Distribution of spontaneous mutants and inferences about the replication mode of the RNA bacteriophage phi6

When a parent virus replicates inside its host, it must first use its own genome as the template for replication. However, once progeny genomes are produced, the progeny can in turn act as templates. Depending on whether the progeny genomes become templates, the distribution of mutants produced by an infection varies greatly. While information on the distribution is important for many population genetic models, it is also useful for inferring the replication mode of a virus. We have analyzed the distribution of mutants emerging from single bursts in the RNA bacteriophage phi6 and find that the distribution closely matches a Poisson distribution. The match suggests that replication in this bacteriophage is effectively by a stamping machine model in which the parental genome is the main template used for replication. However, because the distribution deviates slightly from a Poisson distribution, the stamping machine is not perfect and some progeny genomes must replicate. By fitting our data to a replication model in which the progeny genomes become replicative at a given rate or probability per round of replication, we estimated the rate to be very low and on the on the order of 10(-4). We discuss whether different replication modes may confer an adaptive advantage to viruses.     

Adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats

Adrenomedullin (AM) is a potent vasodilating peptide and is involved in cardiovascular and renal disease. In the present study, we investigated the role of AM in cardiac and renal function in streptozotocin (STZ)-induced diabetic rats. A single tail-vein injection of adenoviral vectors harboring the human AM gene (Ad.CMV-AM) was administered to the rats 1-wk post-STZ treatment (65 mg/kg iv). Immunoreactive human AM was detected in the plasma and urine of STZ-diabetic rats treated with Ad.CMV-AM. Morphological and chemical examination showed that AM gene delivery significantly reduced glycogen accumulation within the hearts of STZ-diabetic rats. AM gene delivery improved cardiac function compared with STZ-diabetic rats injected with control virus, as observed by decreased left ventricular end-diastolic pressure, increased cardiac output, cardiac index, and heart rate. AM gene transfer significantly increased left ventricular long axis (11.69 +/- 0.46 vs. 10.31 +/- 0.70 mm, n = 10, P < 0.05) and rate of pressure rise and fall (+6,090.1 +/- 597.3 vs. +4,648.5 +/- 807.1 mmHg/s), (-4,902.6 +/- 644.2 vs. -3,915.5 +/- 805.8 mmHg/s, n = 11, P < 0.05). AM also significantly attenuated renal glycogen accumulation and tubular damage in STZ-diabetic rats as well as increased urinary cAMP and cGMP levels, along with increased cardiac cAMP and Akt phosphorylation. We also observed that delivery of the AM gene caused an increase in body weight along with phospho-Akt and membrane-bound GLUT4 levels in skeletal muscle. These results suggest that AM plays a protective role in hyperglycemia-induced glycogen accumulation and cardiac and renal dysfunction via Akt signal transduction pathways.     

Proteomic analysis reveals alterations in the renal kallikrein pathway during hypoxia-induced hypertension

Obstructive sleep apnea syndrome (OSAS), a disorder characterized by episodic hypoxia (EH) during sleep, is associated with systemic hypertension. We used proteomic analysis to examine differences in rat kidney protein expression during EH, and their potential relationship to EH-induced hypertension. Young male Sprague-Dawley rats were exposed to either EH or sustained hypoxia (SH) for 14 (EH14/SH14) and 30 (EH30/SH30) days. Mean arterial blood pressure was significantly increased only in EH30 (p < 0.0002). Kidney proteins were resolved by two-dimensional-PAGE and were identified by MALDI-MS. Renal expression of kallistatin, a potent vasodilator, was down-regulated in all animals. Expression of alpha-1-antitrypsin, an inhibitor of kallikrein activation, was up-regulated in EH but down-regulated in SH. Western blotting showed significant elevation of B(2)-bradykinin receptor expression in all normotensive animals but remained unchanged in hypertensive animals. Proteins relevant to vascular hypertrophy, such as smooth muscle myosin and protein-disulfide isomerase were up-regulated in EH30 but were down-regulated in SH30. These data indicate that EH induces changes in renal protein expression consistent with impairment of vasodilation mediated by the kallikrein-kallistatin pathway and vascular hypertrophy. In contrast, SH-induced changes suggest the kallikrein- and bradykinin-mediated compensatory mechanisms for prevention of hypertension and vascular remodeling. To test the hypothesis suggested by the proteomic data, we measured the effect of EH on blood pressure in transgenic hKLK1 rats that overexpress human kallikrein. Transgenic hKLK1 animals were protected from EH-induced hypertension. We conclude that EH-induced hypertension may result, at least in part, from altered regulation of the renal kallikrein system.     

