A block in glycoprotein processing correlates with small plaque morphology and virion targetting to cell-cell junctions for an oral and an anal strain of herpes simplex virus type-1

Summary The characteristics of two clinical isolates of HSV-1 obtained from an oral (424) and an anal (490) lesion were compared with the highly passaged KOS strain. In contrast to KOS, the clinical isolates produced small plaques, were more cell-associated and the predominant viral glycoprotein species for gC and gD in infected cell lysates was the precursor, high mannose glycoform. Total virus production in Vero cells was equivalent for the three virus strains in one-step growths. Pulse-chase studies of glycoprotein C processing showed a reduction in rate at 7.5h post infection and a significant block in processing at 10.5h post infection for 424 and 490 but not KOS. Similar results were obtained for gD. The significant reduction in glycoprotein processing for 424 and 490 suggests a block in transport of viral glycoproteins or virions to and through the Golgi apparatus. Extracellular virions and the cell surface, prior to cell lysis, contained the processed gC glycoform suggesting a competent cellular glycan processing system. Upon co-infection of 424 or 490 with KOS or a gC^– KOS strain, gC was processed to levels equivalent to KOS indicating that 424 and 490 are not inhibitory but that an activity(s) encoded by KOS facilitates maturation of gC from 424 and 490. Unlike KOS infected Vero cells, virion-containing vacuoles were observed in the cytoplasm at 12h p.i. and extracellular virions were concentrated at cell-cell junctions of 424 or 490 infected cells but not in the perinuclear region. These results suggest that intracellular transport of viral glycoproteins and virions in 424 and 490 infected cells is different from KOS infected cells. The reduced level of viral glycoprotein maturation, virus release, cell surface presence and presence of virions at cell-cell junctions are consistent with small plaque production in tissue culture cells.Portions of this work were presented in the 17th International Herpesvirus Workshop, Pittsburgh, PA, 1993.     

The partition of sodium fluxes in isolated toad oocytes

1. The rate constant for Na efflux from the oocyte calculated from (d/dt) (ln [Na^*]_i]) is only approximately 52% of that calculated from (d/dt)[(ln(d[Na^*]_i)dt)]. The difference may be interpreted by supposing that 48% of the internal Na of the oocyte is either bound to proteins or sequestered in cell organelles.2. The mean rate constant for Na efflux was 6·4 × 10^-3 min^-1 corresponding to an apparent Na efflux rate of 13·3 p-mole/cm².sec. When this is corrected for the increase in surface area produced by microvilli the true efflux rate is 1·1-1·3 p-mole/cm².sec.3. The action of ouabain (1-5 μM) appears to involve two different effects: (a) there is 48-65% inhibition of the membrane Na pump, and (b) there is a release of some of the sequestered Na in the cell.4. Removal of external K causes a 40% reduction in Na efflux although this value may be an underestimation owing to the presence of K which has leaked from the cell and may be retained near the cell surface.5. Raising the external K concentration to 15 mM reduces the inhibitory effect of ouabain by approximately a half.6. It was concluded that the Na pump in the toad oocyte may have a slightly lower level of activity than that in frog muscle, but that its general properties are similar to those in frog muscle and some other animal cells.     

Apparent replication of suggestive linkage on chromosome 16 in the NIMH genetics initiative bipolar pedigrees

Analyses of a replication sample of families collected as part of the National Institute of Mental Health (NIMH) Genetics Initiative for bipolar disorder provide further evidence for linkage to a region of chromosome 16. Families who had a bipolar I (BPI) proband and at least one BPI or schizoaffective, bipolar type (SABP) first-degree relative were ascertained for the purpose of identifying genes involved in bipolar affective disorder. A series of hierarchical models of affected status was used in linkage analyses. Initial genetic analyses of chromosomes 3, 5, 15, 16, 17, and 22, completed at Indiana University in 540 subjects from 97 families, suggested evidence of linkage to chromosomes 5, 16, and 22 [Edenberg et al., 1997: Am J Med Genet 74:238-246]. Genotyping was subsequently performed on these chromosomes in a replication sample of 353 individuals from 56 families. Nonparametric linkage analyses were performed using both affected relative and sibling pair methods. Analyses in the new sample on chromosome 16, using the broadest model of affected status, corroborate previously reported suggestive linkage to the marker D16S2619. Combining the initial and replication samples further increased the evidence of linkage to this region, with a peak lod score of 2.8.     

