Structural characterization of Tobacco etch virus coat protein mutants.

The assembly of Tobacco etch potyvirus (TEV) coat protein (CP) and truncated mutants in Escherichia coli was studied. CP from which 28, 63 or 112 amino acids were deleted from the N-terminus polymerized into potyvirus-like particles (PVLPs). These structures were more rigid and progressively smaller in diameter than those produced by full length TEV-CP. CP from which 175 N-terminal amino acids were removed, failed to polymerize. A fragment containing amino acids 131 to 206 of TEV-CP is sufficient for PVLP assembly in E. coli. To determine the function of the highly conserved amino acids Ser152, Arg154, and Asp198 point mutants were generated. The mutant CPDelta63(Asp198Glu) exhibited different spectral properties following circular dichroism analysis showing a lower amount of alpha-helix compared to the wild type molecule. No differences were observed in spectra obtained from fluorescence spectroscopy. The point mutants bind RNA in vitro to the same degree as the wild type protein. However, while the wild type and the Arg154Gln mutant CP were each able to form PVLPs in E. coli, the Asp198Glu and the double mutant Ser152Pro/Arg154Gln mutants did not. These results suggest that the Asp198Glu mutation has an altered secondary structure which affects the capacity of the protein to polymerize but did not affect in vitro protein-RNA interactions.

Wire-bottom versus solid-bottom rodent caging issues important to scientists and laboratory animal science specialists.

Recent emphasis in the National Research Council's Guide for the Care and Use of Laboratory Animals and by the Association for Assessment and Accreditation of Laboratory Animal Care, International, related to the availability of bedding in rodent cages raises regulatory and accreditation issues in the toxicology-laboratory setting. This article reviews the results of a recent survey of 12 United States-based pharmaceutical and contract toxicology laboratories. The perceived benefits and issues related to the use of wire-bottom and bedded caging for rodent studies are presented. The 1999 survey showed that more than 80% of the rodents in surveyed toxicology facilities were housed in wire-bottom cages. Long-term budget expenses related to supplies and waste disposal are assessed. Considerable short-term and long-term costs to programs would be associated with a change from wire-bottom to solid-bottom caging. A review of the past and recent literature related to animal preferences and cage-associated animal lesions is included. The importance of IACUC review of caging chosen by the investigative staff is emphasized.

Short-Term and medium-term effects of spinal cord tract transections on soleus H-reflex in freely moving rats.

Spinal cord function is normally influenced by descending activity from supraspinal structures. When injury removes or distorts this influence, function changes and spasticity and other disabling problems eventually appear. Understanding how descending activity affects spinal cord function could lead to new means for inducing, guiding, and assessing recovery after injury. In this study, we investigated the short-term and medium-term effects of spinal cord bilateral dorsal column (DC), unilateral (ipsilateral) lateral column (LC), bilateral dorsal column ascending tract (DA), or bilateral dorsal column corticospinal tract (CST) transection at vertebral level T8-T9 on the soleus H-reflex in freely moving rats. Data were collected continuously for 10-20 days before and for 20-155 days after bilateral DC (13 rats), DA (10 rats), CST (eight rats), or ipsilateral LC (seven rats) transection. Histological examination showed that transections were 98(+/- 3 SD)% complete for DC rats, 80(+/- 20)% complete for LC rats, 91(+/- 13 SD)% complete for DA rats, and 95(+/-13)% complete for CST rats. LC, CST, and DA transections produced an immediate (i.e., first-day) increase in H-reflex amplitude. LC transection also produced a small decrease in background activity in the first few posttransection days. Other than this small decrease, none of the transections produced evidence for the phenomenon of spinal shock. For all transections, all measures returned to or neared pretransection values within 2 weeks. DA and LC transections were associated with modest increase in H-reflex amplitude 1-3 months after transection. These medium-term effects must be taken into account when assessing transection effects on operant conditioning of the H-reflex. At the same time, the results are consistent with other evidence that, while H-reflex rate dependence and H-reflex operant conditioning are sensitive measures of spinal cord injury, the H-reflex itself is not.

Fungal pathogen protection in potato by expression of a plant defensin peptide.

