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.     

The effect of hypoxia on performance during 30 s or 45 s of supramaximal exercise

The purpose of this study was to evaluate the effect of hypoxia (10.8 +/- 0.6% oxygen) on performance of 30 s and 45 s of supramaximal dynamic exercise. Twelve males were randomly allocated to perform either a 30 s or 45 s Wingate test (WT) on two occasions (hypoxia and room air) with a minimum of 1 week between tests. After a 5-min warm-up at 120 W subjects breathed the appropriate gas mixture from a wet spirometer during a 5-min rest period. Resting blood oxygen saturation was monitored with an ear oximeter and averaged 97.8 +/- 1.5% and 83.2 +/- 1.9% for the air (normoxic) and hypoxic conditions, respectively, immediately prior to the WT. Following all WT trials, subjects breathed room air for a 10-min passive recovery period. Muscle biopsies from the vastus lateralis were taken prior to and immediately following WT. Arterialized blood samples, for lactate and blood gases, were taken before and after both the warm-up and the performance of WT, and throughout the recovery period. Open-circuit spirometry was used to calculate the total oxygen consumption (VO2), carbon dioxide production and expired ventilation during WT. Hypoxia did not impair the performance of the 30-s or 45-s WT. VO2 was reduced during the 45-s hypoxic WT (1.71 +/- 0.21 l) compared with the normoxic trial (2.16 +/- 0.26 l), but there was no change during the 30-s test (1.22 +/- 0.11 vs 1.04 +/- 0.17 l for the normoxic and hypoxic conditions, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of peritoneal ascites fluid by Freund's adjuvant: Genetic and hormonal influences.

Strain differences among inbred mice is demonstrated for the relative amounts of peritoneal ascites fluid induced by the injection of Freund's adjuvant and antigen. Females tend to produce more fluid than males; the lower level in males is attributable to the action of male hormone. Both genetic and epigenetic factors are determinants in the response levels; genetic factors differentiate the inbred strains of mice, epigenetic factors underlie the variations within a strain and the difference between sexes.     

Structural chromosome abnormalities in Down syndrome: a study of two families.

Two families were ascertained through a proband with Down syndrome and a structural rearrangement involving two chromosomes 21. It is suggested that in one patient the chromosome is an isochromosome formed by misdivision of the centromere of a maternal telocentric chromosome 21 and that in the other a Robertsonian translocation involving chromosome 21 was inherited from the mother, who is a 46,XX/46,XX, -21,+t(21q21q) mosaic. The origin of the mosaicism is discussed and considered to be likely to be the result of breakage and reunion at the chromatid, rather than the chromosome, level.     

THE ROLE OF CARBONIC ANHYDRASE IN CERTAIN IONIC EXCHANGES INVOLVING THE ERYTHROCYTE

1. The acceleration by bicarbonates of the swelling and hemolysis of erythrocytes in solutions of ammonium salts, first reported by Ørskov, is strikingly dependent upon carbonic anhydrase, being almost abolished by inhibitors of this enzyme such as KCN and sulfanilamide, and under suitable conditions being enhanced by its addition to the external solution. This behavior gives support to the theory of "catalyzed diffusion" as an explanation of the Ørskov effect. 2. The inhibitory effects of both sulfanilamide and KCN seem to be capable of complete reversal on washing the erythrocytes in isotonic salt solutions. The full effect of KCN appears almost instantly; that of sulfanilamide requires a period measured in seconds, or possibly even in minutes, to reach its maximum, the delay presumably being due to the slower penetration of the erythrocyte by this substance. Under favorable conditions the effect of concentrations of sulfanilamide of a few hundredths of a milligram per cent can be demonstrated. No similar effects have been obtained with sulfapyridine. 3. Bicarbonates also have a "catalytic" effect on the response of the internal pH of erythrocytes to changes in that of their surroundings. The resulting volume changes of the cell, which otherwise frequently require many minutes for their completion, may take place within a few seconds in the presence of low concentrations of bicarbonates. At a given pH value the effect of the latter substances is chiefly on the rate of the change and only to a minor extent on its magnitude. It may be further accelerated under appropriate conditions by the addition to the cell suspension of carbonic anhydrase, and can be almost abolished by KCN and by sulfanilamide. 4. Volume changes of erythrocytes associated with exchanges of Cl'' for SO₄'''' ions are greatly accelerated by low concentrations of bicarbonates, this effect being likewise dependent upon carbonic anhydrase. There is some evidence that in this case the exchange takes place, at least in part, in two steps: Cl'' for HCO₃'' and HCO₃'' for SO₄''''.