Huntington's disease: the challenge for cell biologists.

Huntington's disease (HD) is one of eight inherited neurodegenerative diseases caused by expansions of (CAG)(n) tracts that encode polyglutamine segments in expressed proteins. Studies of pathogenic mechanisms for all these late-onset diseases suffer from a common drawback: experimental studies require massive acceleration of a process that, in affected humans, usually takes decades. But is the rapid-onset disease of transgenic mouse models and in cells the same as the slow-onset disease in humans? We review recent work on HD, noting several issues whose significance is likely to be crucial - but which are as yet unresolved. We discuss these in light of the distinction between disease-specific pathogenic mechanisms and artifacts of polyglutamine overexpression. We suggest that the initial stages of HD result from dysfunction rather than death, and we consider the potential discovery of compounds that might interfere with early pathogenic events.

Glycine-cysteine substitution at codon 13 of the N-ras proto-oncogene in a human T cell non-Hodgkin's lymphoma.

Tumor-derived DNA from a non-Hodgkin's (T cell) lymphoma patient, assayed by NIH3T3 transfection followed by inoculation of cells into nude mice, was found to contain an activated N-ras proto-oncogene. The mode of activation was determined by hybridization with N-ras-specific oligonucleotide probes detecting mutations at codons 12, 13 and 61. A transversion in codon 13 (GGT----TGT) resulting in replacement of glycine13 by cysteine13 in ras p21 protein was found. The mutation was detected in DNA from mouse tumors induced by transfected NIH3T3 cells and in DNA from patient tumor lymphoblasts. The patient was heterozygous for this mutation. These data identify the first base of codon 13 as a novel mutation site in ras genes and indicate that cysteine at position 13 of the ras p21 is a transforming substitution.

Somatic and germinal recombination of a direct repeat in Arabidopsis.

Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings. Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast. Southern analysis showed that recombinants could be formed by either crossing over or gene conversion. A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events. Some evolutionary implications are discussed.

Treatment of severe aplastic anemia.

A total of 100 patients with severe aplastic anemia were treated and evaluated in a prospective study at our hospital between January 1976 and October 1983: 28 patients had an HLA-identical sibling donor and were treated with bone marrow transplantation, and 72 patients without an HLA-identical sibling donor were given antilymphocyte globulin followed by oral low-dose androgen therapy. At 1 1/2-9 years after treatment, 13 patients (46%) survive in the transplant group and 45 patients (75%) survive in the second group. All except one in the second group have self-sustaining hematopoiesis without need for transfusions. There is one major difference between the two therapies. Marrow transplantation restores bone marrow function completely and no late hematologic complications have been seen in this group. The majority of patients treated with antilymphocyte globulin in contrast have residual abnormalities of hematopoiesis: macrocytosis, mild granulocytopenia, and mild thrombopenia. Relapse (11 of 72 patients) and clonal hematologic disorders, such as paroxysmal nocturnal hemoglobinuria (four patients) and leukemia (one patient) can occur years after complete bone marrow reconstitution with antilymphocyte globulin. These late disorders are of concern. Despite this, we conclude that antilymphocyte globulin treatment is an effective therapy with low early mortality and morbidity and a high chance for a long, sustained remission. Results are better or at least equivalent to bone marrow transplantation and patients with donors should be given the option of transplant or antilymphocyte globulin.

Interaction of nod and exo Rhizobium meliloti in alfalfa nodulation.

Among the genes of Rhizobium meliloti SU47 that affect nitrogen-fixing symbiosis with alfalfa are nod genes, in which mutations block nodule induction, and exo genes, in which mutations allow nodule formation but block rhizobial exopolysaccharide production as well as nodule invasion and nitrogen fixation. To investigate whether an exo+ bacterium can "help" (that is, reverse the symbiotic defect of) an exo mutant in trans, we have coinoculated alfalfa with pairs of rhizobia of different genotypes. Coinoculant genotypes were chosen so that the exo+ helper strain was nif while the exo "indicator" strain was nif+, and thus any fixation observed was carried out by the exo coinoculant. We find that a nod exo+ coinoculant can help an exo mutant both to invade nodules and to fix nitrogen. However, a nod+ exo+ coinoculant cannot help an exo mutant: Few exo bacteria are recovered from nodules, some bacteroids differentiate into bizarre aberrant forms, and the nodules fail to fix nitrogen. In a triple coinoculation, the effect of nod+ helper supersedes that of nod helper. Implications of these results for interaction of nod and exo gene products are discussed.

