Parathyroidectomy for parathyroid carcinoma in renal transplantation.

INTRODUCTION: Hyperparathyroidism (HPT) is a highly prevalent pathology in the chronic renal disease population, which is associated with considerable morbidity, and mortality. The histopathological findings most often reported are solitary adenoma, diffuse hyperplasia, and autonomous hyperplasia. Carcinoma is an unusual cause of primary parathyroid hyperfunction (0.5% to 4% according to data); in renal transplanted patients it is exceptional. We sought to analyze parathyroid gland histology from renal transplant patients in comparison with nontransplanted patients and to report a parathyroid carcinoma case in a renal transplant patient. METHODS: We retrospectively analyzed parathyroidectomies (PTX) and histopathological reports between March 1989 and December 2003. RESULTS: Among 72 PTXs 41 were performed because of primary HPT; 26, secondary HPT; and five, tertiary HPT. Among the 41 primary HPT cases there were two carcinomas (4.88% primary HPT operated patients), one of whom was in a kidney transplant recipient. Among the total number of surgeries, seven were performed in six renal transplant patients, including five diffuse hyperplasia cases; one, nodular hyperplasia with an adenoma focus; and one, parathyroid carcinoma. CONCLUSIONS: Parathyroidectomy indications in the renal transplant population are usually associated with the clinical picture of tertiary HPT, which does not resolve after a functional renal transplant. In spite of this, diffuse hyperplasia, which is associated with secondary HPT, was the most frequent hystological finding. Two carcinomas were observed: one in a renal transplant patient (16.6% parathyroidectomies) and the other in a patient who did not show renal failure. These data coincide with international records.

Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate.

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.

Being-with, doing-with: a model of the nurse-client relationship in mental health nursing.

The authors describe a model of the nurse-client relationship within mental health nursing. This is preceded by a consideration of the literature in reference to the efficacy of nursing models in terms of their use within mental health nursing. A statement of philosophical beliefs is the starting point from which the model is described. The terms 'Doing-With' and 'Being-With' form the nucleus of the nurse-client relationship model that is founded upon the philosophy of Martin Buber. Buber explicated a philosophy of human relationships founded on his concepts of the 'I-Thou' and 'I-It' relationships. Two examples of the model illustrate its principles in clinical practice. The authors draw upon the work of Fawcett to argue the limitations and potential for the future development of the model described.

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.

Analysis of the steroid binding domain of rat steroid 5alpha-reductase (isozyme-1): the steroid D-ring binding domain of 5alpha-reductase.

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5alpha-androst an-17beta-carboxamide is an effective probe of rat steroid 5alpha-reductase (isozyme-1) (5alphaR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5alphaR-1 activity were ultraviolet (UV)-photolyzed with [3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5alphaR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55-56 min. Rechromatography of this fraction using a modified gradient (elution 54-55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15-18 of the 5alphaR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an approximately 12-fold increase in the Km for testosterone, whereas the Km for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.

Site-directed mutagenesis studies of the NADPH-binding domain of rat steroid 5alpha-reductase (isozyme-1) I: analysis of aromatic and hydroxylated amino acid residues.

Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.

Voltage-gated sodium channel genes hscp and hDSC1 of Heliothis virescens F. genomic organization.

We report the genomic sequence of hscp, a sodium channel alpha subunit gene for Heliothis virescens. A 32-kb genomic clone and six independent RT-PCR products covering almost the entire coding region of the gene, contained thirty-one deduced exons with a translation of 1695 residues. Overall amino acid similarity to the para locus of Drosophila melanogaster was 86%. The transcription of the gene was complex. Alternate splicing was evident for five optional exons and a pair of mutually exclusive exons. A number of alternatively spliced mRNA revealed a deduced translation product that included only the first homology domain. We also report the first partial sequence for hDSC1, a presumed orthologous of the DSC1 sodium channel alpha subunit gene of D. melanogaster.

Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes.

