Circadian and/or circahemidian rhythms in nine lymphocyte-related variables from peripheral blood of healthy subjects

Circadian variations were investigated for nine lymphocyte-related variables in the peripheral blood of healthy subjects. Monoclonal antibodies targeted at membrane immunoglobulins (anti-Ig, anti-kappa, anti-lambda) or differentiation antigens (anti-IA and OKT3) were used to characterize respectively mature B cells (SIg+, kappa +, lambda +), cells expressing HLA-DR antigen (IA+), and T cells (OKT3+). Blood (33 ml) was drawn every 4 hr for 24 hr starting at 8.30 hr, on seven occasions in five apparently healthy male volunteers, recumbent from 23.00 hr to 07.00 hr. Leukocyte and differential counts were measured. Mononuclear cells were isolated on Ficoll-Hypaque before being incubated with monoclonal antibodies. The proportion of fluorescent cells per 100 microscopically determined cells was multiplied by the number of circulating lymphocytes per milliliter of venous blood. Temporal variations were validated by both paired t-test and cosinor. Rhythms with a period (tau) identical to 24 hr were validated with statistical significance (p less than 0.05) for total lymphocytes, OKT3+ cells and OKT3+:SIg+ ratio, and suggested (0.05 less than or equal to p less than or equal to 0.10) for lambda + and (kappa + + lambda +) cells. Rhythms with tau identical to 12 hr were also found (p less than 0.05) for OKT3+, SIg+, kappa +, and IA+ cells as well as for the OKT3+:SIg+ and the kappa +:lambda + ratios. Validated rhythms exhibited a large amplitude, e.g., peak-through differences were 40% of the 24-hr mean. This circadian and circahemidian temporal structure of immunologic variables constitutes a time-qualified reference system for investigating immune regulations and a tool for optimizing both diagnostic criteria and effectiveness of immunotherapeutic attempts.     

Inhibition of thermolysin by bifunctional N-carboxyalkyl dipeptides

A series of N-carboxyalkyl derivatives of L-leucyl-L-alanine was synthesized and tested as inhibitors of the zinc endoproteinase thermolysin. The purpose of the study was to determine whether bifunctional N-carboxyalkyl compounds with secondary metal coordinating groups are more potent inhibitors than analogs lacking such an additional binding function. Reductive condensation of L-leucyl-L-alanine (LA) with pyruvic, oxalacetic, alpha-ketoglutaric, 2-oxopentanoic, 4-ethyloxalacetic, or imidazoylpyruvic acids gave N-[1(R, S)-carboxyethyl]-LA (I), N-[1(R, S)-carboxy-2-carboxyethyl]-LA (II), N-[1(R, S)-carboxy-3-carboxypropyl]-LA (III), N-[1(R, S)-carboxy-n-butyl]-LA (IV), N-[1(R, S)-2-ethylcarboxyethyl]-LA (V), and N-[1(R, S)-carboxy-2-(4-imidazoyl-ethyl]-LA (VI), respectively. Values of KI determined with furylacryloyl-Gly-Leu-NH2 as substrate were 116 +/- 21, 7.4 +/- 1.8, 6.3 +/- 0.5, 19.7 +/- 1.5, 17.0 +/- 1.0, and 3.3 +/- 0.1 microM for compounds I-VI, respectively. Although bifunctional inhibitors II, III, and VI were indeed more potent than I, they were not much more effective than analogs IV and V that contained noncoordinating functionalities of comparable size. The results do not provide strong evidence for chelation of the active site zinc ion as proposed, although such interactions do not appear to be ruled out altogether.     

Activity of UDP-GlcNAc:alpha-mannoside beta(1,6)N-acetylglucosaminyltransferase (GnT V) in cultured cells using a synthetic trisaccharide acceptor

N-acetylglucosaminyltransferase V activity has been measured under saturating conditions in the extracts of seven cultured cell lines using as substrates, UDP-[3H]-GlcNAc and a synthetic 8-methoxylcarbonyloctyl trisaccharide. The unreacted sugar-nucleotide and its breakdown products were separated from the radiolabeled tetrasaccharide product by reverse-phase chromatography. Enzyme activity was present in six of the cell lines, which were derived originally from either human, mouse, or hamster tissues, with the highest activity in mouse lymphoma BW5147 cells. The PHAR 2.1 variant cell line, derived from the BW5147 line, expressed no detectable activity.     

