Molecular counting of low-density lipoprotein particles as individuals and small clusters on cell surfaces.

We employ the intensely fluorescent analogue diI-LDL (Barak, L. S., and W. W. Webb, 1981, J. Cell Biol. 90:595-604) as a counting marker to determine the numbers of LDL-receptor complexes that are contained in clusters on the surfaces of human fibroblasts and human epidermoid carcinoma cells. The application of quantitative digital intensified video optical microscopy allows the measurement of the fluorescence power collected from individual fluorescent spots on a cell with sufficient accuracy that the number of optically unresolved particles producing the fluorescence in the spot can be estimated. We demonstrate that isolated individual diI-LDL particles are detected on the surface of all cells investigated. Analysis of the LDL cluster size distributions on the various cell lines shows clear differences that correlate with efficiency of LDL metabolism. We find that normal fibroblasts (GM3348) have LDL-receptor complex populations dominated by large cluster sizes (greater than 4 LDL), while internalization-deficient J.D. mutant fibroblasts (GM2408A) and epidermoid carcinoma cells (A-431) show predominantly small clusters (1-3 LDL). No evidence for large-scale ordering or "superclustering" of clusters is found.     

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.     

Structural analyses of a monoclonal heterodimeric suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10

A GAT-specific, MHC-restricted "second-order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 was biosynthetically radiolabeled with 35S-methionine and was isolated from cell extracts. The isolation procedure involved two-dimensional nonreducing/reducing SDS-PAGE and electroelution of the reduced off-diagonal polypeptide chains from the gel. Biochemical characterization studies revealed that TsF2 is a disulfide-linked heterodimer composed of a basic and an acidic polypeptide chain, both having m.w. of 30,000. Both chains are glycosylated and contain sialic acid residues. The basic polypeptide reacts with anti-I-J antisera, whereas the acidic chain contains the antigen-binding capacity. Monoclonal antibodies induced by immunizing rats with TsF2 purified from hybridoma supernatants were selected for the ability to block immunosuppression mediated by TsF2 in vitro. These antibodies, but not irrelevant antibodies, immunoprecipitated the 35S-methionine-labeled protein that migrates off the diagonal in two-dimensional gels. Thus, we have verified that the immunosuppressive protein that migrates off the diagonal in two-dimensional gels binds to antibodies that are known to inhibit the biologic activity of unpurified TsF2.     

Endocrine differences in rams after genetic selection for testis size

Testis diameter and body weight were recorded from 6 to 76 weeks of age in ram lambs from two established lines selected for high (H) and low (L) testis size. While testis growth was greater in the H line up to 14 weeks of age (P less than 0.001), body weight was significantly lower, with the L line rams being 10 kg heavier by 76 weeks. There were no differences in plasma LH up to 20 weeks of age, but FSH concentrations were significantly lower at 14 and 20 weeks in the H line. Testosterone concentrations were not significantly higher in the H line from 6 to 20 weeks. In lambs castrated at birth, significantly higher FSH values were recorded from 6 to 20 weeks of age in the H line (P less than 0.001) whereas there was no difference in LH concentration at 6 and 10 weeks of age between the lines. At 14 and 20 weeks, however, the concentrations of LH were greater in the H than L line lambs (P less than 0.05). After hemicastration at 6 weeks of age, the rate of growth of the remaining testis in the L line lambs was significantly faster than in entire lambs of that line from 10 to 20 weeks (P less than 0.05 at 10 weeks to P less than 0.001 at 20 weeks). There was no difference in the rate of testis growth between the the entire and hemicastrated lambs from the H line from 6 to 12 weeks of age. It can be concluded that there is an underlying genetic difference in pituitary gland and/or hypothalamic activity in ram lambs from the two selected lines.     

Is vegetation in equilibrium with climate? How to interpret late-Quaternary pollen data

Current methods for estimating past climatic patterns from pollen data require that the vegetation be in dynamic equilibrium with the climate. Because climate varies continuously on all time scales, judgement about equilibrium conditions must be made separately for each frequency band (i.e. time scale) of climatic change. For equilibrium conditions to exist between vegetation and climatic changes at a particular time scale, the climatic response time of the vegetation must be small compared to the time scale of climatic variation to which it is responding. The time required for vegetation to respond completely to climatic forcing at a time scale of 10⁴ yr is still unknown, but records of the vegetational response to climatic events of 500-to 1000-yr duration provide evidence for relatively short response times. Independent estimates for the possible patterns and timing of late-Quaternary climate changes suggest that much of the vegetational evidence previously interpreted as resulting from disequilibrium conditions can instead be interpreted as resulting from the individualistic response of plant taxa to the different regional patterns of temperature and precipitation change. The differences among taxa in their response to climate can lead a) to rates and direction of plant-population movements that differ among taxa and b) to fossil assemblages that differ from any modern assemblage. An example of late-Holocene vegetational change in southern Quebec illustrates how separate changes in summer and winter climates may explain the simultaneous expansion of spruce (Picea) populations southward and beech (Fagus) populations northward.     