Programmed cell death is an important regulatory event in spermatogenesis. However, the molecular events governing apoptosis have not been characterized. Using the Leydig cell-specific toxin ethane dimethanesulfonate (EDS) to withdraw androgen support, we have investigated the relationship between apoptosis and apoptosis-related genes. Adult male Sprague-Dawley rats were injected (i.p.) with 100 mg/kg EDS and killed at times of androgen depletion 2, 5, and 8 days postinjection. A 24-fold increase in the apoptotic index 8 days after EDS administration was demonstrated in tissue sections by in situ end-labeling of fragmented DNA. Leydig cell death and androgen withdrawal were confirmed by the absence of 3beta-hydroxysteroid dehydrogenase in testes from animals treated with EDS for 2 days. After androgen withdrawal, there were no significant changes in the levels of clusterin, Bcl-xl, Bak, and Bad. However, the expression of Bcl-2 and Bax was up-regulated at 8 days after EDS administration. The induction of Bax at this time suggests that it may play a role in germ cell apoptosis following androgen withdrawal. The concomitant elevation in Bcl-2 expression may represent a survival mechanism for the remaining germ cells. There was also a decline in the expression of Fas-L and Fas-R in the pachytene spermatocytes and spermatids. Fas-R was also present in Sertoli cells, although Fas-L staining was minimal. As the colocalization of Fas-L and Fas-R correlates with the germ cell types that die in response to androgen withdrawal, the potential exists for apoptosis in the rat spermatogenic epithelium to be regulated by the Fas pathway.

Antisense oligonucleotides for target validation and gene function determination.

Antisense technology is attracting attention from the biotechnology and pharmaceutical industries because it provides a high-throughput and systematic approach to drug target validation and gene function discovery. Antisense represents a logical approach to gene function analysis and discovery as it is specific, broadly applicable, and can be designed with minimal information (ie, expressed sequence tags). This technology in combination with other emerging technologies (eg, microarray technology), will enable efficient 'mining' of the sequence data generated by the human genome project. This review addresses recent advances in the antisense field and discusses the potential use of antisense technology for functional genomics approaches.

Apoptosis and expression of apoptotic regulators in the human testis following short- and long-term anti-androgen treatment.

Apoptosis and its augmentation by androgen withdrawal is an important event in the testis. In other tissues apoptosis is regulated by genes belonging to the bcl-2 family. However, little is known about these pathways in the human testes. Human testes were obtained from patients with prostate cancer, undergoing orchidectomy for permanent androgen ablative treatment. The patients were either untreated or had previously received short- or long-term anti-androgen therapy by cyproterone acetate or GnRH agonist (goserelin). In comparison with untreated patients, testicular testosterone concentrations were reduced by 83% in patients treated with cyproterone acetate and by 99% in patients treated with goserelin. Apoptotic cells were identified in tissue sections by in-situ end labelling of fragmented DNA. The expression of Bcl-2, Bcl-xl, Bax, p53 and poly(ADP) ribose polymerase (PARP) was demonstrated in tissue extracts by Western blotting. Apoptotic germ cells were present in the spermatogenic epithelium of untreated patients and patients who received short-term anti-androgen treatment. There were few or no apoptotic cells in the seminiferous tubules following long-term anti-androgen treatment. Following short-term treatment, the concentrations of the apoptosis-related proteins examined did not change. However, in the long-term treated testes, Bcl-xl and PARP expression declined, Bax and p53 protein concentrations were unchanged, and Bcl-2 was up-regulated. In conclusion, apoptosis occurs in spermatogenic cells of the human testis and may contribute to the regulation of germ cell populations. The apoptosis-related gene products which have been described in other tissues are present in the human testis and are modulated by androgenic stimuli.

Leydig cell apoptosis in the rat testes after administration of the cytotoxin ethane dimethanesulphonate: role of the Bcl-2 family members.

Ethane dimethanesulphonate (EDS) is cytotoxic to Leydig cells in the adult rat. To investigate the role and regulation of apoptosis in the Leydig cell, EDS (100 mg/kg i.p.) was administered to adult male rats and the testes examined 6, 12, 18, 24, 48 and 72 h later. Numbers of Leydig cells, identified by 3 beta-hydroxysteroid dehydrogenase immuno-histochemistry started to fall by 12 h after EDS injection and were almost undetectable by 72 h. Apoptotic cells in the interstitium, visualised by in situ end labelling of DNA, increased in number to reach a maximum 24 h after injection of EDS, and were undetectable by 72 h. In many tissues the apoptosis-related gene products act in cohort: Bcl-2 and Bcl-xl promoting survival of a cell, whilst Bax promotes cell death often positively regulated by the tumour-suppressor gene p53. Western blot analysis showed that: (1) Bcl-2 and p53 were absent from interstitial Leydig cells but were expressed in the seminiferous tubules. (2) Bax protein although expressed in the interstitium was not present in the Leydig cells. (3) Bcl-xl in Leydig cells was transiently increased after EDS. In conclusion, EDS kills Leydig cells by apoptosis; however the control of Leydig cell death does not involve p53 or the Bcl-2 family members but may require other gene products yet to be identified.

