Mechanism of neurogenesis during the embryonic development of a tunicate.

Ascidian and vertebrate nervous systems share basic characteristics, such as their origin from a neural plate, a tripartite regionalization of the brain, and the expression of similar genes during development. In ascidians, the larval chordate-like nervous system regresses during metamorphosis, and the adult's neural complex, composed of the cerebral ganglion and the associated neural gland is formed. Classically, the homology of the neural gland with the vertebrate hypophysis has long been debated. We show that in the colonial ascidian Botryllus schlosseri, the primordium of the neural complex consists of the ectodermal neurohypophysial duct, which forms from the left side of the anterior end of the embryonal neural tube. The duct contacts and fuses with the ciliated duct rudiment, a pharyngeal dorsal evagination whose cells exhibit ectodermic markers being covered by a tunic. The neurohypophysial duct then differentiates into the neural gland rudiment whereas its ventral wall begins to proliferate pioneer nerve cells which migrate and converge to make up the cerebral ganglion. The most posterior part of the neural gland differentiates into the dorsal organ, homologous to the dorsal strand. Neurogenetic mechanisms in embryogenesis and vegetative reproduction of B. schlosseri are compared, and the possible homology of the neurohypophysial duct with the olfactory/adenohypophysial/hypothalamic placodes of vertebrates is discussed. In particular, the evidence that neurohypophysial duct cells are able to delaminate and migrate as neuronal cells suggests that the common ancestor of all chordates possessed the precursor of vertebrate neural crest/placode cells.

Neurogenic role of the neural gland in the development of the ascidian, Botryllus schlosseri (Tunicata, Urochordata).

In adult ascidians, the neural complex consists of a cerebral ganglion (the brain) and the associated neural gland. We have studied the development of the neural complex during the vegetative reproduction of the colonial ascidian Botryllus schlosseri, the buds of which arise from the atrial mantle of the parental zooid. Each bud develops into a new organism within which a neural complex becomes differentiated. We found that the presumptive (pioneer) nerve cells that ultimately form the cerebral ganglion of the adult arise as migratory cells from a primordial cluster of rudimentary gland cells. Hence, the neural gland appears to be neurogenic in that it serves as the cellular source of components that differentiate into conventional nerve cells. In the adult, these cells take on the form of a typical invertebrate ganglion with an outer cortex of nerve cell bodies and an internal medulla. This medulla consists of a neuropile of neuronal processes making classical synaptic contacts. The adult neural gland differentiates into a structure with a ciliated duct that opens into the branchial chamber, the body of the gland, and the dorsal organ, which is quite distinct from the dorsal strand of other ascidians. The rudimentary neural gland cells, therefore, differentiate into one of two distinct pathways: the first, glandular, is possibly involved in the evaluation of environmental signals, and the other, nervous, leads to brain formation. This compares with the vertebrate situation in which the olfactory-pituitary placodes are thought to originate from a common cellular source. Thus, these data support the earlier contention of a homology between the tunicate neural gland and the vertebrate adenohypophysis.

The ultrastructural effects of beta-amyloid peptide on cultured PC12 cells: changes in cytoplasmic and intramembranous features.

The fine structural features of cultured PC12 cells were investigated after treatment for 1, 3, or 5 days with different concentrations of the vascular form of beta-amyloid 1-40 (beta-AP). PC12 cells treated with beta-AP showed time- and concentration-dependent lysosomal system activation and cell toxicity. We observed increases in the number and size of cytoplasmic lysosomes as indicated by increased acid phosphatase reactivity. Some lysosomes were in the form of multivesicular bodies or large residual bodies that appeared to arise by autophagia or by endocytotic uptake. Double-sided plasma membrane invaginations were observed to give rise to increasingly extensive intracytoplasmic vacuolization that was correlated with duration of beta-AP treatment. Freeze-fracture studies of the intramembranous particle (IMP) population in the plasma membrane P-face showed that both control and beta-AP treated cells had two major P-face IMP populations, small-diameter (4-8 nm) IMPs, and large-diameter (> or = 9 nm) IMPs. The larger category of IMPs was found to possess a greater average diameter in the beta-AP treated cells than in the control cells. These IMPs could represent modifications to existing transmembranous receptors, channels, or transducing molecules by the beta-AP. These results demonstrate that beta-AP can induce time- and concentration-dependent ultrastructural changes in PC12 cell membranes.