MAVG: locating non-overlapping maximum average segments in a given sequence

SUMMARY: MAVG is a software tool for finding k non-overlapping maximum-average segments that are sufficiently long in a given sequence of real numbers, for any k > 0. It has applications in several areas of biomolecular sequence analysis including locating GC-rich regions and CpG islands in a genomic sequence, and annotating multiple sequence alignments. AVAILABILITY: http://iubio.bio.indiana.edu/soft/molbio/pattern/cpg_islands/.     

The optimal sequence of microvascular repair during prolonged clamping in free flap transfer

During free flap transfer, the surgeon may decide to begin with repair of the artery or the vein(s) and to unclamp the first vessel as soon as repair is completed or maintain the clamping of both vessels until completion of all repairs. Complications can lead to prolonged clamping times, potentially increasing the risk of tissue ischemia, vascular damage, and thrombosis. The goals of the present study were to determine whether the sequence of vessel repair and the duration of clamping affect the success of free flap transfer in cases requiring prolonged clamping. Sixty abdominal fasciocutaneous free flaps based on the superficial inferior epigastric vessels were created in Sprague-Dawley rats. To model clinical situations in which prolonged clamping is necessary, the study used a 1-hour delay before the repair of the second vessel. Flaps were randomized into four groups. In group I (n = 15), the artery was repaired first, and the arterial clamp was removed immediately to allow arterial inflow. In group II (n = 15), the arterial repair was first, and the arterial clamp was maintained until completion of venous repair. In group III (n = 15), venous repair was first, with venous clamping maintained until completion of the arterial repair. In group IV (n = 15), initial venous repair was followed by immediate unclamping, before arterial repair. On release of all clamps, the patency of arteries and veins was confirmed immediately and after 1 hour using a "milking" test. On the fifth postoperative day, each flap was assessed for necrosis and for patency of the anastomoses. Of 15 flaps in each group, five (33 percent) failed in group I, four (27 percent) failed in groups II and III, and six (40 percent) failed in group IV. Differences between groups were not statistically significant (p = 0.8). These results demonstrate that in cases requiring prolonged occlusive clamping (2 to 3 hours), factors such as venous congestion, possible clamp injury, and presence of static blood in contact with the new anastomosis have relatively equivalent contributions to the risk of failure. Accordingly, no advantage seems to be gained by beginning with the artery or the vein or by using early or delayed unclamping of the first vessel repaired.     

Purification of industrial hydantoinase in one chromatographic step without affinity tag

Hydantoinase is used in industry as a biocatalyst for the production of optically pure D- or L-amino acids. Previously, homogeneous hydantoinase was obtained by multi-chromatographic purification procedures. Here, we reported a process that contained only a single chromatographic step to purify a recombinant hydantoinase to homogeneity. Hydantoinase from Agrobacterium radiobacter NRRL B11291 was expressed in Escherichia coli. The recombinant enzyme was purified following heat treatments, high concentration alcohol precipitation, and chelating Sephacel chromatography. The recombinant hydantoinase did not contain any affinity tags from the plasmid. This simplified procedure provided a convenient way to obtain hydantoinase in high yield (71%) and high purity. It should be very useful for further industrial application and for the study of the structure-function of hydantoinase.     

Effect of synthetic oligopeptides on osteoporosis

The objective of this study was to establish the solution method of GHRPS, the synthetic oligopeptides Tyr-Gly-Gly-Phe-Met-NH2, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Gly-NH2, and Tyr-Gly-Gly-Phe-Gly-OH, to verify their effect on osteoporosis. Male ICR mice (20 +/- 2 g) were used. The intramuscular injection dose of 6.3 mg/kg prednisone induced a significant decrease of body and femur weight of the animals. The subcutaneous injection dose of 18 microg/kg synthetic peptide was not effective to prevent the decrease of body and femur weight of the animals. The subcutaneous injection dose of 6.3 mg/kg prednisone elicited a decrease in content of femur calcium and in the level of serum calcium of the animals. The subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Leu-NH2, or Tyr-Gly-Gly-Phe-Leu-OH, or Tyr-Gly-Gly-Phe-Gly-NH2, significantly increased the content of femur calcium and decreased the level of serum calcium of the animals. It was also observed that the subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Gly-OH, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Met-NH2 significantly increased the content of femur phosphorous and decreased the activity of ALP of the animals.     

Constrained multiple sequence alignment tool development and its application to RNase family alignment

In this paper, we design a heuristic algorithm of computing a constrained multiple sequence alignment (CMSA for short) for guaranteeing that the generated alignment satisfies the user-specified constraints that some particular residues should be aligned together. If the number of residues needed to be aligned together is a constant alpha, then the time-complexity of our CMSA algorithm for aligning K sequences is O(alphaKn(4)), where n is the maximum of the lengths of sequences. In addition, we have built up such a CMSA software system and made several experiments on the RNase sequences, which mainly function in catalyzing the degradation of RNA molecules. The resulting alignments illustrate the practicability of our method.