Defensins are small cysteine-rich peptides with antimicrobial activity. We demonstrate that the alfalfa antifungal peptide (alfAFP) defensin isolated from seeds of Medicago sativa displays strong activity against the agronomically important fungal pathogen Verticillium dahliae. Expression of the alfAFP peptide in transgenic potato plants provides robust resistance in the greenhouse. Importantly, this resistance is maintained under field conditions. There have been no previous demonstrations of a single transgene imparting a disease resistance phenotype that is at least equivalent to those achieved through current practices using fumigants.

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.

Purification and characterization of recombinant tomato fruit (Lycopersicon esculentum Mill.) fructokinase expressed in Escherichia coli.

Fructokinase (FK; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) cloned from a tomato fruit cDNA library has been expressed in Escherichia coli. The recombinant protein was purified 159-fold to greater than 99% purity, based on SDS-PAGE analysis. The subunit molecular mass is estimated to be 35 kDa and the nondissociated molecular mass is 72.4 kDa, indicating that the functional form is a dimer. Two-dimensional IEF/SDS-PAGE analyses combined with immunodetection show that both native and recombinant proteins exhibit the same pattern of six closely grouped peptides with pI values ranging from 5.66 to 6.17. Biochemical characterization of the purified recombinant enzyme shows properties essentially identical to those of the native fructokinase purified from young tomato fruit: the pH optimum is 8.0, the K(m) for fructose is 0.22 mM, and severe substrate inhibition is observed when fructose concentration is greater than 0.5 mM (Ki = 3.0 mM). ATP is the preferred phosphate donor (K(m) = 0.13 mM and Vmax/K(m) = 212), followed by GTP (K(m) = 0.45 mM and Vmax/K(m) = 76) and UTP (K(m) = 1.68 mM and Vmax/K(m) = 20), but Vmax values are slightly greater with GTP and UTP. Product inhibition analyses show that the inhibition by ADP with respect to ATP is dependent on fructose concentration [Ki (ADP) = 0.41 mM with 0.5 mM fructose and decreased to 0.12 mM with 3 mM fructose]. Inhibition by fructose 6-P shows weak noncompetitive inhibition with respect to fructose; however, the recombinant protein is slightly more sensitive to fructose 6-P than the native FK.

Pre-exercise glycerol hydration improves cycling endurance time.

The effects of glycerol ingestion (GEH) on hydration and subsequent cycle ergometer submaximal load exercise were examined in well conditioned subjects. We hypothesized that GEH would reduce physiologic strain and increase endurance. The purpose of Study I (n = 11) was to determine if pre-exercise GEH (1.2 gm/kg glycerol in 26 ml/kg solution) compared to pre-exercise placebo hydration (PH) (26 ml/kg of aspartame flavored water) lowered heart rate (HR), lowered rectal temperature (Tc), and prolonged endurance time (ET) during submaximal load cycle ergometry. The purpose of Study II (n = 7) was to determine if the same pre-exercise regimen followed by carbohydrate oral replacement solution (ORS) during exercise also lowered HR, Tc, and prolonged ET. Both studies were double-blind, randomized, crossover trials, performed at an ambient temperature of 23.5-24.5 degrees C, and humidity of 25-27%. Mean HR was lower by 2.8 +/- 0.4 beats/min (p = 0.05) after GEH in Study I and by 4.4 +/- 1.1 beats/min (p = 0.01) in Study II. Endurance time was prolonged after GEH in Study I (93.8 +/- 14 min vs. 77.4 +/- 9 min, p = 0.049) and in Study II (123.4 +/- 17 min vs. 99.0 +/- 11 min, p = 0.03). Rectal temperature did not differ between hydration regimens in both Study I and Study II. Thus, pre-exercise glycerol-enhanced hyperhydration lowers HR and prolongs ET even when combined with ORS during exercise. The regimens tested in this study could potentially be adapted for endurance activities.

Discovering the invisible Puerto Rican slave family: demographic evidence from the eighteenth century.

