Human herpesvirus 7 infection of lymphoid and myeloid cell lines transduced with an adenovirus vector containing the CD4 gene.

It has been reported recently that CD4 is a major component of the receptor for human herpesvirus 7 (HHV-7), which has been newly identified as a T-lymphotropic virus. To investigate further the role of CD4 in HHV-7 infection, we examined the susceptibility to HHV-7 infection of various CD4-negative or weakly positive cell lines into which the cDNA for CD4 was transferred using an adenovirus vector (Adex1CACD4). Of 13 cell lines transduced with Adex1CACD4, including T-lymphoid, B-lymphoid, monocytoid, and myeloid cell lines, one T-lymphoid cell line, one monocytoid cell line, and two cell lines established from the blast crisis of chronic myelogenous leukemia showed high susceptibility to HHV-7 infection. Taken together with the results of previous studies, these data suggest strongly that CD4 is a major component of the binding receptor for HHV-7. This study also shows that HHV-7 may be able to infect CD4-positive hematopoietic precursor cells as well as T lymphocytes.     

Infectious particles derived from Semliki Forest virus vectors encoding murine leukemia virus envelopes.

Semliki Forest virus vectors encoding murine leukemia virus (MLV) envelope protein with a truncated cytoplasmic tail generate submicrometer, cell-associated, membranous particles that transmit replication-competent vector RNA specifically to cells bearing the MLV receptor. Such "minimal" viruses could have applications as retroviral vaccines or in the study of virus evolution.     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

Regulation of Synthesis of PSI in the Cyanophytes Synechocystis PCC6714 and Plectonema boryanum during the Acclimation of the Photosystem Stoichiometry to the Light Quality

The effects of light quality on the formation of the PSI complexwere examined in Synechocystis PCC6714 and in Plectonema boryanum.The rate of increase in levels of core polypeptides of PSI,PsaA/B, doubled upon shift from Chl a-absorbed light (PSI light)to phycobilisome-ab-sorbed light (PSII light). The elevatedrate was decreased upon the reverse shift. Half time of theacceleration was approximately 10 min, and that of the decreasewas approximately 4 min. The rate of degradation of the polypeptideswas far lower than the rate of the increase under either lightregime. Neither synthesis nor degradation of the PsbA and PsbCpolypeptides of PSII was significantly altered by the lightquality. We conclude that synthesis of the PSI complex is chromaticallyregulated to allow adjustments in photosystem stoichiometry.The level of mRNA for PsaA/B was not altered by the light regime.Anomalous inhibition by chloramphenicol suggested that the regulationoccurs at a step(s) other than the peptide elongation step,perhaps at the initiation of the ribosome cycle or at the insertionof Chl a for the stabilization of the polypeptides. The pho-toreductionof protochlorophyllide (Pchlide) was compared with the synthesisof the polypeptides in a mutant of Plectonema boryanum thatlacked Pchlide dark reductase (YFC1004). The results indicatedthat the synthesis of stable PsaA/B polypeptides was not limitedby the reduction of Pchlide, although the synthesis did dependon a supply of Chl a. ¹Present address: Department of Plant Biology, University ofMaryland at College Park, MD 20742, U.S.A. ²Present address: Department of Marine Bioscience, Fukui Pre-fecturalUniversity, Obama, Fukui, 917 Japan     

The Role of DNase and EDTA on DNA Degradation in Formaldehyde Fixed Tissues

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.     

Lag period for phototropism inPilobolus crystallinus sporangiophores

The lag period for the second positive curvature was examined inPilobolus crystallinus sporangiophores. The lag period for curvature development was 20–30 min at lower fluence rates than 6.32 nmol/m²s but greatly extended at higher fluence rates. When a 20-min symmetrical irradiation with blue light was applied before a 20-min unilateral blue light irradiation, sporangiophores bent as much as those unilaterally and continuously irradiated for 40 min. However, when a 20-min unilateral irradiation was followed by a 20-min symmetrical irradiation, sporangiophores did not show any curvature. That is, the reaction during the first 20 min of the lag period is independent of light direction. This light-direction-independent lag period is considered to be the duration required for adaptation. The lag period for phototropism was also extended when fluence rate was reduced after the start of irradiation. These results suggested that an adaptation process is involved in phototropism ofPilobolus.     

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells This revised version was published online in November 2006 with corrections to the Cover Date.     

PIG-B,a membrane protein of the endoplasmic reticulum with a large lumenal domain,is involved in transferring the third mannose of the GPI anchor.

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.     

Effect of Macrolide Antibiotics on Macrophage Functions

Macrolide antibiotics have a variety of actions other than antimicrobial activities. Recently, it has been suggested that macrolide antibiotics act as immunomodulators. In this study, we evaluated the effects of macrolide antibiotics on macrophage functions. For the macrophage, we used the mouse macrophage cell line J774.1. The following effects of macrolide antibiotics on macrophage functions were evaluated: the effect of macrolide antibiotics on macrophage growth; the phagocytosis of beads; cytocidal activity against Candida albicans; and chemotaxis to lipopolysaccharide (LPS). Macrolide antibiotics except for azithromycin significantly stimulated the growth of the macrophage. In addition, pretreatment with macrolide antibiotics except for roxithromycin significantly stimulated the macrophage phagocytosis of beads, macrophage chemotaxis to LPS, and macrophage cytocidal activity against Candida albicans. These results suggest that macrolide antibiotics stimulate macrophage functions.