Non-linear dynamics of human periodic breathing and implications for sleep apnea therapy.

A mathematical model of non-obstructive human periodic breathing (Cheyne-Stokes respiration) or central sleep apnea (CSA) is described which focused on explaining recently reported non-linear behavior. Evidence was presented that CHF (chronic heart failure)-CSA and ICSA (idiopathic central sleep apnea) both involved limit cycle oscillations. The validity of applying linear control theory for stabilization must then be re-examined. Critical threshold values and ranges of parameters were predicted which caused a change (bifurcation) from limit cycle periodic breathing to stable breathing. Changes in lung volume were predicted to form a bifurcation during CHF-CSA where stability and instability can involve a lung volume change as small as 0.1 l. CSA therapy based on reducing control loop gain was predicted to be relatively ineffective during stable limit cycle oscillation. The relative ratios of durations of ventilation to apnea (T(v)/T(a)) during periodic breathing were primarily determined by peripheral chemoreceptor dynamics during crescendo, de-crescendo, and apnea phases of CSA.

Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis.

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-gamma in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-gamma. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-gamma upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-gamma only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.

[Spinal cord protection during thoracoabdominal aortic aneurysm repair; efficacy of distal aortic perfusion and segmental aortic clamping].

Despite improvement in adjuncts for thoracoabdominal aortic aneurysms (TAAA) repairs, many devastating complications remains after the surgery. Our experience with these aneurysms has been reviewed in order to identify those methods at risk of major morbidity, as well as which further improvements required. During last 16 years, 53 consecutive patients were operated on TAAA. The mean age was 58 years. Twenty patients had dissecting aneurysms and 13 patients had had prior aortic surgery. A femoro-femoral bypass was used to maintain distal aortic perfusion in most patients. Reimplantation of intercostal or lumbar arteries under the multi-segmental aortic clamping is consistent in our technique. Motor evoked potentials (MEP) were measured to monitor spinal cord protection since 2000. The hospital mortality was 9.4% (5/53), 22.2% (2/9) for emergency operation and 15.4% (2/13) for patients with prior aortic surgery. The mortality for the first and elective operations was 3.2% (1/31). No any neurologic dysfunction was observed in all patients including the hospital deaths. In view of clinical results, our adjuncts and techniques are useful for prevention of spinal cord ischemia during the TAAA surgery.

Ultrastructural and immunologic characteristics of mouse x cattle xenogeneic hybridomas originating from bovine leukemia virus-infected cattle.

Nine percent of xenogeneic hybridomas originating from a bovine leukemia virus (BLV)-infected cow secreted monoclonal IgM antibodies with multispecific reactivity. Similar reactivity was evident in some antibodies with an unusually long (> 50 amino acids) third complementarity-determining region of the heavy chain. Electron microscopy of hybridomas demonstrated the presence of c-type virus particles consistent with polymerase chain reaction detection of BLV env gene. Some hybridomas contained dilated rough endoplasmic reticulum and cisternae filled with moderately electron-dense granular substance compatible with plasma cells at presecretory stage. The number of chromosomes in xenogeneic hybridomas corresponded to the sum total of mouse and bovine chromosomes. None of the hybridomas showed polyploidy. The immunochemical and genetic analysis of stable bovine immunoglobulin-secreting xenogeneic hybridomas confirms that BLV infection causes polyclonal B cell activation regardless of antigen specificity. Presence of c-type particles in hybridomas suggests that T cell-derived cytokines are not required for sustained BLV expression.

Role of tetrahydrobiopterin on ischemia-reperfusion injury in isolated perfused rat hearts.

