Secreted Frizzled-Related Protein 4 (SFRP4) is Elevated in Patients with Diabetes Mellitus.

Recently, SFRP4 was identified as a molecular link between islet inflammation and defective insulin secretion. Gene co-expression analysis detected a molecule associated with type 2 diabetes mellitus (T2D), elevated HbA1c, and reduced insulin secretion in mice as well as in a pilot sample of humans. To our knowledge SFRP4 has never been investigated in patients with different types of diabetes. We included 179 patients: 46 with type 1 diabetes (T1D), 30 age matched healthy controls for patients with T1D (CO-T1D), 55 with T2D, 37 with latent autoimmune diabetes of the adult (LADA) and 30 healthy controls (CO) for patients with T2D and LADA. Apart from anthropometric data, lipids and renal parameters were assessed. SFRP4 levels were measured by a commercial ELISA. Patients with diabetes had significant higher SFRP4 levels than CO: T2D vs. CO: 37.1±26.7 vs. 8.8±3.0 ng/ml, p<0.001; LADA vs. CO: 15.6±6.2 vs. 8.7±3.0 ng/ml, p<0.001; T1D vs. CO-T1D: 24.6±17.9 vs. 16.9±4.5 ng/ml, p=0.011. SFRP4 levels were correlated with age, BMI, HbA1c, HDL-cholesterol, and triglycerides. A multivariate model revealed HDL-cholesterol, triglycerides and BMI as predictors for SFRP4. This is the first study demonstrating that SFRP4 is significantly increased in patients with different types of diabetes suggesting that this protein is generally involved in islet dysfunction and potentially subclinical inflammation irrespective of type of diabetes.

Decrease in microvesicle-associated tissue factor activity in morbidly obese patients after bariatric surgery.

BACKGROUND: Tissue factor (TF) is the main in vivo initiator of the blood coagulation cascade. Active circulating TF was detected on small, negatively charged membrane vesicles, the so-called microvesicles (MVs), which are released upon cell activation and apoptosis from a variety of cells. Increased coagulation activation was found in morbidly obese patients, and elevated levels of TF-bearing MVs may contribute to the prothrombotic state in these patients. AIM: To determine MV-associated TF activity levels in morbidly obese patients before and after weight loss due to bariatric surgery. METHODS: MV-TF activity was measured with a factor Xa generation assay in morbidly obese patients before and 2 years after bariatric surgery. In addition, clinical parameters were determined. RESULTS: Seventy-four morbidly obese patients (mean age: 42 (±11) years; 61 females) were included in this study. After bariatric surgery, the body mass index decreased from (median, 25-75th percentile) 45.5 (42.3-50.2) to 30.5 (28.0-34.4 kg m(-2); P<0.001), and a significant improvement in metabolic parameters was observed. Preoperative MV-TF activity correlated with C-reactive protein levels (r=0.3; P=0.02). Postoperatively, the mean MV-TF activity decreased significantly from 0.20 pg ml(-1) (0.18-0.47) to 0.02 (0.00-0.28; P<0.01). CONCLUSION: We could demonstrate a significant decrease in MV-TF activity after weight loss in morbidly obese patients. Decreased MV-TF activity might contribute to an improved coagulation profile in these patients after weight loss.

The soluble form of the receptor of advanced glycation endproducts increases after bariatric surgery in morbid obesity.

