Metabolically inactive 3T3 cells can substitute for marrow stromal cells to promote the proliferation and development of multipotent haemopoietic stem cells

When highly enriched multipotential spleen colony forming cells (CFU-S) obtained following fluorescence activated cell sorting (FACS-CFU-S) are cultured on marrow stromal cells, they undergo proliferation and development to produce mature haemopoietic cells (Spooncer et al., Nature, 316:62-64, 1985). We now show that FACS-CFU-S behave in a similar way when cultured on monolayers of 3T3 cells, indicating that the 3T3 cells can supply at least part of the environment which is representative of marrow stromal cells and provide, therefore, a system for studying stromal cell: haemopoietic cell interactions. We also demonstrate that IL-3-dependent multipotential stem cell lines (FDCP-Mix), but not a variety of other "committed" IL-3-dependent cell lines, resemble FACS-CFU-S in terms of their ability to proliferate and differentiate when cultured on 3T3 cells in the absence of IL-3. In this system, attachment of the FDCP-Mix to the 3T3 cells is critical for the subsequent maintenance of viability and stimulation of development of the cells. When the FDCP-Mix cells are physically separated from the 3T3 cells, they die and their death cannot be prevented by using 3T3-cell-conditioned medium. The extracellular matrix generated by 3T3 cells is not sufficient for promoting attachment or viability of the FDCP-Mix cells, indicating the importance of integral membrane components. However, attachment and development of FDCP-Mix cells occurs on 3T3 cells that have been lightly fixed with glutaraldehyde indicating that active metabolism is not essential for the effects promoted by the 3T3 cells. We suggest that the ability of FACS-CFU-S and FDCP-Mix cells to respond to 3T3 cells involves specific ligand/receptor interactions.     

Characterization of the phospholipid and fatty acid composition of Sendai virus

The lipid composition of Sendai virus, propagated in chicken eggs, was analyzed by high performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and gas-liquid chromatography (GLC). Phosphatidylcholine was found to be the dominant phospholipid (37.3%) with phosphatidylethanolamine (26.8%) and phosphatidylserine (12.0%) also present in significant amounts. Analysis of the fatty acid methyl esters revealed that the dominant fatty acids in total phospholipid were: C16:0 (17.6%), C18:0 (15.4%), C18:1 (n-9) (22.0%), and C24:0 (6.0%). Cardiolipin, phosphatidylserine, and sphingomyelin contained higher levels of saturated fatty acids relative to phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine.     

Behavioral responses of red hake,Urophycis chuss,to decreasing concentrations of dissolved oxygen

Synopsis Laboratory experiments were conducted to examine changes in behavior of red hake,Urophycis chuss, under decreasing concentrations of dissolved oxygen (DO). Since the ecological requirements of this species change with age, responses were measured for three different groups: (1) age 0+, = 89 mm total length (TL); (2) age 1+, = 238 mm TL; and (3) age 2–3+, = 397 mm TL. As DO decreased from 8–10 mg l^-1 to < 0.5 mg l^-1, changes were evident in active time, water column activity, range of horizontal movement, food searching, and agonistic behavior. Age 0+ fish were most sensitive, moving up into the water column and swimming continuously as DO levels fell below 4.2 mg l^-1. Age 2–3+ fish were the least responsive, remaining on the substrate and increasing only their range of movement at concentrations below 3 mg l^-1. Responses of age 1 + fish were variable, possibly reflecting a transition stage between the younger and older fish. Common to all groups was the decrease and eventual cessation of food searching.     

Sex determination from the radius in humans

The discriminatory capacity of the radius in sex determination was investigated in a Dutch skeletal collection of recent origin. Midshaft subperiosteal diameter, maximum length and maximum transverse distal width were measured from roentgenograms. The discriminatory capacity of the radius was found to be of the same order as that reported in the literature for the other long bones. Maximum transverse distal width showed the highest consistency (85%) between estimated and documented sex. This method requires only the presence of the distal fragment of the radius.     

Pathophysiology of pH and Ca2+ in bloodstream and brain

The highlights of the literature and our work on tetany and hyperventilation are reviewed. Our studies concern the following: (1) the changes of [Ca2+] in circulating plasma caused by respiratory and "metabolic" acidosis and alkalosis; (2) critical plasma [Ca2+] levels associated with signs of tetany and neuromuscular blockade; (3) changes in cerebral [Ca2+]o caused by hypo- and hyper-calcaemia, and the changes in cerebral [Ca2+]o and pHo caused by acute systemic acidosis and alkalosis; and (4) effects of changing [Ca2+]o and pHo levels on synaptic transmission in hippocampal formation. Our main conclusions are (1) changes of plasma [Ca2+] caused by "metabolic" pH changes are greater than those associated with varying CO2 concentration; (2) acute systemic [Ca2+] changes are associated with small cerebral [Ca2+]o changes; (3) the decreases in systemic and cerebral [Ca2+]o caused by hyperventilation are too small to account for the signs and symptoms of hypocapnic tetany; (4) moderate decrease of [Ca2+]o depresses and its increase enhances synaptic transmission in hippocampal formation; and (5) H+ ions in extracellular fluid have a weak depressant effect on neuronal excitability. CO2 is a strong depressant, which is only partly explained by the acidity of its solution. CO2 concentration is a significant factor in controlling cerebral function.     

In vivo cytogenetic effects of cooked food mutagens

Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat. C57Bl/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells. PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow. In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested. PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood. However, dose responses were not observed. With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.     

