PubMed Health⌕ Search

Biomedical subjects

J Gustafsson

Publications and source records attributed to J Gustafsson.

At least 37 records · Page 2Linked to original sources

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Adaptor Proteins, Signal Transducing↗

Growth hormone therapy in young children with Down syndrome and a clinical comparison of Down and Prader-Willi syndromes.

The genetic disorders Prader-Willi syndrome and Down syndrome have a number of features in common, for example, both growth and mental retardation. Growth hormone (GH) treatment is becoming part of the clinical management of children with Prader-Willi syndrome, but in children with Down syndrome, such therapy is still on a research level. In this review, we compare the clinical phenotypes of the two syndromes, and report the effects of long-term GH treatment on the linear growth and psychomotor development of 15 young children with Down syndrome (mean age at start of treatment, 7.4 months). The mean height of the treated children with Down syndrome increased significantly from -1.8 to -0.8 SDS (Swedish standard) during the 3 years of GH therapy (P < 0.001). The mean height of a corresponding control group fell from -1.7 to -2.2 SDS. After the cessation of treatment, growth velocity declined in the treated group. Growth of the head did not increase during GH treatment. There was no effect on mental or gross-motor development, although some improvement in fine-motor development was noted in the GH-treated group (P < 0.01). At present, treatment with GH is not recommended in children with Down syndrome who have not been diagnosed with GH deficiency. Long-term studies with an emphasis also on the metabolic effects of GH therapy are necessary before routine treatment can be considered in such children.

Body Height↗

Profound changes in the GH-IGF-I system in adolescent girls with IDDM: can IGFBP1 be used to reflect overall glucose regulation?

Disturbances in the relations between insulin, growth hormone (GH) and insulin-like growth factor I (IGF-I) may be a major cause behind deteriorated metabolic control in adolescent girls with type I diabetes. These patients have increased GH secretion and low IGF-I concentrations. The aim of this study was to identify possible endocrine mechanisms behind good and poor glycaemic control in such girls, focusing on the insulin-GH-IGF-I axis. Ten girls with well-controlled insulin-dependent diabetes mellitus (IDDM), hemoglobin A1c (HbA1c) 6.5+/-0.4% (normal range 3.9-5.2%) and nine healthy controls were investigated and compared with 11 girls with poor glucose regulation, HbA1c 10.9+/-0.4%, and their corresponding controls. Serum profiles of glucose, insulin, GH and IGF-binding protein 1 (IGFBP1) were analysed in addition to IGF-I and HbA1c. Two interesting observations were made. GH concentrations were equally elevated in the two diabetic groups regardless of metabolic control (mean 24 h GH - girls with poorly controlled diabetes 10.0+/-1.0 mU/L vs 9.8+/-1.7 - girls with well-controlled diabetes; p=ns). Likewise, the IGF-I concentrations were reduced to the same extent (233+/-19 vs 242+/-23 microg/L; p=0.75). Secondly, despite similar insulin concentrations (mean 24 h insulin - girls with poorly controlled diabetes 22.9+/-2.6 and girls with well-controlled diabetes 27.3+/-2.9 mU/L, respectively; p=0.26), there was a marked difference in IGFBP1 concentrations between the two groups with IDDM (mean IGFBP1 - girls with poorly controlled diabetes 70.5+/-9.1 microg/L vs girls with well-controlled diabetes 28.6+/-3.3; p<0.001). Despite equally elevated GH concentrations that may induce insulin resistance, the markedly lower concentrations of IGFBP1 in the well-controlled group indicate a higher hepatic insulin sensitivity in these girls compared with those with a poor control. Furthermore, in spite of similar total IGF-I concentrations, the lower IGFBP1 concentrations may result in higher IGF-I bioactivity in the well-controlled group. This may be reflected in better growth of the well-controlled group whose height of 168.7+/-0.9 vs 163.6+/-1.2 cm was significantly different (p<0.004). IGFBP1 may be a marker of overall insulinization in adolescents with type 1 diabetes, independent of the absolute insulin dose used for therapy.

Journal Article↗

Pteridin-dependent hydroxylases as autoantigens in autoimmune polyendocrine syndrome type I.

