Dynamic coupling of residues within proteins as a mechanistic foundation of many enigmatic pathogenic missense variants

Many pathogenic missense mutations are found in protein positions that are neither well-conserved nor fall in any known functional domains. Consequently, we lack any mechanistic underpinning of dysfunction caused by such mutations. We explored the disruption of allosteric dynamic coupling between these positions and the known functional sites as a possible mechanism for pathogenesis. In this study, we present an analysis of 591 pathogenic missense variants in 144 human enzymes that suggests that allosteric dynamic coupling of mutated positions with known active sites is a plausible biophysical mechanism and evidence of their functional importance. We illustrate this mechanism in a case study of β-Glucocerebrosidase (GCase) in which a vast majority of 94 sites harboring Gaucher disease-associated missense variants are located some distance away from the active site. An analysis of the conformational dynamics of GCase suggests that mutations on these distal sites cause changes in the flexibility of active site residues despite their distance, indicating a dynamic communication network throughout the protein. The disruption of the long-distance dynamic coupling caused by missense mutations may provide a plausible general mechanistic explanation for biological dysfunction and disease.     

Molecular cloning and characterization of ovine homeo-box-containing genes

The sheep genome contains at least eleven homeo-boxes (hox). Using two hox-specific 36-mer oligodeoxynucleotides to screen a sheep genomic library, constructed in lambda Charon28, clones of nine of the hox were identified. Six of the hox clones were analysed by nucleotide sequencing, Southern-blot hybridization and Northern-blot analysis. Two of the hox appear to be cognates of the human Hu-1 (or mouse Hox 2.1) and the mouse Hox 1-3, while another is closely related to the mouse Hox 1-4. These results suggest that there is strong sequence conservation in the hox-containing genes of different mammals, and highlight the possible occurrence of an ubiquitous set of hox-containing genes in mammals. Northern-blot analysis of four sheep hox-containing genes indicates that they are all expressed during embryogenesis and that expression is temporally regulated allowing hierarchical-regulatory interaction. Interestingly, none of the cloned hox-containing sequences contain repetitive sequences.     

Posthatching development of Alligator mississippiensis ovary and testis

We investigated ovary and testis development of Alligator mississippiensis during the first 5 months posthatch. To better describe follicle assembly and seminiferous cord development, we used histochemical techniques to detect carbohydrate‐rich extracellular matrix components in 1‐week, 1‐month, 3‐month, and 5‐month‐old gonads. We found profound morphological changes in both ovary and testis. During this time, oogenesis progressed up to diplotene arrest and meiotic germ cells increasingly interacted with follicular cells. Concomitant with follicles becoming invested with full complements of granulosa cells, a periodic acid Schiff's (PAS)‐positive basement membrane formed. As follicles enlarged and thecal layers were observed, basement membranes and thecal compartments gained periodic acid‐methionine silver (PAMS)‐reactive fibers. The ovarian medulla increased first PAS‐ and then PAMS reactivity as it fragmented into wide lacunae lined with low cuboidal to squamous epithelia. During this same period, testicular germ cells found along the tubule margins were observed progressing from spermatogonia to round spermatids located within the center of tubules. Accompanying this meiotic development, interstitial Leydig cell clusters become more visible and testicular capsules thickened. During the observed testis development, the thickening tunica albuginea and widening interstitial tissues showed increasing PAS‐ and PAMS reactivity. We observed putative intersex structures in both ovary and testis. On the coelomic aspect of testes were cell clusters with germ cell morphology and at the posterior end of ovaries, we observed “medullary rests” resembling immature testis cords. We hypothesize laboratory conditions accelerated gonad maturation due to optimum conditions, including nutrients and temperature. Laboratory alligators grew more rapidly and with increased body conditions compared with previous measured, field‐caught animals. Additionally, we predict the morphological maturation observed in these gonads is concomitant with increased endocrine activities. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Analysis of breast implant capsular tissue for crystalline silica and other refractile phases

