Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

CHARACTERIZATION AND LOCALIZATION OF ACID HYDROLASE ACTIVITY IN THE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

Synaptosomes were prepared from the cerebral cortex of adult rats by a rapid technique of centrifugation in a Ficoll-sucrose discontinuous gradient. The synaptosomal fraction contained 40 per cent of the total gradient activity of acid α-naphthyl phosphatase (EC 3.1.3.2). Quantitative electron microscopy of this fraction revealed rare, typical, extrasynaptosomal dense body lysosomes. pH-activity profiles of free and Triton X-100 (total) activities were prepared for α-naphthyl phosphatase, β-glucuronidase (EC 3.2.1.31), β-galactosidase (EC 3.2.1.23), arylsulfatase (EC 3.1.6.1) and N-acetylglucosaminidase (EC 3.2.1.30). The ratios of total to free activity varied in the order: arylsulfatase > β-galactosidase > β-glucuronidase > N-acetylglucosaminidase > acid phosphohydrolase. Incubation of synaptosomal fractions at pH 5 and 37°C produced significant activation of β-galactosidase and N-acetylglucosaminidase but no activation of cryptic lactate dehydrogenase (EC 1.1.1.27). Hyposmotic suspension and subfractionation of the synaptosomal fraction produced considerable solubilization of lactate dehydrogenase, arylsulfatase and β-galactosidase but only partial liberation of α-naphthyl phosphatase, the remainder being associated with synaptosomal membrane fragments. Incomplete equilibrium sedimentation of synaptosomes in a continuous sucrose gradient (0·55-1·5 M) provided a broad lactate dehydrogenase and Na + K ATPase (EC 3.6.1.4) peak (peak I) at low sucrose densities. β-Glucuronidase, β-glucosidase and α-naphthyl phosphatase were significantly present in peak I. Conversely, N-acetylglucosaminidase, arylsulphatase and β-galactosidase were predominantly located in denser particles sedimenting through 1·2 M sucrose (peak II). Electron microscopy confirmed the heterogeneity of this second peak and the presence of numerous extrasynapto-somal dense body lysosomes.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

AGE-DEPENDENT VARIATIONS OF THE HUMAN N-ACETYL-β-D-HEXOSAMINIDASES

The total N-acetyl-β-d -hexosaminidase (hexosaminidase A plus B) activity of the human brain increases during life until the age of 70 years to about twice the activity found in the foetal brain. This activity roughly parallels the increasing level of brain ganglio-sides. Simultaneously, the ratio between the hexosaminidases A and B changes from approx. 3.4 to 1.1 as established by the microscale isoelectric focussing of the enzymes from crude extracts. Age-dependent variations can be also demonstrated for other glycosidase activities: The β-d -glucosidase is shown to increase, the α-d -mannosidase and α-l -fucosidase to decrease during life. The enzyme values are related to the protein content, the age-dependent shift of which we have determined in the human brain. In different organs and body liquids distinct differences are found for the levels of the total hexosaminidase activity as well as for the hexosaminidase pattern. The relation of the hexosaminidase activity to the ganglioside degradation during the human development is discussed.     

QUANTITATIVE LYSOSOMAL ENZYME ACTIVITY CHANGES IN THE NEURAL LOBE OF THE RAT FOLLOWING WATER DEPRIVATION AND LACTATION

—The activities of five lysosomal enzymes, acid phosphatase, β-glucosidase, β-glucuronidase, β-galactosidase and N-arylamidase (classified according to Marks (1970)) were measured by means of sensitive microchemical techniques in frozen-dried rat neural lobe tissue after experimental and physiological stimulation of hormone release from the hypothalamo–neurohypophysial system i.e. water deprivation (3 and 6 days), delivery and lactation (10 days). During all conditions of stimulation increases of 29 to 106 per cent were measured for lysosomal enzyme activity, expressed as mmol/ng DNA/h. With histochemical staining methods the acid phosphatase activity appears to be mainly localized in the pituicytes, but it was impossible to visualize the microchemically measured acid phosphatase activity increase within the two main compartments of the neurohypophysis, i.e. axonal endings and the neurohypophysial glial cells, the pituicytes.     

