Monoclonal anti-mouse macrophage antibodies recognize the globular portions of C1q, a subcomponent of the first component of complement

One of seven monoclonal antibodies generated against mouse macrophages (M phi) was found to recognize isolated heterologous C1q. This antibody was shown to be cytotoxic and to react in a strain-independent way with mouse M phi derived from bone marrow cells as well as with M phi from the peritoneal cavity; it did not react, however, with mouse granulocytes, thymocytes, or T and B lymphocytes. The hemolytic activity of fluid phase C1q was inhibited to 50% at a 2 X 10(-4) dilution of hybridoma supernatant, whereas a 100-fold higher concentration was required to inhibit C1q bound to immune complexes ( EAC1q ) to the same extent. It was demonstrated that this antibody recognizes the isolated globular, Fc-binding portions of the C1q molecule and reacts with the A and B chains. Because M phi have been shown to synthesize C1q, the Fc-recognizing subcomponent of the first component of complement, evidence was provided that endogeneous C1q can serve as an Fc receptor on M phi during secretion. This fact was demonstrated by a dose-dependent inhibition of Fc-receptor activity for EIgG by the F(ab')2 fragment of this monoclonal antibody. These experiments further support the concept that C1q produced by M phi functions on the surface as an Fc-recognizing molecule before it is released and incorporated into the macromolecular complex of serum C1.     

Inactive-state preassembly of G(q)-coupled receptors and G(q) heterotrimers

G protein-coupled receptors (GPCRs) transmit signals by forming active-state complexes with heterotrimeric G proteins. It has been suggested that some GPCRs also assemble with G proteins before ligand-induced activation and that inactive-state preassembly facilitates rapid and specific G protein activation. However, no mechanism of preassembly has been described, and no functional consequences of preassembly have been demonstrated. Here we show that M(3) muscarinic acetylcholine receptors (M3R) form inactive-state complexes with G(q) heterotrimers in intact cells. The M3R C terminus is sufficient, and a six-amino-acid polybasic sequence distal to helix 8 ((565)KKKRRK(570)) is necessary for preassembly with G(q). Replacing this sequence with six alanine residues prevents preassembly, slows the rate of G(q) activation and decreases steady-state agonist sensitivity. That other G(q)-coupled receptors possess similar polybasic regions and also preassemble with G(q) suggests that these GPCRs may use a common preassembly mechanism to facilitate activation of G(q) heterotrimers.     

In vitro plant regeneration from callus derived from root explants of Lathyrus sativus

Protocols have been developed for the in vitro production of plants from callus derived from root explants of Lathyrus sativus cv. P-24. Callus and shoot regeneration were achieved only in MS medium supplemented with 10.7 M naphthaleneacetic acid and an increased concentration of kinetin (0.9 M for 14 days to 1.4 M for 18 days) during callusing. The shoots obtained rooted in 1/2 MS supplemented with 0.5 M indolebutyric acid. During the year plants have been regenerated several times. The requirement for growth regulators is very specific and narrow.Abbreviations IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - BA benzyladenine     

Expression,characterization, and reaction of recombinant monkey metallothionein-1 and its C33M mutant

After we modified the protocol of purification, monkey metallothionein-1 (mkMT-1) and its mutant at position 33 (C33M mutant) were efficiently expressed and purified by using the glutathione-S-transferase fusion protein system. The protein yield has been considerably improved (8 mg/L culture for mkMT-1 and 10 mg/L culture for C33M mutant). The recombinant MT-1 and C33M mutant were characterized by ESI-MS, UV, and CD spectra. The reactions of MI-1 and C33M mutant with 5,5-dithiobis(2-nitrobenzoic acid) and EDTA also have been carefully studied. The pH titration of MT-1 and C33M mutant has been studied by UV and CD spectra. The mutation of cysteine-to-methionine at position 33 mostly maintains the -domain structure similar to that in wild-type mkMT-1, but the C33M mutant has significant loss of stability and cooperative properties of the domain.     