Studies on thymocyte subpopulations in guinea pigs: in vivo differentiation of bromodeoxyuridine labelled cells, with special reference to rosette-forming ability

Cycling thymocytes were labelled by an intracardial injection of bromodeoxyuridine (BrdUrd) in a total of 32 guinea pigs and the incorporation into DNA studied in subpopulations of cells defined by buoyant density and rosette-forming ability. The labelling pattern was compared at different times after injection (0.5 h to 120 h). A marked shift of labelled cells from large, low density cells (population 1a) to small, high density cells (population 2) was observed. During the first 48 h, the ratio between labelling indices of cell populations 1a and 2 decreased from 10 to 0.5. The number of labelled cells forming rosettes with rabbit erythrocytes (RFC+) increased while the number of labelled non-rosetting cells (RFC-) decreased from 0.5 h to 48 h, probably representing transformation of RFC- to RFC+. Then, after a decreased labelling in all cell populations at 72 h, an increase in both RFC+ and RFC- populations occurred at 96 h. The labelling in RFC- cells at 96 h was nearly as high as immediately after labelling. This second labelling of RFC- cells could represent immigration of precursor cells, a wave of proliferation in initially labelled precursors, and/or the formation of mature cells from the initially labelled precursors. The results indicate that a great majority of proliferating cells differentiate into small, high density cells within 48 h and that rosetting ability is acquired in many cells during this period. A model of intrathymic differentiation which fits the observations is presented.     

The effects of cortisol infusion upon hormone secretion from the anterior pituitary and subjective mood in depressive illness and in controls.

The aims of this study were to determine whether the administration of cortisol has a significant effect on mood in patients with depression and whether the effects of cortisol on changes in plasma hormone concentrations are like those of synthetic corticosteroids. Twelve patients had major depression and one each had dysthymic disorder and a depressive adjustment disorder. Five were male and nine were female. All were in-patients. Eight normal subjects, two females and six males, were used as controls. Basal beta-endorphin concentrations were 2- to 3-fold higher in depressed patients than in control subjects, but there were no significant differences between the patient and control groups in the basal (pre-infusion) plasma concentrations of ACTH, cortisol, growth hormone or prolactin. Cortisol, but not saline infusion resulted in a significant improvement in self rated mood. Surprisingly, cortisol infusion at first increased plasma beta-endorphin concentrations. At later times after cortisol infusion, plasma beta-endorphin concentrations decreased as did the plasma concentrations of ACTH and growth hormone; prolactin levels were increased. These results show (i) that cortisol infusion raises mood significantly in major depression, (ii) that plasma beta-endorphin concentration is a potential marker of major depression (iii) that rather than blunting of corticosteroid effects, responses to cortisol may even be enhanced in depressive illness. The unexpected, initial increase in beta-endorphin stimulated by cortisol, suggests that the action of cortisol is not simply one of negative feedback inhibition, but may involve mineralocorticoid, as well as glucocorticoid receptors.     

Metabotropic glutamate receptors are associated with non-synaptic appendages of unipolar brush cells in rat cerebellar cortex and cochlear nuclear complex

Unipolar brush cells (UBCs) are a class of small neurons that are densely concentrated in the granular layers of the vestibulocerebellar cortex and dorsal cochlear nucleus. The UBCs form giant synapses with individual mossy fibre rosettes on the dendrioles which make up their brush formations and are provided with numerous, unusual non-synaptic appendages. In accord with the glutamatergic nature of mossy fibres, our previous post-embedding immunocytochemical studies indicated that various ionotropic glutamate receptor subunits are localized at the post-synaptic densities of the giant synapses, whereas the non-synaptic appendages are immunonegative. On the contrary, the metabotropic glutamate receptors mGluR1 and mGluR2/3 are situated at the non-synaptic appendages and are lacking at the post-synaptic densities. Other authors, however, have shown that antibodies to these metabotropic receptors stain both appendages and post-synaptic densities. In the present study, we have re-evaluated the distribution of metabotropic glutamate receptors in the UBCs of the cerebellum and the cochlear nuclear complex by light and electron microscopic pre-embedding immunocytochemistry with subtype-specific antibodies. We confirm that UBCs dendritic brushes are densely immunostained by antibody to mGluR1 particularly in the cerebellum and that antibody to mGluR2/3 labels at least a percentage of the UBC brushes in both the cerebellum and cochlear nuclei. At the ultrastructural level, it appears that mGluR1 and mGluR2/3 immunoreactivities are not associated with the post-synaptic densities of the giant mossy fibre–UBC synapses, but instead are concentrated on the non-synaptic appendages of the cerebellar UBCs. The non-synaptic appendages, therefore, may be an important avenue for regulating the excitability of UBCs and mediating glutamate effects on their still unknown intracellular signal transduction cascades. We also show that the pre-synaptic densities of UBC dendrodendritic junctions are mGluR2/3 positive. As previously demonstrated, antibodies to mGluR1 and mGluR2/3 label subsets of Golgi cells. Antibody to mGluR5 does not stain UBCs in the cerebellum and cochlear nucleus and reveals the somatodendritic compartment of Golgi cells situated in the core of the cerebellar granular layer, whilst cochlear nucleus Golgi cells are mGluR5 negative.     