Effects of cortisone and thyroxine on suckling rat liver N-acetyl-ß-glucosaminidase isozymes: Comparison with adult reactivity

Suckling rat liver N-acetyl-β-glucosaminidase (hexosaminidase) activity undergoes considerable fluctuation during the first two weeks of life. As two major forms of hexosaminidase (A, heat-labile, and B, heat-stable) are known to exist in both human and adult rat liver, we choose to examine the effect of the maturative hormones, thyroxine and cortisone, upon these isozymes during the suckling period. Between days 7 and 15, the observed developmental change is attributable solely to an increase in the ‘A-like’ (heat-labile) form of the enzyme; an enhanced response is seen in thyroxine-injected 11–15-day old animals. The response may be considered ‘age-independent’, as adult animals react in the same manner. In contrast, cortisone-injected sucklings show a decrease in both A and B isozymes, while in adults no changes in total activity or isozyme distribution are evoked. The ratio of hexosaminidase A to hexosaminidase B in suckling rat liver appears to shift in favor of the labile (A) isozyme early in development.     

Hepatitis C Virus Diversification in Argentina: Comparative Analysis between the Large City of Buenos Aires and the Small Rural Town of O'Brien

Background

The estimated prevalence of HCV infection in Argentina is around 2%. However, higher rates of infection have been described in population studies of small urban and rural communities. The aim of this work was to compare the origin and diversification of HCV-1b in samples from two different epidemiological scenarios: Buenos Aires, a large cosmopolitan city, and O''Brien, a small rural town with a high prevalence of HCV infection.

Patients and Methods

The E1/E2 and NS5B regions of the viral genome from 83 patients infected with HCV-1b were sequenced. Phylogenetic analysis and Bayesian Coalescent methods were used to study the origin and diversification of HCV-1b in both patient populations.

Results

Samples from Buenos Aires showed a polyphyletic behavior with a t_MRCA around 1887–1900 and a time of spread of infection approximately 60 years ago. In contrast, samples from ÓBrien showed a monophyletic behavior with a t_MRCA around 1950–1960 and a time of spread of infection more recent than in Buenos Aires, around 20–30 years ago.

Conclusion

Phylogenetic and coalescence analysis revealed a different behavior in the epidemiological histories of Buenos Aires and ÓBrien. HCV infection in Buenos Aires shows a polyphyletic behavior and an exponential growth in two phases, whereas that in O''Brien shows a monophyletic cluster and an exponential growth in one single step with a more recent t_MRCA. The polyphyletic origin and the probability of encountering susceptible individuals in a large cosmopolitan city like Buenos Aires are in agreement with a longer period of expansion. In contrast, in less populated areas such as O''Brien, the chances of HCV transmission are strongly restricted. Furthermore, the monophyletic character and the most recent time of emergence suggest that different HCV-1b ancestors (variants) that were in expansion in Buenos Aires had the opportunity to colonize and expand in O’Brien.     

UTILIZATION OF ACETATE AND PYRUVATE FOR THE SYNTHESIS OF'TOTAL','BOUND'AND'FREE'ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

—Slices of tissue of the electric organ of Torpedo marmorata were incubated in vitro in a salineurea-sucrose solution containing a labelled precursor of the acetyl moiety of ACh ([1-¹⁴C]glucose, [2-¹⁴C]pyruvate, or [1-¹⁴C]acetate) either alone or in the presence of another unlabelled precursor. The incorporation of ¹⁴C from [1-¹⁴C]acetate into ACh was considerably higher than from the other two substrates. The specific radioactivities (SRA) of the‘total',‘bound’and‘free’ACh were compared in experiments with [2-¹⁴C]pyruvate and [1-¹⁴C]acetate. With both precursors, the SRA of the‘bound’ACh were lower than those of‘total’ACh; consequently, the‘free’ACh pool was more labelled than the‘bound’pool. After short incubations with [2-¹⁴C]pyruvate the SRA of'bound’ACh were closer to the SRA of‘total’ACh than with [1-¹⁴C]acetate. A simple method is described for the labelling of ACh and its separation from other labelled compounds in experiments with the electric organ using [¹⁴C]acetate as the labelled precursor.     