Apoptosis and related genes in the rat ventral prostate following androgen ablation in response to ethane dimethanesulfonate.

BACKGROUND: Following androgen withdrawal, regression of the prostate is characterized by apoptotic cell death. The molecular events governing this process have not been fully characterized. METHODS: Using ethane-1,2-dimethanesulfonate (EDS) to induce androgen ablation, we investigated the role of the Bcl-2 family members and Fas pathway in this phenomenon. Prostates were examined from adult male rats injected with 100 mg/kg EDS and killed 2, 5, and 8 days later. RESULTS: Regression of the prostate was evident as a time-dependent decrease in weight. The number of apoptotic cells identified by in situ end labeling was maximal after 5 days of treatment. There was no statistically significant change in the expression of Bax, Bcl-xl, Bcl-2, or p53 following androgen withdrawal. In contrast, 5 days post-EDS treatment, testosterone-repressed prostate message (TRPM-2) and Fas-R expression were induced. There was a decline in Fas-L levels 8 days after EDS administration. CONCLUSIONS: This study extends previous work which has shown that androgen withdrawal induces apoptosis in the prostate. We have shown that although p53 and the Bcl-2 family members examined in this study do not seem to be important in this process, the Fas pathway may play a role in apoptosis of the ventral prostate in response to androgen ablation.

Effect of TNF-alpha antisense oligomers on cytokine production by primary murine alveolar macrophages.

Antisense oligomers can inhibit expression of a single gene in a sequence-specific manner. As a result, these sequences are being developed both as powerful experimental tools in the laboratory and as a novel class of therapeutic agents. In this study, we evaluated a panel of morpholino antisense (M-AS) oligomers for their ability to inhibit tumor necrosis factor-alpha (TNF-alpha) production by primary murine alveolar macrophages (AMs) and compared them with the more commonly used phosphorothioate oligonucleotides (S-AS). We found that 25 microM of morpholino oligomers whose sequence spanned the AUG (M-AS 2, M-AS 2me, and M-AS 5) start codon of TNF-alpha significantly inhibited TNF production on stimulation by both lipopolysaccharides (LPS) (36.6 +/- 3.2%, 27.3 +/- 3.0%, and 37.7 +/- 2.0% inhibition, respectively), whereas S-AS targeted toward the same region were ineffective. M-AS 2 and M-AS 2me also significantly inhibited TNF production in AMs stimulated by adherence to a solid substrate (28.7 +/- 2.2% and 29.4 +/- 8.3% inhibition, respectively). Increasing the concentration of M-AS 2 and M-AS 2me to 50 microM improved their efficacy in both LPS-stimulated (42.7 +/- 1.5% and 45.9 +/- 2.1% inhibition, respectively) and adherence-stimulated (52.6 +/- 0.7% and 41.7 +/- 2.9% inhibition, respectively) AMs. In contrast, we showed that neither an antisense sequence targeted to a region upstream of the AUG site (M-AS 4) nor the nonsense control sequences M-NS 1 and M-NS 2 significantly inhibited TNF-alpha production by AMs on exposure to either stimulus. The data indicate that morpholino oligomers inhibit TNF-alpha production by murine AMs in a sequence-dependent and dose-dependent manner.

Uptake of suramin by human microvascular endothelial cells.

We have demonstrated for the first time that suramin is taken up by human dermal microvascular endothelial (HMEC-1) cells by an active process involving the caveolae system. The uptake of suramin was time-dependent and reduced by more than 90% when incubated in the presence of albumin or at 4 degrees C. Suramin uptake was also inhibited when incubated in the presence of filipin and digitonin, both potent cholesterol-binding agents, but not in the presence of probenecid. The [3H]suramin taken up by the HMEC-1 cells was located primarily within the nucleus, followed by the cytoplasmic fraction. The presence of suramin in these cellular compartments suggests that this drug may act through intracellular mechanisms.