Increased protoplast yield from oat leaves and bean internodes by non-injurious mechanical perturbation

Summary A simple new technique has been developed to greatly increase the yield of protoplasts from plant organs without injury to the plant. Mechanical perturbation (MP) by non-stressful rubbing of oat leaf segments and bean internodes yielded ten to twenty times more viable protoplasts than did controls. The increase in protoplast yield due to MP is best manifested, if the organs are excised and transferred to the cellulytic enzymes immediately after MP is given to the intact organ. The enzymes begin digesting from the lower end of the bean internodes and proceed acropetally. Vacuum infiltration of control oat leaf segments for 15 min with enzyme solution resulted in increased yield but less than due to MP. Increased levels of calcium (10 mM) in the medium decreased the yield of protoplasts from both control and MP-treated plant organs. EGTA significantly increased the yield of protoplasts from control oat leaf segments and marginally over that found in the control bean internodes. Cycloheximide increased the yield of protoplasts from oat leaf segments but not from bean internodes. It is suggested that MP may increase the susceptibility of cell wall polymers to cellulytic enzymes by reducing calcium cross linking. MP is thus a tool for increasing the yield of protoplasts from plant organs without causing injury.Abbreviations CHI cycloheximide - EGTA ethyleneglycol-bis-(ß-aminoethyl ether)-N,N-tetraacetic acid - FDA fluorescein diacetate - MP mechanical perturbation     

The effect of pregnancy and foaling on intravaginal pressure in pony mares

Pressures were recorded monthly at two sites in the vagina of each of five pregnant and five nonpregnant pony mares; pressures in five mares were also recorded weekly after foaling. The developing pregnancy did not influence pressure, and after foaling the integrity of the seal formed by the caudal reproductive tract was rapidly restored.     

State transitions,photosystem stoichiometry adjustment and non-photochemical quenching in cyanobacterial cells acclimated to light absorbed by photosystem I or photosystem II

Cells of the cyanobacterium Synechococcus 6301 were grown in yellow light absorbed primarily by the phycobilisome (PBS) light-harvesting antenna of photosystem II (PS II), and in red light absorbed primarily by chlorophyll and, therefore, by photosystem I (PS I). Chromatic acclimation of the cells produced a higher phycocyanin/chlorophyll ratio and higher PBS-PS II/PS I ratio in cells grown under PS I-light. State 1-state 2 transitions were demonstrated as changes in the yield of chlorophyll fluorescence in both cell types. The amplitude of state transitions was substantially lower in the PS II-light grown cells, suggesting a specific attenuation of fluorescence yield by a superimposed non-photochemical quenching of excitation. 77 K fluorescence emission spectra of each cell type in state 1 and in state 2 suggested that state transitions regulate excitation energy transfer from the phycobilisome antenna to the reaction centre of PS II and are distinct from photosystem stoichiometry adjustments. The kinetics of photosystem stoichiometry adjustment and the kinetics of the appearance of the non-photochemical quenching process were measured upon switching PS I-light grown cells to PS II-light, and vice versa. Photosystem stoichiometry adjustment was complete within about 48 h, while the non-photochemical quenching occurred within about 25 h. It is proposed that there are at least three distinct phenomena exerting specific effects on the rate of light absorption and light utilization by the two photoreactions: state transitions; photosystem stoichiometry adjustment; and non-photochemical excitation quenching. The relationship between these three distinct processes is discussed.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - F relative fluorescence intensity at emission wavelength nm - F _o fluorescence intensity when all PS II traps are open - light 1 light absorbed preferentially by PS I - light 2 light absorbed preferentially by PS II - PBS phycobilisome - PS photosystem     

Heat-shock cognate protein (hsc71) and related proteins in mouse spermatogenic cells

A monoclonal antibody (13D3) has been developed that recognizes a 71 kilodalton (71 kDa) protein on two-dimensional immunoblots of proteins extracted from a mixture of mouse spermatogenic cells (mainly pachytene spermatocytes and spermatids). This protein was shown by immunoblotting and adenosine triphosphate (ATP)-binding characteristics to be identical to a 71 kDa mouse heat-shock cognate (hsc) protein, hsc71, present in 3T3 cells. Along with a 70 kDa heat-shock inducible protein (hsp70), and a 74 kDa heat-shock cognate protein (hsc74), hsc71 is a product of the mouse HSP70 multigene family. Although antibody 13D3 reacted strongly with hsc71, it reacted only faintly with hsp70 in 3T3 cells, and not at all with hsc74 or a germ cell-specific hsp70-like protein (P70) on immunoblots of mixed germ cells. Antibody 13D3 is unique among known antibodies in its pattern of reaction with these heat-shock proteins. In immunofluorescence studies on isolated germ cells, 13D3 reacted uniformly with the cytoplasm of pachytene spermatocytes, round spermatids, and residual bodies, but only with the midpiece of spermatozoa. Antibody 13D3 recognizes other proteins in addition to hsc71 on two-dimensional immunoblots of condensing spermatids and spermatozoa. Two of the proteins (70 kDa/pI 6.4 and 70 kDa/pI 6.5) were present in condensing spermatids and spermatozoa, and another protein (69 kDa/pI 7.0) was detected only in spermatozoa. The new proteins also were recognized by monoclonal antibody 7.10, which reacts specifically with hsp70, hsc71, hsc74, and P70. Although [35S]methionine was incorporated into the new proteins in condensing spermatids, hsc71, hsc74, and P70 were not labeled. These results suggest that unique heat-shock proteins are synthesized late in spermatogenesis.