Growth-stimulatory action of plasma membrane-associated growth factor. A synergistic effect with platelet-poor plasma

Growth factor activity was partially purified from mouse liver plasma membranes and its growth-stimulatory action on cultured mouse fibroblasts was studied. The plasma membrane-associated growth factor (PMGF) was unable to support the proliferation of mouse fibroblasts in monolayer when added as the sole source of growth factor. However, it stimulated the growth of fibroblasts in the presence of CM-Sephadex-treated human platelet-poor plasma (h-CMP) which by itself is not growth-stimulatory. The stimulation of DNA synthesis in quiescent fibroblasts was also observed upon the addition of PMGF and h-CMP. Under the same conditions, both platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) showed the same effect as did PMGF. The synergistic action of h-CMP with PMGF on quiescent cells was partially reproduced by insulin at microgram quantities or by insulin-like growth factor I(IGF-I) at nanogram quantities. Thus, the data presented here indicates that the action of PMGF is similar to that of the family of growth factors termed 'competence factor', and distinct from that of plasma growth factors termed 'progression factor'.     

Isolation and Characterization of a Cyanophage Infecting the Toxic Cyanobacterium Microcystis aeruginosa

We isolated a cyanophage (Ma-LMM01) that specifically infects a toxic strain of the bloom-forming cyanobacterium Microcystis aeruginosa. Transmission electron microscopy showed that the virion is composed of anisometric head and a tail complex consisting of a central tube and a contractile sheath with helical symmetry. The morphological features and the host specificity suggest that Ma-LMM01 is a member of the cyanomyovirus group. Using semi-one-step growth experiments, the latent period and burst size were estimated to be 6 to 12 h and 50 to 120 infectious units per cell, respectively. The size of the phage genome was estimated to be ca. 160 kbp using pulse-field gel electrophoresis; the nucleic acid was sensitive to DNase I, Bal31, and all 14 restriction enzymes tested, suggesting that it is a linear double-stranded DNA having a low level of methylation. Phylogenetic analyses based on the deduced amino acid sequences of two open reading frames coding for ribonucleotide reductase alpha- and beta-subunits showed that Ma-LMM01 forms a sister group with marine and freshwater cyanobacteria and is apparently distinct from T4-like phages. Phylogenetic analysis of the deduced amino acid sequence of the putative sheath protein showed that Ma-LMM01 does not form a monophyletic group with either the T4-like phages or prophages, suggesting that Ma-LMM01 is distinct from other T4-like phages that have been described despite morphological similarity. The host-phage system which we studied is expected to contribute to our understanding of the ecology of Microcystis blooms and the genetics of cyanophages, and our results suggest the phages could be used to control toxic cyanobacterial blooms.     

Different effects of gellan gum and agar on change in root elongation in Arabidopsis thaliana by polyploidization: the key role of aluminum

Journal of Plant Research - Agar and gellan gum have been considered to have different effects on polyploidy-dependent growth in plants. We aim to demonstrate that agar and gellan gum differently...     

Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization

We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min. The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.     

Establishment of a monoclonal antibody to human myeloma cell: relation to chemotherapy and extramedullar infiltration

Resistance of myeloma cells to melphalan (L-PAM) is a serious problem. To investigate mechanisms of drug resistance, we generated a monoclonal antibody, clone O3, to melphalan-resistant myeloma cells, KHM-11R. Western blot analysis showed that molecular weight of O3 antigen was approximately 90 kDa. Expression of O3 antigen was approximately two times higher in KHM-11R than in parental melphalan sensitive cell line, KHM-11. O3 was preferentially expressed in plasma cell, B-cell, and monocytic cell lines, but not in T-cell lines. Analysis of bone marrow samples from myeloma patients revealed that 13 of 23 samples expressed O3 antigen at various levels, and that O3 antigen expression in patients correlate with preceding chemotherapy, advanced clinical stage and extramedullar invasion of myeloma cells. Furthermore, patients expressing O3 antigen at the time of diagnosis tended to have poor prognosis. The investigation of O3 antigen in myeloma cells will be useful to reveal the pathophysiology of extramedullar invasion and the mechanism of cell killing by melphalan.     

Fast release of calcium from sarcoplasmic reticulum vesicles monitored by chlortetracycline fluorescence

Rapid Ca2+ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free Ca2+ concentration. Ca2+ efflux was activated by extravesicular Ca2+ with an apparent dissociation constant of 25 microM and was inhibited with an inhibition constant of 120 microM in the absence of Mg2+. Caffeine enhanced the Ca2+ release rate by increasing only the affinity of Ca2+ for the activation site. Mg2+ reduced the Ca2+ release rate by competitive binding to the activation site. ATP increased the Ca2+ release rate very much without changing the affinities of Ca2+ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca2+ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca2+ release in the presence of ATP reached 80 s-1. This value is considered to be sufficient to cause muscular contraction.     

Ryanodine sensitivity of the calcium release channel of sarcoplasmic reticulum

Ryanodine modulates Ca2+ permeability in isolated terminal cisternae of sarcoplasmic reticulum, suggesting that it is a specific ligand for the calcium release channel. Our laboratory has purified the ryanodine receptor and demonstrated it to be equivalent to the feet structures, which are involved in the junctional association of the transverse tubule with the terminal cisternae. Recently, Smith, Coronado and Meissner have incorporated sarcoplasmic reticulum into bilayers and found a high conductivity channel (approximately .100 pS) which has a number of characteristics expected of the Ca2+ release channels in SR. We now find that the high conductivity channel in the bilayer is sensitive to ryanodine. Low concentrations of ryanodine (sub microM): (1) lock the channels in an open state; (2) prevent the action of ruthenium red (microM) to completely close the channel; and (3) much higher concentrations of ryanodine (300 microM) close the channel. In these three respects ryanodine acts similarly on the channel in the bilayer as in vesicles. Further, the bilayer studies provide new insight into the action of ryanodine on the channel in that: (1) ryanodine locks the channel in the open state, but the conductivity is reduced to about 40%; (2) ryanodine prevents ruthenium red from closing the channel, although there is a further decrease in the open current. These studies provide support that the high conductivity calcium channel in sarcoplasmic reticulum is involved in excitation-contraction coupling. By the same token the pharmacological action of ryanodine is pinpointed to the calcium release channel.     

Existence of phosphoenolpyruvate: carbohydrate phosphotransferase systems in Lactobacillus fermentum, an obligate heterofermenter.

The presence or absence of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) in obligately heterofermentative group III lactobacilli including Lactobacillus brevis (3 strains), L. buchneri (2 strains) and L. fermentum (3 strains) was surveyed systematically for a series of sugars utilizable by these organisms. Contrary to common expectation, PTSs were found in two strains of L. fermentum: sucrose-PTS in one strain; sucrose- and mannose-PTSs in the other. All these activities were found to be constitutive.