Autoimmune polyendocrine syndrome type I (APS I) is characterized by autoantibodies, often directed towards tissue-specific enzymes in the affected organs. We have earlier reported the identification of tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) as autoantigens in APS I associated with intestinal dysfunction and alopecia, respectively. These two enzymes, together with phenylalanine hydroxylase (PAH), constitute the group of biopterin-dependent hydroxylases, which all are involved in the biosynthesis of neurotransmitters. A clone encoding PAH was used for in vitro transcription/translation, followed by immunoprecipitation with sera from 94 APS I patients and 70 healthy controls. Of the APS I patients, 25% had PAH antibodies, and no reactivity was detected in the controls. No association with the main clinical components of APS I was found with PAH antibodies. Altogether, 59 sera from the 94 APS I patients reacted with at least one of TPH, TH, or PAH, whereas 35 showed no reactivity. Nineteen of the sera contained antibodies towards all enzymes, 12 to TPH only and 12 to TH only. No sera showed antibodies that reacted to only PAH. An immunocompetition assay demonstrated that the reactivity against PAH represents a cross-reactivity with TPH, whereas antibodies against TPH and TH are directed towards epitopes unique for the two enzymes.

Autoantibodies↗

Specific Adsorption of Nickel and zeta Potential of Silica at Various Solid-to-Liquid Ratios.

Theelectrokinetic curves of silica obtained at given total concentrations of heavy metal cations depend on the solid-to-liquid ratio; namely, at low solid-to-liquid ratios the sign of the zeta potential of silica is reversed to positive at relatively low concentrations of heavy metal cations, while at higher solid-to-liquid ratios the electrokinetic curves are not substantially influenced at much higher concentrations. The surface complexation model fails to properly reflect the Ni effect on the course of electrokinetic curves of silica. The course of electrokinetic curves [positions of the isoelectric point(s) and of the maxima in mobility] is correlated with a linear combination of the surface charge density in the absence of heavy metal cations at given ionic strength and the adsorption density of Ni. Copyright 1999 Academic Press.

Journal Article↗

Phase Behavior and Aggregate Structure in Aqueous Mixtures of Sodium Cholate and Glycerol Monooleate.

The phase behavior of the glycerol monooleate (GMO)-sodium cholate-water (or 0.9 wt% NaCl) system has been examined in the solvent-rich part, using small-angle X-ray scattering and conventional methods. Addition of cholate up to 7% of the total amphiphile swells the cubic phase of the binary GMO-water system so that it takes up almost 70% of water in the salt-free case and 55% in salt. With more bile salt the lamellar phase also appears highly swollen (up to 85% in water, 75% in brine). In the salt solution a small isotropic L3-phase region replaces the lamellar phase at a solvent content of about 79%. The lamellar phase can accept only about 0.2 cholate molecule per GMO, in both water and brine, and a phase with globular micelles (L1) follows and dominates the diagram. No threadlike micelles appear in this system. Investigation of the particle structures with cryo-transmission electron microscopy (TEM) in dilute systems (99% solvent) show globular micelles and coexisting vesicles and globular micelles. In the presence of salt, dilution of the L3 phase results in dispersed globular particles with an irregular internal morphology that suggests they are a dispersed L3 phase. These particles coexist with faceted particles having an inner structure giving a hexagonal pattern in projection, suggested to derive from the cubic phase. The cubic phase in the salt-free systems did not give dispersions stable enough for cryo-TEM examination. Copyright 1999 Academic Press.

Journal Article↗

Correlation between the Zeta Potential and Rheological Properties of Anatase Dispersions.

The viscosity at an arbitrarily selected shear rate of 1.16 s-1 and the yield stress of anatase dispersions are linearly correlated, and both show a maximum at the isoelectric point (IEP) at pristine conditions. The rheological properties of anatase dispersions respond to the shift in the IEP to higher pH values, which is observed in concentrated solutions of 1-1 electrolytes and in the presence of the organic cosolvents, but the shift in the pH corresponding to a maximum viscosity does not exactly match the shift in the IEP. In contrast with the electrokinetic curves, the rheological curves at high ionic strengths are rather insensitive to the nature of the supporting electrolyte. The acidic branch of the viscosity(pH) curves is relatively insensitive to the ionic strength while the slope of the basic branch decreases when the ionic strength increases. Copyright 1999 Academic Press.

