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Assessment is the undergirding of palliative care and of geriatrics care. Both disciplines insist on a comprehensive assessment that includes personal and social aspects of the patient's illness experience. At the same time, both face challenges due to the amount of time and skill needed to encompass such a broad scope and the often heavy illness burden of the patients, which makes interaction stressful or difficult. This article examines question-based assessment instruments in palliative care for elders. Important in all aspects of medicine, reliance on verbal assessments is of special importance in palliative care.

Caspases are critical proapoptotic proteases that execute cell death signals by selectively cleaving proteins at Asp residues to alter their function. Caspases trigger apoptotic chromatin degradation by activating caspase-activated DNase and by inactivating a number of enzymes that sense or repair DNA damage. We have identified the mismatch repair protein MLH1 as a novel caspase-3 substrate by screening small pools of a human prostate adenocarcinoma cDNA library for cDNAs encoding caspase substrates. In this report, we demonstrate that human MLH1 is specifically cleaved by caspase-3 at Asp(418) in vitro. Furthermore, MLH1 is rapidly proteolyzed by caspase-3 in cancer cells induced to undergo apoptosis by treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the topoisomerase II inhibitor etoposide, which damages DNA. Importantly, proteolysis of MLH1 by caspase-3 triggers its partial redistribution from the nucleus to the cytoplasm and generates a proapoptotic carboxyl-terminal product. In addition, we demonstrate that a caspase-3 cleavage-resistant D418E MLH1 mutant inhibits etoposide-induced apoptosis but has little effect on TRAIL-induced apoptosis. These results indicate that the proteolysis of MLH1 by caspase-3 plays a functionally important and previously unrecognized role in the execution of DNA damage-induced apoptosis.

Men with acquired hypogonadotropic hypogonadism treated with testosterone are generally assumed to be infertile. The finding of two such patients with unexpected fertility and normal sperm counts prompted an evaluation of spermatogenesis in additional men with this condition. Case records were initially searched and one similar case with fertility was found. Subsequently, 12 consecutive men with acquired hypogonadotropic hypogonadism were evaluated for gonadal function and sperm production while receiving testosterone. In five of the cases with proven spermatogenesis, exon 10 of the FSH receptor was sequenced to look for activating mutations. The original three cases and four of the subsequent 12 men had sperm concentrations > or = 15 million/ml. Two additional men had concentrations of 1 million/ml and six were azoospermic. Residual LH and FSH levels were slightly higher in those with maintained spermatogenesis prior to testosterone replacement. No activating mutations were found in exon 10 of the FSH receptor in the five cases studied. We conclude that men with acquired hypogonadotropic hypogonadism being treated with testosterone should not be assumed to be sterile, as we have found that more than half have been shown to have persistent spermatogenesis with more than one-third having sperm concentrations > or = 15 million/ml. This may be related to fact that gonadotropin levels in such patients are present, albeit low. Semen analyses in such men should be routinely carried out so that they can be appropriately counseled regarding potential fertility.

Cats are the only nonhuman mammalian species with a high incidence of hyperthyroidism, and a better understanding of the pathogenesis of feline hyperthyroidism is of clinical relevance for veterinary medicine. The etiology of this disease in cats remains controversial. Both an intrinsic autonomy of growth and function of follicular cells as well as an autoimmune-related mechanism have been proposed. To explore the role of the autologous TSH receptor (TSHR) in the pathogenesis of hyperthyroidism in cats, we cloned the coding sequence of the feline TSHR by RT-PCR. The open reading frame consists of 2292 nucleotides and encodes a 763-amino acid protein, one amino acid less than the human TSHR. Species comparison reveals that the cat TSHR is most closely related to the canine TSHR, with 96% identity and 97% similarity in amino acid sequence. cAMP accumulation, inositol phosphate production, and TSH binding were similar in the feline TSHR, compared with the human receptor. Analogous to the human TSHR, the cat TSHR also displays basal constitutive activity. To test the possibility that hyperthyroid cats develop antibodies that stimulate the autologous receptor, transfected cells expressing the feline TSHR were treated with sera or purified IgG obtained from 16 hyperthyroid cats. There was no increase in cAMP-dependent luciferase activity in the hyperthyroid cats, suggesting the absence of stimulatory autoantibodies. These sera were also negative for TSH-binding inhibitory Igs in an RRA. At least in the animals included in this study, there is no evidence for the presence of circulating thyroid stimulating factors as a mechanism underlying the pathogenesis of feline hyperthyroidism, and the findings support a model involving intrinsic autonomy of thyroid follicular cell growth and function.