Circannual rhythm in natural killer activity and mitogen responsiveness of murine splenocytes

Seasonal variations were observed in murine splenic natural killer (NK) cell activity and also in murine lymphocyte responsiveness to mitogens, namely, concanavalin A, phytohemagglutinin, and lipopolysaccharide. The maximum and minimum splenic NK cell activities were observed in January-February and July-August, respectively. Conversely, maxima and minima of lymphoproliferative responses to all the three mitogens occurred in April-June and January-February, respectively. Such variations, when inferential statistics are used, appeared to be accounted for by circannual and other low-frequency (infradian) bioperiodicities. More specifically, the circannual rhythm in murine NK cell activity was demonstrated in data from a total of 356 mice collected over a period of 5 years. The various components of the immune system are characterized by a multifrequency time structure. The understanding of the organization of the immune system along the yearly scale may have bearings on that of the seasonal incidence of numerous infectious diseases and on the success/failure of immunotherapy.     

Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport.

X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer.     

Induction of voluntary prolonged running by rats

The rat is widely used in studies of the metabolic and physiological effects of physical exercise. The most commonly used form of exercise is running on treadmills or mechanically driven running wheels. Rats will not voluntarily run significant distances, under normal circumstances. If rats are exposed to running wheels with food freely available, only very limited activity normally occurs. When rats with access to a running wheel are restricted to a fixed amount of food, presented once per day, consistent running occurs. The running is spontaneous and very sensitive to the amount of food provided. Six 6-wk-old rats of 197 g mean body wt were induced to run for 139 days. The distance run increased rapidly over a 20-day initial period on a food supply of 15 g/day (vs. 19.5 g/day consumption by sedentary controls). From day 20 to day 139 the mean distance run was described by the regression equation distance (m/day) = 10,410 - 37.9 X days. Food provided was varied according to distance run, ranging from 15 to 18 g/day, and was normally 17.5 g/day. Thus a food deprivation of 10% of normal consumption will result in mean distances run of approximately 8,000 m/day. The use of pair-fed control animals without access to a wheel allows the conduct of experiments to test the effects of chronic long-distance running. The running is spontaneous; thus the technique avoids the complications accompanying techniques that force running.     

Sepharose-linked concanavalin A in the purification and characterization of glycoprotein hormones of the bovine pituitary

Affinity chromatography on concanavalin A-Sepharose is a time saving step in both large and small scale isolations of the bovine pituitary glycoprotein hormones. After ion-exchange chromatography, the final yield of purified lutropin is 40-50% of material in starting concentrates and of purified thyrotropin is approximately 20%. The final products have the same electrophoretic and immunological properties and amino acid compositions as previous preparations. Less than 3% of the immunoreactive lutropin, follitropin and thyrotropin are present as non-glycosylated forms in either crude pituitary extracts or concentrates. Thyrotropin and follitropin elute from the immobilized lectin as a single fraction, whereas lutropin separates into two glycosylated fractions. Gel filtration of both crude extracts and the glycoprotein fractions shows that less than 5% of the immunoreactivity of the hormones is present as material of apparently high molecular weight. Substantial alpha subunit immunoreactivity, however, is in three fractions (as found by others in human pituitary extracts) corresponding to "high molecular weight material" (7%), intact hormones (46%) and free subunit (47%).     

Azasterol inhibitors in yeast. Inhibition of the 24-methylene sterol delta24(28)-reductase and delta24-sterol methyltransferase of Saccharomyces cerevisiae by 23-azacholesterol.

