Immunochemical Studies on Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase in the Chloroplasts of the Marine Alga Bryopsis maxima

The localization of ribulose 1,5-bisphosphate carboxylase/oxygenase(RuBisCO) in chloroplasts of the green alga Bryopsis maximawas examined by immunological techniques. Three strains of hybridomaswere established between myeloma cells and the spleen cellsfrom mouse immunized against B. maxima RuBisCO. The antibodiesreacted with the large subunit of B. maxima RuBisCO but notwith spinach RuBisCO. Immunofluorescence and immunoenzymaticstudies showed that the large subunit of B. maxima RuBisCO wasconcentrated in pyrenoids and on the surface of starch grainssurrounding the pyrenoids. (Received September 22, 1987; Accepted March 2, 1988)     

Isolation of Native Pyrenoid Core Matrix Having Ribulose 1,5-bisphosphate Caxrboxylase Activity from the Green Alga Bryopsis maxima

The native pyrenoid core matrix of the green alga Bryopsis maximawas isolated by diethyl ether treatment and sucrose densitygradient centrifugation using 1.8 M phosphate buffer. The purityof the pyrenoids was examined by microscopy, polyacrylamidegel electrophoresis and marker materials. The purified pyrenoidscontained the large subunit and the small subunit of ribulose1,5-bisphosphate carboxylase (RuBPCase) and more than 10 minorpolypeptides. They also showed RuBPCase activity when solubilizedon being transferred to a low-concentration buffer. The specificactivity was 0.62 µmol CO₂ fixed (mg protein)^–1min^–1. This isolation method is suitable for obtainingintact pyrenoids not covered by starch sheaths or membraneswithout the need for chloroplast fixation. (Received July 27, 1987; Accepted October 20, 1987)     

Adenylate cyclase and phosphodiesterase activities in relation to a rapid increase in cellular cAMP concentration by hypotonic shock indunaliella viridis

An adenylate cyclase activity of 16.02±1.03 pmol cAMP produced min⁻¹ (mg protein)⁻¹ was detected in a cell homogenate ofDunaliella viridis, a unicellular halotolerant green alga. It was present in both the membrane fraction and soluble fraction separated from the homogenate. Adenylate cyclase activity in the homogenate was activated by 1μM GTPγS but not by Ca²⁺+calmodulin, suggesting this enzyme to be regulated by a G-protein. A phosphodiesterase activity of 23.12±15.03 pmol cAMP decomposed min⁻¹ (mg protein)⁻¹ was found in the homogenate. These activities suggest the presence of a cAMP mediated signal transduction system inDunaliella. Cells, transferred from 1.7 M NaCl medium to 1 M NaCl, showed rapid increase in cAMP within 2 min to about 1.5 times the original concentration (from 2.4±0.2 to 3.9±0.2 pmol per 10⁸ cells) which was recovered in 30 min.     

Characterization of Leptospira species isolated from soil collected in Japan

Leptospira were isolated from soil obtained from Hokkaido, the northernmost island, to Okinawa, the southernmost island, of Japan using sulfamethoxazole, trimethoprim, amphotericin B, fosfomycin, and 5‐ fluorouracil. Fifty of 132 soil samples (37.9%) were culture‐positive. On the basis of 16S‐rDNA sequences, 12 of the isolated Leptospira were classified into a pathogenic species clade that is closely associated with L. alstonii and L. kmetyi. Nine isolates were classified as intermediate species and were found to be similar to L. licerasiae. Twenty‐seven isolates were classified as non‐pathogenic species, of which 23 were found to be related to L. wolbachii. Non‐pathogenic Leptospira are commonly distributed in environmental soil.     

High-efficiency transduction of human lymphoid progenitor cells and expression in differentiated T cells.

Gene therapy strategies for humans have been limited by low transduction efficiencies and poor expression of retroviral vectors in differentiated progeny cells carrying the transduced vector. Here we describe a strategy utilizing a cell surface reporter gene, murine thy-1.2, selectable by fluorescence-activated cell sorting (FACS), to achieve higher gene marking efficiencies. Human CD34-positive cells were transduced by a murine retroviral vector bearing the thy-1.2 marker and pseudotyped with vesicular stomatitis virus G protein, followed by FACS to enrich for CD34-positive cells that express Thy-1.2 on the cell surface. Gene marking and expression after differentiation into thymocytes were assessed in a SCID-hu Thy/Liv mouse model for human lymphoid progenitor cell gene therapy. We found that virtually all of the differentiated T-cell progeny were marked with vector sequences. It is of particular importance that reconstitution with the selected cells resulted in expression of Thy-1.2 in up to 71% of donor-derived thymocytes. It is of note that the donor-derived thymocytes that did not express Thy-1.2 still harbored vector thy-1.2 sequences, suggesting repression of transgene expression in some cells during progenitor cell differentiation into thymocytes. These studies provide a proof of concept for efficient expression of transgenes through T-lymphoid differentiation and a potential basis for utilizing similar strategies in human gene therapy clinical trials.     

