Diabetes may affect the expression of matrix metalloproteinases and their inhibitors more than smoking in chronic periodontitis.

BACKGROUND AND OBJECTIVE: No previous study has directly compared the levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) between smokers and individuals with diabetes mellitus (DM) with periodontitis. Therefore, the aim of this study was to evaluate the gene expression of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 in tissues with chronic periodontitis (ChP) of smokers and individuals with type 2 DM. MATERIAL AND METHODS: Gingival biopsies were harvested from: non-smokers and non-diabetic individuals with ChP (n = 18) (ChP group); non-diabetic smokers (≥ 10 cigarettes per day for at least the past 5 years) with ChP (n = 18) (SChP group); non-smoking individuals with type 2 diabetes (glycated hemoglobin levels ≥ 7.5%) and ChP (n = 18) (DMChP group). The tissue levels of mRNA of MMP-1, MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 were evaluated by quantitative real-time polymerase chain reaction. RESULTS: The MMP-8 expression was the lowest in the ChP group (p < 0.05). The DMChP group presented increased mRNA levels of MMP-2 and MMP-9, when compared to the SChP group (p < 0.05). MMP-1 expression and the MMP-1/TIMP-1, MMP-2/TIMP-1, MMP-8/TIMP-1, MMP-9/TIMP-1, MMP-1/TIMP-2 and MMP-2/TIMP-2 ratios were higher in the DMChP group than in the ChP and SChP groups (p < 0.05). The DMChP group presented lower mRNA levels of TIMP-1 than the ChP group (p < 0.05). The MMP-8/TIMP-2 ratio was the highest in the SChP group (p < 0.05). CONCLUSION: Uncontrolled type 2 DM upregulates the ratio of MMP/TIMPs in sites with ChP more than smoking, which may contribute to a greater extracellular matrix degradation and periodontal breakdown in DM-related periodontitis.

Evaluation of chondrocyte growth and function subjected to 21% and 6% oxygen levels.

In osteoarthritis, the metabolic activity of the chondrocytes is shifted toward a state where new matrix synthesis is outweighed by breakdown of matrix constituents. The result is degeneration and gradual loss of articular cartilage. Although osteoarthritis is frequently regarded as a non-inflammatory form of arthritis, considerable data implicates a role for pro-inflammatory cytokines in the cartilage destruction associated with osteoarthritis. The best studied pro-inflammatory cytokines in osteoarthritis are interleukin-1ß (IL-1ß), tumor necrosis factor-a (TNF-a), and interleukin-6 (IL-6). Since articular cartilage is not vascularized, it must rely on diffusion from the articular surface for nutrient and metabolic exchange. Consequently, the entire metabolism of the cell is geared towards operating at a low oxygen tension. In this study, chondrocytes were challenged with pro-inflammatory cytokines at 21% O2 and 6% O2. Chondrocyte proliferation, membrane integrity, oxidative stress, matrix metalloproteinase – 9 (MMP-9) and hypoxia inducible factor-1a (HIF-1a) production were measured. Our results showed that there was less of a decrease in cell number at 6% O2 compared to 21% O2 after they were challenged with pro-inflammatory cytokines. In addition, there was less of an increase in oxidative stress, membrane damage and MMP-9 production at 6% O2 compared to21% O2. The significance of this study represents the first attempt to replicate a diseased inflammatory environment characterized by an osteoarthritic joint in vitro and to examine these effects on the growth and stability of chondrocytes.

Protein recovery from several paper types used to collect gingival crevicular fluid.

The purpose of this study was to compare the efficiency of protein elution from several types of gingival crevicular fluid (GCF) collection papers when the volume of the inoculated protein and the elution methods were constant. Various concentrations of bovine serum albumin (BSA) and 14C-BSA were placed onto strips of Whatman #1 [W1], Whatman 3 MM chromatographic [W3], Periopaper (ProFlow) [P] and Periopaper (Harco) [H], and recovered proteins measured following a non-optimized centrifugal elution technique. There were significant differences in % recovery of BSA and 14C-BSA from the papers, which was dependent on both the type of paper and the concentration of the inoculated protein; that is, proteins at the lowest concentrations were less efficiently eluted from GCF collection papers than those at higher concentrations. Equations for regression lines of elution efficiency were quadratic. Thus, our data suggest significant differences in the efficiency for elution of BSA from absorbent papers when the volume of the inoculated fluid and the elution technique were constant. Previous variable or conflicting experimental data between research groups may have resulted from incomplete elution of proteins from GCF collection papers, possibly due to entrapment within, or binding of GCF proteins to the paper.