AIM: It has recently been shown that nitric oxide synthase in the presence of suboptimal levels of tetrahydrobiopterin (BH(4)), an essential cofactor of nitric oxide synthase, may favor increased production of oxygen free radicals. This study was designed to define the role of BH(4) in myocardial ischemia-reperfusion injury. METHODS: Isolated perfused rat hearts were subjected to 37 degrees C ischemia and reperfusion. Hearts were received with BH(4) or vehicle for 5 min just before ischemia and during the first 5 min of the reperfusion period. The effects of BH(4) on left ventricular function, myocardial contents of lipid peroxidation and high energy phosphates, and levels of lactate dehydrogenase and nitrite plus nitrate in perfusate before ischemia and after reperfusion were estimated. Moreover, the effect of BH(4) given with 2,4-diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of BH(4) production, intraperitoneally 24 h before the experiments were estimated. RESULTS: BH(4) improved contractile and metabolic abnormalities in reperfused hearts. Furthermore, BH(4) significantly alleviated ischemic contracture during ischemia, and restored diminished perfusate levels of nitrite plus nitrate after reperfusion. On the other hand, DAHP-treatment aggravated ischemia-reperfusion induced functional and metabolic abnormalities. Administration of BH(4) improved DAHP-induced functional and metabolic abnormalities. CONCLUSION: Results demonstrated that BH(4) lessens ischemia-reperfusion injury in isolated perfused rat hearts. Conversely, deficiency of BH(4) seems to accelerate endothelial dysfunction and myocardial ischemia-reperfusion injury. Present data may be compatible with the hypothesis that nitric oxide synthase in the presence of insufficiency of BH(4) serve as the cause of oxidative injury.

Blood distribution to the anterior spinal artery from each segment of intercostal and lumbar arteries.

AIM: Prevention of paraplegia, a serious complication of surgery for thoracoabdominal aortic aneurysm, has been well documented. However no assured prophylaxis against this complication has yet been found. Spinal ischemia is believed to be the major cause of paraplegia. We conducted an experimental study to define the development of paraplegia with regard to the blood supply to the spinal cord. METHODS: A porcine model was used to evaluate blood distribution to the anterior spinal artery. Colored silastic agent was selectively injected into the intercostal and lumbar arteries, and distribution to the anterior spinal artery was evaluated on 50 animals. The intercostal and lumbar arteries were ligated in the segments where the blood supply to the anterior spinal artery would be interrupted. Whether or not paraplegia developed was checked 2 days later. RESULTS: Colored silastic agent arrived at the anterior spinal artery from all segments of the 8th intercostal to 4th lumbar arteries. Two of 9 pigs (22.2%) that underwent ligation of the segments from the 9th intercostal to 2(nd) lumbar artery suffered paraplegia. In 3 non-paraplegic pigs, colored silastic agent injected into the preserved arteries was found to have covered a wider range. CONCLUSION: All the intercostal and lumbar arteries supplied blood to the anterior spinal artery. When large segments of intercostal and lumbar arteries were ligated, the blood flow from the preserved segments acquired increased dominance. The possibility exists that any intercostal and lumbar artery can supply blood to the spinal cord and become collateral circulation to the anterior spinal artery.

[Impact of multi-segmental aortic clamping and distal aortic perfusion on the prevention of postoperative paraplegia during thoracoabdominal aortic graft replacement]. / Multisegmentale Aortenabklemmung und distale Aortenperfusion zur Verhinderung der postoperativen Paraplegie bei thorakoabdomineller Aortenprothesenimplantation.

OBJECTIVE: We present the impact of multisegmental aortic clamping under distal aortic perfusion and segmental artery reimplantation on the prevention of postoperative paraplegia during thoracoabdominal aortic graft replacement. PATIENTS: During the last 14 years in 47 patients (age range: 22 to 82 years; average: 57,9 +/- 13,2 years; 16 females and 31 males) with thoracoabdominal aortic aneurysm a graft replacement was performed with adjuncts of normothermic partial bypass and multisegmental aortic clamping. As many patent segmental arteries as possible were reimplanted. RESULTS: Five patients died during hospitalization, for an in-hospital mortality rate of 10,6 %. In the elective patients (n = 40), the hospital mortality rate was 7,5 %. The average number of segmental aortic clampings per patient was 2,83 +/- 1,19 times. In 39 patients (82,9 %), 117 segmental arteries were reimplanted or preserved by beveled anastomosis. Eighty-three out of 117 segmental arteries (70,9 %) were located between TH9 and L2. Postoperative paraplegia/paraparesis did not occur in any patient. CONCLUSION: In view of our results reimplantation of as many segmental arteries as possible under multisegmental aortic clamping with adequate distal aortic perfusion can be recommended for effective prevention of spinal cord ischemia in TAAA surgery.