OBJECTIVE: The increased cardiovascular (CV) disease risk in patients with morbid obesity (MO) cannot be fully explained by traditional CV risk factors. Activation of the receptor of Advanced Glycation Endproducts (RAGE) leads to inflammation via the NF κß (nuclear factor κß) pathway. The soluble form of RAGE (sRAGE), which is present in plasma, can bind to ligands of RAGE and avoids interaction of RAGE with proinflammatory ligands. We investigated sRAGE levels in patients with MO and compared them with healthy lean controls (CO), before and after bariatric surgery. DESIGN: We conducted a cross-sectional study and a 24-month longitudinal study. SUBJECTS: We included 85 patients (mean age: 41 ± 12 years; mean body mass index (BMI): 45.4 ± 7.9 kg m(-2)) with MO in comparison with 40 CO (mean age: 42 ± 13 years; mean BMI: 26.0 ± 5.5 kg m(-2)). All patients were investigated before and 2 years after bariatric surgery. Apart from weight and CV risk markers (blood pressure, lipids), a glucose tolerance test (75 g), renal and inflammation parameters were assessed. sRAGE levels were assessed by a commercial ELISA. To investigate the associations of the observed reductions of values, delta (Δ) of parameters were calculated. RESULTS: Patients with MO had significant lower sRAGE levels than CO: 1010 ± 514 vs 1501 ± 674 pg ml(-1); P<0.001. In the longitudinal study, sRAGE levels increased significantly after bariatric surgery from 1010 ± 514 to 1261 ± 710 pg ml(-1); P=0.008. In the correlation analysis, ΔsRAGE levels were associated with Δ1-h and Δ2-h postprandial glucose, Δfasting insulin, Δ2-h postprandial insulin, ΔHOMA (homeostatic model assessment)-insulin resistance (ΔHOMA-IR), Δγ-glutamyl transferase and Δtriglycerides. In a multivariate model, Δ1-h and Δ2-h postprandial glucose, Δ2-h postprandial insulin and ΔHOMA-IR predicted ΔsRAGE. CONCLUSION: Patients with MO have significantly lower sRAGE levels compared with non-obese CO, but sRAGE levels increase significantly after weight loss induced by bariatric surgery. As high sRAGE levels inhibit the activation of inflammatory pathways, our results might help understand the beneficial effects of bariatric surgery regarding CV morbidity and mortality.

Thrombin generation in morbid obesity: significant reduction after weight loss.

BACKGROUND: Patients with morbid obesity (MO; body mass index > 40 kg m(-2)) suffer from an increased risk of cardiovascular disease, stroke, venous thromboembolism and all-cause mortality. OBJECTIVES: Because weight loss by bariatric surgery reduces cardiovascular and all-cause mortality, we hypothesized that the plasmatic clotting system might be involved in cardiovascular risk. PATIENTS/METHODS: Thirty-six MO patients [mean age 42 (+/-13) years; 29 female) were investigated before and 2 years after bariatric surgery. Thrombin generation was measured with a commercially available assay (Technothrombin-TGA,Technoclone). Metabolic parameters and parameters of the hemostatic system, such as tissue factor (TF), TF pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1) and prothrombinfragment 1.2 (F1.2), were determined. To investigate associations of changing parameters, deltas were calculated. RESULTS: Metabolic parameters improved with a mean weight loss of 41 (+/-19) kg. Postoperatively, the lag phase was significantly extended compared with preoperative values [median (25th-75th percentile), 7 (4-12) vs. 12 (7-19) min, P = 0.005]. Peak thrombin decreased after weight loss from 345 (232-455) to 282 (111-388) nm (P = 0.015) and the area under the curve from 3962 (3432-5023) to 3227 (2202-4030) nm thrombin (P < 0.001). TF, PAI-1 and F1.2 significantly decreased after weight loss. Analyses of the deltas showed a significant correlation between peak thrombin and total cholesterol (r = 0.50), triglycerides (r = 0.46) and HbA1c (r = 0.55). Moreover, an inverse correlation was found between insulin resistance and the lag phase (r = -0.46). CONCLUSION: Thrombin generation, a marker of the overall coagulation potential, decreased significantly with weight reduction. This might, at least in part, explain the decreased risk of cardiovascular disease after bariatric surgery.

Serum concentrations of retinol-binding protein 4 in women with and without gestational diabetes.