Rhizobium meliloti homologs of Escherichia coli mur genes.

The pectate-lyase-encoding gene pelB of Erwinia chrysanthemi Ec16 was used as a probe for hybridization to Rhizobium meliloti Rm1021 chromosomal DNA under low-stringency conditions. An Rm1021 DNA fragment that hybridized to this probe was cloned and sequenced. Results of RNA hybridization indicate that a portion of the cloned fragment is transcribed in R. meliloti. Although the Rm1021 fragment shares no significant nucleotide sequence identity with Ec16 pelB, it includes an ORF (open reading frame) that shares a high degree of nt sequence identity with the Escherichia coli murD gene. This gene codes for UDP-N-acetylmuramoyl-L-alanyl-D-glutamate synthetase, which catalyzes a step in the synthesis of the E. coli cell wall. The R. meliloti putative murD sequence is preceded by a partial ORF that shares sequence identity with mraY. The orientation of the two ORFs in R. meliloti is similar to that of the E. coli murD and mraY genes.

The chicken CP49 gene contains an extra exon compared to the human CP49 gene which identifies an important step in the evolution of the eye lens intermediate filament proteins.

The gene structure for chicken CP49 gene is presented. It differs from the human CP49 gene with the presence of an extra exon in helix IB and the apparent loss of an intron, intron H. The CP49 gene localises to chromosome 2 in the chicken genome where it is flanked by homologues that map to human chromosome 10p13 (VIM) 6p24-p23 (BMP6). Two transcripts, CP49 and CP49ins, are produced from the single chicken CP49 gene. The difference is a 49-amino-acid insertion in helix IB of CP49 that is encoded by a novel exon found in the chicken CP49 gene. An extended helix IB is believed to be a characteristic of the ancestral intermediate filament protein as it is found in many invertebrate intermediate filament proteins but has been lost from all vertebrate intermediate filament proteins except the nuclear lamins. Although the intron position and length of the helix IB insert sequences in CP49ins differ to those found both in the invertebrate intermediate filament proteins and the vertebrate lamins, the CP49 gene is the first vertebrate cytoplasmic intermediate filament protein to be described with an extended helix IB. The chicken CP49 gene is also the first where differential splicing can remove such a feature. Human and bovine CP49 appear to have lost the helix IB insert sequences, and so the avian CP49 gene provides an interesting evolutionary link between the eye lens proteins and the ancestral intermediate filament protein.

Positive-negative selection for homologous recombination in Arabidopsis.

In plants gene knock-outs and targeted mutational analyses are hampered by the inefficiency of homologous recombination. We have developed a strategy to enrich for rare events of homologous recombination in Arabidopsis using combined positive and negative selection. The T-DNA targeting construct contained two flanking regions of the target alcohol dehydrogenase gene as homologous sequences, and neomycin phosphotransferase and cytosine deaminase as positive and negative markers, respectively. A root explant transformation procedure was used to obtain transgenic calli. Among 6250 transformants isolated by positive selection, 39 were found to be resistant to negative selection as well. Of these 39, at least one had undergone homologous recombination correlated with a unidirectional transfer of information. Although the ADH locus was not changed, our data demonstrate that a homologous recombination event can be selected by positive negative selection in plants.

Chronic cyclosporine-associated nephrotoxicity in bone marrow transplant patients.