PROPERTIES OF ACID β-d-GALACTOSIDASE ISOLATED FROM I-CELL DISEASE BRAIN AND SPLEEN

Abstract— Acid 4-methylumbelliferyl β- d -galactosidase activity from autopsied I-cell disease brain and spleen tissues was 28% and 35% respectively of normal activity. Acid β- d -gatactosidase (β- d -galactoside galactohydrolase, EC 3.2.1.23) from two I-cell disease brains demonstrated a 5-fold increase over normal for the proportion of enzyme activity which did not adsorb to Concanavalin A-Sepharose 4B, while acid β- d -galactosidase from two I-cell disease spleens demonstrated a 21–35-fold increase in the proportion of unadsorbed enzyme activity. Normal and I-cell disease acid β- d -galactosidase present in crude brain and spleen supernatant fluids and in preparations partially purified on Concanavalin A-Sepharose 4B had similar apparent K _m values with respect to 4-methylumbelliferyl β- d -galactopyranoside and G_M1-ganglioside. Isoelectric focusing profiles of normal and I-cell disease acid β- d -galactosidase from crude brain and spleen-supernatant fluids and partially purified preparations were similar. Neuraminidase treatment and subsequent isoelectric focusing of the partially purified normal and I-cell disease enzyme preparations from brain and spleen revealed increases in the proportion of I-cell β- d -galactosidases found at neutral pH values, suggesting that the electrophoretic variations observed for the I-cell enzymes may not be attributed solely to changes in sialic acid composition.     

Demonstration of Various Acid Hydrolases and Preliminary Characterization of Acid Phosphatase in Naegleria fowleri1

ABSTRACT. Extracts of the pathogenic ameba Naegleria fowleri, prepared by freeze-thawing and sonication, were analyzed for their content of various hydrolytic enzymes that have acid pH optima. The organism is rich in acid phosphatase activity as well as a variety of glycosidases which include β-glucosidase, β-galactosidase, β-fucosidase, α-mannosidase, hexosaminidase, arylsulfatase A, and β-glucuronidase. The crude extract contained only negligible levels of sphingomyelinase, neuraminidase, or arylsulfatase B. All of the hydrolases exhibited higher activity at pH 5.5 than at 7.0, indicating that they are truly “acid” hydrolases. In general, after centrifugation (100,000 g, 1 h), except for arylsulfatase B, more than half of the activity of each of the various hydrolases was recovered in the supernatant fraction. The acid phosphatase in the high-speed supernatant was purified 45-fold (32% yield) by chromatography on QAE-Sephadex and Sephadex G-200 and shown to have the following properties: 1) pH optima, 5.5; 2) K_m (4-methylumbelliferyl phosphate), 0.60 mM; 3) molecular weight (estimated by gel filtration chromatography), 92,000; 4) inhibited by heteropolymolybdate complexes but not by L(+) sodium tartrate (0.5 mM) or sodium fluoride (0.5 mM). In addition, unlike the tartrate-resistant acid phosphatase of Leishmania donovani, the major acid phosphatase of N. fowleri is less than 5% as effective in inhibiting superoxide anion production by f-Met-Leu-Phe-stimulated human neutrophils. The finding of high levels of a number of acid hydrolases in Naegleria fowleri raises several questions that merit further study: Do the hydrolases perform a housekeeping function in this single cell eukaryote or do they play some role in the pathogenic process that ensues when the organism infects a suitable host?     

RETRACING JOSEPH HOOKER'S ROUTES IN THE SIKKIM HIMALAYA

Joseph Hooker was one of the most widely travelled botanists of the Victorian period, having explored regions as far afield as Antarctica, Morocco and North America, though it is for his pioneering exploration of the Sikkim Himalaya that he is perhaps best remembered. Seamus O'Brien led four expeditions to this remote corner of the eastern Himalaya, and the results of these trips will appear in a new biography due to be published by Kew in 2018. He summarises that story here.     

Characterization of lysosomal hydrolases that are elevated in Gaucher's disease.