Histopathological prognostic factors of adult granulosa cell tumors of the ovary.

BACKGROUND: The prognostic factors of adult granulosa cell tumor (AGCT) have not been well defined. METHODS: In 27 AGCT patients, we examined clinical stage, microscopic patterns, mitotic index (MI), and lymph-vascular space invasion (LVSI) to determine whether these factors were related to disease-free survival (DFS) of patients with AGCT. We also performed immunohistochemical examination for p53. RESULTS: Seventeen cases represented stage I tumors, four stage II, five stage III, and one stage IV. Patients with stage I disease had more favorable prognosis than those with stage II to IV disease (p=0.034). There was no relation between the microscopic patterns and the DFS. The MI, which was categorized into < or =3/10 high power field (HPF) and > or =4/10 HPF, was significantly related to patients DFS (p<0.0005). The DFS time for patients with moderate or prominent LVSI was significantly shorter than that for patients with no or minimal LVSI (p<0.0001). By multivariate analysis, MI and LVSI were shown to be independent prognostic factors. Five of seven patients with recurrent tumor had extrapelvic spread; two in the abdominal cavity and three in the liver. CONCLUSION: The results of this study suggest that prognosis for patients with AGCT depends on the MI and LVSI. During the follow-up period of patients, they need to be examined for distant metastasis including liver.

Discoidin domain receptor 1 isoform-a (DDR1alpha) promotes migration of leukocytes in three-dimensional collagen lattices.

Although integrins are crucial for migration of leukocytes through endothelium, integrin-independent mechanisms appear to take over and mediate the migration of leukocytes through extracellular matrix (ECM) in a three-dimensional tissue microenvironment. Discoidin domain receptor (DDR) 1 is a receptor tyrosine kinase activated by collagen, the most abundant ECM protein. In the present study, we detected that peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophils were induced to express DDR1 after incubation in RPMI 1640. The expression level of DDR1 in PBMC was increased further by stimulation with tumor necrosis factor-alpha, interleukin-1beta, granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, or phytohemagglutinin, but not with interferon-gamma. In vivo, DDR1 mRNA was detectable in mononuclear leukocytes infiltrating human renal tumor tissue. Among three DDR1 isoforms, DDR1alpha was the major transcript in leukocytes. Functionally, overexpression of either DDR1alpha or DDR1beta in THP-1 cells resulted in increased adherence to collagen-coated plates in a beta1-integrin independent manner. However, only DDR1alpha-, but not DDR1beta-, overexpressing cells exhibited marked pseudopod extension and migrated successfully through three-dimensional collagen lattices. Consequently, we propose that the interaction of DDR1alpha with collagen of the ECM results in a requisite intracellular signaling that enables leukocytes to migrate in a tissue microenvironment and participate in host defense.

The gelsolin/fragmin family protein identified in the higher plant Mimosa pudica.

Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)-dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca(2+)-dependent manner.

One-stage thoracic aortic aneurysm treatment and coronary artery bypass grafting.

OBJECTIVE: The treatment of thoracic aortic aneurysm accompanied by ischemic heart disease presents a surgical challenge and has up to now shown a high hospital mortality rate. This report discusses the factors contributing to improved results in these cases. METHODS: We conducted a retrospective analysis of the records of 24 consecutive patients who had undergone replacement of thoracic aorta with combined coronary artery bypass grafting (CABG) between May 1991 and October 1998. Fifteen patients received total arch replacement (Arch-with-CABG Group), and the other 9 patients received the Bentall operation (Bentall-with-CABG Group). These results were compared with those patients who had undergone replacement of the thoracic aorta without CABG (Without-CABG Group). RESULTS: In the combined CABG groups, the overall operative mortality rate was 16.7%. In comparison with the Arch-without-CABG Group, there was a significantly longer cardiopulmonary bypass time and longer selective cerebral perfusion time in the Arch-with-CABG Group. However, no significant difference was observed in postoperative complications between the two groups. In addition, there was no significant difference in either actuarial survival or the cardiac-event-free rate at 5 years between the replacement of thoracic aorta with- and without-CABG Groups (83.1% vs. 90.4%, and 78.5% vs. 77.7%, respectively). No reoperation and no late death were observed during the follow-up period (mean 21.3 months). CONCLUSIONS: We concluded that replacement of the thoracic aorta combined with CABG can be carried out safely, and that revascularization for coronary artery disease is useful for preventing any occurrence of cardiac event.

