Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors

Summary Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids. Offprint requests to: R. Schröder     

Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase.

The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.     

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2

Summary Monoclonal antibody 14G2a (anti-GD₂) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD₂. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagentN-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor and interferon and chemotherapeutic agents such as cisplatinum andN,N-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.Research conducted, in part, by the Clayton Foundation for Research     

Seasonal cycles of reserves in relation to reproduction inSebastes

Synopsis Seasonal cycles of reserve deposition and utilization in many fishes, amphibians and reptiles alleviate temporal mismatches of energy supply and demand. Utilization of reserves can be related to maintenance during periods of reduced food supply, to reproduction, particularly during periods of poor food availability, and to migration. Published data on the seasonal cycles of reserves and reproduction inSebastes suggest that reserves are important for maintenance during wintertime periods of low food availability. Unlike many other ectothermic vertebrates, some species ofSebastes deposit fat reserves at the same time as gametogenesis, a pattern consistent with the longevity and iteroparity evident in the genus. Other species ofSebastes have similar seasonal timing of fat cycles, but since reproduction takes place later in the year, the decline in reserves during winter coincides with the main period of reproductive activity. The significance of this is not clear. Interspecific differences in amounts of reserves may be related to geographical differences in the seasonality or abundance of food. Intraspecific variation in reserves, marked most strongly by allometry of reserves with regard to fish legth, bears further study, since it may link the proces of sexual maturation and the responses of repeat spawners to variability in food supply and other environmental factors.     

Relationship between sensitivity to 4'-(9-acridinylamino)methanesulfon-m-anisidide and DNA topoisomerase II in a cold-sensitive cell-cycle mutant of a murine mastocytoma cell line

The cold-sensitive (proliferating at 39.5 degrees C, reversibly arrested in GI-phase at 33 degrees C) cell-cycle mutant 21-Fb of the murine mastocytoma cell line P815 was used to study the effect of amsacrine on non-cycling cells. The sensitivity of arrested 21-Fb cells decreased less than 2-fold in cell survival experiments when compared to proliferating cells. In contrast, DNA breakage and stimulation of protein-DNA complex formation in intact or lysed cells was reduced approx. 10-fold in arrested cells and DNA topoisomerase II activity in arrested cells was only 5% of the activity in proliferating cells. Thus, there was no correlation between cell survival and DNA damage or DNA topoisomerase II activity in drug-treated cells.     

Stimulation of macrophage antibody-dependent killing of tumor targets by recombinant lymphokine factors and M-CSF

Macrophage colony-stimulating factor (M-CSF) was investigated as a stimulator of ADCC to the murine R1.1 thymoma target by murine peritoneal exudate macrophages which were elicited by proteose peptone. Both an 125IUdR release and a viable cell count assay were used. The latter assay avoids radiation damage, and the fate of the targets can be determined over a long period. Pretreatment of macrophages for several days in culture with lymphokine (LK) from concanavalin A-induced mouse spleen cells moderately stimulated ADCC. Preincubation of macrophages with conventional or recombinant human M-CSF or immunoaffinity-purified mouse M-CSF alone had little effect. However, M-CSF greatly enhanced ADCC to the tumor target when used as a costimulant with LK, IFN-gamma, IFN-alpha, IFN-beta, or IL-2 to pretreat macrophages. Incubation of macrophages with LK or LK plus M-CSF for 2 days generated stronger ADCC than 1- or 3-day incubations. Enhancement of LK-stimulated ADCC by M-CSF appeared to plateau at about 1000 U/ml. The enhancement of macrophage cytotoxicity when stimulated with IFNs or IL-2 was most effective at the lowest active concentration of these LKs. At 1 U/ml IFN-gamma or IL-2, or 5 U/ml IFN-alpha or IFN-beta, M-CSF boosted ADCC activity to that using 10-fold of the LK alone. IL-1, IL-4, and TNF had little or no stimulating activity for ADCC alone or with M-CSF, and the other hemopoietic growth factors IL-3 and GM-CSF did not promote this effector function alone or with IFN-gamma. We previously showed that M-CSF boosted macrophage antibody-independent killing of TU5 sarcoma targets with or without LK (Cell. Immunol. 105, 270, 1987). These studies thus show that M-CSF is a positive regulator of both macrophage-nonspecific tumor lysis and ADCC.     

Seed morphology under SEM and light microscopy in Kansas Juncus (Juncaceae)

Seed morphology was studied in 15 species of four subgenera ofJuncus occurring in Kansas, to determine if seeds provide traits useful in assessing systematic relations within the genus. In this study seed size and shape were of limited value, while surface ornamentation of the hard inner seed coat provided encouraging results. SubgenusPoiophylli showed little variation in surface ornamentation among taxa; similar ornamentation was observed in subgenusGenuini. SubgeneraGraminifolii andSeptati were separately distinct with the taxa in theSeptati forming a continuum of variation.     

Improved Agar Bottle Plate for Isolation of Methanogens or Other Anaerobes in a Defined Gas Atmosphere

A bottle plate for the cultivation of methanogens or other organisms in a defined pressurized-gas atmosphere was developed. The bottle provides the convenience of an agar streak plate, solves the problem of the water exudate from agar medium, and provides a convenient way of adding or sampling a defined gas atmosphere.     

Life Cycles in the Methanogenic Archaebacterium Methanosarcina mazei

Methanosarcina mazei S6 and LYC were used to study the structure and differentiation of the aggregating methanogens. Cultures harvested under various conditions are described at the ultrastructural level. Cells of strain S6 are enclosed by a layer 12 nm thick in contact with the plasma membrane. In sarcinal colonies, cells are held in close association by a fibrous matrix up to 60 nm thick. Colony maturation was examined in strain S6 over a period of 1 year. Changes occurred in the shape and staining of individual cells. Also, various inclusion bodies were observed that either persist throughout colony maturation or are only found at certain growth stages. Two types of cores that are composed of double membranes in M. mazei S6 are described. One has a 90-nm diameter and contains electron-dense granules similar to those found in the cytoplasm. The other core type does not contain granules, is more numerous, and is found in older cultures. Two life cycles are described for M. mazei based on electron microscope examinations. A complex life cycle involving the release of single cells is described with two variations for strains S6 and LYC. When released cells of strain S6 are placed in fresh medium they can repeat the cycle. In addition, a limited cycle is described for both strains of M. mazei. This limited cycle contains the only sarcinal morphotypes observed in M. barkeri. When M. mazei S6 remains in the limited cycle and does not disaggregate in stationary phase, several types of possible resting forms are found.