Rapid physiological assays for nutrient demand by the plankton. I. Nitrogen

Three assays for nitrogen demand were compared on samples ofnatural plankton and on green and blue-green algal species inculture. The most reliable guide was selective luxury uptakeof nitrogen by the plankton after enrichment with a 10-to-1(wt/wt) mixture of inorganic N and P. Ammonium transport capacity,measured either directly (cultures) or by V_max for uptake ofthe NH₄⁺ analogue methylammonium (lakewaters), was generallyhigh in lakewaters with low dissolved inorganic N-to-P ratiosand in N-deficient cultures of green algae. By contrast, transportcapacity was much reduced in both natural and cultured populationsof heterocystous blue-green algae growing under conditions oflow combined inorganic nitrogen. Ammonium enhancement assays(heterotrophic CO₂, fixation after NH₄⁺ enrichment) were conductedat monthly intervals on eutrophic Lake Rotorua. There was astrong, negative correlation between this indicator of N deficiencyand dissolved inorganic N-to-P ratios below a threshold of 6:1.Ammonium enhancement was not, however, a reliable indicatorof combined inorganic N-demand by populations of heterocystousblue-green algae. All three assays provided strong evidenceof a persistent shortage of nitrogen relative to phosphorusfor algal production in Lake Rotorua.     

The Yale Affiliated Hospital Program in Internal Medicine

In July, 1975, the Departments of Internal Medicine at the Yale University School of Medicine and eight community hospitals in southern and western Connecticut formed the Yale Affiliated Hospital Program (YAHP) in Internal Medicine. The YAHP provides a planned and focused program of continuing education for medical staff and housestaff at the affiliated hospitals. Six formats for the over 1,000 rounds, lectures, and conferences given annually are used. The members of the YAHP also cooperate in housestaff and faculty recruiting, evaluation of quality of care and evaluation of the process of continuing medical education itself. This report summarizes the organization, goals and future plans of the YAHP.     

Flexibility of the pyrrolidine ring in proline peptides

A survey of over 50 crystal structures indicates that both imino acid and peptide derivatives of proline populate ring conformers consistent with the torsional potentials about single bonds. In both cases, lower barriers for rotation about C? N bonds relative to those about C? C bonds favor smaller values for dihedral angles about the former bonds. In peptides a minimum in the torsional potential about C? N bonds occurs at zero dihedral angle, further favoring small angles. The pyrrolidine-ring dihedral angles of the proline compounds in the solid state obey a cyclopentane-type pseudorotation function. Thus the puckering of the five-membered ring can be quantitatively described by two parameters. Consistent with small dihedral angles about C? N bonds, C^β and/or C^γ are puckered out of the mean plane of the ring in nearly all of the nonstrained compounds. Utilizing the consistent force-field method of Lifson and coworkers [see A. Warshel, M. Levitt, and S. Lifson (1970) J. Mol. Spectrosc. 33 , 84] the intramolecular energy of five proline peptides was minimized with respect to all internal coordinates. In addition, the energy surface near minima was explored by constraining a particular dihedral angle and reminimizing the energy with respect to all remaining variables. In linear peptides two types of pyrrolidine-ring conformers have identical predicted energies. In the cyclic dipeptide cyclo (Pro-Gly) one of the ring conformers is favored by about 3 kcal/mol, while the cyclic tripeptide cyclo(Pro-Gly-Gly) favors the other conformer by a comparable margin. In agreement with observations in the solid state and in solution, C^β and/or C^γ are puckered in the predicted conformers. A correlation between proline Φ and the details of the puckered conformation was predicted and found to match precisely conformers observed in crystals. For the diamides N-acetyl-L -proline-N′-methyl-amide and N-acetyl-L -proline-N′,N′-dimethylamide (AcProMe₂A) 30% and 60% cis acetyl peptide bonds were predicted in good agreement with observations in nonpolar solvents for the respective compounds. The conformational distributions with respect to proline Ψ are also in accord with experimental observations. For AcProMe₂A, a model for a -Pro-Pro-sequence in a peptide chain, this study is the first to predict stable conformers for proline Ψ either ca. ?50° or 140° for both cis and trans peptides.     

Quinine-induced agranulocytosis: toxic effect of quinine bisulphate on bone marrow cultures in vitro.

In a case of quinine-induced agranulocytosis marrow culture studies confirmed the inhibitory effect on the patient''s cells of equivalent therapeutic plasma concentrations of quinine. Similar concentrations had no effect on normal marrow cells. Quinidine, the stereoisomer of quinine, had no effect on either cells from the patient or normal cells. The results encourage the use of in-vitro bone marrow cultures for identifying drugs responsible for agranulocytosis.     

Neurotensin and Neuromedin N Are Differently Metabolized in Ventral Tegmental Area and Nucleus Accumbens

Whole homogenates and membrane-bound and cytosoluble fractions prepared from rat ventral tegmental area (VTA) and nucleus accumbens were examined for their content of peptidasic activities and for their ability to metabolize neurotensin and its natural related hexapeptide neuromedin N. No qualitative differences were observed between these two brain regions concerning the presence and the subcellular distribution of a series of activities able to hydrolyze various specific fluorimetric enzymatic substrates. However, aminopeptidase B, endopeptidase 24-15, and endopeptidase 24-11 were significantly lower in the VTA than in the nucleus accumbens membrane preparations, while proline endopeptidase was detected in significantly higher amount only in the cytosolic fraction prepared from nucleus accumbens. Both neurotensin and neuromedin N were metabolized more rapidly in the nucleus accumbens than in the VTA. Furthermore, the degradation rate of neuromedin N was considerably faster than that of neurotensin whatever the cerebral area examined. Studies carried out with highly specific peptidase inhibitors revealed that endopeptidase 24-15 mainly contributed to the catabolism of neurotensin in homogenates and membrane-bound preparations of nucleus accumbens and VTA, while aminopeptidase B appeared predominantly responsible for the rapid disappearance of neuromedin N in both cerebral tissues. The possibility that the different metabolic processes of the two peptide congeners could explain their distinct pharmacological profiles observed after their microinjection in the nucleus accumbens and in the VTA is discussed.     

Interleukin-6 administration has no acute hemodynamic or hematologic effect in the dog

To investigate the possible hemodynamic effects of interleukin-6 (IL-6), a single dose of 15 mcg/kg of recombinant IL-6 isolated from Escherichia coli was injected intravenously in six pentobarbital-anesthetized dogs. After 30 min, saline infusion was performed to maintain the pulmonary artery balloon-occluded pressure at baseline level. The animals were observed for up to 5 hours. No other hemodynamic alteration was observed than a gradual decline in cardiac output attributed to anesthesia. Hematologic variables, blood glucose, and total serum proteins were also constant. IL-6 levels were markedly elevated in the blood, but no tumor necrosis factor activity was detected. Thus a primary role for IL-6 in the early cardiovascular alterations associated with septic shock seems unlikely.     

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.