B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

Regulation of Histamine Turnover via Muscarinic and Nicotinic Receptors in the Brain

To clarify the regulation of central histaminergic (HAergic) activity by cholinergic receptors, the effects of drugs that stimulate the cholinergic system on brain histamine (HA) turnover were examined, in vivo, in mice and rats. The HA turnover was estimated from the accumulation of tele-methylhistamine (t-MH) during the 90-min period after administration of pargyline (65 mg/kg, i.p.). In the whole brain of mice, oxotremorine, at doses higher than 0.05 mg/kg, s.c., significantly inhibited the HA turnover, this effect being completely antagonized by atropine but not by methylatropine. A large dose of nicotine (10 mg/kg, s.c.) also significantly inhibited the HA turnover. This inhibitory effect was antagonized by mecamylamine but not by atropine or hexamethonium. A cholinesterase inhibitor, physostigmine, at doses higher than 0.1 mg/kg, s.c., significantly inhibited the HA turnover. This effect was antagonized by atropine but not at all by mecamylamine. None of these cholinergic antagonists used affected the steady-state t-MH level or HA turnover by themselves. In the rat brain, physostigmine (0.1 and 0.3 mg/kg, s.c.) also decreased the HA turnover. This inhibitory effect of physostigmine was especially marked in the striatum and cerebral cortex where muscarinic receptors are present in high density. Oxotremorine (0.2 mg/kg, s.c.) and nicotine (1 mg/kg, s.c.) also decreased the HA turnover in the rat brain. However, these effects showed no marked regional differences. These results suggest that the stimulation of central muscarinic receptors potently inhibits the HAergic activity in the brain and that strong stimulation of central nicotinic receptors can also induce a similar effect.     

Purification and characterization of the active serine: pyruvate aminotransferase of rat liver mitochondria expressed in Escherichia coli

In the previous study (Oda, T., et al. (1985) Eur. J. Biochem. 150, 415-421), we isolated a cDNA clone which expressed in Escherichia coli a specific size of product having the activity of rat liver serine:pyruvate aminotransferase (SPTm). This specific product (SPT10) was purified to homogeneity through three different column chromatographies. The amino acid composition and N-terminal amino acid sequence of the purified enzyme agreed with those predicted from the nucleotide sequence of cDNA and showed that SPT10 consists of the whole amino acid sequence of mature SPTm and several extra amino acid residues at the N-terminus. The catalytic and physical properties of SPT10, such as substrate specificity, Km for alpha-keto acids, electric charge, and quaternary structure, were all very similar to those of SPTm. Using several cDNA clones which lack a 5'-terminal sequence corresponding to a portion of the N-terminal amino acid sequence of SPTm, we examined the expression profile of the specific product in bacteria transformed with each cDNA clone. The products encoded by these cDNAs were segregated into inclusion bodies and were neither catalytically active nor easily solubilized by sonication. In contrast, the inclusion bodies were not formed in the bacteria transformed with the cDNA clone for SPT10.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Rapid Increase of Pneumococcal Resistance to β-Lactam and Other Antibiotics in Isolates from the Respiratory Tract (Nagasaki,Japan: 1975–1994)

The susceptibility of 101 pneumococcal isolates from the respiratory tract during 1991–1994 was examined and compared with the susceptibility of isolates over the period of 1975–1990. A rapid increase of resistance was seen not only to penicillin but also other antimicrobial agents. During 1991–1994, 38% of all the isolates were resistant to penicillin. The rates of resistance during this period were 16–23% for three newer cephalosporins, 18% for imipenem, 69% for tetracycline, 31% for erythromycin, 20% for chloramphenicol and 9% for clindamycin. The use of antibiotics within one month prior to pneumococcal isolation was correlated with penicillin resistance (P < 0.05). Serotyping of the isolates by antiserum revealed differences in predominant types between penicillin-resistant (19F, 23F, 4) and -susceptible isolates (15, 4, 11A). Our data suggests that anti-pneumococcal antibiotics should be carefully chosen on the basis of susceptibility tests.     

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.     

Parental investment and offspring sex ratio in a solitary bee,Anthidium septemspinosum lepeletier (Hymenoptera: Megachilidae)

Anthidium septemspinosum is a solitary, tube-nesting bee. Studies onA. septemspinosum were made to investigate how offspring sex ratios were influenced by maternal conditions. Males were generally larger than females, indicating that parental investment between sexes differed. Body size was related to male mating success, but was not related to nesting success of females. Large and young mother bees, who had more ability to invest, invested more in male offspring while small and old mothers invested more in female offspring. These results indicate that mother bees of this species are able to adaptively manipulate offspring sex ratio in relation to their ability to invest in offspring, as predicted in mammals by Trivers & Willard (1973).