The relationship between concentrations of proinflammatory cytokines within gingiva and the adjacent sulcular depth.

The objective of this study was to determine and compare concentrations and ratios of 3 proinflammatory cytokines, interleukin (IL) IL-1beta, IL-6, and IL-8 within gingival tissue biopsies adjacent to < or = 3, 4 to 6, or >6 mm sulci. All gingiva adjacent to > or = 4 mm sulci had clinical evidence of active inflammation. Factorial analysis of variance suggested significant effects of sulcus depth on the type and concentration of the three cytokines in the adjacent gingiva (P < 0.001). IL-8 concentrations were highest in gingiva adjacent to < or = 3 and lowest adjacent to >6 mm sulci (P < 0.001). In contrast, IL-6 concentrations were lowest in gingiva adjacent to < or = 6 mm and highest adjacent to >6 mm sites. IL-1beta concentrations were highest in gingiva adjacent to >6 mm and lowest adjacent to 4 to 6 mm sites; they were also higher adjacent to < or = 3 mm than adjacent to 4 to 6 mm sites (P < 0.01). Multiple regression analysis suggested that sulcular depth, type of cytokine, and cytokine concentration were significantly correlated (P < 0.001). Ratios of gingival cytokines changed with increased sulcular depth. In gingiva adjacent to < or = 6 mm sites, IL-8 was the most and IL-6 the least prevalent. In gingiva adjacent to > or = 6 mm sites, IL-8 was the least and IL-1-beta the most prevalent. The data suggest that the characteristics of the gingival cytokine network are affected by adjacent sulcular depth. These data could be used to design adjunct diagnostic tests for progression of periodontal diseases.

Factors for progression of periodontal diseases.

Progression factors for periodontal diseases have been suggested by in vitro study of peripheral blood and gingival cells; however, those factors are not established in vivo. This investigation assessed biopsies of three groups of gingival tissues: those adjacent to a 1) < or =3 mm (normal), 2) 4-6 mm, and 3) >6 mm gingival sulcus, to determine changes in the gingival microenvironment coincident to the progression of periodontal disease. Superoxide dismutase (SOD) and catalase activity, and IL-12 and bcl-2 levels, were decreased at >6 mm; total protein and IL-6 concentrations were increased adjacent to >6 mm, as compared to < or =3 and 4-6 mm, sites. Apoptotic cells were evident only within gingiva adjacent to >6 mm sites. These data suggest that IL-12 is an important factor in the shift from a TH1 to TH2 cell profile and that a favorable gingival microenvironment for hyperinflammation may develop coincident to progression of periodontal diseases due to decreased bcl-2 and increased IL-6 concentrations within gingiva. These changes in the gingival microenvironment could impair apoptosis and promote enhanced release of reactive oxygen species (ROS) by phagocytes; decreased catalase and SOD activity could promote accumulation of ROS and result in additional tissue destruction.

Synovial tissues collected from rheumatoid patients undergoing total joint arthroplasty express markers for acute inflammation.

Rheumatoid arthritis is an autoimmune disease that causes inflammation mainly in synovial tissues. RA manifests as a chronic polyarthritis with intermittent acute inflammatory episodes. The inflammatory sites are characterized by infiltration of activated lymphocytes and macrophages into the synovial membrane, and the proliferation of synovial cells. The local production of a number of cytokines by proliferative synovial cells as well as by infiltrating cells appears to account for many of the pathological and clinical manifestations in rheumatoid arthritis. Tissues were collected from twelve RA patients undergoing joint replacement surgery. The synovium was collected and the cell types were identified, and markers for chronic and acute inflammatory mediators were measured. The cells types found in the synovium are capable of secreting cytokines which are capable of both acute inflammation (IL-1, IL-6, IL-8, MCP-1 and TNF), as well as chronic inflammation (IL-2, IL-10, and IL-4). The results obtained showed that the macrophages-derived acute inflammatory cytokines (IL-1, IL-6 and IL-8) were easily detected at levels of 22.6 +/- 12 pg/mg protein; 48.5 +/- 42 pg/mg protein, and 76 +/- 31 pg/mg protein; respectively. T-cell derived chronic inflammation cytokines (IL-2, IL-4 and IL-10) were rarely detected. Retrieved tissues that immunostained positive for IL-6, IL-1 and IL-8 also is suggestive of an acute inflammatory response. The results clearly demonstrate that the acute response may be responsible for the subsequent need for joint arthroplasties.

