Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis

Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.     

Technical knockout, a Drosophila model of mitochondrial deafness

Mutations in mtDNA-encoded components of the mitochondrial translational apparatus are associated with diverse pathological states in humans, notably sensorineural deafness. To develop animal models of such disorders, we have manipulated the nuclear gene for mitochondrial ribosomal protein S12 in Drosophila (technical knockout, tko). The prototypic mutant tko(25t) exhibits developmental delay, bang sensitivity, impaired male courtship, and defective response to sound. On the basis of a transgenic reversion test, these phenotypes are attributable to a single substitution (L85H) at a conserved residue of the tko protein. The mutant is hypersensitive to doxycyclin, an antibiotic that selectively inhibits mitochondrial protein synthesis, and mutant larvae have greatly diminished activities of mitochondrial redox enzymes and decreased levels of mitochondrial small-subunit rRNA. A second mutation in the tko gene, Q116K, which is predicted to impair the accuracy of mitochondrial translation, results in the completely different phenotype of recessive female sterility, based on three independent transgenic insertions. We infer that the tko(25t) mutant provides a model of mitochondrial hearing impairment resulting from a quantitative deficiency of mitochondrial translational capacity.     

Grain processing and nutrition

Whole grains provide a wide range of nutrients and phytochemicals that optimize health. Epidemiologic studies support the protectiveness of whole grain consumption for cardiovascular disease and cancer. Dietary guidance endorses increased whole grains in our diet. A crucial question remaining is the effect of processing of whole grains on their content of nutrients and phytochemicals. Although processing is often considered to be a negative attribute in nutrition, and some forms of processing reduce nutritional value, many factors support the importance of processing of grains to enhance grain consumption. First, whole grains as harvested are generally not consumed directly by humans but require some processing prior to consumption. While refining, that is, removal of the bran and the germ, reduces the nutrient content of grain, milling of grains otherwise concentrates desirable grain components and removes poorly digested compounds and contaminants. Cooking of grains generally increases digestibility of nutrients and phytochemicals. Studies in both animal models and humans support the notion that processed grains are often nutritionally superior to unprocessed grains, probably because of enhanced nutrient bioavailability in processed grains. Processing of grains also provides shelf-stable products that are convenient and good tasting for consumers.     

Protein flexibility predictions using graph theory

Techniques from graph theory are applied to analyze the bond networks in proteins and identify the flexible and rigid regions. The bond network consists of distance constraints defined by the covalent and hydrogen bonds and salt bridges in the protein, identified by geometric and energetic criteria. We use an algorithm that counts the degrees of freedom within this constraint network and that identifies all the rigid and flexible substructures in the protein, including overconstrained regions (with more crosslinking bonds than are needed to rigidify the region) and underconstrained or flexible regions, in which dihedral bond rotations can occur. The number of extra constraints or remaining degrees of bond-rotational freedom within a substructure quantifies its relative rigidity/flexibility and provides a flexibility index for each bond in the structure. This novel computational procedure, first used in the analysis of glassy materials, is approximately a million times faster than molecular dynamics simulations and captures the essential conformational flexibility of the protein main and side-chains from analysis of a single, static three-dimensional structure. This approach is demonstrated by comparison with experimental measures of flexibility for three proteins in which hinge and loop motion are essential for biological function: HIV protease, adenylate kinase, and dihydrofolate reductase.     

Cytological characterization of heterochromatin and rDNA inPinus radiata andP. taeda

Fluorochrome C-banding ofPinus radiata andP. taeda metaphase chromosomes showed many pericentromeric DAPI bands and interstitial CMA bands inP. radiata, and centromeric and interstitial CMA bands inP. taeda. Giemsa C-band patterns differed between the species with centromeric bands inP. radiata but no consistent bands inP. taeda. A karyotype ofP. radiata was developed based on banding patterns that distinguished all but two of the 12 pairs of chromosomes. In situ hybridization (ISH) using probes for high-copy ribosomal DNA (rDNA) showed 10 pairs of 18S–25S sites and two pairs of 5S sites in both species. Most of the sites were interstitial or centromeric.     

