Distinct stability of recombinant L and H subunits of human ferritin: calorimetric and ANS binding studies

Thermodynamic and pH stability of recombinant human L- and H-ferritins wereprobed by differential scanning calorimetry and 8-anilino-1-naphthalenesulfonate (ANS) binding in the pH range 2-7. At pH 2.0-2.8 theywere dissociated into subunit monomers and in this pH interval theH-subunit displayed a single calorimetrically-revealed domain withproperties of a molten globule-like state: low enthalpy (6.3-8.0 J/g or169-172 kJ/mol) and Tm of thermal unfolding (approximately 50 degrees C), awide transition range (approximately 20 degrees C) and high ANS binding. Incontrast, at pH 2 the L-ferritin subunit showed two calorimetric domainswith Tm of 35 and 40 degrees C with similar unfolding enthalpies and withmoderate extent of interactions, as indicated by the ratio of calorimetricenthalpy (293.9 kJ/mol) and van't Hoff enthalpy (174.2 kJ/mol) for thethermal transition. A pH increase from 2.0 to 2.8 determined the couplingof the two domains into a single cooperative folding unit and drasticincrease of the transition temperature (from 37 to 80 degrees C). Thecontacts between the two domains in the L-subunit appeared to contribute toabout 30% of the total stabilization free energy. The unfolding enthalpies,heat capacity changes and pronounced ANS binding of the L-subunit at pH2.0- 2.8 indicated that part of the structure lacked 'meltable' tertiaryinteractions. The results indicate that H- and L-subunits are stabilized bylargely different intra-chain interactions with a critical contribution toL-subunit stability of embedded salt bridge(s) absent in the H-subunit.     

Endocrine and reproductive function in ewes exposed to the organochlorine pesticides lindane or pentachlorophenol

The effects of lindane (LIN, gamma-hexachlorocyclohexane) and pentachlorophenol (PCP) on reproduction and general endocrine function were examined in breeding ewes as a model for wild and domestic ungulates, which may be exposed to low levels of pesticides that are potential endocrine-disrupting chemicals. Ewes (n = 13/group) were fed either a control untreated diet (CON), or a diet treated with LIN (1 mg/kg/d) or PCP (1 mg/kg/d) during the 5 wk prior to mating and throughout pregnancy and lactation. Mating response, ovulation rate, follicle and corpus luteum size, gestation length, pregnancy rate, lambing rate, and lamb birth weight were recorded. After weaning, 6 ewes from each group were bled at 15-min intervals for 8 h during the day and night and for 1 h before and 5 h after i.v. administration of gonadotropin-releasing hormone, thyroid-stimulating hormone (TSH), and adrenocorticotropin, to measure serum concentrations of luteinizing hormone, follicle-stimulating hormone, thyroxine (T4), and cortisol. Ewes were then killed and endocrine tissues examined histologically. Pregnancy rate as a result of matings taking place at the synchronized estrus was significantly decreased by the lindane treatment However, PCP and lindane did not markedly affect any other aspect of reproductive function studied. In PCP-treated ewes, serum concentrations of T4 were significantly reduced compared to control ewes during the day and night; however, the T4 response to TSH was not altered by PCP treatment. No other measured endocrine parameters were consistently affected by lindane or PCP. Thyroid follicle size was significantly increased in the LIN and PCP ewes compared to the control ewes. Low serum concentrations of T4 in the PCP ewes may have resulted in increased TSH secretion and increased thyroid follicle size. In conclusion, although pesticide treatments had no serious adverse effects on reproductive function in breeding ewes, PCP reduced T4 concentration, which in the long term could influence reproductive and general performance.     

An endogenous calcium oscillator may control early embryonic division

Transient elevations in the concentration of free cytosolic calcium ion ([Ca2+]i) promote cell phase transitions in early embryonic division and persist even if these transitions are blocked. These observations suggest that a [Ca2+]i oscillator is an essential timing element of the early embryonic "master clock." We explore this possibility by coupling a [Ca2+]i oscillator model to an early embryonic cell cycle model based on the protein interactions that govern the activity of the M-phase-promoting factor (MPF). We hypothesize three dynamical states of the MPF system and choose parameter sets to represent each. We then investigate how [Ca2+]i dynamics may control early embryonic division in both sea urchin and Xenopus embryos. To investigate both systems, distinct [Ca2+]i profiles matching those observed in sea urchin embryos (in which [Ca2+]i exhibits sharp transients) and Xenopus embryos (in which [Ca2+]i is elevated and oscillates sinusoidally) are imposed on each of the hypothesized dynamical states of MPF. In the first hypothesis, [Ca2+]i oscillations entrain the autonomous MPF oscillator. In the second and third hypotheses, where the MPF system rests in excitatory and bistable states, respectively, [Ca2+]i oscillations drive MPF activation cycles. Simulation results show that hypotheses two and three, in which a [Ca2+]i oscillator is a fundamental timing element of the master clock, best account for key experimental observations and the questions that they raise. Finally, we propose experiments to elucidate further [Ca2+]i regulation and the fundamental components of the early embryonic master clock.     

Adrenergic receptors and secretory responses of the rat submandibular salivary gland after periodic incisor reduction

While loss-of-function mutations in Gsalpha are invariably associated with the short stature and brachydactyly of Albright hereditary osteodystrophy (AHO), the association with hormone resistance (to parathyroid hormone and thyrotropin) typical of pseudohypoparathyroidism type Ia (PHP-Ia) is much more variable. Observational studies and DNA polymorphism analysis suggest that maternal transmission of the Gsalpha mutation may be required for full expression of clinical hormone resistance. To test this hypothesis, we studied transmission of a frameshift mutation in Gsalpha through three generations of a pedigree affected by AHO and PHP-Ia. While all family members carrying this loss-of-function mutation in one Gsalpha allele had AHO, neither the presence of the mutation nor the degree of reduction of erythrocyte Gsalpha bioactivity allowed prediction of phenotype (AHO alone versus AHO and PHP-Ia). Paternal transmission of the mutation (from the patriarch of the first generation to three members of the second generation) was not associated with concurrent PHP-Ia, but maternal transmission (from two women in the second generation to four children in the third generation) was invariably associated with PHP-Ia. No expansion of an upstream short CCG nucleotide repeat region was detected, nor was there evidence of uniparental disomy by polymorphism analysis. This report, the first to document the effects across three generations of both paternal and maternal transmission of a specific Gsalpha mutation, strongly supports the hypothesis that a maternal factor determines full expression of Gsalpha dysfunction as PHP-Ia.     

The methodology of Corrosion inhibitor development for CO2 systems

Three fundamental criteria are defined by means of which test data are being judged relative to their information content. They must be UNIQUE, RELEVANT, and PREDICTIVE. The degree to which these criteria are met depends on the mechanistic understanding of the underlying corrosion or inhibition processes. It will be shown that of all the routine tests the Wheel Test procedure is the worst in that it does not meet is main objectives and cannot rank different inhibitors predictively. Comparative tests to establish the corrosion kinetics and performance/concentration curves for inhibitors were run in highly agitated kettles and small autoclaves. Corrosion rates were determined by iron analyses and weightloss determinations. The problem of the constantly changing pH in solution was eliminated by pH-stating. The inhibitor performance in some cases is strongly dependent on pH and CO₂ partial pressure. All evaluations were done on AISI-1020 or AISI-1018 metal.