Site-specific variations in metabolism by human fibroblasts exposed to nifedipine in vitro.

Nifedipine induces overgrowth of gingival tissues in some patients. However, other collagenous tissues in their body do not overgrow. The purpose of this study was to compare effects of serial dilutions of nifedipine on the in vitro metabolism of fibroblasts derived from normal gingiva, nifedipine-induced hyperplastic gingiva, knee capsular ligament, and dermis. The data suggested that nifedipine affects the metabolism of fibroblasts derived not only from gingiva, but also from other collective connective tissues. Thus, nifedipine-responder cells are present in tissues other than gingiva. There was an inverse relationship between in vivo tissue levels of IL-1-beta and in vitro responsiveness to nifedipine of fibroblasts derived from that tissue. Nifedipine-induced overgrowth of connective tissues, other than gingiva, probably does not occur either because of the relatively slow rate of collagenous protein synthesis by resident fibroblasts or because of alterations in collagen deposition/resorption within susceptible tissues produced by nifedipine on collagenase synthesis.

Biochemical and histological analysis of the flexor tenosynovium in patients with carpal tunnel syndrome.

Carpal tunnel release is the most common hand operation performed in this country. In the absence of specific systemic diseases, the etiology and persistence of pain and dysfunction even after surgical decompression is poorly understood. The focus of this investigation was to investigate the biological factors present within the patients serum that may lead to increased sensitivity to pain. Tissue was collected from patients during surgery. The tissue was homogenized and the homogenate analyzed for the presence of IL-1, IL-6, prostaglandin E series (PGE2). The levels were compared with volunteers that had no evidence of carpal tunnel syndrome or pain. The results showed similar levels of IL-1 (range 42-26 ng/ml) in tissue homogenates, and a significant increase in levels of IL-6 and malionaldehyde bis-(diethyl acetal) in CTS patients in comparison to control tissues. This increase may be associated with oxidative changes occurring as a result of ischemia and reperfusion. Tissue homogenates were also evaluated for PGE2. The CTS tissues showed a five fold elevation in PGE2 compared to control tissues. Levels of PGE2 in CTS tissues were statistically different using a two-tailed student T-test. Increased levels of PGE2 can enhance vascular permeability at the site of injury, and can play an important role in activating adenylate cyclase which increases intracellular cyclic adenosine monophosphate (cAMP). This increase in cAMP levels can inhibit functional responses to other inflammatory stimuli. Increases in PGE2 can also cause sensitization of the nerve endings so that a normal stimulus that would not necessarily cause pain will now be experienced as painful. The results of this study demonstrate that arachidonic acid metabolites PGE2 may be responsible for both the pathological changes and clinical symptomatology in carpal tunnel syndrome.

Biochemical and immunochemical evaluation of tissues and synovial fluid from patients undergoing total joint arthroplasty.

Cytokines are inflammatory mediators responsible for numerous clinical conditions, and are thought to lead to the resorption of bone. Understanding the nature of the cells producing these factors which control the resorption of bone will ultimately lead to a better understanding of why implants fail or integrate. In this study, synovial tissues and synovial fluids were processed for biochemical as well as histochemical and immunohistochemical determination cytokines responsible for bone resorption. The results from this study showed by both quantitative enzyme linked immunoassay (ELISA) and qualitatively by immunohistology a marked increase (twofold) in interleukin-1 (IL-1), and tumor necrosis factor-beta (TNF beta) in synovial tissues in comparison to control tissues of cartilage, ligament and meniscus. Evaluation of tissues both immunochemically and by Hematoxylin and Eosin demonstrated the presence of fibroblast and cells such as macrophages, and multinucleated giant cells in the synovium that are capable of producing bone resorption. Synovial fluid from primary and revision patients were evaluated for TNF beta and IL-1 were not statistically different. Overall, the results indicate that the inflammatory cells of the synovium are secreting factors which may act to mediate aseptic loosening of implants.

Case conference: complete heart block in a young man.