Constitutively activating mutations of the thyrotropin (TSH) receptor have been identified as a molecular cause of toxic adenomas, nonautoimmune familial hyperthyroidism, and sporadic congenital hyperthyroidism. By analyzing genomic DNA from a toxic adenoma, we detected a novel somatic mutation in codon 601, tyrosine to asparagine (Y601N), a residue located in the carboxyterminal part of the fifth transmembrane helix. This codon is also notable for the presence of a polymorphic variant, Y601H. These two naturally occurring substitutions (Y601N and Y601H) were analyzed together with an artificial mutation, Y601F, to study the role of this residue for receptor function further. Transient transfection assays revealed that the Y601N mutation results in constitutive activation of the cyclic adenosine monophosphate (cAMP) pathway, but that it is unable to couple to Gq/11. Y601H and Y601F do not display basal activity while retaining responsiveness to TSH, but also lose the ability to induce inositol phosphate accumulation in response to TSH. These studies define Y601N as a mutation that selectively activates the cAMP pathway, and they confirm that Y601H is not a silent polymorphism. In conclusion, residue Y601 has an important role for the characteristic constitutive basal activity of the TSH receptor and coupling to Gq/11.

Constitutively activating mutations in the thyrotropin (TSH) receptor have been identified as a major molecular cause of hyperfunctioning thyroid adenomas. A smaller subset of these benign tumors is caused by constitutive activation of the adenylyl cyclase cascade by somatic mutations in the Gsalpha gene. In this study, we analyzed hyperfunctioning thyroid adenomas from seven Brazilian patients for TSH receptor and G(s)alpha gene mutations. Solitary autonomous thyroid adenomas were identified by ultrasound and scintigraphy, and DNA was extracted from adenomatous and periadenomatous tissue. Exons 9 and 10 of the TSH receptor gene, and exons 8 and 9 of the G(s)alpha gene, were amplified by polymerase chain reaction (PCR) and subjected to direct sequence analysis. Six of seven adenomas harbored heterozygous mutations known to confer constitutive activity to the TSH receptor. In one case, aspartate 619 was substituted by glycine (D619G). In four adenomas, alanine 623 was replaced by valine (A623V). Both residues are located in the third intracellular loop. In one instance, aspartate 633 located in the sixth transmembrane domain was replaced by tyrosine (D633Y). In this patient, one allele also contained a change of aspartate 727 to glutamate (D727E). This substitution is thought to be a polymorphic variant of the wild-type but it has also been associated with toxic multinodular goiters. Functional comparison of D727 with E727 did not reveal differences in basal or TSH-stimulated cyclic adenosine monophosphate (cAMP)-dependent luciferase activity in transiently transfected cells. These results demonstrate a high prevalence of activating TSH receptor mutations in toxic adenomas in this small series from Brazil (approximately 86%). These findings are in agreement with reports from other countries with a marginal iodine intake but contrast with studies from regions with a high iodine intake where these mutations appear to be less prevalent.

