Expression of thyroid hormone receptor isoform alpha1 in pancreatic islets.

Thyroid hormone receptors (TR) mediate the action of thyroid hormones. Genetic studies revealed that the individual TR isoforms possess different functions. In the present paper we studied the expression of the isoforms TRalpha1 and TRbeta1 in the murine pancreatic islet. TRalpha1 and TRbeta1 mRNA transcripts and proteins were detected in islets using reverse transcription-polymerase chain reaction and Western blotting analyses, respectively. In immunohistochemical studies individual cells in the periphery of islets were labelled using an anti-TRalpha1 antibody. No labelled cells were detected in the exocrine pancreas. A similar staining pattern was obtained with an anti-glucagon antibody, but not with an anti-insulin antibody, which suggests that TRalpha1 is mainly expressed in alpha-cells. In order to address a potential function of TRalpha1 in this cell type, the regulation of glucagon gene expression by triiodothyronine was studied in a glucagon-producing cell line by Northern blot analysis and transient transfection assays using glucagon promoter luciferase fusion gene constructs. In these assays, triiodothyronine did not regulate the glucagon mRNA level or the glucagon promoter activity. The predominant localization of TRalpha1 in pancreatic alpha-cells suggests that this receptor isoform mediates a specific, yet unknown, function of thyroid hormones in this cell type.

Complete virilization in congenital adrenal hyperplasia: clinical course, medical management and disease-related complications.

OBJECTIVE: In girls with congenital adrenal hyperplasia (CAH), genital ambiguity usually leads to a rapid neonatal diagnosis. Rarely, CAH causes complete virilization and male sex assignment with a delayed diagnosis. After being confronted with very specific problems in two of such patients, we collected data of patients with CAH and complete virilization in a nationwide study to delineate specific problems of these rare patients in order to improve their management. DESIGN AND PATIENTS: Through the German Working Group of Paediatric Endocrinology (Arbeitsgemeinschaft Pädiatrische Endokrinologie, APE), questionnaires were sent to all members caring for patients with CAH and complete virilization in their endocrine clinics. Data from 16 patients from 10 paediatric endocrine centres were assessed by questionnaire. RESULTS: The following problems have been encountered. (1) Sex assignment/gender identity: initially all patients had a male sex assignment. Six patients were diagnosed during the first month of life. Five were reassigned to female sex immediately, one at the age of 19 months. Except in one girl demonstrating some tomboyish behaviour, gender role behaviour in these patients did not differ from unaffected girls. Ten patients were diagnosed late at 3.4--7 years of age. In seven patients with a late diagnosis, male sex assignment was maintained; one of them expressed some concerns about living as a male. In three patients late sex reversal was performed, gender identity is very poor in one and new sex assignment is currently under consideration. (2) SURGERY: irrespective of the sex assigned, all patients had between one and three surgical procedures, including clitoris reduction and (repeated) vaginoplasties in patients with female sex assignment. Hysterectomy and ovarectomy were performed in patients with male sex assignment. (3) Short stature: patients with a late diagnosis of CAH had extremely advanced bone ages of +6.3 to +9.5 years, leading to severely reduced final height of 137 to 150 cm in adult patients. Patients tended to follow height percentiles of genetic females. One pubertal patient was suicidal due to short stature. (4) Central precocious puberty (CPP): prolonged exposition to adrenal androgens led to CPP in one patient. He was treated with GnRH analogues until gonadectomy. CONCLUSIONS: Patients with CAH and complete virilization have a high risk of being diagnosed late. There are major problems and uncertainties of the patients' families and the treating physicians concerning gender assignment. Gender identity is disturbed in some patients. In addition, multiple surgical procedures are necessary and short stature as well as central precocious puberty might be important to avoid late sequelae. While some surgical interventions are probably unavoidable, most of these issues could be resolved with an early diagnosis. Thus, especially for these patients, a neonatal screening programme for CAH would be of paramount importance.

A novel karyoskeletal protein: characterization of protein NO145, the major component of nucleolar cortical skeleton in Xenopus oocytes.

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

Identification of a sequence element directing a protein to nuclear speckles.

