Heat cleavage of bacteriophage T4 gene 23 product produces two peptides previously identified as head proteins.

During studies on the intracellular protein pools of bacteriophage T4, we found that amber mutants in gene 23 blocked the synthesis of a 20-kilodalton (kDa) protein. Radiolabeled amino acid pulses showed that the protein appears at 8 min postinfection with kinetics similar to those of other major late species. Pulse-chase experiments demonstrated that the 20-kDa protein behaves like a primary product and also revealed a 29-kDa protein which, like other proteins cleaved during head assembly, appeared only after a long chase. Both species have been identified as constituents of the T4 head and have resisted previous efforts to identify their genetic origin. The dependence of the 20- and 29-kDa head proteins on the presence of gene 23 protein (gp23) and the observation that the sum of their masses equalled that of mature cleaved gp23 suggested that these two proteins were derived from this major capsid species. Evidence is presented demonstrating that heating samples before electrophoresis causes peptide bond cleavages in gp23, leading to the formation of the two peptides. As predicted by the results of Rittenhouse and Marcus (Anal. Biochem. 138:442-448, 1984), the cleavage occurs at Asp-336-Pro-337 and at two other Asp-Pro sites. Limited heat-induced proteolysis followed by two-dimensional gel analysis provided a peptide map of gp23 useful in the characterization of its assembly-related cleavages.     

Characterizing genetic integrity of rear-edge trout populations in the southern Appalachians

Vertebrate populations at the periphery of their range can show pronounced genetic drift and isolation, and therefore offer unique challenges for conservation and management. These populations are often candidates for management actions such as translocations that are designed to improve demographic and genetic integrity. This is particularly true of coldwater species like brook trout (Salvelinus fontinalis), whose numbers have declined greatly across its historic range. At the southern margin, remnant wild populations persist in isolated headwater streams, and many have a history of receiving translocated individuals through either stocking of hatchery reared fish, relocation of wild fish, or both during restoration attempts. To determine current genetic integrity and resolve the genetic effects of past management actions for brook trout populations in SC, USA, we genetically assessed all 18 documented remaining brook trout populations along with individuals acquired from six hatcheries with recorded stocking events in SC. Our results indicated that six of the 18 streams showed signs of hatchery admixture (range 57–97%) and restored patches retained genetic signatures from multiple source populations. Populations had among the lowest genetic diversity (min average H_E?=?0.147) and effective number of breeders (mean N_b?=?31.2) estimates observed throughout the native brook trout range. Populations were highly differentiated (mean pair-wise F_ST?=?0.396), and substantial genetic divergence was evident across major river drainages (max pair-wise F_ST?=?0.773). The lowest local genetic diversity and highest genetic differentiation ever reported for this species make its conservation a challenging task, particularly when combined with other threats such as climate change and non-native species. We offer recommendations on managing peripheral populations with depleted genetic characteristics and provide a reference for determining which existing populations will best serve as sources for future translocation efforts aimed at enhancing or restoring wild brook trout genetic integrity.     

PH regulation of connexin43: molecular analysis of the gating particle.

Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent "gating particle," which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amino acids 274 and 285. Furthermore, site-directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amino acids 364-373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.     

Modeling and measuring lateral line excitation patterns to changing dipole source locations

In order to determine excitation patterns to the lateral line system from a nearby 50 Hz oscillating sphere, dipole flow field equations were used to model the spatial distribution of pressures along a linear array of lateral line canal pores. Modeled predictions were then compared to pressure distributions measured for the same dipole source with a miniature hydrophone placed in a small test tank used for neurophysiological experiments. Finally, neural responses from posterior lateral line nerve fibers in the goldfish were measured in the test tank to demonstrate that modeled and measured pressure gradient patterns were encoded by the lateral line periphery. Response patterns to a 50 Hz dipole source that slowly changed location along the length of the fish included (1) peaks and valleys in spike-rate responses corresponding to changes in pressure gradient amplitudes, (2) 180° phase-shifts corresponding to reversals in the direction of the pressure gradient and (3) distance-dependent changes in the locations of peaks, valleys and 180° phase-shifts. Modeled pressure gradient patterns also predict that the number of neural amplitude peaks and phase transitions will vary as a function of neuromast orientation and axis of source oscillation. The faithful way in which the lateral line periphery encodes pressure gradient patterns has implications for how source location and distance might be encoded by excitation patterns in the CNS. Phase-shift information may be important for (1) inhibitory/excitatory sculpting of receptive fields and (2) unambiguously encoding source distance so that increases in source distance are not confused with decreases in source amplitude.     