AIMS/HYPOTHESIS: Pregnancy is characterised by temporarily increased insulin resistance. Gestational diabetes occurs when pancreatic beta cell function is unable to compensate for this insulin resistance. Retinol-binding protein 4 (RBP4) could be related to insulin resistance. We hypothesised that RBP4 is elevated in gestational diabetes. METHODS: Serum RBP4, transthyretin and retinol were cross-sectionally measured in 42 women with gestational diabetes and 45 pregnant controls. Of these, 20 women with and 22 without gestational diabetes were included in an additional longitudinal study. RBP4 was determined by enzyme immunometric assay (EIA) and western blot. RESULTS: Women with gestational diabetes had lower RBP4 EIA and western blot levels than controls (median 6.8 [interquartile range, 3.9-14.3] vs 11.3 [7.8-19.9] microg/ml, p < 0.001 and 25.1 [21.7-29.6] vs 26.6 [23.5-32.2] microg/ml, p = 0.026). Transthyretin and the RBP4:transthyretin molar ratio were comparable between the groups. Serum retinol was lower (p < 0.001) and the RBP4 Western blot level: retinol molar ratio was higher in women with gestational diabetes (p = 0.044). RBP4 was not associated with the glucose or homeostasis model assessment of insulin resistance (HOMA-IR), but in gestational diabetes the RBP4:retinol molar ratio correlated with blood glucose and negatively with 2 h post-load insulin. The RBP4:transthyretin ratio correlated with HOMA-IR and fasting insulin in controls. In women with gestational diabetes RBP4 EIA and western blot levels increased after delivery. Retinol increased in both groups, while transthyretin and the RBP4:transthyretin ratio were not altered after parturition. CONCLUSIONS/INTERPRETATION: RBP4 measured by two different techniques is not elevated, but the RBP4:retinol molar ratio is higher and correlates with fasting blood glucose in women with gestational diabetes. Thus, the RBP4:retinol ratio and the RBP4:transthyretin ratio are more informative than RBP4 levels alone when assessing insulin-glucose homeostasis during pregnancy.

[Effects of active and passive movement stimuli on cerebral hemodynamics and the cerebral metabolism]. / Wirkungen kinetischer Stimuli auf die zerebrale Hämodynamik und den zerebralen Stoffwechsel am Beispiel von aktiven und passiv ausgeführten Bewegungen.

INTRODUCTION: In contrast to the well-examined cardiovascular changes during movement stimuli, up to now changes of cerebral hemodynamics and cerebral metabolism have rarely been studied. We investigated the question if active and passive movement stimuli cause changes in the cerebral hemodynamics and the cerebral metabolism. METHOD: Active and passive repetitive movement stimuli on 14 volunteers (8 females, 6 males, age 35 +/- 8 years) were examined. As a parameter of cerebral hemodynamics the mean and the peak blood flow velocity (mCBFV(MCA), pCBFV(MCA)) in the middle cerebral artery (MCA) were recorded by transcranial Doppler sonography. At the same time the noninvasive blood pressure (Penaz method) and the CO(2) expiration concentration were investigated on 8 volunteers of the collective. As cerebral metabolic parameters we examined in 4 volunteers additionally the cerebral respiratory chain enzyme cytochrome aa3 (ccytaa3) and the cerebral oxygen saturation (cHbO(2)) by the transcranial near infrared spectroscopy. With each volunteer 4 measurement series were carried out with a special active and passive exercise program for the right upper as well as the right lower extremity. Each measurement series was formed according to the evoked flow test (R. Aaslid): Exercises were carried out for 20 s, followed by a break of 20 s; this was repeated 10 times for each series. RESULTS: During active exercises of the right lower extremity we found an increase of 13.6% (p < 0.001) of pCBFV(MCA) and an increase of 3.8% (p = 0.003) of mCBFV(MCA). During passive exercises of the lower extremity the increases ran up to 12.3% (p < 0.001) for pCBFV(MCA) and 3.4% (p = 0.004) for mCBFV(MCA). The increases of pCBFV(MCA) came up to 12.5% (p < 0.001) at active exercises of the right upper extremity, those of mCBFV(MCA) to 3.5% (p = 0.15). During passive exercises of the upper extremity the pCBFV(MCA) increased by 12.2% (p < 0.001) and the mCBFV(MCA) by 4.6% (p = 0.007). Significant increases of ccytaa3 were measured during active exercises of the upper extremity (1.6%; p = 0.04) and of the lower extremity (2.7%, p = 0.007). We also found an increase of ccytaa3 during passive exercises of the upper extremity (1.5%, p = 0.04). Significant changes of cHbO(2) were measured with 2.5% (p < 0.05) at active exercises of the lower extremity. CONCLUSION: These studies show that active as well as passive clinical exercises cause an increase of cerebral blood flow velocity. We attribute the increase of cerebral hemodynamics and cerebral metabolism to cerebral activation and autoregulative mechanisms.

Measurement of the individual exposure to 50 and 16 2/3 Hz magnetic fields within the Bavarian population.

