Effects of an S6 strain of Mycoplasma gallisepticum inoculation before beginning of lay on the leukocytic characteristics of commercial layers.

A clinical study was conducted on commercial layers housed in biological isolation units, within which exogenous stress factors potentially affecting bird performance were minimized. This set-up was devised in order to assess how a pre-lay inoculation of S6 strain Mycoplasma gallisepticum affects the leukocytic properties of laying chickens. Previous studies have demonstrated relative decreases in lymphocyte and relative increases in heterophil percentages in birds infected with other strains of Mycoplasma gallisepticum. However, current results showed that the differential percentages of lymphocytes were decreased, whereas those of heterophils were increased, in both sham-inoculated control birds and birds inoculated with S6 Mycoplasma gallisepticum between 19 and 26 wk of age. This study clearly shows that a pre-lay inoculation of S6 Mycoplasma gallisepticum alone had no apparent effect on the leukocyte profile of commercial layers housed in biological isolation units.

Immune system and cardiac functions of progeny chicks from dams fed diets differing in zinc and manganese level and source.

This research was conducted to evaluate immunity (experiments 1 to 3), cardiac function, and ascities resistance (experiment 4) of progeny chicks from broiler breeders fed diets differing in trace metal level and source. Broiler breeders received a control diet (75 mg of Zn and 83 mg of Mn added/kg of diet), the control diet supplemented with inorganic Zn (75 mg/kg of diet) and Mn (80 mg/kg of diet), the control diet supplemented with organic Zn (75 mg/kg of diet) and inorganic Mn (80 mg/kg of diet), or the control diet supplemented with organic Zn (75 mg/kg of diet) and Mn (80 mg/kg of diet) in experiments 1, 2, and 3. In experiment 4, the control diet and diet supplemented with organic sources of Zn and Mn were fed to broiler breeders. Immune organ weights, circulating granulocytes vs. agranulocytes, CD4 and CD8 positive T cells, cutaneous basophil hypersensitivity, and antibody titers to SRBC and breeder vaccinations were measured in progeny. Some supplemental mineral treatments increased (P < or = 0.05) cutaneous basophil hypersensitivity and relative bursa weight. All supplemental mineral treatments increased (P < or = 0.05) relative thymus weight. In experiment 4, electrocardiograph, pulse oximetry, heart rate, hematocrits, ventricle weights, and ascites incidence were measured in progeny reared in a cold-stress environment. The supplemental organic minerals increased (P < or = 0.05) left ventricle plus septum and total ventricular weights. Although progeny ascites incidence did not differ between breeder mineral treatments, breeders fed supplemental Zn and Mn sired progeny with improved cardiac functional capacity and some improvements in immunity.

Effects of an S6 strain of Mycoplasma gallisepticum challenge at onset of lay on digestive and reproductive tract characteristics in commercial layers.

Mycoplasma gallisepticum (MG), a reproductive/respiratory pathogen in poultry, has been implicated in suboptimum egg production and decreased hatchability. Commercial layer hens raised in a controlled environment were inoculated with the S6 strain of MG at 20 wk of age. The S6 inoculation had no effect on bird weight, egg production, digestive tract weight and length, or histopathologic lesion scores, although significant differences were noted in the lengths and weights of various portions of the reproductive tract. This study shows that S6MG inoculation does not detrimentally affect layer hen performance when in the absence of environmental stressors customary to a caged layer facility.

Effects of an S6 strain of Mycoplasma gallisepticum challenge before beginning of lay on various egg characteristics in commercial layers.

Mycoplasma gallisepticum (MG) is a reproductive/respiratory disease in poultry implicated in suboptimum egg production and decreased hatchability. Commercial layer hens raised in a controlled environment were inoculated with the S6 strain of MG at 10 wk of age. Egg production and selected egg and egg quality parameters were quantitated over the entire lay cycle for inoculated and control birds. The S6 inoculation had no effect on bird weight, egg production, associated egg quality parameters, or histopathologic lesion scores. This study shows that in the absence of environmental stressors a prelay S6 MG inoculation does not produce detrimental effects on layer hen performance.

Caprine serum fraction immunomodulator as supplemental treatment of lower respiratory disease in the horse.

