Mikael Holst

Naturally Occurring Mutants of Human Steroid 21-Hydroxylase (P450c21) Pinpoint Residues Important for Enzyme Activity and Stability

Three mutants (deletion of E196, G291S, and R483P) of steroid 21-hydroxylase (P450c21) from patients with inherited congenital adrenal hyperplasia had reduced activity toward progesterone and 17-hydroxyprogesterone after transient expression in cultured mammalian cells. In addition, both the E196 deletion and the R483P mutant had shorter half-lives than the wild-type enzyme, whereas the half-life of the G291S mutant was comparable with that of the normal protein. These results directly link the clinical situation with the three mutations and suggest that G291 is important for the catalytic activity of P450c21.

No overrepresentation of congenital adrenal hyperplasia in patients with adrenocortical tumours

The development and progression of sporadic adrenocortical tumours are poorly understood. In autopsy studies adrenocortical tumours are found in between 2 and 9% of the general population. In congenital adrenal hyperplasia (CAH), decreased production of cortisol leads to increased secretion of ACTH from the pituitary, resulting in hyperplasia of the adrenals. More than 95% of all cases of CAH are due to steroid 21‐hydroxylase deficiency, resulting from mutations in the CYP21 gene. In subjects homozygous and heterozygous for CYP21 mutations, adrenocortical tumours have been found in a high frequency compared to the general population, suggesting that chronic ACTH stimulation may play a role in the development of this tumour form. In order to test whether mild undiagnosed CAH is a common predisposing factor, we screened 27 patients with sporadic adrenocortical tumours for CYP21 mutations.

Gelatinase A and Membrane-type 1 Matrix Metalloproteinase mRNA: Expressed in Adrenocortical Cancers but Not in Adenomas

In an attempt to understand the mechanism behind the invasion and metastasis in adrenocortical cancer we performed mRNA in situ hybridization on 30 tumors for three matrix metalloproteinases (MMPs): gelatinase A, membrane type 1 matrix metalloproteinase (MT1-MMP), and collagenase-3. All are known to participate in the invasion and metastasis of other tumor forms by degrading the extracellular matrix. Thirteen of sixteen cancers, but only one of fourteen benign lesions showed expression of gelatinase A, which was localized in stromal cells. MT1-MMP is thought to assist in tumor invasion and metastasis by activating the zymogen gelatinase A. Of 14 malignant tumors analyzed, 12 showed MT1-MMP mRNA expression, which in 7 cases was detected in both neoplastic and stromal cells. The benign tumors showed MT1-MMP expression in only 3 of 11 cases, and it was restricted to tumor cells. Fourteen tumors (11 cancers, 3 adenomas) were also analyzed for collagenase-3 mRNA, but no expression was detected. In conclusion, our data show that gelatinase A mRNA is expressed in most malignant adrenocortical tumors but not in the benign tumors. Gelatinase A mRNA expression is restricted to stromal cells, whereas its activator, MT1-MMP, is expressed in both stromal and neoplastic cells. Inhibition of gelatinase A and other proteinases may in the future become important as a form of cancer treatment.

Single Subcutaneous Administration of Chorionic Gonadotropin to Rats Induces a Rapid and Transient Increase in Testicular Expression of Pro-Inflammatory Cytokines

hCG has been reported to cause an inflammation-like effect in the testis, although the background and consequences of this phenomenon remain to be understood. This investigation reveals that a single injection of hCG (100 U) induces a transient surge in pro-inflammatory cytokine expression in the adult rat testis. Reverse transcriptase PCR analysis demonstrated onset of testicular expression of IL-1β and IL-6 mRNA and increases in the levels of mRNA encoding the constitutively expressed cytokines IL-1α, IL-1 receptor antagonist, and tumor necrosis factor-α 4 h after hCG injection and a maximal response after 8–12 h. These increases were accompanied by a transient increase in testicular IL-1 bioactive protein. Twenty-four hours after administration of hCG, the levels of all cytokine mRNA had decreased, although most were still elevated above control. Immunohistochemical staining revealed that the IL-1β protein was undetectable in normal testes but was seen to be localized to interstitial macrophages but not Leydig cells after hCG treatment. Testes devoid of Leydig cells after pretreatment with ethane dimethane sulphonate exhibited normal staining for interstitial macrophages but failed to respond to hCG with increases in IL-1β mRNA and protein expression. We conclude that hCG induces testicular inflammation via local activation by Leydig cells of the production of pro-inflammatory cytokines by resident macrophages. It remains to be investigated whether the high-dose hCG regimens used for treatment of boys with cryptorchidism could induce similar increases of pro-inflammatory cytokines in the human testis and if such treatments could adversely affect future testicular function.

Serotonin and its 5-HT1 receptor in human mastocytosis.

Context: Human mastocytosis is a rare disease, in which the serotonergic system may be involved. Objective: The objective of the present study was to examine the possible presence of serotonin (5-HT) and its 5-HT1A receptor (R) in the skin of patients with mastocytosis. In addition, the effect of the 5-HT1AR was tested on human mastocytosis cells, cultured in vitro. Materials and methods: The expression of 5-HT and 5-HT1AR in patients with urticaria pigmentosa and mastocytoma was studied using immunohistochemistry. The effects of 8-OH-DPAT, an agonist of 5-HT1AR, on the proliferation (cell number), viability, apoptosis, spontaneous release of histamine, as well as a possible 5-HT metabolism, in the human HMC-1 mast cell line, were investigated. Results: Both 5-HT and 5-HT1AR were expressed in the mast cells in biopsies of mastocytoma and urticaria pigmentosa, as well as in HMC-1 cells. However, no metabolism of 5-HT by the cell line could be detected by the methodology used. The 5-HT1AR agonist had no significant effect on the viability and number of HMC-1 cells, and was without effect on the apoptosis. At concentrations of 10−6 mol/L and 10−8 –10−10 mol/L (i.e. also at physiological concentrations), the agonist inhibited histamine release by these cells by as much as 30%. Conclusion: These findings indicate that 5-HT and its 5-HT1AR are expressed in human mastocytosis and that an agonist of the 5-HT1AR might be of value in the treatment of these patients.

Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with 32Pi. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of 32P) and not sedimentable (supernatant) material. About 90% of 32P in the supernatant fraction was sedimentable at 100,000 g (“disperse chromatin”). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of 32P. The 32P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the 32P-labeled nuclear proteins into 12 major bands in the Mr range of 12,000–120,000. The incorporation of 32P into most bands reached a steady-state within 5–10 min of incubation with 32Pi and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an Mr 95,000 phosphoprotein. Two of the fastest moving 32P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving 32P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.

Polymorphisms in the serotonin transporter gene of patients with atopic dermatitis-association with personality traits related to high level of anxiety.

Context: The symptoms of atopic dermatitis (AD) are often aggravated by anxiety, and the serotonin transporter (5-HTT) has been shown to be of importance in this context. Three polymorphisms affecting transcription of this gene are known: a repetitive element, in the promoter region (5HTTLPR), a variable number tandem repeats (VNTR) within intron 2 referred to as STin2, and a single-nucleotide (A/G) polymorphism (SNP) located within the 5-HTTLPR. Objective: To examine for possible relationships between these polymorphisms and aggravation of AD by stress. Materials and methods: Thirty-three patients with a history of such aggravation, together with 33 age- and gendermatched healthy control subjects, were recruited. The Karolinska Scales of Personality questionnaire was employed to evaluate anxiety-related personality traits and genomic DNA was extracted from blood samples and analyzed using the polymerase chain reaction. Results: Although the prevalence of the short and long alleles of 5-HTTLPR did not differ between the patients and healthy controls, there was a tendency towards high prevalence of the short (10-copy) variant of STin2 among the patients. When the study population was further analysed by subdivision into subgroups all AD patients with high- anxiety traits carried the short variant of STin2. In the corresponding healthy control group, the prevalences of the 10-and 12-copy variants were 62% and 38%, respectively (P < 0.01). Conclusion: These findings indicate a possible association between the 10-copy variant of STin2 and aggravation of AD by anxiety.

Steroid 21-hydroxylase in the kidney: Demonstration of levels of messenger RNA which correlate with the level of activity

Steroid 21-hydroxylase activity was assayed in low-speed supernatants prepared from whole cell homogenates of mouse and rat tissues. Kidney supernatants had an activity which was approximately 2-5% that of adrenal preparations while heart muscle was found to be without 21-hydroxylase activity. When the enzyme kinetics were characterized, both adrenal and kidney low-speed supernatants demonstrated saturation kinetics, but with very different Vmax and Km values. Using polymerase chain reaction amplification after reverse transcriptase synthesis of cDNA from isolated RNA (RT-PCR), we found low levels of mRNA for steroid 21-hydroxylase in mouse kidney, but none in heart muscle. Thus, extra-adrenal steroid 21-hydroxylase activity in the kidney may be mediated by the same enzyme as found in adrenals.

The rapidly phosphorylated chromosomal 42-kDa protein is a subunit of larger protein complexes

We have isolated, purified and characterized a 42-kDa phosphoprotein which has been found to be preferentially associated with active gene loci of salivary gland cells of Chironomus tentans. The rapidly phosphorylated form of this protein could be extracted with 0.2 M NaCl. Chromatographic analysis by gel filtration revealed that a significant fraction of labelled 42-kDa polypeptide elutes with an apparent molecular mass of 150 to 200 kDa. The result suggests that a portion of the phosphorylated 42-kDa polypeptide in native state forms a multisubunit protein complex consisting of rapidly phosphorylated 42-kDa polypeptide chains alone.

Differential kinase systems are involved in the rapidly turning over phosphorylation of prominent nuclear proteins

The activity of endogenous nuclear protein kinases has been probed in an vitro assay system of isolated nuclei from Chironomus salivary gland cells. The phosphorylation of a set of seven prominent rapidly phosphorylated non-histone proteins and of histones H3, H2A and H4 was analyzed using ATP or GTP as phosphoryl donor and heparin as protein kinase effector. The core histones H2A and H3 both incorporate 32P from [gamma-32P]ATP as well as from [gamma-32P]GTP but their phosphorylation is differentially affected by heparin. The phosphorylation of H2A is blocked by heparin while that of H3 is even stimulated in the presence of heparin when ATP is used as phosphate donor. H4 is unable to incorporate phosphate groups from GTP but its ATP-based phosphorylation is heparin sensitive. Of the non-histone protein kinase substrates, we could only detect two, the 44-kDa and 115-kDa proteins, which are heparin sensitive with either ATP or GTP and, thus, strictly meet the criteria for casein kinase type II-specific phosphorylation. The investigated histones and non-histone proteins can be grouped into three broad categories on the basis of their phosphorylation properties. (A) Proteins very likely affected by casein kinase NII. (B) Proteins phosphorylated by strictly ATP-specific protein kinases