Although Gaucher's disease occurs in three distinct forms with greatly varying degrees of severity, there is no correlation between the clinical course of the disease and levels of residual glucocerebrosidase, the fundamental enzymatic deficiency. In an effort to study secondary changes which might contribute to the pathology of Gaucher's disease, homogenates of spleen, liver, and brain tissue, as well as serum from patients with Gaucher's disease were analyzed for their content of a number of lysosomal enzymes. Extracts of 8 Gaucher spleens contained 3- to 4-fold increases in acid phosphatase activity as well as 5-to 10-fold increases in galactocerebrosidase⁵ activity. The marked elevation in galactocerebrosidase activity in Gaucher spleen was documented using various [³H]galactose labeled galactocerebrosides as substrates and with [³H]galactose labeled lactocerebroside under the “lactosylceramidase I”⁵ assay conditions established by Suzuki (Tanaka, H., and Suzuki, K., 1975, J. Biol. Chem., 250, 2324–2332) that measure galactocerebrosidase activity specifically in the presence of G_mi-ganglioside β-galactosidase. Acid phosphatase determinations using extracts of liver from a case of infantile, neuropathic Gaucher's disease revealed a 2-fold elevation in this activity, whereas brain acid phosphatase activity in this case was similar to that of control tissue. Separation of hexosaminidase A and B activities on DEAE-Sephadex columns indicated increases in both forms of the enzyme in Gaucher tissue with the major increase occurring in the hexosaminidase B component. Glucuronidase and nonspecific esterase were observed to be elevated approximately 2-fold. However, not all lysosomal enzyme activities were increased. Levels of splenic arylsulfatase A and B, α-arabinosidase, sphingomyelinase, α-mannosidase, and G_mi-ganglioside β-galactosidase activities in Gaucher spleen were unremarkable. G_mi-ganglioside β-galactosidase was measured using 4-methylumbelliferyl-β-d-galactopyranoside and [³H]galactose labeled lactocerebroside under the specific assay conditions described by Suzuki for the determination of “lactosylceramidase II” activity. Although levels of arylsulfatase A and B in Gaucher spleen were similar to those of control tissue, arylsulfatase A activity was markedly reduced (20% of control) in homogenates of brain from the case of infantile (type 2) Gaucher's disease. The metabolic and pathologic consequences of these changes in lysosomal enzymes in Gaucher's disease are discussed.     

Testing a Primary and a Secondary Endpoint in a Group Sequential Design

Summary We consider a clinical trial with a primary and a secondary endpoint where the secondary endpoint is tested only if the primary endpoint is significant. The trial uses a group sequential procedure with two stages. The familywise error rate (FWER) of falsely concluding significance on either endpoint is to be controlled at a nominal level α. The type I error rate for the primary endpoint is controlled by choosing any α‐level stopping boundary, e.g., the standard O'Brien–Fleming or the Pocock boundary. Given any particular α‐level boundary for the primary endpoint, we study the problem of determining the boundary for the secondary endpoint to control the FWER. We study this FWER analytically and numerically and find that it is maximized when the correlation coefficient ρ between the two endpoints equals 1. For the four combinations consisting of O'Brien–Fleming and Pocock boundaries for the primary and secondary endpoints, the critical constants required to control the FWER are computed for different values of ρ. An ad hoc boundary is proposed for the secondary endpoint to address a practical concern that may be at issue in some applications. Numerical studies indicate that the O'Brien–Fleming boundary for the primary endpoint and the Pocock boundary for the secondary endpoint generally gives the best primary as well as secondary power performance. The Pocock boundary may be replaced by the ad hoc boundary for the secondary endpoint with a very little loss of secondary power if the practical concern is at issue. A clinical trial example is given to illustrate the methods.     

Neurochemical abnormality in I-cell disease: chemical analysis and a possible importance of beta-galactosidase deficiency.