Genetic analysis of cholinergic nerve terminal function in invertebrates.

Genetic analysis of nerve terminal function is proving fruitful and studies on invertebrates are making a substantial impact. In this survey, particular emphasis has been placed on cholinergic chemical synaptic transmission. The advanced genetics of Drosophila melanogaster and Caenorhabditis elegans with their rich diversity of behavioural and biochemical mutants is providing new insights into the functions of key molecular components of synapses. A 'space-invader' mutant of Periplaneta americana permits investigations of competition between neurons during synaptogenesis and its impact on neurotransmitter release. The growing importance of the C. elegans genome as a major research resource is emphasized in this survey.

A fine structural study of the hippocampus and dorsal root ganglion in mouse trisomy 16, a model of Down's syndrome.

Mouse trisomy 16 (Ts16) appears to provide an animal model of Down's syndrome in that a portion of mouse chromosome 16 is syntenic with part of human chromosome 21. Trisomy 21 in human beings leads to the mental retardation of Down's syndrome and in middle age, to some presenile anatomic and clinical features of Alzheimer's disease. Neural tissue from aging Ts16 mice is unavailable, however, as Ts16 mouse embryos die late in utero. We studied these embryos looking at the ultrastructure of neurons from the hippocampus and dorsal root ganglion in normal control mice embryos (diploid) and in Ts16 late embryonic litter mates after day 15 of gestation. The organelles in the Ts16 neurons looked similar to those in control neurons, fixed and processed under similar conditions. No obvious neuropathological structures were observed. These results, when compared to reports on electrophysiological abnormalities of cultured fetal Ts16 neurons and on abnormalities in neurotransmitter markers in the Ts16 fetal brain, lead us to suggest that the mental retardation of Down's syndrome is likely to result from functional and chemical defects not directly related to abnormal neuronal ultrastructure. When related to fine structural studies of transplanted embryonic Ts16 hippocampus which have been maintained for long periods of time, these results indicate that the trisomic mouse brain would not be useful as a structural model for Down's syndrome and hence presenile Alzheimer's disease, as it is not associated with any detectable morphological abnormality.

A novel adhering junction in the apical ciliary apparatus of the rotifer Brachionus plicatilis (Rotifera, Monogononta).

Cultures of the rotifer Brachionus plicatilis were examined with regard to their interepithelial junctions after infiltration with the extracellular tracer lanthanum, freeze-fracturing or quick-freeze deep-etching. The lateral borders between ciliated cells have an unusual apical adhering junction. This apical part of their intercellular cleft looks desmosome-like, but it is characterized by unusual intramembranous E-face clusters of particles. Deep-etching reveals that these are packed together in short rows which lie parallel to one another in orderly arrays. The true membrane surface in these areas features filaments in the form of short ribbons; these are produced by projections, possibly part of the glycocalyx, emerging from the membranes, between which the electron-dense tracer lanthanum permeates. These projections appear to overlap with each other in the centre of the intercellular cleft; this would provide a particularly flexible adaptation to maintain cell-cell contact and coordination as a consequence. The filamentous ribbons may be held in position by the intramembranous particle arrays since both have a similar size and distribution. These contacts are quite different from desmosomes and appear to represent a distinct new category of adhesive cell-cell junction. Beneath these novel structures, conventional pleated septate junctions are found, exhibiting the undulating intercellular ribbons typical of this junctional type, as well as the usual parallel alignments of intramembranous rows of EF grooves and PF particles. Below these are found gap junctions as close-packed plaques of intramembranous particles on either the P-face or E-face. After freeze-fracturing, the complementary fracture face to the particles shows pits, usually on the P-face, arrayed with a very precise hexagonal pattern.

