Testing the accuracy of methods for reconstructing ancestral states of continuous characters.

Many methods are available for estimating ancestral values of continuous characteristics, but little is known about how well these methods perform. Here we compare six methods: linear parsimony, squared-change parsimony, one-parameter maximum likelihood (Brownian motion), two-parameter maximum likelihood (Ornstein-Uhlenbeck process), and independent comparisons with and without branch-length information. We apply these methods to data from 20 morphospecies of Pleistocene planktic Foraminifera in order to estimate ancestral size and shape variables, and compare these estimates with measurements on fossils close to the phylogenetic position of 13 ancestors. No method produced accurate estimates for any variable: estimates were consistently less good as predictors of the observed values than were the averages of the observed values. The two-parameter maximum-likelihood model consistently produces the most accurate size estimates overall. Estimation of ancestral sizes is confounded by an evolutionary trend towards increasing size. Shape showed no trend but was still estimated very poorly: we consider possible reasons. We discuss the implications of our results for the use of estimates of ancestral characteristics.     

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14–28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10⁶ cryptococci, while no mortality was observed in mice exposed to 10³ or 10⁴ cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.     

Human inter-alpha-trypsin inhibitor: localization of the Kunitz-type domains in the N-terminal part of the molecule and their release by a trypsin-like proteinase

The N-terminal amino-acid sequence of human ITI has been found to be identical with that of the acid-stable human 30-kDa inhibitors (HI-30) from urine, serum, and those released from inter-alpha-trypsin inhibitor by trypsin or chymotrypsin. Serum HI-30 and HI-30 released by trypsin differ from the urinary inhibitor by an additional C-terminal arginine residue. Compared to these two inhibitors the inhibitor released by chymotryptic proteolysis is elongated C-terminally by an additional phenylalanine residue. These results strongly favour HI-30 as the N-terminus of the inter-alpha-trypsin inhibitor and its release from this inhibitor in vivo by cleavage of the Arg123-Phe124 peptide bond by trypsin-like proteinases.     

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.     

Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states

The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.