SF3b(155) is an essential spliceosomal protein, highly conserved during evolution. It has been identified as a subunit of splicing factor SF3b, which, together with a second multimeric complex termed SF3a, interacts specifically with the 12S U2 snRNP and converts it into the active 17S form. The protein displays a characteristic intranuclear localization. It is diffusely distributed in the nucleoplasm but highly concentrated in defined intranuclear structures termed "speckles," a subnuclear compartment enriched in small ribonucleoprotein particles and various splicing factors. The primary sequence of SF3b(155) suggests a multidomain structure, different from those of other nuclear speckles components. To identify which part of SF3b(155) determines its specific intranuclear localization, we have constructed expression vectors encoding a series of epitope-tagged SF3b(155) deletion mutants as well as chimeric combinations of SF3b(155) sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified (i) a functional nuclear localization signal of the monopartite type (KRKRR, amino acids 196--200) and (ii) a molecular segment with multiple threonine-proline repeats (amino acids 208--513), which is essential and sufficient to confer a specific accumulation in nuclear speckles. This latter sequence element, in particular amino acids 208--440, is required for correct subcellular localization of SF3b(155) and is also sufficient to target a reporter protein to nuclear speckles. Moreover, this "speckle-targeting sequence" transfers the capacity for interaction with other U2 snRNP components.

A novel helicase-type protein in the nucleolus: protein NOH61.

We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is a member of the family of "DEAD-box" proteins, representing a subgroup of putative ATP-dependent RNA helicases. ATPase activity of the recombinant protein is evident and stimulated by a variety of polynucleotides tested. Immunolocalization studies revealed that protein NOH61 (nucleolar helicase of 61 kDa) is highly conserved during evolution and shows a strong accumulation in nucleoli. Biochemical experiments have shown that protein NOH61 synthesized in vitro sediments with approximately 11.5 S, i.e., apparently as homo-oligomeric structures. By contrast, sucrose gradient centrifugation analysis of cellular extracts obtained with buffers of elevated ionic strength (600 mM NaCl) revealed that the solubilized native protein sediments with approximately 4 S, suggestive of the monomeric form. Interestingly, protein NOH61 has also been identified as a specific constituent of free nucleoplasmic 65S preribosomal particles but is absent from cytoplasmic ribosomes. Treatment of cultured cells with 1) the transcription inhibitor actinomycin D and 2) RNase A results in a complete dissociation of NOH61 from nucleolar structures. The specific intracellular localization and its striking sequence homology to other known RNA helicases lead to the hypothesis that protein NOH61 might be involved in ribosome synthesis, most likely during the assembly process of the large (60S) ribosomal subunit.

Effect of leptin on CYP17 enzymatic activities in human adrenal cells: new insight in the onset of adrenarche.

CYP17 is a microsomal enzyme embodying two distinct activities, 17alpha-hydroxylase and 17,20-lyase, essential for the synthesis of cortisol and sex hormone precursors, respectively. The two activities are differentially regulated in a tissue and developmental stage-dependent fashion. Leptin might play a role in such differential control. Low dose leptin caused a significant increase in 17,20-lyase activity in adrenal NCI-H295R cells expressing leptin (OB) receptor (OB-R), without significant sustained influence on the 17alpha-hydroxylase activity. To analyze the time dependence of this leptin effect, the impact of long and short-term leptin treatment was studied. To assess the relationship with the OB-R signal transduction pathway, the same experiments were performed in intact cells and in a reconstituted system. The long- and short-term studies in intact cells and in microsomes suggest that the 17alpha-hydroxylase activity of CYP17 can be promptly stimulated by leptin, but that the effect is transient. In contrast, physiological doses of leptin steadily enhance 17,20-lyase activity. This influence is direct, OB-R specific and dependent on the integrity of the signal transduction pathway. The 17,20-lyase activity stimulation relies on phosphate incorporation, as demonstrated by the loss of leptin-dependent 17,20-lyase stimulation after phosphate removal, and by the fact that the DHEA production appears to be related exclusively to the presence of phosphorylated CYP17, independently from novel protein synthesis. The mechanism underlying the observed events seems to involve CYP17 phosphorylation, a feature of the OBR signal transduction pathway, and a process already shown to be crucial for 17,20-lyase activity.

17alpha-hydroxylase/17,20-lyase deficiency as a model to study enzymatic activity regulation: role of phosphorylation.

Cytochrome P450 17alpha-hydroxylase (CYP17) is a single gene-encoded protein with two activities: 17alpha-hydroxylase and 17,20-lyase. The two catalytic activities are differentially regulated in health and disease. We took advantage of naturally occurring human mutations to understand the molecular bases of this differential regulation. We identified eight novel mutations in the CYP17 gene, different in nature and spread throughout the gene. As posttranslational modifications appear to be important for activity control, we investigated the phosphorylation state of wild-type and mutant CYP17 proteins. Although phospholabeled protein was seen when the wild-type and most mutant proteins were expressed, no phosphorylation was detected for the F417C mutant. F417C is the only 17,20-lyase deficiency case confirmed at the molecular level and represents the first phosphorylation CYP17-deficient mutant. In search of the physiological agents involved in this process, the effect of cAMP was tested on activity and phosphorylation state of our mutant CYP17 proteins. cAMP stimulates activity and phosphorylation in all cases, except in the F417C and R35L mutants. The lack of response to the physiological second messenger might explain the different phenotypes. The F417C mutant protein, which is already shown to be associated with the lack of electron transfer, provides for the first time a link between the electron transfer system and the phosphorylation state of the CYP17 enzyme in the control of 17,20-lyase activity.