A valine421 to methionine mutation in IS6 of the hscp voltage-gated sodium channel associated with pyrethroid resistance in Heliothis virescens F.

Multiple mutations in a locus encoding a voltage-gated sodium channel have been predicted for pyrethroid resistance in insects. Previously we reported a mutation associated with pyrethroid resistance, Leu1029 to His, in domain II transmembrane segment S6 (IIS6) of the Heliothis virescens F. sodium channel (para homologue) hscp locus. Sequence analysis of additional resistance haplotypes 5' to this mutation in the hscp locus has uncovered a G to A transition leading to a Val to Met mutation at amino acid position 421 in IS6 (V421M, numbering from Drosophila para). The V421M mutation is found only in a unique resistant haplotype, but not in two susceptible and a distinct resistant haplotype carrying the L1029H mutation. Implications of this finding in the evolution and mechanisms of pyrethroid resistance are discussed.

Comparison of efficacy of antisense oligomers directed toward TNF-alpha in helper T and macrophage cell lines.

The authors investigated the use of antisense oligomers specific for TNF-alpha (AS-2) and nonsense control oligomers (NS) in T cells (HT2) and macrophages (RAW264.7), comparing three distinct chemical formulations. Phosphorothioate antisense (S-AS) caused sequence-specific inhibition of TNF-alpha production by activated HT2s (0.5 microM S-AS 2 vs S-NS: 31.4 +/- 1.2%, 4.2 +/- 3.2% inhibition, respectively). In contrast, S-AS were ineffective in RAW264.7, despite greater uptake as measured with fluorescent S-oligonucleotides. Furthermore, differences in efficacy of S-AS (HT2 > RAW) were not attributable to differences in the pinocytic (HT2 = RAW) or adsorptive endocytic (RAW > HT2) pathways implicated in oligonucleotide uptake, suggesting an important role for intracellular events after antisense uptake. Morpholino oligomers (M-AS), in contrast, were more effective in RAW264.7 than in HT2 (32.6 +/- 2.6% vs 12.3 +/- 0.5% inhibition), consistent with uptake experiments using fluorescent M-oligomers. Phosphodiester oligonucleotides were ineffective in both cell types. It was concluded that antisense efficacy in leukocytes varies according to type of oligomer, cell target and intracellular processing event(s).

Leydig cell apoptosis in response to ethane dimethanesulphonate after both in vivo and in vitro treatment.

The biological effects of ethane dimethanesulphonate (EDS) are unique since cytotoxicity in the adult rat is almost exclusively confined to the Leydig cells. For this reason, EDS has been used extensively to investigate the physiological role of the Leydig cell and its products. Experiments were conducted to determine whether the Leydig cell will undergo apoptosis in response to EDS or methylprednisolone (MP), a glucocorticoid known to cause apoptosis in a number of other cell types. Percoll-purified Leydig cells were incubated for 24 hours with EDS (750 micrograms/ml), at which time the cells attached to the culture plate became rounded up while control cells were flattened and polyhedral. Following incubation with EDS or MP (10 microM), cells that became detached from the plate were characteristically apoptotic when stained with the fluorescent DNA dye, acridine orange. These cells had shrunk and the nuclear chromatin had become condensed, which is an early characteristic of apoptosis in other cells; eventually, apoptotic bodies formed, reflecting a later apoptotic stage. Electrophoresis of DNA extracted from the treated Leydig cells exhibited the characteristic ladder of the apoptotic process. Increasing the concentration of EDS or MP resulted in a dose-dependent increase in the incidence of apoptosis that reached a maximum of 25% (EDS) or 12% (MP) of detached cells. Administration of EDS in vivo caused a 20-fold increase in the number of apoptotic cells observed in interstitial cell preparations. In conclusion, the data indicates that programmed cell death, apoptosis, can occur in the Leydig cell and that this is the likely mechanism by which EDS kills the cells in vivo and in vitro.

Expression of rat steroid 5 alpha-reductase (isozyme-1) in Spodoptera frugiperda, SF21, insect cells: expression of rat steroid 5 alpha-reductase.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.