A previously healthy 32-year-old man presented to the ED in complete heart block. Ischemic, infectious, and inflammatory conditions were considered in the differential diagnosis. Management options for complete heart block, the etiology of heart block in young adults, and treatment guidelines are reviewed.

Quantitative analysis of the effect of surface impregnation with biomedical polymers on the proliferation rate of HeLa cells in culture.

The use of biomaterials in biological system was extensively conducted in an in vivo environments. This method limits the evaluation and imposes an obstacle for quantitative analysis of several parameters. The development of tissue culture techniques to evaluate the bioactivity of potential organic compounds alleviated these problems and provided a reliable prediction regarding the biocompatibility of biomaterials when compared with parallel animal testing. The objective of this study is to investigate the effect of various biomedical polymers on the adhesion rate of transformed HeLa cells as a model. The HeLa cells used in this study seeded by following our standard laboratory procedure. A total of 1.5 x 10(5) cells were plated in each of the pretreated wells with various concentrations of (0.01 0.1 and 1% wt/vol) polyvaline (P-Val), polyalanine (P-Ala), polytryptophan (P-Trp), polyasparagine (P-Asn), polyaspartate (P-Asp), poly glycine (P-Gly), and buffered control. At the end of 1,4, and 24 hours the cell viability was determined by hexoamindase activity. The data obtained from this study suggest that (I) the ease of adhesion of HeLa cells were in the following order: P-Val = P-Ala > P-Gly = P-Trp = P-Asp = P-Asn > Control, (II) the rate of HeLa cells spreading was strongly influenced by both incubation time and the polymer concentration, and (III) the surface attachment of HeLa to the polymer were demonstrated to vary depending on their chemical structure and level of microporosity. Thus, overall observation led us to conclude that the surface reactivity of polymer materials be always taken into account in discussing their biocompatibility in vivo.

Identification of a gene essential for piliation in Haemophilus influenzae type b with homology to the pilus assembly platform genes of gram-negative bacteria.

Haemophilus influenzae type b (Hib) pili are complex filamentous surface structures consisting predominantly of pilin protein subunits. The gene encoding the major pilin protein subunit of Hib adherence pili has been cloned and its nucleotide sequence has been determined. In order to identify specific accessory genes involved in pilus expression and assembly, we constructed isogenic Hib mutants containing insertional chromosomal mutations in the DNA flanking the pilin structural gene. These mutants were screened for pilin production, pilus expression, and hemagglutination. Pili and pilin production were assessed by immunoassays with polyclonal antisera specific for pilin and pili of Hib strain Eagan. Hemagglutination was semiquantitatively evaluated in a microtiter plate assay. Six Hib mutants produced proteins immunoreactive with antipilin antiserum but no longer produced structures reactive with antipilus antiserum. In addition, the mutants were unable to agglutinate human erythrocytes. Nucleotide sequence analysis localized the insertion sites in the six mutants to 2.5-kb open reading frame upstream of the pilin structural gene and immediately downstream of an Hib pilin chaperone gene. The amino acid sequence encoded by this open reading frame has significant homology to members of the pilus assembly platform protein family, including FhaA of Bordetella pertussis, MrkC of Klebsiella pneumoniae, and the Escherichia coli assembly platform proteins FimD and PapC. This open reading frame, designated hifC, appears to represent a gene essential to Hib pilus biogenesis that has genetic and functional similarity to the pilus platform assembly genes of other gram-negative rods.

The role of different biomaterials on adhesion of nontransformed buccal epithelial cells.

The specific objective of this study was to investigate the effect of various biomedical polymers on the adhesion rate of buccal epithelial cells (BEC) as a model for biocompatibility. The BEC in this study were isolated, biotin labeled and seeded by following standard laboratory procedures. A total of 2.5 x 10(5) cells was plated in each microtiter-well pretreated with various concentrations of (0.01, 0.10 and 1% wt/vol) poly-L-valanine (P-Val), poly-L-alanine (P-Ala), poly-glycine (P-Gly), poly-L-tryptophan (P-Trp), poly-L-asparagine (P-Asn) and buffered control. At the end of 1, 4, and 24 hours the assay was developed by utilizing avidin-horseradish peroxidase and o-phenylenediamine dihydrochloride as substrate to measure biotin-labeled-BEC. Cell number was then determined using a standard curve prepared by using known BEC number vs. absorbance units. The data from this study suggest that: (I) the ease of adhesion of BEC was in the following order: P-Val > P-Gly = P-Trp = P-Ala > P-Asn > P-Asp = Control, (II) the rate of BEC spreading was strongly influenced by both incubation time and the polymer concentration, (III) the surface attachment of BEC to the polymer were demonstrated to vary depending on their chemical structure and level of microporosity. Thus, overall observation led us to conclude that the surface reactivity of polymer materials must always be taken into account in discussing their biocompatibility in vivo.