Basal inhibin B and the testosterone response to human chorionic gonadotropin correlate in prepubertal boys.

During childhood, the quiescent phase of testicular activity, the hCG stimulation test is widely used to evaluate testicular function. Inhibin B, a gonadal peptide regulating FSH secretion, is an established marker of Sertoli cell function and spermatogenesis in adults. In contrast to the other hormones of the hypothalamo-pituitary-gonadal axis, inhibin B is also secreted in detectable amounts during childhood. The aim of this study was to determine whether basal inhibin B levels are able to predict prepubertal testicular function, so as to avoid a stimulation test. Inhibin B and testosterone before and after hCG stimulation were measured in 54 male children with various testicular disorders by an immunoassay specific for inhibin B. Basal inhibin B was compared to the testosterone increase after hCG. Inhibin B and the hCG-induced testosterone increment correlated strongly (r = 0.84; P<0.0001). Patients with anorchia were clearly distinguishable from those with abdominal testes, having undetectable (inhibin B, <15 pg/mL) respective normal inhibin B levels for age. Inhibin B and the testosterone response to hCG were low in boys with testicular damage (delayed diagnosis of cryptorchidism; after testicular torsion) and in patients with gonadal dysgenesis, but were normal or increased in children with androgen insensitivity syndrome. We conclude that basal inhibin B predicts the testosterone response to hCG in boys and therefore gives reliable information about both the presence and function of the testes. The diagnostic procedure in cryptorchidism may be reduced to a single inhibin B measurement. Furthermore, inhibin B levels show specific alterations in patients with sexual ambiguity, adding a valuable diagnostic tool to the complex differential diagnosis of male pseudohermaphroditism.

Apparent cortisone reductase deficiency: a rare cause of hyperandrogenemia and hypercortisolism.

A 55-year-old woman presented with androgenetic alopecia which had started at age 40. Her clinical history revealed that, unlike her younger sister, she was unable to conceive and was diagnosed as being sterile at age 30. At age 45, 21-hydroxylase deficiency (late-onset CAH) was assumed and glucocorticoid treatment suggested, but not initiated. There was slight hirsutism, but no other sign of virilization. Retesting of plasma steroids revealed elevated 17-OH-progesterone and free testosterone. Treatment with prednisone, cyproterone acetate, and spironolactone was started with significant clinical success. Surprisingly, the analyses of urinary steroid metabolites revealed a pattern that did not support the diagnosis of 21-hydroxylase deficiency (pregnanetriolone absent, pregnanediol, 17-OH-pregnanolone and pregnanetriol not increased). Abdominal CT showed bilateral adrenal hyperplasia and masses in both ovaries. Bilateral adnexectomy was performed, and cystic teratomas were diagnosed. Postoperative urinary steroid analyses showed a decreased tetrahydrocortisol/tetrahydrocortisone ratio (values around 0.08 as compared to age- and sex-matched controls with a ratio of about 0.5-0.8). Plasma cortisol appeared to be repeatedly elevated with exogenous sources excluded. Mass spectrometry showed that, while the tetrahydro metabolites were mainly cortisone-derived, the metabolites not reduced in A ring were mostly cortisol derivatives. This constellation clearly indicates cortisone reductase deficiency, a defect of hepatic 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1). This enzyme catalyzes the oxidation of cortisol to cortisone and the reduction of cortisone to cortisol. In contrast to the corresponding kidney enzyme (11 beta-HSD2), its primary activity is, however, reductive. Although this is only the fifth reported case of that defect, more attention should be paid to this condition in hyperandrogenic women, even if elevated 17-OH-progesterone and testosterone suggest a more frequent cause.

Maternal uniparental disomy 14 as a cause of intrauterine growth retardation and early onset of puberty.

Uniparental disomy for particular chromosomes is increasingly recognized as a cause of abnormal phenotypes in humans either as a result of imprinted genes or, in the case of isodisomy, homozygosity of mutated recessive alleles. We report on the occurrence of maternal uniparental disomy for chromosome 14 (matUPD 14) in a 25-year-old woman with a normal karyotype, normal intelligence but low birth weight, short stature, small hands, and early onset of puberty. Comparison of her phenotype with those of 15 previously described liveborn patients with matUPD14 gives further evidence for an imprinted gene region on chromosome 14 and highlights the necessity to consider this cause in children with intrauterine growth retardation and early onset of puberty caused by acceleration of skeletal maturation.