Journal Article↗

Antibodies against hair follicles are associated with alopecia totalis in autoimmune polyendocrine syndrome type I.

In the autosomal recessively inherited autoimmune polyendocrine syndrome type I (APS I) patients have autoantibodies directed against several endocrine and nonendocrine organs. Alopecia areata is present in about one-third of the patients and usually in the more severe forms, alopecia universalis or totalis. Sera from 39 patients with APS I, diluted 1:150, were used in indirect immunofluorescence staining of cryo-sections from normal human scalp. Two hair follicle staining patterns were observed. A cytoplasmic staining of the differentiating matrix, cuticle, and cortex keratinocytes in the anagen hair follicle was seen in five (13%) APS I sera. All these five patients had alopecia totalis, representing 63% of the eight patients with alopecia totalis (p < 0.0001). Furthermore, four (10%) of the APS I sera stained the nuclei of the melanocytes in the hair follicle. Two of these patients had vitiligo. None of 20 healthy control sera stained the keratinocyte cells or the melanocyte nuclei. These data show that many patients with APS I have high-titer autoantibodies directed against the anagen matrix, cuticle, and cortex keratinocytes and a melanocyte nuclear antigen, and also that the hair follicle keratinocyte staining is associated with alopecia, especially alopecia totalis. This study emphasizes the role of the differentiating anagen keratinocytes as an important structure in the autoimmune etiology of alopecia, both in APS I and at least in a subgroup of patients with alopecia areata unrelated to APS I.

Adult↗

Growth hormone treatment during suppression of early puberty in adopted girls. Swedish Growth Hormone Advisory Group.

Girls adopted from developing countries often have early or precocious puberty, requiring treatment with gonadotropin-releasing hormone (GnRH) analogues. During such treatment decreased growth velocity is frequent. The aim of this investigation was to study whether the addition of growth hormone (GH) to GnRH analogue treatment improves height velocity and final height in girls with early or precocious puberty. Forty-six girls with early or precocious puberty adopted from developing countries were randomized for treatment with GnRH analogue or a combination of GH and GnRH analogue. After 2 y of treatment the mean growth in the GH/GnRH analogue group was significantly higher, 14.6 cm, compared to 10.9 cm in the control group. The increase in bone age did not differ, while the difference in predicted adult height increased by 2.7 cm in favour of the combination group. Although data on final height are not yet available, combined GH/GnRH analogue treatment for 2 y resulted in a higher growth velocity and predicted final height compared to GnRH analogue treatment alone.

Adoption↗

Prevalence of coeliac disease in Turner syndrome.

This study was undertaken to investigate the prevalence of coeliac disease in children and adolescents with Turner syndrome. Eighty-seven children and adolescents with Turner syndrome were screened for IgA-antiendomysium antibodies (EMA) and IgA-antigliadin antibodies (AGA), 5% (4/87) being found to be EMA-positive, and 15% (13/87) to have AGA levels above normal. Of the 10 patients who were either AGA- or EMA-positive and further investigated with intestinal biopsy, four manifested villous atrophy (i.e. all three of the EMA-positive patients, but only one of the seven AGA-positive patients). The results suggest EMA-positivity to be a good immunological marker for use in screening for coeliac disease, and such screening to be justified in patients with Turner syndrome.

Adolescent↗

Effect of growth hormone (GH) during puberty in GH-deficient children: preliminary results from an ongoing randomized trial with different dose regimens.