Traditional notions that family life among slaves during the pre-plantation period in the non-Hispanic Caribbean was necessarily unstable are fading in light of new research. Although marriage among this segment of the population in Caguas, Cayey, San Germán, and Yauco--rural parishes in Puerto Rico--involved only a fraction of the overall number of marriages in these communities, the marriage of slaves was much more frequent than previously assumed. Family life among the eighteenth-century Puerto Rican slave population appears to have been quite stable, as shown by the reconstruction of birth intervals for both married and unmarried mothers. Married and unmarried mothers exhibited similar reproductive behavior. These results strongly suggest that a majority of the unmarried slave mothers lived in unions that were not institutionally recognized, but that were nevertheless stable, as indicated by the high percentage of their children born at intervals comparable to those of married mothers. If unmarried mothers were living in stable consensual unions, then our understanding of these slave family units during the colonial period must be reassessed not only for Puerto Rico but possibly for the rest of the Caribbean and Latin America.

Hemodynamic effects of nasal and face mask continuous positive airway pressure.

Studies of the hemodynamic effects of nasal continuous positive airway pressure (n-CPAP) in normal subjects have had conflicting results. The largest study (n = 19) found no effect of up to 15 cm H2O on heart rate (HR), cardiac stroke volume (SV), or cardiac index. We hypothesized that n-CPAP, by increasing intrathoracic pressure, should decrease SV and cardiac output (CO) in a dose-dependent fashion in normal subjects. We also hypothesized that mouth position, i.e., open or closed, could affect intrathoracic pressure and thus SV and CO. Six normal subjects were tested with four levels of CPAP (5, 10, 15, and 20 cm H2O) under three mask conditions-face mask and nasal mask with the mouth open (mo) or with the mouth closed (mc). Noninvasive pulsed Doppler measurements of SV and HR were made under each condition. N-CPAP (mc) and face mask CPAP (f-CPAP) resulted in significant dose-dependent decreases of SV-24 +/- 5 ml (21%) and 33 +/- 5 ml (28%), respectively--from baseline to 20 cm H2O (p < 0.05). HR were unchanged and CO significantly decreased with n-CPAP(mc) and with f-CPAP, 1.6 +/- 0.38 L/min (23%) and 2.29 +/- 0.54 L/min (31%), respectively, from baseline to 20 cm H2O (p < 0.05). Esophageal pressure measurements verified increasing intrathoracic pressure with increasing levels of f-CPAP and n-CPAP (mc) but not with n-CPAP (mo). In conclusion, n-CPAP (mc) and f-CPAP resulted in significant and similar dose-dependent decreases in SV and CO.

Hyperoxic training increases work capacity after maximal training at moderate altitude.

High-intensity training may be difficult to sustain due to limitations in systemic oxygen transport, particularly at high altitudes. The purpose of this study was to examine the effects of a high-intensity training protocol using hyperoxic gas breathing in athletes "maximally trained" at an altitude of 1,600 m. Five subjects underwent progressive cycle training until they reached a plateau of aerobic capacity, maximal workload, and endurance time at 85 percent maximal workload. Significant decreases (2 to 6 percent) in arterial oxygen saturation were found after the 85 percent maximal workload tests. Training intensity was then increased to 95 percent maximal workload while the subjects breathed a gas mixture containing at least 70 percent oxygen. After 6 weeks of hyperoxic training, exercise parameters were compared with the plateau values obtained during the baseline training period. Total time during maximal cycle testing increased from 19.1 to 19.6 min (p = 0.015), heart rate at 85 percent maximal workload decreased from 168 to 163 bpm (p = 0.047), and endurance time at 85 percent maximal workload increased from 6.2 to 8.2 min (p = 0.012). There was a trend toward improvement of maximal workload. We conclude that hyperoxic training increases work capacity after attainment of "maximal training" at moderate altitude.

Plastid targeting of E. coli ß-glucuronidase and ADP-glucose pyrophosphorylase in maize (Zea mays L.) cells.

