Cytoskeletal control of polar growth in plant cells.

There are two quite different modes of polar cell expansion in plant cells, namely, diffuse growth and tip growth. The direction of diffuse growth is determined by the orientation of cellulose microfibrils in the cell wall, which in turn are aligned by microtubules in the cell cortex. The orientation of the cortical microtubule array changes in response to developmental and environmental signals, and recent evidence indicates that microtubule disassembly/reassembly and microtubule translocation participate in reorientation of the array. Tip growth, in contrast, is governed mainly by F-actin, which has several putative forms and functions in elongating cells. Longitudinal cables are involved in vesicle transport to the expanding apical dome and, in some tip growers, a subapical ring of F-actin may participate in wall-membrane adhesions. The structure and function of F-actin within the apical dome may be variable, ranging from a dense meshwork to sparse single filaments. The presence of multiple F-actin structures in elongating tips suggests extensive regulation of this cytoskeletal array.

Electrophysiological properties of Achlya hyphae: ionic currents studied by intracellular potential recording.

The electrical properties of the water mold Achlya bisexualis were investigated using intracellular microelectrodes. Hyphae growing in a defined medium maintained a membrane potential (Vm) of -150 to -170 mV, interior negative. Under the conditions used here, this potential was insensitive to changes in the inorganic ion composition of the medium. Changes in external pH did affect Vm, but only outside the physiological pH range. By contrast, the addition of respiratory inhibitors caused a rapid depolarization without affecting the conductance of the plasma membrane. Taken together these findings strongly suggest that the membrane potential is governed by an electrogenic ion pump rather than by an ionic diffusion potential. Previous work from this laboratory showed that Achlya hyphae generate a transcellular proton current that enters the growing tip, flows along the hyphal length, and exits distally from the trunk. These initial experiments used an extracellular vibrating electrode, and I now report intracellular electrical recordings which support the hypothesis that protons enter the tip by symport with amino acids and are expelled distally by a proton-translocating ATPase. Most significantly, current flowing intracellularly along the hyphal length is associated with a cytoplasmic electric field of 0.2 V/cm or greater. Conditions that inhibit the current also abolish the internal field, suggesting that these two phenomena are closely linked.

Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry.

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.

Cell wall is required for fixation of the embryonic axis in Fucus zygotes.

Establishment of a primary developmental axis generally is thought to involve rearrangements in the plasma membrane or cytoplasm of the egg. In this report the additional requirement for cell wall in polarization of Fucus zygotes was investigated. Protoplasts of fertilized eggs were tested for their ability to establish an axis in accordance with an orienting vector of unilateral light. The results demonstrate that cell wall is not required for axis formation. However, the orientation of the axis remains labile until new cell wall is synthesized. The presence of a cell wall is an absolute requirement for axis fixation.

Cytosolic pH Gradients Associated with Tip Growth.

The presence of a cytosolic pH gradient and its relation to polar tip growth was investigated in rhizoid cells of Pelvetia embryos with the use of pH-sensitive microelectrodes and by ratio imaging. Growing rhizoid cells generated a longitudinal pH gradient in which the apical cytosol was 0.3 to 0.5 units more acidic than the cytosol at the base of the cell. Treatment with a membrane-permeant weak acid, propionic acid, dissipated the cytosolic pH gradient and inhibited growth. The magnitude of the pH gradient correlated well with the rate of tip elongation. The pH gradient spatially superimposed on the cytosolic calcium gradient, and inhibition of calcium fluxes by treatment with lanthanum abolished the pH gradient and inhibited growth.

Cell polarity: endogenous ion currents precede and predict branching in the water mold achyla.

The hyphae of the water mold Achyla bisexualis generate electrical currents that enter the growing tips and leave farther back. An inward-moving current also precedes branching and predicts the site of branch emergence; during the branching process, the current at the original tip declines or even reverses transiently without any change in growth rate. The inward current probably acts as an early signal during branch differentiation. The flow of specific ions rather than the flow of electrical charge probably serves to localize growth.

Instrumental reflectance values of fresh pork are dependant on aperture size.