This paper reports results from an ongoing, randomized, multicentre national trial. The aim is to elucidate whether a dose of growth hormone (GH) of 0.2 IU/kg (0.07 mg/kg), given either as once-daily or twice-daily injections during puberty, is more effective than a once-daily dose of 0.1 IU/kg/day (0.03 mg/kg/day) in improving final height in children with GH deficiency (GHD). The twice-daily regimen comes closer to the spontaneous GH secretion pattern in puberty. Ninety-two children with GHD who had been receiving GH therapy for at least 1 year, and with spontaneous puberty or who were prepubertal and due to be started on replacement therapy to induce puberty, were randomly assigned to receive GH as follows: group A, 0.1 IU/kg/day (0.03 mg/kg/day), administered once daily; group B, 0.2 IU/kg/day (0.07 mg/kg/day), administered once daily; and group C, 0.2 IU/kg/day (0.07 mg/kg/day), divided into two equal injections given at 12-hour intervals. Pubertal height gain was 0.7, 0.7 and 1.3 SDS for groups A, B and C, respectively. The gain in height during puberty was thus most marked in group C. Mean final height, when corrected for parental height, was between 0 and 1 SDS in all treatment groups. All but seven children reached a final height within +/- 2 SD of the general population. There was a wide range of final heights in all three treatment groups. This variation in response suggests the need to individualize treatment in order to achieve an appropriate final height for most individuals.

Adolescent↗

Growth hormone treatment in young children with Down's syndrome: effects on growth and psychomotor development.

BACKGROUND: Learning disability and short stature are cardinal signs of Down's syndrome. Insulin-like growth factor I (IGF-I), regulated by growth hormone (GH) from about 6 months of age, may be involved in brain development. AIMS: To study long term effects of GH on linear growth and psychomotor development in young children with Down's syndrome. Study design-Fifteen children with Down's syndrome were treated with GH for three years from the age of 6 to 9 months (mean, 7.4). Linear growth, psychomotor development, skeletal maturation, serum concentrations of IGF-I and its binding proteins (BPs), and cerebrospinal fluid (CSF) concentrations of IGF-II were studied. RESULTS: The mean height of the study group increased from -1.8 to -0.8 SDS (Swedish standard) during treatment, whereas that of a Down's syndrome control group fell from -1.7 to -2.2 SDS. Growth velocity declined after treatment stopped. Head growth did not accelerate during treatment. No significant difference in mental or gross motor development was found. The low concentrations of serum IGF-I and IGFBP-3 became normal during GH treatment. CONCLUSIONS: GH treatment results in normal growth velocity in Down's syndrome but does not affect head circumference or mental or gross motor development. Growth velocity declines after treatment stops.

Age Determination by Skeleton↗

Effect of theophylline on glucose production and lipolysis in preterm infants (< or = 32 weeks).

Apnea occurs commonly in preterm infants. Theophylline is used as prophylaxis and treatment. Apart from improving ventilatory function, theophylline may also have metabolic effects, including an effect on glucose metabolism and lipolysis. No data are available on the effect of theophylline on glucose production and lipolysis in preterm infants at start of medication. Ten preterm infants with gestational ages of < or = 32 wk, postnatal ages of 16-84 h, and birth weights > 900 g were recruited. Hepatic glucose production and lipolysis were measured by use of gas chromatography/mass spectrometry after constant rate infusion of [6,6-2H2]glucose and [2-13C]glycerol tracers. Plasma glucose levels increased after theophylline administration (mean +/- SD, 4.0 +/- 1.9 mmol/L before and 4.7 +/- 2.1 mmol/L after start of therapy), whereas the rate of glucose production decreased (6.0 +/- 2.5 mg.kg-1.min-1 and 4.3 +/- 1.9 mg.kg-1.min-1, respectively). The plasma glycerol concentration did not show any change after theophylline administration (154 +/- 257 mumol/L before and 217 +/- 258 mumol/L after), and the same was true for the rate of glycerol production (5.9 +/- 2.6 mumol.kg-1.min-1 before and 6.7 +/- 3.0 mumol.kg-1.min-1 after). The fraction of glycerol converted into glucose did not change significantly, although the percentage of glucose derived from glycerol increased after theophylline administration. The results are in line with the lack of adverse metabolic effects at start of theophylline treatment in the preterm infant.

