Design of bivalent ligands using hydrogen bond linkers: synthesis and evaluation of inhibitors for human beta-tryptase

We exploit the concept of using hydrogen bonds to link multiple ligands for maintaining simultaneous interactions with polyvalent binding sites. This approach is demonstrated by the syntheses and evaluation of pseudo-bivalent ligands as potent inhibitors of human β-tryptase.     

C-terminal site-specific PEGylation of a truncated thrombomodulin mutant with retention of full bioactivity

Addition of poly(ethylene glycol) to bioactive proteins (PEGylation) improves their plasma half-life, enhances stability against proteolytic cleavage, and may also decrease protein immunogenicity. Characteristically, PEGylation usually involves a reaction to available lysine amino groups, some of which may be within or near a bioactive site. Thus, most protocols are nonspecific and result in a loss of protein activity. We report herein a strategy for site-specific PEGylation of a thrombomodulin (TM) derivative at the C terminus. A truncated TM mutant consisting of epidermal growth factor (EGF)-like domains 4-6 was expressed in Escherichia coli with a C-terminal azido-methionine. The TM mutant was site-specifically conjugated to a methyl-PEG-triarylphosphine compound via the Staudinger reaction. Enzymatic activity of the TM construct before and after PEGylation was unchanged, which confirms the utility of this site-specific PEGylation scheme.     

UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340–400 nm), UVB (295–320 nm), UVC (254 nm) or simulated sunlight (SSL; λ > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was ~28, ~26, ~16 and ~30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of ‘UV signature’ mutational hotspots consisting primarily of C→T and CC→TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.     

Signals that dictate nuclear localization of human papillomavirus type 16 oncoprotein E6 in living cells

Human papillomavirus (HPV) type 16 E6 (16E6) is an oncogenic, multifunctional nuclear protein that induces p53 degradation and perturbs normal cell cycle control, leading to immortalization and transformation of infected keratinocytes and epithelial cells. Although it is unclear how 16E6 disrupts the epigenetic profile of host genes, its presence in the nucleus is a key feature. The present report describes intrinsic properties of 16E6 that influence its nuclear import in living cells. When the coding region of full-length 16E6 was inserted in frame into the C terminus of green fluorescent protein (GFP), it effectively prevented the 16E6 pre-mRNA from being spliced and led to the expression of a GFP-E6 fusion which localized predominantly to the nucleus. Further studies identified three novel nuclear localization signals (NLSs) in 16E6 that drive the protein to accumulate in the nucleus. We found that all three NLS sequences are rich in positively charged basic residues and that point mutations in these key residues could abolish the retention of 16E6 in the nucleus as well as the p53 degradation and cell immortalization activities of the protein. When inserted into corresponding regions of low-risk HPV type 6 E6, the three NLS sequences described for 16E6 functioned actively in converting the normally cytoplasmic HPV type 6 E6 into a nuclear protein. The separate NLS sequences, however, appear to play different roles in nuclear import and retention of HPV E6. The discovery of three unique NLS sequences in 16E6 provides new insights into the nuclear association of 16E6 which may reveal other novel activities of this important oncogenic protein.     

A transgene carrying an A2G missense mutation in the SMN gene modulates phenotypic severity in mice with severe (type I) spinal muscular atrophy

5q spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans and the leading genetic cause of infantile death. Patients lack a functional survival of motor neurons (SMN1) gene, but carry one or more copies of the highly homologous SMN2 gene. A homozygous knockout of the single murine Smn gene is embryonic lethal. Here we report that in the absence of the SMN2 gene, a mutant SMN A2G transgene is unable to rescue the embryonic lethality. In its presence, the A2G transgene delays the onset of motor neuron loss, resulting in mice with mild SMA. We suggest that only in the presence of low levels of full-length SMN is the A2G transgene able to form partially functional higher order SMN complexes essential for its functions. Mild SMA mice exhibit motor neuron degeneration, muscle atrophy, and abnormal EMGs. Animals homozygous for the mutant transgene are less severely affected than heterozygotes. This demonstrates the importance of SMN levels in SMA even if the protein is expressed from a mutant allele. Our mild SMA mice will be useful in (a) determining the effect of missense mutations in vivo and in motor neurons and (b) testing potential therapies in SMA.     