Primary human immunodeficiency virus type 1 viremia and central nervous system invasion in a novel hu-PBL-immunodeficient mouse strain.

We established four new mouse strains with defective T and B cells as well as defects in innate immunological reactions using an NK cell depletion antibody and showed that all mutant mouse strains efficiently received human peripheral blood leukocyte (PBL) engraftment (hu-PBL-scid mice). Higher levels of human immunodeficiency virus type 1 (HIV-1) replication were observed in these new hu-PBL-scid mice than in conventional hu-PBL-C.B-17-scid mice. In one particular strain, hu-PBL-NOD-scid mice, high levels of HIV-1 viremia (more than 10(6) 50% infectious doses per ml) were detected after infection with HIV-1. The plasma viral load was about 100 to 1,000 times higher than that observed in other hu-PBL-scid mice infected with HIV-1. Although high-level viremia did not correlate with the total amount of HIV-1 RNA in cells from infected mice, high levels of free virions were detected only in hu-PBL-NOD-scid mice. HIV-1 viremia induced systemic HIV-1 infection involving the liver, lungs, and brain. PCR in situ hybridization confirmed that HIV-1-infected cells invaded the brain tissue of the hu-PBL-NOD-scid mice. Our results suggest that the genetic background, including innate immunity, is critical in the development of primary HIV-1 viremia and subsequent central nervous system invasion with HIV-1. The hu-PBL-NOD-scid mouse represents a useful model for the study of the pathogenesis of HIV-1 in vivo, especially brain involvement, and therapy of primary HIV-1 viremia.     

Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A,an evolutionarily and structurally conserved protein,to degrade ovine and caprine APOBEC3 proteins

Mammals have co‐evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti‐viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core‐binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi–Visna virus [MVV]). However, the co‐evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif‐mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co‐factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co‐factor in degradation of ovine and caprine APOBEC3.     

Interleukin-1 and tumor necrosis factor alpha can be induced from mononuclear phagocytes by human immunodeficiency virus type 1 binding to the CD4 receptor.

Cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) are important in normal immune processes. In this study, we demonstrate that human immunodeficiency virus type 1 (HIV-1) virions induce normal peripheral blood mononuclear phagocytes to produce both IL-1 and TNF within a few hours after their exposure to virus. The induction of these cytokines by HIV-1 does not require a productive infection. Blocking studies with soluble CD4 indicate that the effect is mediated through the CD4 molecule. In addition, the treatment of mononuclear phagocytes with OKT4A monoclonal antibody mimics the effects of HIV-1. Thus, these results indicate that induction of IL-1 and TNF alpha can occur via signals mediated through the CD4 molecule on mononuclear phagocytes. TNF has been shown by other investigators to induce HIV-1 expression. Therefore, TNF alpha may play a role in autocrine and paracrine regulation of HIV-1 expression. In addition, the induction of IL-1 and TNF by HIV-1 may also contribute to some of the neurologic and physiologic disorders associated with acquired immunodeficiency syndrome.     

A new light-induced absorbance change at 561 nm in chloroplasts of green alga, Bryopsis maxima

A new light-induced absorbance change having a maximum at 561nm was discovered in the thalli, as well as in isolated chloroplastsof a green alga, Bryopsis maxima Okamura. Another simultaneous change also occurred at 515 nm. The magnitudeof the 561 nm change was several-fold larger than that at 515nm and much larger than could be explained by an oxidation-reductionchange in cytochromes contained in chloroplasts. There was noabsorbance change in the Soret region that may be correlatedto the 561 nm change. Both 561 and 515 nm changes showed a spike-liketime course pattern, both having a half-rise time of about 20msec. Effects of inhibitors and uncouplers such as DCMU, Cl-CCPand gramicidin J on the absorbance change were also similarat 561 and at 515 nm. We inferred that the 561 nm change is related to photophosphorylationand possibly to the membrane potential in a way similar to the515 nm change. (Received March 27, 1974; )