Biofilms and biodiversity: an interactive exploration of aquatic microbial biotechnology and ecology

The study of biofilms provides a unique educational opportunity to examine ecosystems, biodiversity and applications of environmental biotechnology. There are many variables that could be studied for measuring the interactions between bacterial biofilms and invertebrate biodiversity as a method for assessing the health of aquatic ecosystems. These interactions also lend themselves to an easily replicated model system which can be used to reach a wide audience with an educational opportunity for students as well as a professional development opportunity for teachers. At the foundation of the research are invaluable basic microbiology skills: strain collection, isolation, cultivation and characterization. Through the additional process of characterizing, identifying and enumerating invertebrate organisms that attach to bacterial biofilms in aquatic ecosystems, there evolved a multidisciplinary class laboratory activity that has found broad application. This activity is captivating not only to undergraduate microbiology students but to middle and high school students and their teachers. The demand for information about the activity has led to the development of a truly interactive web-based lesson, which in turn has resulted in additional inquiries and further refinement of the lesson as an undergraduate independent research course. Both of these are freely accessible on the web, with growing international participation and data exchange. Journal of Industrial Microbiology & Biotechnology (2000) 24, 334–338. Received 19 April 1999/ Accepted in revised form 17 February 2000     

Mechanisms of Hearing Loss after Blast Injury to the Ear

Given the frequent use of improvised explosive devices (IEDs) around the world, the study of traumatic blast injuries is of increasing interest. The ear is the most common organ affected by blast injury because it is the body’s most sensitive pressure transducer. We fabricated a blast chamber to re-create blast profiles similar to that of IEDs and used it to develop a reproducible mouse model to study blast-induced hearing loss. The tympanic membrane was perforated in all mice after blast exposure and found to heal spontaneously. Micro-computed tomography demonstrated no evidence for middle ear or otic capsule injuries; however, the healed tympanic membrane was thickened. Auditory brainstem response and distortion product otoacoustic emission threshold shifts were found to be correlated with blast intensity. As well, these threshold shifts were larger than those found in control mice that underwent surgical perforation of their tympanic membranes, indicating cochlear trauma. Histological studies one week and three months after the blast demonstrated no disruption or damage to the intra-cochlear membranes. However, there was loss of outer hair cells (OHCs) within the basal turn of the cochlea and decreased spiral ganglion neurons (SGNs) and afferent nerve synapses. Using our mouse model that recapitulates human IED exposure, our results identify that the mechanisms underlying blast-induced hearing loss does not include gross membranous rupture as is commonly believed. Instead, there is both OHC and SGN loss that produce auditory dysfunction.     

Cytogenetic analysis of lymphoblastoid cell lines

Cytogenetic abnormalities were discovered in more than half of 16 lymphoblastoid cell lines established from fragile X individuals and their relatives upon routine cytogenetic analysis of early passage cultures. Subsequently, a second series of lymphoblastoid lines was analyzed to determine if the aneuploidy was a characteristic of lymphoblastoid cell lines derived from fragile X families or a result of the use of cyclosporin A in the establishment of these lines. In the second series of 33 lymphoblastoid cultures, no aneuploid clones were found in the fragile X group, while two were detected in the control cultures, one in a line initiated with cyclosporin A and the other in a line established without cyclosporin A. We conclude that the abnormal clones in our preliminary series were not a characteristic of lines derived from fragile X families and probably not due to the use of cyclosporin A. However, the finding of chromosome abnormalities in a large proportion of lines during the first 3 mo of culture contrasts with previous reports of chromosome stability for the first 12-18 mo of cultivation and indicates that the chromosomes of lymphoblastoid lines should be monitored in any experiment in which a normal diploid complement is critical.