The cell growth-promoting factor.

It was recently shown that macromolecular serum proteins [1-3] as well as some of their hydrolyzed products, especially peptides of molecular weight around 5000[4] and even much less[5,6], are able to promote the growth of cells. This paper describes how the serum proteins were separated by salt precipitation and polyacrylamide electrophoresis into various albumin and globulin fractions and their growth-promoting activities ascertained. Subsequently, these macromolecules were treated with alkali, acids or proteolytic enzymes, and the activity of the products obtained was determined. We also isolated growth-promoting peptides from the liver by enzymatic hydrolysis, followed by gel filtration, or by ultrafiltration through Diaflo membranes.     

Development of a Quantitative Immunological Assay for the Study of Spore Coat Synthesis and Morphogenesis

A quantitative assay employing (125)I-labeled antibody has been developed for Bacillus cereus T spore coat protein. Populations of antibody molecules with various affinities for inner or outer coat can be prepared by selective adsorption to and elution from different coat preparations. Adsorption to and elution from intact spores results in an antibody preparation at least 15 times more reactive to outer coat. This antibody is useful for measuring the time and extent of spore coat maturation, i.e., outer spore coat formation. Rifampin inhibits the increase in content of this coat antigen.     

Depletion of NK cells with the lysosomotropic agent L-leucine methyl ester and the in vitro generation of NK activity from NK precursor cells

Human natural killer (NK) cell activity in peripheral blood lymphocytes (PBL) is totally inhibited by pretreatment of the effector cells with a lysosomotropic agent, L-leucine methyl ester (LeuOMe). This treatment specifically eliminates cells expressing the NK cell markers HNK-1, OKM1, B73.1, or Leu-11b, but has minimal effect on viability of cells with T cell markers Leu-1, OKT3, Leu-2a, or Leu-3a. LeuOMe also drastically decreased the proportion of K562 target-binding lymphocytes among PBL. PBL pretreated with LeuOMe respond normally in thymidine uptake to stimulation by phytohemagglutinin or allogeneic lymphocytes as long as irradiated autologous accessory cells are provided, indicating that the treatment is not toxic to T cells. NK activity can be regenerated in the NK cell-depleted PBL population by incubation with IL 2 or by mixed lymphocyte cultures, but not by alpha-interferon. Cells responsible for regeneration of such NK activity are probably large agranular lymphocytes, because they are resistant to LeuOMe treatment but have the same low buoyant density as NK cells in Percoll density gradient separation. The in vitro-generated NK is still sensitive to LeuOMe inhibition, but a higher concentration of the reagent is required to achieve total inhibition of the activity.     

Inhibition of mitogen-induced lymphocyte proliferation correlated to anticomplementary activity in sera from melanoma patients

Summary Sera from 98 melanoma patients and 90 normal donors were analyzed for antibody (Ab) to melanoma extracts, melanoma-associated antigen (Ag) and anticomplementary (Ac) activities by the microcomplement consumption technique. Sera were also tested for their ability to inhibit mitogen(phytohemagglutinin: PHA)-ininduced blastogenesis of normal lymphocytes. The results of the complement consumption assays were correlated with the results of inhibition of PHA-induced blastogenesis. Of 98 melanoma sera, 22% were Ab-positive, 30% were Ag-positive, and 44% were Ac-positive, in contrast to only 6% Ab-positive, no Ag-positive, and 7% Ac-positive in 90 normal sera. Fifty-nine percent of melanoma sera were inhibitory to PHA-induced blastogenesis, as against 12% of normal donors' sera. Ac-positive melanoma sera were significantly more inhibitory than Acnegative melanoma sera. The inhibitory activity of Acpositive sera was potentiated by the simultaneous presence of detectable Ag activity and was diminished by detectable Ab activity. Presence of Ab or Ag activity alone did not correlate with the inhibitory activity of the melanoma sera. Increasing incidence of Ac activity and ability to inhibit PHA-induced blastogenesis was observed with increasing tumor burden or advancing clinical stage. Sera from patients who displayed a delayed cutaneous hypersensitivity reaction to 2,4-dinitrochlorobenzene (DNCB) were less inhibitory to PHA-induced blastogenesis than the sera from patients who did not respond to this contact allergen. Thus, Ac activity may be one of the circulating factors responsible for the immunosuppressive effect of cancer patients' sera.     