Suppurative lower airway disease is a common debilitating disease in performance horses and, while rarely fatal, is often recalcitrant to conventional therapy. A variety of treatments have been used to combat this condition and we conducted two types of studies to determine if caprine serum fraction--immunomodulator (CSFI), a nonspecific immunomodulator, improved recovery from lower respiratory disease. Two dose response studies were performed to ascertain the efficacy of CSFI. Horses were maintained daily on conventional antibiotic therapy. Respiratory tract exudate, nasal discharge, dyspnoea, chest auscultation and cough frequency were monitored weekly. One hundred percent of the horses treated with 2 i.m. injections of either 60 or 120 mg CSFI one week apart showed significant improvement with each weekly evaluation and were fully recovered by week 3. Horses treated with 15 or 30 mg CSFI did not differ significantly from the control group. Only 10% of the control horses responded to conventional antibiotic therapy. An expanded field trial utilising 80 horses diagnosed with lower respiratory disease and housed at 4 equine clinics was conducted. Thirty-five percent of the 40 control horses, treated solely by conventional antibiotic therapies, recovered while 75% of the horses treated with a supplemental administration of 60 mg CSFI as described above recovered. The combined data from these studies showed that CSFI was able to promote an overall recovery from lower respiratory disease of 86%.

Reduction of mortality in specific-pathogen-free layer chickens by a caprine serum fraction after infection with Pasteurella multocida.

Caprine serum was fractionated by size, and its proteinaceous material <8,000 Da [caprine serum fraction immunomodulator 2 (CSF-I2)] was evaluated for its ability to impart immunoresistance to specific-pathogen-free (SPF) layer chickens. The SPF layers were challenged with 18 to 30 cfu of Pasteurella multocida X-73 (serotype 1) at 5 wk of age. A high degree of mortality was apparent 24 and 48 h later (62+/-14% and 88+/-7%, respectively). Mortality observed after 48 h was minimal. Noting the rapid onset of mortality, we administered CSF-I2 (material that expressed no direct antimicrobial activity but was believed to be an immunostimulant) 1 d before challenge and coincident to time of challenge. The group of birds that received CSF-I2 (either 5 or 10 mg per administration) expressed significant reduction in mortality throughout the 1-wk study period. Reduction in mortality appeared to be dose dependent. Birds that received two administrations of 10 mg CSF-I2 had significantly fewer deaths than did the group of birds that received half that amount. No deaths were recorded through 24 h, whereas, at 48 h, the percentage mortality was 13 in CSF-I2-treated birds. This study demonstrates that one or more small molecular weight compounds isolated from caprine serum were able to reduce mortality in SPF layers infected with Pasteurella multocida.

Modulation of proteolytic activity associated with persistent corneal ulcers in dogs.

Canines affected with corneal lesions demonstrate increased proteolytic activity at the affected site. Canines that develop persistent corneal ulcers and maintain elevated levels of proteolytic activity respond to protease inhibitor therapy with polysulpated glycosaminoglycan, PSGAG. In this study, the proteolytic activity expressed in lacrimal fluid of canines was evaluated as normal (1.15 U mL-1) or healed (1.19 U mL-1). Six of the 26 dogs affected with persistent corneal ulcers, however, had a protease level consistent with the unaffected control animals. These dogs did not appear to respond to PSGAG therapy. Two pathophysiologies for persistent corneal ulcers are indicated. Identifying which mode is active could help determine the therapeutic treatment needed.

Association of a 33-kilodalton cysteine proteinase found in corn callus with the inhibition of fall armyworm larval growth.

Protein patterns of callus from corn (Zea mays L.) inbreds that are either resistant or susceptible to fall armyworm (Spodoptera frugiperda [J.E. Smith]) were analyzed by two-dimensional electrophoresis. Fall armyworm larvae reared on callus initiated from resistant inbreds were significantly smaller than those reared on callus of susceptible inbreds. A 33-kD protein found in callus from the resistant inbreds Mp704 and Mp708 was absent in callus from the susceptible inbreds Tx601 and Ab24E. However, a 36-kD protein found in Ab24E callus immunoreacted with polyclonal antibody raised against the 33-kD protein. When Mp704 nonfriable callus changed to friable, larval growth was not inhibited and the 33-kD protein was absent. There was a significant negative correlation between the concentration of the 33-kD protein in the callus and the weight of the larvae feeding on the callus in the F2 progeny of Mp704 x Tx601. The N-terminal amino acid sequence of the 33-kD protein suggested that it was cysteine proteinase. Purification of the 33- (Mp708) and 36-kD (Ab24E) proteins indicated that they were both cysteine proteinases. The 33-kD cysteine proteinase had 7-fold higher specific activity than the 36-kD enzyme.