Differential requirements for cellular cytoskeleton in human macrophage complement receptor- and Fc receptor-mediated phagocytosis.

We investigated the requirement for cellular cytoskeleton in CR- and FcR-mediated phagocytosis by human monocyte-derived macrophages (M phi). Inhibition of actin microfilament (MF) assembly and stability by cytochalasins B and D completely inhibited M phi phagocytosis of sheep E coated with C3b (EC3b), iC3b (EC3bi), and IgG (EIgG) via CR1, CR3, and FcR, respectively. Ligand-binding to either CR or FcR was not effected by cytochalasins. Nocodazole (NOC), which prevents microtubule (MT) polymerization, and taxol, which causes random polymerization of MT inhibited M phi phagocytosis of EC3b(i) but not EIgG. However, the combination of taxol (5 x 10(-4) M) and NOC (2 x 10(-6) M) augmented M phi CR-mediated phagocytosis. In addition, agents known to increase intracellular cGMP augmented phagocytosis of EC3b(i). Conversely, agents that increase intracellular cAMP inhibited CR-mediated phagocytosis. These agents had no effect on FcR-mediated phagocytosis, and did not effect ligand-binding to CR or FcR. PMA markedly enhanced CR- but not FcR-mediated phagocytosis, and augmentation of CR-mediated phagocytosis by PMA was inhibited by both CD and NOC. In contrast, the synthetic diacylglycerol, 1-oleoyl-2-acetoyl-sn-3-glycerol augmented, and inhibitors of protein kinase C inhibited M phi phagocytosis via CR and FcR. These data indicate that for adherently cultured human M phi: 1) binding of ligand-coated E to CR or FcR does not require an intact cytoskeleton; 2) intact actin microfilament are required for phagocytosis via CR and FcR; 3) phagocytosis via CR1 and CR3 but not FcR is dependent on MT assembly; 4) PMA most likely augments CR-mediated phagocytosis through promotion of MT assembly; and 5) PKC activity is involved in the phagocytic signal generated by both CR and FcR.

A colorimetric assay for measuring the lysis of a plasma clot.

The present study describes a simple, quantitative assay for measuring the lysis of a plasma clot. The principle of the assay is based on the release of Coomassie brilliant blue R-250 dye from the clot. Thirty microliters of freshly prepared Coomassie brilliant blue R-250 (1 mg/ml) was added to 200 microliters of diluted human plasma (1:5). After mixing, 100 microliters of thrombin (2.5 NIH units/ml) were added to mediate a plasma clot. One milliliter of streptokinase (0.1 mg/ml) was used as a plasminogen activator to initiate clot lysis. During the course of lysis, 100 microliters of soluble material were transferred to microtiter wells and the absorbance at 540 nm was determined as a measure of clot lysis. This assay was used to measure clot lysis in 18 human plasma samples. The colorimetric method (X) developed in this report correlated well with that determined using a conventional 125I-fibrinogen method (Y): Y = 0.83X + 7.98 (r = 0.91).

Regulation of human monocyte/macrophage function by extracellular matrix. Adherence of monocytes to collagen matrices enhances phagocytosis of opsonized bacteria by activation of complement receptors and enhancement of Fc receptor function.

In inflammation monocytes emigrate from the peripheral circulation into an extravascular area rich in extracellular matrix proteins. In this milieu, phagocytes ingest and kill invading pathogens. In the present studies, we found that monocytes adhered to type I collagen gels phagocytized 2.5-12-fold more opsonized Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae than plastic-adherent monocytes. The rate of phagocytosis and the number of bacteria ingested by collagen-adherent monocytes was equal to, or greater than, the number of bacteria ingested by 7-d cultured macrophages (M phi). Although both collagen- and plastic-adherent monocytes were bactericidal for E. coli and S. aureus, more bacteria were killed by collagen-adherent monocytes by virtue of their enhanced phagocytic capacity. Cultured M phi only were bacteriostatic. Adherence of monocytes to collagen gels activated C receptors (CR) types 1 and 3 for phagocytosis, and enhanced Fc receptor (FcR)-mediated phagocytosis. Collagen- and plastic-adherent monocytes produced equivalent amounts of superoxide anion in response to phorbol myristate acetate and opsonized zymosan. Thus, the enhanced phagocytosis and killing of opsonized bacteria by collagen-adherent monocytes appear to be by regulation of the function of membrane CR and FcR, without apparent enhancement of the respiratory burst. These data suggest that adherence of monocytes to the extracellular matrix during inflammation may rapidly activate these cells for enhanced phagocytic bactericidal activity.