Sporadic congenital hypothyroidism is most commonly caused by developmental abnormalities of the thyroid gland. More rarely, it is due to defects in gene products involved in the regulation of the hypothalamic-pituitary-thyroid axis or thyroid hormone synthesis. Loss of function mutations in the thyrotropin (TSH) receptor have been shown to result in resistance to biologically active TSH. In complete resistance to TSH, the thyroid gland is hypoplastic and unable to synthesize and secrete sufficient amounts of thyroid hormones. In partial resistance, referred to as euthyroid hyperthyrotropinemia, the size of the gland and the thyroid hormone levels are normal at the expense of an elevated TSH. Four patients with sporadic congenital hypothyroidism and properly located hypoplastic thyroid glands were included in this study. Serum TSH concentrations were 150 mU/L or higher, serum thyroglobulin levels were within normal limits (6.1 to 8.2 ng/mL; normal range: 2.1 to 32 ng/mL), and thyroid autoantibodies were absent. The coding region of the TSHbeta subunit gene, the TSH receptor gene, and exons 8 and 9 of Gsalpha were analyzed by direct sequencing and found to be normal in all patients. One patient was heterozygous for a G to A transition in the TSHbeta gene resulting in a substitution of alanine by threonine at position -7 of the signal peptide. This substitution was also found in her euthyroid father. In addition, Southern analysis of the TSH receptor gene excluded major structural alterations. These findings support previous reports that indicate that TSH resistance is genetically heterogeneous. In addition to mutations in the TSH receptor or the Gsalpha genes, other genetic defects can lead to an identical phenotype. These observations also suggest that TSH receptor mutations might be a relatively rare cause of congenital thyroid hypoplasia.

Pendred's syndrome is an autosomal recessive disease characterized by goiter, impaired iodide organification, and congenital sensorineural deafness. The gene mutated in Pendred's syndrome, PDS (Pendred's syndrome gene), was cloned very recently and encodes the putative sulfate transporter pendrin. Pendred's syndrome may account for up to 10% of the cases with hereditary hearing loss, and pendrin mutations have also been found in a kindred with non-syndromic deafness. In this study, 41 individuals from a large, highly inbred pedigree from Northeastern Brazil were examined for features of Pendred's syndrome. Linkage studies and sequence analysis of the coding region of the PDS gene were performed with DNA from 36 individuals. The index patient, with the classical triad of deafness, positive perchlorate test, and goiter, was found to be homozygous for a deletion of thymidine 279 in exon 3, resulting in a frameshift and a premature stop codon at amino acid 96. This alteration resulted in truncation of the protein in the first transmembrane domain. Two other patients with deafness were found to be homozygous for this mutation; 19 were heterozygous and 14 were homozygous for the wild type allele. Surprisingly, 6 deaf individuals in this kindred were not homozygous for the PDS gene mutation; 3 were heterozygous and 3 were homozygous for the wild type allele, suggesting a probable distinct genetic cause for their deafness. All 3 homozygous individuals for the PDS mutation had goiters. However, goiters were also found in 10 heterozygous individuals and in 6 individuals without the PDS mutation and are most likely caused by iodine deficiency. In conclusion, we identified a novel mutation in the PDS gene causing Pendred's syndrome. The comparison of phenotype and genotype reveals, however, that phenocopies generated by distinct environmental and/or genetic causes are present in this kindred and that the diagnosis of Pendred's syndrome may be difficult without molecular analysis.

The thyroid hormone receptor splice variant, alpha2, is unable to bind thyroid hormone (T3) and has been proposed to function as an endogenous inhibitor of T3 action. In this report, we examined further the DNA sequence requirements for alpha2 binding to thyroid hormone response elements (TREs) in an attempt to identify response elements that mediate potent inhibition by alpha2. Heterodimers of alpha2 and retinoid X receptor were found to bind to a subset of TREs (DR4, direct repeats spaced by 4 bp) in which selected flanking and spacer sequences enhanced interactions with the AGGTCA core binding sequence. Despite the optimization of the TRE-binding sites, alpha2 remained a weak dominant negative inhibitor of TRE-driven transcription. A promoter interference assay was also developed for testing inhibition by alpha2. In these studies, alpha2 blocked gene transcription, but it required cotransfected retinoid X receptor, and it was not as potent as unliganded thyroid hormone receptors. These results led to the hypothesis that alpha2 might be deficient in interactions with nuclear receptor corepressors. Consistent with this view, alpha2 did not silence basal transcription in its native form or when linked to Gal4. Alpha2 also failed to interact with corepressors (NCoR and SMRT) in both gel shift assays and mammalian two-hybrid assays. We conclude that alpha2 is a weak antagonist of thyroid hormone action because it binds weakly to a limited repertoire of response elements, and it does not interact with corepressors. Thus, alpha2 may be able to compete with thyroid hormone receptors for binding to a limited group of target sites, but it is not able to actively inhibit transcription.