In vitro selection of miltefosine resistance in promastigotes of Leishmania donovani from Nepal: genomic and metabolomic characterization

In this study, we followed the genomic, lipidomic and metabolomic changes associated with the selection of miltefosine (MIL) resistance in two clinically derived Leishmania donovani strains with different inherent resistance to antimonial drugs (antimony sensitive strain Sb‐S; and antimony resistant Sb‐R). MIL‐R was easily induced in both strains using the promastigote‐stage, but a significant increase in MIL‐R in the intracellular amastigote compared to the corresponding wild‐type did not occur until promastigotes had adapted to 12.2 μM MIL. A variety of common and strain‐specific genetic changes were discovered in MIL‐adapted parasites, including deletions at the LdMT transporter gene, single‐base mutations and changes in somy. The most obvious lipid changes in MIL‐R promastigotes occurred to phosphatidylcholines and lysophosphatidylcholines and results indicate that the Kennedy pathway is involved in MIL resistance. The inherent Sb resistance of the parasite had an impact on the changes that occurred in MIL‐R parasites, with more genetic changes occurring in Sb‐R compared with Sb‐S parasites. Initial interpretation of the changes identified in this study does not support synergies with Sb‐R in the mechanisms of MIL resistance, though this requires an enhanced understanding of the parasite's biochemical pathways and how they are genetically regulated to be verified fully.     

Production and characterization of polyclonal and monoclonal antibodies against Aflatoxin B1 oxime-BSA in an enzyme-linked immunosorbent assay

From a single aflatoxin B₁ oxime — bovine serum albumin conjugate, polyclonal and monoclonal antibody preparations were produced. The four rabbit polyclonal antisera were specific for aflatoxin Bi in a microtitration plate enzyme — linked immunosorbent assay. The monoclonal antibodies showed a wide range of differing specificities, recognizing, for example, aflatoxins B₁, B₂, G₁ and G₂; B₁ and B₂; B₁ and G₁; and G₁ alone. No antibody preparations reacted with aflatoxin M₁. The significance of these results to the strategy of anti-aflatoxin antibody production for use in quantitative enzyme immunoassays is discussed.     

Effects of combined nitrogen on anapleurotic carbon assimilation and bleeding sap composition in Phaseolus vulgaris L.

Dwarf french beans, Phaseolus vulgaris L., were grown with or without inoculation with rhizobia (strain 3644), and with or without a combined nitrogen source (nitrate or ammonium ions). The distribution of radioactivity into products of dark ¹⁴CO₂ assimilation was studied in roots or nodules from these plants. A detailed study was also made of the distribution and rates of excretion of nitrogen in xylem bleeding sap in 28 day old plants grown on the various sources of nitrogen. Whereas detached nodules accumulated radioactive glycine, serine and glutamate when incubated with ¹⁴CO₂, bleeding sap from plants root fed ¹⁴CO₂ contained low levels of radioactivity in these compounds but higher levels in allantoin. Chemical analysis showed allantoin to be the major compound transported in the xylem of nodulated plants, whether or not they were fed on combined nitrogen. In contrast uninoculated plants accumulated mainly amino acids in the bleeding sap, the amount and chemical composition of which depended on the combined nitrogen source.Abbreviations PEP phosphoenol pyruvate - OAA oxaloacetate     

A lead phthalocyanin method for the demonstration of acid hydrolases in plant and animal tissues

Summary A new method of synthesis of the lead phthalocyanin and the chemical properties of its diazotate are described. The use of this reagent for high resolution studies of the localization of intra- and extralysosomal acid phosphatases and esterases in plant and animal cells has been assessed.