Oligomerization and regulation of higher plant phosphoenolpyruvate carboxylase.

The specific activity of phosphoenolpyruvate (PEP) measured at a saturating level of substrate diminishes as the enzyme is diluted at about the same rate that specific light scattering by the diluted enzyme decreases. The presence of PEP in the assay causes an increase in activity with increasing dilution. This is accompanied by an increase in light scattering of the diluted enzyme. The reverse situation obtains with the addition of malate to assays: the activity decreases with increasing dilution but light scattering is not substantially changed, indicating that the enzyme is already brought to a smaller aggregate by the dilution itself. In this case, the inhibition by malate in the assay probably is the noncompetitive type not involved in regulatory control by malate. Glucose-6-phosphate in the range from 1 to 6 millimolar causes an increase in activity of the enzyme run at a substrate level less than K(m), and an associated increase in light scattering is found, indicating an increase in the mean size of the enzyme. When PEP is added to a 1/80 diluted enzyme, light scattering increases and is associated with a more rapid activity of the enzyme. When malate is added to the same cuvette, the activity decreases and the light scattering diminishes, thus showing that the ligand response is immediately reversible. When malate is added first, followed by PEP, the reverse sequence of activity and light scattering change is observed.

Regulation of Phosphoenolpyruvate carboxylase from Crassula argentea: effect of incubation with ligands and dilution on oligomeric state, activity, and allosteric properties.

The relationship between the aggregation state and allosteric properties of purified phosphoenolpyruvate carboxylase from Crassula argentea was examined using both kinetic and physical techniques. Analysis by native polyacrylamide gel electrophoresis showed that dilution induced a dissociation of the active tetramer to a less active dimer. Kinetic assays showed that inhibition of phosphoenolpyruvate carboxylase by 5 mM malate measured at a saturating phosphoenolpyruvate concentration rose to nearly 80% with increasing preassay dilution while the activity in the absence of malate remained constant. Kinetic bursts were observed when enzyme-initiated assays were measured at a subsaturating phosphoenolpyruvate concentration. At saturating phosphoenolpyruvate concentrations, however, increasing lags developed in response to increasing the preassay dilution of the enzyme. Further, dynamic laser-light scattering measurements showed that preincubation of the dilute enzyme with phosphoenolpyruvate stabilized the tetramer while the presence of malate induced dimer formation. These observations confirm and extend earlier work with the extracted active malate insensitive night and less active, malate-sensitive day forms of the enzyme (Wu and Wedding [1985] Plant Physiol. 77, 667-675). Activity measured at subsaturating phosphoenolpyruvate concentrations dropped with increasing preassay dilution of enzyme, while activation by 3.2 mM glucose 6-phosphate, assayed at a low phosphoenolpyruvate concentration (0.044 mM), increased with dilution to nearly 400%. In this case activation results from a decrease in the control rate as the activity measured in the presence of glucose 6-phosphate was nearly constant, similar in effect to saturating phosphoenolpyruvate in the assay. Glucose 6-phosphate induced tetramer formation of the dilute enzyme as measured by light-scattering similar to the effects induced by PEP. In addition, when diluted (dimeric) PEPC was preincubated with PEP or glucose 6-phosphate the enzyme became less sensitive to malate inhibition, while the active-site directed ligand 2-phosphoglycolate had no effect on malate inhibition. These results indicate that both the substrate PEP and the activator glucose 6-phosphate stabilize the active tetramer via binding and interaction at an activator site separate from the active site.

Regulation of the aggregation state of maize phosphoenolpyruvate carboxylase: evidence from dynamic light-scattering measurements.

The molecular weights of different aggregational states of phosphoenolpyruvate carboxylase purified from the leaves of Zea mays have been determined by measurement of the molecular diameter using a Malvern dynamic light scattering spectrometer. Using these data to identify the monomer, dimer, tetramer, and larger aggregate(s) the effect of pH and various ligands on the aggregational equilibria of this enzyme have been determined. At neutral pH the enzyme favored the tetrameric form. At both low and high pH the tetramer dissociated, followed by aggregation to a "large" inactive form. The order of dissociation at least at low pH appeared to be two-step: from tetramer to dimers followed by dimer to monomers. The monomers then aggregate to a large aggregate, which is inactive. The presence of EDTA at pH 8 protected the enzyme against both inactivation and large aggregate formation. Dilution of the enzyme at pH 7 at room temperature results in driving the equilibrium from tetramer to dimer. The presence of malate with EDTA stabilizes the dimer as the predominant form at low protein concentrations. The presence of the substrate phosphoenolpyruvate alone and with magnesium and bicarbonate induced formation of the tetramer, and decreased the dissociation constant (Kd) of the tetrameric form. The inhibitor malate, however, induced dissociation of the tetramer as evidenced by an increase in the Kd of the tetramer.