Treatment with human growth hormone in patients with Prader-Labhart-Willi syndrome reduces body fat and increases muscle mass and physical performance.

UNLABELLED: Twelve children with documented Prader-Labhart-Willi syndrome were treated with human growth hormone (24 U/m2/week) during 1 year. The children were divided into three groups: group 1: overweight and prepubertal (n=6, age 3.8-7.0 years); group 2: underweight and prepubertal (n=3, age 0.6-4.1 years); group 3: pubertal (n=3, age 9.2-14.6 years). In group 1, height increased from -1.7 SD to -0.6 SD, while weight decreased from 1.1 SD to 0.4 SD, with a dramatic drop in weight for height from 3.8 SD to 1.2 SD. Hand length increased from -1.5 SD to -0.4 SD and foot length from -2.5 SD to -1.4 SD. Body fat, measured by dual X-ray energy absorptiometry, dropped by a third, whereas muscle mass increased by a fourth. Physical capability (Wingate test) improved considerably. The children were reported to be much more active and capable. In group 2, similar changes were seen, but weight for height increased, probably because muscle mass increase exceeded fat mass decrease. Changes in group 3 were similar as in group 1, even though far less distinct. CONCLUSION: Growth hormone treatment in Prader-Labhart-Willi syndrome led to dramatic changes: distinct increase in growth velocity, height and muscle mass, as well as an improvement in physical performance. Fat mass and weight for height decreased in the initially overweight children, and weight for height increased in underweight children.

Molecular characterization of a novel, widespread nuclear protein that colocalizes with spliceosome components.

We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a approximately 12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.

Acute metabolic effects of human growth hormone on 15N-nitrogen balance in patients with thalassaemia as compared to patients with other types of short stature.

The acute response to various doses of human growth hormone (hGH) was determined in short patients with thalassaemia and compared to that in patients with classic growth hormone deficiency and Turner's syndrome. Nitrogen balance was analyzed using the stable isotope 15N. While patients with growth hormone deficiency responded with a marked nitrogen retention (+2.9 +/- 0.4 to +6.1 +/- 0.6 mg 15N/kg) to small doses of hGH (2 x 3 IU/m2), those with Turner's syndrome had a higher basal balance, but responded much less (+3.1 +/- 0.7 to +3.7 +/- 1.8 mg 15N/kg). They required a double dose of hGH (2 x 6 IU/m2) to achieve a significant retention (+4.1 +/- 1.0 to +7.1 +/- 0.4 mg 15N/kg). The thalassaemic patients responded still less than the patients with Turner's syndrome to 2 x 6 IU/m2 (+7.7 +/- 0.3 to +8.0 +/- 0.4 mg 15N/kg), and even hGH doses up to 2 x 12 IU/m2 had little effect, indicating a relative resistance to hGH. In conclusion, no or little effect is to be expected from long-term hGH treatment at low doses in thalassaemic patients. When it is decided to treat these patients, the dose should be about 4 times higher than a regular replacement dose in growth hormone deficiency.

A single amino acid substitution in the putative redox partner-binding site of P450c17 as cause of isolated 17,20-lyase deficiency.

The molecular basis of isolated 17,20-lyase deficiency was clarified in a newborn male patient from Israel with micropenis, undescended testes, and hormonal pattern consistent with isolated 17,20-lyase deficiency. Analysis of the CYP17 gene revealed the presence of a compound heterozygosity. One allele carries a single base pair deletion (T at position 198 in exon 1) leading to a frame shift with the introduction of a premature stop codon, TGA, at residue 74 in place of Val. The other allele bears a missense mutation due to a single base change, T to G, which substitutes Phe417 with Cys. The proof of heterozygosity was possible via amplification and direct sequencing of genomic DNA fragments from the parents and the healthy brother of the index case. We could demonstrate that the mother is the carrier of the nonsense mutation and the father of the missense mutation. The brother carries two normal alleles for the CYP17 gene. The nonsense mutation gives no functional product. The missense mutation causes the synthesis of a protein that retains 17 alpha-hydroxylase activity but virtually no 17,20-lyase activity. Experiments based on the use of an electron donor independent from enzyme binding (iodosobenzene) demonstrated that the addition of electrons restores, at least in part, in vitro 17,20-lyase activity, with no significant influence on the 17 alpha-hydroxylase activity. This suggests that the electron transfer system plays a major role in the differential regulation of the two P450c17 activities. This is the first case of mutated CYP17 in which the in vitro model corresponds to the in vivo situation.