IS1245 Restriction Fragment Length Polymorphism Typing of Mycobacterium avium Isolates: Proposal for Standardization

Mycobacterium avium has become a major human pathogen, primarily due to the emergence of the AIDS epidemic. Restriction fragment length polymorphism (RFLP) typing, using insertion sequence IS1245 as a probe, provides a powerful tool in the molecular epidemiology of M. avium-related infections and will facilitate well-founded studies into the sources of M. avium infections in animal and environmental reservoirs. The standardization of this technique allows computerization of IS1245 RFLP patterns for comparison on a local level and the establishment of M. avium DNA fingerprint databases for interlaboratory comparison. Moreover, by combining international DNA typing results of M. avium complex isolates from a broad spectrum of sources, long-lasting questions on the epidemiology of this major agent of mycobacterial infections will be answered.     

Axonal projections of single bulbospinal inspiratory neurons revealed by spike-triggered averaging and antidromic activation

Activity was recorded extracellularly from 26 inspiratory bulbospinal neurons in anesthetized, paralyzed, artificially ventilated cats. All but one were located in the ventral respiratory group. A neuron was classified as either I alpha or I beta by comparing its firing pattern during inspiratory cycles with lung inflation to its pattern when lung inflation was withheld during the central inspiratory phase (2, 14, 15). In this study, the projection and conduction velocity of these axons were determined using two methods: antidromic activation (AA) of the bulbospinal neurons and spike-triggered averaging (STA) of the extracellular field potentials. These methods have been compared directly because the same electrode was used both for stimulating the axon of the bulbospinal neuron and recording its axonal potential in the same location. Axonal projections from these neurons were mapped in the contralateral spinal cord with a mobile electrode by determining where the lowest stimulus threshold occurs for AA and greatest axonal potential can be recorded with STA. The locations of these axons were in the ventral and lateral funiculi. Each method determined a similar location for an axon. Positions of 10 axons were determined at both the third (C3) and fourth (C4) cervical segments. Single axons maintained their positions in either descending tract from rostral C3 to mid-C4. In five of six cases where two "neighboring" medullary units were characterized, the axons of each pair projected together within 350 micron of each other in the cervical spinal cord. Estimates of mean axonal conduction velocity (CV) from antidromic activation from a single stimulus site, "single-point AA," were as much as 42% less than corresponding estimates from STA extracellular field potentials at that point (P less than 0.001). Such single-point estimates were less than determinations that were calculated from the difference in conduction time and the difference in conduction distance from two points in the spinal cord. These two-point determinations averaged 55.4 +/- 13.1 m/s (using AA) and 53.3 +/- 13.1 (using STA) for 10 neurons. These values were not significantly (P greater than 0.2) different from each other and are greater than most earlier reports, which used the single-point AA method. Either method, AA or STA, can be used to determine axonal position and CV. The advantages and disadvantages of each method are discussed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromosomes and causation of human cancer and leukemia. LIV. Near-tetraploidy in acute leukemia

Near-tetraploid cell populations were observed in a case of T-cell acute lymphoblastic leukemia (T-ALL) and in one of acute myeloblastic leukemia (AML). In the ALL case, hyperdiploid chromosomal changes, characterized by an isochromosome 17q [i(17q)], as well as other changes, were seen at the onset of the disease. At the first relapse, hypertetraploid cells appeared in about 10% of the mitoses in the bone marrow (BM), and by the second and third relapses, the hypertetraploidy was present in more than 90% of the mitoses in the BM. Even though karyotypic instability was evident, all abnormal karyotypes contained one or two i(17q) at every sampling. In spite of karyotypic instability at each relapse, karyotypic evolution was observed whenever relapse occurred. A normal female karyotype was confirmed in the BM of each period. Immunologic examinations performed at each sampling revealed no recognizable changes before and after the appearance of tetraploidy. In the AML case, which was classified as FAB M2, cytogenetic examination was performed at diagnosis and relapse. In both, hypotetraploid cells were observed in over 60% of the BM cells; the modal chromosome number was 90. Banding analysis was successful at relapse, and a pseudodiploid clone characterized by t(8;21) and a hypotetraploid clone with two t(8;21) and a loss of two Y chromosomes were observed in the same BM sample. A normal male karyotype was also observed in BM cells. In both cases, giant and bizarre blasts were seen in the BM. A close correlation between near-tetraploid mitoses and giant and bizarre blast cells in BM smears of the same samples was observed. Previously published tetraploid acute leukemia cases analyzed with banding methods were accumulated and compared with our two cases.