Effect of mannitol and polyethylene glycol on the action of frusemide during renal storage and transplantation.

Hypoxic injury is a major cause of tubular necrosis in the corticomedullary junction of isolated perfused kidneys, and is ameliorated by inhibitors of active reabsorption, such as frusemide. Our objective was to determine whether frusemide has a similar effect on hypothermically stored transplanted kidneys and whether this effect is modulated by impermeant solutes included in the preservation solution. The effect of frusemide on cytochrome oxidase (cyt aa3) oxidation, renal hemodynamics, and morphology was investigated in the New Zealand White rabbit renal autograft model using near-infrared spectroscopy and light microscopy. A total of 30 kidneys were autografted in six groups. Kidneys were transplanted with or without frusemide either (1) without storage (groups 1 and 2) or after 72 hr of storage in: (2) hypertonic citrate containing mannitol (groups 3 and 4); and (3) hypertonic citrate containing polyethylene glycol (PEG) (groups 5 and 6). In unstored transplanted kidneys, frusemide infusion stimulated a significant (P < 0.05) increase in hemoglobin oxygenation, compared with untreated controls. There was a tendency for cyt aa3 to become reduced, but there were no significant differences between groups 1 and 2. After 72 hr of storage, frusemide infusion stimulated a significant increase in hemoglobin oxygenation in kidneys stored in mannitol (P < 0.005), but there was no significant change in the kidneys stored in PEG. There was a corresponding reduction in cyt aa3 in kidneys stored in mannitol (P < 0.05) but no change in those stored in PEG. These results suggest that frusemide has a significant effect on cortical hemoglobin oxygenation in transplanted kidneys and on active reabsorption in the corticomedullary junction. The selection of impermeant is important and mannitol is significantly superior to PEG in this model.

Hemoglobin oxygenation kinetics and secondary ischemia in renal transplantation.

The significance of poor medullary reperfusion in the etiology of acute tubular necrosis during renal transplantation is poorly understood. Our objective was to determine the kinetics of renal hemoglobin oxygenation using near-infrared spectroscopy during renal transplantation, to provide a framework against which the timing of mitochondrial dysfunction could be considered. New Zealand White rabbit kidneys were flushed with hypertonic citrate solution (0-2 degrees C and autografted immediately (group 1) or stored at 0-2 degrees C for 72 hours before autografting (group 2). Changes in oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) were monitored by near-infrared spectroscopy for 3 hours of reperfusion. Intrarenal perfusion was evaluated separately by barium sulfate angiography. Reperfusion resulted in rapid increases in HbO2 within 1 minute in both groups. Group 1 HbO2 fell sharply to a minimum at 3 minutes but recovered by 20 minutes; group 2 changes were similar, but there was no recovery (P<0.05 by 10 minutes). Hb increased rapidly in both groups upon reperfusion but in group 2 was significantly greater after 10 minutes (P<0.05). Total hemoglobin levels were similar in both groups. Renal hemoglobin saturation was 69% at 1 minute in both groups; there was no significant change in group 1 but a profound desaturation in group 2 to 25% at 10 minute (P<0.005) and no recovery thereafter. Barium sulfate distribution was normal in all group 1 kidneys; cortical distribution was normal in all group 2 kidneys, but medullary perfusion was poor for the first 60 minutes. Renal hemoglobin oxygenation kinetics as determined here do not correlate with the timing of mitochondrial dysfunction previously reported (Thorniley et al., Kidney International, 1994; 45: 1489). We conclude that secondary ischemia during reflow is not the only mechanism leading to acute tubular necrosis.

Two different forms of gap junctions within the same organism, one with cytoskeletal attachments, in tunicates.