The role of oligomerization in regulation of maize phosphoenolpyruvate carboxylase activity. Influence of Mg-PEP and malate on the oligomeric equilibrium of PEP carboxylase.

A purification procedure which yields a near homogenous preparation of phosphoenolpyruvate (PEP) carboxylase from the leaves of Zea mays is reported. The enzyme had a final specific activity of 33.3 micromoles per minute per milligram protein. Size exclusion high performance liquid chromatography and dynamic laser-light scattering spectroscopy showed that PEP carboxylase exists in an equilibrium of aggregates. Enzyme predominantly in the dimeric configuration is less active (when assayed at sub-optimal Mg-PEP concentrations, less than 0.4 millimolar) than when in its tetrameric arrangement. The difference in activity diminishes and disappears as the concentration of the substrate Mg-PEP increases. The substrate drives the equilibrium toward the tetramer, while malate, an inhibitor of PEP carboxylase, shifts the equilibrium toward the dimer. It thus appears that the quaternary structure (oligomeric state) of maize PEP carboxylase can be regulated by the naturally occurring effector molecules Mg-PEP and malate which in turn can control the enzyme's activity.

Evidence for Chloroplastic Succinate Dehydrogenase Participating in the Chloroplastic Respiratory and Photosynthetic Electron Transport Chains of Chlamydomonas reinhardtii.

A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure ([1983] Plant Physiol 72: 481-487). Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K(m) for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H(2) adapted Chlamydomonas (M Gibbs, RP Gfeller, C Chen [1986] Plant Physiol 82: 160-166).

Localization of the Enzymes Involved in the Photoevolution of H(2) from Acetate in Chlamydomonas reinhardtii.

The localization of a series of enzymes involved in the anaerobic photodissimilation of acetate in Chlamydomonas reinhardtii F-60 adapted to a hydrogen metabolism was determined through the enzymic analyses of the chloroplastic, cytoplasmic, and mitochondrial fractions obtained with a cellular fractionation procedure that incorporated cell wall removal by treatment with autolysine, digestion of the plasmalemma with the detergent digitonin, and fractionation by differential centrifugation on a Percoll step gradient. The sequence of events leading to the photoevolution of H(2) from acetate includes the conversion of acetate into succinate via the extraplastidic glyoxylate cycle, the oxidation of succinate to fumarate by chloroplastic succinate dehydrogenase, and the oxidation of malate to oxaloacetate in the chloroplast by NAD dependent malate dehydrogenase. The level of potential activity for the enzymes assayed were sufficient to accommodate the observed rate of the photoanaerobic dissimilation of acetate and the photoevolution of H(2).

Aerobic and anaerobic respiration in the intact spinach chloroplast.

Aerobic and anaerobic chloroplastic respiration was monitored by measuring (14)CO(2) evolution from [(14)C]glucose in the darkened spinach (Spinacia oleracea) chloroplast and by estimating the conversion of fructose 1,6-bisphosphate to glycerate 3-phosphate in the darkened spinach chloroplast in air with O(2) or in N(2) with nitrite or oxaloacetate as electron acceptors. The pathway of (14)CO(2) evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide and glycolate 2-phosphate under air or N(2) were those expected from the oxidative pentose phosphate cycle and glycolysis. Of the electron acceptors, O(2) was the best (2.4 nanomoles CO(2) per milligram chlorophyll per hour), followed by nitrite and oxaloacetate. With respect to glycerate 3-phosphate formation from fructose 1,6-bisphosphate, methylene blue increased the aerobic rate from 3.7 to 5.4 micromoles per milligram chlorophyll per hour. A rate of 4.8 micromoles per milligram chlorophyll per hour was observed under N(2) with nitrite and oxaloacetate.

Inhibition of chloroplastic respiration by osmotic dehydration.