Sphingolipid composition in both gray and white matter of a patient with I-cell disease was normal except for the higher proportion.of G_MI-ganglioside in gray and white matter. In the patient's liver and kidney there was a significant accumulation of ceramide dihexoside and ceramide trihexoside and of sulphatide in kidney. Non-lipid hexosamine and sialic acid concentration in brain was increased 1.2-1.5 times above normal. Recovery of myelin from I-cell's white matter was 80-100%, suggesting that demyelination, if present, is minimal. Myelin lipid and myelin specific glycoprotein patterns were normal. Except for β-galactosidase activity the activity of other brain lysosomal enzymes were within the normal range. This finding was similar to that of Hurler's syndrome. Only β-galactosidase activity was reduced to less than 10% of normal in the patient's brain. To examine the possible metabolic significance of β-galactosidase deficiency in I-cell disease the physical characteristics of this enzyme, isolated from tissues from I-cell, Hurler and control patients, were compared using isoelectric focusing, Con A-Sepharose and Sephadex G-150 chromatography. The isoelectric point and the binding affinity of I-cell β-galactosidase with Con A-Sepharose was comparable to normal. However, the isoenzyme patterns of brain and liver I-cell β-galactosidase with Sephadex G-150 gel filtration revealed decreased acid β-galactosidase. Effects of the addition of sodium chloride on each fraction of β-galactosidase isoenzymes isolated from I-cell tissues were markedly different from controls, whereas the pH optimum of these enzymes were similar to normal. These enzyme characteristics in I-cell tissues were different from normal and Hurler's syndrome. These findings suggest that β-galactosidase deficiency in I-cell disease is a more specific phenomenon rather than secondary inhibition as found in the mucopolysaccharidoses and thus may have an important role for the pathogenesis of brain damage and disease occurrence.     

Group Sequential Monitoring with Arbitrary Inspection Times

The classical group sequential test procedures that were proposed by Pocock (1977) and O'Brien and Fleming (1979) rest on the assumption of equal sample sizes between the interim analyses. Regarding this it is well known that for most situations there is not a great amount of additional Type I error if monitoring is performed for unequal sample sizes between the stages. In some cases, however, problems can arise resulting in an unacceptable liberal behavior of the test procedure. In this article worst case scenarios in sample size imbalancements between the inspection times are considered. Exact critical values for the Pocock and the O'Brien and Fleming group sequential designs are derived for arbitrary and for varying but bounded sample sizes. The approach represents a reasonable alternative to the flexible method that is based on the Type I error rate spending function. The SAS syntax for performing the calculations is provided. Using these procedures, the inspection times or the sample sizes in the consecutive stages need to be chosen independently of the data observed so far.     

LOCALIZATION OF DEHYDROGENASE AND ACID PHOSPHATASE IN THE ABSCISSION ZONE OF BEAN LEAVES

Leaf abscission in Phaseolus vulgaris L. cv. ‘Contender’ is associated with enzymatic changes during and prior to separation. Deblading resulted in a localized increase in dehydrogenase and acid phosphatase in the abscission zone. Increased enzyme activities were observed 24–48 hr after deblading. In debladed plants separation was complete in 6–8 days. At separation, dehydrogenase activity appeared to decrease and localization was specific to the protective layer, while the petiole side had no activity. In contrast, acid phosphatase activity was observed in some layers of cells on the petiole side after separation. Ethylene treatment promoted abscission and separation occurred in 24–48 hr in both debladed and intact plants. No protective layer was formed during ethylene-induced abscission. Enzymatic changes similar to those observed in debladed control plants were observed with ethylene treatment. Ethylene induced an additional abscission layer between the pulvinus and petiole, where an abscission layer normally does not form. In this ethylene-induced abscission layer, similar enzyme activities were detected.     