Apnea↗

Identification of tryptophan hydroxylase as an intestinal autoantigen.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS1) is an autosomal recessive disorder with both endocrine and non-endocrine features. Periodic gastrointestinal dysfunction occurs in 25-30% of APS1 patients. We aimed to identify an intestinal autoantigen. METHODS: A human duodenal cDNA library was immunoscreened with serum samples from APS1 patients. A positive clone was identified and used for in-vitro transcription and translation, followed by immunoprecipitation with serum samples from 80 APS1 patients from Norway, Finland, and Sweden. Sections of normal and APS1-affected small intestine were immunostained with serum from APS1 patients and specific antibodies. An enzyme-inhibition assay was used to characterise the autoantibodies. FINDINGS: We isolated a cDNA clone coding for tryptophan hydroxylase. 48% (38/80) of APS1 patients had antibodies to tryptophan hydroxylase, whereas no reactivity to this antigen was detected in patients with other autoimmune diseases (n=372) or healthy blood donors (n=70). 89% (17/19) of APS1 patients with gastrointestinal dysfunction were positive for antibodies to tryptophan hydroxylase, compared with 34% (21/61) of patients with no gastrointestinal dysfunction (p<0.0001). Serum from antibody-positive APS1 patients specifically immunostained tryptophan-hydroxylase-containing enterochromaffin cells in normal duodenal mucosa. No serotonin-containing cells were seen in duodenal biopsy samples from APS1 patients. Serum from antibody-positive APS1 patients almost completely inhibited activity of tryptophan hydroxylase. INTERPRETATION: Tryptophan hydroxylase is an endogenous intestinal autoantigen in APS1, and there is an association between antibodies to the antigen and gastrointestinal dysfunction. Analysis of antibodies to tryptophan hydroxylase may be a valuable diagnostic tool to predict and monitor gastrointestinal dysfunction in APS1.

Adolescent↗

Is growth hormone hypersecretion in diabetic adolescent girls also a daytime problem?

OBJECTIVES: Glycaemic control often deteriorates during puberty in girls with insulin dependent diabetes mellitus (IDDM). This may be due in part to the normal psychosocial changes associated with adolescence. Puberty is, however, also characterized by rapid somatic development, orchestrated by hormonal changes. Some of these hormones play a major role in glucose homeostasis. We have examined the insulin-GH-IGF-I axis in 11 adolescent girls with poorly controlled insulin dependent diabetes and compared the data with those of 10 non-diabetic girls matched for age, pubertal stage and body mass index (BMI). METHODS: Serum profiles of glucose, insulin, GH and IGF binding protein 1 (IGFBP1) were analysed in addition to IGF-I in serum and nocturnal urinary excretion of GH. MEASUREMENTS: Serum glucose, insulin and IGFBP1 were measured every hour for 24 h, whereas GH in serum was measured every 30 minutes during the same period. Nocturnal urinary GH was analysed as a mean of three consecutive nights. RESULTS: The insulin profiles of the IDDM patients were flat with low post-prandial peaks, corresponding to only one-third of the peaks of the non-diabetic girls. The integrated insulin levels, both during 24-h sampling and during daytime, were significantly lower in the diabetic group. There were no differences during night-time. The diabetic patients had elevated mean baseline levels of serum GH (IDDM 2.8 +/- 0.5 mU/l, controls 0.7 +/- 0.2; P < 0.001), a higher 24-h mean serum GH level (9.8 +/- 1.7 mU/l vs. 4.4 +/- 0.7; P < 0.001), significantly more peaks and a urinary GH excretion twice as high as in the non-diabetic group. An interesting observation was the finding of marked differences in daytime GH concentrations between the groups, both regarding overall integrated levels (GH AUC 103 +/- 15.8 and 35.9 +/- 7.1 mU/l x 12 h, respectively; P < 0.005) as well as baseline levels (3.8 +/- 0.6 mU/l vs. 0.7 +/- 0.2; P < 0.001). In contrast, during night-time only the mean basal levels of GH differed. The level of IGF-I was reduced in the diabetic group compared with the healthy controls (IDDM 233 +/- 19 micrograms/l vs. controls 327 +/- 21; P < 0.005). In addition, the IDDM patients had significantly increased concentrations of IGFBP 1, but kept a normal diurnal rhythm with a pronounced night peak. CONCLUSION: Hypoinsulinaemia in adolescent IDDM patients, particularly in the portal hepatic circulation, results in decreased IGF-I and increased IGFBP 1 production in the liver. High levels of IGFBP 1 may, in turn, reduce the bioactivity of IGF-I even further. Low levels of IGF-I will lead to increased GH secretion. Earlier studies on the relationship between GH and diabetic control have focused on elevated GH levels during the night. In this study we have observed markedly elevated levels of GH also during daytime in adolescent IDDM patients. This indicates increased insulin resistance and insulin demand also during the day in diabetic subjects. The increased insulin resistance may result in hyperglycaemia leading to additional insulin resistance. A vicious circle may thus be induced, accelerating metabolic impairment in poorly controlled adolescent IDDM girls.