The effect of aperture size on instrumental pork color measurements was studied. Pork longissimus chops (n=30) were cut, aerobically packaged, and allowed to bloom overnight. Chops were measured once with each of four aperture ports (4.45, 2.54, 1.27, and 0.64cm diameter) on a HunterLab LabScan. Reflectance values measured with smaller aperture sizes were reduced (P<0.05) for most visual wavelengths (10nm intervals), and the percent reduction was greatest in the range of the longer, redder wavelengths. Values for L(∗), a(∗), b(∗), and saturation index were reduced (P<0.05) with decreasing aperture size under illuminants A, C, and D(65). Using illuminant A, hue angle increased (P<0.05) as aperture size decreased. For illuminants C and D(65), hue angles were similar (P>0.05) for the three largest aperture sizes, but negative a(∗) values found with the smallest aperture size skewed the hue angle data.

Actin Localization during Fucus Embryogenesis.

Embryogenesis in the Fucales serves as a model system for studying the acquisition of cellular and developmental polarity. Fertilized eggs bear no asymmetry, yet within 16 hours, a developmental axis is formed and the unicellular zygote germinates in accordance with this axis. Microfilaments (actin) play a crucial role in establishing the axis as evidenced by the inhibitory effects of cytochalasins on axis fixation. The cellular content of actin was determined by immunoblot, whereas the localization of F-actin was investigated using the fluorescent probe rhodamine phalloidin. Three isoforms of actin were detected in constant amounts at all developmental stages. Actin networks were found to be distributed uniformly in eggs and zygotes through the period of early zygote development when the polar axis was formed. However, as the polar axis became irreversibly fixed in space, actin was localized at the presumptive germination site by a cytochalasin-sensitive process. This correlation supports the proposal that actin networks play a critical role in axis fixation, and is consistent with our hypothesis that this process involves stabilization of membrane components by transmembrane bridges from the cell wall to the microfilament cytoskeleton.

A comparison in the composition of recovered meat produced from beef neckbones processed using hand boning, a traditional Advanced Meat Recovery (AMR) system, and a Desinewated Minced Meat system.

Beef neckbones were processed through a traditional Advanced Meat Recovery system (TAMR), a Desinewated Minced Meat machine with/without prior use of Jarvis saw for removal of spinal cord (DMMJ/DMMNJ), or hand boned with/without Jarvis saw (HJ/HNJ). This study investigated the composition of meat recovered by these five methods. Ranking from the most to least total fat percentage was TAMR (22.02%), HNJ (18.37%), HJ (14.69%), DMMNJ (11.14%), and DMMJ (9.76%); higher fat was related to less moisture. Protein was most for HJ (18.32%) and least for TAMR (15.79%). TAMR and HJ were similar (P>0.05) in ash content. Calcium was most in DMMJ (79.81mg); the least was found in the hand boned (HJ, 20.86mg/100g and HNJ, 23.66mg) lean. All samples contained calcium below the required limits set by USDA-FSIS. Total iron was the highest in TAMR (5.28mg of iron/100g), followed by DMMJ (3.65mg), DMMNJ (3.46mg), HJ (2.77mg), and HNJ (2.18mg).

Effects of ascorbic and citric acid on beef lumbar vertebrae marrow colour.

Citric acid was evaluated as a way of improving ascorbic acid's ability to stabilize beef lumbar vertebrae colour in high-oxygen packaging (MAP; 80% O(2)/20% CO(2)). Vertebrae were treated with citric acid (1%, 3%, or 10%), ascorbic acid (1%, 3%, or 10%), or a combination of both. Citric acid demonstrated no positive effects (P>0.05), compared with ascorbic acid, which inhibited (P<0.05) discolouration throughout the 7d display. Although ascorbic acid inhibited discolouration (visual colour and a(∗); P<0.05), 3% and 10% ascorbic acid were most effective. However, if vertebrae are displayed for less than 7d, there may be no significant colour-stabilizing advantages associated with increasing ascorbic acid from 3 to 10%. The significant oxidizing effects of citric acid at 10% were reversed (P<0.05) by ascorbic acid. Combining citric and ascorbic acid had no synergistic affect (P>0.05) on vertebrae colour.

Comparison of ascorbic acid and sodium erythorbate: Effects on the 24h display colour of beef lumbar vertebrae and longissimus lumborum packaged in high-oxygen modified atmospheres.

