High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.     

Ergatoid reproductives in Nasutitermes columbicus (Isoptera,Termitidae)

Numerous functional ergatoid replacement reproductives were found in one colony of Nasutitermes columbicus in Panama. Their morphology was mainly workerlike, although several imaginal characters such as the compound eyes and variable wing buds were more or less developed. The sex organs were fully mature and the fat body of the females, not of the males, was of the “royal” type. The development of the eyes was not accompanied by the differentiation of the optic lobes of the brain, nor was the presence of wing buds correlated with a development of the wing muscles.     

Differential effects of aldosterone and ADH on intracellular electrolytes in the toad urinary bladder epithelium

Summary Quantitative electron microprobe analysis was employed to compare the effects of aldosterone and ADH on the intracellular electrolyte concentrations in the toad urinary bladder epithelium. The measurements were performed on thin freeze-dried cryosections utilizing energy dispersive x-ray microanalysis. After aldosterone, a statistically significant increase in the intracellular Na concentration was detectable in 8 out of 9 experiments. The mean Na concentration of granular cells increased from 8.9±1.3 to 13.2±2.2 mmol/kg wet wt. A significantly larger Na increase was observed after an equivalent stimulation of transepithelial Na transport by ADH. On average, the Na concentration in granular cells increased from 12.0±2.3 to 31.4±9.3 mmol/kg wet wt (5 experiments). We conclude from these results that aldosterone, in addition to its stimulatory effect on the apical Na influx, also exerts a stimulatory effect on the Na pump. Based on a significant reduction in the Cl concentration of granular cells, we discuss the possibility that the stimulation of the pump is mediated by an aldosterone-induced alkalinization.Similar though less pronounced concentration changes were observed in basal cells, suggesting that this cell type also participates in transepithelial Na transport. Measurements in mitochondria-rich cells provided no consistent results.     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Electron microprobe analysis of intracellular electrolytes in resting and isoproterenol-stimulated exocrine glands of frog skin

Summary In the intact, in vitro frog skin, isoproterenol (ISO) stimulates and amiloride-insensitive increase in short-circuit current (SCC) that can be localized to the exocrine glands and is associated with secretion of chloride. To determine which cells in the glands respond to stimulation we measured the intracellular electrolyte concentrations of the various cell types of the mucous and seromucous glands of the skin using freeze-dried cryosections and electron microprobe analysis. In the resting state, the various cell types of the glands have intracellular electrolyte concentrations similar to the epithelial cells of the skin. Exposure to amiloride (10^–4 m) has little effect on the concentration of Na and Cl in the cells of the glands. The effect of isoproterenol has two distinct phases. Analysis of glands in tissues frozen at the peak of the SCC response (13 min after addition of isoproterenol) shows that the only significant change is an increase in Na and Ca in a group of cells at the ductal pole of the acini of both gland types. These are termed gland cells. The duct cells and cells that secrete macromolecules did not show any significant changes at this timepoint. In the gland cells, after a one-hour exposure to isoproterenol the Na concentration is at prestimulation levels while Cl drops. There is also a smaller drop in Cl in the duct and skin epithelial cells. Ouabain, which can completely block the isoproterenol SCC response, has little short-term effect on Na and Cl in the control gland but accentuates the gain of Na and drop in Cl in the isoproterenol-treated condition. Bumetanide and, to a lesser extent, furosemide, also blocks the isoproterenol SCC response and causes a further drop in Cl. The results provide indirect evidence that a major portion of the ionic component of the gland secretion is produced by a distinct group of cells separate from those producing the macromolecular component and that the mechanism of secretion involves a Na:Cl coupled transport system linked to the activity of the basolateral Na pump.     

Inhalation anesthesia in adult cattle

An inhalation technique was used for anesthesia during ileal cannulation in five adult cows. Following sedation with intravenous acepromazine, anesthesia was induced intravenously with thiopental sodium in 5% glyceryl guaiacolate solution. Endotracheal intubation was performed and anesthesia maintained with halothane in oxygen via a circle system with a precision vaporizer. In all cases, induction was smooth and no difficulties were experienced during the maintenance of anesthesia. Total anesthesia time was 1.5 to 2.5 hours. Following completion of the surgical procedure, which was performed with the animal in left lateral recumbency, each cow was rolled to a sternal position and supported, if necessary. The endotracheal tube was left in place, with oxygen administration continued, until the animal was able to swallow. Recoveries were rapid and all animals were ambulatory within 30 minutes after completion of the surgery. The only post-operative complication due to anesthesia was transient mouth soreness in two cases, attributed to the use of a mouth speculum during intubation.     

Chromosomal mapping of Bacillus thuringiensis by transduction.

Three groups of linked markers were mapped in Bacillus thuringiensis 4042B by using two-, three-, and four-factor crosses mediated by the temperate bacteriophages TP-13 and TP-18. The order of markers was (trp-11, trp-2)-(leu-1, leu-2)-his-1-(lys-1, lys-2)-cys-1 in the first group; met-1-(argCl, argOl)-met-2-(pyr-1, pyrA2) in the second group; and met-3-pur-1-(nal-1, nal-2)-str-1-(pur-2, pur-4)-pur-3 in the third group. Electron microscopic measurements of head sizes suggested that the volume of the TP-13 phage head is seven times greater than that of the TP-18 phage head. The TP-18 genome was shown by DNA restriction analysis to have a molecular mass of 36 megadaltons. TP-13 was useful for scanning large segments of the B. thuringiensis chromosome, and TP-18 was effective for ordering markers too closely linked for simple resolution with TP-13.     

Possible evidence for novel metabolism of nitrogen gas by a green alga

We report the isolation of a cukaryotic green alga ( Chlorella , strain WPI-2) which accumulates large stores of nitrogen (N) during growth in N-free medium and seems to incorporate¹⁴N₂, yet does not reduce acetylene to ethylene. Total N accumulation during growth on N-free medium and in gases free of combined N was measured by three methods: Kjeldahl, oxidative pyrolysis via chemiluminescence (Antek N analyzer), and Dumas (Coleman N analyzer). Increases in N ranging from 22–64%± 1% were observed. Isotope dilution studies using cells labelled with ¹⁵NO ₃- and then shifted to ¹⁴N₂ in N-free medium showed dilution of the ¹⁵N isotope by ¹⁴N from 5.67 to 5.32%± 0.05%. Using a variety of conditions, we were unable to demonstrate the reduction of acctylene to ethylene by WPI-2, although diazotrophic cyanobacteria gave positive results. Although the data on WPI-2 are not conclusive in establishing this alga as a diazotroph, the data do suggest that within the Chlorophyceae there may exist a novel form of nitrogen gas metabolism.