Comparative investigation of structural and gene somatic mutations in workers of nuclear chemical plants. I. A study of stable and unstable chromosome aberrations

A study of frequency of unstable chromosome aberrations in 50 workers of nuclear chemical plants in remote period after beginning or finishing professional contact with ionizing radiation was carried out. 14 persons from this cohort were mainly whole-body exposed to external gamma-rays and 36 were exposed to combined external and internal radiation from incorporated Pu nuclides. In results of this irradiating practically every subject had a chronical radiation sickness. In the 1-st group the frequency of unstable aberrations varied from 0.2 to 3.6 per 100 cells and exceeded reliably control level in 5 persons. In the 2-nd group the frequency of unstable aberrations varied from 0 to 11.6 per 100 cells and exceeded reliably control level in 20 examined workers. The FISH study of frequency of stable aberrations was performed in 13 subjects who were exposed to combined external and internal radiation. Total frequency of complete and incomplete translocations varied from 0.6 to 18.5 aberrations per genome per 100 cells and reliable exceeded control level in 9 subjects. Non-random participation in exchange rearrangements (translocations) was revealed for used set of chromosomes (2, 3 and 8).     

PI(4,5)P2-dependent microdomain assemblies capture microtubules to promote and control leading edge motility

The lipid second messenger PI(4,5)P(2) modulates actin dynamics, and its local accumulation at plasmalemmal microdomains (rafts) might mediate regulation of protrusive motility. However, how PI(4,5)P(2)-rich rafts regulate surface motility is not well understood. Here, we show that upon signals promoting cell surface motility, PI(4,5)P(2) directs the assembly of dynamic raft-rich plasmalemmal patches, which promote and sustain protrusive motility. The accumulation of PI(4,5)P(2) at rafts, together with Cdc42, promotes patch assembly through N-WASP. The patches exhibit locally regulated PI(4,5)P(2) turnover and reduced diffusion-mediated exchange with their environment. Patches capture microtubules (MTs) through patch IQGAP1, to stabilize MTs at the leading edge. Captured MTs in turn deliver PKA to patches to promote patch clustering through further PI(4,5)P(2) accumulation in response to cAMP. Patch clustering restricts, spatially confines, and polarizes protrusive motility. Thus, PI(4,5)P(2)-dependent raft-rich patches enhance local signaling for motility, and their assembly into clusters is regulated through captured MTs and PKA, coupling local regulation of motility to cell polarity, and organization.     

Analysis of phosphorescence in heterogeneous systems using distributions of quencher concentration.

A continuous distribution approach, instead of the traditional mono- and multiexponential analysis, for determining quencher concentration in a heterogeneous system has been developed. A mathematical model of phosphorescence decay inside a volume with homogeneous concentration of phosphor and heterogeneous concentration of quencher was formulated to obtain pulse-response fitting functions for four different distributions of quencher concentration: rectangular, normal (Gaussian), gamma, and multimodal. The analysis was applied to parameter estimates of a heterogeneous distribution of oxygen tension (PO2) within a volume. Simulated phosphorescence decay data were randomly generated for different distributions and heterogeneity of PO2 inside the excitation/emission volume, consisting of 200 domains, and then fit with equations developed for the four models. Analysis using a monoexponential fit yielded a systematic error (underestimate) in mean PO2 that increased with the degree of heterogeneity. The fitting procedures based on the continuous distribution approach returned more accurate values for parameters of the generated PO2 distribution than did the monoexponential fit. The parameters of the fit (M = mean; sigma = standard deviation) were investigated as a function of signal-to-noise ratio (SNR = maximum signal amplitude/peak-to-peak noise). The best-fit parameter values were stable when SNR > or = 20. All four fitting models returned accurate values of M and sigma for different PO2 distributions. The ability of our procedures to resolve two different heterogeneous compartments was also demonstrated using a bimodal fitting model. An approximate scheme was formulated to allow calculation of the first moments of a spatial distribution of quencher without specifying the distribution. In addition, a procedure for the recovery of a histogram, representing the quencher concentration distribution, was developed and successfully tested.     

Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates

Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chiken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis do not appear to be species specific, but intrinsic to the meiotic process.     

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE³⁷³ neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.