Afterdrop of body temperature during rewarming: an alternative explanation

Afterdrop, the continued fall of deep body temperatures during rewarming after hypothermia, is thought to endanger the heart by further cooling from cold blood presumed to be returning from the periphery. However, afterdrop is not always observed, depending on the circumstances. To explore this phenomenon, mild hypothermia was induced quantitatively with a suit calorimeter, using several patterns of cooling and rewarming. When cooling was rapid and followed immediately by rewarming, there were typical afterdrops in the temperatures measured in the rectum, auditory canal, and esophagus. However, when rewarming was delayed, or when cooling had been slow and prolonged, afterdrop was not seen. Afterdrops were then observed in two physical models that had no circulation: a bag of gelatin and a leg of beef. Central layers continued to give up heat as long as the surrounding layer was cooler. These results, together with recent findings by others that peripheral blood flow is low until afterdrop is complete, make this circulatory explanation of afterdrop improbable. Alternatively, afterdrop can be explained by the way heat moves through a mass of tissue.     

Immunological detection of the messenger RNA cap-binding protein

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.     

Purification and partial characterization of a monoclonal "second order" suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10

A GAT-specific "second order" suppressor T cell factor (TsF2) from the hybridoma 762 B3.7 has been purified and biochemically characterized. The protein has a m.w. of approximately 66,000, an isoelectric point of 6.8 to 6.9, and elutes from a reversed phase HPLC column in two peaks, one in 55% acetonitrile, the other in 70% propanol. Amino acid analysis of both forms gave similar molar ratios, suggesting that the two forms are closely related and may differ mainly in the degree of posttranslational modification. SDS-PAGE electrophoresis under reducing conditions gave two chains of the apparent m.w. of 42,000 and 35,000.     

Deficiency of immunoreactive somatostatin in the median eminence of Snell dwarf mice

Snell dwarf mice (dw/dw) are characterized by a genetically determined, congenital lack of pituitary GH, TSH and prolactin. Given that hypothalamic somatostatin is involved in the regulation of pituitary GH and TSH release, it was decided to investigate the content of immunoreactive somatostatin (IRS) in the median eminence of dw/dw and phenotypically normal mice of the same strain. The content of IRS in the pyloric antrum and pineal gland of these animals was also examined. The effects of ovariectomy and of hyperprolactinemia (induced by a pituitary graft under the kidney capsule) on the median eminence content of IRS were also studied in both normal and dwarf mice. Median eminence IRS content was significantly lower in the dw/dw (23.6 +/- 1.8 ng) than in normal mice (57.4 +/- 7.1 ng); no difference was found in the pyloric IRS content of dw/dw (16.9 +/- 1.6 ng/mg of protein) and normal animals (13.8 +/- 1.9 ng/mg of protein), nor in the pineal content of IRS (639.4 +/- 64.4 pg/gland in the dw/dw; 732 +/- 265 pg/gland in normals). Neither ovariectomy nor hyperprolactinemia were found to affect the IRS content in the tissues studied in normal or dwarf mice. Treatment of an additional group of 9 dwarf mice with L-thyroxine (L-T4 2 micrograms/48 h. s.c. for 2 weeks) significantly increased the animals weight (10.2 +/- 0.4 g versus 7.4 +/- 0.3 g) and produced maturation of facial features; however, it did not change the IRS content in any of the tissues studied. It is concluded that the content of IRS in the median eminence of mice with a congenital lack of GH, TSH and prolactin is significantly reduced and that this is unlikely to be related to the deficiency of thyroid hormones in these animals.     

Development of three copper metalloenzymes in clover leaves

Subterranean clover (Trifolium subterraneum L. cv Seaton Park) was grown in solution cultures containing adequate nitrogen both with and without Cu. After Cu deficiency had developed, Cu²⁺ was added to some deficient plants and Cu content, protein content, and activities of three Cu metalloenzymes (diamine oxidase [EC1.4.3.6], ascorbate oxidase [EC1.10.3.3] and o-diphenol oxidase [EC1.10.3.1]) were assayed in young and recently matured leaf blades over 11 days during the development of the next three leaves.

Copper deficiency had little effect on protein concentrations, but markedly depressed enzyme activities and Cu concentration in all leaf blades assayed. Within 4 d of adding Cu²⁺ to Cu-deficient plants, Cu concentrations of all the leaf blades increased to adequate values. Enzyme activities only increased to control levels in leaves which had not yet emerged at the time that Cu²⁺ was added.

The results suggest that active holoenzymes of diamine oxidase, ascorbate oxidase, and o-diphenol oxidase can only be synthesized in leaf blades during very early stages of their development.