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The wheat germ translation system was programmed with soluble RNA extracted from foliar abscission zones of Phaseolus vulgaris, These extracts were taken at various times after the induction of abscission. A translation product with a molecular weight of 42 kilodalton (kD) was only present after this treatment, though three other species 32, 27 and 17 kD increased substantially. The isozyme of cellulase with a pi of 9.5 could not be conclusively identified amongst the products though the 32 kD protein is probably chitinase. Comparison of the abscission zone translatable RNA with that from adjacent petiole and stem tissues showed the 17 kD protein developed in all these location. The 42, 32 and 27 kD bands were found predominantly in the zone and petiole.

Egypt is one of the most populous countries in Africa. Most of Egypt 82.2 million people live near the banks of the Nile River, in an area of about 40,000 km2, where the only arable land is found. The large areas of the Sahara Desert are sparsely inhabited. About half of Egypt’s residents live in urban areas, with most people spread across the densely populated centers of greater Cairo, Alexandria and other major cities in the Nile Delta. Egypt’s fertile area totals about 3.3 million ha, about one-quarter of which is land reclaimed from the desert. However, the reclaimed lands only add 7% to the total value of agricultural production. Even though only 3% of the land is arable, it is extremely productive and can be cropped two or even three times annually. Most land is cropped at least twice a year, but agricultural productivity is limited by salinity, which afflicts an estimation of 35% of cultivated land, and drainage issues. Climate change is a natural phenomenon, but humankind has drastically altered the process. Climate change has the potential to affect agriculture through changes in temperature, rainfall timing and quantity, CO2, and solar radiation. Agriculture can both mitigate or worsen global warming. Some of the increase in CO2 in the atmosphere comes from the decomposition of organic matter in the soil, and much of the methane emitted into the atmosphere is caused by the decomposition of organic matter in wet soils such as rice paddies. Egypt’s agricultural development has been constrained by, among other factors, the need to conserve scarce natural resources, the pressures of rapid urbanization, the onslaught of the desert, and, not least important, technological limitations and restrictive economic structures.

The occurrence of enzymes associated with bean leaf abscission was investigated in bean (Phaseolus vulgaris) flower reproductive organs in which catabolic cell wall events are essential during anther and pistil development. Cellulase activity was detected in high levels in both pistil and anthers of bean flowers before anthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified a protein in anthers and pistil with the same size (51 kilodaltons) and serologically closely related to the abscission cellulase. The accumulation of 9.5 cellulase protein in the anther is developmentally regulated and increases from undetectable levels at very young stages of anther development to high levels as the anther matures. In the pistil, the 9.5 cellulase was localized in the upper part of the pistil where the stigma and the stylar neck reside and was detected in the youngest developmental stage analyzed. Antibodies against basic chitinase, which accumulates to high levels in abscission zones after exposure to ethylene, identified a protein with the same size (33 kilodaltons) and serologically closely related, in both anthers and upper portion of the pistil. In contrast, a 45-kilodalton protein and the basic beta-1,3-glucanase associated with abscission were undetected in bean reproductive organs. Interestingly, beta-1,3-glucanase activity was detected in young bean anthers and decreased at anthesis, but the anther beta-1,3-glucanase is serologically unrelated to the basic beta-1,3-glucanase. Thus, it appears that the basic cellulase and chitinase occur in combination in many plant processes that require major cell wall disruption, whereas hemicellulases such as beta-1,3-glucanase are specific to each process.

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.

A generalized case study of the failure of an oxygen-acetylene cutting torch is presented with discussion of the failure and investigation. The theory that an oxygen-acetylene flashback reaction caused the explosive failure of the torch mixing tube was investigated. The investigation led to the conclusion that abusive treatment of the torch was the primary cause of the failure. The mechanical strength of the cutting torch was determined and the dynamic overpressures due to oxygen-acetylene flashback reactions were measured by means of fast-response piezoelectric pressure transducers. The dynamic pressure measurements were compared and related to predictions of the NASA Gordon and McBride "Computer Program for Calculations of Complex Chemical Equilibrium Compositions, Rocket Performance, Incident and Reflected Shocks, and Chapman-Jouquet Detonations" for oxygen-acetylene combustion. Based on this investigation, a reconstruction of the accident is proposed.

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.

A cellulase (β(1:4)4-glucan hydrolase) has been purified 700-fold by cellulose affinity chromatography from anthers of Lathyrus odoratus L. Two forms are present. The major species has a molecular weight of 49 kDa and a p I value of 8.0; the minor a molecular weight of 51 kDa and a p I value of 7.8. Both proteins are recognized by antibodies raised against the ethylene/IAA regulated bean ( Phaseolus vulgaris L.) abscission zone cellulase which has a more basic p I value and some unique antigenic determinants. Ethylene induced Lathyrus flower bud abscission zones contain two forms of cellulase apparently identical to those in the anthers. The possible role of cellulase in the development of anthers is discussed.