Myopathic Mutations Affect Differently the Inactivation of the Two Gating Modes of Sodium Channels

Three groups of mutations of the subunit of the rat skeletal muscle sodium channel (rSkM1),homologous to mutations linked to human muscle hereditary diseases, have been studied byheterologous expression in frog oocytes: S798F, G1299E, G1299V, and G1299A, linked withpotassium-aggravated myotonia (PAM); T1306M, R1441C and R1441P, linked withparamyotonia congenita (PC); T698M and M1353V, linked with the hyperkalemic periodic paralysis(HyPP). Wild-type rSkM1 channels (WT) show two gating modes, M1 and M2, which differmainly in the process of inactivation. The naturally most representative mode M1 is tenfoldfaster and develops at 30 mV less depolarized potentials. A common feature ofmyopathy-linked mutants is an increase in the mode M2 probability, P_M2, but phenotype-specific alterationsof voltage-dependence and kinetics of inactivation of both modes are also observed. Thecoexpression of the sodium channel ₁ subunit, which has been studied for WT and for thefive best expressing mutants, generally caused a threefold reduction of P_M2 without changingthe properties of the individual modes. This indicates that the mutations do not affect the – ₁ interaction and that the phenotypic changes in P_M2 observed for the enhanced mode M2behavior of the sole subunits, although largely depressed in the native tissue, are likely to bethe most important functional modification that causes the muscle hyperexcitability observed inall patients carrying the myotonic mutations. The interpretation of the more phenotype-specificchanges revealed by our study is not obvious, but it may offer clues for understanding the differentclinical manifestations of the diseases associated with the various mutations.     

Regeneration and large-scale propagation of Phragmites communis through somatic embryogenesis

A protocol for large-scale propagation of Phragmites communis Trin. by somatic embryogenesis has been established. Plants were regenerated through somatic embryogenesis from stem segments of R5002-12, a salt-tolerant variant line of Phragmites communis Trin. Stem segment explants produced hard white callus on the semi-solid Murashige and Skoog (MS) medium supplemented with 9.05 M 2,4-dichlorophenoxy acetic acid (2,4-D) for 4 weeks. The induction frequency was 36.7%. Then, the callus was transferred to MS medium supplemented with 4.52 M 2,4-D. After 4 weeks in culture, yellow embryogenic callus with some nodular structures was formed. When the embryogenic callus was transferred to differentiation medium (MS supplemented with 0.45 M 2,4-D), differentiation was initiated to form small green islands on the surface of the callus after 2 weeks in culture. Within 4 weeks, a large number of somatic embryos were formed with a frequency of 86.7%. Six weeks later, they developed into strong plantlets. When the plantlets (about 1 cm in length) were cultured on propagation medium (MS supplemented with 13.31 M BA+5.37 M NAA), a great number of regenerated plants were obtained. After the plants were cultured on liquid 1/2 MS medium with 2.69 M NAA added 2.46 M IBA roots developed. The rooted plants were transferred to soil with over 85% survival. Using this methodology, more than 20000 regenerated plants of salt tolerant variant line of Phragmites communis Trin. have been produced.     

Asymmetric diffusion into the postsynaptic neuron: An extremely efficient mechanism for removing excess GABA from synaptic clefts on the Deiters' neurone plasma membrane

Microdissected Deiters' neuron plasma membranes have been used for studying the passage of GABA through the membrane both in the inward and outward direction. Working with 0.2 mM GABA in the compartment simulating the outside of the neurone and with 2.0 mM GABA in the one simulating the inside we found a net transport of GABA towards the inside. This mechanism does not require a Na⁺ ion gradient across the membrane. The nature of the transport process involved was studied by determining the rate of [³H]-GABA inward passage as a function of GABA concentration (1 nM–800 M) on the outward side of the membrane. The results have shown that until 50 M a diffusion process (v=D₁×C, where D₁=3.1×10^–11 1/m²×sec) is the sole mechanism involved. Above 50 M a second diffusion process is activated v=D₂×(C–50×10^–6), where D₂=2.8×10^–11 1/m²×sec. Taking in account both inward and outward directed diffusion, one can calculate 16 M as the equilibrium concentration of GABA on the outward side of the membrane. From a kinetic point of view, these diffusion processes are able to reduce GABA concentration in a synaptic cleft from 3 mM to 20 M within 3 sec. These diffusion systems are discussed as extremely efficient in removing the excess of released GABA in the synaptic cleft.     

Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q

The localization to 19q of the gene causing myotonic dystrophy (DM) has been defined more precisely by refinement of the physical location of several linked markers. A somatic cell hybrid mapping panel from cells with t(1;19), t(12;19), and t(X;19) translocation products was constructed to define five different intervals across 19q. In addition, we have derived a series of cell hybrids by irradiation of a der(19)-only hybrid to further subdivide the cen-q13.1 region. Using an array of 36 cloned genes, anonymous DNAs, and enzyme markers, we have tested the location of the panel breakpoints and refined the regional assignment of several of these markers. All markers tightly linked to DM are localized mainly within 19q13.2, thus suggesting that the DM gene is also close to this region.     

三种龟类动物的细胞遗传研究

本文以外周血淋巴细胞为材料,首次报道马来闭壳龟和地龟的核型,Ｇ带,Ｃ带和Ａｇ－ＮＯＲｓ,以及平胸龟的Ｇ带和Ｃ带,发现平胸龟的核型与前人报道的有差异。研究结果表明：平胸龟２ｎ＝５４（１４Ｍ＋４ＳＴ＋８Ｔ＋２８ｍ）,Ｎ．Ｆ．＝７２,７＋６＋１４。Ａ组Ｎｏ．６ｑ ｐｅｒ有一次缢痕。Ａｇ－ＮＯＲｓ位于Ａ组的Ｎｏ．７ ｑ ｔｅｒ。其全部染色体的着丝粒区均显示阳性Ｃ带,并且Ａ组的Ｎｏ．７整条异染色质化;马来闭     

Callus induction and plantlet regeneration in ornamental Alocasia micholitziana

Callus induction from petiole explants has been achieved in Alocasia micholitziana `Green Velvet'. The highest percentage (71%) of explants inducing callus was obtained on MS medium supplemented with 0.5 M 2,4-D and 0.5 M kinetin in the dark after 4 months of culture. Shoots were regenerated at the highest frequency of 33.3% under light condition when 0.5 M BA was added to MS medium with the average of 7.8±2.3 shoots per callus explant. The callus-derived shoots rooted on hormone free MS medium and within 4 weeks the plantlets were ready for acclimatization. The regenerated plants appeared morphologically similar to mother plants.     

Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into palsma cells. CD19 is a member of Ig gnee superfamily with two extreacellular Ig-like domains separated amino acid cytoplasmic domain. In this study, Southern blot analysis revelaed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned 8 kilobases (kb) of DNA in human and 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5 of the putative translation initiation AUG codon as well conserved in sequence between human and mouse and contained potential trasncription regulatory elements. In addition, the 3 untranslated regions (UT) of the CD19 genes following the termination codon were conservedf in sequence. The high level conservation of nucleotide sequences between species in all exons and 5 and 3 UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.The nucleotide sequence database reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: human CD19 gnee, M62544 to M62550; mouse CD19 gene, M62551 to M62553.     

Comparative gene assignment in Ateles paniscus chamek (Platyrrhini, Primates) and man: association of three separate human syntenic groups and evolutionary considerations

Regional assignment of eight markers to chromosome 2 of Ateles paniscus chamek (APC) confirmed a syntenic association similar to human (HSA) 12q + 14q + 15q. Three HSA 12q markers (RAP1B, PAH and ALDH2) were allocated to a shortest region of overlap (SRO) in APC 2p and found to be syntenic to other HSA 12q markers (PEPB and TCF1). Five HSA 14q markers (CTLA, PAX9, NSP, FOS and CHGA) were allocated to APC 2q and found to be syntenic to other HSA 14q markers (NP, TGM1, and CALM1) and to four HSA 15q markers (THBS1, B2M, HEXA and MPI) but dissociated from markers close to HSA 14qter (CKB) and HSA 15qter (FES-IDH2). Karyotypic comparisons showed an evident homoeology between APC 2p and HSA 12q while APC 2q was similar to an HSA 14qter::HSA 15qter fusion product. Comparative gene mapping data show that the HSA 14q + HSA 15q syntenic association is an ancestral mammalian gene cluster that has been maintained in several primate taxa. Conversely, in Ateles, it has been further associated with HSA 12q while, in Hominoids and Cebus, it has been independently dissociated into two separate syntenic groups, similar to HSA 14q and HSA 15q. Received: 24 October 1997; in revised form: 10 December 1997 / Accepted: 20 December 1997     