The cells of the intestinal tract and the stigmatal cells of the branchial basket have been studied in a range of tunicates including phlebobranch, aplousobranch and stolidobranch ascidians, as well as the doliolid and pyrosomatid thaliaceans. The intercellular gap junctions between gut cells appear conventional in thin section as do those found in the lower part of adjacent stigmatal cells. However, save for the stolidobranchs, the stigmatal cells also have a second kind of gap junction which exhibit an unusual fibrous density in association with their junctional cytoplasmic surfaces; these are found in the apical region of the cells. The fibrous density is particularly well demonstrated in specimens treated with tannic acid during fixation, and subsequent en bloc uranyl acetate staining. In the branchial basket the position of these apical gap junctions is at regular intervals between adhaering junctions, which have a more substantial paramembranous fibrous mat; these two kinds of junctions alternate along deeply undulating membrane appositions. With freeze-fracture, after chemical or cryo-fixation, the gap junctions of the gut and those of the lower part of the stigmatal cells appear typical, with P-face connexons, while in the apical part of cells of the branchial basket the two faces of the gap junctions are very difficult to cleave apart. Frequently the P- and E-faces are found to adhere together in replicas, so that in these apical gap junctional regions, plaques of E-face with pits overlie the PF particles. In addition, regions of cytoplasm, into which the dense fibres project, often cleave over these adhaering E-faces of the apical gap junctions. The presence of these unusual gap junctional features in the apical region of the stigmata in the vicinity of cilia is discussed as regards their functional role.

Fine-structural investigation of rat brain microvascular endothelial cells: tight junctions and vesicular structures in freshly isolated and cultured preparations.

A comparison was made between endothelial cells in freshly-isolated rat brain microvessels, and following culture of the cells for 1-10 days during growth to confluence. Attention focused on tight junctions and vesicular structures, as seen in thin sections and freeze-fracture replicas. Freshly-isolated vessels had an abnormal appearance, with a profusion of luminal microvillar processes, and extensive cytoplasmic vacuolation. There were numerous vesicular profiles, reaching a density of approximately 60 microns-2, and with a large proportion open to the surface, as shown by labelling with cationized ferritin at 4 degrees C for 5 min. Junctional zones were relatively loosely organized, with evidence for some cell:cell separation, as well as some residual tight junctional sites within zonula adhaerens junctions. In freeze-fracture replicas, junctional strands showed segments of tightly packed intramembrane particles, generally on the P face. After 1 day in culture, the cells appeared more normal, with no vacuolation or luminal processes. Vesicles were still numerous, some associated with junctional zones, while tight junctions were relatively sparse; freeze-fracture showed some incomplete tight junctional strands, with some of the intramembrane particles fracturing onto the E face. The double offset fibrillar nature of the strands could occasionally be seen. Cells cultured for 4 and 10 days showed a progressive increase in the completeness of the junctional zone, with more tight junctional contacts within the length of the adhaerens junction, and an aggregation of microfilaments in the underlying cytoplasm. The number of vesicular profiles declined, and they were progressively excluded from the junctional zone. These observations have relevance for studies on the physiology of the brain endothelium in vitro, and for comparisons with the in vivo condition.

Certain HLA antigens are associated with specific morphologic and cytogenetic subsets of acute myeloid leukemia.