This study investigates the individual magnetic field exposures at 16 2/3 and 50 Hz of 1952 people, selected from the Bavarian population. Personal flux density meters ("Field Watcher FW2A") were worn by the participants for 24 h. Every second, the flux density was recorded for both frequencies and for the three spatial axes (dynamic range per axis: several nT up to 100 microT at 50 Hz, 150 microT at 16 2/3 Hz). For 50 Hz fields, the mean of the 1,952 individual means was 0.101 microT and that of the individual medians was 0.047 microT. High level exposures occurred mainly during working hours. Only 2.4% of the subjects showed individual medians higher than 0.2 microT. About 53% of all volunteers were working on the day of recording. Levels for craftsmen (n = 148; mean individual mean: 0.166 microT) were generally higher than those for office workers (n = 624; mean individual mean: 0.107 microT). Flux densities exceeding 100 microT at 50 Hz were measured in 31 persons. The total time with such extreme exposures amounts to nearly 21 min, less than 0.001% of the total time for all measurements (5.3 years). To our knowledge, this is the first exposure study where 16 2/3 Hz magnetic fields (caused by electrified railways) have additionally been monitored over 24 h. For persons living next to railway lines, the mean individual mean (0.156 microT) and mean individual median (0.102 microT) were calculated. Over all, the mean exposures are only 0.1% of the magnetic flux density limit for 50 Hz (100 microT) and about 0.05% of the limit (300 microT) for 16 2/3 Hz recommended by the International Commission on Non-Ionizing Radiation Protection.

The mitochondrial import receptor Tom70: identification of a 25 kDa core domain with a specific binding site for preproteins.

The mitochondrial import receptor of 70 kDa, Tom70, preferentially recognizes precursors of membrane proteins with internal targeting signals. We report the identification of a stably folded 25 kDa core domain located in the middle portion of Tom70 that contains two of the seven tetratricopeptide repeat motifs of the receptor. The core domain binds non-cleavable and cleavable preproteins carrying internal targeting signals with a specificity indistinguishable from the full-length receptor. Competition studies indicate that both types of preproteins interact with overlapping binding sites of the core domain and that at least one additional interaction site is present in the full-length receptor. We suggest a model of Tom70 function in import of membrane proteins whereby a hydrophobic preprotein concomitantly interacts with several binding sites of the receptor.

Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein.

Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.

External stimuli in the form of vibratory massage after heart or lung transplantation.

Manual vibratory massage is part of the preventive physiotherapeutic activities performed in intensive care units. The vibratory massage can be performed manually or as electrovibratory massage. The manual massage is a fast rhythmical vibration performed by the arm and shoulder muscles of the masseur and transferred to the patient's thorax by the hand. The hand of the masseur has to achieve a tremor with a frequency of 8 to 11 tremors/s. The aim of the pilot study was to examine the influence of manual vibratory massage on the pulmonary function of postoperative patients who were receiving mechanical ventilation, with special interest being focused on pulmonary ventilation and perfusion and cerebral blood flow velocity. Manual vibratory massage was performed postoperatively in the intensive care unit on eight patients: three patients had undergone heart transplantation, three had undergone lung transplantation, and two had undergone coronary artery bypass grafting (mean age, 53.6+/-8 yr). With the aid of continuous monitoring, we examined the changes of the respiration parameters and the cerebral blood flow velocity (measured by transcranial Doppler sonography). The vibratory massage was performed with a frequency of 8 to 10 vibrations/s for 15 min, 7.5 min on each side of the thorax, starting from the lower costal arch and progressing to the upper thoracic aperture. For 10 min before, during, and 10 min after the massage, the parameters of peripheral oxygen saturation, central venous pressure, mean arterial pressure, heart rate, lung resistance and compliance, tidal volume, respiration rate, and cerebral blood flow velocity were recorded at 2-min intervals. Moreover, before and after vibratory massage, arterial blood gases were determined. In four of the eight patients, it was possible to determine pulmonary arterial pressure, pulmonary capillary wedge pressure, as well as pulmonary vascular resistance. During the vibratory massage, we could prove a significant increase of the mean tidal volume by 30% (P = 0.008). The percutaneous oxygen saturation significantly increased also, from 92 to 93.6% (P = 0.002). Central venous pressure significantly decreased by 11% (P = 0.04), and pulmonary vessel resistance was reduced by 18.3% (P = 0.001). The pulmonary resistance decreased from 10.5 to 9.2 H2O/l/s (P < 0.05) by the end of the observation period. Cerebral blood flow velocity showed no significant change. Vibratory massage seems to improve pulmonary mechanism and perfusion, thus, reducing ventilation perfusion mismatch and increasing oxygen saturation.

