optic nerve axotomy.

Damage to the optic nerve in mammals induces retrograde degeneration and apoptosis of the retinal ganglion cell (RGC) bodies. The mechanisms that mediate the response of the neuronal cells to the axonal injury are still unknown. We have previously shown that semaphorins, axon guidance molecules with repulsive cues, are capable of mediating apoptosis in cultured neuronal cells (Shirvan, A., Ziv, I., Fleminger, G., Shina, R., He, Z., Brudo, I., Melamed, E., and Brazilai, A. (1999) J. Neurochem. 73, 961-971). In this study, we examined the involvement of semaphorins in an in vivo experimental animal model of complete axotomy of the rat optic nerve. We demonstrate that a marked induction of type III semaphorin proteins takes place in ipsilateral retinas at early stages following axotomy, well before any morphological signs of RGC apoptosis can be detected. Time course analysis revealed that a peak of expression occurred after 2-3 days and then declined. A small conserved peptide derived from semaphorin 3A that was previously shown to induce neuronal death in culture was capable of inducing RGC loss upon its intravitreous injection into the rat eye. Moreover, we demonstrate a marked inhibition of RGC loss when axotomized eyes were co-treated by intravitreous injection of function-blocking antibodies against the semaphorin 3A-derived peptide. Marked neuronal protection from degeneration was also observed when the antibodies were applied 24 h post-injury. We therefore suggest that semaphorins are key proteins that modulate the cell fate of axotomized RGC. Neutralization of the semaphorin repulsive function may serve as a promising new approach for treatment of traumatic injury in the adult mammalian central nervous system or of ophthalmologic diseases such as glaucoma and ischemic optic neuropathy that induce apoptotic RGC death.

Localization of two enzymes of the tetrahydrobiopterin biosynthetic pathway in embryonic chick retina.

Tetrahydrobiopterin (BH4) is an essential co-factor for the biosynthesis of catecholamine-type neurotransmitters and of nitric oxide (NO). The expression of the enzymes catalyzing the first two steps of the BH4 biosynthetic pathway was studied in the developing chicken retina by in situ hybridization and immunocytochemistry. GTP-cyclohydrolase-I (GTP-CH-I) and 6-pyruvoyl-tetrahydropterin synthase (PTPS) were already expressed in the undifferentiated and proliferating retina of E7. At stage E11 both enzymes were expressed in photoreceptors, amacrine cells, displaced amacrine cells, and ganglion cells, and in the plexiform layers in which synaptic connections take place. At stage E18 the labeling was comparable to E11 but appeared to be more concentrated in photoreceptors and ganglion cells.