This study questions previous reports of the presence of micrometer-sized areas of crystalline silica in pathologic tissue sections that are based exclusively on polarized-light microscopy. By using optical principles, it can be argued that it is impossible to identify unambiguously or to detect the birefringence of crystalline silica in 5-microm-thin sections. To clarify whether silicone, amorphous silica, or crystalline silica occurs in micrometer-sized moieties in standard 5-microm-thick tissue sections, one needs to apply a structural means of analysis in addition to optical microscopy. This study recommends the use of the laser Raman spectroscopic technique, which is very well suited to clarify this highly controversial issue in future pathologic studies.     

Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their correlation with fertility

The objectives of this study were to 1) identify proteins found in stallion seminal plasma utilizing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) in conjunction with Western blot analysis; and 2) to determine if any of these individual proteins were correlated with stallion fertility utilizing regression analysis. Fertility was quantified by assigning a breeding score for each stallion. Each score was calculated by dividing the number of conceptions by the number of breedings for each stallion for four successive breeding seasons (1992-1995). Ejaculates from stallions of known fertility (n = 6) were collected with a Missouri-style artificial vagina. Immediately after collection, the semen sample was filtered and the gel fraction removed. The resultant sperm-rich fraction was centrifuged in a Beckman Microfuge E at 10,000 x g and the seminal plasma aspirated from the pelleted sperm cells. Two-dimensional PAGE of the seminal plasma was performed under denaturing conditions which revealed that 14 proteins were common in all stallions in the research population. Four of these proteins (SP-1, SP-2, SP-3, and SP-4) were found to be significantly (P < 0.05) correlated with the breeding score assigned for each stallion. Regression analysis of protein optical densities with breeding score indicated that SP-1 (72 kDa, pI 5.6) was positively correlated with fertility (P < 0.05, r2 = 0.706), while SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 4.3), and SP-4 (16 kDa, pI 6.5) were found to be negatively correlated (P < 0.05, r2 = 0.762, 0.730, 0.775 respectively) with fertility. Western blot analysis of SP-1 indicated there was an antigenic homology with a bovine 55 kDa fertility-associated seminal plasma protein identified in a study by Killian et al. (19). This suggests that the two proteins may have a similar physiological role and therefore common biological properties. These results indicate that analysis of stallion seminal plasma proteins can be used as an indicator of fertilizing capacity. Identification of such proteins in stallion seminal plasma could lead to better insight into the nature of subfertility or infertility in the horse, as well as to indicate better cryopreservation strategies.     

An in vivo method for measuring turbulence in mechanical prosthesis leakage jets

This work introduces a method for the in vivo measurement and analysis of turbulence within the leakage of a mechanical heart valve. Several analysis techniques were applied to ultrasound measurements acquired within the atrium of a pig, and error associated with these techniques was analyzed. The technique chosen applies cyclic averaging to mean and maximum velocity measurements within small, normalized phase windows to calculate Reynolds normal stresses in the direction of the ultrasound beam. Maximum shear stresses are estimated from these normal stresses using an analytical technique. The stresses observed were smaller than those reported from previous in vitro simulations.     

Synthesis of a bromotyrosine-derived natural product inhibitor of mycothiol-S-conjugate amidase

Recently we described the structures of two new bromotyrosine-derived alkaloids that inhibit the detoxification enzyme mycothiol-S-conjugate amidase (MCA) from Mycobacterium tuberculosis. Here we describe a concise total synthesis of bromotyrosine oxime 1. The six-step synthesis of 1 utilized a trifluoromethyloxazole intermediate, whose hydrolysis product underwent alkylation and coupling to agmatine to give the inhibitor in approximately 40% overall yield. Oxime 1 inhibited MCA and its homolog AcGI deacetylase with IC(50) values of 30 and 150 microM, respectively.     

Mastermind recruits CycC:CDK8 to phosphorylate the Notch ICD and coordinate activation with turnover

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.