Lipoprotein(a) enhances plasma clot lysis in vitro.

Human plasma lipoprotein(a) (Lp(a)) is a macromolecular complex that contains a protein homologous to plasminogen, the precursor of plasmin. We confirmed recent reports that Lp(a) is not activated by streptokinase or tissue plasminogen activator (t-PA) to yield plasmin-like activity. In testing the hypothesis that Lp(a) can competitively inhibit plasma clot lysis mediated by plasmin, the present study shows that Lp(a) significantly enhanced plasma clot lysis mediated by streptokinase or t-PA. The enhancement, however, was not observed in a plasmin-mediated clot lysis using a purified fibrinogen system. The addition of alpha-2-antiplasmin (alpha 2-plasmin inhibitor) to this system inhibited fibrinolysis; however, no inhibition was observed in the presence of Lp(a). One potential explanation for the Lp(a)-enhanced plasma clot lysis is that Lp(a) neutralizes the activity of alpha 2-antiplasmin present in plasma, thereby restoring the activity of plasmin to lyse the clot.

Leaf Carbon Metabolism and Metabolite Levels during a Period of Sinusoidal Light.

Photosynthesis rate, internal CO(2) concentration, starch, sucrose, and metabolite levels were measured in leaves of sugar beet (Beta vulgaris L.) during a 14-h period of sinusoidal light, which simulated a natural light period. Photosynthesis rate closely followed increasing and decreasing light level. Chloroplast metabolite levels changed in a manner indicating differential activation of enzymes at different light levels. Starch levels declined during the first and last 2 hours of the photoperiod, but increased when photosynthesis rate was greater than 50% of maximal. Sucrose and sucrose phosphate synthase levels were constant during the photoperiod, which is consistent with a relatively steady rate of sucrose synthesis during the day as observed previously (BR Fondy et al. [1989] Plant Physiol 89: 396-402). When starch was being degraded, glucose 1-phosphate level was high and there was a large amount of glucose 6-phosphate above that in equilibrium with fructose 6-phosphate, while fructose 6-phosphate and triose-phosphate levels were very low. Likewise, the regulatory metabolite, fructose, 2,6-bisphosphate was high, indicating that little carbon could move to sucrose from starch by the triose-phosphate pathway. These data cast doubt upon the feasibility of significant carbon flow through the triose-phosphate pathway during starch degradation and support the need for an additional pathway for mobilizing starch carbon to sucrose.

A Method for Calculating Sucrose Synthesis Rates throughout a Light Period in Sugar Beet Leaves.

Sucrose synthesis rate in an exporting sugar beet (Beta vulgaris L.) leaf was calculated from simultaneous measurements of export and changes in leaf sucrose level. The amount of recently fixed carbon exported was determined from net carbon assimilated minus the tracer carbon accumulated in the leaf. The relative amount of (14)C accumulated in the leaf supplied with (14)CO(2) throughout an entire light period was recorded continuously with a Geiger-Mueller detector. To produce a continuous time course for tracer carbon accumulated in the leaf during the light period, the latter curve was superimposed on values for tracer carbon accumulated in leaves sampled at hourly intervals. Validity of the method requires that nearly all of the carbon that is exported be sucrose and that nearly all of the sucrose that is synthesized be either exported or accumulated as sucrose in the exporting leaves. These conditions appeared to be fulfilled in the situations where the method was applied. The method was used to study the effect of increasing atmospheric CO(2) concentration on the rate of sucrose synthesis. Further, the method can be used in conjunction with the gathering of other data such as gas exchange, metabolite levels, and enzyme activities in a set of leaves of a similar age on the same plant. This assemblage of data was found to be useful for understanding how rates of photosynthesis, sucrose synthesis, and translocation are regulated in relation to each other in an intact plant.