Adolescent↗

Growth hormone treatment of short children born small-for-gestational-age: the Nordic Multicentre Trial.

The aims of this study were to evaluate the efficacy and safety of different doses of growth hormone (GH) treatment in prepubertal short children born small-for-gestational-age (SGA). Forty-eight children born SGA from Sweden, Finland, Denmark and Norway were randomly allocated to three groups: a control group of 12 children received no treatment for 2 y, one group was treated with GH at 0.1 IU/kg/d (n=16), and one group was treated with GH at 0.2 IU/kg/d (n=20). In total 42 children completed 2 y of follow-up, and 24 children from the treated groups completed 3 y of treatment. Their mean (SD) age at the start of the study was 4.69 (1.61) y and their mean (SD) height was -3.16 (0.70) standard deviation scores (SDS). The children remained prepubertal during the course of the study. No catch-up growth was observed in the untreated group, but a clear dose-dependent growth response was found in the treated children. After the third year of treatment, the group receiving the higher dose of GH, achieved their target height. The major determinants of the growth response were the dose of GH used, the age at the start of treatment (the younger the child, the better the growth response) and the family-corrected individual height deficit (the higher the deficit, the better the growth response). Concentration of insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 increased during treatment. An increase in insulin levels was found without negative effects on fasting glucose levels or glycosylated haemoglobin levels. GH treatment was well tolerated. In conclusion, short prepubertal children born SGA show a dose-dependent growth response to GH therapy, and their target height SDS can be achieved within 3 y of treatment given GH at 0.2 IU/kg/d. However, the long-term benefit of different regimens of GH treatment in children born SGA remains to be established.

Body Height↗

Identification of CYP2A3 as a major cytochrome P450 enzyme in the female peripubertal rat breast.

On isolation of rat breast cytochrome P450, one of the proteins whose amino terminus was sequenced was CYP2A3. CYP2A3 was detected by Western blotting in cytochrome P450 fractions isolated from breast of 3-, 6-, and 9-week-old rats but was low during pregnancy and lactation. Reverse transcription-polymerase chain reaction analysis and sequencing of the PCR product confirmed the presence and identity of CYP2A3 in the rat breast. Breast microsomal coumarin-7-hydroxylase activity paralleled the developmental pattern observed for CYP2A3 on Western blots. In the lung, coumarin-7-hydroxylase activity was 10-fold higher than that in the breast, but the developmental pattern was similar to that in the breast. Lung microsomes from 9-week-old rats activated the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline to mutagenic metabolites which could be detected with the Ames test. This activation could be inhibited by the CYP2A3 antiserum. With breast microsomes, which contain approximately 10% of the cytochrome P450 in the lung, activation of 2-amino-3-methylimidazo[4, 5-f]quinoline could not be reliably measured. Immunohistochemical localization revealed that CYP2A3 was expressed in a limited number of epithelial cells in the ducts of 6-week-old rat breast. Double staining with smooth muscle actin, a marker for myoepithelial cells, showed no staining of CYP2A3 immunoreactive cells, indicating that these cells were not myoepithelial. The data clearly show that a cytochrome P450 that can activate environmental procarcinogens is developmentally regulated and concentrated in specific cells in the breast. The peripubertal period seems to be a window in time when the breast may be more sensitive to procarcinogens that are substrates for CYP2A3.

Amino Acid Sequence↗