Cerebral autoregulation during whole-body hypothermia and hyperthermia stimulus.

The purpose of the study contained herein was to investigate the effects of old traditional physiotherapeutic treatments on cerebral autoregulation. Treatment consisted of complete body immersion in cold or warm water baths. Fifteen volunteers were investigated by means of transcranial Doppler sonography and a servo-controlled noninvasive device for blood pressure measuring. One group of 8 volunteers (mean age, 27.2+/-3.5 yr; gender, 3 females/5 males) was subjected to cold baths of 22 degrees C for 20 min Another group of 7 volunteers (mean age, 52.1+/-8.5 yr; gender, 4 females/3 males) took hyperthermic baths at rising water temperatures from 36 degrees to 42 degrees C, increased by 1 degree C every 5 min. Each volunteer in both groups underwent autoregulation tests two to four times before, during, and after the thermic bath. Dynamic autoregulation was measured by the response of cerebral blood flow velocity to a transient decrease of the mean arterial blood pressure, induced by rapid deflation of thigh cuffs. The autoregulation index, i.e., a measure of the speed of change of cerebral autoregulation, was used to quantify the response. Further parameters were core temperature, blood pressure (mm Hg) and CO2et. During hypothermic baths, core temperature decreased by 0.3 degrees C (P = 0.001), measured between preliminary phase and the end of the bath; the autoregulation index decreased significantly (P < 0.05) from 5.3 before the bath to 4.25 during the bath. During hyperthermic baths, the autoregulation index increased from 6.0 to 7.5 and 8.9 (P < 0.001), with an increase of core temperature of 0.4 degrees C. The main cerebral autoregulation system is dependent on changes of core temperature, provoked by hypothermic or hyperthermic whole-body thermostimulus. Application of hyperthermic baths increased the autoregulation index, and hypothermic baths decreased the autoregulation index. Further studies are needed to prove the positive effects of thermo-stimulating water applications on cerebral hemodynamics in patients with cerebral diseases.

The preprotein translocase of the outer mitochondrial membrane: receptors and a general import pore.

Cytosol-synthesized preproteins destined for the mitochondria are transported across the outer membrane by the translocase of the mitochondrial outer membrane (TOM complex). This dynamic transport machinery can be divided into receptors that recognize preprotein targeting signals and components of the general import pore complex that mediate preprotein transport across the outer membrane. This review focuses on recent studies dealing with the central questions regarding the pore-forming subunits, and architecture and gating of the translocation channel of the outer membrane.

Passive and active exercises increase cerebral blood flow velocity in young, healthy individuals.

Unlike the well-examined cardiovascular changes during movement stimuli, changes of cerebral hemodynamics and cerebral metabolism in physical exercises have, as yet, rarely been studied. Our objective was to investigate whether there are changes in cerebral hemodynamics and cerebral metabolism caused by active and passive movement stimuli. Response to repetitive active and passive movement stimuli was studied in 14 volunteers (8 females, 6 males; mean age, 35.2+/-8 yr). Each volunteer was subjected to four measurement series while performing a defined active and passive exercise program of the right upper or right lower limb. Measurement series were designed according to Aaslid's "evoked flow test"; exercises were performed for 20 s, followed by a rest of 20 s. This sequence was repeated 10 times in each series. As a measure of cerebral hemodynamics mean and peak blood flow velocity of the middle cerebral artery were recorded by transcranial Doppler sonography (Multidop-X-DWL). In addition, cerebral metabolism was quantified in a subsample by means of oxygenic cytochrome aa3 (respiratory chain enzyme), cerebral oxygen saturation using near infrared spectroscopy (Critikon). As well, noninvasive blood pressure (Penaz method) and expiratory pCO2 were measured. Compared with resting measures, an increase in mean cerebral blood flow velocity of the middle cerebral artery of 3.8% (P = 0.003, paired t test) was observed after active exercises of the right lower limb and 3.5% after active exercises of the right upper limb. Respective changes were 3.4% (P = 0.004) for passive exercises of the lower limb and 4.6% (P = 0.007) for passive exercises of the right upper limb. Peak cerebral blood flow velocity of the middle cerebral artery showed an even more pronounced increase during passive and active exercises in all cases, with values of between 12.2% (P < 0.001) and 13.6% (P < 0.001). Significant increases (1.5-3%) of cytochrome aa3 and cerebral oxygen saturation were observed during active and passive exercises. These studies show that active as well as passive exercises are followed by an increase of cerebral blood flow velocity. We attribute the increase of cerebral hemodynamic and cerebral metabolism to cerebral activation and autoregulative mechanisms.

Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of approximately 400,000 ( approximately 400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of approximately 100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.

Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70.

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins. We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt. Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner. Tom70 preferentially binds preproteins with internal targeting information. A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70. We conclude that each of the three import receptors binds preproteins independently and by a different mechanism. Both Tom20 and Tom22 function as presequence receptors.

Cerebral hemodynamics and cerebral metabolism during cold and warm stress.

The purpose of this study was to examine if local thermo-applications affect central nervous reactions. In a crossover study, six normal, healthy volunteers at first received cold packs (Cryogel, 8-12 degrees C; Pino GmbH, Hamburg, Germany) and afterwards hot packs (Parafango, 50-60 degrees C; Pino GmbH), and another six volunteers started with the hot packs and had the cold packs later; both groups administered the hot and cold packs to their thighs. Before, during, and after treatment, cerebral blood flow velocity (CBFV) in the middle cerebri-artery (MCA) was measured continuously by transcranial Doppler sonography, whereas cerebral respiratory chain enzyme cytochrome aa3 (cCytaa3) and cerebral oxygen saturation (cHbO2) were measured by transcranial near infrared spectroscopy in frontal brain tissue. Furthermore, CO2 end-tidal and arterial blood pressure (noninvasive) were also measured. Six other volunteers received only one treatment; therefore, 15 measurements with cold and 15 measurements with hot packs were performed. During application of cold packs, a decrease of cHbO2 of 10.5% (P < 0.001) and cCytaa3 of 6.7% (P < 0.001) was found, whereas the CBFV(MCA) increased significantly (3.9%; P < 0.001) between preliminary and post-stimulus periods. When cold packs were removed, a significant increase of the cHbO2 (16.9%; P < 0.001) and cCytaa3 (9.7%; P < 0.001) was measured. With these values, cHbO2 and cCytaa3 showed an overshooting counterreaction beyond the initial level. When applying the hot packs, a contrary course of the parameters was found. cCytaa3 showed a significant increase of 9.3% (P < 0.001) at the end of the stimulus phase and a decrease of 1.9% (P = 0.02) during the post-stimulus period. The correlating increase of cHbO2 was significant at 13.7% (P < 0.005). At the end of the post-stimulus phase, a significant decrease of cHbO2 at 1.9% (P = 0.004) was recorded. With Parafango applications, a significant decrease of CBFV(MCA) at 6.9% (P < 0.001) was measured at the end of the stimulus in comparison with the preliminary phase. Crossover analysis showed no significant period effects and intraindividual changes between period and treatment. Therefore, both treatments can be compared within the individual using paired t test. Local cold and warm stimuli influence the cerebral hemodynamics and cerebral metabolism. Cerebral hemodynamics (CBFV(MCA) in comparison with cerebral metabolism (cCytaa3, cHbO2) show opposite reactions under thermo-stress. Of special interest is the overshooting counter-regulation of cerebral metabolism after cold stimulation. These effects may open new thermotherapeutic aspects in central nervous system diseases.

Mechanical and electromechanical properties of the stereovillar bundles of isolated and cultured hair cells of the chicken.

Isolated single chicken hair cells and pieces of epithelium without the tectorial membrane, either freshly isolated or in tissue culture, were studied using water-jet stimulation of their stereovillar bundles and current injection. Responses were measured under enhanced video-microscopic observation or while using a differential photodiode technique sensitive down into the nanometer range. When stimulated with a water jet at low displacement amplitudes up to about 200 nm, the stereovillar bundle displacement was asymmetrical, indicating a lower stiffness in the excitatory direction, but the reverse was true at higher displacement amplitudes. Undamaged bundles showed no mechanical resonances below 1 kHz. In damaged bundles, however, such resonances were prominent and accompanied by splaying of the stereovilli. Hair cells in the epithelium showed small bundle movements (0.6 nm/mV) whose polarity depended on the polarity of the injected current. These movements probably resulted from activation of the bundle's adaptation motors.