Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating succinyl hydroxamate and iminodiacetic acid hydroxamate moieties

A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC). The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.     

Crystal structure and refolding properties of the mutant F99S/M153T/V163A of the green fluorescent protein

The mutant F99S/M153T/V163A of the Green Fluorescent Protein (c3-GFP) has spectral characteristics similar to the wild-type GFP, but it is 42-fold more fluorescent in vivo. Here, we report the crystal structure and the refolding properties of c3-GFP and compare them with those of the less fluorescent wt-GFP and S65T mutant. The topology and the overall structure of c3-GFP is similar to the wild-type GFP. The three mutated residues, Ser99, Thr153, and Ala163, lie on the surface of the protein in three different beta-strands. The side chains of Ser99 and Thr153 are exposed to the solvent, whereas that of Ala163 points toward the interior of the protein. No significant deviation from the structure of the wild-type molecule is found around these positions, and there is not clear evidence of any distortion in the position of the chromophore or of the surrounding residues induced by the mutated amino acids. In vitro refolding experiments on urea-denatured c3-GFP reveal a renaturation behavior similar to that of the S65T molecule, with kinetic constants of the same order of magnitude. We conclude that the higher fluorescence activity of c3-GFP can be attributed neither to particular structural features nor to a faster folding process, as previously proposed.     

Susceptibility of the prion protein to enzymic phosphorylation

Ten protein kinases have been assayed for their ability to phosphorylate in vitro the recombinant bovine PrP (25-242) (rbPrP). Substantial phosphorylation was observed with PKC, CK2, and two tyrosine kinases, Lyn and c-Fgr. With regard to CK2, phosphorylation occurs at Ser 154 with a stoichiometry of about 0.1 mol phosphate/mol rbPrP, which is doubled by mild heat treatment of rbPrP. Heat also reduces the overall protein ellipticity, suggesting that reversibly unfolded conformers are more susceptible to phosphorylation. Our data disclose the possibility that phosphorylation might modulate PrP biological activity.     

Temperature-dependent functional expression of a plant K(+) channel in mammalian cells

The Arabidopsis thaliana potassium channel KAT1 was expressed and characterized in Chinese hamster ovary cells. KAT1-GFP fusion protein was successfully targeted to the plasma membrane and electrophysiological analysis revealed functional expression of KAT1 only in cells cultured at 30 degrees C. The main biophysical characteristics of KAT1 are similar to those described for the channel expressed in other systems. CHO cells represent an advantageous expression system and may be the system of choice to study the expression, assembly, function, and regulation of plant potassium channels in general.     

Evidence for a novel bursting mechanism in rodent trigeminal neurons.

We investigated bursting behavior in rodent trigeminal neurons. The essential mechanisms operating in the biological systems were determined based on testable predictions of mathematical models. Bursting activity in trigeminal motoneurons is consistent with a traditional mechanism employing a region of negative slope resistance in the steady-state current-voltage relationship (Smith, T. G. 1975. Nature. 253:450-452). However, the bursting dynamics of trigeminal interneurons is inconsistent with the traditional mechanisms, and is far more effectively explained by a new model of bursting that exploits the unique stability properties associated with spike threshold (Baer, S. M., T. Erneux, and J. Rinzel. 1989. SIAM J. Appl. Math. 49:55-71).     

Expression Study on D123 Gene Product: Evidence for High Positivity in Testis

A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared withD123,three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in α-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.     

Estimating the cumulative incidence of COVID-19 in the United States using influenza surveillance,virologic testing,and mortality data: Four complementary approaches

Effectively designing and evaluating public health responses to the ongoing COVID-19 pandemic requires accurate estimation of the prevalence of COVID-19 across the United States (US). Equipment shortages and varying testing capabilities have however hindered the usefulness of the official reported positive COVID-19 case counts. We introduce four complementary approaches to estimate the cumulative incidence of symptomatic COVID-19 in each state in the US as well as Puerto Rico and the District of Columbia, using a combination of excess influenza-like illness reports, COVID-19 test statistics, COVID-19 mortality reports, and a spatially structured epidemic model. Instead of relying on the estimate from a single data source or method that may be biased, we provide multiple estimates, each relying on different assumptions and data sources. Across our four approaches emerges the consistent conclusion that on April 4, 2020, the estimated case count was 5 to 50 times higher than the official positive test counts across the different states. Nationally, our estimates of COVID-19 symptomatic cases as of April 4 have a likely range of 2.3 to 4.8 million, with possibly as many as 7.6 million cases, up to 25 times greater than the cumulative confirmed cases of about 311,000. Extending our methods to May 16, 2020, we estimate that cumulative symptomatic incidence ranges from 4.9 to 10.1 million, as opposed to 1.5 million positive test counts. The proposed combination of approaches may prove useful in assessing the burden of COVID-19 during resurgences in the US and other countries with comparable surveillance systems.     

Is Preening Behaviour Sexually Selected? An Experimental Approach

Elaborate or colourful feathers are important traits in female mate choice in birds but little attention has been given to potential costs of maintaining these traits in good condition with preening behaviour. Recent studies indicate that the time and energy required to maintain ornamental plumage in good condition reinforces the honesty of plumage trait. It has been proposed that some behaviours, whose primary function is not to transfer information, can also evolve as signalling components. Here we investigate whether the preening behaviour intensity has a signalling component: we hypothesized that if only high quality males can invest a lot of time in preening, this behaviour may be used by females as a quality signal (attractive preening hypothesis). We tested this hypothesis by using female budgerigars in mate‐choice tests in captivity. We tried to experimentally manipulate the preening behaviour of two groups of budgerigar males (treatment and control group). The proportion of time in which treated males preened in front of females was statistically higher than for control males, however, females spent similar amounts of time with treated males and control males. Moreover, males did not show significant quantitative changes in preening (for both groups) when females were present, suggesting that male budgerigars did not use this behaviour to convey information. These results are inconsistent with the ‘attractive preening’ hypothesis which predicts that preening behaviour itself provides information on condition and is used in female choice.     

Dual Utility of a Melanin‐Based Ornament in Bearded Tits

A general mechanism to account for the maintenance of sexually selected traits is the armament–ornament model, which predicts that females exploit signals used in male–male aggressive contests. Melanin‐based ornaments are good candidates to be dual utility traits because they are usually involved in male–male competition and function as signals of male dominance or fighting ability. Despite this, very few studies have investigated the dual utility of melanin‐based plumage traits and results remain generally ambiguous. In this study, we investigated the dual function of a melanin‐based trait (the black beard) in the male bearded tit, Panurus biarmicus. In a controlled aviary experiment, beard length was the best predictor of dominance in male–male competition. In a choice experiment, females showed a proximity preference for males whose beard length was experimentally elongated. Our results indicate that beard length plays an important role in both male–male competition and female choice in bearded tits, as proposed by the dual utility model.