The effects of 23-azacholesterol on sterol biosynthesis and growth of Saccharomyces cervisiae were examined. In the presence of 0.2, 0.5, and 1 micron 23-azacholesterol, aerobically-growing yeast produced a nearly constant amount of ergosta-5,7,22,24(28)-tetraenol (approx. 36% of total sterol) and slowly accumulated zymosterol with a concommitant decline in ergosterol synthesis. Growth and total sterol content of yeast cultures treated with 0.2-1 micron 23-azacholesterol were similar to that of the control culture. Yeast cultures treated with 5 and 10 micron 23-azacholesterol produced mostly zymosterol (58-61% of total sterol), while ergosta-5,7,22,24(28)-tetraenol production declined to less than 10% of total sterol. The observed changes in the distribution of sterols in treated cultures are consistent with inhibition of 24-methylene sterol 24(28)-sterol reductase (total inhibition at 1 micron 23-azacholesterol) and of 24-sterol methyltransferase (71% inhibition at 10 micron 23-azacholesterol). Yeast cultures treated with 10 micron 23-azacholesterol were found to contain 4,4-dimethylcholesta-8,14,24-trienol and 4alpha-methylcholesta-8,14,24-trienol, which were isolated and characterized for the first time.     

N-acetyl-beta-d-glucosaminidase in marmoset kidney, serum and urine.

N-Acetyl-beta-D-glucosaminidase activities were determined in homogenates of marmoset kidney, in serum and in urine by using the 4-methylumbelliferyl substrate. The enzyme activity was separated into several components by DEAE-cellulose ion-exchange chromatography, starch-gel electrophoresis and isoelectric focusing. The kidney contained two major forms of the enzyme, A and B, which had similar pH optima and Km values. The A-form bound to DEAE-cellulose at pH 6.8, migrated towards the anode on starch-gel electrophoresis and had a pI of 5.0. The B-form did not bind to DEAE-cellulose at pH 6.8, remained near the origin on starch-gel electrophoresis and had a pI of 7.64. The isoenzymes also differed in heat stability, the B-form being the more stable. Serum contained B-form activity and, in addition, two intermediate forms (I1 and I2) were loosely bound to DEAE-cellulose. The serum A-form activity was less firmly bound to DEAE-cellulose than was the tissue A-form and was designated As. Serum from a pregnant marmoset contained a form which may be analogous to the human P-isoenzyme. Urine contained only a small amount of B-form activity, the majority being present in the A-form. The kidney A- and B-forms both had mol.wts. of 96000--100000 and the activity was predominantly lysosomal. Partial purification of the kidney A isoenzyme was undertaken. Immunoprecipitation studies indicated a relationship between marmoset kidney A-form and human liver A-form activity.     

Selective photodestruction of α-amino acids

A problem typically encountered in the analysis of amino acids in chemical evolution experiments and in extracts of meteorites is the large number present. For example, α-, β-, and γ-amino acids, N-mono substituted α-amino acids, and dicarboxylic α-amino acids have been found in extracts of the Murchison meteorite, and many more amino acids are present than have been positively identified by computerized gas chromatographic mass spectrometry. This paper reports an analytical method to selectively destroy the α-amino acids, with only the β- and γ-amino acids remaining in the solution. It is based on the ability of Cu²⁺ to complex with amino acids, the order of stability of these complexes being α > β > γ, = δ, = ε = 0. Aqueous solutions of α-amino acid-Cu²⁺ chelates are known to be decomposed by 254 nm light as well as by nonmonochromatic uv light, yielding a precipitate of Cu₂O. This paper shows that at 254 nm (ligand-metal charge transfer band) the rate of destruction of amino acids in Cu²⁺ aqueous solutions is in the following order, dicarboxylic α-amino acids > α-amino acids > N-monosubstituted α-amino acids β-amino acids ≈ γ-amino acids. Thus by irradiation with 254 nm light in the presence of Cu²⁺ all the amino acids can be destroyed except the β- and γ-amino acids. When almost 100% of the α-amino acids are destroyed, 80% of the β- and γ-amino acids still exist in solution. With this procedure, complex mixtures of amino acids can be simplified to make identification by gas chromatographic mass spectrometry casier.