Phenotypic and functional change of cytokine-activated neutrophils: inflammatory neutrophils are heterogeneous and enhance adaptive immune responses.

Polymorphonuclear leukocytes (PMN) are the most abundant leukocytes, comprising about two-thirds of peripheral blood leukocytes, and play major roles in innate immunity. In addition, PMN play critical roles in the development of adaptive immunity. Recently, defensins and other peptides pre-stored in PMN granules were shown to attract monocytes, dendritic cells, and T cells, leading to the hypothesis that the release of PMN granular peptides may link innate and adaptive immunity. During the past several years, we have focused on an alternative hypothesis that activated PMN further differentiate and acquire new phenotypes and functions that enable them to link the two responses. To test our hypothesis, we have taken local and global approaches and have shown several key findings that support the hypothesis. The findings include the requirement for priming PMN by cytokines to induce the delayed expression of MCP-1/CCL2, a signal for mononuclear cells, and the expression of new cell-surface markers by such cytokine-activated PMN. In the present manuscript, we focus on the phenotypic and functional changes that occur during PMN activation with selected cytokines. The results of our study indicate that inflammatory PMN are heterogeneous and play roles in not only innate but also adaptive immunity in response to stimuli released in injured tissues.

Effect of 1-week treatment with erythropoietin on the vascular endothelial function in anaesthetized rabbits.

Chronic administration of erythropoietin (EPO) is often associated with hypertension in animals and humans. The aim of this study was to estimate whether 1-week treatment with EPO can affect the vascular endothelial function. Rabbits were given with EPO (400 iu kg(-1) s.c.) or saline each other day for 1 week. Hypotensive responses to intravenously given acetylcholine (ACh), endothelium-independent nitric oxide donors (NOC7, nitroprusside and nitroglycerin) and prostaglandin I2 were tested before and after administration of N(G)-nitro-L-arginine methyl ester (L-NAME), a specific nitric oxide synthase inhibitor, under pentobarbitone anaesthesia. Blood haemoglobin concentration in EPO group was significantly higher than that in control group, whereas baseline values of aortic pressure, heart rate and femoral vascular resistance were similar. The dose of ACh (172 ng kg(-1)) requiring for a 15 mmHg hypotension from the baseline in EPO group was apparently higher than that (55 ng kg(-1)) in control group. On the contrary, hypotensive responses to NOC7, nitroprusside, nitroglycerin and prostaglandin I2 were comparable between two groups. The extent of ACh-induced hypotension did not correlate with haemoglobin concentration. L-NAME significantly inhibited the ACh-induced vasodilating response in control group but did not in EPO group. In another set of rabbits, the same treatment with EPO also decreased vasodilating responses to carbachol, bradykinin and substance P besides ACh as compared with control group. These results indicate that 1-week treatment with EPO selectively attenuates depressor responses to endothelium-dependent vasodilators in anaesthetized rabbits, most likely due to inhibition of endothelial nitric oxide synthase.

Mutant Caldesmon lacking cdc2 phosphorylation sites delays M-phase entry and inhibits cytokinesis.

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division of Xenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia.

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.

Management of the heart rate during coronary artery bypass grafting on the beating heart: newly devised methods of decreasing heart rate--a preliminary report-.