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of beta-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.

Lymphocyte subset analysis was performed on 114 healthy children and 84 healthy adults. Samples were prepared by a whole blood lysis technique and analyzed by flow cytometry. The percentage and total number of CD3+, CD4+, CD8+, and CD19+ lymphocytes were calculated for each of six age groups. A direct correlation with age was seen in the percentages of CD3+, CD4+, and CD8+ lymphocytes. The absolute number and percentage of total lymphocytes, the percentage and absolute number of CD19+ lymphocytes, and the absolute number of CD3+ lymphocytes decreased with age. No significant correlation with age was observed for white blood cells, the absolute number of CD4+ and CD8+ lymphocytes, and the CD4+/CD8+ ratio.

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10(-5) or 10(-2)m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10(-2)m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10(-5)m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.

The purification of a cellulase isoenzyme with a pI of 9.5 from kidney bean abscission zones is described. An important step in the purification involved the adsorption of the cellulase isoenzyme onto an affinity column of CF-11 cellulose and the subsequent elution with cellobiose. Native and SDS polyacrylamide gel electrophoresis established that there was only one component in the purified cellulase samples. Antibodies raised against the purified pI 9.5 cellulase precipitated this isoenzyme from crude or purified solutions but did not cross react with pI 4.5 cellulase from 2,4-D-treated abscission zones. The antibody was shown to be monospecific by immunoelectrophoresis and by the fact that it precipitated only a single 14C-labeled protein from an abscission zone extract heavily labeled with 14C amino acids.

The petals of young flowers ofGeranium robertianum L. start to be shed 2.25 hours after exposure to 20 ppm ethylene whilst controls kept in air take approximately 8 hours longer. The detachment of the petal takes place at its junction with the receptacle. The cells in the region show evidence of cell wall degradation and fracture takes place by loss of cell cohesion along the line of the middle lamella. The petal base is surrounded by a canal of receptacle tissues which alter shape either during or immediately after fracture. It is proposed that these structural changes may produce stresses which facilitate fracture.

Antibodies raised against 9.5 cellulase (β 1,4 glucan-4-glucanohydrolase) were used to investigate the distribution of the enzyme in abscising bean petioles (Phaseolus vulgaris L. cv. Red Kidney).

Membranes isolated from abscission zones of Phaseolus vulgaris L., cv. Red Kidney, contained cellulase activity. This particulate activity was enhanced 10- to 20-fold by treatment with Triton X-100. Sucrose density gradient analyses of cell fractions showed that the membranes with which cellulase was associated had a peak equilibrium density of 1.16 to 1.17 g/cm(3) which coincided with that of ion-activated ATPase, a marker for plasma membranes. The membrane fraction having the highest cellulase activity also contained a high proportion of plasma membranes as shown by electron microscopy of sucrose density gradient fractions after staining by periodic acid-chromic acid-phosphotungstic acid. It was concluded that the particulate cellulase was associated with the plasma membrane.

Optimum conditions for the extraction and purification of avocado cellulase have been established. The best quantiative recovery of the cellulase from avocado tissue was obtained with a 0.04M acetate buffer, pH 5.0, containing 0.4M NaCl and 0.25% (v/v) Triton X-100. The isoelectric point of the enzyme was 4.7 and the molecular weight, based on SDS gel electrophoresis, was 49,000. The enzyme was purified on a cellulose column to a single protein band on SDS gel electrophoresis. Rabbit antibody produced to the cellulase gave further proof of the enzyme's purity.

The changes in the levels of two different isozymes of cellulase (EC 3.2.1.4) have been followed during the abscission of the primary leaves of bean (Phaseolus vulgaris c.v. Red Kidney), using antibodies raised against the 9.5 form of the enzyme. Data from both radioimmune and direct assay show that the 9.5 form of cellulase is undetectable prior to the induction of abscission. After a 12 h lag this isozyme increases in activity, the increase preceding a decrease in integrity of the abscission zone cell walls. The results are consistent not only with the view that this specific isozyme is involved in wall hydrolysis but also with previous data which showed that cellulase is synthesized ‘de novo’. The 4.5 isozyme of cellulase is more widely spread throughout the plant, being most active in young tissues. During abscission the activity of this isozyme in the abscission layer falls and consequently it is not thought to be involved directly in the abscission process.

Cotyledons of germinating kidney beans contain two forms of a carboxy methyl cellulase which can be separated by ammonium sulfate fractionation and isoelectric focusing. The two cellulases are similar in their molecular weight but differ in isoelectric points, pH and temperature optimum, pH and temperature stability and sensitivity to thiol inhibitors and metal ions. One cellulase (isoelectric point 4.8) has been purified 100-fold to give a major protein band on acrylamide gel electrophoresis.