Identification of a small, very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family.

We report the discovery and molecular characterization of a small and very acidic nucleolar protein of an SDS/PAGE mobility corresponding to Mr 29,000 (NO29). The cDNA-deduced sequence of the Xenopus laevis protein defines a polypeptide of a calculated molecular mass of 20,121 and a pI of 3.75, with an extended acidic region near its C terminus, and is related to the major nucleolar protein, NO38, and the histone-binding protein, nucleoplasmin. This member of the nucleoplasmin family of proteins was immunolocalized to nucleoli in Xenopus oocytes and diverse somatic cells. Protein NO29 is associated with nuclear particles from Xenopus oocytes, partly complexed with protein NO38, and occurs in preribosomes but not in mature ribosomes. The location and the enormously high content of negatively charged amino acids lead to the hypothesis that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles.

Subnormal cortisol response to adrenocorticotropin in isolated partial 17,20-lyase deficiency.

We describe three male children from a Bedouin clan, two of whom are siblings, who have various degrees of incomplete virilization due to isolated 17,20-lyase deficiency. The patients have low (basal and post ACTH or hCG stimulation) plasma testosterone and androstenedione levels. An abnormally high plasma 17-hydroxyprogesterone concentration was detected. A favorable response following local testosterone administration was seen in two patients. Surprisingly, an unexplained flat cortisol response to ACTH test was also noted. Although no biochemical model can yet adequately explain the impairment in cortisol response to ACTH in these patients, it seems prudent to take this lack of cortisol response into consideration. We therefore recommend hydrocortisone supplement during moderate to severe stress.

AND-1, a natural chimeric DNA-binding protein, combines an HMG-box with regulatory WD-repeats.

Using a specific monoclonal antibody (mAb AND-1/23-5-14) we have identified, cDNA-cloned and characterized a novel DNA-binding protein of the clawed toad, Xenopus laevis, that is accumulated in the nucleoplasm of oocytes and various other cells. This protein comprises 1,127 amino acids, with a total molecular mass of 125 kDa and a pI of 5.27. It is encoded by a mRNA of approximately 4 kb and contains, in addition to clusters of acidic amino acids, two hallmark motifs: the amino-terminal part harbours seven consecutive 'WD-repeats', which are sequence motifs of about 40 amino acids that are characteristic of a large group of regulatory proteins involved in diverse cellular functions, while the carboxy terminal portion possesses a 63-amino-acid-long 'HMG-box', which is typical of a family of DNA-binding proteins involved in regulation of chromatin assembly, transcription and replication. The DNA-binding capability of the protein was demonstrated by DNA affinity chromatography and electrophoretic mobility shift assays using four-way junction DNA. Protein AND-1 (acidic nucleoplasmic DNA-binding protein) appears as an oligomer, probably a homodimer, and has been localized throughout the entire interchromatinic space of the interphase nucleoplasm, whereas during mitosis it is transiently dispersed over the cytoplasm. We also identified a closely related, perhaps orthologous protein in mammals. The unique features of protein AND-1, which is a 'natural chimera' combining properties of the WD-repeat and the HMG-box families of proteins, are discussed in relation to its possible nuclear functions.

Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.

To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture.

A childhood fibrolamellar hepatocellular carcinoma with increased aromatase activity and a near triploid karyotype.

We report a 15-year-old boy with hepatocellular carcinoma (HCC) of the fibrolamellar type. He presented with advanced disease and a non-resectable tumor. Clinical features included marked gynecomastia which had been present for 3 years, failure to enter puberty, and failure to thrive. These features might have been due to a high aromatase activity of the tumor. The course of the illness suggested that the tumor had been present for at least 3 years prior to diagnosis. At diagnosis the patient had multiple metastases which included infiltrated ascites. Cytogenetic analysis of the ascites revealed a near triploid karyotype with cell-to-cell variation and an abnormality of chromosome 1 q. This to our knowledge is the first karyotype report of fibrolamellar HCC in a child.

Psychiatric, neuropediatric, and neuropsychological symptoms in a case of hypomelanosis of Ito.

This case report presents a thirteen year-old boy who was diagnosed as having Hypomelanosis of Ito. The developmental history includes severe failure to thrive, and moderate atypical autism as well as diverse clinical and neuropsychological symptoms are present. The pattern of neuropsychological functioning, which can be partially related to the neurophysiological findings, is discussed within the context of existing neuropsychological theories about autistic disorders.