Sodium erythorbate and ascorbic acid were compared as a means to stabilize surface colour of bone-in beef steaks in high-oxygen modified atmosphere (80% oxygen and 20% carbon dioxide). Bone-in strip loins (n=8) were fabricated into 1.9-cm thick steaks, of which both the lumbar vertebrae and longissimus lumborum were topically treated with either ascorbic acid or sodium erythorbate (0, 0.05, 0.1, 0.5, 1.0, or 1.5%, wt/wt basis). Colour (L(∗)a(∗)b(∗)) was evaluated before treatment and 24h after packaging (display at 1°C). Sodium erythorbate was as effective as ascorbic acid for inhibiting vertebrae discolouration (P>0.05). Either reducing agent at 0.5, 1.0, or 1.5% improved (P<0.05) vertebrae redness (compared with 0%, 0.05% and 0.1%). No detrimental effects on muscle colour were observed. When selecting antioxidants intended for bone-in beef steaks displayed in high-oxygen packaging, sodium erythorbate may be a cost effective substitute for ascorbic acid.

Effects of ascorbic acid, rosemary, and Origanox™ in preventing bone marrow discoloration in beef lumbar vertebrae in aerobic and anaerobic packaging systems.

Treatments of 1.25% or 2.5% ascorbic acid (AA); 0.1% or 0.2% rosemary; or a combination of 0.15% Origanox™+0.3% AA on beef lumbar vertebrae (n=72) were evaluated for their effectiveness in preventing bone discoloration in retail packages. Vertebrae were held either 6 or 14d postmortem; cut into 2.54-cm sections; and packaged into either polyvinyl chloride film overwrap (PVC), high-oxygen (80% O(2)/20% CO(2)) modified-atmosphere packages (MAP), or ultra-low-oxygen (70% N(2)/30% CO(2)) MAP. Controls and vertebrae treated with 0.1% or 0.2% rosemary discolored significantly, whereas 0.15% Origanox™+0.3% AA was effective through d 2 of display in PVC and high-oxygen MAP. In both high- and ultra-low-oxygen MAP, 1.25% AA was as effective as 2.5% AA in preventing bone discoloration. Vertebrae treated with AA or packaged in ultra-low-oxygen MAP had the largest a* values over display time. Bone discoloration was effectively prevented with AA, especially at the 2.5% concentration.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Exclusion of oxygen from modified atmosphere packages limits beef rib and lumbar vertebrae marrow discoloration during display and storage.

Visual and instrumental color (L*a*b* and reflectance from 400 to 700 nm) were used to evaluate packaging atmosphere as a way of minimizing beef marrow discoloration. In experiment 1, rib ends (n=24) packaged in 80% O(2)/20% CO(2) discolored more than ribs packaged in 100% N(2), which resulted in a relatively stable purplish marrow color through a 7-day display at 1 °C. In experiment 2, lumbar vertebrae (n=10) packaged in 80% O(2)/20% CO(2) had a rapid and significant discoloration within 24 h after packaging, likely because of the formation of methemoglobin. Conversely, vertebrae packaged in 80% N(2)/20% CO(2) and 0.4% CO/30% CO(2)/69.6% N(2) remained color stable during 2 and 6 weeks of storage at 4 °C, respectively. Exclusion of oxygen from MAP packages and the addition of low concentrations of CO minimized beef rib and lumbar vertebrae discoloration compared with high-oxygen MAP.

Measurement and manipulation of cytosolic pH in polarizing zygotes.

The role of cytosolic pH (pHc) in establishment and expression of developmental polarity was examined in zygotes of the brown alga Pelvetia. pHc was measured and manipulated at specific developmental stages during the first zygotic cell cycle. pHc was measured using pH-sensitive microelectrodes and by confocal ratio imaging of dextran-conjugated SNARF 1 (dc SNARF 1) loaded cells. The two techniques yielded very similar values of pHc in the cellular cortex, but ratio imaging was not effective in measuring endoplasmic pHc values. As zygotes formed a developmental axis, cortical pHc decreased abruptly by approximately 0.1 units, and a small but significant difference in pHc was detected at the thallus and rhizoid poles. The cortical cytosol was relatively acidic at the presumptive rhizoid pole. The magnitude of the pHc difference increased following initiation of rhizoid growth. pHc was manipulated by treating zygotes with membrane-permeant weak acids (propionic and benzoic acid) or bases (methylamine and procaine), which effectively clamp pHc to specific values in a concentration-dependent manner. pHc values in treated zygotes were measured for each concentration of acid or base, and a dose response curve was generated. Zygotes in which pHc had been clamped were examined for their ability to form a developmental axis and to initiate rhizoid outgrowth (germination). Both developmental processes were inhibited by relatively small (0.2-0.3 pH units) perturbations of pHc. The permissive ranges of pHc were slightly different, germination (permissive pHc range-pH 7.0 to 7.7) being more acid tolerant than axis formation (permissive pHc range-7.2 to 7.8).