Extra copy of 20q deletion, acute myelomonocytic leukemia (M4) and Niemann-Pick disease

A 59-year-old hypertensive white male was diagnosed with acute myelogenous leukemia (AML), M4. A bone marrow aspirate showed a karyotype of 46,XY,del(20)(q11.2q13.3)[12]/ 47,XY,del(20)(q11.2q13.3)x2[8]. The majority of cases with 20q deletion are associated with myeloid disorders; however, an extra copy of the 20q deletion has rarely been reported. The patient expired seven days after admission to the hospital. At autopsy hepatosplenomegaly was present. Many foamy macrophages with bubbling cytoplasm in the spleen, liver, bone marrow and lymph nodes were suggestive of Niemann-Pick disease, type E. AML has not previously been reported with Niemann-Pick disease.     

Growth inhibition of hopanoid synthesizing bacteria by squalene cyclase inhibitors

2,3-Dihydro-2-azasqualene, its N-oxide and its N,N-diethyl analogue, as well as 2,3-dihydro-2,3-iminosqualene are potent inhibitors of the squalene to hopanoid (diplotene and diplopterol) cyclases in cell-free systems from Acetobacter pasteurianus ssp pasteurianus, Methylobacterium organophilum and Zymomonas mobilis. The inhibitory concentration giving 50% inhibition at a 120 M squalene concentration was determined in each cases. The growth of hopanoid producing prokaryotes (with the exception of Acetobacter pasteurianus ssp pasteurianus and Pseudomonas syringae probably capable of degrading the drugs) was inhibited by these squalene analogues at concentrations in the M range, whereas the growth of hopanoid non-producers was not affected at the highest tested concentration (200 M). Thus hopanoids which have been shown to possess similar properties to those of sterols in membrane reinforcement are probably essential for the cells producing them. Furthermore, all tested hopanoid producers are very sensitive to trimethyloctadecyl ammonium bromide which does not inhibit the squalene to hopane cyclases at a 50 M concentration, and do not grow after 24 h in its presence at a 1 M minimal inhibitory concentration. Growth of hopanoid non-producers was however not affected by this ammonium salt (highest tested concentration: 200 M). The mode of action of this cationic detergent is still unknown, but might be related to specific desorganization of hopanoid containing membranes.Abbreviation TLC thin layer chromatography     

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.     

Chromosomal Mapping of the Human M6 Genes

M6 is a neuronal membrane glycoprotein that may have an important role in neural development. This molecule was initially defined by a monoclonal antibody that affected the survival of cultured cerebellar neurons and the outgrowth of neurites. The nature of the antigen was discovered by expression cDNA cloning using this monoclonal antibody. Two distinct murine M6 cDNAs (designated M6a and M6b) whose deduced amino acid sequences were remarkably similar to that of the myelin proteolipid protein were previously isolated. We have isolated partial human cDNA and genomic clones encoding M6a and M6b and have characterized them by restriction mapping, Southern hybridization with cDNA probes, and sequence analysis. We have localized these genes within the human genome by FISH (fluorescencein situhybridization). The human M6a gene is located at 4q34, and the M6b gene is located at Xp22.2. A number of human neurological disorders have been mapped to the Xp22 region, including Aicardi syndrome (MIM 304050), Rett syndrome (MIM 312750), X-linked Charcot–Marie–Tooth neuropathy (MIM 302801), and X-linked mental retardation syndromes (MRX1, MIM 309530). This raises the possibility that a defect in the M6b gene is responsible for one of these neurological disorders.     

A novel translocation,t(9;21)(q13;q22) rearranging the RUNX1 gene in acute myelomonocytic leukemia

A novel translocation t(9;21)(q13;q22) associated with trisomy 4 has been detected in a patient with acute myelomonocytic leukemia (AML,M4) in relapse. The chromosomal translocation results in rearrangement of the RUNX1 gene at 21q22. The DNA sequence rearranged on chromosome 9 remains unidentified. The diversity of the partners involved in translocations implicating RUNX1 suggests that the functional consequences of the abnormality are more due to the truncation of RUNX1 than to the identity of its partner in the rearrangement.     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid