D2 receptor imaging in neonates using I-123 iodobenzamide brain SPECT.

PURPOSE: Hypoxic-ischemic injury induces early changes in cerebral energy that later lead to the presence and extension of brain damage and subsequently to severe neurodevelopmental impairments such as the dyskinetic form of cerebral palsy, which is associated with damage to the striatum. The purpose of the current study was to evaluate the viability of D2 receptors in the perinatal period using I-123 iodobenzamide brain SPECT and to correlate this with early neurologic status. METHODS: After obtaining informed parental consent, 12 full-term neonates with hypoxic-ischemic events were included. I-123 iodobenzamide brain SPECT was performed 1 week after birth, corresponding to a gestational age of 39.2+/-1.7 weeks. Images were acquired using a brain-dedicated gamma camera 1 hour after intravenous injection of 30 MBq (0.8 mCi) I-123 iodobenzamide. Magnetic resonance images (T2 weighted sequence: repetition time/echo time: 2,000/30 to 150) of the brains of the same neonates were acquired on the same day. RESULTS: The right and left striatum:cerebellum activity ratios were between 1.28 and 2.25, with the greatest concentration of I-123 iodobenzamide occurring in the striatum area. A tendency of the striatum:cerebellum ratio to decrease was observed as the severity of the perinatal hypoxic-ischemic event increased despite striatal hypersignal on magnetic resonance imaging in only two neonates. CONCLUSIONS: This study, which confirms that I-123 iodobenzamide could be used in the neonatal period, shows the biochemical maturation of D2 receptors as early as 1 week after birth and also suggests the deleterious effect of perinatal hypoxic-ischemic events on D2 receptors.

Nucleotide sequence of the 3' terminal region of the genome of four lettuce mosaic virus isolates from Greece and Yemen.

Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3' terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3' non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1(1) and mo1(2) and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR.

Comparison of monoclonal antibodies and polymerase chain reaction assays for the typing of isolates belonging to the d and m serotypes of plum pox potyvirus.

ABSTRACT Plum pox potyvirus (PPV) isolates may be divided into four groups separated by serological, molecular, and epidemiological differences. Monoclonal antibodies specific for the two major groups of isolates, represented by the D and M serotypes of the virus, have been obtained. Polymerase chain reaction (PCR)-based assays allowing the direct detection and differentiation of PPV isolates have also been developed. We now report on a large-scale comparison of these two typing approaches. The results obtained show an overall excellent correlation between the results obtained in indirect double-antibody sandwich enzyme-linked immunosorbent assay using PPV-D- and PPV-M-specific monoclonal antibodies and those derived from either specific PCR assays or restriction fragment length polymorphism analysis of PCR fragments. Without exception, all isolates reacting positively with the PPV-M-specific monoclonal antibody were found to belong to the M serotype using the PCR-based assays, while 51 out of 53 isolates recognized by the D-specific monoclonal antibodies belonged to the D serotype according to the PCR typing results. However, failure to react with a specific monoclonal antibody did not prove as effective a predictor of the serotype of the isolate analyzed. In a few cases, the results obtained with the various techniques diverged, indicating low level variability of the epitopes recognized by the serotype-specific monoclonal antibodies. Isolates belonging to the two minor groups of PPV (El Amar and Cherry) also gave divergent results, indicating that the current typing assays are not suited for the analysis of such isolates.

In vitro translation of apple chlorotic leaf spot virus (ACLSV) RNA.

The genomic RNA of apple chlorotic leaf spot virus was translated in a rabbit reticulocyte lysate system, yielding a large, 190 K product as well as several other polypeptides of smaller size (60, 56, 22 and 15 kDa). The 22 kDa product was immunoprecipitated by an anti-ACLSV serum and comigrated with purified ACLSV coat protein. In vitro translation of RNA transcripts prepared from cloned ACLSV cDNAs demonstrates that the coat protein is synthesised by initiation on the second in frame AUG codon of the 28 kDa open reading frame located at the 3' end of the genome. In the in vitro translation system used, the ability of various ACLSV-derived RNAs to direct the synthesis of the coat protein appears to be the result of initiation on the internal AUG codon.

The nucleotide sequence of the RNA-2 of an isolate of the English serotype of tomato black ring virus: RNA recombination in the history of nepoviruses.

The RNA-2 of a carrot isolate from the English serotype of tomato black ring nepovirus (TBRV-ED) has been sequenced. It is 4618 nucleotides long and contains one open reading frame encoding a polypeptide of 1344 amino acids. The 5' non-coding region contains three repetitions of a stem-loop structure also conserved in TBRV-Scottish and grapevine chrome mosaic nepovirus (GCMV). The coat protein domain was mapped to the carboxy-terminal one-third of the polyprotein. Sequence comparisons indicate that TBRV-ED RNA-2 probably arose by an RNA recombination event that resulted in the exchange of the putative movement protein gene between TBRV and GCMV.

Genetically engineered resistance against grapevine chrome mosaic nepovirus.

Nepoviruses are a group of isometric plant viruses with a genome divided between two-single-stranded, positive-sense, RNA molecules. They are usually transmitted by nematodes and a number of them have significant economic impact, especially in perennial crops such as grapevine and fruit trees. Like all other picorna-like viruses, nepoviruses express their coat protein (CP) as part of a larger polyprotein which is further processed by a virus-encoded protease, a feature which poses specific problems when trying to express the viral coat protein in transgenic plants. A hybrid gene, driving the high-level expression of the CP of grapevine chrome mosaic nepovirus (GCMV) has been constructed and transferred to the genome of tobacco plants. Progeny of CP-expressing transformants show resistance against GCMV. When compared to control plants, fewer inoculated plants become infected and those that become infected accumulate reduced levels of viral RNAs. This protection was also shown to be efficient when plants are inoculated with purified viral RNA.

Analysis of the dsRNAs of apple chlorotic leaf spot virus.

Double-stranded RNAs were isolated from plants infected with five different isolates of apple chlorotic leaf spot virus (ACLSV). Analysis by PAGE and by Northern blot hybridization showed that six major species of viral dsRNA of approximately 7.5, 6.4, 5.4, 2.2, 1.1 and 1.0 kbp can be detected in infected plants, irrespective of the ACLSV isolate used. In addition to the dsRNA of 7.5 kbp corresponding to the full-length genome, the size and position on the genome of the 2.2 and 1.1 kbp species indicate that these are very probably double-stranded forms of subgenomic RNAs allowing the expression of the internal open reading frames coding respectively for the ACLSV 50K and coat proteins. The subgenomic messenger for the coat protein was indeed detected in total RNA preparations from infected plants. Surprisingly, the two most abundant dsRNA species, of 6.4 and 5.4 kbp, were found to be 5'-coterminal with the genomic RNA. A model for the expression of the genome of ACLSV and for the production of the molecules 5'-coterminal with the genomic RNA is presented.

Nucleotide sequence and genomic organization of apple chlorotic leaf spot closterovirus.

The nucleotide sequence of Apple chlorotic leaf spot closterovirus (ACLSV) genomic RNA has been determined from cDNA clones. It is 7555 nucleotides in length excluding the 3' terminal poly(A) tail and contains three putative open reading frames capable of encoding proteins of 216.5, 50, and 28 kDa. ACLSV RNA has untranslated regions of 151 and 190 nucleotides at its 5' and 3' termini, respectively. The 216.5-kDa ORF encodes a protein which contains the conserved "signature" sequences and has significant homology with the proteins suspected to be involved in viral RNA replication of members of the "Sindbis-like" supergroup of viruses. On the basis of distant homologies with viral movement proteins (M proteins), the 50-kDa ORF is suspected to encode a protein responsible for virus cell-to-cell spread. The 28-kDa ORF contains, in frame, a smaller 21.5-kDa ORF encoding the coat protein of ACLSV. These results show that ACLSV and probably at least the subgroup A of closteroviruses should be regarded as members of the "Sindbis-like" supergroup of RNA viruses.

High mobility group proteins in ram spermatids.