Dicot and monocot chloroplast targeting peptides (CTPs) were evaluated for their effect on targeting, processing, and expression of two reporter proteins in maize cells. When tested transiently in maize leaf protoplasts, the maize ribulose bisphosphate carboxylase small subunit CTP required the inclusion of the amino terminus of mature small subunit protein to target ß-glucuronidase (GUS) to the plastid. To remove this amino terminal extension from GUS after import and processing, a repeat of the native processing site was inserted between the native mature protein and the reporter protein. This repeat processing site was used with less efficiency than the native site. Parallel constructs using the Arabidopsis thaliana small subunit and maize granule-bound starch synthase CTPs also localized GUS, but varied in repeat site use and GUS expression levels. Data from the CTP fusions with GUS were generally confirmed with fusions to an allosteric variant of E. coli ADP-glucose pyrophosphorylase. Plastid targeting of this enzyme was required for starch enhancement of transgenic BMS cells.

Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase.

Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step.

Prevalence of feline immunodeficiency virus and feline leukemia virus infections in random-source cats.

Retroviral serologic profiles were generated for 506 random-source cats (Felis catus) that were received by our facility during a twenty-month period. Feline leukemia virus antigens were detected in plasma samples from 26 (5.1%) of the cats. Antibodies to feline immunodeficiency virus were present in 24 (4.7%) of the samples tested. A single cat (0.2%) was positive for both viruses. Neither gender nor vendor correlation with retroviral seropositivity could be demonstrated.

Screening rabbit colonies for antibodies to Pasteurella multocida by an ELISA.

Rabbit serum samples from eleven different research facilities were evaluated for the presence of immunoglobulin G against Pasteurella multocida by using an enzyme-linked immunosorbent assay (ELISA). Each facility which submitted serum samples also provided a brief history of each rabbit colony tested. Rabbits from colonies reported to have endemic P. multocida or of undetermined status had 83 (58.9%) of 141 rabbits that were positive. Colonies reported to be free from P. multocida had 110 (92.4%) of 119 rabbits that were negative by ELISA. The ELISA test described here showed a high degree of agreement (92-94%) with two other P. multocida ELISAs at different diagnostic facilities. This study confirms that an ELISA testing for serum antibodies against the P. multocida is a reliable diagnostic tool to screen colonies for P. multocida.

Rigid internal fixation for shoulder arthrodesis.

Fifteen patients underwent a shoulder arthrodesis utilizing standard dynamic compression plate fixation, but with limited postoperative immobilization with only an abduction pillow. In each case, the position of the extremity relative to the scapula and trunk was recorded immediately postoperatively, at regular intervals until fusion, and at follow-up evaluations. Thirteen of 15 shoulders fused without change of intraoperative position after an average postoperative period of 4 months. One patient lost position in the early postoperative period secondary to inadequate fixation, but subsequently fused. Another who demonstrated a persistent non-union at 2 1/2 years was subsequently explored and underwent a bone graft. Four patients complained of residual symptomatic hardware, with two requiring surgical removal of the plate and screws. All but one patient were satisfied with the clinical result at follow up. Only two patients were within 5 degrees of the preoperatively determined position of 30 degrees abduction, 30 degrees forward flexion, and 30 degrees internal rotation. However, almost all were able to function satisfactorily. The authors concluded that shoulder arthrodesis utilizing rigid internal fixation without postoperative cast or brace immobilization maximizes patient comfort without compromising the success of arthrodesis. However, control of arm position remains inexact and additional modifications are needed to ensure fusion position and to minimize disability.

cDNA cloning and nucleotide sequence of the wheat streak mosaic virus capsid protein gene.

The 3'-terminal region of wheat streak mosaic virus (WSMV) genomic RNA was cloned and a cDNA sequence of 1809 nucleotides upstream of the poly(A) tract was determined. The sequence contains a single open reading frame of 1662 nucleotides and a 3' untranslated region of 147 nucleotides. Translation products from WSMV RNA and WSMV cDNA transcripts were immunoprecipitated by WSMV capsid protein antiserum, indicating that the 3'-terminal region of WSMV RNA encodes the capsid protein. Five potential N-terminal capsid protein protease cleavage sites were identified, which would yield proteins ranging from 31.7K to 46.8K. Alignment of the deduced amino acid sequence of the WSMV capsid protein with those of other potyviruses showed significant, but limited, identity as compared to the alignment of two or more aphid-transmitted potyviruses. Although WSMV has characteristics distinct from potyviruses, because of its particle morphology, translation strategy apparently based on polyprotein processing, the ability to form cytoplasmic cylindrical inclusions and the degree of capsid protein homology with aphid-transmitted potyviruses, it should be considered a member of the potyvirus group.