Although many associations have been found between specific HLA antigens and an increased susceptibility to various diseases, previous attempts to associate class I and II antigens with acute myeloid leukemia (AML) have been inconclusive, probably due in part to the heterogeneity of AML. We subdivided 165 consecutive adults with AML de novo into distinct clinical, morphological, and cytogenetic subsets and then tested for statistically significant associations with specific HLA antigens. Both morphology and cytogenetic pattern identified subsets of patients with important clinical features and different outcomes. Ten statistically significant (P < 0.05) HLA cytogenetic associations were observed: HLA-A11 with t(8;21), A26 with t(15;17), B7 with 11q23 abnormalities, B44 with +8, Cw2 with -20/del(20q), DR3 with t(15;17) and FAB-M3, DR4 with inv(16) and FAB-M4Eo, DQ2 with +8, and DQ6 with +22. HLA-DQ1 had a negative association with -5/del(5q), which was present in 13% of the 165 AML patients overall but in none of the 27 with DQ1. Certain HLA antigens were significantly correlated with more favorable remission rates, remission duration and survival. Possible mechanisms for the association of HLA antigens with particular subtypes of AML include the linkage or co-inheritance of an oncogene, the facilitation of binding of a transforming virus, toxin, or cytokine, or a permissive role involving impaired immune recognition of an emerging neoplasm. Given the heterogeneity of both the HLA system of immune recognition genes and the cytogenetic subtypes of AML, however, larger numbers of patients must be studied to have confidence that biologically important relationships truly exist.

Microtubule systems associated with the septate junctions of the gill cells of four gammarid amphipods.

The microtubular systems associated with the septate junctions of the gill epithelial cells of four species of gammarid amphipod are described. The four species examined included two relatively stenohaline marine forms, Chaetogammarus marinus and Gammarus locusta; a highly euryhaline species, Gammarus duebeni, and a stenohaline freshwater species, Gammarus pulex. Of these amphipods, G. locusta and C. marinus maintain only a limited osmotic gradient between their haemolymph and the medium and have a poorly developed junctional microtubular system; G. pulex has haemolymph which is some 300 mOsmol hypertonic to freshwater and has a well ordered system of microtubules on both sides of fairly long septate junctions; G. duebeni from brackish water tend to have a somewhat shorter length of septate junctions lined by one or occasionally by a double row of microtubules. The most complex junctional microtubular systems are shown by specimens of the freshwater race of G. duebeni celticus which have been acclimated to seawater. These can take the form of multiple arrays in which some microtubules are linked to the plasma membrane by dense strands. It is suggested that these findings are consistent with the hypothesis that one role of these microtubules is to provide mechanical stability to enable the integrity of the septate junctions to be maintained during osmotic stress.

A phase I trial of concomitant chemoradiotherapy with cisplatin dose intensification and granulocyte-colony stimulating factor support for advanced malignancies of the chest.

UNLABELLED: Concomitant chemoradiotherapy with cisplatin and combination chemotherapy in the neoadjuvant setting have both shown promising results. PURPOSE: To identify a locally and systemically active concomitant chemoradiotherapy regimen incorporating high-dose cisplatin, interferon alfa-2a (IFN), fluorouracil (5-FU), hydroxyurea (HU) and radiotherapy. METHODS: Phase I cohort design establishing the maximal tolerated dose (MTD) of cisplatin with and without granulocyte colony stimulating factor (GCSF). For the first six dose levels, a 4-week cycle consisted of escalating doses of cisplatin during weeks 1 and 2, IFN (week 1), and 5-FU and HU (week 2) with single daily radiation fractions of 200 cGy during days 1-5 of weeks 1-3 and no treatment in week 4. When dose-limiting neutropenia was encountered. GCSF was added during weeks 1, 3, and 4. Finally, to decrease esophagitis, the radiotherapy schedule was altered to 150 cGy twice daily during weeks 1 and 2, followed by a 2-week break (level 7). RESULTS: Forty-nine patients with refractory chest malignancies were treated. The MTD of this regimen without GCSF was cisplatin 50 mg/m2 in weeks 1 and 2, IFN 5 million Units (MU)/m2 per day on days 1-5 in week 1, 5-FU 800 mg/m2 per day for 5 days by continuous infusion, and HU 500 mg every 12 h for 11 doses during week 2. The addition of GCSF during weeks 1, 3, and 4 allowed for escalation of cisplatin to 100 mg/m2 during weeks 1 and 2, with a decreased dose of IFN at 2.5 MU/m2 per day to avoid renal toxicity. Dose-limiting toxicity (DLT) included severe neutropenia, thrombocytopenia, and esophagitis in 5 of 13 patients. Increased thrombocytopenia in patients receiving GCSF was not observed. During hyperfractionated radiotherapy (level 7) chemotherapy doses were as above except for a reduction of 5-FU to 600 mg/m2 per day. While severe esophagitis was reduced, grade 4 thrombocytopenia became more prevalent and was seen in 6 of 7 patients. In-field tumor responses were observed in 17 of 28 evaluated patients with non-small-cell lung cancer. The median times to progression and survival were 4 and 6 months, respectively. When only patients with all known disease confined to the radiotherapy field were considered the corresponding times were 6 and 15 months, respectively. Most treatment failures occurred outside of the irradiated field. CONCLUSIONS: (1) This intensive multimodality regimen can be given with aggressive supportive care incorporating GCSF. The recommended phase II doses for a 4-week cycle are cisplatin 50 mg/m2 week 1, and 100 mg/m2 week 2, IFN 2.5 MU, HU 500 mg every 12 h x 11 and 5-FU 800 mg/m2 per day with single fraction radiotherapy during weeks 1-3 and GCSF during weeks 1, 3, and 4. (2) GCSF can be safely administered and provides effective support of neutrophils when administered simultaneously with IFN, cisplatin, and chest radiotherapy. (3) There is synergistic renal toxicity when high doses of IFN and cisplatin are given together. (4) Hyperfractionated radiotherapy decreases the severity of esophagitis but increases thrombocytopenia. (5) Although highly toxic, response rates, time to progression and survival figures with this regimen are encouraging and support its investigation in the phase II setting.

