The structure of B-helical C-G-A-T-C-G-A-T-C-G and comparison with C-C-A-A-C-G-T-T-G-G. The effect of base pair reversals

The crystal structure of the DNA decamer C-G-A-T-C-G-A-T-C-G has been solved to a resolution of 1.5 A, with a final R-factor of 16.1% for 5,107 two-sigma reflections. Crystals are orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 38.93 A, b = 39.63 A, c = 33.30 A, and 10 base pairs/asymmetric unit. The final structure contains 404 DNA atoms, 142 water molecules treated as oxygen atoms, and two Mg(H2O)6(2+) complexes. Decamers stack atop one another to simulate continuous helical columns through the crystal, as with three previously solved monoclinic decamers, but the lateral contacts between columns are quite different in the orthorhombic and monoclinic cells. Narrow and wide regions of the minor groove exhibit a single spine or two ribbons of hydration, respectively, and the minor groove is widest when BII phosphate conformations are opposed diagonally across the groove. Phosphate conformation, in turn, appears to have a base sequence dependence. Twist, rise, cup, and roll are linked as has been observed in the three monoclinic decamers and can be characterized by high or low twist profiles. In all five known decamer crystal structures and eight representative dodecamers, a high twist profile is observed with G-C and G-A steps whereas all other R-R steps are low twist profiles (R = purine). A-T and A-C steps are intermediate in character whereas C-A and C-G exhibit behavior that is strongly influenced by the profiles of the preceding and following steps. When sufficient data are in hand, sequence/structure relationships for all helix parameters probably should be considered in a 4-base pair context. At this stage of limited information the problem is compounded because there are 136 unique 4-base steps x-A-B-y in a double helix as compared with only 10 2-base steps A-B.     

Synthesis of cyclic peptides on solid support. Application to analogs of hemagglutinin of influenza virus

In order to mimic a well-known loop structure (site A) of the hemagglutinin of influenza virus, a series of cyclic peptides derived from the region 139-147 were synthesized. The lactam analogs cyclised between the N-terminus Cys 139 and the beta-carboxyl of aspartic acid 148 (small loop) or the epsilon-NH2 of lysine 148 via succinimidyl linker (large loop) were synthesized by the solid phase method. Cyclisation was directly performed on the solid support prior to final cleavage of the peptide. We describe two protection schemes which allow us to obtain different loop sizes derived from the same sequence. Eight of the analogs contained relatively large ring structures (up to 38 membered). For protection of the side chain of aspartic acid in combination with N-alpha-Fmoc protection, the cyclohexyl ester was more satisfactory than the benzyl ester with respect to imide formation. When the rate of cyclodimerisation, as a function of resin substitution, was compared to the rate of cyclic monomer formation, it was found that dimerisation was proportional to the charge of the resin. Furthermore, a comparison of the recently reported BOP reagent over the classical DIPC/HOBt method for the cyclisation reaction shows that in our case the reaction proceeded more rapidly by the BOP procedure although it gave a less pure crude product.     

Comparison of performances of four ELISA kits in the detection of BLV antibodies in bulk tank milk or concentrated lactoserum from herds with low prevalence of infection

Performances of four ELISA kits in the detection of BLV antibodies in bulk tank milk was studied in 76 non-infected herds and 44 herds with low prevalence of BLV infection. None of the kits gave false positive results. On the other hand, there was an important variation in sensitivity. The kits with the highest sensitivity identified 43% of infected herds, which included 65% of infected cows. When concentrated lactoserum was tested, 59% of infected herds, which included 73% of infected cows, could be identified.     

Fluorescence banding in four species of Microtidae: an analysis of the evolutive changes of the constitutive heterochromatin

Three different fluorochrome and specific counterstain combination (DAPI/AMD, DA/DAPI and CMA/DA) treatments were applied to the chromosomes of four Microtidae (Rodentia) species. The results complete the data obtained in our previous paper (Burgos, M., Jiménez, R., & Dìaz de la Guardia, R., Genome 30:540–546, 1988) and prove that the changes in the constitutive heterochromatin in the evolution of the karyotypes of these species are not only due to gain or loss of heterochromatin, but are qualitative with respect to their nucleotide composition, repeated base pair organization or DNA-protein complex modification. These variations lead to the differential response to the fluorescence dye combinations used.     

Real-time gait assessment utilizing a new way of accelerometry

Real-time registration of body segment angles is essential in artificial body position control. A new method is presented for the real-time calculation of the lower extremity angles using data obtained from pairs of two one-dimensional accelerometers. It is shown that, assuming rigid-body dynamics and simple hinge joints, relative angles (i.e. angles between segments) can be calculated without integration, thereby solving the problem of integration drift normally associated with accelerometry. During the stance phase of walking, the relative angles can be transformed to absolute angles (i.e. relative to the gravitational field direction) for the different leg segments. The feasibility of relative angle calculation is demonstrated by calculation of the knee angle of a healthy subject. Stability and resolution were demonstrated with measurements during standing. Measurements during standing up, sitting down and walking showed that shock (heel-strike) and skin movements, due to movements of the underlying muscle tissue, are the main error sources. Additional signal processing, e.g. low-pass filtering, can be used to diminish this error. The accuracy of the knee angle found is shown to be high enough to be used in a feedback controller for functional electrostimulation of the lower extremities.     

Immunohistological detection of Saruplase (recombinant single-chain urokinase-type plasminogen activator) in normal rat tissue

Summary Saruplase — a recombinant single-chain urokinase-type plasminogen activator was identified immunohistochemically in normal rat tissue after intravenous administration by means of a polyclonal antibody. For this purpose, rat tissues were fixed in various ways (liquid nitrogen, ethanol, formaldehyd solution). Saruplase could be detected by the PAP method, streptavidinbiotin system and indirect immunofluorescence in the kidney (proximal tubule), liver (hepatocytes, Kupffer cells) and spleen (reticular cells). Saruplase was not localized in the rat endothelium. It is discussed that the ratspecific receptors for urokinase-type plasminogen activator on endothelial cells cannot bind Saruplase due to the extreme species specificity.     

Comparative studies on the degradation of guanidino and ureido compounds by Pseudomonas

The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Multiple routes of agmatine catabolism were found. All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine. In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase. A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidinobutyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate. Novel amidino-hydrolases were characterized in P. putida for the utilization of arcaine and audouine, and in P. cepacia for arcaine, homoarginine and guanidinovalerate. Guanidinovalerate amidinohydrolase was also detected in P. doudoroffii. Some of these amidinohydrolases accept more than one substrate, e.g., guanidinobutyrate and guanidinovalerate utilization by P. doudoroffii and P. cepacia, the catabolism of arcaine and audouine by P. putida, and the degradation of arcaine and homoarginine by P. cepacia.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

Decarboxylation of branched-chain alpha-ketoacids in hepatocytes from alloxan-diabetic rats. The effect of insulin

The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.