SIALIC ACID AND THE SOCIAL BEEIAVIOUR OF CELLS

1. It is suggested that specific carbohydrate side-chains of membrane glycoproteins are the sites for cell recognition or adhesion when the terminal sugar, sialic acid, is absent. 2. It is suggested that sialic acid plays a ‘protective’ or ‘blocking’ role in cell interactions so that addition of sialic acid to asialo side-chains converts them to forms inactive for recognition. This principle of ‘blocking’ by sialic acid has been observed in other situations as in covering tumour antigens and in protecting glycoproteins from uptake by the liver. It is here extended to cell-cell adhesions. 3. It is to be expected that specific ‘protective’ actions of sialic acid in membrane-bound glycoproteins will be difficult to detect. As a charged residue, sialic acid is likely to have a strong influence both on the glycoproteins on which it is borne and on their interactions with each other at the cell surface. Removal of sialic acid by enzymes could therefore perturb the structure of the cell surface in several ways and so obscure the ‘protective’ effects of sialic acid. Sialic acid is therefore suggested to have a structural role also. 4. Evidence is assembled in favour of a model in which sialysation of specific adhesive receptors affects the social behaviour of cells. This may be an effect associated with growing cells since the contact properties of mitotic cells (and populations rich in dividing cells) are decreased by the increased sialysation of receptors. One of the factors associated with malignant behaviour could be that adhesive receptors are permanently blocked by sialic acid. 5. A schematic representation of some of the points is given in Fig. 4.     

Wall-bound enzymes in callus of Convolvulus arvensis

Isolated cell walls of Convolvulus callus contain α- and β-galactosidase, α- and β-glucosidase, α- and β-mannosidase, acid invertase and acid phosphatase activities. No neutral invertase or alkaline phosphatase activities could be detected. Acid invertase activity per mg cell wall increased considerably during incubation of callus fragments in nutrient solution, as opposed to the activities of the other enzymes mentioned.     

FLORAL DEVELOPMENT OF POTAMOGETON RICHARDSONII

The inception and development of the sterile floral appendages of Potamogeton richardsonii have been re-investigated with a refined dissection technique (Sattler, 1968) and improved microtechnical methods (Feder and O'Brien, 1968). The results obtained by Sattler (1965) are confirmed, i.e., the sterile appendages are initiated at the flanks of the floral apex before the stamen primordia are formed. Consequently, they may be homologized with tepals or perianth members, although in the mature flower they are inserted at the stamen connective, due to growth between and at the base of each developing tepal and stamen. Each carpel arises as a radial primordium which becomes peltate immediately after its inception. One ovule primordium is initiated at the cross-zone. The stigma becomes bilobed. A slight outgrowth develops at the abaxial side of the style. The floral apex has a two-layered tunica. The primordia of the tepals, carpels, and ovules arise by periclinal divisions in the second tunica layer, whereas the stamen primordia are initiated by periclinal divisions in the corpus and second tunica layer. Variation in floral pattern, especially with regard to the number of appendages, has been observed in flowers near the tip of the inflorescence axis.     

The composition of pericarp,cell aging,and changes in water absorption in two tomato genotypes: mechanism,factors, and potential role in fruit cracking

Cracking, a serious problem in many fruits, may cause significant economic losses. It may occur when internal pressure cannot sustain by the epidermis any longer. Water absorption and epidermis are among the most important factors that associated with cracking. To determine whether pericarp composition and its mechanical performance, endogenous cell wall disassembly, and water-absorbing capacity influences tomato fruit cracking, we grew a cracking-resistant genotype ‘LA1698’ and susceptible genotype ‘LA2683’. The results illustrated that the cuticle and subcutaneous layer were thicker in ‘LA1698’ than in ‘LA2683’. Compared with ‘LA2683’, the fruit firmness, consistency, and bursting strength of ‘LA1698’ were all higher. Fruits of ‘LA1698’ had decreased activities of polygalacturonase, β-galactosidase, and cellulose, which can disassemble the polysaccharide network. As a result, it had reduced water-soluble pectin and more covalently and ionically bound pectin that can crosslink with Ca²⁺ and B. These fruits also have a greater abundance of hemicelluloses. In addition, ‘LA1698’ had higher SOD activities and lower relative conductivity, meaning its cells might have a better biological activity to resist changes of the external environment (such as water variation) and to prevent fruit cracking. However, POD in ‘LA2683’ was more abundant than in ‘LA1698’. ‘LA1698’ produced juice with lower total soluble solids, which led to a lower initial water-absorbing ability and difference between the exocarp and mesocarp. In conclusion, a stronger pericarp and cells with a better biological activity in addition to the lower water-absorbing difference between the exocarp and mesocarp made ‘LA1698’ more resistant to cracking.