Clinical and prognostic significance of chromosomal abnormalities in childhood acute myeloid leukemia de novo.

We report on the chromosomal pattern of 120 patients with childhood AML de novo. One hundred and fifteen patients (96%) had adequate samples for analysis; 98 (85%) of these showed clonal karyotypic abnormalities. They were classified into cytogenetic subgroups which were closely correlated with FAB subtypes: t(8;21) and M2 (n = 9); t(15;17) and M3 (n = 12); inv(16) and M4Eo (n = 9); t(9;11) and M5a (n = 10); t(11q23) other than t(9;11) and M4-M5 (n = 11); and t(1;22) and M7 (n = 4). In patients with -7/del(7q) (n = 6), leukemia was preceded by MDS in half of the cases, although they had diverse FAB subtypes. Thirty-seven patients had miscellaneous abnormalities. Despite a high CR rate, patients with t(8;21) had a very poor survival: only one child was event-free at 3 years from diagnosis. One third of patients with t(15;17) died during induction. Those eight who achieved CR fared well: only two relapsed, and six were event-free survivors. Patients with inv(16) had a high remission rate and a long survival: five children were in CR 20 to 136 months. Both groups with t(9;11) and t(11q23) had a high remission rate: however, outcome was superior for the t(9;11) group when compared to either the t(11q23) group (EFS at 3 years +/- SE, 56 +/- 17% vs. 11 +/- 10%, p = 0.07) or to the remaining patients (p = 0.06). Both -7/del(7q) and t(1;22) groups had low CR rates (50%) and poor survival. Cytogenetic analysis identifies clinically distinct subsets of childhood AML and is useful in tailoring treatment for these patients. Favorable cytogenetic groups (t(15;17), inv(16), and t(9;11)) may do well with current therapy protocols, whereas unfavorable groups (t(11q23), t(8;21), -7/del(7q), and t(1;22)) require more effective therapies.

Inhibition of leukotriene B4 synthesis does not prevent development of acute renal failure following storage and transplantation.