Electromyographic analysis of selected muscles during sitting push-ups. Effects of position and sex.

The purpose of this study was to determine if differences in EMG activity of the latissimus dorsi, pectoralis major and triceps brachii occurred between muscles and between the 16 male and 16 female subjects performing push-ups from three different sitting positions. Surface electrodes and associated instrumentation recorded a linear envelope during seated push-ups performed 1) in a wheelchair, 2) in a long-sit position with elbows at 90 degrees, and 3) in a long-sit position with maximum elbow flexion and shoulder abduction. Results showed that women produced greater mean EMG activity than men in all muscles at all positions. Altering the exercise position did not have a consistent effect on level of activity recorded from either sex. The study concludes that use of these exercises should be based on a knowledge of the differences in muscle activity in exercise positions for men and women before treatment objectives can be effectively accomplished.

Porcine somatotropin and dietary lysine influence bone mineralization and mechanical properties of bones in finishing swine.

The femur, a rib, and a third metacarpal were collected from 108 barrows (initial weight = 57 kg) and analyzed to determine the effects of porcine somatotropin (pST) and dietary lysine on bone mineralization and mechanical properties. In Exp. 1, pigs were injected daily with 4 mg of pST and fed diets containing 1.0% Ca and .9% P and either .6, .8, 1.0, 1.2, or 1.4% lysine. Control pigs (placebo injection) received the .6% lysine diet. Bone wall thickness (BWT) of the femur increased (linear, P < .05) but ash content decreased (linear, P < .10) as dietary lysine level increased. Stress and ash content of the rib also decreased (linear, P < .05) with increasing dietary lysine level. In Exp. 2, pigs were injected with either 4 or 8 mg/d of pST and fed diets containing 1.1% Ca and 1.0% P and either .8, 1.0, 1.2, or 1.4% lysine. Control pigs received the .8% lysine diet. Increasing pST dosage increased BWT of the femur (linear, P < .10) but decreased ash content (linear, P < .10). In the rib, increasing pST dosage reduced stress, modulus of elasticity (linear, P < .10), and ash content (linear, P < .01). Increasing lysine level resulted in increased BWT of the femur and decreased ash content of rib, femur, and metacarpal (linear, P < .10). These data indicate that pST administration in conjunction with increasing lysine levels decreases bone ash content but increases BWT of finishing pigs.

The effects of porcine somatotropin and dietary lysine on growth performance and carcass characteristics of finishing swine fed to 105 or 127 kilograms.

One hundred twenty barrows (initially 59 kg) were used to determine the effects of dietary lysine and porcine somatotropin (pST) on growth performance and carcass characteristics of finishing pigs fed to heavy market weights (127 kg). Pigs were injected daily with either 4 mg of pST or a placebo and fed diets containing either .8, 1.0, 1.2, or 1.4% lysine in a 2 x 4 factorial arrangement. Performance data were collected and evaluated for the weight ranges: 59 to 105, 105 to 127, and 59 to 127 kg. In addition, daily accretion rates of protein (DPA), lipid (DLA), moisture, and ash were determined by slaughtering six randomly selected pigs at the start of the experiment, then one pig per pen when pigs reached mean weights of 105 and 127 kg. Pigs injected with pST had greater (P < .05) ADG than control pigs at all weight ranges. Increased dietary lysine had no effect (P > .20) on ADG during the entire trial (59 to 127 kg). Although control pigs showed no increase in ADG with increasing lysine, ADG of pST-treated pigs tended to increase. This resulted in a pST x lysine interaction (P < .07) for ADG from 59 to 127 kg. Feed consumption decreased (P < .05) in pigs from 59 to 105 kg and 59 to 127 kg with pST treatment and with increasing dietary lysine. Feed conversion (G/F) was improved (P < .01) by pST administration and a tendency for a pST x lysine interaction (P < .12) was observed. At 105 kg, average backfat thickness, kidney fat, longissimus muscle area, and DPA were unaffected by dietary lysine but were improved by pST treatment (P < .01). At a slaughter weight of 127 kg, average backfat thickness decreased and DPA increased (linear, P < .05) with increasing dietary lysine and pST treatment (P < .01). Longissimus muscle area was increased and DLA was decreased (P < .01) by injections of pST but both were unchanged by dietary lysine. These data indicate that growth performance and carcass characteristics at 105 and 127 kg were improved by daily administration of 4 mg of pST. At both slaughter weights, increasing dietary lysine resulted in increased carcass leanness (increased carcass protein and decreased carcass lipid). Although increasing lysine did not improve overall (59 to 127 kg) ADG and feed efficiency, pST-treated pigs showed a greater response to increasing dietary lysine. The data indicate that pST-treated pigs (4 mg/d) require approximately 28 g/d of lysine.