The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of (14)CO(2) evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with [(14)C]glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO(2) evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84% respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH(4)Cl (a stromal alkylating agent), iodoacetamide) an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction.

pH Effects on the Activity and Regulation of the NAD Malic Enzyme.

The NAD malic enzyme shows a pH optimum of 6.7 when complexed to Mg(2+) and NAD(+) but shifts to 7.0 when the catalytically competent enzyme-substrate (E-S) complex forms upon binding malate(-2). This is characteristic of an induced conformational change. The slope of the V(max) or V(max)/K(m) profiles is steeper on the alkaline side of the pH optimum. The K(m) for malate increases markedly under alkaline conditions but is not greatly affected by pH values below the optimum. The loss of catalysis on the acidic side is due to protonation of a single residue, pK 5.9, most likely histidine. Photooxidation inactivation with methylene blue showed that a histidine is required for catalytic activity. The location of this residue at or near the active site is revealed by the protection against inactivation offered by malate. Three residues, excluding basic residues such as lysine (which have also been shown to be vital for catalytic activity, must be appropriately ionized for malate decarboxylation to proceed optimally. Two of these residues directly participate in the binding of substrates and are essential for the decarboxylation of malate. A pK of 7.6 was determined for the two residues required by the E-S complex to achieve an active state, this composite value representing both histidine and cysteine suggests that both have decisive roles in the operation of the enzyme. A major change in the enzyme takes place as protonation nears the pH optimum, this is recorded as a change in the enzyme's intrinsic affinity for malate (K(m pH6.7) = 9.2 millimolar, K(m pH7.7) = 28.3 millimolar). Similar changes in K(m) have been observed for the NAD malic enzyme as it shifts from dimer to tetramer. It is most likely that the third ionizable group (probably a cysteine) revealed by the V(max)/K(m) profile is needed for optimal activity and is involved in the association-dissociation behavior of the enzyme.

Evidence for a multiple subunit composition of plant NAD malic enzyme.

Malate dehydrogenase (decarboxylating) (EC 1.1.1.39) was purified to near homogeneity from both a C3 plant, Solanum tuberosum, and a CAM plant, Crassula argentea. Sodium dodecyl sulfate-gel electrophoresis of both enzymes revealed an alpha,beta subunit composition with corresponding molecular mass assignments of 61,000 and 55,000 daltons. Isoelectric focusing under native conditions showed only one constituent malic enzyme form with an isoelectric point of 5.1. No evidence of additional isoenzymes was found. Urea isoelectric focusing showed the alpha subunit to be more acidic than the beta subunit. Peptide mapping by limited proteolysis with Staphylococcus aureus V-8 protease, trypsin, and endoproteinase Arg-C eliminated the possibility that a precursor-product relationship may have existed between the two subunits and demonstrated that they each possess unique primary sequences. Further support for this conclusion was obtained when significant differences in the contents of glutamic acid, isoleucine, and arginine were revealed by amino acid analysis of the isolated subunits. There was no apparent activity associated with the separated subunits (as resolved by urea-DEAE chromatography), but activity could be found in a reconstituted system, thereby indicating an (alpha,beta)n protomeric configuration. This is the first case where malic enzyme has been conclusively shown to be constructed from nonidentical subunits. This phenomenon has been observed only for the NAD malic enzyme isolated from plants.

Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula.

The 13C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on Vmax. This indicates a stepwise conversion of malate to pyruvate and CO2 with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylate oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean acid metabolism while maintaining the catalytic events found in malic enzymes from animal sources.

Regulation of the NAD Malic Enzyme from Crassula.

Using size exclusion chromatography, the nicotinamide adenine dinucleotide malic enzyme purified to near homogeneity from leaves of Crassula argentea was found to exist in at least three aggregational states (dimer, tetramer, and octamer). These forms differ in their apparent kinetic characteristics in initial rate assays, but all display similar characteristics at the steady state. The presence of 50 millimolar malate during chromatography causes a shift in favor of the smaller forms with the tetramer predominating. The native enzyme, when diluted 1/1000 and incubated 18 hours in buffer of high ionic strength, changes its steady state kinetic parameters to ones which indicate a low activity and low affinity for malate. When 50 millimolar malate or 50 micromolar coenzyme A are present the loss of activity and increase in K(m) is reduced. When both malate and coenzyme A are present the effects in minimizing the change in kinetic characteristics are additive.