Compound BW B70C, a selective 5-lipoxygenase inhibitor was tested for its ability to reduce inflammatory damage in an in vivo rabbit model of renal storage and transplantation. Kidneys were stored at 0-2 degrees C for 48 hr prior to autografting. In controls, renal vein LTB4 levels rose significantly after 30 min reperfusion but fell after 2 hr to baseline. TxB2 levels remained at baseline for the 6 hr measured. 6-k-PGF1 alpha levels rose significantly after 1 hr of reperfusion and remained elevated thereafter. Histology after 6 hr reperfusion showed moderate-to-severe cortical edema and mild congestion. Infused colloidal carbon was retained in the perivascular area in a narrow band at the corticomedullary junction, indicating a zone of vascular permeability. At 3 days after transplant, kidneys exhibited widespread tubular necrosis and calcification but little inflammation. Serum creatinine and urea peaked between days 3 and 5. 3/6 rabbits showed no symptoms of renal failure after 3 weeks. Pretreatment with BW B70C prevented the increase in LTB4 but had little effect on TxB2 and 6-k-PGF1 alpha levels. Histology showed no amelioration of cortical edema at 6 hr and congestion and hemorrhage were exacerbated. BW B70C had no effect on either colloidal carbon retention or distribution but did significantly reduce tubular necrosis and calcification at day 3. There was very little inflammatory infiltrate. BW B70C treatment did not improve the long-term viability of transplanted kidneys: 2/6 rabbits showed no symptoms of renal failure after 3 weeks. These data indicate that inhibition of LTB4 synthesis by BW B70C does not prevent the development of acute renal failure following 48 hr hypothermic storage and transplantation.

Structural changes in the gill cells of Gammarus duebeni (Crustacea, Amphipoda) under osmotic stress; with notes on microtubules in association with the septate junctions.

The amphipod crustacean Gammarus duebeni (Lilljeborg) tolerates salinities in the range freshwater to seawater. Such tolerance requires the ability to respond to the varying degrees of osmotic stress imposed on the single layer of epithelial cells separating the outside medium from the haemolymph space in the gills. Following transfer of individuals from low salinities to seawater, changes occur in the fine structure of the epithelial cells. These changes involve the configuration of the apical border of the cells, the mitochondria and cytoplasmic lacunae. Despite such variation in cell organisation, the association between neighbouring epithelial cells appears unaffected. Attention is drawn to a well developed system of microtubules in association with the septate junctions. The possible mechanical role of these microtubules in protecting the integrity of the septate junctions from the effects of osmotically induced changes in cell volume is discussed.

Long-term intracerebral transplants of fetal hippocampus from mouse trisomy 16, a model for Down's syndrome (trisomy 21), do not exhibit Alzheimer's disease neuropathology by ultrastructural criteria .

Murine trisomy 16 (Ts16) is an animal model for Down's syndrome (human trisomy 21), because mouse chromosome 16 is genetically homologous to human chromosome 21. Down's syndrome patients, older than 35 years, demonstrate the neuropathological and neurochemical defects characteristic of Alzheimer's disease and Ts16 mouse fetuses exhibit phenotypic abnormalities similar to those in Down's syndrome fetuses. Trisomic mouse fetuses, however, die in utero, and so do not survive long enough for their brains to develop the degenerative changes of aging. This can be overcome by grafting the fetal Ts16 hippocampus (an early site for the development of the pathological features characteristic of Alzheimer's disease), into the cerebral ventricle or striatum of a normal adult mouse host. We have made such transplants, which have survived for up to 12 months. Examining these grafts ultrastructurally at various stages from 1 to 12 months, and comparing them with normal control grafts, reveals no structural difference that could be deemed characteristic of Alzheimer disease; no neurofibrillary tangle or senile plaque was observed. These observations, together with the normal structure of the neuronal organelles in trisomic hippocampal tissue, suggest that trisomic mouse grafts are not a useful model for Alzheimer's disease, despite earlier reports to the contrary.

Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H(+)-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila.

The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H(+)-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus. Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992). We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated. Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit. Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions. The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain. A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene. Although without phenotypic consequences, these can serve as a starting point for generation of null alleles.