Protection against tobacco mosaic virus in transgenic plants that express tobacco mosaic virus antisense RNA.

Transgenic tobacco plants that express RNA sequences complementary to the tobacco mosaic virus (TMV) coat protein (CP) coding sequence with or without the tRNA-like structure at the 3' end of the TMV RNA were produced. Progeny of self-pollinated plants were challenged with TMV to determine their resistance to infection. Plants that expressed RNA sequences complementary to the CP coding region and the 3' untranslated region, including the tRNA-like sequences, were protected from infection by TMV at low levels of inoculum. However, plants that expressed RNA complementary to the CP coding sequence alone were not protected from infection. These results indicate that sequences complementary to the terminal 117 nucleotides of TMV, which include a putative replicase binding site, are responsible for the protection. However, the level of protection in these plants was considerably less than in transgenic plants that expressed the TMV CP gene and accumulated CP. Since the mechanisms of protection in the two systems are different, it may be possible to increase protection by introducing both sequences into transgenic plants.

The nucleotide sequence of a soybean mosaic virus coat protein-coding region and its expression in Escherichia coli, Agrobacterium tumefaciens and tobacco callus.

A DNA complementary to the 3'-terminal 1168 nucleotides of the genome of the N strain of soybean mosaic virus (SMV) has been cloned and sequenced. cDNA sequence and coat protein analyses indicate that the SMV coat protein-coding region is at the 3' end of the genome, and that the coat protein is processed from a larger protein. The coat protein-coding sequence is predicted to be 795 nucleotides in length, encoding a protein of 265 amino acids with a calculated Mr of 29,857. The 3' untranslated region is 259 nucleotides in length and is followed by a polyadenylate tract. The SMV coat protein-coding region, along with a small amount of upstream sequence, has been expressed in Escherichia coli as a beta-galactosidase fusion protein. The size of the protein was less than predicted for the fusion protein, suggesting processing in E. coli. The coat protein-coding region has also been expressed in Agrobacterium tumefaciens and transgenic tobacco callus as an unfused protein under the control of the cauliflower mosaic virus 35S promoter. The coat protein produced in transgenic tobacco callus had an electrophoretic mobility identical to that of SMV coat protein and constituted approximately 0.05% (w/w) of the total extracted protein.

Identification of nonspecific reactions in laboratory rodent specimens tested by Rotazyme rotavirus ELISA.

Fecal specimens from several laboratory animal species were tested for rotavirus antigen by Rotazyme II, a commercially available enzyme-linked immunosorbent assay (ELISA) that is widely used in human diagnostic studies. Fecal samples from rabbits, hamsters, guinea pigs, dogs, and cats tested negative; whereas those from rats and mice yielded a high proportion of positive results. Rats had the highest rate with 82% of the samples being positive. However, the presence of rotavirus in positive rodent samples could not be confirmed by virus isolation, electron microscopy or blocking ELISA using anti-EDIM mouse rotavirus serum. Several lines of evidence indicated that these positive reactions were false positives, apparently due to a non-specifically reacting substance in the diet of rats and mice. All the positive fecal samples were from rats and mice that had been fed nonautoclaved diet. Samples from rodents fed autoclaved diet were consistently negative in the Rotazyme test. When rats fed autoclaved diet were subsequently fed nonautoclaved diet, their stool converted from negative to positive within 6 hours. Conversely, rats with positive stool samples converted to negative within 15 hours when fed autoclaved diet. Similar results were found with mice. Positive fecal specimens and nonautoclaved rodent diet both contained a substance that apparently attached nonspecifically to the antibody coated beads used in the ELISA and reacted directly with the substrate in the absence of the conjugate. This substance was heat labile and trypsin sensitive, suggesting that it was a protein.