Importance of ambient saturation deficits in an epizootic of the fungus Neozygites floridana in cassava green mites (Mononychellus tanajoa)

The mite-pathogenic fungus Neozygites floridana Fisher (Entomophthorales: Neozygitaceae) is considered to have potential for the biological control of the cassava green mite, Mononychellus tanajoa (Bondar). However, its activity is sporadic and laboratory data suggest a strong dependence on night-time saturation deficits for transmission. We report on an epizootic of this fungus in a mite population in northeastern Brazil. During the epizootic, host populations appeared to be limited by a combination of the pathogen and a predatory mite Neoseiulus idaeus (Acari: Phytoseiidae). When temperatures increased, the epizootic finished and the host population began to grow. Abiotic conditions could not explain the variation in host mortality following pickup of infective propagules in this epizootic. However, night-time saturation did help to explain the variation in transmission from infective cadavers to newly killed hosts. This supports laboratory observations that horizontal transmission between hosts is determined mainly by saturation deficits, while the process of infection is little affected by abiotic conditions. A further field observation was the near-absence of resting spores in dead mites (ca. 0.1% of cadavers), suggesting that the pathogen population was unsuccessful in producing inoculum to infect future M. tanajoa populations. The implications are that this pathogen will only be effective as a biological control agent in periods of high relative humidity, and establishment in new areas may be limited by resting spore formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

An evaluation of the assembly of an approximately 15-Mb region on human chromosome 13q32-q33 linked to bipolar disorder and schizophrenia

The human 13q32-q33 region has been linked to both bipolar disorder and schizophrenia. Before completion of the draft sequences, we developed an approximately 15-Mb comprehensive map for the region extending from D13S1300 to ATA35H12. This map was assembled using publicly available mapping data and sequence-tagged site (STS)-based PCR confirmation. We then compared this map with the NCBI, Celera Genomics, and UCSC Golden Path data in February, June, and September 2001. All data sets showed gaps, misassignment of STSs, and errors in orientation and marker order. Surprisingly, the completed sequences of many bacterial artificial chromosomes (BACs) had been truncated. Of 21 gaps that were detected, 4 were present in both the NCBI and Celera databases. All gaps could be filled using 1-2 BAC clones. A total of 39 loci mapped to additional sites within the human genome, providing evidence of segmental duplications. Additionally, 61 unique cDNA clones were sequenced to increase available transcribed sequence, and 11,353 reference single-nucleotide polymorphisms (SNPs) with an average density of 1 SNP/3720 bases were identified. Overall, integration of the data from multiple sources is still needed for complete assembly of the 13q32-q33 region. (c)     

One-dimensional viscoelastic behavior of fibroblast populated collagen matrices

Bio-artificial tissues are being developed as replacements for damaged biologic tissues. Their mechanical properties are critical for load bearing applications. Current testing protocols for bio-artificial tissues vary widely and often do not consider viscoelasticity. Uniaxial stretch tests were performed on fibroblast populated collagen matrices (FPCMs) to determine the influence of specific test protocols on the mechanical behavior. The peak force, hysteresis and shape of the force-stretch curve are affected by the stretch rate, rest period, stretch amplitude and the number and magnitude of preconditioning cycles.     

Simulated human diving and heart rate: making the most of the diving response as a laboratory exercise

Laboratory exercises in which students examine the human diving response are widely used in high school and college biology courses despite the experience of some instructors that the response is unreliably produced in the classroom. Our experience with this exercise demonstrates that the bradycardia associated with the diving response is a robust effect that can easily be measured by students without any sophisticated measurement technology. We discuss measures that maximize the success of the exercise by reducing individual variation, designing experiments that are minimally affected by change in the response over time, collecting data in appropriate time increments, and applying the most powerful statistical analysis. Emphasis is placed on pedagogical opportunities for using this exercise to teach general principles of physiology, experimental design, and data analysis. Data collected by students, background information for instructors, a discussion of the relevance of the diving reflex to humans, suggestions for additional experiments, and thought questions with sample answers are included.