This study describes the prevalence and degree of chronic cyclosporine-associated nephropathy and its risk factors. For this purpose we reviewed all available renal histology specimens in 169 bone marrow transplant recipients treated during an eight year period with cyclosporine for prevention of graft-versus-host-disease, and determined their pattern and degree of histomorphological changes. A total of 51 specimens obtained from 49 patients by biopsy (n = 12) or autopsy (n = 39) was evaluated. The pattern of histomorphological changes was compared with diagnosis, age, sex, and potential risk factors--such as cyclosporine dose, levels, duration of therapy, changes in serum creatinine and onset of hypertension. Morphological lesions of chronic cyclosporine-associated nephropathy were found in 67% of the specimens. They were more frequent and more severe with increasing duration of cyclosporine therapy, in patients with a higher increase in serum creatinine during the first 3 months and in patients given total-body irradiation for conditioning. These latter findings suggest that additional damage sensitizes the kidney to irreversible toxic effects of cyclosporine.

Application of human minisatellite probes to the development of informative DNA fingerprints and the isolation of locus-specific markers in animals.

In this study, the alpha-globin 3' HVR (Jarman et al., 1986), the RNA transcripts of 33.15 and 33.6 (Carter et al., 1989), and the human locus-specific minisatellites MS1, MS8, MS31, MS32 (Wong et al., 1987), MS51, MS228A (Armour et al., 1989) and g3 (Wong et al., 1986) were applied to domestic pigs, common marmoset monkeys (Callithrix jacchus jacchus) and Waldrapp ibises (Geronticus eremita) and evaluated for their suitability firstly for isolating polymorphic VNTR markers from genomic libraries (pigs), and secondly for producing informative DNA fingerprints (pigs, marmosets and Waldrapp ibises).

Celiac disease transmitted by allogeneic non-T cell-depleted bone marrow transplantation.

We observed the occurrence of celiac disease following allogeneic bone marrow transplantation in a patient transplanted for acute leukemia. The marrow donor was his HLA-identical sister, who had suffered from celiac disease since birth. The post-transplant period was characterized by recurrent episodes of diarrhea. Detailed workup showed atrophic intestinal mucosa on histology and anti-gliadin and anti-endomysium antibodies in the serum, features that were not present before transplantation. GVHD was absent at that time. The patient remains free of symptoms on gluten-free diet and slight immunosuppression. This case suggests transmission of celiac disease by bone marrow transplantation and supports the T cell concept in celiac disease.

Bone marrow graft versus ALG in patients with aplastic anaemia.

Eighty-six successive patients with severe aplastic anaemia were admitted to this hospital between January 1976 and October 1982. They were treated and evaluated in a prospective study according to one protocol. 26 patients with an HLA identical sibling underwent bone marrow transplantation. Overall survival calculated according to Kaplan and Meier in this group is 47%, 60 patients without an HLA identical sibling were given antilymphocyte globulin with or without an infusion of HLA-haploidentical marrow. All these 60 patients received low dose androgens after the procedure. In this study the marrow infusion did not significantly improve the results and overall survival was 74% compared to 70% in patients receiving only antilymphocyte globulin and androgens. In a current pilot study combining ALG and high dose prednisone we were able to further increase remission rates. Between HLA-identical siblings we improved the results of marrow transplants significantly by the use of cyclosporin-A instead of methotrexate for prophylaxis against GvHD.

Bleeding gastric ulcer in a newborn infant diagnosed by transumbilical aortography.

Transumbilical aortography has been utilized for the first time to diagnose the cause of massive upper gastrointestinal hemorrhage in a newborn infant. We consider it a safe and accurate method in cases that meet the following criteria: massive hemorrhage uncontrollable by blood transfusions and supportive medical therapy, and hemorrhage active at the time of study as indicated by fresh blood draining from nasogastric tube.

DNA profiling reveals remarkably low genetic variability in a herd of SLA homozygous pigs.

Inbred strains of rodents have become indispensable for a wide range of biological studies. It has generally been accepted that genetic uniformity is unlikely to be achieved before 20 generations of brother x sister matings discouraging attempts to inbreed larger mammals. Nevertheless, pigs, homozygous for the swine MHC haplotype SLA b/b, have been inbred at the Babraham Institute for almost thirty years and used for immunological studies. Since the herd had not been studied at the DNA level, DNA profiling at multiple hypervariable loci was performed and surprisingly little genetic polymorphism and extremely high inter-individual resemblance were observed reminiscent of that observed in inbred strains of mice.