BACKGROUND: To develop new methods for achieving bradycardia, we studied the feasibility of producing transient, reversible bradycardia with atrial stimulation and cooling of the sinoatrial node. METHODS: In an animal study, the atrium was stimulated electrically during the refractory period of the atrioventricular node. Alternatively, an area of the sinoatrial node was cooled regionally. The two methods were also performed in combination. In a clinical study, atrial stimulation was applied in seven consecutive patients who underwent coronary artery bypass grafting (CABG). RESULTS: In the animal study, atrial stimulation was effective only when 2 mg/kg of diltiazem was administered. Such atrial stimulation decreased heart rate (beats/minute) from 95.8+/-16.9 to 64.2+/-20.0 (the average reduction from the control value 66.1+/-10.3%). Cooling the sinoatrial node decreased heart rate, and was effective with or without administration of diltiazem. Heart rate was decreased from 156.6 31.7 to 110.7+/-21.7 (average reduction from control value 71.3+/-9.2%) before using diltiazem and from 102.0+/-11.9 to 63.5+/-13.9 (average reduction from control value 62.0+/-10.4%) after administration of diltiazem. By combining the two methods, heart rate was decreased from 102.0+/-12.3 to 44.6+/-9.1 (average reduction from control value 43.5+/-6.3%). In our clinical study, the atrial stimulation method was effective. CONCLUSION: Atrial stimulation or regional cooling of the sinoatrial node slowed the heart rate. By combining the two methods, the heart rate was slowed to 40. Clinically, atrial stimulation was effective in CABG patients.

Role of myosin light chain phosphorylation in the regulation of cytokinesis.

Phosphorylation of regulatory light chain (RMLC) of myosin II at Ser19/Thr18 is likely to play important roles in controlling the morphological changes seen during cell division of cultured mammalian cells. Phosphorylation of RMLC regulates the activity of myosin II, an essntial motor for cytokinesis, and phosphorylation of RMLC shows dramatic changes during mitosis. Two exzymes, myosin phosphatase and kinase, control phosphorvlation of RMLC. Myosin phosphatase is activated during mitosis, apparently as a result of mitosis-specific phosphorylation of the myosin phosphatase targeting subunit (MYPT). This activation of myosin phosphatase is likely to result in RMLC dephosphorylation, causing the disassemly of stress fibers and focal adhesions during prophase. The phosphorylation of MYPT is lost in cyotokinesis, which would decrease myosin phosphatase activity. At the same time, ROCK (Rho-kinase) probably phosphorylates MYPT at its inhibitory sites, further decreasing the activity of myosin phosphatase. These changes in MYPT phosphorylation would raise RMLC phosphorylation, leading to the activation of myosin II for cyotokinesis. RMLC phosphorylation is also regulated by several RMLC kinases including ROCK (Rho-kinase), MLCK and citron kinase, all of which are localized at cleavage furrows. Future studies should examine whether these multiple kinases are redundant or whether they control distinct aspects of cell division.

Expression of CCR6 and CD83 by cytokine-activated human neutrophils.

Polymorphonuclear leukocytes (PMNLs) are thought to be terminally differentiated, short-lived, and unable to actively synthesize new proteins or to interact with T cells. In the current study, it was found that PMNLs incubated with supernatants of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-sup) expressed high levels of CCR6 mRNA. Neutralization with IgG against several cytokines revealed that tumor necrosis factor (TNF)-alpha was largely responsible for the PHA-sup-induced CCR6 mRNA expression. Among recombinant cytokines, TNF-alpha induced high levels of CCR6 mRNA expression, whereas interferon (IFN)-gamma induced low levels. The 2 cytokines together exhibited a considerable synergy. Cytokine-activated PMNLs expressed functional CCR6, as detected by the binding of sodium iodide I 125-labeled liver and activation-regulated chemokine (LARC) and dose-dependent migration toward LARC. The induction of CCR6 suggested that these cytokine-activated PMNLs have more similarities with dendritic cells (DCs) that express CCR6 in an immature stage. In fact, the activation of PMNLs with TNF-alpha and IFN-gamma induced the expression of CD83, a dominant cell-surface marker of DCs. When PMNLs were activated with granulocyte macrophage-colony-stimulating factor, TNF-alpha, and IFN-gamma, these cells expressed CD40 and HLA-DR in addition to CD83. Taken together, PMNLs, under appropriate conditions, can undergo a differentiation process characterized by the acquisition of new phenotypes and functions, and such differentiated PMNLs may play more active roles in the adaptive immune response. (Blood. 2000;96:3958-3963)