The use of urea dilution for estimation of carcass composition of Holstein steers at 3, 6, 9, and 12 months of age.

Every 3 mo for a 2-yr period, two weaned Holstein steer calves (94.5 kg) were randomly assigned to each of four slaughter age groups (3, 6, 9, and 12 mo). Urea dilution was performed before slaughter, and urea space (US) was calculated as total volume and as a percentage of body weight (BW) and empty body weight (EBW). The relationships between US (kg, % EBW and % BW), BW, and EBW and carcass soft tissue composition (protein, fat, moisture, and ash) were studied. One- and two-pool models were fitted using the urea dilution data and the coefficients of those equations (zero time, A + B), and the intercepts of compartments A and B were used to estimate body volume. Body weight and EBW effectively predicted the amount of water, fat, and protein in the carcass soft tissue. Equations expressed in kilograms were more accurate than those expressed as percentages. Urea space overestimated body water, probably because of the fast rate of urea disappearance in plasma. Correlation coefficients between US and carcass soft tissue water (kg) based on the pooled data ranged from .74 at 6 min to .48 at 42 min after infusion. The biexponential models coefficients explained more of the variation of carcass soft tissue composition than US; correlation coefficients using volume B and the soft tissue composition (in kg) with pooled data were .78 (water), .68 (fat), .69 (ash), and .76 (protein). The relationships between A and soft tissue composition were weaker (water .59, fat .51, ash .58 and protein .59). The highest correlation coefficients were obtained when A + B was used for water, fat, ash, and protein (.83, .70, .74 and .81, respectively). Equations combining BW, EBW, and two-model coefficients (A, B, A + B) explained much of the variation of soft tissue composition. No significant benefit was found in using the urea space at various times after infusion over BW or EBW alone to estimate carcass soft tissue composition in Holstein steers.

Effects of omitting vitamin and trace mineral premixes and(or) reducing inorganic phosphorus additions on growth performance, carcass characteristics, and muscle quality in finishing pigs.

Three experiments were conducted to determine the effects of omitting vitamin and trace mineral premixes and(or) reducing inorganic phosphorus additions to finishing diets on growth performance, carcass characteristics, and muscle quality in pigs. In Exp. 1, a corn-soybean meal-based diet (.70% lysine, .65% Ca, and .55% P) was used as the control. Pigs (n = 128; average initial BW of 85.7 kg) were fed the control diet or the control diet without 1) the vitamin premix, 2) the trace mineral premix, or 3) both premixes. Omitting the premixes had no effect on ADG (P>.39); gain/feed (P>.17); carcass backfat thickness (P>.42); and marbling, color, and firmness of the longissimus muscle (P>.11). In Exp. 2, pigs (n = 128; average initial BW of 86.2 kg) were fed the control diet (.65% Ca and .53% P) used in Exp. 1 and the control diet without 1/3 (.56% Ca and .46% P), 2/3 (.51% Ca and .40% P), or all (.47% Ca and .31% P) of the added monocalcium phosphate (MCP). Omitting up to 2/3 of the MCP increased ADG (quadratic effect, P<.02) and had no effect on meat quality (P>.12), but backfat thickness increased slightly (quadratic effect, P<.02). In Exp. 3, pigs (n = 160; average initial BW of 86.6 kg) were fed the control diet used in Exp. 1 or the control without 1) the vitamin and trace mineral premixes, 2) 2/3 of the MCP, or 3) the premixes and 2/3 of the MCP. Treatment had no effects on ADG (P>.23), gain/feed (P>.94), stomach lesions (P>.37), or serum gamma globulins (P>.08). In conclusion, vitamin and trace mineral premixes and up to 2/3 of the supplemental MCP can be omitted during late finishing (i.e., approximately the final 30 d) to reduce nutrient excesses that increase cost of feeding and nutrients excreted in waste material.