The four major high mobility group proteins HMG 1, 2, 14 and 17, HMG 19B and histone H1(0) were identified in the ram testis by their extraction and solubility characteristics and by their electrophoretic mobilities. HMG 14 and 17 were isolated by chromatography and amino acid analysis revealed that they were similar to their calf thymus analogues. A protein, named 2R and co-extracted with HMG 14, was also purified and analysed. Electrophoretic analyses of the proteins extracted by 0.75 M perchloric acid (PCA) or by 0.35 M NaCl from round and non-round spermatids, separated by centrifugal elutriation, showed that the four major HMG proteins disappear from nuclei in the oldest round spermatids, at the time the nuclear content of protein 2R and histone H1(0) increases in spermatids. Ubiquitin and HMG 19B were present in the round and elongating spermatids, but not in elongated spermatids which contained only protamine. The relation was considered between several protein changes and genetic inactivation and structural reorganization of the spermatid chromatin.

Isolation and characterization of the ram spermatidal nuclear proteins P1, 3 and T.

Ram spermatidal proteins P1, 3 and T were isolated from non-round spermatid nuclei and characterized by amino acid analysis. Spermatidal proteins are small arginine- and cysteine-rich basic proteins. Proteins P1 and T are unusually rich in serine and the histidine content of P1 is particularly high. The NaCl molarities required to dissociate these proteins from the spermatid nuclei were determined. These proteins are present only during the reorganization of the spermatid chromatin.

Interactions of nuclear proteins with DNA, during sperm differentiation in the ram.

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nucleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous "knobby fibers" that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30-200 A region.

Purification and characterization of ubiquitin from mammalian testis.

Ubiquitin was extracted from testis of 4 mammals and purified to homogeneity by gel filtration chromatography. Amino acid compositions and NH2-terminal sequences were found to be identical in the 4 species and with calf thymus ubiquitin. Ubiquitin conformation was shown to be very sensitive to oxidation. Improved methods for radioimmunoassay of ubiquitin in tissue extracts are also discussed.

Structural function of the basic nuclear proteins in ram spermatids.

The function of the cysteine-containing spermatidal proteins and of protamine in the packaging and stabilization of chromatin during ram spermiogenesis was investigated. Extractions of the histones and spermatidal proteins from the nonround spermatid nuclei decreases the nuclear stability (sonication resistance), decondenses the chromatin, and reduces the diameter of the largest chromatin threads (100-200 A vs. 380 A in the control nuclei). Extractions by acid, salt, or heparin have no effect on the protamine-containing electron-opaque chromatin. In contrast, treatment by dithiothreitol alone decondenses all the nonround spermatid nuclei at a rate which decreases with the maturation state of the nuclei. The electron-opaque chromatin is then resolved in 35-A-thick filaments. Experimentally induced fluctuations of the level of SS bonding appear to influence the chromatin stabilization and ultrastructure in most of the nonround spermatid nuclei. These data evidence that noncovalent interactions play a main structural role at the beginning of chromatin reorganization, and SS bonding between spermatidal proteins and then between protamine molecules increases progressively and becomes mainly responsible for the chromatin stabilization in the protamine-containing nuclei.

Evolution of Ca2+- and cAMP-dependent regulatory mechanisms during ram spermatogenesis.

Calmodulin level and cAMP-dependent protein kinase activity of ram germ cells at different stages of spermatogenesis have been determined. Calmodulin levels decrease during maturation. Simultaneously, calmodulin localization changes during cell differentiation. In round, elongating, and elongated spermatids, calmodulin is closely associated with the developing acrosome; in spermatozoa, it becomes present in the postacrosome, the neck region and the tail. Protein kinase activity is relatively low in testicular cells but increases dramatically during epididymal maturation of spermatozoa. A concerted regulation by cAMP and Ca2+ of biochemical events in spermatogenic cells and spermatozoa is suggested.

An electrophoretic investigation of mammalian spermatid-specific nuclear proteins.

Using standardized methods for protein extraction and analysis, the testes of rams, bulls, goats, boars, stallions, rats, cats, hedgehogs, European mink and ferrets were examined for basic spermatid nucleoproteins by electrophoresis. The results suggest that differences exist in the total number of these proteins as well as in the number and amount of the cross-linked cystein-containing proteins. These differences appear to be more family-specific than species-specific.