Conformational relaxations of urea- and guanidine hydrochloride-unfolded ferricytochrome c.

Several recent studies of protein the unfolded proteins. In urea- and guanidine HCl-unfolded ferricytochrome c (horse heart), an acid-induced spin state transformation of the heme group has been detected by the heme absorptions, Trp-59 fluorescence, and the intrinsic viscosity of protein. Kinetics of this second conformational transition, by the temperature jump and stopped flow methods, are complex. One rapid reaction (tau1), pH-independent, occurs in a 50-mus range; the second reaction (tau2), in a 1-ms range, depends linearly upon pH and is faster at the alkaline side; a third reaction (tau3), in a 1-s range, shows a sigmoidal transition at pH 5.1 and is faster at the acidic side. The results are consistent with a kinetic scheme which involves protein conformational changes in the transformation of the heme coordination state. The kinetics, along with previous equilibrium studies, indicate that ligand or charge interactions within a protein molecule are not completely prohibited even in strongly denaturing conditions, such as in high concentrations of urea and guanidine HCl. Thus, local structures of peptide chain associated with these interactions can exist in the unfolded protein.     

Carbon Metabolism in Seagrasses: I. THE UTILIZATION OF EXOGENOUS INORGANIC CARBON SPECIES IN PHOTOSYNTHESIS

Four species of seagrasses, Halophila stipulacea, Thalassodendronciliatum, Halodule uninervis, and Syringodium isoetifolium,were investigated for their ability to utilize and CO₂ as exogenous carbon sources for photosynthesis. Ratesof photosynthesis were measured as rates of O₂ evolution ina closed system in which the pH was continuously controlled.A computer program was written to calculate the concentrationsof different carbon species as a function of pH and other specifiedexperimental conditions. Bicarbonate as well as CO₂ were readily assimilated by all fourseagrass species. Saturating concentrations of , at saturating light intensities, were 0.5–1.8 mM dependingon the species. Rates of photosynthesis under such conditionswere 0.1–0.55 µmol O₂ min^–1 mg^–1 chlorophyll.At saturating CO₂ concentrations, i.e. 0.5–1.3 mM, ratesof photosynthesis were 0.22–1.4 µmol CO₂ min^–1mg^–1 chlorophyll. Photosynthetic rates in each specieswere considerably higher when CO₂ rather than was supplied at saturating concentrations. The concentration of in natural seawater was found to be saturating, and that of CO₂ insufficient forconsiderable photosynthetic rates in these plants under thegiven conditions It was thus concluded that is the major carbon source for photosynthesis in seagrasses.     

Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

Partial tetrasomy 9(9pter to 9q2101) due to an extra iso-dicentric chromosome.

A 3-year-old boy with partial No. 9 tetrasomy is described. The patient showed markedly retarded physical and mental development as well as multiple congenital anomalies. Routine chromosome analysis revealed an extra C-group chromosome. It had a pronounced secondary constriction at the proximal part of its long arm. Based on studies by a variety of banding techniques, the extra chromosome was identified to be an iso-dicentric No. 9 chromosome with inactivation of one of the two centromeres, the karyotype being 47,XY, + DIC (9)(Q2101). The value of BrdUrd treatment was emphasized in the detection of a very small piece of euchromatin within a long stretch of constitutive heterochromatin.     

Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, C1r-, and C1 esterase.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.     

In vitro construction of the tufB-lacZ fusion: analysis of the regulatory mechanism of tufB promoter

Summary Genetic studies suggest that the so-called phosphorus-family of enzymes inN. crassa are controlled by a complex system of regulatory genes which are responsive to the level of phosphorus in the growth medium. The intracellular metabolite(s) that interact with this system to signal changes in the external phosphorus concentration has not been identified. In this study the pools of acid-soluble, phosphorus-containing, compounds are measured in wild-type and phosphorus-family enzyme regulatory mutant strains ofN. crassa before and during phosphorus starvation.Prolonged phosphorus starvation of wild-typeN. crassa failed to alter significantly the pre-starvation level of intracellular orthophosphate, suggesting that intracellular P_i would be a poor effector signal for the control of the phosphorus family enzymes. However, inorganic pyrophosphate (PP_i) decreased 15-fold, and tri- and tetrapolyphosphate (PPP_i and PPPP_i) increased 3- to 5-fold within 15 minutes after transfer of the wild-type strain to phosphorus-free medium. Phosphate starvation of seven different regulatory gene mutant strains resulted in a rapid decrease in the PP_i pool similar to that which occurred in the wild-type. However, only two of these seven strains showed increased PPP_i and PPPP_i pools following phosphate starvation. Additional experiments demonstrated that PP_i pools, but not PPP_i and PPPP_i pools, were unaffected by several starvation regimens other than phosphorus starvation. Metabolic studies employing H₃ ³²PO₄ showed that the pool of PP_i was labeled to steady-state levels after two minutes of continuous labeling of a phosphate-sufficient culture. Furthermore, long-term steady-state labeling showed that the intracellular PP_i pool was directly responsive to the decrease in the extracellular P_i concentration of the medium resulting from cell growth. Growth on phosphoethanolamine, a phosphorus source that allows a modest degree of derepression even in growing cells, resulted in lower levels of PP_i than were seen in phosphate-grown cells. These observations suggest that PP_i may be involved in the mechanism responsible for the control of phosphorus-family enzyme regulatory gene product activity.